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Similar anxiety-like responses in male and female rats exposed to repeated withdrawals from ethanol
Abstract
Previous research indicated that male rats exhibited anxiety-like behavior following withdrawal from chronic ethanol exposure. This behavior, as well as other symptoms of withdrawal such as seizure susceptibility, may be sensitized in male rats repeatedly withdrawn from chronic ethanol exposure. Because there are sex differences in some alcohol effects, the present study explored whether male and female rats would respond differently to a course of repeated ethanol withdrawals. Similarly aged male and female rats were exposed to a control liquid diet or a diet containing ethanol (7% w/v). Ethanol-exposed rats had only one 5-day cycle of exposure or three cycles, with 2 days of control diet (CD) between cycles. At 5 h after the final ethanol was removed, the rats were placed as same-sexed pairs in the social interaction test; approximately half of the rats were tested later in the elevated plus maze. Rats withdrawn from ethanol after three cycles exhibited reduced the time spent in social interaction and general activity in the social interaction test and reduced the open and closed arm entries in the elevated plus maze. There were no sex differences in these effects. However, male rats exhibited a small anxiety-like response after one cycle of 5 days' exposure to ethanol and female rats did not. Thus, there are no sex differences in the three-cycle multiple-withdrawal paradigm, but there may be differences after briefer exposures.
... Men are also more likely than women to report anxiety during alcohol withdrawal (Deshmukh et al., 2003). Although withdrawal-induced anxiety-like behavior has been demonstrated in male and female rats using the EPM and social interaction test (Morales et al., 2015(Morales et al., , 2018Overstreet et al., 2004), females may require longer or more intense ethanol exposures to produce anxiogenisis during withdrawal (Overstreet et al., 2004). In other models of anxiety like the novelty-suppressed feeding task, withdrawal-induced anxiety-like behavior is observed exclusively in male mice (Jury et al., 2017). ...
... Men are also more likely than women to report anxiety during alcohol withdrawal (Deshmukh et al., 2003). Although withdrawal-induced anxiety-like behavior has been demonstrated in male and female rats using the EPM and social interaction test (Morales et al., 2015(Morales et al., , 2018Overstreet et al., 2004), females may require longer or more intense ethanol exposures to produce anxiogenisis during withdrawal (Overstreet et al., 2004). In other models of anxiety like the novelty-suppressed feeding task, withdrawal-induced anxiety-like behavior is observed exclusively in male mice (Jury et al., 2017). ...
... Preclinical studies have yielded mixed results regarding sex differences in withdrawal-induced anxiety-like behavior. Some studies have found that chronic ethanol does not induce anxiety-like behavior in female mice using the novelty-suppressed feeding test (Jury Field Code Changed et al., 2017) or that female rats require longer alcohol exposures to increase anxiety-like behavior using the social interaction test (Overstreet et al., 2004), consistent with the delayed neurophysiological changes in the BLA. However, other studies have showed that rats of both sexes develop anxiety-like behavior in the EPM after only 3 days of CIE and 24 hours of withdrawal (Morales et al., 2015(Morales et al., , 2018. ...
The basolateral amygdala (BLA) is intimately involved in the development of neuropsychiatric disorders such as anxiety and alcohol use disorder (AUD). These disorders have clear sex biases with women more likely to develop an anxiety disorder and men more likely to develop AUD. Preclinical models have largely confirmed these sex-specific vulnerabilities and emphasize the effects of sex hormones on behaviors influenced by the BLA. This review will discuss sex differences in BLA-related behaviors and highlight potential mechanisms mediated by altered BLA structure and function, including the composition of GABAergic interneuron subpopulations, glutamatergic pyramidal neuron morphology, glutamate/GABA neurotransmission, and neuromodulators. Further, sex hormones differentially organize dimorphic circuits during sensitive developmental periods (organizational effects) and initiate more transient effects throughout adulthood (activational effects). Current literature indicates that estradiol and allopregnanolone, a neuroactive progestogen, generally reduce BLA-related behaviors through a variety of mechanisms including activation of estrogen receptors or facilitation of GABAA-mediated inhibition, respectively. This enhanced GABAergic inhibition may protect BLA pyramidal neurons from the excitability associated with anxiety and alcohol withdrawal. Understanding sex differences and the effects of sex hormones on BLA structure and function may help explain sex-specific vulnerabilities in BLA-related behaviors and ultimately improve treatments for anxiety and AUD.
... Intracerebroventricular or systemic CRF receptor antagonist administration also blocked the potentiation of anxiety-like responses to stressors that were observed during protracted abstinence from chronic alcohol (Valdez et al., 2003;Breese et al., 2005;Sommer et al., 2008). A CRF 1 receptor antagonist prevented the sensitization of withdrawal-induced anxiety, supporting a between-system neuroadaptation (Overstreet et al., 2004a). Additionally, systemic CRF 1 receptor antagonist administration blunted the hyperalgesic response to alcohol withdrawal (Edwards et al., 2012b). ...
... Rats drink alcohol excessively during acute and protracted abstinence from intermittent, high, sustained alcohol via a liquid diet or vapor exposure. Systemic injections of small-molecule CRF 1 receptor antagonists blocked the increase in alcohol intake that was associated with acute withdrawal (Knapp et al., 2004;Overstreet et al., 2004a;Funk et al., 2007) and protracted abstinence (Gehlert et al., 2007). Corticotropin-releasing factor receptor antagonists also reduced binge-like, excessive drinking and stress-induced increases in alcohol intake (Lowery et al., 2008;Cippitelli et al., 2012;Lowery-Gionta et al., 2012;Simms et al., 2014). ...
... Male rats also exhibit greater physical signs of withdrawal, such as greater seizure susceptibility, compared with female rats (Devaud and Chadda, 2001). However, male rats 184 Koob also exhibit greater motivational signs of withdrawal, such as anxiety-like responses in the social interaction test (Varlinskaya and Spear, 2004) and elevated plus maze after one cycle of exposure to an alcohol liquid diet (Overstreet et al., 2004a), and greater increases in acoustic startle responses than female rats (Reilly et al., 2009). Male rats also exhibit greater hangoverlike anxiety-like responses (acute alcohol withdrawal; Varlinskaya and Spear, 2004). ...
Compulsive drug seeking that is associated with addiction is hypothesized to follow a heuristic framework that involves three stages (binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation) and three domains of dysfunction (incentive salience/pathologic habits, negative emotional states, and executive function, respectively) via changes in the basal ganglia, extended amygdala/habenula, and frontal cortex, respectively. This review focuses on neurochemical/neurocircuitry dysregulations that contribute to hyperkatifeia, defined as a greater intensity of negative emotional/motivational signs and symptoms during withdrawal from drugs of abuse in the withdrawal/negative affect stage of the addiction cycle. Hyperkatifeia provides an additional source of motivation for compulsive drug seeking via negative reinforcement. Negative reinforcement reflects an increase in the probability of a response to remove an aversive stimulus or drug seeking to remove hyperkatifeia that is augmented by genetic/epigenetic vulnerability, environmental trauma, and psychiatric comorbidity. Neurobiological targets for hyperkatifeia in addiction involve neurocircuitry of the extended amygdala and its connections via within-system neuroadaptations in dopamine, enkephalin/endorphin opioid peptide, and γ-aminobutyric acid/glutamate systems and between-system neuroadaptations in prostress corticotropin-releasing factor, norepinephrine, glucocorticoid, dynorphin, hypocretin, and neuroimmune systems and antistress neuropeptide Y, nociceptin, endocannabinoid, and oxytocin systems. Such neurochemical/neurocircuitry dysregulations are hypothesized to mediate a negative hedonic set point that gradually gains allostatic load and shifts from a homeostatic hedonic state to an allostatic hedonic state. Based on preclinical studies and translational studies to date, medications and behavioral therapies that reset brain stress, antistress, and emotional pain systems and return them to homeostasis would be promising new targets for medication development. SIGNIFICANCE STATEMENT: The focus of this review is on neurochemical/neurocircuitry dysregulations that contribute to hyperkatifeia, defined as a greater intensity of negative emotional/motivational signs and symptoms during withdrawal from drugs of abuse in the withdrawal/negative affect stage of the drug addiction cycle and a driving force for negative reinforcement in addiction. Medications and behavioral therapies that reverse hyperkatifeia by resetting brain stress, antistress, and emotional pain systems and returning them to homeostasis would be promising new targets for medication development.
... Rodents provide an important model species for studying the neural and genetic basis of AUDs, and sex differences in ethanol drinking and other behavioral responses to ethanol have been well documented both in rats and mice (reviewed in Becker & Koob, 2016;Lynch et al., 2002). For example, previous studies have shown that, as compared to males, female rodents exhibit enhanced place-preference for ethanol (Torres, Walker, Beas, & O'Dell, 2014) and reduced signs of withdrawal, including attenuated anxiety-like behavior and plasma corticosterone levels (e.g., Alele & Devaud, 2007;Devaud & Chadda, 2001;Janis, Devaud, Mitsuyama, & Morrow, 1998;Lopez, Grahame, & Becker, 2011;Overstreet, Knapp, & Breese, 2004;Reilly, Koirala, & Devaud, 2009;Strong, Kaufman, Crabbe, & Finn, 2009;Tanchuck-Nipper et al., 2015;Varlinskaya & Spear, 2004;Veatch, Wright, & Randall, 2007) (but see Morales, McGinnis, & McCool, 2015). Furthermore, many (e.g., Aufrère, Le Bourhis, & Beaugé, 1997;Lancaster & Spiegel, 1992;Lancaster, Brown, Coker, Elliott, & Wren, 1996;Li & Lumeng, 1984;McKinzie et al., 1998;Moore & Lynch, 2015;Vengeliene, Vollmayr, Henn, & Spanagel, 2005;Vetter, Doremus-Fitzwater, & Spear, 2007), although not all (e.g., Schramm-Sapyta et al., 2014;Varlinskaya & Spear, 2002), prior studies have shown that females drink more ethanol than males. ...
... A heightened level of anxiety during withdrawal is thought to be a major factor underlying relapse and can be observed in rodents following chronic ethanol exposure (Koob, 2003). However, consistent with prior studies in rats using other chronic ethanol models (Overstreet et al., 2004;Reilly et al., 2009), we found that female C57BL/6J mice were resistant to the anxiety-like effects of CIE. Specifically, while CIE-exposed males exhibited increased anxiety-like behavior in two separate assays, NSF and marble burying, which persisted for up to six days after exposure, females did not. ...
... interpretation of these data is that female mice are protected from chronic ethanol effects on drinking, as they are from its effects on anxiety-like behavior and withdrawal hyperexcitability (Alele & Devaud, 2007;Devaud & Chadda, 2001;Janis et al., 1998;Lopez et al., 2011;Overstreet et al., 2004;Reilly et al., 2009;Strong et al., 2009;Tanchuck-Nipper et al., 2015;Varlinskaya & Spear, 2004;Veatch et al., 2007). Indeed, it is possible that these various behavioral effects are related, such that elevated withdrawal severity and anxiety-like behavior is a driver of elevated drinking in males that is absent in females. ...
Rates of alcohol use disorders (AUDs) differ between men and women, and there is also marked variation between sexes in the effects of acute and chronic alcohol. In parallel to observations in humans, prior studies in rodents have described male/female differences across a range of ethanol-related behaviors, including ethanol drinking. Nonetheless, there remain gaps in our knowledge of the role of sex in moderating the effects of ethanol, particularly in models of chronic ethanol exposure. The goal of the current study was to assess various behavioral sequelae of exposing female C57BL/6J mice to chronic intermittent ethanol (CIE) via ethanol vapors. Following four weeks of CIE exposure, adult male and female mice were compared for ethanol drinking in a two-bottle paradigm, for sensitivity to acute ethanol intoxication (via loss of righting reflex [LORR]) and for anxiety-like behaviors in the novelty-suppressed feeding and marble burying assays. Next, adult and adolescent females were tested on two different two-bottle drinking preparations (fixed or escalating ethanol concentration) after CIE. Results showed that males and females exhibited significantly blunted ethanol-induced LORR following CIE, whereas only males showed increased anxiety-like behavior after CIE. Increased ethanol drinking after CIE was also specific to males, but high baseline drinking in females may have occluded detection of a CIE-induced effect. The failure to observe elevated drinking in females in response to CIE was also seen in females exposed to CIE during adolescence, regardless of whether a fixed or escalating ethanol-concentration two-bottle procedure was employed. Collectively, these data add to the literature on sex differences in ethanol-related behaviors and provide a foundation for future studies examining how the neural consequences of CIE might differ between males and females.
... Whereas withdrawal from three cycles of CIA exposure facilitated anxiety-like behavior, continuous exposure to alcohol did not (Overstreet et al., 2002). This facilitation of anxiety during a shorter repeated exposure to CIA (Overstreet et al., 2002;Overstreet, Knapp, & Breese, 2004a, 2004b but not following withdrawal from continuous alcohol provided a further conceptual foundation for an accumulated adaptation being induced by repeated CIA exposures that could contribute to the etiology of alcoholism (Ballenger & Post, 1978;. Based upon this framework, the present review describes our current understanding of how such CIA exposures interact with CRF and neuroimmune neural mediation of stress to sustain this persistent adaptation that follows chronic repeated alcohol exposures. ...
... Finding that a CRF1RA mitigated the sensitization of withdrawal anxiety induced by the CIA exposure (Knapp, Overstreet, Angel, et al., 2007;Knapp et al., 2004;Overstreet et al., 2003Overstreet et al., , 2004aOverstreet et al., , 2004bOverstreet et al., , 2005 and that a BZD inverse agonist (DMCM) substituted for the earlier withdrawals of CIA exposure (Knapp, Overstreet, Angel, et al., 2007) raised the possibility that the initial withdrawals from the CIA protocol induced a "stress-like" response that initiated CRF release (Bale & Vale, 2004;Dunn & Berridge, 1990a, 1990bImaki, Shibasaki, Hotta, & Demura, 1993;Vale, Spiess, Rivier, & Rivier, 1981). To determine whether activation of a neural "stress" pathway was a critical component in the CIA-induced anxiety during withdrawal (Overstreet et al., 2002), single bi-weekly stresses were substituted for the two initial weekly repeated withdrawals of CIA exposure. ...
... Error bars, letters above bars, and CMC are described in A above. Adapted from Overstreet et al., 2004aOverstreet et al., , 2004b. 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 whether sensitization of negative symptoms (anxiety) would occur upon withdrawal from the single 5 days of alcohol exposure that alone induced no behavioral change during withdrawal (Breese et al., , 2008. ...
... Stress can be a key factor in inducing alcohol dependence. Clinical studies have shown that stressful events increase alcohol intake [23] and the risk of relapse to alcohol use by enhancing craving [21]. Experimentally studies on the relationship between stress and alcohol seeking usually use a variety of types of stress, such as footshock stress, immobilization stress, and isolation stress [17]. ...
... In the present study, we found that 24 [23]. The possible mechanisms underlying the observed delayed fear memory and anxiety may be the enhanced HPA negative feedback occurring on the seventh day [2,8,21]. It is worth mentioning that, EPM is commonly used in anxiety behavior study with a single evaluation, and tolerance can occur easily when making repeated measurements. ...
Aims:
Previous studies showed that exposure to certain types of stressors enhance the rewarding effects of many drugs of abuse, including alcohol; however, no systematic study has investigated the role of single prolonged stress (SPS) in acquisition of alcohol conditioned place preference (CPP). The purpose of this study was to examine whether SPS would facilitate the acquisition of alcohol CPP in rats.
Main methods:
Male Sprague-Dawley rats were randomly assigned to either SPS exposure condition or no exposure condition. Freezing behavior and Elevated plus maze (EPM) were employed to evaluate PTSD-like symptoms induced by SPS. Further, using unbiased procedure, CPP conditioning was conducted with alcohol (2g/kg).
Key findings:
SPS significantly enhanced freezing behavior of rats, decreased percentages (%) of both time spent and number of entry into the open arms, and facilitated the acquisition of alcohol CPP without inhibiting rats' activity.
Significance:
Our findings suggest that SPS plays an important role in alcohol dependence, and CPP paradigm with SPS may be useful for exploring the rewarding mechanism of alcohol with regard to the interaction between alcohol and post-traumatic stress disorder (PTSD).
... These models have traditionally been used to model the somatic symptoms associated with withdrawal in alcohol dependence in humans (e.g., Isbell et al., 1955; Victor & Adams, 1953). Over time, refinements have been made to facilitate the study of motivational symptoms associated with withdrawal in alcohol dependence (e.g., O'Dell et al., 2004; Overstreet et al., 2004; Roberts et al., 1996 Roberts et al., , 2000). Perhaps the two most frequently used methods of producing alcohol dependence are alcohol vapor inhalation and alcohol liquid diet. ...
... More recently, it was shown that an indirect GABA agonist, gabapentin suppresses alcohol drinking in rats made dependent on alcohol via vapor inhalation and liquid diet (Roberto et al., 2008), and a subsequent human laboratory study confirmed that gabapentin reduces alcohol craving in dependent individuals (Mason et al., 2009). Other labs have developed elegant models for dependence induction via alcohol liquid-diet, and use this model for high-throughput screening of compounds that target the high anxiety state produced by chronic alcohol exposure and repeated withdrawals (Breese et al., 2004Breese et al., , 2005 Overstreet et al. 2002 Overstreet et al. , 2003 Overstreet et al. , 2004). For example, the GABA B agonist baclofen, an effective anxiolytic in PTSD and alcoholic humans (Addolorato et al., 2002; Drake et al., 2003), blocks sensitization of anxiety-like behavior in rats consuming intermittent alcohol liquid-diet (Knapp et al., 2007), and also selectively suppresses alcohol drinking in rats made dependent on alcohol via vapor inhalation (Walker & Koob, 2007). ...
The purpose of the present investigation was to more clearly define blood-alcohol parameters associated with alcohol dependence produced by alcohol vapor inhalation and alcohol-containing liquid diet.
Alcohol levels in blood and brain were compared during and after 4 hours of acute alcohol vapor exposure; also, brain-alcohol levels were assessed in alcohol-exposed (14-day alcohol vapor) and alcohol-naïve rats during and after 4 hours of acute alcohol vapor exposure. A separate group of rats were implanted with i.v. catheters, made dependent on alcohol via vapor inhalation, and tested for operant alcohol responding; blood-alcohol levels (BALs) were measured throughout operant alcohol drinking sessions during alcohol withdrawal. A final group of rats consumed an alcohol-liquid diet until they were dependent, and those rats were then tested for operant behavior at various withdrawal time points; BALs were measured at different withdrawal time points and after operant sessions.
Blood- and brain-alcohol levels responded similarly to vapor, but brain-alcohol levels peaked at a higher point and more slowly returned to zero in alcohol-naïve rats relative to alcohol-exposed rats. Alcohol vapor exposure also produced an upward shift in subsequent operant alcohol responding and resultant BALs. Rats consumed large quantities of alcohol-liquid diet, most of it during the dark cycle, sufficient to produce high blood-alcohol levels and elevated operant alcohol responding when tested during withdrawal from liquid diet.
These results emphasize that the key determinants of excessive alcohol drinking behavior are the BAL range and pattern of chronic high-dose alcohol exposure.
