[Show abstract][Hide abstract] ABSTRACT: Despite impressive progress in understanding the molecular, cellular and circuit-level correlates of major depression, the biological mechanisms that causally underlie this disorder are still unclear, possibly due to excessive focus on the dysfunctioning of neurons, as compared with other types of brain cells. Therefore, we examined the role of dynamic alterations in microglia activation status in the development of chronic unpredictable stress (CUS)-induced depressive-like condition in rodents. We found that following an initial period (2–3 days) of stress-induced microglial proliferation and activation, some microglia underwent apoptosis, leading to reductions in their numbers within the hippocampus following 5 weeks of CUS exposure. At that time, microglia displayed reduced expression of activation markers as well as dystrophic morphology. Blockade of the initial stress-induced microglial activation by minocycline, imipramine or by transgenic interleukin-1 receptor antagonist over-expression rescued the subsequent microglial apoptosis and decline, as well as the CUS-induced depression and suppressed neurogenesis. Treatment of CUS-exposed mice with LPS (endotoxin), M-CSF or GM-CSF, which all stimulated hippocampal microglial proliferation, reversed the depressive-like behavior and dramatically increased hippocampal neurogenesis, whereas treatment with imipramine or minocycline had minimal anti-depressive effects in these mice. These findings provide direct causal evidence that disturbances in microglial functioning has an etiological role in chronic stress-induced depression, suggesting that microglia stimulators could serve as fast-acting anti-depressants in some forms of depressive and stress-related conditions.
Brain Behavior and Immunity 01/2014; 40:e2–e3. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microgliosis occurs after morphine and peripheral nerve injury alone, but the behavioral and molecular impact in tandem is unknown. We hypothesized that sciatic chronic constriction injury (CCI)-allodynia would be enhanced by subsequent repeated morphine in rats, involving TLR4, P2X7 receptor (P2X7R) and caspase-1, facilitating release of interleukin (IL)-1β. Beginning 10 days after CCI, morphine (5 mg/kg b.i.d.) or saline was administered for 5 days. Compared to vehicle, morphine significantly prolonged the duration of CCI-induced allodynia (n = 6/group; p < 0.05). Morphine also significantly elevated TLR4 mRNA, P2X7R, NFkappaB, NLRP3 and caspase-1 protein levels (p < 0.05) in the ipsilateral lumbar dorsal quadrant (iLDQ), 5 weeks after dosing conclusion. Supporting a causal role for NLRP3 inflammasome activation in morphine-prolonged CCI-allodynia, continuous intrathecal infusion of inhibitors of TLR4 ([+]-naloxone; 60 micrograms/h), P2X7R (Brilliant Blue G; 30 ng/h), or caspase-1 (ac-YVAD-cmk; 1 μg/h) prevented prolonged allodynia when administered concomitantly with morphine, and abolished established morphine-prolonged CCI-allodynia when administered 5 weeks after morphine dosing (n = 6/group; p < 0.05). A single intrathecal IL-1 receptor antagonist dose (100 micrograms) also attenuated morphine-prolonged CCI-allodynia (n = 6/group; p < 0.05). In keeping with known pro-nociceptive roles for IL-1β, phosphorylation of the NR1 NMDA subunit was elevated, while GRK2 levels and GLT-1 mRNA were decreased in iLDQ 5 weeks after dosing conclusion (p < 0.05). These data suggest that morphine and the products of nerve injury interact, resulting in prolonged neuropathic pain via sustained inflammasome signaling.
