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The influence of endogenous dopamine levels on the density of [3H]SCH23390-binding sites in the brain of the honey bee, Apis mellifera L
Abstract
This paper examines the relationship between endogenous dopamine (DA) levels and the density of [3H]SCH23390-binding sites in the brain of the adult worker honey bee. DA levels were reduced pharmacologically using a single 10 microl injection of either alpha-methyl-DL-p-tyrosine (AMT; 250 microg or 500 microg) or alpha-methyl-DL-tryptophan (AMTP; 250 or 500 microg) into the haemolymph of the bee. In all cases, maximum depletion of DA was observed 3 h after treatment, but in bees treated with AMTP (250 or 500 microg) or with 250 microg AMT, DA levels returned to normal within 24 h of treatment. Neither AMT nor AMTP was selective for DA: both drugs also reduced serotonin (5-hydroxytryptamine, 5HT) levels in the brain. However, AMTP was more effective than AMT at depleting 5HT, whereas for DA, the reverse was true. Depletion of DA levels, using 250 microg AMT, led to a dramatic decline in the levels of specific binding of [3H]SCH23390, defined in this study as binding in the presence of 5x10(-6) M cis-(Z)-flupentixol (see Ref. [28] ). In contrast, naturally occurring diel fluctuations in DA levels, identified in the optic lobes of the brain, and changes in brain DA levels resulting from queenlessness, had no significant effect on the density of [3H]SCH23390-binding sites in the brain of the bee. Overall, these results indicate that under normal physiological conditions, there is no direct link in honey bees between changes in endogenous brain DA levels and the density of D(1)-like receptors labelled by [3H]SCH23390.
... Marked drones were returned to the original colony and kept until the required age. All drones used to quantify biogenic amines were collected in the hive at the same time (14:00–16:00) because biogenic amine levels might fluctuate within a day (Purnell et al., 2000). To investigate the relationship between age and brain DA level, drones at various ages were collected, frozen in liquid nitrogen within 30 min after collection, and stored at À80 1C until analysis. ...
... Perhaps the density or sensitivity of receptors compensates for differences in total levels of brain DA. As in other animals, honeybees can adjust receptor density in response to the level of ligands (Purnell et al., 2000). However, this does not explain why drones of some colonies produce more DA despite its metabolic costs. ...
Dopamine (DA) is a major functional biogenic amine in insects and has been suggested to regulate reproduction in female honeybees. However, its function has not been investigated in male drones. To clarify developmental changes of DA in drones, brain DA levels were investigated at various ages and showed a similar pattern to the previously reported juvenile hormone (JH) hemolymph titer. The DA level was lowest at emergence and peaked at day 7 or 8, followed by decline. Application of JH analog increased brain DA levels in young drones (2-4-days-old), suggesting regulation of DA by JH in drones. In young drones, maturation of male reproductive organs closely matched the increase in brain DA. The dry weight of testes decreased and that of seminal vesicles increased from emergence to day 8. The dry weight of mucus glands increased up to day 4. Consequently, DA regulated by JH might have reproductive behavior and/or physiological functions in drones.
... There are reports that the circadian clock controls many aspects of the brain including the neuron cell size, 12 regulation of neuromodulation, 13 as well as learning and memory. 14 We are interested in the question whether daily foraging activity involves the activation of specific neuromodulator systems. ...
Honey bee foragers show a strong diurnal rhythm of foraging activity and such behavioral changes are likely under the control of specific neuromodulators. To identify and quantify neuromodulators involved in regulating rest and arousal in honey bees, we established a mass spectrometric method for quantifying 14 different neurochemicals and precursor molecules. We measured forager type and brain region specific differences in amine levels from individual honey bee brains and brain regions. The observed differences in amine levels between resting and aroused foragers resemble findings in other species indicating a conserved molecular mechanism by glutamate and GABA in regulating arousal. Sub-esophageal ganglion specific changes in the histaminergic system and global increases in aspartate during arousal suggest a possible role of histamine and aspartate in feeding and arousal, respectively. More aminergic systems were significantly affected due to arousal in nectar foragers than in pollen foragers, implying that forager phenotypes differ not only in their food preference, but also in their neuromodulatory signaling systems (brain states). Finally, we found that neurotransmitter precursors were better at distinguishing brain states in the central brain, while their end products correlated with arousal associated changes in sensory regions like the optic and antennal lobes.
... DA levels in the brain of young bees performing tasks within the colony are generally lower than the levels found in foragers [43,47]. If a colony is made queenless, however, DA levels in the brain of young workers increase to a level similar to that seen in foragers [16,38,40]. The queen's influence on brain DA levels has been traced to QMP. ...