... Although no animal model of addiction fully emulates the human condition, the models do permit the investigation of the specific elements of the addiction process [1]. Multiple methods have been developed to produce BALs in the 100-300 mg% range for 12+ h per day, associated with somatic and affective withdrawal symptoms, but they either use forced alcohol liquid diet [2][3][4], intragastric ethanol intubation [5][6][7][8], passive exposure to ethanol vapor [9][10][11][12][13][14][15], or selected inbred lines of ethanol-preferring rats or mice [16,17]. ...
A major limitation of the most widely used current animal models of alcohol dependence is that they use forced exposure to ethanol including ethanol-containing liquid diet and chronic intermittent ethanol (CIE) vapor to produce clinically relevant blood alcohol levels (BAL) and addiction-like behaviors. We recently developed a novel animal model of voluntary induction of alcohol dependence using ethanol vapor self-administration (EVSA). In this model, naive outbred rats given intermittent access to alcohol vapor self-administration exhibit BAL in the 150-300 mg% range and develop somatic signs of withdrawal during acute abstinence. However, it is unknown whether EVSA leads to an escalation of alcohol drinking per se , and whether such escalation is associated with neuroadaptations in brain regions related to stress, reward, and habit. To address these issues, we compared the levels of alcohol drinking during withdrawal between rats passively exposed to alcohol (CIE) or voluntarily exposed to EVSA and measured the number of Fos+ neurons during acute withdrawal (16 h) in the central nucleus of the amygdala (CeA), dorsomedial striatum (DMS), dorsolateral striatum (DLS), nucleus accumbens core (Nacc), periaqueducal grey area (PAG), lateral Habenula (HbL), and the paraventricular nucleus of the thalamus (PVT). The rats were first trained to orally self-administer alcohol in standard operant chambers and then divided in 4 groups (CIE, CI-Air, EVSA and Air-SA) and exposed to intermittent ethanol vapor (passive or active) or intermittent air (passive or active) for 8 h/day, 3 days a week. CIE and EVSA rats exhibited similar BAL (150-300 mg% range) and similar escalation of alcohol drinking during withdrawal, while no changes in terms of drinking were observed in the air exposed rats. CIE and EVSA also increased the motivation for alcohol compared to their respective air control groups under a progressive ratio schedule of reinforcement. Acute withdrawal from EVSA and CIE recruited a similar number of Fos+ neurons in the CeA, however, acute withdrawal from EVSA recruited a higher number of Fos+ neurons in every other brain region analyzed compared to acute withdrawal from CIE. Moreover, acute withdrawal from EVSA specifically recruited the DMS and PVT, a pattern not observed in CIE rats.
In summary, these results demonstrate that EVSA produces similar escalation of alcohol drinking, motivation to drink, and blood-alcohol levels than the CIE model, while letting animals voluntary initiate alcohol exposure and maintain alcohol dependence. Moreover, while the behavioral measures of alcohol dependence between the voluntary (EVSA) and passive (CIE) model was similar, the recruitment of neuronal ensembles during acute withdrawal was very different with a higher recruitment of Fos+ neurons in key brain regions important for stress, reward and habit-related processes. The EVSA model may be particularly useful to unveil the neuronal networks and pharmacology responsible for the voluntary induction and maintenance of alcohol dependence and may improve translational studies by providing preclinical researchers with an animal model with better face validity for alcohol use disorder.
... Although no animal model of addiction fully emulates the human condition, the models do permit the investigation of the specific elements of the addiction process (Rodd et al., 2004). Multiple methods have been developed to produce BALs in the 100-300 mg/dL range for 12+ h per day, associated with somatic and affective withdrawal symptoms, but they use either forced alcohol liquid diet (Gilpin et al., 2009;Lieber and DeCarli, 1982;Overstreet et al., 2004), intragastric ethanol intubation (Aujla et al., 2013;Braconi et al., 2010;de Guglielmo et al., 2015;Sidhpura et al., 2010), passive exposure to ethanol vapor (Gilpin et al., 2008;Goldstein and Pal, 1971;O'Dell et al., 2004;Rimondini et al., 2002;Roberts et al., 1996;Schulteis et al., 1995;Vendruscolo and Roberts, 2014), or selected inbred lines of ethanol-preferring rats or mice (Ciccocioppo et al., 2006;McBride and Li, 1998). ...
A major limitation of the most widely used current animal models of alcohol dependence is that they use forced exposure to ethanol including ethanol-containing liquid diet and chronic intermittent ethanol (CIE) vapor to produce clinically relevant blood alcohol levels (BAL) and addiction-like behaviors. We recently developed a novel animal model of voluntary induction of alcohol dependence using ethanol vapor self-administration (EVSA). However, it is unknown whether EVSA leads to an escalation of alcohol drinking per se, and whether such escalation is associated with neuroadaptations in brain regions related to stress, reward, and habit. To address these issues, we compared the levels of alcohol drinking during withdrawal between rats passively exposed to alcohol (CIE) or voluntarily exposed to EVSA and measured the number of Fos+ neurons during acute withdrawal (16 h) in key brain regions important for stress, reward, and habit-related processes. CIE and EVSA rats exhibited similar BAL and similar escalation of alcohol drinking and motivation for alcohol during withdrawal. Acute withdrawal from EVSA and CIE recruited a similar number of Fos+ neurons in the Central Amygdala (CeA), however, acute withdrawal from EVSA recruited a higher number of Fos+ neurons in every other brain region analyzed compared to acute withdrawal from CIE. In summary, while the behavioral measures of alcohol dependence between the voluntary (EVSA) and passive (CIE) model were similar, the recruitment of neuronal ensembles during acute withdrawal was very different. The EVSA model may be particularly useful to unveil the neuronal networks and pharmacology responsible for the voluntary induction and maintenance of alcohol dependence and may improve translational studies by providing preclinical researchers with an animal model that highlights the volitional aspects of alcohol use disorder.
... In alcohol dependent rats, chronic treatment with a CRF1 antagonist blocked alcohol withdrawal-induced increases in alcohol drinking, and in non-dependent rats the CRF1 antagonist tempered moderate increases in alcohol consumption [17]. In addition, the escalation of alcohol self-administration and anxiety typically observed during protracted abstinence can be blocked by competitive CRF1 antagonists [18][19][20][21]. ...
Alcohol use disorder (AUD) is a chronically relapsing disease characterized by loss of control in seeking and consuming alcohol (ethanol) driven by recruitment of brain stress systems. However, AUD differs among the sexes: men are more likely to develop AUD, but women progress from casual to binge drinking and heavy alcohol use more quickly. The central amygdala (CeA) is a hub of stress and anxiety, with corticotropin releasing factor (CRF)-CRF1 receptor and GABAergic signaling dysregulation occurring in alcohol dependent male rodents. However, we recently showed that GABAergic synapses in female rats are less sensitive to the acute effects of ethanol. Here, we used patch clamp electrophysiology to examine the effects of alcohol dependence on the CRF-modulation of rat CeA GABAergic transmission of both sexes. We found that GABAergic synapses of naïve female rats were unresponsive to CRF application compared males, although alcohol dependence induced a similar CRF responsivity in both sexes. In situ hybridization revealed that females had less CeA neurons containing mRNA for the CRF1 receptor (Crhr1) than males, but in dependence, the percentage of Crhr1-expressing neurons in females increased, unlike males. Overall, our data provide evidence for sexually dimorphic CeA CRF system effects on GABAergic synapses in dependence.
... The vast majority of preclinical studies have found that female rodents have higher baseline alcohol consumption compared to males and chronic intermittent ethanol and withdrawal (CIE/WD) only increases alcohol consumption in male rodents, suggesting that males are potentially more sensitive to changes in reward systems (Almeida et al., 1998;Vetter-O'Hagen and Spear, 2011;Morales et al., 2015;Amodeo et al., 2018;Scott et al., 2020). In contrast, WDinduced anxiety-like behavior is independent of sex (Overstreet et al., 2004;Morales et al., 2015Morales et al., , 2018, and thus CIE/WD alters aversion systems in a similar fashion between male and female rats. ...
Chronic intermittent ethanol and withdrawal (CIE/WD) produces alcohol dependence, facilitates anxiety-like behavior, and increases post-CIE alcohol intake. The basolateral amygdala (BLA) is one of several brain regions that regulates anxiety-like behavior and alcohol intake through downstream projections to the nucleus accumbens (NAC) and bed nucleus of the stria terminalis (BNST), respectively. Previous studies revealed that CIE/WD induces input- and sex-specific adaptations to glutamatergic function in the BLA. The BLA receives information from two distinct input pathways. Glutamatergic afferents from medial structures like the thalamus and prefrontal cortex enter the BLA through the stria terminalis whereas lateral cortical structures like the anterior insula cortex enter the BLA through the external capsule. CIE/WD increases presynaptic glutamatergic function at stria terminalis synapses and postsynaptic function at external capsule synapses. Previous studies sampled neurons throughout the BLA, but did not distinguish between projection-specific populations. The current study investigated BLA neurons that project to the NAC (BLA-NAC neurons) or the BNST (BLA-BNST neurons) as representative “reward” and “aversion” BLA neurons, and showed that CIE/WD alters glutamatergic function and excitability in a projection- and sex-specific manner. CIE/WD increases glutamate release from stria terminalis inputs only onto BLA-BNST neurons. At external capsule synapses, CIE/WD increases postsynaptic glutamatergic function in male BLA-NAC neurons and female BLA-BNST neurons. Subsequent experiments demonstrated that CIE/WD enhanced the excitability of male BLA-NAC neurons and BLA-BNST neurons in both sexes when glutamatergic but not GABAergic function was intact. Thus, CIE/WD-mediated increased glutamatergic function facilitates hyperexcitability in male BLA-NAC neurons and BLA-BNST neurons of both sexes.
... Another important finding was that neither PDYN nor KOR knockout did not protect against ethanol withdrawal-induced increases in anxiety. While ethanol vapor exposure and ethanol liquid diet have been shown to increase anxiety-like behavior in rodent models (Overstreet, Knapp, & Breese, 2004;Rose et al., 2016;Valdez & Harshberger, 2012), increased anxiety after DID typically has not been observed (Cox et al., 2013). However, these studies assessed anxiety using the EPM and open-field paradigms. ...
Excessive alcohol drinking has been shown to modify brain circuitry to predispose individuals for future alcohol abuse. Previous studies have implicated the central nucleus of the amygdala (CeA) as an important site for mediating the somatic symptoms of withdrawal and for regulating alcohol intake. In addition, recent work has established a role for both the Kappa Opioid Receptor (KOR) and its endogenous ligand dynorphin in mediating these processes. However, it is unclear whether these effects are due to dynorphin or KOR arising from within the CeA itself or other input brain regions. To directly examine the role of preprodynorphin (PDYN) and KOR expression in CeA neurons, we performed region-specific conditional knockout of these genes and assessed the effects on the Drinking in the Dark (DID) and Intermittent Access (IA) paradigms. We then examined the effects of DID on PDYN and KOR modulation of CeA circuit physiology. Conditional gene knockout resulted in sex-specific responses wherein PDYN knockout decreased alcohol drinking in both male and female mice, whereas KOR knockout decreased drinking in males only. We also found that neither PDYN nor KOR knockout protected against anxiety caused by alcohol drinking. Lastly, a history of alcohol drinking did not alter synaptic transmission in PDYN neurons in the CeA of either sex, but excitability of PDYN neurons was increased in male mice only. Taken together, our findings indicate that PDYN and KOR signaling in the CeA plays an important role in regulating excessive alcohol consumption and highlight the need for future studies to examine how this is mediated through downstream effector regions.
... Although some literature has suggested that women are more vulnerable than men to the pathological effects of alcohol [39][40][41][42], other studies found that alcoholic women do not show disproportionately greater deficits than alcoholic men [43][44][45]. The rodent literature suggests that female relative to male rats of several strains drink more alcohol [46, 47, but see: [48][49][50]; but that male relative to female rats show greater evidence for withdrawal [51][52][53][54]. Thus, a second aim of the current study was to evaluate sex effects on brain structural and biochemical changes in response to alcohol exposure. ...
An overarching goal of our research has been to develop a valid animal model of alcoholism with similar imaging phenotypes as those observed in humans with the ultimate objective of assessing the effectiveness of pharmacological agents. In contrast to our findings in humans with alcohol use disorders (AUD), our animal models have not demonstrated enduring brain pathology despite chronic, high ethanol (EtOH) exposure protocols. Relative to healthy controls, older individuals with AUD demonstrate accelerating brain tissue loss with advanced age. Thus, this longitudinally controlled study was conducted in 4-month old (equivalent to ~16-year-old humans) and 17-month old (equivalent to ~45-year-old humans) male and female Fisher 344 rats to test the hypothesis that following equivalent alcohol exposure protocols, older relative to younger would exhibit more brain changes as evaluated using in vivo structural magnetic resonance imaging (MRI) and MR spectroscopy (MRS). At baseline, total brain volume as well as the volumes of each of the three constituent tissue types (i.e., cerebral spinal fluid (CSF), gray matter, white matter) were greater in old relative to young rats. Baseline metabolite levels (except for GSH) were higher in older than younger animals. Effects of binge ethanol (EtOH) exposure on brain volumes and neurometabolites replicated our previous findings in Wistar rats and included ventricular enlargement and reduced MRS-derived creatine levels. Brain changes in response to binge EtOH treatment were more pronounced in young relative to older animals, negating our hypothesis. Additional metabolite changes including low inositol levels in response to high blood alcohol levels suggest a mechanism of reversible osmolarity disturbances due to temporarily altered brain energy metabolism. Higher baseline GSH levels in female than male rats suggest that female rats are perhaps protected against the more pronounced changes in CSF and gray matter volumes observed in male rats due to superior metabolic homeostasis mechanisms.
... Together, these results support a transient increase in anxiety in the female, EtOH-exposed animals following the first EtOH-exposure cycle and correspond with neuroimaging findings in indicating a higher sensitivity of the exposure paradigm in the female animals. This is in contrast to published evidence suggesting male relative to female rats are more sensitive to withdrawal-induced anxiety as quantified on the elevated plus maze following one 5 day EtOH-exposure cycle (Overstreet et al., 2004), on the social interaction test following a single dose of EtOH (Varlinskaya and Spear, 2004), and greater acoustic startle response following 2 weeks of an EtOH liquid diet (Reilly et al., 2009). ...
A generally accepted framework derived predominately from animal models asserts that repeated cycles of chronic intermittent ethanol (EtOH; CIE) exposure cause progressive brain adaptations associated with anxiety and stress that promote voluntary drinking, alcohol dependence, and further brain changes that contribute to the pathogenesis of alcoholism. The current study used CIE exposure via vapor chambers to test the hypothesis that repeated episodes of withdrawals from chronic EtOH would be associated with accrual of brain damage as quantified using in vivo magnetic resonance imaging (MRI), diffusion tensor imaging (DTI), and MR spectroscopy (MRS). The initial study group included 16 male (∼325g) and 16 female (∼215g) wild-type Wistar rats exposed to 3 cycles of 1-month in vapor chambers + 1 week of abstinence. Half of each group (n = 8) was given vaporized EtOH to blood alcohol levels approaching 250 mg/dL. Blood and behavior markers were also quantified. There was no evidence for dependence (i.e., increased voluntary EtOH consumption), increased anxiety, or an accumulation of pathology. Neuroimaging brain responses to exposure included increased cerebrospinal fluid (CSF) and decreased gray matter volumes, increased Choline/Creatine, and reduced fimbria-fornix fractional anisotropy (FA) with recovery seen after one or more cycles and effects in female more prominent than in male rats. These results show transient brain integrity changes in response to CIE sufficient to induce acute withdrawal but without evidence for cumulative or escalating damage. Together, the current study suggests that nutrition, age, and sex should be considered when modeling human alcoholism.
... Another important finding was that neither PDYN nor KOR knockout protected against ethanol withdrawalinduced increases in anxiety. While ethanol vapor exposure and ethanol liquid diet have been shown to increase anxiety-like behavior in rodent models [15,35,44,45], increased anxiety after DID typically has not been observed [46]. However, these studies assessed anxiety using the EPM and open-field paradigms. ...
Excessive alcohol drinking has been shown to modify brain circuitry to predispose individuals for future alcohol abuse. Previous studies have implicated the central nucleus of the amygdala (CeA) as an important site for mediating the somatic symptoms of withdrawal and for regulating alcohol intake. In addition, recent work has established a role for both the Kappa Opioid Receptor (KOR) and its endogenous ligand dynorphin in mediating these processes. However, it is unclear whether these effects are due to dynorphin or KOR arising from within the CeA itself or other input brain regions. To directly examine the role of preprodynorphin (PDYN) and KOR expression in CeA neurons, we performed region-specific conditional knockout of these genes and assessed the effects on the Drinking in the Dark (DID) and Intermittent Access (IA) paradigms. Conditional gene knockout resulted in sex-specific responses wherein PDYN knockout decreased alcohol drinking in both male and female mice, whereas KOR knockout decreased drinking in males only. We also found that neither PDYN nor KOR knockout protected against anxiety caused by alcohol drinking. Lastly, a history of alcohol drinking did not alter synaptic transmission in PDYN neurons in the CeA of either sex, but excitability of PDYN neurons was increased in male mice only. Taken together, our findings indicate that PDYN and KOR signaling in the CeA plays an important role in regulating excessive alcohol consumption and highlight the need for future studies to examine how this is mediated through downstream effector regions.
... While the mechanisms underlying this vulnerability remain largely unexplored, there is evidence that females may be more sensitive to the rewarding properties of drugs of abuse and less sensitive to the aversive consequences 35,54 . For example, while female rodents consume more alcohol than males, males develop more robust conditioned taste aversions to alcohol and signs of alcohol withdrawal 47,[55][56][57][58][59] . ...
Acute early life stress (ELS) alters stress system functioning in adulthood and increases susceptibility to posttraumatic stress disorder (PTSD) and alcohol use disorder (AUD). The current study assessed the effects of acute, infant ELS on alcohol drinking, including aversion-resistant drinking, in male and female Long Evans rats. Acute ELS was induced using a stress-enhanced fear learning (SEFL) protocol that consisted of 15 footshocks delivered on postnatal day (PND) 17. Alcohol drinking during adolescence and adulthood was measured with a two-bottle choice intermittent alcohol access paradigm. Aversion-resistant drinking was assessed in adulthood by adding quinine (0.01, 0.1, and 1.0 g/L) to the alcohol bottle after 5 to 6 weeks and 11 to 12 weeks of drinking. ELS had minimal influences on adolescent and adult alcohol consumption and preference. However, ELS, sex, and alcohol exposure history all influenced aversion-resistant alcohol drinking in an additive fashion. Higher concentrations of quinine were tolerated in females, ELS-exposed rats, and after 11 to 12 weeks of drinking. Tests of quinine sensitivity in a separate cohort of animals found that rats can detect concentrations of quinine as low as 0.001 g/L in water and that quinine sensitivity is not influenced by sex or ELS exposure. These results agree with reports of sex differences in aversion-resistant drinking and are the first to demonstrate an influence of ELS on this behavior. Our results also suggest that a single traumatic stress exposure in infancy may be a promising model of comorbid PTSD and AUD and useful in studying the interactions between ELS, sex, and alcohol dependence.