Brain Behavior and Immunity 01/2014; 40:e9–e10. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The limited success in understanding the pathophysiology of major depression may result from excessive focus on the dysfunctioning of neurons, as compared with other types of brain cells. Therefore, we examined the role of dynamic alterations in microglia activation status in the development of chronic unpredictable stress (CUS)-induced depressive-like condition in rodents. We report that following an initial period (2-3 days) of stress-induced microglial proliferation and activation, some microglia underwent apoptosis, leading to reductions in their numbers within the hippocampus, but not in other brain regions, following 5 weeks of CUS exposure. At that time, microglia displayed reduced expression of activation markers as well as dystrophic morphology. Blockade of the initial stress-induced microglial activation by minocycline or by transgenic interleukin-1 receptor antagonist overexpression rescued the subsequent microglial apoptosis and decline, as well as the CUS-induced depressive-like behavior and suppressed neurogenesis. Similarly, the antidepressant drug imipramine blocked the initial stress-induced microglial activation as well as the CUS-induced microglial decline and depressive-like behavior. Treatment of CUS-exposed mice with either endotoxin, macrophage colony-stimulating factor or granulocyte-macrophage colony-stimulating factor, all of which stimulated hippocampal microglial proliferation, partially or completely reversed the depressive-like behavior and dramatically increased hippocampal neurogenesis, whereas treatment with imipramine or minocycline had minimal or no anti-depressive effects, respectively, in these mice. These findings provide direct causal evidence that disturbances in microglial functioning has an etiological role in chronic stress-induced depression, suggesting that microglia stimulators could serve as fast-acting anti-depressants in some forms of depressive and stress-related conditions.Molecular Psychiatry advance online publication, 17 December 2013; doi:10.1038/mp.2013.155.
[Show abstract][Hide abstract] ABSTRACT: Studies on the biological basis of major depression usually focus on abnormalities in neuronal functions. Glia cells, particularly astrocytes, have also been implicated in the pathophysiology of depression, however the role of microglia in this disease is still elusive. To elucidate the involvement of microglia in depression we examined the role of dynamic alterations in microglia activa- tion status on the development of chronic unpredictable stress (CUS)-induced depressive-like condition in rodents. We report that following an initial period (2–3 days) of stress-induced microglial activation (reflected by proliferation, assumption of activated mor- phology and mRNA expression of activation markers), some microglia underwent apoptosis (reflected by activated caspase-3 and TUNEL staining), leading to reductions in their numbers within the hippocampus (but not in other brain regions) following 5- weeks of CUS exposure. At that time, microglia displayed reduced expression of activation markers as well as dystrophic morphology. The effects of CUS on microglia were blocked by chronic treatment with the tricyclic antidepressant drug imipramine. Furthermore, blockade of the initial stress-induced microglia activation by the microglial inhibitor minocycline or by transgenic interleukin-1 receptor antagonist over-expression rescued the subsequent microglia apoptosis and decline, as well as the CUS-induced depressive-like behavior and suppressed neurogenesis. These find- ings provide direct causal evidence that disturbances in microglial functioning have an etiological role in chronic stress-induced depression.
PsychoNeuroImmunology Research Society's 20th Annual Scientific Meeting, Stockholm, Sweden; 09/2013
[Show abstract][Hide abstract] ABSTRACT: Many studies have shown that minocycline, an antibacterial tetracycline, suppresses experimental pain. While minocycline's positive effects on pain resolution suggest that clinical use of such drugs may prove beneficial, minocycline's antibiotic actions and divalent cation (Ca(2+); Mg(2+)) chelating effects detract from its potential utility. Thus, we tested the antiallodynic effect induced by a non-antibacterial, non-chelating minocycline derivative in a model of neuropathic pain and performed an initial investigation of its anti-inflammatory effects in vitro. Intraperitoneal minocycline (100mg/kg) and 12S-hydroxy-1,12-pyrazolinominocycline (PMIN; 23.75, 47.50 or 95.00mg/kg) reduce the mechanical allodynia induced by chronic constriction injury of mouse sciatic nerve. PMIN reduces the LPS-induced production of PGE2 by primary microglial cell cultures. Human embryonic kidney cells were transfected to express human toll-like receptors 2 and 4, and the signaling via both receptors stimulated with PAM3CSK4 or LPS (respectively) was affected either by minocycline or PMIN. Importantly, these treatments did not affect the cell viability, as assessed by MTT test. Altogether, these results reinforce the evidence that the anti-inflammatory and experimental pain suppressive effects induced by tetracyclines are neither necessarily linked to antibacterial nor to Ca(2+) chelating activities. This study supports the evaluation of the potential usefulness of PMIN in the management of neuropathic pain, as its lack of antibacterial and Ca(2+) chelating activities might confer greater safety over conventional tetracyclines.