Dopamine (DA) is a signaling molecule derived from the amino acid tyrosine. It is an important neuromodulator, neurotransmitter and neurohormone in invertebrates as well as in vertebrates and numerous studies suggest roles for this amine in motor function, learning and memory, aggression, arousal and sleep, and in a number of other behaviors. A growing body of evidence suggests that DA plays a diversity of roles also in Apis mellifera. Three honey bee DA receptor genes have been cloned and characterized. In this chapter we focus on their likely involvement in the regulation of locomotor activity, ovary development, and olfactory learning and memory.
... Behavioral experiments were carried out between 10 a.m. and 2 p.m. in an attempt to avoid diel changes in brain amine levels that could potentially influence the learning performance of the bees (Purnell et al. 2000). Bees in the three treatment groups described above (control, vehicle, 20-E) were restrained in individual harnesses as described elsewhere (Vergoz et al. 2007a) and then left for 1 -1.5 h to adjust to their environment. ...
Here, we examine effects of the steroid hormone, 20-hydroxyecdysone (20-E), on associative olfactory learning in the honeybee, Apis mellifera. 20-E impaired the bees' ability to associate odors with punishment during aversive conditioning, but did not interfere with their ability to associate odors with a food reward (appetitive learning). The steroid had a significant impact also on the expression of amine-receptor genes in centers of the brain involved in the formation and recall of associative olfactory memories (mushroom bodies). 20-E increased expression of the dopamine receptor gene, Amdop2, and reduced the expression of the putative dopamine/ecdysone receptor gene, Amgpcr19. Interestingly, Amgpcr19 tended to be highly expressed in the brains of foragers that exhibited strong aversive learning, but expressed at lower levels in bees that performed well in appetitive learning assays. In 2-d-old bees, transcript levels of the same gene could be reduced by queen mandibular pheromone, a pheromone that blocks aversive learning in young worker bees. As ecdysteroid levels rise to a peak ∼2 d after adult emergence and then fall to low levels in foragers, we examined aversive learning also in young worker bees. Aversive learning performance in 2-d-old bees was consistently poor. The results of this study indicate that learning in honeybees can be modulated by ecdysteroids. They highlight, in addition, a potential involvement of the putative dopamine/ecdysone receptor, AmGPCR19, in hormonal regulation of associative olfactory learning in the honeybee.
... Laying queens of approximately 1 year of age were collected from nucleus and standard hives for measurement of brain DA. These adult queens were collected from hives between 10.00 h and 12.00 h on the experiment day because DA levels might fluctuate diurnally ( Purnell et al. , 2000 ). They were frozen in liquid N 2 and stored at −80 °C. ...
Abstract Dopamine has been suggested to be involved in physiological and/or behavioural changes triggered by mating in European honeybee (Apis mellifera) queens but its specific role remains unclear. In the present study, the amounts of dopamine (DA) and its metabolite, N-acetyldopamine (NADA) are measured, in queens of various ages to clarify the association with locomotor activity. The effects of DA receptor agonist/antagonist drugs on locomotor activity are further investigated. Brain levels of DA and NADA are relatively constant during the period before mating when locomotor activity reportedly increases with age but decreases in 1-year-old laying queens with low locomotor activity. Reduced DA and NADA levels are also found in haemolymph of 1–3-month-old laying queens. When a DA receptor agonist or antagonist is injected into 6-day-old virgin queens, locomotor activity levels increase significantly with 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (agonist), and decreased with cis(Z)-flupenthixol (antagonist). These results suggest that DA systems are involved in the motor control of honeybee queens, and that the decline in DA levels reduces locomotor activity after mating but increased locomotor activity before mating may be independent of DA levels.
... Additionally, task experience may trigger neural development or result in learning. Future work uncoupling experience and age and using pharmacological agents to alter levels of neuromodulators and neural development (Malun 1998;Harris & Woodring 1999;Purnell et al. 2000) will enable the investigation of causal associations of worker age and labour efficiency in P. dentata. ...
Age-related task performance, or temporal polyethism, is a prominent characteristic of labour organization in most social insects. In many species, including almost all ants, young workers perform within-nest tasks such as brood rearing and queen attendance and transition to tasks outside the nest as they age. We examined temporal polyethism in the ant Pheidole dentata by constructing single-age cohort subcolonies and directly determining the ability of young and old minor workers to care for the queen and developing brood. In contrast to the predictions of most temporal polyethism models, larvae reared by old workers gained significantly more mass than those reared by young workers, and did not differ significantly in mass from larvae reared by groups of workers of intermediate age. Additionally, old workers were more responsive to brood care and queen care needs than young workers: old workers approached, cared for and retrieved brood more frequently than young workers, and engaged in more queen-directed behaviours even though young workers tended to remain in closer proximity to the queen. These results do not support the traditional characterization of young workers as a discrete temporal caste specialized to respond to queen and brood needs. Although brood and queen care are performed earlier in development than foraging, older workers retain the ability to perform these tasks and do so with high efficiency. Our study suggests that the apparent specialization of young P. dentata minor workers on nursing may be an artefact of their age-related repertoire expansion and development of task proficiencies.