... Consistent with previous reports 44,45 , AW from alcohol liquid diet resulted in a small but significant increase in operant responding for alcohol (Fig. 2c, d). Similar alcohol diet schedules are known to produce somatic and emotional withdrawal symptoms in rats 46,47 . The observed increase in operant responding, evident from the initial No S-(alcohol) sessions under AW (as in refs. ...
Drug addiction is a chronic relapsing disorder of compulsive drug use. Studies of the neurobehavioral factors that promote drug relapse have yet to produce an effective treatment. Here we take a different approach and examine the factors that suppress—rather than promote—relapse. Adapting Pavlovian procedures to suppress operant drug response, we determined the anti-relapse action of environmental cues that signal drug omission (unavailability) in rats. Under laboratory conditions linked to compulsive drug use and heightened relapse risk, drug omission cues suppressed three major modes of relapse-promotion (drug-predictive cues, stress, and drug exposure) for cocaine and alcohol. This relapse-suppression is, in part, driven by omission cue-reactive neurons, which constitute small subsets of glutamatergic and GABAergic cells, in the infralimbic cortex. Future studies of such neural activity-based cellular units (neuronal ensembles/memory engram cells) for relapse-suppression can be used to identify alternate targets for addiction medicine through functional characterization of anti-relapse mechanisms. Drug addiction is a chronic disorder and many sufferers experience relapses even after a period of successful abstinence. Here, the authors reveal a subset of neurons in the rat infralimbic cortex that suppresses relapse into cocaine or alcohol use by responding to drug-omission cues.
... Generally alcohol elevates serotonin in brain regions (Ding et al., 2012) by increasing the activity of serotonin receptors 5HT3, 5HT1A, 5HT1B, and serotonin transporters therefore low serotonin turnover accounts for the negative mood states, aggressive behavior, feeling of insecure and threat that leads to the development of anxiety behavior (Sari et al., 2011;Virkunen et al., 1994;Johnson, 2004). Therefore an AW-induced reduction in serotonin turnover in all the regions in the present results confirm the hypothesis consistent with the development of agitation, tremors, stereotype behavior, WDS and audiogenic seizures (Overstreet et al., 2004) (Table I). 78 Volume 30 Issue 2 (2019) ...
The present study aims to investigate the neurochemical and
behavioral effects of alcohol withdrawal (AW) syndrome in rats. Serotonergic
antidepressants such as Citalopram and tianeptine at a dose of 20mg/ kg were
tested separately for their ability to prevent seizures in AW groups of rats.
Brain regional tryptophan (TRP), 5-hydroxytryptamine (5-HT; serotonin),
5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined using
high-performance liquid chromatography connected to the fluorimetric
detector. Data analyzed showed increases brain TRP concentrations in all the
three regions (amygdala, hippocampus and hypothalamus) after citalopram
administered alcohol withdrawn rats; however, tianeptine increases brain
TRP concentrations only in amygdala and hippocampus. Increases in brain
serotonin concentrations were seen in all the regions only in citalopram
administered alcohol withdrawn rats. Further, tianeptine was shown to
increase 5-HT turnover in all the regions, however, citalopram appeared to
increase 5-HT turnover only in the hippocampus. It is concluded that the
citalopram and tianeptine behave differently on the intrinsic pathway of
serotonin metabolism that appeared to be compensated by the pattern of
5-HT release. Further, it could be inferred that exposure of serotonergic
agents might restore serotonin reuptake mechanism that is desensitized
following chronic alcohol exposure. Thus the inclusive approach of the
serotonergic system plays an undoubted role in the pharmacological
management of AW syndrome.
Keywords: Serotonin, antidepressants, tryptophan, tianeptine, citalopram
... Generally alcohol elevates serotonin in brain regions (Ding et al., 2012) by increasing the activity of serotonin receptors 5HT3, 5HT1A, 5HT1B, and serotonin transporters therefore low serotonin turnover accounts for the negative mood states, aggressive behavior, feeling of insecure and threat that leads to the development of anxiety behavior (Sari et al., 2011;Virkunen et al., 1994;Johnson, 2004). Therefore an AW-induced reduction in serotonin turnover in all the regions in the present results confirm the hypothesis consistent with the development of agitation, tremors, stereotype behavior, WDS and audiogenic seizures (Overstreet et al., 2004) (Table I). 78 Volume 30 Issue 2 (2019) ...
The present study aims to investigate the neurochemical and behavioral effects of alcohol withdrawal (AW) syndrome in rats. Serotonergic antidepressants such as Citalopram and tianeptine at a dose of 20mg/ kg were tested separately for their ability to prevent seizures in AW groups of rats. Brain regional tryptophan (TRP), 5-hydroxytryptamine (5-HT; serotonin), 5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined using high-performance liquid chromatography connected to the fluorimetric detector. Data analyzed showed increases brain TRP concentrations in all the three regions (amygdala, hippocampus and hypothalamus) after citalopram administered alcohol withdrawn rats; however, tianeptine increases brain TRP concentrations only in amygdala and hippocampus. Increases in brain serotonin concentrations were seen in all the regions only in citalopram administered alcohol withdrawn rats. Further, tianeptine was shown to increase 5-HT turnover in all the regions, however, citalopram appeared to increase 5-HT turnover only in the hippocampus. It is concluded that the citalopram and tianeptine behave differently on the intrinsic pathway of serotonin metabolism that appeared to be compensated by the pattern of 5-HT release. Further, it could be inferred that exposure of serotonergic agents might restore serotonin reuptake mechanism that is desensitized following chronic alcohol exposure. Thus the inclusive approach of the serotonergic system plays an undoubted role in the pharmacological management of AW syndrome.
... Generally alcohol elevates serotonin in brain regions (Ding et al., 2012) by increasing the activity of serotonin receptors 5HT3, 5HT1A, 5HT1B, and serotonin transporters therefore low serotonin turnover accounts for the negative mood states, aggressive behavior, feeling of insecure and threat that leads to the development of anxiety behavior (Sari et al., 2011;Virkunen et al., 1994;Johnson, 2004). Therefore an AW-induced reduction in serotonin turnover in all the regions in the present results confirm the hypothesis consistent with the development of agitation, tremors, stereotype behavior, WDS and audiogenic seizures (Overstreet et al., 2004) (Table I). 78 Volume 30 Issue 2 (2019) ...
... Female vulnerability to drug abuse may be due to increased sensitivity to the rewarding properties of drugs combined with decreased sensitivity to the aversive properties (Anker and Carroll, 2011;Lynch et al., 2002). There is some evidence for the latter phenomenon in rodent models of alcohol intake, with male rats exhibiting more robust alcohol-induced conditioned taste aversion (Cailhol and Morm ede, 2001;Schramm-Sapyta et al., 2014;Sherrill et al., 2011) and symptoms of alcohol withdrawal (Gatch and Lal, 2001;Overstreet et al., 2004;Varlinskaya and Spear, 2004). This combination of factors could potentially make females more likely to engage in compulsive drug seeking, where negative or aversive outcomes are ignored. ...
... Although no animal model of addiction fully emulates the human condition, the existing models permit investigations of specific elements of the addiction process (Rodd et al. 2004). Multiple methods have been developed to produce BALs in the 100-300 mg% range for ≥12 h per day, associated with somatic and affective withdrawal symptoms, but they use a forced alcohol liquid diet (Gilpin et al. 2009;Lieber and DeCarli 1982;Overstreet et al. 2004), intragastric alcohol intubation (Aujla et al. 2013;Braconi et al. 2010;de Guglielmo et al. 2015;Sidhpura et al. 2010), passive exposure to alcohol vapor (Becker and Lopez 2004;de Guglielmo et al. 2016;Gilpin et al. 2008;Goldstein and Pal 1971;Kallupi et al. 2014;Lopez and Becker 2014;O'Dell et al. 2004;Rimondini et al. 2002;Roberts et al. 1996;Schulteis et al. 1995;Vendruscolo and Roberts 2014), or selected inbred lines of alcohol-preferring rats or mice (Bell et al. 2012;Ciccocioppo et al. 2006;Colombo et al. 2006;Crabbe et al. 2014;Matson and Grahame 2013;McBride and Li 1998;Quintanilla et al. 2006;Sommer et al. 2006). Forced and passive administration models have limited face validity and make it difficult to unveil the neuronal networks that mediate the voluntary induction and maintenance of alcohol dependence. ...
RationaleA major issue in the addiction field is the limited number of animal models of the voluntary induction and maintenance of alcohol dependence in outbred rats. Objectives
To address this issue, we developed a novel apparatus that vaporizes alcohol for 2–10 min after an active nosepoke response. Methods
Male Wistar rats were allowed to self-administer alcohol vapor for 8 h/day every other day for 24 sessions (escalated) or eight sessions (non-escalated). Escalated and non-escalated rats were then tested for progressive ratio responding. Anxiety-like behavior, somatic signs of withdrawal, and hyperalgesia were assessed during acute withdrawal. ResultsThe results showed that rats exhibited excellent discrimination between the active and inactive operanda (>85%), and the escalated rats quickly increased their blood alcohol levels from ~50 to >200 mg% in ~6 weeks. Compared with non-escalated rats, escalated rats exhibited severe addiction-like behavior, including somatic signs of withdrawal, anxiety-like behavior, hyperalgesia, and higher responding on a progressive ratio schedule of reinforcement. Conclusions
These results demonstrate that outbred rats will voluntarily self-administer alcohol vapor to the point of dependence without the use of forced alcohol administration, sweeteners, food/water restriction, operant pretraining, or behavioral/genetic selection. This novel animal model may be particularly useful for medication development to help unveil the neuronal circuitry that underlies the voluntary induction of alcohol addiction and identify novel molecular targets that are specifically recruited after the voluntary induction and maintenance of alcohol dependence.
... Despite the fact that ethanol abuse and dependence increased among women during the last decades (Greenfield, 2002), there is little information in the literature about the effects on anxiety of female rats subjected to chronic ethanol. Under repeated ethanol withdrawals, similar anxiogenic effects were observed in male and female rats (Overstreet, Knapp, & Breese, 2004). Further behavioral studies aimed to comparing the withdrawal's effect on anxiety after chronic ethanol exposure in male and female rats are in progress by our research group. ...
Anxiety disorders are associated with increased impairments in psychosocial functioning, work productivity and health-related quality of life. In addition, anxiety is a common symptom of ethanol withdrawal and it strongly contributes to relapse. Benzodiazepines are frequently prescribed for relief of anxiety and ethanol withdrawal symptoms but considerable side effects, such sedation, tolerance and dependence, are observed during treatment. Therefore, better drugs are needed for the treatment of anxiety states. The purpose of this study was to investigate whether topiramate would reduce basal levels of anxiety and ethanol-withdrawn induced anxiety in male rats; the elevated plus maze (EPM) was used as an animal model of anxiety. In Experiment 1, topiramate (0, 10, and 40 mg/kg, i.g.) and diazepam (1 mg/kg, i.p.) was acutely and repeatedly administered to naive rats. In Experiments 2 and 3, topiramate (0 or 40 mg/kg, i.g.) was acutely and chronically administered in early (72 hr after ethanol removal) and protracted (21 days after ethanol removal) ethanol-withdrawn rats, respectively. Acute and repeated topiramate treatment induced anxiolytic-like effects in naive rats. Early ethanol withdrawal increased anxiety, and acute topiramate administration counteracted the anxiogenic-like effects of ethanol removal. Protracted withdrawal did not produce lasting changes in anxiety but topiramate was equally effective at reducing anxiety in ethanol-withdrawn and control animals. Importantly, no signs of tolerance to the anxiolytic effects of topiramate were observed. In conclusion, these data support a role for topiramate in the treatment of basal levels of anxiety and ethanol withdrawal-induced anxiety.
... Generally alcohol elevates serotonin in brain regions (Ding et al., 2012) by increasing the activity of serotonin receptors 5HT3, 5HT1A, 5HT1B, and serotonin transporters therefore low serotonin turnover accounts for the negative mood states, aggressive behavior, feeling of insecure and threat that leads to the development of anxiety behavior (Sari et al., 2011;Virkunen et al., 1994;Johnson, 2004). Therefore an AW-induced reduction in serotonin turnover in all the regions in the present results confirm the hypothesis consistent with the development of agitation, tremors, stereotype behavior, WDS and audiogenic seizures (Overstreet et al., 2004) (Table I). 78 Volume 30 Issue 2 (2019) ...
... The notion that repeated withdrawal experience progressively intensifies withdrawal symptoms is also supported by animal models. For example, animals chronically exposed to alcohol were shown to exhibit enhanced withdrawal-related anxiety when submitted to repeated withdrawal experiences Overstreet, Knapp, & Breese, 2004Zhang, Morse, Koob, & Schulteis, 2007). Indeed, multiple animal models support the notion that repeated ethanol withdrawal experiences increase the severity and duration of subsequent withdrawal seizures (Becker, Diaz-Granados, & Weathersby, 1997;Becker & Hale, 1993). ...
... For example, male rats, compared with females, that were withdrawn from alcohol had greater physical signs of withdrawal, such as greater seizure susceptibility (Devaud and Chadda, 2001). Perhaps more relevant, male rats also showed greater motivational signs of withdrawal, such as anxiety-like responses in the social interaction test (Varlinskaya and Spear, 2004) and elevated plus maze after one cycle of exposure to a liquid diet (Overstreet et al., 2004). Males showed greater increases in acoustic startle responses than either intact female rats or ovariectomized female rats (Reilly et al., 2009), and males showed greater hangover-like anxiety-like responses (Varlinskaya and Spear, 2004). ...
The purpose of this review is to discuss ways to think about and study sex differences in preclinical animal models. We use the framework of addiction, in which animal models have excellent face and construct validity, to illustrate the importance of considering sex differences. There are four types of sex differences: qualitative, quantitative, population, and mechanistic. A better understanding of the ways males and females can differ will help scientists design experiments to characterize better the presence or absence of sex differences in new phenomena that they are investigating. We have outlined major quantitative, population, and mechanistic sex differences in the addiction domain using a heuristic framework of the three established stages of the addiction cycle: binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. Female rats, in general, acquire the self-administration of drugs and alcohol more rapidly, escalate their drug taking with extended access more rapidly, show more motivational withdrawal, and (where tested in animal models of "craving") show greater reinstatement. The one exception is that female rats show less motivational withdrawal to alcohol. The bases for these quantitative sex differences appear to be both organizational, in that estradiol-treated neonatal animals show the male phenotype, and activational, in that the female phenotype depends on the effects of gonadal hormones. In animals, differences within the estrous cycle can be observed but are relatively minor. Such hormonal effects seem to be most prevalent during the acquisition of drug taking and less influential once compulsive drug taking is established and are linked largely to progesterone and estradiol. This review emphasizes not only significant differences in the phenotypes of females and males in the domain of addiction but emphasizes the paucity of data to date in our understanding of those differences.
... There is a close link between alcoholism and anxiety, since increased anxiety is a behavioral trait predisposing to engage in alcohol intake (Hayton et al., 2012;Spanagel et al., 1995) and alcoholics show a high psychiatric comorbidity for anxiety disorders, so the anxiety disorder and the alcohol use disorder can both serve to initiate and maintain the other (Kushner et al., 2000). In relation to this, alcohol withdrawal symptoms are anxiogenic and the anxiety experienced in the alcohol-deprivation periods predicts relapse in alcohol use (Overstreet et al., 2004;Willinger et al., 2002); while animal models for high alcohol consumption frequently display an increased anxiety-like response [e.g. (Huot et al., 2001;Pandey et al., 2006Pandey et al., , 2005Thiele et al., 1998)]. ...
Background:
Lysophosphatidic acid species (LPA) are lipid bioactive signaling molecules that have been recently implicated in the modulation of emotional and motivational behaviors. The present study investigates the consequences of either genetic deletion or pharmacological blockade of lysophosphatidic acid receptor-1 (LPA1) in alcohol consumption.
Methods:
The experiments were performed in alcohol-drinking animals by using LPA1-null mice and administering the LPA1 receptor antagonist Ki16425 in both mice and rats.
Results:
In the two-bottle free choice paradigm, the LPA1-null mice preferred the alcohol more than their wild-type counterparts. Whereas the male LPA1-null mice displayed this higher preference at all doses tested, the female LPA1-null mice only consumed more alcohol at 6% concentration. The male LPA1-null mice were then further characterized, showing a notably increased ethanol drinking after a deprivation period and a reduced sleep time after acute ethanol administration. In addition, LPA1-null mice were more anxious than the wild-type mice in the elevated plus maze test. For the pharmacological experiments, the acute administration of the antagonist Ki16425 consistently increased ethanol consumption in both wild-type mice and rats; while it did not modulate alcohol drinking in the LPA1-null mice and lacked intrinsic rewarding properties and locomotor effects in a conditioned place preference paradigm. In addition, LPA1-null mice exhibited a marked reduction on the expression of glutamate-transmission-related genes in the prefrontal cortex similar to those described in alcohol-exposed rodents.
Conclusions:
Results suggest a relevant role for the LPA/LPA1 signaling system in alcoholism. In addition, the LPA1-null mice emerge as a new model for genetic vulnerability to excessive alcohol drinking. The pharmacological manipulation of LPA1 receptor arises as a new target for the study and treatment of alcoholism.
... Aside from the withdrawal-related effects, additional work has shown that while no differences were detected with respect to gender, there may be an age related effect, with adolescents experiencing less severe withdrawal-related anxiety compared to adults [185,186]. ...
Alcohol (ethanol) has a prominent role in society and is one of the most frequently used and abused drugs. Despite the pervasive use and abuse of ethanol, the molecular mechanisms of ethanol action remain unclear. What is well known is that ethanol intoxication elicits a range of behavioral effects. These effects most likely occur through the direct action of ethanol on targets in the central nervous system. By studying behavioral effects, the role of individual targets can be determined. The function of γ-amino butyric acid type A (GABAA) receptors is altered by ethanol, but due to multiple receptor subunits the exact role of individual GABAA receptor subunits in ethanol action is not known. This dissertation focused on the role of α1-containing GABAA receptors in ethanol action using gene knockin mice with ethanol insensitive α1 GABAA receptors. In the second chapter, knockin mice were molecularly characterized and ethanol-induced behavioral effects were assessed. α1 was found to mediate acute tolerance to the motor ataxic effects of ethanol. In the third chapter, α1 involvement in ethanol induction of neuronal activity was assessed in discrete neuroanatomic regions using the immediate early gene c-fos. Specifically, c-fos immunohistochemistry was characterized after acute ethanol exposure, after chronic ethanol exposure, and finally during the ethanol withdrawal phase. α1 was found to be involved in ethanol-mediated effects in the dentate gyrus. In the fourth chapter, α1 involvement in chronic tolerance to ethanol as well as physical dependence on ethanol was characterized. Results demonstrated that α1-GABAA-Rs play a role in the development of tolerance to chronic ethanol in motor ataxia. Intriguingly, α1 was implicated in dependence as assessed with ethanol withdrawal-related hyperexcitability. Knockin mice were more sensitive to ethanol's withdrawal-related hyperexcitability effects. In summary, this dissertation further supports α1 GABAA-Rs in the mechanism of ethanol action. By chiseling away at the various components of ethanol action we are beginning to elucidate the mechanism of ethanol action. Further elucidation of the mechanism of action of α1 GABAA-Rs in tolerance and dependence could deepen our understanding of the molecular mechanisms behind alcohol abuse and alcoholism. By understanding the molecular mechanisms of ethanol, alcohol abuse may be lessened and alcoholism could potentially be cured.