[Show abstract][Hide abstract] ABSTRACT: Opioid action was thought to exert reinforcing effects solely via the initial agonism of opioid receptors. Here, we present evidence for an additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, toll-like receptor 4 (TLR4), and its MyD88-dependent signaling. Blockade of TLR4/MD2 by administration of the nonopioid, unnatural isomer of naloxone, (+)-naloxone (rats), or two independent genetic knock-outs of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned place preference. (+)-Naloxone also reduced opioid (remifentanil) self-administration (rats), another commonly used behavioral measure of drug reward. Moreover, pharmacological blockade of morphine-TLR4/MD2 activity potently reduced morphine-induced elevations of extracellular dopamine in rat nucleus accumbens, a region critical for opioid reinforcement. Importantly, opioid-TLR4 actions are not a unidirectional influence on opioid pharmacodynamics, since TLR4(-/-) mice had reduced oxycodone-induced p38 and JNK phosphorylation, while displaying potentiated analgesia. Similar to our recent reports of morphine-TLR4/MD2 binding, here we provide a combination of in silico and biophysical data to support (+)-naloxone and remifentanil binding to TLR4/MD2. Collectively, these data indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the mesolimbic dopamine system that amplifies opioid-induced elevations in extracellular dopamine levels, therefore possibly explaining altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions, and provides further evidence supporting a role for central proinflammatory immune signaling in drug reward.
Journal of Neuroscience 08/2012; 32(33):11187-200. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Uncontrollable stress can interfere with instrumental learning and induce anxiety in humans and rodents. While evidence supports a role for serotonin (5-HT) and serotonin 2C receptors (5-HT(2C)R) in the behavioral consequences of uncontrollable stress, the specific sites of action are unknown. These experiments sought to delineate the role of 5-HT and 5-HT(2C)R in the dorsal striatum (DS) and the lateral/basolateral amygdala (BLA) in the expression of stress-induced instrumental escape deficits and exaggerated fear, as these structures are critical to instrumental learning and fear behaviors. Using in vivo microdialysis, we first demonstrated that prior uncontrollable, but not controllable, stress sensitizes extracellular 5-HT in the dorsal striatum, a result that parallels prior work in the BLA. Additionally, rats were implanted with bi-lateral cannula in either the DS or the BLA and exposed to uncontrollable tail shock stress. One day later, rats were injected with 5-HT(2C)R antagonist (SB242084) and fear and instrumental learning behaviors were assessed in a shuttle box. Separately, groups of non-stressed rats received an intra-DS or an intra-BLA injection of the 5-HT(2C)R agonist (CP809101) and behavior was observed. Intra-DS injections of the 5-HT(2C)R antagonist prior to fear/escape tests completely blocked the stress-induced interference with instrumental escape learning; a partial block was observed when injections were in the BLA. Antagonist administration in either region did not influence stress-induced fear behavior. In the absence of prior stress, intra-DS administration of the 5-HT(2C)R agonist was sufficient to interfere with escape behavior without enhancing fear, while intra-BLA administration of the 5-HT(2C)R agonist increased fear behavior but had no effect on escape learning. Results reveal a novel role of the 5-HT(2C)R in the DS in the expression of instrumental escape deficits produced by uncontrollable stress and demonstrate that the involvement of 5-HT(2C)R activation in stress-induced behaviors is regionally specific.