The aim of this study was to analyse the consumption habits of bee products such as: honey, bee pollen, royal jelly and propolis in the elderly of different places of Spain. Honey is an energetic food of its own, and it can also help to overcome several diseases; due to its components that have a beneficial effect in our health. These facts turn honey into a valuable constituent of one's nutrition specially in ancient people. This report demonstrates the knowledge and consumption habits of these persons. Date came from a questionnaire about preferences, places where they buy these products, time and amount of consumption, knowledge of the quality of these products, etc. The results suggest that the vast majority of the people inquired are regular consumers of honey and we also would like to underline the high rate of knowledge of its nutritional value and its medicinal qualities. However, most of the elderly inquired do not know about other apiary products such as bee propolis.
The expression patterns of two dopamine receptor genes, Amdop1 and Amdop2, in the developing mushroom bodies of the honeybee brain were determined by using in situ hybridisation. Both genes were expressed throughout pupal development, but their patterns of expression in the three major divisions of mushroom body intrinsic neurons (outer compact cells, noncompact cells, and inner compact cells) were quite distinct. Amdop1 expression could be detected in all three mushroom body cell groups throughout development. Staining for Amdop1 mRNA was particularly intense in newly born Kenyon cells, suggesting that levels of Amdop1 expression are higher in newborn cells than in more mature mushroom body neurons. This was not the case for Amdop2. Amdop2 expression in the mushroom bodies was restricted to inner and outer compact cells during most of pupal development, appearing in noncompact cells only late in metamorphosis or at adult eclosion. In contrast to the case with Amdop1, staining for Amdop2 mRNA was observed in glial cells. Expression of Amdop2 in glial cells was detected only at early stages of glial cell development, when the cells are reported to be actively dividing. This study not only implicates dopamine in the development of honeybee mushroom bodies but also suggests different roles for the two dopamine receptors investigated.
This study examines the relationship between cyclical variations in optic-lobe dopamine levels and the circadian behavioural rhythmicity exhibited by forager bees. Our results show that changing the light-dark regimen to which bees are exposed has a significant impact not only on forager behaviour, but also on the levels of dopamine that can be detected in the optic lobes of the brain. Consistent with earlier reports, we show that foraging behaviour exhibits properties characteristic of a circadian rhythm. Foraging activity is entrained by daily light cycles to periods close to 24 h, it changes predictably in response to phase shifts in light, and it is able to free-run under constant conditions. Dopamine levels in the optic lobes also undergo cyclical variations, and fluctuations in endogenous dopamine levels are influenced significantly by alterations to the light/dark cycle. However, the time course of these changes is markedly different from changes observed at a behavioural level. No direct correlation could be identified between levels of dopamine in the optic lobes and circadian rhythmic activity of the honey bee.
Honey bee queens produce a sophisticated array of chemical signals (pheromones) that influence both the behavior and physiology of their nest mates. Most striking are the effects of queen mandibular pheromone (QMP), a chemical blend that induces young workers to feed and groom the queen and primes bees to perform colony-related tasks. But how does this pheromone operate at the cellular level? This study reveals that QMP has profound effects on dopamine pathways in the brain, pathways that play a central role in behavioral regulation and motor control. In young worker bees, dopamine levels, levels of dopamine receptor gene expression, and cellular responses to this amine are all affected by QMP. We identify homovanillyl alcohol as a key contributor to these effects and provide evidence linking QMP-induced changes in the brain to changes at a behavioral level. This study offers exciting insights into the mechanisms through which QMP operates and a deeper understanding of the queen's ability to regulate the behavior of her offspring.
• Apis mellifera
• biogenic amines
• neuroethology
• neuromodulation
• pheromonal communication
Brain levels of octopamine (OA), dopamine (DA) and serotonin (5HT) were compared between worker honey bees (Apis mellifera L.) kept with or without queens in various experiments. Levels of DA were significantly higher in queenless bees than in their sisters that had been kept with queens either (1) in small field colonies for 30 days or (2) in incubator cages through 18 days. This difference was not observed in a separate incubator experiment after 6 and 12 days. A correlation between ovariole width and brain DA levels was found in groups of caged queenless workers after 18 days. In pairs of caged workers kept in an incubator through 10 days, the worker having the largest ovariole width was significantly more likely to have higher brain DA levels. The increased DA levels in queenless bees probably reflect changes in behavioral states that occur after onset of oogenesis.