... The notion that repeated withdrawal experience progressively intensifies withdrawal symptoms is also supported by animal models. For example, animals chronically exposed to alcohol were shown to exhibit enhanced withdrawal-related anxiety when submitted to repeated withdrawal experiences Overstreet, Knapp, & Breese, 2004Zhang, Morse, Koob, & Schulteis, 2007). Indeed, multiple animal models support the notion that repeated ethanol withdrawal experiences increase the severity and duration of subsequent withdrawal seizures (Becker, Diaz-Granados, & Weathersby, 1997;Becker & Hale, 1993). ...
Background
To date, no screening tools for alcohol withdrawal syndromes (AWS) have been validated in the medically ill. Although several tools quantify the severity of AWS (e.g., Clinical Institute Withdrawal Assessment for Alcohol [CIWA]), none identify subjects at risk of AWS, thus missing the opportunity for timely prophylaxis. Moreover, there are no validated tools for the prediction of severe AWS in the medically ill.
Objectives
Our goals were (1) to conduct a systematic review of the published literature on AWS to identify clinical factors associated with the development of AWS, (2) to use the identified factors to develop a tool for the prediction of alcohol withdrawal among patients at risk, and (3) to conduct a pilot study to assess the validity of the tool.
Methods
For the creation of the Prediction of Alcohol Withdrawal Severity Scale (PAWSS), we conducted a systematic literature search using PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines for clinical factors associated with the development of AWS, using PubMed, PsychInfo, MEDLINE, and Cochrane Databases. Eligibility criteria included: (i) manuscripts dealing with human subjects, age 18 years or older, (ii) manuscripts directly addressing descriptions of AWS or its predisposing factors, including case reports, naturalistic case descriptions, and all types of clinical trials (e.g., randomized, single-blind, or open label studies), (iii) manuscripts describing characteristics of alcohol use disorder (AUD), and (iv) manuscripts dealing with animal data (which were considered only if they directly dealt with variables described in humans). Obtained data were used to develop the Prediction of Alcohol Withdrawal Severity Scale, in order to assist in the identification of patients at risk for moderate to severe AWS.
For the Pilot Study
A pilot study was conducted to assess the new tool’s psychometric qualities on patients admitted to a general inpatient medicine unit over a 2-week period, who agreed to participate in the study. Blind to PAWSS results, a separate group of researchers retrospectively examined the medical records for evidence of AWS.
Results
The search produced 2802 articles describing factors potentially associated with increased risk for AWS, increased severity of withdrawal symptoms, and potential characteristics differentiating subjects with various forms of AWS. Of these, 446 articles met inclusion criteria and underwent further scrutiny, yielding a total of 233 unique articles describing factors predictive of AWS. A total of 10 items were identified as correlated with moderate to severe AWS (i.e., withdrawal hallucinosis, withdrawal-related seizures, and delirium tremens) and used to construct the PAWSS. During the pilot study, a total of 68 subjects underwent evaluation with PAWSS. In this pilot sample the sensitivity, specificity, and positive and negative predictive values of PAWSS were 100%, using the threshold score of 4.
Discussion
The results of the literature search identified 10 items which may be correlated with risk for moderate to severe AWS. These items were assembled into a tool to assist in the identification of patients at risk: PAWSS. The results of this pilot study suggest that PAWSS may be useful in identifying risk of moderate to severe AWS in medically ill, hospitalized individuals. PAWSS is the first validated tool for the prediction of severe AWS in the medically ill and its use may aid in the early identification of patients at risk for moderate to severe AWS, allowing for prophylaxis against AWS before severe alcohol withdrawal syndromes develop.
... This result suggests that LoS males in particular may be less vulnerable to morphine withdrawal than LoS females. While relatively little work has been done investigating sex differences in the emotional signs of drug withdrawal, there is some evidence that male rats exhibit greater anxiety during withdrawal from ethanol (Gatch and Lal 2001;Varlinskaya and Spear 2004;Overstreet et al. 2004) and male monkeys (vs. female) decrease responding for food during PCP withdrawal (Perry et al. 2006b). ...
Rationale
Rats bred for high (HiS) and low (LoS) saccharin intake exhibit divergent behavioral responses to multiple drugs of abuse, with HiS rats displaying greater vulnerability to drug taking. Previous research indicates that this effect may be due to increased sensitivity to reward in HiS rats and to the aversive effects of acute drug administration in LoS rats.
Objective
The current study investigated whether HiS and LoS rats also exhibit different behavioral signs of withdrawal following one or repeated opiate exposures.
Methods
Emotional signs of opiate withdrawal were assessed with potentiation of the acoustic startle reflex and conditioned place aversion (CPA) in male and female HiS and LoS rats. Startle was measured before and 4 h after a 10-mg/kg injection of morphine on days 1, 2, and 7 of opiate exposure. CPA was induced with a 2-day, naloxone-precipitated conditioning paradigm. Somatic signs of withdrawal and weight loss were also measured.
Results
Male and female LoS rats exhibited lower startle potentiation than HiS rats on the seventh day of morphine exposure. LoS male rats also failed to develop a CPA to morphine withdrawal. No differences in physical withdrawal signs were observed between HiS and LoS rats, but males of both lines had more physical signs of withdrawal than females.
Conclusions
These results suggest that LoS rats are less vulnerable to the negative emotional effects of morphine withdrawal than HiS rats. A less severe withdrawal syndrome may contribute to decreased levels of drug taking in the LoS line.
... In the present study, we evaluated the effects of alcohol withdrawal following chronic ethanol treatment on the expression of different cannabinoid signaling-related genes in the amygdala. Since there is considerable evidence that repeated cycles of withdrawal contribute to excessive ethanol consumption and progressively worsen withdrawalassociated symptoms (Holter et al., 1998; Lopez and Becker, 2005; Overstreet et al., 2002; Overstreet et al., 2004; Rimondini et al., 2002), we have compared the potential differences existing between continuous and intermittent ethanol exposure on these neuroadaptations. ...
Background:
Endogenous cannabinoids such as anandamide and 2-arachidonoylglycerol (2-AG) exert important regulatory influences on neuronal signaling, participate in short- and long-term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity, and affective control.
Methods:
The consequence of long-term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT-PCR analyses of amygdala tissue. Two groups of ethanol (EtOH)-exposed rats were generated by maintenance on an EtOH liquid diet (10%): the first group received continuous access to EtOH for 15 days, whereas the second group was given intermittent access to the EtOH diet (5 d/wk for 3 weeks). Control subjects were maintained on an isocaloric EtOH-free liquid diet. To provide an initial profile of acute withdrawal, amygdala tissue was harvested following either 6 or 24 hours of EtOH withdrawal.
Results:
Acute EtOH withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and 2-AG (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL], respectively) were evident, as were reductions in mRNA expression for CB(1) , CB(2) , and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent EtOH exposure, alterations in MAGL and cannabinoid receptor-related mRNA (e.g., CB(1) , CB(2) , GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal-associated deficits in mRNA expression were evident following 24 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in 2-AG biosynthesis (e.g., diacylglicerol lipase-α/β) were found in any condition.
Conclusions:
These findings suggest that EtOH dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal-related dysregulation of amygdalar neurotransmission.
... It has been thought that the progression from high alcohol intake to alcohol dependence may be driven by repeated cycles of heavy drinking followed by deprivation (Breese et al., 2005;Meisch, 1983). According to the kindling hypothesis (Ballenger and Post, 1978;Becker, 1998), increasing amounts of ethanol ingested over repeated episodes may intensify ensuing symptoms of withdrawal when alcohol is absent (Begleiter and Porjesz, 1979;Overstreet et al., 2002;Overstreet et al., 2004b), thus generating a compensatory mechanism for relapse (Becker and Hale, 1993;Holter et al., 1998). ...
Intermittent access (IA) to drugs of abuse, as opposed to continuous access, is hypothesized to induce a kindling-type transition from moderate to escalated use, leading to dependence. Intermittent 24-hour cycles of ethanol access and deprivation can generate high levels of voluntary ethanol drinking in rats.
The current study uses C57BL/6J mice (B6) in an IA to 20% ethanol protocol to escalate ethanol drinking levels. Adult male and female B6 mice were given IA to 20% ethanol on alternating days of the week with water available ad libitum. Ethanol consumption during the initial 2 hours of access was compared with a short-term, limited access "binge" drinking procedure, similar to drinking-in-the-dark (DID). B6 mice were also assessed for ethanol dependence with handling-induced convulsion, a reliable measure of withdrawal severity.
After 3 weeks, male mice given IA to ethanol achieved high stable levels of ethanol drinking in excess of 20 g/kg/24 h, reaching above 100 mg/dl blood ethanol concentrations, and showed a significantly higher ethanol preference than mice given continuous access to ethanol. Also, mice given IA drank about twice as much as DID mice in the initial 2-hour access period. B6 mice that underwent the IA protocol for longer periods of time displayed more severe signs of alcohol withdrawal. Additionally, female B6 mice were given IA to ethanol and drank significantly more than males (ca. 30 g/kg/24 h).
The IA method in B6 mice is advantageous because it induces escalated, voluntary, and preferential per os ethanol intake, behavior that may mimic a cardinal feature of human alcohol dependence, though the exact nature and site of ethanol acting in the brain and blood as a result of IA has yet to be determined.
... These results are consistent with the anxiolytic and anxiogenic properties of NPY and CRF, respectively (Dunn and File, 1987;Heilig et al., 1993;Sutton et al., 1982;Thatcher-Britton et al., 1986;Wahlestedt et al., 1993). The evidence of increase anxiety-like behaviors during the acute ethanol withdrawal phase (Overstreet et al., 2004;Zhao et al., 2007) and findings from the present study showing a reduction of NPY levels in the FCTX also support the notion that reductions in NPY levels may play a role in the expression of spontaneous anxiety symptoms during ethanol withdrawal periods. ...
There is evidence to suggest that alterations in neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) contribute to the escalated voluntary ethanol intake seen following long term chronic ethanol exposure. The present study assessed whether the duration of chronic ethanol exposure and abstinence alters brain levels of NPY and CRF in adult Wistar rats. NPY-like immunoreactivity (NPY-LI) and CRF-LI were determined in the amygdala (AMYG), frontal cortex (FCTX), hippocampus (HPC) and parietal cortex (PCTX) of adult Wistar rats after chronic ethanol exposure, and 24-h and 2-weeks following withdrawal (WD). Chronic ethanol exposure consisted of either a 2-week or an 8-week ethanol vapor regimen. No change in brain levels of NPY-LI, CRF-LI and the NPY-LI/CRF-LI ratio was observed 2-weeks following ethanol exposure, whereas, 8-weeks of ethanol exposure produced a significant effect on NPY-LI expression in the AMYG and FCTX. Moreover, an 8-week ethanol vapor regimen significantly increased CRF-LI levels in the HPC and PCTX. Findings from the present study suggest that a longer duration of ethanol vapor, similar to what is required to enhance voluntary drinking, is required to produce changes in NPY-LI and CRF-LI expression in the adult rat brain.
... The social interaction test (SI) was first described by File and Hyde (1978), and a modified version of this test has been used regularly in our laboratory to assess anxiety-like behavior. The results of the social interaction test have also been confirmed with the use of the elevated plus maze (Overstreet et al., 2002Overstreet et al., , 2004b). In the social interaction test, rats were placed into a 60 × 60 cm square open field with 15 × 15 cm squares marked on the floor under low lighting conditions (30 lx). ...
Repeated stress or administration of corticotropin-releasing factor (CRF) prior to ethanol exposure sensitizes anxiety-like behavior in adult rats. Current experiments determined whether adolescent rats were more sensitive to these challenges in sensitizing ethanol withdrawal-induced anxiety and altering CRF levels in brain during withdrawal.
Male adult and adolescent Sprague-Dawley rats were restraint stressed (1 hour) twice 1 week apart prior to a single 5-day cycle of ethanol diet (ED; stress/withdrawal paradigm). Other rats received control diet (CD) and three 1-hour restraint stress sessions. Rats were then tested 5, 24, or 48 hours after the final withdrawal for anxiety-like behavior in the social interaction (SI) test. In other experiments, adolescent rats were given two microinjections of CRF icv 1 week apart followed by 5 days of either CD or ED and tested in social interaction 5 hours into withdrawal. Finally, CRF immunoreactivity was measured in the central nucleus of the amygdala (CeA) and paraventricular nucleus (PVN) after rats experienced control diet, repeated ethanol withdrawals, or stress/withdrawal.
Rats of both ages had reduced SI following the stress/withdrawal paradigm, and this effect recovered within 24 hours. Higher CRF doses were required to reduce SI in adolescents than previously reported in adults. CRF immunohistochemical levels were higher in the PVN and CeA of CD-exposed adolescents. In adolescent rats, repeated ethanol withdrawals decreased CRF in the CeA but was not associated with decreased CRF cell number. There was no change in CRF from adult treatments.
In the production of anxiety-like behavior, adolescent rats have equal sensitivity with stress and lower sensitivity with CRF compared to adults. Further, adolescents had higher basal levels of CRF within the PVN and CeA and reduced CRF levels following repeated ethanol withdrawals. This reduced CRF within the CeA could indicate increased release of CRF, and future work will determine how this change relates to behavior.
... A prominent ethanol withdrawal symptom in humans is anxiety (Driessen et al., 2001). In rodents, anxiety-like behavior measured in several animal models of anxiety is a commonly reported sign of ethanol withdrawal (Baldwin et al., 1991; File et al., 1993; Gatch et al., 1999; Knapp et al., 2004; Overstreet et al., 2004; Valdez et al., 2002; Zhang et al., 2007). With the present procedure, six days of intragastric ethanol intubation were sufficient to produce lasting elevations in spontaneous anxiety-like behavior measured five days post-withdrawal on the elevated plus-maze, one of the most widely used rodent models for measuring anxiety-like behavior (Pellow et al., 1985; Treit et al., 1993). ...
The purpose of this study was to re-examine intragastric ethanol intubation as a dependence induction method that effectively induces physical dependence upon ethanol over a short time period, is devoid of intrinsic stress artifacts, inexpensive, and easy to implement.
Male Wistar rats were subjected to ethanol dependence induction via intragastric ethanol intubation. Ethanol solution (final concentration 20%, made up in a dietary liquid vehicle consisting of powdered milk, sucrose, and water) was intubated 4 times per day, at 4-hour intervals, for 6 consecutive days (for a total of 10 g/kg/day). The utility of this procedure was evaluated for inducing physical dependence, determined by daily and final withdrawal ratings. Anxiety-like behavior associated with ethanol dependence history was examined using the elevated plus-maze (EPM) test, conducted 5 days after ethanol withdrawal. To evaluate whether potential stress-like effects of intragastric intubation per se produce lasting effects on behavior, experimentally naive rats were compared with vehicle-intubated rats for anxiety-like behavior on the EPM.
Blood alcohol levels reached stable levels between 200 and 250 mg%, measured 1 hour after the second and third ethanol intubation on days 2, 4, and 6. Ethanol-treated rats developed significant somatic withdrawal signs, recorded daily between 10 and 12 hours after the last ethanol administration. At 5 days postwithdrawal, ethanol-treated rats showed significant anxiety-like behavior, measured by decreased open arm time and open arm entries on the EPM, compared with vehicle controls. Additionally, ethanol postdependent rats showed decreased open arm time compared with experimentally naive rats. EPM performance did not differ between vehicle-intubated and naive rats. No withdrawal seizures were observed and mortality rate was near zero.
These findings suggest that intragastric ethanol administration produces a behavioral profile consistent with ethanol dependence (i.e., significant withdrawal signs after termination of ethanol exposure and elevated anxiety-like behavior persisting beyond completion of physical withdrawal), and that the intubation procedure itself does not produce lasting nonspecific anxiety-like effects. Thus, under the conditions employed here, this procedure provides an effective tool for inducing and evaluating the consequences of ethanol dependence in animal models of ethanol reward and motivation.
... The SI test was first described by File and Hyde (1978) and has been used regularly in our laboratory to assess anxiety-like behavior 5 to 6 hours following the removal of ED. The results of the SI test have also been confirmed with the use of the elevated plus maze (Overstreet et al., 2002Overstreet et al., , 2004b). In the SI test, rats were placed into a 60 × 60 cm square open field with a 15 × 15 cm square grid floor under low lighting conditions (30 lx). ...
In abstinent alcoholics, stress induces negative affect-a response linked to craving and relapse. In rats, repeated stresses at weekly intervals before 5-day ethanol diet sensitize withdrawal-induced anxiety-like behavior ("anxiety") that is blocked by a corticotrophin-releasing factor 1 (CRF-1)-receptor antagonist. Current experiments were performed to identify brain sites that support CRF involvement in stress sensitization of ethanol withdrawal-induced anxiety-like behavior. First, different doses of CRF microinjected weekly into the central amygdala (CeA) before ethanol exposure produced a dose-related sensitization of anxiety during ethanol withdrawal. Subsequently, CRF microinjection into the basolateral amygdala, dorsal raphe nucleus (DRN), or dorsal bed nucleus of the stria terminalis (d-BNST) also sensitized ethanol withdrawal-induced anxiety. In contrast, sensitization of ethanol withdrawal-induced anxiety was not observed after weekly CRF administration into the ventral-BNST, CA1-hippocampal region, or hypothalamic-paraventricular nucleus. Then, experiments documented the CRF receptor subtype responsible for CRF and stress sensitization of withdrawal-induced anxiety. Systemic administration of a CRF-1 receptor antagonist before CRF microinjection into the CeA, DRN, or d-BNST prevented CRF-induced sensitization of anxiety during ethanol withdrawal. Furthermore, repeated microinjections of urocortin-3, a CRF-2 receptor agonist, into the CRF-positive sites did not sensitize anxiety after withdrawal from ethanol. Finally, microinjection of a CRF-1 receptor antagonist into the CeA, DRN, or d-BNST before stress blocked sensitization of anxiety-like behavior induced by the repeated stress/ethanol withdrawal protocol. These results indicate that CRF released by stress acts on CRF-1 receptors within specific brain regions to produce a cumulative adaptation that sensitizes anxiety-like behavior during withdrawal from chronic ethanol exposure.
... The social interaction test (SI) was first described by File and Hyde (1978), and a modified version of this test has been used regularly in our laboratory to assess anxiety-like behavior. The results of the social interaction test have also been confirmed with the use of the elevated plus maze (Overstreet et al., 2002Overstreet et al., , 2004b). In the social interaction test, rats were placed into a 60 × 60 cm square open field with 15 × 15 cm squares marked on the floor under low lighting conditions (30 lx). ...
Repeated ethanol withdrawal sensitizes anxiety-like behavior in adult rats and causes anxiety-like behavior and decreased seizure thresholds in adolescent rats. Current experiments determined if adolescent rats exhibit sensitized anxiety-like behavior, the duration of this effect, if drug pretreatments blocked these effects, and if these effects differed from those seen in adults.