[Show abstract][Hide abstract] ABSTRACT: Control over an aversive experience can greatly impact the organism's response to subsequent stressors. We compared the effects of escapable (ES) and yoked inescapable (IS) electric tail shocks on the hypothalamic-pituitary-adrenal (HPA) axis hormonal (corticosterone and adrenocorticotropic hormone (ACTH)), neural (c-fos mRNA) and behavioral (struggling) response to subsequent restraint. We found that although the HPA axis response during restraint of both previously stressed groups were higher than stress-naïve rats and not different from each other, lack of control over the tailshock experience led to an increase in restraint-induced struggling behavior of the IS rats compared to both stress-naïve and ES rats. Additionally, c-fos expression in the basolateral amygdala was increased selectively in the IS group, and relative c-fos mRNA expression in the basolateral amygdala positively correlated with struggling behavior. Restraint-induced c-fos expression in the medial prefrontal cortex, a brain area critical for mediating some of the differential neurochemical and behavioral effects of ES and IS, was surprisingly similar in both ES and IS groups, lower than that of stress-naïve rats, and did not correlate with struggling behavior. Our findings indicate that basolateral amygdala activity may be connected with the differential effects of ES and IS on subsequent behavioral responses to restraint, without contributing to the concurrent HPA axis hormone response.
[Show abstract][Hide abstract] ABSTRACT: Glutamate neurotransmission is highly regulated, largely by glutamate transporters. In the spinal cord, the glutamate transporter GLT-1 is primarily responsible for glutamate clearance. Downregulation of GLT-1 can occur in activated astrocytes, and is associated with increased extracellular glutamate and neuroexcitation. Among other conditions, astrocyte activation occurs following repeated opioids and in models of chronic pain. If GLT-1 downregulation occurs in these states, GLT-1 could be a pharmacological target for improving opioid efficacy and controlling chronic pain. The present studies explored whether daily intrathecal treatment of rats with ceftriaxone, a beta-lactam antibiotic that upregulates GLT-1 expression, could prevent development of hyperalgesia and allodynia following repeated morphine, reverse pain arising from central or peripheral neuropathy, and reduce glial activation in these models. Ceftriaxone pre-treatment attenuated the development of hyperalgesia and allodynia in response to repeated morphine, and prevented associated astrocyte activation. In a model of multiple sclerosis (experimental autoimmune encephalomyelitis; EAE), ceftriaxone reversed tactile allodynia and halted the progression of motor weakness and paralysis. Similarly, ceftriaxone reversed tactile allodynia induced by chronic constriction nerve injury (CCI). EAE and CCI each significantly reduced the expression of membrane-bound, dimerized GLT-1 protein in lumbar spinal cord, an effect normalized by ceftriaxone. Lastly, ceftriaxone normalized CCI- and EAE-induced astrocyte activation in lumbar spinal cord. Together, these data indicate that increasing spinal GLT-1 expression attenuates opioid-induced paradoxical pain, alleviates neuropathic pain, and suppresses associated glial activation. GLT-1 therefore may be a therapeutic target that could improve available treatment options for patients with chronic pain.
[Show abstract][Hide abstract] ABSTRACT: Experience with behavioral control over tailshock (escapable shock, ES) has been shown to block the behavioral and neurochemical changes produced by later uncontrollable tail shock (inescapable shock, IS). The present experiments tested, in rats, whether the protective effect of control over tailshock extends beyond reducing the behavioral and neurochemical impact of a subsequent tailshock experience to stressors that are quite different. Social defeat (SD) was chosen as the second stress experience because it has few if any cues in common with tailshock. SD produced shuttlebox escape learning deficits ("learned helplessness") and reduced juvenile social investigation 24 h later, as does IS. IS is notable for inducing a large increase in dorsal raphe nucleus (DRN) serotonergic (5-HT) activity as measured by extracellular levels of 5-HT within the DRN, and SD did so as well. ES occurring 7 days before SD blocked this SD-induced DRN activation, as well as the SD-induced interference with shuttlebox escape and reduction in social investigation. Prior exposure to yoked IS did not reduce the DRN 5-HT activation or later behavioral effects produced by SD, and thus the proactive stress-blunting effects of ES can be attributed to it's controllability. Thus, ES confers a very general protection to the impact of a subsequent stress experience.