To investigate diurnal variations in dopamine receptor binding, the amount of specifically bound 3H-spiroperidol was measured at 4-h intervals over a 24-h period in the striatum of rats which had been housed under a controlled 12-h light-dark cycle (lights on 7 a.m.). A highly significant ultradian rhythm with peaks at 2 a.m. and 2 p.m. was found with an amplitude of about 75%. Chronic imipramine modified the rhythm such that the two peaks occurred 4 h later and amplitude as well as 24-h mean of binding decreased. Scatchard analysis at times of least and greatest binding indicated that the differences in binding were due not to changes in the affinity, but in the number of binding sites. These results are interpreted with regard to the mode of action of psychoactive drugs and to postulated changes of receptor sensitivity in neurological and psychiatric disorders.
The effects of environmental and genetic factors on levels of octopamine, dopamine and serotonin in brains from worker honey bees (Apis mellifera L.) were measured using high-performance liquid chromatography with electrochemical detection. Exiting foragers were stressed by clamping their legs, which resulted in peak elevation of octopamine and serotonin after 10 min. Significant seasonal differences in levels of all three biogenic amines were found for bees sampled from an observation colony during the spring, summer and autumn of 1990. Levels of the amines were highest during June–September, corresponding to high levels of colony foraging activity. These differences may reflect seasonal changes in colony nutrition, population size, or brood-rearing activity. The levels of all three amines were significantly lower in the brains of newly emerged bees than in brains from randomly aged worker bees from the same colony. Since the total protein content in bee brains was not different for bees from these two groups, age-related differences were not related to brain growth. Significantly different levels of octopamine, dopamine and serotonin were detected among workers in colonies that contained unrelated queens.
lntracerebral microdialysis was used to measure the extra- cellular concentration of striatal dopamine (DA) and its me- tabolites in freely moving rats depleted of DA by the bilateral infusion of 6-hydroxydopamine into the substantia nigra ap- proximately 1 month earlier. It was found that the basal ex- tracellular concentration of DA remained within the same range as seen in control animals until the size of the lesion exceeded 60% (estimated by the postmortem tissue con- centration of DA). In animals with an 60-95% lesion there was only a modest decrease in basal extracellular DA, but as lesion size exceeded 95% there was a marked drop in the basal extracellular concentration of DA. In contrast, the basal extracellular concentration of the DA metabolites showed a more steady decline as a function of lesion size. To determine the ability of the residual population of DA terminals to further increase DA release upon increased de- mand, animals were given a challenge injection of 1.5 mg/ kg of d-amphetamine. Amphetamine-evoked DA release re- mained within the control range until lesion size exceeded 95%. These results provide direct confirmation for the hy- pothesis that following recovery from partial bilateral dam- age to the nigrostriatal DA system in adulthood, there are presynaptic compensatory changes in the remaining popu- lation of DA neurons sufficient to "normalize" the extracel- lular concentration of striatal DA. It is suggested that this normalization of extracellular DA is responsible for the spar- ing of function seen following the loss of up to 60% of the DA innervation to the striatum and contributes to the recov- ery of function seen after even more extensive damage (60- 95%). However, at least 5% of the usual dopaminergic in- nervation to the striatum is required to maintain a relatively normal extracellular concentration of DA, and it appears that this 5% is necessary for any significant recovery of function.
In the brain of the honey bee, dopamine receptors have been identified by using the vertebrate D1 dopamine antagonist [3H]-SCH23390 and the vertebrate D2 dopamine antagonist [3H]-spiperone. This study uses light microscope autoradiography to investigate the anatomical distributions of the binding sites labelled by [3H]-SCH23390 and [3H]-spiperone in tissue sections cut at three depths from the anterior surface of the brain. The binding of these radioligands differed significantly, in both density and distribution. Specific binding of [3H]-SCH23390, defined by using 5 x 10(-6) M cis-(Z)-flupentixol, was densest in regions of somata, such as the deutocerebral somatal rind, the somatal layer beneath the calyces of the mushroom bodies and the ventral protocerebral somatal group. High levels of [3H]-SCH23390 binding were also measured in the lateral protocerebrum. [3H]-Spiperone binding site density estimates were consistently lower than those of [3H]-SCH23390. Specific binding of [3H]-spiperone, determined by subtracting binding in the presence of 10(-4) M domperidone from the total binding, was highest in the alpha lobes, beta lobes, and calyces of the mushroom body neuropil. Relatively high binding was also measured in the central body and lateral protocerebral neuropil. These results suggest that the distribution of dopamine receptors in the brain of the bee is subtype specific, and they support the view that dopamine plays many roles in the insect central nervous system.