Male adolescent rats received three 5-day cycles of 2.5% ethanol diet (ED) separated by two 2-day withdrawal periods, continuous 15 days of 2.5%ED, or a single 5-day cycle of 2.5%ED. Male adult rats received three 5-day cycles of either 2.5% or 3.5%ED. These groups were tested 5 hours into the final withdrawal for social interaction (SI) deficits (an index of anxiety-like behavior). Ethanol intake was monitored throughout and blood concentrations were obtained from separate groups of rats. Additionally, adolescent rats were tested for SI 1, 2, 7, 14, and 18 days and adults 1 and 2 days after the final withdrawal. Some adolescent rats were also pretreated with the CRF(1) antagonist CP-154,526, the 5-HT(1A) agonist buspirone, or the benzodiazepine receptor antagonist flumazenil during the first 2 withdrawals.
SI was reduced in adolescent rats following repeated withdrawals of 2.5%ED while neither a continuous or single cycle ED exposure caused this effect. Adult rats also had reduced SI following repeated withdrawals from both 2.5% and 3.5%ED. This effect was present up to 1 week following the final withdrawal in adolescents but returned to baseline by 1 day in adults. CP-154,526, buspirone, or flumazenil prevented this reduction in SI in adolescent rats.
Adolescent rats exhibit sensitized anxiety-like behavior following repeated withdrawals at ED concentrations similar to those used in adults. However, this effect is longer lasting in adolescent rats. Drugs modulating CRF, 5-HT, or GABA systems during initial withdrawals prevent the development of anxiety-like behavior otherwise manifest during a final withdrawal in adolescent rats.
Neuropeptide Y (NPY) signaling regulation of corticolimbic communication is known to modulate binge-like ethanol consumption in rodents. In this work we sought to assess the impact of intra-BLA NPY system modulation on binge-like ethanol intake and to assess the role of the NPY1R+ projection from the BLA to the mPFC in this behavior. We used "drinking-in-the-dark" (DID) procedures in C57BL6J mice to address these questions. First, the impact of intra-BLA administration of NPY on binge-like ethanol intake was assessed. Next, the impact of repeated cycles of DID intake on NPY1R expression in the BLA was assessed with use of immunohistochemistry (IHC). Finally, chemogenetic inhibition of BLA→mPFC NPY1R+ projections was assessed to determine if limbic communication with the mPFC was specifically involved in binge-like ethanol intake. Importantly, as both the BLA and NPY system are sexually dimorphic, both sexes were assessed in these studies. Intra-BLA NPY dose-dependently decreased binge-like ethanol intake in males only. Repeated DID reduced NPY1R expression in the BLA of both sexes. Silencing of BLA→mPFC NPY1R+ neurons significantly reduced binge-like ethanol intake in both sexes in a dose-dependent manner. We provide novel evidence that (1) intra-BLA NPY reduces binge-like ethanol intake in males; (2) binge-like ethanol intake reduces NPY1R levels in the BLA; and (3) chemogenetic inhibition of BLA→mPFC NPY1R+ neurons blunts binge-like drinking in male and female mice. These observations provide the first direct evidence that NPY signaling in the BLA, and specifically BLA communication with the mPFC, modulates binge-like ethanol consumption.
Alcohol use disorder (AUD) is a chronically relapsing disease characterized by loss of control in seeking and consuming alcohol (ethanol) driven by the recruitment of brain stress systems. However, AUD differs among the sexes: men are more likely to develop AUD, but women progress from casual to binge drinking and heavy alcohol use more quickly. The central amygdala (CeA) is a hub of stress and anxiety, with corticotropin-releasing factor (CRF)-CRF1 receptor and Gamma-Aminobutyric Acid (GABA)-ergic signaling dysregulation occurring in alcohol-dependent male rodents. However, we recently showed that GABAergic synapses in female rats are less sensitive to the acute effects of ethanol. Here, we used patch-clamp electrophysiology to examine the effects of alcohol dependence on the CRF modulation of rat CeA GABAergic transmission of both sexes. We found that GABAergic synapses of naïve female rats were unresponsive to CRF application compared to males, although alcohol dependence induced a similar CRF responsivity in both sexes. In situ hybridization revealed that females had fewer CeA neurons containing mRNA for the CRF1 receptor (Crhr1) than males, but in dependence, the percentage of Crhr1-expressing neurons in females increased, unlike in males. Overall, our data provide evidence for sexually dimorphic CeA CRF system effects on GABAergic synapses in dependence.
Background
Early initiation of alcohol drinking has been associated with increased risk of alcohol dependence in adulthood. Although negative affect mediated in part by corticotropin‐releasing factor (CRF) is a strong motivator for alcohol consumption in adults, comparisons of alcohol withdrawal in adolescents and adults generally have not included CRF‐related measures like anxiety. The purpose of the present study was to compare withdrawal signs including anxiety‐like behavior after a brief multi‐day alcohol treatment in adolescent and adult male and female rats.
Methods
Animals were treated with a 5‐day regimen of alcohol injections (3 daily i.p. injections of 1.5 g/kg at 3‐hour intervals, total of 15) starting on PN 28 or PN 70. Spontaneous withdrawal signs and anxiety‐like behavior (light/dark box) were assessed 18 hours after the last injection as described. One cohort of rats was treated with alcohol, killed 18 hours after the last injection, and blood collected to assess corticosterone. Another cohort of rats was treated with alcohol or vehicle, given 1, 2 or 3 alcohol injections (1.5 g/kg) and killed 1 hour after final injection to determine blood alcohol concentration (BAC). Finally, adult and adolescent males and females received 5 days of alcohol or vehicle treatment followed by a final challenge with alcohol (3 g/kg), and blood was collected for corticosterone.
Results
BAC was comparable in adolescents and adults. Spontaneous withdrawal signs were comparable in adolescents and adults, and no sex differences were observed. Anxiety‐like behaviors (time and distance in light, latency to emerge and light entries) differed in alcohol‐ and vehicle‐treated adults but not adolescents. Corticosterone was not elevated at withdrawal. Alcohol increased corticosterone significantly in vehicle‐treated animals, but both adolescents and adults were tolerant to alcohol‐induced elevation of corticosterone after 5 days of alcohol treatment.
Conclusions
These findings suggest that adolescents experience milder negative affect during withdrawal from brief alcohol exposures relative to adults but comparable suppression of HPA axis function.
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Background:
Alcohol use disorders are characterized by a complex behavioral symptomatology, which includes the loss of control over alcohol consumption and the emergence of a negative affective state when alcohol is not consumed. Some of these symptoms can be recapitulated in rodent models, for instance following chronic intermittent ethanol (EtOH; CIE) vapor inhalation. However, the detection of negative affect in mice withdrawn from CIE has proven challenging and variable between strains. This study aimed to detect reliable indices of negative emotionality in CIE-exposed C57BL/6J (C57) and DBA/2J (DBA) mice. Males were used because they are known to escalate their voluntary EtOH consumption upon CIE exposure, which is hypothesized to be driven by negative reinforcement (relief from negative affect).
Methods:
Adult male mice were exposed to 4 to 6 weeks of CIE and were evaluated 3 to 10 days into withdrawal in the social approach, novelty-suppressed feeding, digging, marble burying, and bottle brush tests.
Results:
Withdrawal from CIE decreased sociability in DBA mice but not in C57 mice. Conversely, hyponeophagia was exacerbated by CIE in C57 mice but not in DBA mice. Withdrawal from CIE robustly increased digging activity in both strains, even in the absence of marbles. Aggressive responses to bottle brush attacks were elevated in both C57 and DBA mice following CIE exposure, but CIE had an opposite effect on defensive responses in the 2 strains (increase in C57 vs. decrease in DBA).
Conclusions:
Our results indicate that withdrawal from CIE elicits negative emotionality in both C57 and DBA mice, but different tests need to be used to measure the anxiogenic-like effects of withdrawal in each strain. Increased digging activity and irritability-like behavior represent novel indices of affective dysfunction associated with withdrawal from CIE in both mouse strains. Our findings enrich the characterization of the affective symptomatology of protracted withdrawal from CIE in mice.
Toll-like receptors (TLRs) are components of the innate immune system that respond to exogenous infectious ligands (pathogen-associated molecular patterns, PAMPs) and endogenous molecules that are released during host tissue injury/death (damage-associated molecular patterns, DAMPs). Interaction of TLRs with their ligands leads to activation of downstream signaling pathways that induce an immune response by producing inflammatory cytokines, type I interferons (IFN), and other inflammatory mediators. TLR activation affects vascular function and remodeling, and these molecular events prime antigen-specific adaptive immune responses. Despite the presence of TLRs in vascular cells, the exact mechanisms whereby TLR signaling affects the function of vascular tissues are largely unknown. Cardiovascular diseases are considered chronic inflammatory conditions, and accumulating data show that TLRs and the innate immune system play a determinant role in the initiation and development of cardiovascular diseases. This evidence unfolds a possibility that targeting TLRs and the innate immune system may be a novel therapeutic goal for these conditions. TLR inhibitors and agonists are already in clinical trials for inflammatory conditions such as asthma, cancer, and autoimmune diseases, but their study in the context of cardiovascular diseases is in its infancy. In this article, we review the current knowledge of TLR signaling in the cardiovascular system with an emphasis on atherosclerosis, hypertension, and cerebrovascular injury. Furthermore, we address the therapeutic potential of TLR as pharmacological targets in cardiovascular disease and consider intriguing research questions for future study.
Alcohol is widely used in society for both its social and medicinal benefits. It is readily derived in nature from fermentation. Taken in excess, it is one of the most toxic substances in society from the perspective of both behavioral and physical damage to the body. Alcohol is a sedative hypnotic that has euphoric and disinhibitory effects, explaining why some see it as a “social lubricant.” As the dose of alcohol increases to levels associated with binge drinking, however, disinhibition gives way to motor impairment, muscular incoordination, impairments in reaction time, impairments in judgment, impairments in sensory processing, and impairments in cognitive function – all behavioral effects that contribute to its behavioral toxicity. The chronic use of alcohol can lead to alcoholism, and numerous other medical diseases can also result from chronic alcohol use.
Alcoholism and Anxiety: Animal Models. Animal models for the study of alcoholism has been developed during the last years. These models contribute in revealing the mechanisms involved in this pathology. A synthesis of the main models used with rodents is presented along with studies that show the complex links between anxiety and alcoholism. The results indicate that alcohol acts as anxiolytic during early stages of consumption and as an anxiogenic agent when absent after chronic or acute doses, that the forced administration of ethanol provokes anxiolytic effects, and that the application of stressors alters the voluntary consumption of alcohol. There are few experiments that tested the relationship between alcoholism and frustration, a state considered equal to physical pain and to learned fear. The first results on the later subject obtained by the authors are described.
Experimental studies in laboratory animals discussed in this chapter demonstrate the involvement of various metabotropic glutamate
receptors (mGluRs) in different aspects of drug and alcohol dependence. Postsynaptic mGluR5 antagonists and inhibitory presynaptic
mGluR2/3 agonists decreased drug self-administration and attenuated reinstatement of drug-seeking behavior by reducing the
increases in glutamate transmission induced by drugs of abuse or the presentation of stimuli previously associated with the
drug effects or availability. These findings suggest that medications decreasing glutamatergic transmission may reduce the
reinforcing and motivational properties of drugs of abuse and prevent relapse to drug taking in humans. mGluR2/3 antagonists
may be useful in the treatment of depressive-like affective symptoms of drug withdrawal by reversing the hypothesized decrease
in glutamate transmission that occurs during psychostimulant, but not opiate, withdrawal. The potential of these compounds
as medications for drug and alcohol dependence remains to be evaluated in humans.
Once dependent on alcohol or opioids, negative affect may accompany withdrawal. Dependent individuals are hypothesized to "self-medicate" in order to cope with withdrawal, which promotes escalated alcohol and drug use.
The current study aimed to develop a reliable animal model to assess symptoms that occur during spontaneous alcohol and opioid withdrawal.
Dependence was induced using intermittent alcohol exposure or pulsatile heroin delivery and assessed for the presence of withdrawal symptoms during acute withdrawal by measuring somatic signs, behavior in the forced swim test (FST), and air-puff-induced 22-kHz ultrasonic vocalizations (USVs). Additional animals subjected to 8 weeks of alcohol vapor exposure were evaluated for altered somatic signs, operant alcohol self-administration, and 22-kHz USV production, as well as performance in the elevated plus maze (EPM).
During spontaneous withdrawal from pulsatile heroin or intermittent alcohol vapor, animals displayed increased somatic withdrawal signs, FST immobility, and 22-kHz USV production but did not show any behavioral change in the EPM unless the duration of alcohol exposure was extended to 4 weeks. Following 8 weeks of alcohol vapor exposure, animals displayed somatic withdrawal signs, escalated alcohol self-administration, and increased 22-kHz USVs.
These paradigms provide consistent methods to evaluate the behavioral ramifications, and neurobiological substrates, of alcohol and opioid dependence during spontaneous withdrawal. As immobility in the FST and percent open-arm time in the EPM were dissociable, with 22-kHz USVs paralleling immobility in the FST, assessment of air-puff-induced 22-kHz USVs could provide an ethologically valid alternative to the FST.
Alcohol withdrawal is associated with reduced activity, increased anxiety, and other signs of distress.
The goal of the current studies was to determine whether acute ethanol exposure would alter hypothalamic-pituitary-adrenal (HPA) axis reactivity and cytokine responses to stress challenges imposed during the withdrawal period.
Male Sprague-Dawley rats were intubated with 4 g/kg of ethanol to simulate acute binge-like ethanol intake. After characterizing the blood ethanol concentrations (BECs; Experiment 1), exploratory activity in a novel environment was explored at 10, 14 and 18 h after ethanol (Experiment 2) to characterize altered activity patterns indicative of withdrawal. In Experiment 3, rats were exposed to footshock during withdrawal to examine whether prior ethanol exposure would alter cytokine and HPA axis responses to stress. Experiments 4 and 5 investigated HPA axis sensitivity and gene expression changes during restraint imposed during withdrawal.
Prior ethanol exposure produced a period of stress hyper-reactivity evidenced by an enhanced HPA axis response (increased corticosterone and adrenocorticotropic hormone) observed during withdrawal. While this hyper-reactivity in response to two different stress challenges (novel environment and restraint) was accompanied by profound behavioral changes indicative of withdrawal, no alterations in cytokine changes evoked by stress were observed.
Taken together, these findings provide support for the hypothesis that alcohol withdrawal enhances HPA axis reactivity to stress challenges, though not likely as the result of heightened inflammatory signaling, and may have implications for understanding the mechanisms by which stress impacts relapse drinking in humans.
Increased anxiety is a characteristic of the acute ethanol withdrawal syndrome. Repeated exposure of rats to withdrawal from chronic ethanol increases sensitivity to seizures.
We investigated whether repeated withdrawal experience increases withdrawal-induced anxiety and stress, and if it changes withdrawal-induced activation of related brain areas.
Rats were chronically treated with an ethanol-containing liquid diet either for 24 days continuously (single withdrawal, SWD) or interspersed with 2x3-day withdrawal periods (repeated withdrawal, RWD), or with a control diet. Eight hours after ethanol withdrawal, anxiety-like behaviour was tested in the elevated plus-maze, blood corticosterone levels were measured, and expression level of markers of neuronal activity and plasticity, c-fos and zif268, was assessed.
Eight hours after ethanol withdrawal, SWD rats showed increased anxiety on the elevated plus-maze relative to control rats. Rats given previous withdrawal experiences did not show further increases in measures of anxiety. Corticosterone levels were elevated during withdrawal in SWD rats but not in RWD rats. RWD resulted in marked increases in c-fos expression in amygdala, hippocampus, nucleus accumbens and dorsolateral periaqueductal grey. In contrast, zif268 expression was not increased after RWD, and in central amygdala the marked increase in zif268 seen after SWD was absent after RWD.
The data suggest increased ability of withdrawal to activate neuronal circuits but reduced plasticity after RWD. We suggest parallels between the consequences of repeated ethanol withdrawal and repeated exposure to stress, and discuss implications of withdrawal for brain plasticity.
Alcoholism is a chronic relapsing disorder. Major characteristics observed in alcoholics during an initial period of alcohol abstinence are altered physiological functions and a negative emotional state. Evidence suggests that a persistent, cumulative adaptation involving a kindling/allostasis-like process occurs during the course of repeated chronic alcohol exposures that is critical for the negative symptoms observed during alcohol withdrawal. Basic studies have provided evidence for specific neurotransmitters within identified brain sites being responsible for the negative emotion induced by the persistent cumulative adaptation following intermittent-alcohol exposures. After an extended period of abstinence, the cumulative alcohol adaptation increases susceptibility to stress- and alcohol cue-induced negative symptoms and alcohol seeking, both of which can facilitate excessive ingestion of alcohol. In the alcoholic, stressful imagery and alcohol cues alter physiological responses, enhance negative emotion, and induce craving. Brain fMRI imaging following stress and alcohol cues has documented neural changes in specific brain regions of alcoholics not observed in social drinkers. Such altered activity in brain of abstinent alcoholics to stress and alcohol cues is consistent with a continuing ethanol adaptation being responsible. Therapies in alcoholics found to block responses to stress and alcohol cues would presumably be potential treatments by which susceptibility for continued alcohol abuse can be reduced. By continuing to define the neurobiological basis of the sustained alcohol adaptation critical for the increased susceptibility of alcoholics to stress and alcohol cues that facilitate craving, a new era is expected to evolve in which the high rate of relapse in alcoholism is minimized.
Alcohol withdrawal is associated with affective-behavioral disturbances in both human alcoholics and in animal models. In general, these phenomena are potentiated by increased alcohol exposure duration and by prior withdrawal episodes. Previous studies have also reported locomotor hypoactivity during ethanol withdrawal in rats and mice, but only in novel test environments and not in the home cage. In the present study, we examined the effects of withdrawal from chronic intermittent ethanol (CIE) vapor exposure on the level and circadian periodicity of wheel-running activity in C57BL/6J mice. CIE treatment resulted in reductions in wheel-running activity compared with plain-air controls that persisted for about 1 week after withdrawal. Analysis of circadian waveforms indicated that reduced activity occurred throughout the night phase, but that daily-activity patterns were otherwise unaltered. CIE failed to alter free-running circadian period or phase in animals maintained under constant darkness. These results show that ethanol withdrawal can result in locomotor hypoactivity even in the habitual, home-cage environment, and suggest that withdrawal-related reductions in wheel-running activity may reflect the specific motivational significance of this behavior.
Gender differences in anxiety were examined in a large sample of adolescents that included 1,079 who had never met criteria for any disorder, 95 who had recovered from an anxiety disorder, and 47 who had a current anxiety disorder. Participants were examined on a wide array of psychosocial measures. There was a preponderance of females among current and recovered anxiety disorder cases, but not among those who had never experienced an anxiety disorder. The female preponderance emerges early in life, and retrospective data indicate that at age 6, females are already twice as likely to have experienced an anxiety disorder than are males. Psychosocial variables that were correlated with both anxiety and gender were identified. Statistically controlling for these variables did not eliminate the gender differences in prevalence or anxiety symptom means.