[Show abstract][Hide abstract] ABSTRACT: Morphine-3-glucoronide (M3G) is a major morphine metabolite detected in cerebrospinal fluid of humans receiving systemic morphine. M3G has little-to-no affinity for opioid receptors and induces pain by unknown mechanisms. The pain-enhancing effects of M3G have been proposed to significantly and progressively oppose morphine analgesia as metabolism ensues. We have recently documented that morphine activates toll-like receptor 4 (TLR4), beyond its classical actions on mu-opioid receptors. This suggests that M3G may similarly activate TLR4. This activation could provide a novel mechanism for M3G-mediated pain enhancement, as (a) TLR4 is predominantly expressed by microglia in spinal cord and (b) TLR4 activation releases pain-enhancing substances, including interleukin-1 (IL-1). We present in vitro evidence that M3G activates TLR4, an effect blocked by TLR4 inhibitors, and that M3G activates microglia to produce IL-1. In vivo, intrathecal M3G (0.75 microg) induced potent allodynia and hyperalgesia, blocked or reversed by interleukin-1 receptor antagonist, minocycline (microglial inhibitor), and (+)-and (-)-naloxone. This latter study extends our prior demonstrations that TLR4 signaling is inhibited by naloxone nonstereoselectively. These results with (+)-and (-)-naloxone also demonstrate that the effects cannot be accounted for by actions at classical, stereoselective opioid receptors. Hyperalgesia (allodynia was not tested) and in vitro M3G-induced TLR4 signaling were both blocked by 17-DMAG, an inhibitor of heat shock protein 90 (HSP90) that can contribute to TLR4 signaling. Providing further evidence of proinflammatory activation, M3G upregulated TLR4 and CD11b (microglial/macrophage activation marker) mRNAs in dorsal spinal cord as well as IL-1 protein in the lumbosacral cerebrospinal fluid. Finally, in silico and in vivo data support that the glucuronic acid moiety is capable of inducing TLR4/MD-2 activation and enhanced pain. These data provide the first evidence for a TLR4 and IL-1 mediated component to M3G-induced effects, likely of at least microglial origin.
[Show abstract][Hide abstract] ABSTRACT: We recently described a non-viral gene therapy paradigm offering long-term resolution of established neuropathic pain in several animal models. Here, the requirements for long-term therapeutic effects are described, and evidence is provided for a mechanism of action based on immunological priming of the intrathecal (i.t.) space. Long-term pain reversal was achieved when two i.t. injections of various naked plasmid DNA doses were separated by 5 h to 3 days. We show that an initial DNA injection, regardless of whether a transgene is included, leads to an accumulation of phagocytic innate immune cells. This accumulation coincides with the time in which subsequent DNA injection efficacy is potentiated. We show the ability of non-coding DNA to induce short-term pain reversal that is dependent on endogenous interleukin-10 (IL-10) signaling. Long-term efficacy requires the inclusion of an IL-10(F129S) transgene in the second injection. Blockade of IL-10, by a neutralizing antibody, either between the two injections or after the second injection induces therapeutic failure. These results show that this gene therapy paradigm uses an initial 'priming' injection of DNA to induce accumulation of phagocytic immune cells, allowing for potentiated efficacy of a subsequent 'therapeutic' DNA injection in a time- and dose-dependent manner.
[Show abstract][Hide abstract] ABSTRACT: Traditional approaches to treating chronic neuropathic pain largely focus on manipulations directly altering neuronal activity or neuron-to-neuron communication. Recently, however, it has become clear that glial cells (including microglia and astroglia) play a significant role in pain expression in a variety of neuropathic pain models. Multiple aspects of the inflammatory response of glial cells, commonly observed in neuropathic pain conditions, have been implicated in pain expression. Thus, glial cell inflammation has emerged as a potential therapeutic target in neuropathic pain. Our laboratory has been exploring the use of an anti-inflammatory cytokine, interleukin-10 (IL-10), to control glial inflammatory activation thereby controlling neuropathic pain. IL-10 protein delivery is limited by a short half-life and an inability to cross into the central nervous system from the periphery, making a centrally delivered gene therapy approach attractive. We have recently characterized a non-viral gene therapy approach using two injections of naked DNA to achieve long-term (>3 months) control of neuropathic pain in a peripheral nerve injury model. Timing and dose requirements leading to long-term pain control are discussed in this review, as is recent work using microparticle-encapsulated DNA to achieve long-term therapeutic efficacy with a single injection.