Dopaminergic denervation supersensitivity has been implicated in the pathogenesis of levodopa-induced dyskinesias, the most common and limiting side effect in the drug treatment of Parkinson's disease, yet the mechanisms that mediate altered drug sensitivity remain poorly understood. In animals models, one key component of denervation supersensitivity is the enhanced efficacy of selective D1 agonists to stimulate locomotion. In rats with chronic dopamine depletion induced by 6-hydroxydopamine nigral lesion, the increased ability of D1 agonists to stimulate regional cerebral glucose utilization (RCGU) in the substantia nigra pars reticulata (SNr) has provided a metabolic correlate to the heightened motor response. In this study, we used the stimulation of RCGU in the SNr as a sensitive in vivo assay of D1 agonist effect to examine the time course of development of supersensitivity in rats following acute dopamine depletion with single doses of reserpine (5.0 mg/kg, i.p.) and alpha-methyl-p-tyrosine (AMPT; 100 mg/kg, i.p.). The stimulatory effect of the D1 agonist SKF 38393 (30 mg/kg) on RCGU in the SNr was first enhanced 6 hr after reserpine/AMPT injection and was maximally enhanced at 12-24 hr (relative 2-deoxyglucose uptake increased 32-51%; P less than 0.05). The response to SKF 38393 returned to control values 5 d after reserpine/AMPT injection. The single reserpine/AMPT injections depleted striatal dopamine to 1-2% of control values from 3-48 hr postinjection, whereas D1 and D2 dopamine receptor densities were unchanged at 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)
The striatum, which is the major component of the basal ganglia in the brain, is regulated in part by dopaminergic input from the substantia nigra. Severe movement disorders result from the loss of striatal dopamine in patients with Parkinson's disease. Rats with lesions of the nigrostriatal dopamine pathway caused by 6-hydroxydopamine (6-OHDA) serve as a model for Parkinson's disease and show alterations in gene expression in the two major output systems of the striatum to the globus pallidus and substantia nigra. Striatopallidal neurons show a 6-OHDA-induced elevation in their specific expression of messenger RNAs (mRNAs) encoding the D2 dopamine receptor and enkephalin, which is reversed by subsequent continuous treatment with the D2 agonist quinpirole. Conversely, striatonigral neurons show a 6-OHDA-induced reduction in their specific expression of mRNAs encoding the D1 dopamine receptor and substance P, which is reversed by subsequent daily injections of the D1 agonist SKF-38393. This treatment also increases dynorphin mRNA in striatonigral neurons. Thus, the differential effects of dopamine on striatonigral and striatopallidal neurons are mediated by their specific expression of D1 and D2 dopamine receptor subtypes, respectively.
Circadian adrenoceptor rhythms were measured in the cortex, hypothalamus, cerebellum and pons-medulla of WKY and SHR rats. The 24-h mean binding to alpha 1-, and beta-adrenoceptors was higher in SHR than WKY in all regions:greatest differences were found at the end of the dark phase. The most consistent and large amplitude circadian rhythms in adrenoceptor binding occurred in the hypothalamus; strain differences in wave form of the rhythm were the most noticeable phenomenon. In the pons-medulla SHR rats showed decreased rhythm amplitudes. In the cerebellum, beta-adrenoceptor binding was higher in SHR rats at all time points, with an identical rhythm to that in WKY rats. Thus regional differences between SHR and WKY rats were expressed in changes in many circadian rhythm parameters. There were no differences between SHR and WKY in the ratios of pre-synaptic: post-synaptic receptors. High affinity binding to alpha-adrenoceptors showed similar circadian rhythms to total specific binding, indicating that the short-term fluctuations cannot be attributed to change in affinity state. Whether these modifications in circadian rhythm characteristics are related to the development or maintenance of hypertension in the SHR strain remains to be investigated.
Circadian rhythms in the numbers of alpha- and beta-adrenergic receptors exists in the rat forebrain and hypothalamus. These rhythms are endogenous as they persist in the absence of time cues. The alpha- and beta-receptor rhythms differ both in their shapes (wave form) and in the timing (phase) of the peak numbers. In the course of the year, there are shifts in the timing of both alpha- and beta-receptor peaks. Circadian rhythms in synchronizing both the synaptic events and the behaviors related to them to the photoperiod of the natural environment.
In four experiments, performed at different months throughout the year, a significant daily rhythm in dopamine receptor binding has been observed. This rhythm is endogenous, as it persists in the absence of time cues. Striking differences in wave form, amplitudes, and timing of peaks during the year suggest that the endogenous rhythm undergoes seasonal variations.
Flight activity of honeybees was observed in two locations in California. The total number of outgoing flights from a hive varied between 14 000 and 163 000 a day. Significant diurnal variations were recorded; with high temperature and low humidity in the desert, there were very few flights at midday, and very many at dawn and dusk. The use of relative daily numbers of flights as a measure of foraging potential is discussed.