Repeated withdrawals from chronic ethanol sensitize the withdrawal-induced reduction in social interaction behaviors. This study determined whether stress might substitute for repeated withdrawals to facilitate withdrawal-induced anxiety-like behavior. When two 1-h periods of restraint stress were applied at 1-week intervals to rats fed control diet, social interaction was reduced upon withdrawal from a subsequent 5-day exposure to ethanol diet. Neither this ethanol exposure alone nor exposure to three restraint stresses alone altered this measure of anxiety. Further, the repeatedly stressed singly withdrawn rats continued to exhibit a reduction in social interaction 16 days later, upon withdrawal from re-exposure to 5 days of chronic ethanol, consistent with a persistent adaptation by the multiple-stress/withdrawal protocol. Weekly administration of corticosterone in place of stress induced no significant change in social interaction upon withdrawal from the single chronic ethanol exposure, indicative that corticoid release is not responsible for the stress-induced reduction in anxiety-like behavior during withdrawal. In the multiple-withdrawal protocol, stress applied during withdrawal from voluntary ethanol drinking by P-rats facilitated ethanol drinking sufficiently, to induce a withdrawal-induced reduction in social interaction. Administration of a CRF-1 receptor antagonist, a benzodiazepine receptor antagonist, or a 5-HT 1A receptor agonist prior to each stress minimized sensitization of the withdrawal-induced reduction in anxiety-like behavior. Since these pharmacological consequences on the induction of anxiety-like behavior following the stress/withdrawal protocol are like those previously seen when these drug treatments were given prior to multiple withdrawals, evidence is provided that repeated stresses and multiple withdrawals sensitize the withdrawal reduction in social interaction by similar central adaptive mechanisms.
After 7 day's gradual introduction of ethanol, rats were maintained for a further 4 weeks on a liquid diet containing 10% ethanol (mean daily dose 11.8 +/- 0.2 g/kg/day). Control-treated rats received liquid diet alone. Pairs of rats were tested in the social interaction test of anxiety 8 h after withdrawal. Withdrawal from ethanol significantly reduced the time spent in social interaction compared with controls, indicating an anxiogenic withdrawal response. Nitrendipine (50 mg/kg) had no effect on, whereas flumazenil (4 mg/kg) significantly reversed, this withdrawal response. This reversal appeared to be long-lasting as there was still no evidence of increased anxiety when rats were again withdrawn after 3 more days of ethanol diet.
Rats were fed a liquid diet with concentrations of ethanol increasing over 2 weeks and then maintained for 4 weeks to give a mean ethanol intake of 11.6 g/kg/day. They were withdrawn from ethanol for 12 hr and tested in two tests of anxiety, the social interaction and elevated plus-maze tests, and in the holeboard, which provides measures of exploration and motor activity. Compared with control animals that had received the liquid diet without ethanol, the rats withdrawn from ethanol showed significant reductions in social interaction, in the percentage of entries onto, and time spent on, the open arms of the plus-maze, and in measures of general activity in all three tests. Tianeptine, a tricyclic antidepressant which increases 5-HT uptake, reversed the anxiogenic responses and the hypoactivity detected in the social interaction test, but was without significant effect in the other tests.
On the basis of our previous series of experiments we had postulated that the increased anxiety that occurred during diazepam withdrawal was mediated by increased 5-HT release in the hippocampus. The present series of experiments provide evidence for a major role of the median raphé nucleus (MRN) dorsal hippocampal pathway. Rats were treated once daily for 21 days with diazepam (2 mg/kg IP) and then tested after 24 h withdrawal in the social interaction test of anxiety. Relative to chronically vehicle treated animals, those withdrawn from diazepam were significantly more anxious and had significantly greater K(+)-evoked release of [3H]-5-hydroxytryptamine (5-HT) from slices of dorsal and of ventral regions of the hippocampus. Estimation of extracellular concentrations of 5-HT within the dorsal hippocampus, using in-vivo microdialysis, showed doubling in the levels of 5-HT in the rats withdrawn from chronic diazepam treatment. This just failed to reach significance, but 33% of the rats showed dramatic increases (650%). It was not possible to test these animals in the social interaction test, but it is proposed that only the diazepam-withdrawn rats with raised extracellular levels of 5-HT would have displayed increased anxiety. 5-HT1A receptor agonists injected into the MRN decrease the MRN firing rate, and hence the release of 5-HT in the dorsal hippocampus. As a further test of our hypothesis, we examined the effects of MRN injection of the 5-HT1A receptor agonist, 8-OH DPAT, on animals withdrawn from diazepam and tested in the low light familiar condition of the social interaction test. 8-OH DPAT (50-200 ng) dose-dependently reversed the anxiogenic effect of diazepam withdrawal, while having no effects in chronic vehicle-treated animals. These results provide clear evidence that the MRN-dorsal hippocampal 5-HT pathway is at least one of the pathways playing an important role in mediating diazepam withdrawal-induced anxiety.
In previous research, the drug flumazenil has been categorized both as a pure benzodiazepine antagonist and as a benzodiazepine partial agonist. The following studies used an elevated plus maze to test whether flumazenil would exert any antianxiety action in rats. While chlordiazepoxide (3.0 mg/kg), ethanol (0.75 g/kg), and the atypical benzodiazepine zolpidem (1.0 mg/kg) all significantly increased time spent on the open arms and percent open arm entries, flumazenil (1-10 mg/kg) alone did not produce any anxiolytic effects on the maze. Withdrawal from chronic ethanol treatment led to a decrease in open arm time and percent open arm entries. Flumazenil (3.0 mg/kg) blocked these changes, suggesting that the effects of flumaxenil are at least partially dependent upon the levels of stress or anxiety in the subjects. An anxiolytic action of flumazenil was not seen following the central administration of the neuropeptide corticotropin-releasing factor (CRF), which reduced open arm time on the elevated plus maze. These results support the hypothesis that the mechanism of action for flumazenil effects on the anxiety observed during ethanol withdrawal involves antagonism of an endogenous benzodiazepine inverse agonist, rather than activity as a partial agonist or blockade of CRF-mediated effects.
Administration of flumazenil, a benzodiazepine (BZD) antagonist, has therapeutic efficacy against some anxiogenic effects of ethanol withdrawal. This observation has led to the suggestion that anxiety associated with ethanol withdrawal is related to release in brain of an endogenous BZD inverse agonist.
The present studies further tested this hypothesis by assessing the effect of flumazenil on withdrawal-induced changes in a behavioral task and on the expression of the neuronal protein, Fos.
Male Sprague-Dawley rats were withdrawn from a chronic ethanol regimen and tested, with or without flumazenil pretreatment, for either ultrasonic vocalization in response to air puff or for the induction of Fos protein-like immunoreactivity (Fos-LI) in brain. In addition, flumazenil effects on Fos-LI were measured in a group of animals treated with the BZD inverse agonist DMCM (0.75 and 1.0 mg/kg).
Flumazenil (5.0 mg/kg) significantly reduced the number of ultrasonic vocalizations observed following withdrawal from chronic ethanol. In contrast, flumazenil (5.0 mg/kg), given either 14 h before withdrawal from chronic ethanol, or during hours 3 and 5 following withdrawal, did not attenuate the effects of withdrawal on Fos-LI. Subsequent testing with DMCM confirmed that a benzodiazepine inverse agonist can induce Fos-LI in most of the same brain regions as observed following ethanol withdrawal, and that this change in Fos protein can be attenuated by pretreatment with flumazenil (5.0 mg/kg).
Overall, these results demonstrate that specific behavioral indices of anxiety, but not measures of Fos-LI, support the contribution of an endogenous BZD inverse agonist in the ethanol withdrawal syndrome.
Repeated withdrawals from chronic forced ethanol exposure sensitize animals to withdrawal-induced deficits in social interaction behavior. The deficits in social interaction behavior following withdrawal from continuous ethanol exposure can be reduced following acute treatments with 5-HT(2C) antagonists or 5-HT(1A) agonists.
The present study investigated whether prior treatment with these serotonergic agents during early withdrawals in rats subjected to repeated withdrawals from ethanol exposure would ameliorate the social interaction deficits observed following the final withdrawal.
Sprague-Dawley rats were exposed to three cycles of 5 days forced ethanol (7%, w/v), with 2 days of control diet after the first and second cycles. Drugs were administered IP 4 h after removal of ethanol on the first and second cycles but not the third in one group and 4.5 h after removal of ethanol on the third cycle in another. The social interaction test was performed 5 h after removal of ethanol on the third cycle. Drugs tested included SB-242084, a 5-HT(2C) antagonist; buspirone, a 5-HT(1A) partial agonist; WAY-100635, a 5-HT(1A) antagonist; ketanserin, a 5-HT(2A) antagonist; ritanserin, a mixed 5-HT(2A/2C) antagonist; and Ro-601075, a 5-HT(2C) agonist.
Both SB-242084 and buspirone reduced ethanol withdrawal-induced deficits in social interaction when given either acutely 30 min before the test or at 4 h after withdrawal from the first and second cycles. WAY-100635 and ketanserin were completely ineffective regardless of mode of treatment. In contrast, the 5-HT(2C) agonist, Ro-601075, accentuated the withdrawal-induced deficit in social interaction behavior in rats exposed to either 4.5 or 7% ethanol diet.
These results support the utility of 5-HT(1A) agonists and 5-HT(2C) antagonists in reducing anxiety-like behavior induced by ethanol withdrawal and reducing the adaptive changes associated with repeated withdrawals.
Repeated withdrawals from chronic ethanol sensitize the withdrawal-induced reduction in social interaction behaviors. This study determined whether stress might substitute for repeated withdrawals to facilitate withdrawal-induced anxiety-like behavior. When two 1-h periods of restraint stress were applied at 1-week intervals to rats fed control diet, social interaction was reduced upon withdrawal from a subsequent 5-day exposure to ethanol diet. Neither this ethanol exposure alone nor exposure to three restraint stresses alone altered this measure of anxiety. Further, the repeatedly stressed singly withdrawn rats continued to exhibit a reduction in social interaction 16 days later, upon withdrawal from re-exposure to 5 days of chronic ethanol, consistent with a persistent adaptation by the multiple-stress/withdrawal protocol. Weekly administration of corticosterone in place of stress induced no significant change in social interaction upon withdrawal from the single chronic ethanol exposure, indicative that corticoid release is not responsible for the stress-induced reduction in anxiety-like behavior during withdrawal. In the multiple-withdrawal protocol, stress applied during withdrawal from voluntary ethanol drinking by P-rats facilitated ethanol drinking sufficiently, to induce a withdrawal-induced reduction in social interaction. Administration of a CRF-1 receptor antagonist, a benzodiazepine receptor antagonist, or a 5-HT(1A) receptor agonist prior to each stress minimized sensitization of the withdrawal-induced reduction in anxiety-like behavior. Since these pharmacological consequences on the induction of anxiety-like behavior following the stress/withdrawal protocol are like those previously seen when these drug treatments were given prior to multiple withdrawals, evidence is provided that repeated stresses and multiple withdrawals sensitize the withdrawal reduction in social interaction by similar central adaptive mechanisms.
Clinical studies report an increase in the prevalence of alcohol withdrawal-related seizures in patients with a history of multiple detoxifications. In order to investigate the alcohol withdrawal-related alterations in neural activity that lead to this increase in seizure propensity, basic researchers have examined both spontaneous and elicited seizures in animals undergoing withdrawal from chronic ethanol. This study was designed to further examine alcohol withdrawal-related seizure activity in a rodent model by assessing the development of electrical kindling after chronic ethanol exposure administered in multiple or single treatment episodes. Laboratory rats were exposed to either five periods of 3 days of ethanol, one 15-day period of continuous ethanol, or a period of control handling with no ethanol exposure. Ten days after a final withdrawal episode, all animals were surgically prepared with recording and stimulating electrodes. Twenty days after final withdrawal from ethanol or an equivalent period of similar handling, daily electrical stimulation of hippocampal area CA,was initiated. Animals exposed to ethanol required more daily stimulations to become fully kindled than did ethanol-naive controls, with those animals experiencing five withdrawals requiring the most stimulations overall and more stimulations to progress from focal to generalized seizure behaviors. These results indicate that chronic ethanol exposure and withdrawal alter neuronal mechanisms important for hippocampal kindling in a manner that persists long after cessation of ethanol exposure, and they indicate that this effect is increased by exposure to repeated withdrawal episodes.
Gender differences in anxiety were examined in a large sample of adolescents that included 1,079 who had never met criteria for any disorder, 95 who had recovered from an anxiety disorder, and 47 who had a current anxiety disorder. Participants were examined on a wide array of psychosocial measures. There was a preponderance of females among current and recovered anxiety disorder cases, but not among those who had never experienced an anxiety disorder. The female preponderance emerges early in life, and retrospective data indicate that at age 6, females are already twice as likely to have experienced an anxiety disorder than are males. Psychosocial variables that were correlated with both anxiety and gender were identified. Statistically controlling for these variables did not eliminate the gender differences in prevalence or anxiety symptom means. (PsycINFO Database Record (c) 2012 APA, all rights reserved)
1 Pairs of male rats were placed in a test box for 10 min and the time they spent in active social interaction was scored. Maximum active interaction was found when the rats were tested under low light in a box with which they were familiar. When the light level was increased or when the box was unfamiliar active social interaction decreased. 2 Exploration (time spent sniffing objects) decreased in the same way in relation to test conditions as did social interaction. As these decreased, defecation, and freezing increased. 3 Anosmic controls showed that the decrease in social interaction across test conditions could not be attributed to olfactory changes in the partner. 4 Chlordiazepoxide (5 mg/kg) given chronically prevented or significantly reduced the decrease in social interaction that occurred in undrugged rats as the light level or the unfamiliarity of the test box was increased. Controls showed that this effect could not be entirely attributed to chlordiazepoxide acting selectively to increase low levels of responding. 5 The effect of chronic chlordiazepoxide contrasts with its action when given acutely; in the latter case it has only sedative effects. 6 Whether this test can be used as an animal model of anxiety is discussed and this test is compared with existing tests of anxiety.
The ethanol withdrawal syndrome includes anxiety as a prominent symptom. Because the extent that specific regions of brain are critical to the generation of this emotional state is unknown, Fos-like immunoreactivity (Fos-LI) was used to associate specific regions of the rat brain with the anxiety component of the ethanol withdrawal syndrome exacerbated by an air puff challenge in rats. Chronic ethanol liquid diet was administered intragastrically for 4 days or by having the rats consume the diet for 14 days. During withdrawal from either treatment protocol, Fos-LI was induced most prominently in forebrain areas, although the midbrain and hindbrain were also represented. Included in these Fos-LI positive regions were many cortical regions, septum, accumbens, claustrum, amygdala, paraventricular nucleus of the thalamus and hypothalamus, hippocampus, locus coeruleus, and central gray. Fos-LI expression differed mostly in intensity between the two treatment and withdrawal protocols, with the gastric protocol producing the greatest Fos-LI induction in most brain regions. The threshold for air puff-induced ultrasonic vocalizations was decreased, and the number of vocalizations was increased and the period of vocalization was extended. These behavioral data indicate that aversively motivated responding in rats during ethanol withdrawal can be readily quantified with the ultrasonic vocalizations test without precipitating convulsive activity. Furthermore, a comparison of the effects of the air puff challenge versus withdrawal on Fos-LI indicated that the behavioral state induced in these two situations share functional neuroanatomical features. Some regions–such as the accumbens core, medial septum, subregions of the amygdala, hippocampus, substantia nigra, and cerebellum–exhibited little Fos-LI during withdrawal and also did not exhibit strong increases after the addition of the air puff challenge. However, other regions–such as the cerebral cortex (medial prefrontal, frontal, cingulate and ventrolateral orbital, claustrum, and tenia tecta), hypothalamus, and locus ceoruleus–exhibited Fos-LI at levels higher than that seen after either the ethanol withdrawal or puff challenge alone. These overlapping patterns of Fos-LI in specific regions of the brain, activated by both ethanol withdrawal and an anxiety provoking behavioral challenge, suggest that specific neuroanatomical sites in brain are associated with the symptom of anxiety observed during the “ethanol withdrawal syndrome.”
The present investigation tested the hypothesis that multiple withdrawals from chronic ethanol treatment “kindles” seizure activity. Two animal models of kindled seizure activity–electrical stimulation of the inferior collicular cortex or the amygdala–were used to evaluate this hypothesis. Four withdrawals from a 12-day ethanol-liquid diet regimen facilitated the seizure kindling rate in the inferior collicular cortex, when the stimulation was initiated 7 days after the last withdrawal. In contrast, four withdrawals from this chronic ethanol regimen significantly attenuated the rate of amygdaloid kindling. When the withdrawals were increased to six or 10 using a 5-day chronic ethanol treatment schedule, the kindling rate in the inferior collicular cortex proved directly proportional to the withdrawal number. Continuous ethanol exposure over the same period as the 10 withdrawal group also facilitated the inferior collicular kindling rate, but not to the extent found in the 10 withdrawal group. As before, 10 withdrawals from the 5-day chronic ethanol liquid diet treatment attenuated the rate of amygdaloid kindling. Thus, this kindling action of repeated ethanol withdrawals appears specific to seizures originating from the inferior collicular cortex, not the limbic system. These findings support a previous hypothesis for a kindling etiology of alcoholism related seizures.
Periodic brain stimulation, particularly in the limbic system, at stimulus intensities initially too low to produce any behavioural or EEG effects, progressively produces EEG changes, motor automatisms, and eventually convulsions, an effect called kindling. Data are presented and reviewed that suggest that the severity of alcohol withdrawal symptoms progressively increases over years of alcohol abuse in a stepwise fashion similar to the kindling process. The model is presented that the limbic system hyperirritability which accompanies each alcohol withdrawal serves over time to kindle increasingly widespread subcortical structures. These long-term changes in neuronal excitability might relate to the progression of alcohol withdrawal symptoms from tremor to seizures and delirium tremens, as well as the alcoholic personality changes between episodes of withdrawal.
Pairs of male rats were placed in a test box for 10 min and the time they spent in active social interaction was scored. Maximum active interaction was found when the rats were tested under low light in a box with which they were familiar. When the light level was increased or when the box was unfamiliar active social interaction decreased.
Exploration (time spent sniffing objects) decreased in the same way in relation to test conditions as did social interaction. As these decreased, defecation, and freezing increased.
Anosmic controls showed that the decrease in social interaction across test conditions could not be attributed to olfactory changes in the partner.
Chlordiazepoxide (5 mg/kg) given chronically prevented or significantly reduced the decrease in social interaction that occurred in undrugged rats as the light level or the unfamiliarity of the test box was increased. Controls showed that this effect could not be entirely attributed to chlordiazepoxide acting selectively to increase low levels of responding.
The effect of chronic chlordiazepoxide contrasts with its action when given acutely; in the latter case it has only sedative effects.
Whether this test can be used as an animal model of anxiety is discussed and this test is compared with existing tests of anxiety.
The abilities of ketotifen and other 4-piperidylidene derivatives (HF200-184, HE36-953, SDZ209-321 and SDZ206-703) to inhibit aversive responding were compared in the mouse light/dark test box and in the rat social interaction test. Ketotifen and HF200-184 reduced aversive responding of the mouse to the brightly illuminated area of the test box and facilitated rat social interaction; HF200-184 was approximately 100 times more potent than ketotifen. The chronic administration and withdrawal from treatment with diazepam, ethanol, nicotine and cocaine in the mouse was associated with increased behavioural suppression which was prevented by the administration of ketotifen and HF200-184 during the period of withdrawal. HE36-953 also prevented the behavioural consequences of withdrawal from diazepam and cocaine. The relative potencies of ketotifen and its analogues to inhibit aversive responding did not correlate with their affinities for the 5-HT3 recognition site. It is concluded that compounds within the 4-piperidylidene series can reduce behavioural suppression in rodent models of anxiety and attenuate the behavioural consequences of withdrawing from treatment with drugs of abuse.
Social interaction (SI) between two unfamiliar male rats in a dimly lit, familiar environment has been investigated as a model of anxiety, where novelty of the partner remains as the principal anxiogenic stimulus. A range of centrally acting drugs have been tested in this situation. Chlordiazepoxide, nitrazepam, flunitrazepam, and flurazepam all increase SI, as does buspirone, CL 218872, suriclone, sodium valproate, and nicotinamide in the model described. Anxiogenic agents FG 7142 and yohimbine reduced SI without significant modification of motor activities. However, the stimulant amphetamine increased all behaviours in this condition. Amphetamine also increased all behaviours when rats were tested with their cagemates, when the desire for SI is largely satiated. CL 218872 also increased SI in this second situation, and it is suggested that this agent may have a non-specific component in its action in this test. Additionally, caffeine, theophylline, and piracetam may also have non-specific behavioural actions in this model.
There has been speculation that a kindling model may have applicability to alcohol withdrawal syndromes and seizures, suggesting that repeated alcohol withdrawals may lead to increased severity of subsequent withdrawals. We evaluated historical and clinical variables of a group of male alcoholics with (n = 25) and without (n = 25) alcohol withdrawal seizures. We found that the number of detoxifications appeared to be an important variable in the predisposition to withdrawal seizures. The withdrawal seizure group had 12 of 25 (48%) patients with 5 or more previous detoxifications, compared to only 3 of 25 (12%) of the control group. A relationship between alcohol use history and withdrawal seizures was not supported by the data. These findings support the concept that previous alcohol withdrawals may "kindle" more serious subsequent withdrawal symptomatology, ultimately culminating in withdrawal seizures.
This paper examines the concept of emotionality, particularly in relation to measures taken in ‘novel environment’ tests (e.g. the open field). Evidence of several different types is reviewed to assess the validity of the measures in relation to common assumptions underlying their use. In response to increased light and noise, open field defecation increases and ambulation decreases, and thus on this basis they may be termed emotional responses. In many cases defecation and ambulation in the open field showed a fairly low but significant inverse correlation. Nevertheless, a number of limitations to this relationship were found, depending on the species, strain, sex, sample size, and early experience; other, presumed, measures of emotionality in the open field showed little consistent relationship to defecation, but the evidence here was sparse. Studies measuring heart-rate responses provided insufficient evidence to relate open field defecation to this other response presumed to be under sympathetic nervous control. Ambulation showed little descriptive validity as a measure of exploration; owing to the occurrence of both immobility and escape behaviour as alternative forms of emotional behaviour, ambulation also provided an inadequate indication of emotional responses. Thus, conceptual frameworks which suggested that emotionality energized ambulation in a consistent manner were criticized. Open field defecation showed little relationship to supposed measures of emotionality taken in other types of tests (e.g. emergence tests, active avoidance learning); these findings clearly failed to support the use of emotionality as a consistent constitutional trait, with unitary drive properties. Two alternative methods were suggested, for testing: (a) responses to novel environments, and (b) emotional behaviour, without making assumptions that the measures represent unitary major motivational constructs.
The social interaction test in rats provides a method for detecting anxiolytic activity that does not use food or water deprivation, or electric shock, and therefore obviates difficulties of interpretation that might arise from drug-induced changes in motivation. Since social interaction is measured under more than one test condition any overall increase or decrease in social behaviour can be detected independently from the drug x test condition interaction that characterizes an anxiolytic drug. The Geller-Seifter conflict test was designed with two schedules of reinforcement for the same reasons. Any candidate test for anxiolytic action that examines drug effects under only one experimental condition is open to misinterpretation and may also prove unreliable if the critical experimental factors ( e.g. the level of food deprivation or the shock intensity) are changed. The testing procedure in the social interaction test is relatively time consuming in terms of observer-hours, but no lengthy pretraining of the animals is required. There is no way of fully automating the scoring and therefore it is important that the observers do not know the experimental group of the rats that they are scoring, and that tape recordings are made so that the scores can be checked. It has not so far been fruitful to analyze drug effects on every individual social behaviour, but this method does allow changes in individual behaviours to be detected. By entering the data directly into a computer we are now able to store the frequency and duration of each behaviour as well as the sequence of behaviours. It will then be possible to determine whether a detailed analysis of drug effects on the patterning of social behaviours will prove a useful addition to the social interaction test
Intraperitoneal injection of ethanol (1-2 g/kg) and chlordiazepoxide (2-16 mg/kg) suppressed susceptibility to audiogenically induced, clonic-tonic seizures and antagonized forelimb tremor in rats undergoing ethanol withdrawal, 30 min after treatment. However, a smaller dose of ethanol (0.5 g/kg) actually increased clonic seizure frequency, suggesting that ethanol exerts a biphasic proconvulsant/anticonvulsant action. Direct activation of gamma-aminobutyric acid (GABA) receptors by intracisternal administration of GABA (100-1000 micrograms), muscimol (0.3-1.0 micrograms) or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) (0.3-3.0 micrograms) 5 to 10 min before testing also reduced susceptibility to audiogenic clonic-tonic seizures. In sharp contrast to these anticonvulsant actions, GABA, muscimol and THIP had no effect on withdrawal-induced forelimb tremors. Blockade of GABA uptake with 1-2,4-diaminobutyric acid (300 and 600 mg/kg i.p.) and inhibition of GABA transaminase with aminooxyacetic acid (12.5 and 25.0 mg/kg i.p.) both reduced susceptibility to seizures. However, anticonvulsant doses of these two drugs, unlike GABA, muscimol and THIP, also reduced forelimb tremor. Three other GABA transaminase inhibitors, gamma-vinyl GABA (450 and 900 mg/kg i.p.), gamma-acetylenic GABA (50-150 mg/kg i.p.) and ethanolamine-O-sulfate (250-750 mg/kg i.p.), were inactive against ethanol withdrawal audiogenic seizures and forelimb tremors. These results indicate that direct GABA receptor activation can selectively suppress one type of ethanol withdrawal response (i.e., audiogenic seizure susceptibility) while failing to influence another (forelimb tremors).
The effects of picrotoxin (2 and 4 mg/kg) and of pentylenetetrazole (PTZ 5, 10 and 20 mg/kg) were examined in a social-interaction test of anxiety. Picrotoxin (2 mg/kg) caused a significant reduction in active social interaction, without a concomitant reduction in motor activity, indicating an anxiogenic action. Picrotoxin (4 mg/kg) and pentylenetrazole (20 mg/kg) reduced social interaction by more than 75% and motor activity by 40%. Although it is likely that the reduction in motor activity is secondary to the very low levels of social interaction, an unambiguous interpretation of an anxiogenic effect is not possible. Increased concentrations of corticosterone in plasma following administration of picrotoxin were consistent with the drug being anxiogenic. Chlordiazepoxide (5 mg/kg) significantly reversed the reductions in social interaction and locomotor activity following injection of picrotoxin (4 mg/kg) and PTZ (20 mg/kg).
The effects of short- and long-term administration of the selective serotonin reuptake inhibitor paroxetine were investigated in a rat social interaction test. A single administration of paroxetine at oral doses of 0.3, 1, 3 and 10 mg/kg had no effect on social interaction between pairs of male rats under bright light (high anxiety) conditions. After 21 days of daily administration, paroxetine given orally at 3 mg/kg significantly (p < 0.01) increased the time spent in social interaction by pairs of rats tested under the same conditions, with no effect on locomotor activity, indicating an anxiolytic-like effect. The magnitude of increase (+97%) was comparable to that seen after a single dose of chlordiazepoxide (4 mg/kg orally). Although there was also an increase in time spent in social interaction after 21 days of repeated oral administration of paroxetine at 0.3, 1, and 10 mg/kg (+44, +56, and +54% increases, respectively), statistical significance was not achieved. These results indicate that in the long term paroxetine has an anxiolytic action, and thus support the clinical evidence for its therapeutic use in the treatment of anxiety disorders in addition to its established role as an antidepressant.
Recent studies have suggested an important kindling-like exacerbation of ethanol withdrawal symptoms after repeated cycles of ethanol intoxication and withdrawal. Few studies, however, have evaluated the effect of multiple episodes of intoxication and withdrawal on spontaneous EEG activity after cessation of ethanol intake. In this study, electrographic activity in cortical and subcortical structures male Sprague-Dawley rats was examined after multiple cycles of ethanol intoxication and withdrawal. After surgical implantation of electrodes, animals received repeated cycles of chronic ethanol exposure in vapor inhalation chambers for 10 or 20 days, with 4-day withdrawal periods between each. Upon removal from the inhalation chamber, spontaneous EEG activity was recorded intermittently for 72 hr. These data were then examined for the presence of spikes and sharp waves. Results indicate that the levels of spike and sharp wave activity observed vary with both length of ethanol exposure and with the number of withdrawal cycles, and that these effects varied with neuronal site. Changes in spike and sharp wave activity were first observed within hippocampal areas, with other subcortical and cortical sites showing increased activity after additional ethanol exposure or additional cycles of intoxication and withdrawal. Hippocampal areas CA1 and CA3 differed significantly from one another in their response to chronic ethanol exposure, with area CA1 most affected by changes in amount of ethanol exposure and are CA3 most affected by number of withdrawal cycles. These results indicate an increased severity of the ethanol withdrawal syndrome after repeated ethanol withdrawal episodes and suggest differential, site-specific changes in neuronal excitability.
Repeated ethanol withdrawal experience has been shown to result in an exacerbation of future withdrawal episodes. This sensitization of the withdrawal response has been hypothesized to represent a "kindling" phenomenon. The present study was designed to examine whether a systematic increase in the number of previous ethanol withdrawal experiences increases both the severity and duration of a subsequent withdrawal response. An established model of repeated ethanol intoxication/withdrawal was employed in which adult C3H mice were chronically exposed to ethanol vapor in inhalation chambers. In the first experiment, multiple withdrawal (MW) groups of mice received nine (MW x 9), six (MW x 6), or three (MW x 3) cycles of 16-h ethanol vapor separated by 8-h periods of abstinence prior to testing: a single withdrawal (SW) group was tested following a single bout of 16-h ethanol exposure; and a control (C) group did not receive any ethanol treatment throughout the experiment. In a second experiment, a group of mice (MW1-9) were repeatedly tested over nine cycles of withdrawal. A third experiment was designed to assess the effects of repeated pyrazole administration on the potentiated withdrawal seizure response. Results indicated a positive relationship between the number of previously experienced ethanol withdrawals and the severity and duration of a subsequent withdrawal episode. Blood ethanol levels were similar for all ethanol-exposed groups prior to withdrawal assessment. Further, the intensity of withdrawal seizures (handling-induced convulsions) progressively increased over nine cycles of intoxication/withdrawal and repeated testing did not significantly influence the development of this potentiated response. In addition, repeated administration of pyrazole did not appear to influence this withdrawal sensitization phenomenon. Collectively, these results provide further support for the "kindling" hypothesis of ethanol withdrawal.
The anxiogenic action of caffeine (10, 25 and 50 mg/kg, i.p.) was investigated in rats and compared with that of yohimbine (2 mg/kg, i.p.). The experimental methods used were the open-field, elevated plus-maze, social interaction and novelty-suppressed feeding latency tests. Caffeine produced a dose-related profile of behavioural changes, which were qualitatively similar to those induced by yohimbine and which indicate an anxiogenic activity in rodents. Thus, both the drugs reduced ambulation and rears, and increased immobility and defaecation in the open-field test. They decreased the number of entries and time spent on the open arms of the elevated-plus maze, reduced social interaction in paired rats and increased the feeding latency in an unfamiliar environment in 48-h food-deprived rats. Lorazepam, a well known benzodiazepine anxiolytic agent, attenuated the anxiogenic effects of caffeine and yohimbine. Subchronic administration of caffeine (50 mg/kg, i.p.) for 21 days, in different groups of animals, induced a significant degree of tolerance in the elevated plus-maze test, which was statistically significant after 14 and 21 days' treatment. Yohimbine, however, did not induce similar tolerance. When caffeine (50 mg/kg, i.p.) was withdrawn after 21 days' administration, to a separate group of rats, significant withdrawal anxiety was observed 48 h later as noted in the elevated plus-maze test. The investigations support clinical evidence of caffeine-induced anxiety, tolerance to anxiety on continued use, and withdrawal anxiety in chronic caffeine-containing beverage users.
Selective breeding for high and low sensitivity to the hypothermic response of the 5-HT1A receptor agonist 8-OH-DPAT has established two lines (HDS and LDS, respectively) whose behavior differs in a model of depression, but not in the elevated plus-maze test of anxiety. The lines also differed in postsynaptic, but not presynaptic, 5-HT1A receptors. Based on previous evidence that postsynaptic 5-HT1A receptors mediate anxiogenic effects in the social interaction test of anxiety, but not the elevated plus-maze, we investigated possible differences between the lines in these two tests. The HDS line had a consistently lower level of social interaction compared with the LDS line, but no differences were found on any of the measures of the anxiety on trials 1 or 2 in the elevated plus-maze. To determine whether the line differences in anxiety were mediated by different hippocampal 5-HT1A receptor function, 8-OH-DPAT (50 and 100 ng) was applied bilaterally to the dorsal hippocampus. This elicited anxiogenic effects in the LDS line, as has been previously reported in other rat strains, but there was no response in the HDS line, thus demonstrating an abnormal 5-HT1A receptor function in the hippocampus. The 5-HT1A receptor antagonist WAY100635 (200 ng) was administered to the dorsal hippocampus to test for possible differences between the lines in 5-HT tone. There were no significant changes in social interaction in either the HDS or LDS rats, indicating that the different level of anxiety between lines is not due to differences in hippocampal 5-HT tone. It is proposed that the HDS line may prove a useful model of a type of high trait anxiety linked to a susceptibility to depression.
Repeated ethanol withdrawal experience has been shown to result in exacerbated seizures associated with future withdrawal episodes. This sensitization of the withdrawal response has been postulated to represent a "kindling" phenomenon. The present study employed an established model of repeated ethanol withdrawals to examine the potential role of GABA(A), and NMDA and non-NMDA glutamate receptor systems in mediating enhanced seizure activity, as assessed by sensitivity to seizures induced by pentylenetetrazol (PTZ), NMDA, and kainic acid (KA) i.v. infusions, respectively. Adult C3H mice were chronically exposed to ethanol vapor in inhalation chambers. A multiple withdrawal (MW) group received four cycles of 16-h ethanol vapor exposure interrupted by 8-h periods of abstinence; a single withdrawal (SW) group was tested after a single 16-h bout of ethanol intoxication; and the third group was ethanol-naive, serving as controls (C). Results indicated that the MW group evidenced significantly lower PTZ and NMDA seizure thresholds compared to SW and C groups at 8 and 24 h post-withdrawal. In contrast, MW and SW groups exhibited reduced sensitivity (higher seizure threshold) to KA in comparison to controls, and this effect only emerged at 24 h post-withdrawal. Further, MW mice required significantly less additional PTZ or NMDA to induce more severe convulsions once initial signs of seizures were elicited. Conversely, latency and amount of KA required to transition from initial seizure signs to more severe end-stage convulsions was significantly greater for MW and SW groups compared to controls. Taken together, these results suggest that repeated ethanol withdrawal experience does not result in a global non-specific lowering of threshold to convulsive stimuli, but rather, selective changes in CNS mechanisms associated with neural excitability may underlie potentiated withdrawal responses. Thus, reduced GABA(A) receptor function and increased NMDA receptor activity may become exaggerated as a consequence of repeated withdrawal experience, while reduced sensitivity to KA induced seizures may represent a compensatory response to withdrawal-related CNS hyperexcitability.
Selective breeding for high and low sensitivity to the hypothermic response induced by the 5-HT1A receptor agonist 8- OH-DPAT has established two lines of rat (HDS and LDS, respectively) whose behavior differs in a model of depression and in the social interaction test of anxiety. The HDS line has a higher level of anxiety and, furthermore, does not display the usual anxiogenic response to intrahippocampal administration of 8-OH-DPAT. It was therefore hypothesized that this line of rat might be a useful model of high trait anxiety with a susceptibility to depression. We thus investigated whether chronic treatment with fluoxetine would result in an anxiolytic effect in the social interaction test in the LDS and HDS lines of rat. In both lines, acute fluoxetine (10 mg/kg) produced an anxiogenic effect in the social interaction test; when rats were tested 24 h after 14 days of fluoxetine treatment there were no anxiolytic effects in either line. In the social interaction test, chronic fluoxetine treatment did not change either the anxiogenic effect of 8-OH-DPAT (100 ng) injected bilaterally into the dorsal hippocampus in the LDS line or the lack of response in the HDS line. In the elevated plus-maze, chronic fluoxetine treatment resulted in a significant anxiogenic effect in the HDS line, but was without effect in the LDS line. Intrahippocampal 8-OH-DPAT was without effect in the plus-maze in either line. These results suggest that chronic treatment with fluoxetine did not modify the hippocampal 5-HT1A receptor in either line. The anxiogenic effects observed in the plus-maze in the HDS line after chronic fluoxetine might relate to line differences in 5-HT1A receptors in other brain regions.
Clinical studies report an increase in the prevalence of alcohol withdrawal-related seizures in patients with a history of multiple detoxifications. In order to investigate the alcohol withdrawal-related alterations in neural activity that lead to this increase in seizure propensity, basic researchers have examined both spontaneous and elicited seizures in animals undergoing withdrawal from chronic ethanol. This study was designed to further examine alcohol withdrawal-related seizure activity in a rodent model by assessing the development of electrical kindling after chronic ethanol exposure administered in multiple or single treatment episodes. Laboratory rats were exposed to either five periods of 3 days of ethanol, one 15-day period of continuous ethanol, or a period of control handling with no ethanol exposure. Ten days after a final withdrawal episode, all animals were surgically prepared with recording and stimulating electrodes. Twenty days after final withdrawal from ethanol or an equivalent period of similar handling, daily electrical stimulation of hippocampal area CA3 was initiated. Animals exposed to ethanol required more daily stimulations to become fully kindled than did ethanol-naive controls, with those animals experiencing five withdrawals requiring the most stimulations overall and more stimulations to progress from focal to generalized seizure behaviors. These results indicate that chronic ethanol exposure and withdrawal alter neuronal mechanisms important for hippocampal kindling in a manner that persists long after cessation of ethanol exposure, and they indicate that this effect is increased by exposure to repeated withdrawal episodes.