1.1. Liquid chromatography with electrochemical detection was used to determine concentrations of dopamine, N-acetyldopamine, 5-hydroxytryptamine, and N-acetyl-5-hydroxytryptamine in cerebral ganglion of the American cockroach, Periplaneta americana.2.2. Neuroactive drugs were injected into the cockroaches and their effects on biogenic amine levels in cerebral ganglion assessed. Drugs tested included α-methyltyrosine, p-chlorophenylalanine methyl ester, D( + )-amphetamine, reserpine, pargyline, harmaline, and the formamidine pesticides chlordimeform, N-demethylchlordimeform, and N,N-didemethylchlordimeform.3.3. Although N-acetyldopamine was easily demonstrable in P. americana cerebral ganglion, N-acetyl-5-hydroxytryptamine could not be detected in cerebral ganglion from normal animals. However, when cerebral ganglia were incubated with 5-hydroxytryptophan or 5-hydroxytryptamine in vitro, a peak with the low oxidation potential and retention time of N-acetyl-5-hydroxytryptamine appeared.4.4. p-Chlorophenylalanine methyl ester and α-methyltyrosine, which have dramatic effects on the levels of 5-hydroxytryptamine and catecholamines respectively in mammalian brain, failed to significantly affect the levels of 5-hydroxytryptamine and dopamine in cerebral ganglia from cockroaches pretreated with the drugs.5.5. Two possible products of monoamine oxidase activity, dihydroxyphenylacetic acid and 5-hydroxy-indoleacetic acid, were not detected in cerebral ganglion from P. americana.6.6. Results of the present study do not support the hypotheses that either MAO inhibition or N-acetyl-transferase inhibition are important modes of insecticidal action of the formamidine pesticides.
HPLC with electrochemical detection was used to determine the levels of p-hydroxyphenylethanolamine (octopamine), 3,4-dihydroxyphenylethylamine (dopamine), and 5-hydroxytryptamine (5-HT) in the brains of control, reserpine, and d-amphetamine-treated blow flies, Phormia regina Meigen. Parallel studies were carried out to assess the effects of the two drugs on fly feeding behavior, measured as mean acceptance threshold: the minimum sucrose concentration to which the average fly in a population will respond by proboscis extension when its tarsi contact the solution. In saline-injected control flies, all three amines were found at levels of approximately 2 pmol/brain. Thirty minutes after injection with d-amphetamine (12 μg/fly), brain octopamine was depleted by 85%, whereas dopamine and 5-HT were depleted by 70%. Reserpine (5 μg/fly) caused 70% depletion of dopamine and >90% depletion of both octopamine and 5-HT 24 h after injection. However, the effect of reserpine was much slower in onset (hours versus minutes) and more persistent (days versus hours) than was the effect of d-amphetamine. With either drug, the time course of amine depletion closely matched the time course of the increase in feeding threshold observed in drug-treated flies. These results suggest that CNS pools of the biogenic amines, octopamine, dopamine, and 5-HT are important in governing blow fly responsiveness to food stimuli.
Changes in biogenic amine levels associated with the morphological and behavioural development of the worker honeybee are examined.
A significant increase in amine levels in the head of the honeybee is associated with transition from the larval to pupal stage. Adult emergence is also accompanied by a significant increase in 5-HT levels in the brain, but no significant change in brain dopamine (DA) levels. NADA (N-acetyldopamine) levels increase during larval and pupal development, but in contrast to both DA and 5-HT, drop significantly during the transition from pupa to adult.
Levels of DA in the brain of nectar and pollen forager bees, presumed to be among the oldest adults sampled, were found to be significantly higher than in nurses, undertakers or food storers. These results suggest that an age-dependent change in amine levels occurs in the brain of the worker bee. In the optic lobes, levels of DA and 5-HT were found to be significantly higher in pollen forager bees than in all other behavioural groups. Significant differences in amine levels in the optic lobes of nectar foragers and pollen foragers indicate that some differences in amine levels occur independent of worker age. The functional significance of differences in brain amine levels and whether or not biogenic amines play a direct role in the control of honeybee behaviour has yet to be established.
Changes in the levels of binding of 3H-SCH-23390, a vertebrate D1 dopamine receptor ligand, and 3H-spiperone, a vertebrate D2 dopamine receptor ligand were investigated in the brain of the worker honey bee during metamorphic
adult development and during the lifetime of the adult bee. Age-related fluctuations in binding levels were markedly different
for these two ligands. 3H-SCH-23390 and 3H-spiperone binding sites were present at low levels during metamorphic adult development. After adult emergence, however,
3H-SCH-23390 binding levels, in contrast to those of 3H-spiperone, increased significantly. Within the first 48 h of adult life 3H-SCH-23390 binding reached a level not significantly different from that detected in forager bees. No significant fluctuations
in the levels of 3H-spiperone binding were observed during the adult lifetime of the bee. Measurements of dopamine levels in the brains of pupal
and adult bees revealed no direct correlation between fluctuations in endogenous amine levels and the amount of binding of
either 3H-SCH-23390 or 3H-spiperone. These results provide evidence for subtype-specific patterns of expression of dopamine receptors in the insect
brain and show that D1- and D2-like receptors are expressed not only in the adult CNS, but also in the developing brain of
the bee.
1. In the brains of worker honeybees (Apis mellifera carnica) corresponding to different stages in the life span, we measured the content of GABA, glutamate, acetylcholine, eholine, norepinephrine, dopamine and serotonin.2. The highest concentrations were found for GABA, glutamate and acetylcholine.3. Biogenic amines occur in considerably lower concentrations in comparison to the above mentioned transmitters.4. Age-correlated changes were found for different neuroactive substances.5. GABA and glutamate show a well marked rise and fall of their concentrations with a maximum at day 10.6. The results are discussed in comparison to other species and with respect to age polyethism of worker honeybees.
Behavioural studies have shown that stimulation of D1 receptors, which is uneffective in normal rats, induced strong hypermotility in rats pretreated with reserpine for 5 days. On this basis, we investigated D1 receptor plasticity using the 5-day treatment with reserpine (1 mg/kg; s.c.) as an experimental model. The function of striatal D1 receptors was determined both in binding studies with [3H]SCH 23390 and by measuring formation of cAMP in response to the selective agonist, SKF 82526. The results indicate that the responsiveness of adenylate cyclase (AC) to D1 receptor stimulation was markedly increased after reserpine administration, while no significant changes were found in [3H]SCH 23390 binding site density. Moreover, formation of cAMP after stimulation of Gs protein with GppNHp was markedly enhanced in dopamine (DA)-depleted rats; the responsiveness of AC to forskolin, which directly stimulates the AC catalytic unit, was not affected by reserpine administration. These data indicate that reserpine-induced D1 receptor up-regulation is apparently mediated by a marked enhancement of the coupling efficiency of Gs protein, suggesting that the D1 behavioral supersensitivity does not correlate with the density of D1 receptors, but is reflected by a selective up-regulation of their transduction mechanisms.
Phenomena consistent with postsynaptic supersensitivity developed in the rat neostriatum following the destruction of dopaminergic afferent neurons. A gradual in-increase in the densoty of binding sites for [3H]spiperone occurred over a 2–3 week period. This increase was apparent only after the almost complete loss of dopamine-containing nerve terminals as measured by the depletion of endogenous dopamine. The properties of the receptor labeled by [3H]spiperone were not altered by denervation. Elimination of dopamine-containing nerve endings in the neostriatum was accompanied by the gradual development of an increase in dopamine-sensitive adenylate cyclase activity in homogenates of the caudate ipsilateral to the lesion as compared to the contralateral side. The administration of apomorphine led to pronounced circling behavior. This effect occurred rapidly and was maximal within 3 days following destruction of dopaminergic neurons. The increase in the density of dopamine receptors and in a receptor-mediated function may partially account for the development of enhanced electrophysiological responses to dopamine agonists in the neostriatum. However, the results do not explain the drug-induced rotational behavior which develops after destruction of the dopamine-containing nigrostriatal pathway. This behavioral phenomenon clearly preceded the appearance of receptor alterations in the corpus striatum.
Chronic treatment with SCH 23390, a selective D-1 dopamine receptor antagonist, increased (40%) the density of [3H]SCH 23390 binding sites in striatal membrane preparations but failed to change the apparent KD of the ligand for its binding sites. Haloperidol, which preferentially blocks D-2 receptors, induced only a slight, not significant increase in the total number of [3H]SCH 23390 binding sites. (−)Sulpiride, a selective D-2 receptor blocker, also failed to change either Bmax or KD of [3H]SCH 23390 binding. Thus, chronic blockade of D-1 receptor sites by SCH 23390 can lead to an increase in their total number.
The precise topographical changes in striatal D2 dopamine receptors that occur after neurotoxic lesion of the mesostriatal dopaminergic pathway have been studied autoradiographically in the rat through the use of [3H]spiperone as a ligand. 6-Hydroxydopamine-induced lesion of the dopaminergic afferents to the striatum caused an increase in [3H]spiperone binding in the ventro- and dorsolateral but not in the ventro-and dorsomedian aspects of the striatum. This lesion caused a loss of tyrosine hydroxylase-like immunoreactivity in all striatal subregions. These results demonstrate that not all striatal D2 dopamine receptors are able to proliferate after dopaminergic denervation.