Women are more likely than men to develop anxiety disorders. Yet, relatively few studies have investigated whether women with anxiety disorders have characteristics that are distinct from those of men with the same disorders. The cause of the enhanced vulnerability to anxiety for women remains largely undetermined. Recent data suggest that female reproductive hormones and related cycles may play an important role. In addition to etiologic functions, reproductive hormones may substantially influence the clinical course of preexisting anxiety conditions in women. Psychotropic medications are more likely to be prescribed to women, and gender differences have been identified in the pharmacokinetics of psychotropic medication. Yet, relatively few systematic data are available concerning the potential clinical relevance or possible treatment implications of gender differences in the treatment of women with anxiety disorders. This article reviews the unique characteristics of primary anxiety disorders in women, summarizes the neurobiological effects associated with estrogen and progesterone, discusses gender differences in medication metabolism and the potential relevance of these differences in the pharmacologic management of women with anxiety disorders, and reviews issues specific to women (e.g., hormone therapy, oral contraceptives, menstrual cycle, pregnancy, lactation) that may impact treatment with psychotropic medication.
This experiment explored sex differences in behaviour using factor analysis to describe the relationship between different behavioral variables. A principal component solution with an orthogonal rotation of the factor matrix was used, ensuring that the extracted factors are independent of one another, and thus reflect separate processes. In the elevated plus-maze test of anxiety, in male rats factor 1 accounted for 75% of the variance and reflected anxiety, factor 2 represented activity, and accounted for 24% of the variance. This contrasted with the finding in female rats in which factor 1 was activity, accounting for 57% of the variance, with the anxiety factor accounting for only 34% of the variance. When behaviour in both the plus-maze and holeboard were analysed, a similar sex difference was found with anxiety emerging as factor 1 in males and holeboard activity as factor 1 in females. Locomotor activity in the inner portion of the holeboard loaded on the anxiety factor for males, but on activity for females. When behaviours in the plus-maze and sexual orientation tests were analysed, anxiety emerged as factor 1 in males, sexual preferences factor 2, and activity factor 3. In females, activity was factor 1, sexual preference factor 2, anxiety factor 3, and social interest factor 4. These results suggest caution should be exercised in interpreting the results from female rats in tests validated on males because the primary controlling factor may be different.
Research has demonstrated a relationship between the number of previous alcohol detoxifications and increased severity of the alcohol withdrawal syndrome (AWS) that is hypothesized to be similar to an electrophysiologic "kindling process." Application of a "kindling" model to AWS suggests that neuroadaptation of the central nervous system to repeated detoxifications may also cause neurobehavioral alterations that may affect "craving." This study examined craving as assessed by the Obsessive Compulsive Drinking Scale (OCDS) in 67 adult outpatients meeting DSM-IV criteria (American Psychiatric Association, 1994) for alcohol dependence and AWS having either < 2 and > or = 2 previous detoxifications. Results of ANCOVA revealed that patients with > or = 2 previous detoxifications had higher scores on a scale that measures obsessive thoughts about alcohol, drinking urges and behaviors, and a composite of these scores after controlling for alcohol dependence severity, depressive symptoms and number of drinks 2 weeks prior to the study. Findings emphasize the need to address craving and other psychological variables with respect to treatment of AWS.
Background:
It has been proposed that the alcohol-preferring P rat meets many of the criteria for an animal model of alcoholism. However, the development of alcohol dependence has not been explored in rats that self-administer ethanol for less than 15-20 weeks. The present study investigated the development of physical dependence upon alcohol after 2-6 weeks of voluntary alcohol intake. Changes in bicuculline-induced seizure thresholds, microstructure of alcohol drinking, and anxiety-related behavior were used as indices of alcohol dependence. In addition, we evaluated the microstructure of alcohol drinking associated with the development of physical dependence upon alcohol.
Methods:
Alcohol (10% ethanol solution) was measured in graduated drinking tubes with both alcohol and water available continuously. Microstructure of alcohol intake was monitored by a computerized drinkometer. Physical dependence upon alcohol was determined by measuring bicuculline-induced seizure thresholds after alcohol withdrawal. Anxiety-related behavior of P rats after alcohol withdrawal was determined by the social interaction and elevated plus maze tests.
Results:
Initial alcohol intake in the alcohol-preferring P rat was relatively modest (3.9 +/- 0.4 g/kg/day). Four days of forced alcohol exposure (initiation) followed by 6 weeks of voluntary drinking resulted in an increase of alcohol intake to 5.5 +/- 0.2 g/kg/day. Ethanol self-administration for 6 weeks, but not for 2 or 4 weeks, produced a significant reduction (30%; p < 0.05) in bicuculline-induced seizure thresholds during alcohol withdrawal. Alterations in the microstructure of alcohol intake (i.e., 90% increase in the size of alcohol drinking bouts compared to the baseline [p < 0.001] with no change in bout frequency) were associated with the development of alcohol dependence. Termination of alcohol intake after 6 weeks of voluntary alcohol consumption resulted in increased anxiety according to both the social interaction and elevated plus maze tests.
Conclusions:
The results of this study indicate that 6 weeks of voluntary alcohol intake are sufficient for the development of physical dependence upon alcohol in the alcohol-preferring P rats as measured by susceptibility to bicuculline-induced seizures. This time is much shorter than the 15-20 weeks reported earlier. Development of physical dependence to alcohol was associated with an increase in daily alcohol intake (40% over the baseline), an increase in alcohol intake during each drinking bout (90% over the baseline), and elevated anxiety during alcohol withdrawal.
Background: Current clinical treatment of alcohol detoxification commonly includes pharmacotherapy to lessen the potential for seizures, especially in those patients undergoing repeated treatment. Basic research continues to study the alcohol withdrawal-related “kindling” of seizures both to understand the mechanisms involved and to identify alternative treatments. Ethanol withdrawal has been shown to result in the delay of electrical kindling at several brain sites, which suggests a long-lasting disruption of neuronal function.
Methods: This study focused on the participation of the l-type voltage-gated calcium channels in this process by the treatment of animals during withdrawal with nifedipine, an agent that blocks these channels. Animals were randomly assigned to ethanol (ethanol-exposed/ethanol-naive) and drug treatment (nifedipine/vehicle) groups. Subjects receiving ethanol were exposed to five cycles of 3 days’ ethanol exposure, with each exposure cycle separated by a 1-day withdrawal period. Drug treatment was administered twice during each withdrawal period. Twenty days after completion of ethanol exposure, animals received daily kindling stimulations to hippocampal area CA3 until the kindling criterion was attained.
Results: Ethanol-exposed animals that received vehicle treatment during ethanol withdrawal required more daily stimulations to become fully kindled than did ethanol-naive controls. This delay in seizure development was most pronounced in the progression from focal seizure behaviors to more generalized seizures. Animals that received the same ethanol exposure but that were treated with nifedipine required significantly fewer stimulations than did ethanol-exposed animals that received vehicle. Ethanol-exposed/nifedipine-treated animals did not differ from ethanol-naive controls that received vehicle or nifedipine.
Conclusions: Alcohol withdrawal-related alterations in seizure-sensitive neural circuitry such as the hippocampus persist long after cessation of ethanol exposure. Furthermore, the L-type voltage-gated calcium channels are involved in this effect in that blockade of these channels during acute withdrawal alleviates alterations in seizure mechanisms on a long-term basis.
Investigators have found a relationship between the number of previous alcohol withdrawals (AWs) and severity of withdrawal. We evaluated patients with multiple previous AWs, as compared to those with 0-1 previous withdrawals, in an outpatient detoxification trial comparing lorazepam (LZ) to carbamazepine (CBZ). A mixed model analysis of covariance was used to analyze Clinical Institute Withdrawal Assessment for Alcohol-Revised (CIWA-Ar) scores as a function of detoxification history (0-1 vs. 2 or more), drug group (CBZ vs. LZ), assessment day, and hours since last drink. The mixed model analysis of covariance (ANCOVA) indicated a significant detoxification history by assessment day interaction (P< or =.03). Least square means associated with this interaction suggested that the CIWA-Ar scores for the multiple detox patients declined more slowly than those with 0-1 previous detoxifications. Patients with multiple detoxes were 150% more likely to experience a heavy drinking day during treatment (P< or =.03). The multiple detox group drank more each drinking day (P=.001) and a greater proportion of this group had early heavy drinking (P=.0002). In the present study, intensity of AW symptoms and early heavy drinking were independent of treatment medications and were more common in patients who had previously undergone multiple treatments for AW.
Most smokers report smoking has an anxiolytic effect, which may contribute to nicotine dependence.
To examine effects in the social interaction test (SI) of anxiety after 4 weeks' self-administered nicotine (15 infusions of 0.03 mg/kg, totalling 0.45 mg/kg per day), and after 24 and 72 h of withdrawal. The effect of exposure to the operant chamber on withdrawal responses was also examined.
Animals were trained to self-administer saline or nicotine and after 4 weeks they were tested in SI after their daily self-administration session. Animals were retested after 24 and 72 h withdrawal, when they were either taken directly from the home cage or were tested 5 min after a 30-min exposure to the operant chamber.
Compared with the saline control group, the animals that had been self-administering nicotine for 4 weeks showed decreased social interaction with no decrease in locomotor activity, indicating a significant anxiogenic effect of the nicotine infusions. There was no change in social interaction after 24 and 72 h withdrawal from chronic nicotine, regardless of whether or not the rats were exposed to the operant chamber just prior to being tested.
Nicotine self-administration is not maintained because of its anxiolytic effect, but despite, or because of, its anxiogenic effect. There was no evidence of an anxiogenic response after either 24 or 72 h of withdrawal and thus increased anxiety on withdrawal from nicotine does not seem to contribute to nicotine self-administration.
Previous investigations have found sex differences in rats in response to chronic ethanol exposure. The most dramatic differences were observed with anticonvulsant treatment during ethanol withdrawal, when seizure susceptibility is significantly increased. Sex differences in this response were found for both GABAergic and glutamatergic compounds. This study was aimed at exploring whether sex also influences the timing for the development of and recovery from ethanol dependence.
Ethanol was administered in a liquid diet, with pair-fed animals receiving dextrose, substituted isocalorically for the ethanol. Ethanol dependence and withdrawal were assessed by measurement of seizure thresholds after abrupt removal of the ethanol diet. Seizure thresholds were determined by slow, tail vein infusion of the gamma-aminobutyric acidA-receptor antagonist bicuculline.
Male and female rats displayed differences in timing for both onset and recovery from ethanol dependence, as determined by changes in ethanol withdrawal seizure susceptibility. Female rats were slower to develop dependence and quicker to recover compared with male rats. Furthermore, acute ethanol administration did not alter seizure susceptibility in pair-fed control animals, but it was anticonvulsant in ethanol-withdrawn rats. Ethanol-withdrawn female rats showed a greater response to acute ethanol administration than did male rats.
This set of experiments uncovered additional sex differences in one measure of ethanol dependence and withdrawal. Proposed mechanisms for the development of ethanol dependence involve alterations in subunit assembly of gamma-aminobutyric acidA and NMDA receptors or various posttranslational modifications. In consideration of these findings, whatever mechanisms underlie the development of ethanol dependence, there is a different sequence of events in male compared with female rats. Studies are ongoing to determine associations between behavioral measures of ethanol dependence/withdrawal and selective neuronal adaptations.
Previous work has shown that repeated withdrawals from chronic ethanol exposure can kindle seizures in rodents. In this article, the effects of a three-cycle model of ethanol exposure and withdrawal on the social interaction test of anxiety are summarized.
Rats were exposed to ethanol (7% or 4.5%) diets over three periods of 5 days, with 2 days of withdrawal between cycles. Between 5 and 6 hr after the ethanol was removed, pairs of rats were placed in open field chambers for the assessment of social interaction behavior and locomotor activity.
After the third cycle of ethanol (7%) presentation, both male and female rats exhibited lower social interaction behavior (more anxiety) and activity than after a single cycle. Rats exposed to a similar amount of ethanol but tested while ethanol was still available did not exhibit a reduction in social interaction. The decrease in social interaction was still present for up to 24 hr but had disappeared by 48 hr after ethanol was withdrawn. When rats were allowed 8 or 16 days to recover from the effects of the three-cycle protocol, a further exposure to 5 days of 7% ethanol diet resulted in a reduction in social interaction on withdrawal similar to that seen from the three-cycle protocol. In contrast, rats exposed continuously to 7% ethanol diet for 15 consecutive days exhibited higher levels of social interaction when maintained on control diet for 8 or 16 days and then reexposed to ethanol. Rats that were exposed to the three-cycle protocol and allowed 32 days to recover before being reexposed to ethanol still had a partial deficit in social interaction. Finally, animals subjected to repeated withdrawals from 4.5% ethanol exhibited a reduction in social interaction without a change in activity after the final withdrawal from ethanol, whereas rats exposed continuously to a 4.5% ethanol diet did not exhibit a reduction in social interaction or activity. Neither blood ethanol concentrations nor changes in body weight could account for these behavioral differences.
Repeated withdrawal from ethanol can lead to accentuated or more persistent anxiety-like behavior in rats, as indicated by a decrease in social interaction. The withdrawal-induced decrease in locomotor activity is not accentuated by repeated withdrawals. This model of repeated withdrawals from ethanol may prove useful in defining the neurochemical basis of this accentuation.
The social interaction test of anxiety was developed 25 years ago to provide an ethologically based test that was sensitive to both anxiolytic and anxiogenic effects. It is sensitive to a number of environmental and physiological factors that can affect anxiety. It has detected anxiogenic effects of peptides such as corticotropin-releasing factor (CRF) and adrenocorticotropic hormone (ACTH), and anxiolytic effects of neuropeptide Y and substance P receptor antagonists. It has successfully identified neuropharmacological sites of action of anxiogenic compounds and drug withdrawal. Effects of compounds acting on the gamma-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) systems have been extensively investigated after both systemic administration and microinjection into specific brain regions. The use of this test has, thus, played a crucial role in unravelling the neural basis of anxiety. It is hoped that in the next 25 years, the test will play a crucial role in determining the genetic basis of anxiety disorders.
For many years, researchers have avoided including females in their research because of the poorly understood influences of cycling hormones. However, we are becoming increasingly aware that sex matters, showing that it is important to conduct studies in females as well as males. This review will focus on the central nervous system (CNS) actions of alcohol (ethanol) because we have found significant sex differences in ethanol actions at the molecular as well as the behavioral level. Most recently, in our studies of ethanol dependence and withdrawal, we found that female rats displayed a shorter time for recovery from ethanol withdrawal, assessed by measuring seizure susceptibility. We now report that this finding was confirmed with a second convulsant agent. Moreover, GABAA receptor function was differentially altered in ethanol-withdrawn female compared to male rats. Studies by other investigators have reported additional significant sex differences in ethanol seeking and drinking behaviors and across several measures of ethanol dependence and withdrawal. We are gaining a better understanding of how the actions of ethanol in the CNS overlay sex differences in brain architecture and the hormonal milieu. Therefore, it is not surprising to observe sex-selective effects on cellular and behavioral outcomes from ethanol consumption. While current research is focused on characterizing sex differences in the actions of ethanol, it has not yet reached the point where we can integrate our findings into a unifying concept of how being female differentially regulates CNS responses to ethanol. This is likely a result of the complexity of ethanol actions, involving multiple neurotransmitter systems and responses covering the spectrum from drug seeking behaviors to neuropathological consequences of ethanol misuse. Regardless, the observed sex differences in ethanol withdrawal are noteworthy because they suggest that treatment of alcoholism should be managed differently in women than in men. Finally, it remains important to compare and contrast responses in males and females because recent studies of sex differences in basic physiology have made it clear that being female impacts health and disease.
Previous work demonstrated that rats subjected to multiple withdrawals from chronic ethanol exhibit a sensitization of anxiety-like behavior compared to animals withdrawn from treatment with an equal but continuous amount of ethanol. This study sought to examine whether corticotropin-releasing factor (CRF) could modulate this ethanol-withdrawal-induced anxiety-like behavior. Initially, rats were administered with CRF (1 microg) or vehicle intraventricularly on two occasions 5 days apart while on control diet (CD) followed by exposure to 7% ethanol diet (ED) for 5 days, with social interaction assessed 5 h into withdrawal. Social interaction was significantly reduced in the CRF-treated animals compared to vehicle-treated rats and vehicle- and CRF-treated rats maintained on CD, indicative that CRF given before ethanol exposure was capable of inducing an adaptive change that sensitized withdrawal-induced anxiety-like behavior. Next, the CRF(1) receptor antagonist CRA1000 (3 mg/kg, systemically), the CRF(2) receptor antagonist antisauvagine-30 (20 microg intraventricularly), or vehicle was injected 4 h after the ethanol was removed following the first and second cycles of chronic ethanol exposure and the effect on the multiple-withdrawal-induced anxiety-like behavior determined after the third withdrawal cycle. The CRF(1) receptor antagonist blocked the reduced social interaction behavior, whereas the CRF(2) receptor antagonist was without effect. Similar pretreatment with another CRF(1) receptor antagonist CP-154,526 (10 mg/kg systemically) during the first and second withdrawals also counteracted anxiety-like behavior. These findings indicate that the CRF system and CRF(1) receptors play key roles in the adaptive change responsible for the anxiety-like behavior induced by repeated withdrawals from chronic ethanol.
Anxiety-like behaviors are integral features of withdrawal from chronic ethanol exposure. In the experiments in the current study, we tested the hypothesis that anxiety can be regulated independently of other withdrawal signs and thus may be responsive to selective pharmacological agents. For 17 days, rats were fed ethanol (8-12 g/kg/day) in a liquid diet. Between 5 and 6 h after cessation of ethanol treatment, rats were tested in either the social interaction or plus-maze test of anxiety-like behavior after treatment with drugs hypothesized to have anxiolytic action. SB242084, flumazenil, and CRA1000-antagonists for 5-hydroxytryptamine (serotonin) (5-HT) 2C (5-HT(2C)), benzodiazepine, and corticotropin-releasing factor type 1 (CRF(1)) receptors, respectively-attenuated decreased social interaction without concomitant effects on activity measures. In contrast, ifenprodil, MDL 72222, and zolpidem-antagonists for N-methyl-d-aspartate (NMDA) and 5-HT(3) receptors, and agonist for benzodiazepine type 1 receptors, respectively-did not share this effect. Results for SB242084, flumazenil, and ifenprodil in the elevated plus-maze test were comparable to those in the social interaction test. These results support the suggestion that multiple neuronal systems (CRF(1), 5-HT(2C), and benzodiazepine receptors) contribute to the ethanol withdrawal sign of decreased social interaction. Furthermore, the selective effects of pharmacological agents on social interaction seem to indicate that this behavior can be dissociated from other signs. Because anxiety may be a complicating factor in alcohol withdrawal and relapse, future studies of this type are needed to provide focus for the effort to define selective and novel antianxiety agents for these disorders.
Repeated ethanol withdrawal experience increases the severity and duration of subsequent withdrawal seizures in mice
- Hc Becker
- H Diaz-Granados
- Rt Weathersby
Becker HC, Diaz-Granados H, Weathersby RT. Repeated ethanol withdrawal experience increases the
severity and duration of subsequent withdrawal seizures in mice. Alcohol 1997;14:319–26. [PubMed:
9209546]
Accentuated decreases in social interaction in rats subjected to repeated ethanol withdrawals
- Overstreet
Repeated ethanol withdrawal experience increases the severity and duration of subsequent withdrawal seizures in mice
- Becker