3ollowing unilateral 6-hydroxydopamine nigrostriatal lesions in rats, the binding of both 3H-apomorphine and 3H-spiroperidol in the striatum is increased. In rats with incomplete lesions or at early time points after lesion, binding is not significantly different from control levels.
The biogenic amine receptor genes constitute an ancient and highly divergent family within the larger superfamily of G-protein-coupled receptors. These receptors play a central role in modulating nerve cell activity and thus behaviour. Because the honey bee offers numerous advantages for behavioural studies we endeavoured to isolate as many members of this gene family as possible from the bee. We compared numerous approaches to gene isolation and found that PCR amplification from small subfractions of cDNA or genomic DNA libraries enabled us to isolate clones that are otherwise undetectable. In total we isolated seven biogenic amine receptor clones and identified five additional related sequences by low-stringency Southern hybridization. Two clones, AmBAR4 and AmBAR6, are 84% and 72% identical to the Drosophila 5-HT2 and D1b receptors, respectively, and probably represent orthologous genes. Phylogenetic analysis indicates that AmBAR5 clusters loosely with a variety of tyramine and octopamine receptors with which it shares <66% identity. The other four clones, AmBAR1, AmBAR2, AmBAR3 and AmBAR7, are weakly to moderately related (28-45% identical) to Drosophila dopaminergic or mammalian adrenergic receptors and probably represent receptors of these classes whose orthologues have not previously been isolated from any insect. The honey bee clones expand the size of the known insect biogenic amine receptor gene family to sixteen members. Therefore the size of the biogenic amine receptor gene family of insects approaches that of vertebrates. This is true despite the reduced behavioural and genetic complexity of the insects relative to vertebrate animals.
Modifications in the sensitivity of target-cells to DA or agonists in mouse striatum have been assesses both behaviorally, by scoring a stereotyped behaviour elicited by apomorphine and biochemically, by measurement of HVA and DA levels to evaluate the activity of the feedback loop controlling nigro-striatal neurons.Following the acute administration of α-MPT, hypersensitivity develops in a few hours and decays progressively in 3 days. Since the development of hypersensitivity is prevented by the administration of cycloheximide or anisomycin, two inhibitors of protein synthesis, it is hypothesized that it migth be accounted for by synthesis of new receptor sites.
A decrease in specific [3H]spiroperidol binding to rat caudate tissue and a parallel decrease in sensitivity to apomorphine
in eliciting stereotyped behavior was observed in the offspring of rat mothers treated with either haloperidol or alpha-methyl-p-tyrosine-methyl
ester during pregnancy. In contrast, evidence of increased dopamine-receptor sensitivity was observed in the pups if haloperidol
was administered to their mothers postpartum during nursing rather than during pregnancy.
Some recent modifications of the protein assay by the method of Lowry, Rosebrough, Farr, and Randall (1951, J. Biol. Chem.193, 265–275) have been reexamined and altered to provide a consolidated method which is simple, rapid, objective, and more generally applicable. A DOC-TCA protein precipitation technique provides for rapid quantitative recovery of soluble and membrane proteins from interfering substances even in very dilute solutions (< 1 μg/ml of protein). SDS is added to alleviate possible nonionic and cationic detergent and lipid interferences, and to provide mild conditions for rapid denaturation of membrane and proteolipid proteins. A simple method based on a linear log-log protein standard curve is presented to permit rapid and totally objective protein analysis using small programmable calculators. The new modification compared favorably with the original method of Lowry et al.
Chronic treatment of rats with the neuroleptic drugs haloperidol, fluphenazine, and reserpine elicits a 20 to 25% increase in striatal dopamine receptor binding assayed with [3H]haloperidol. This increase in receptor sites may account for behavioral supersensitivity to dopamine receptor stimulants in such animals and for tardive dyskinesia in patients treated with these drugs.
The binding of [3H]haloperidol to rat striatal dopamine receptors increases after lesion (made by injection of 6-hydroxydopamine) of the nigrostriatal dopamine pathway in those rats which are behaviorally supersensitive, as reflected by apomorphine-induced contralateral rotations. The enhanced binding is associated with an increased number of receptor sites with no change in their affinity.
The changes in sensitivity of striatal dopamine (DA) receptors, following pharmacological treatments modifying their level of stimulation, have been studied in mice both biochemically by measurement of DA neurone activity and behaviourally by scoring a stereotyped behaviour. In control mice, at least two classes of DA receptors can be deduced from the biphasic effects of apomorphine, a specific DA agonist, on the climbing behaviour as well as on the striatal homovanillic acid (HVA) level.The blockade of DA receptors induced by a single haloperidol administration results in a state of hypersensitivity of presumably postsynaptic receptors indicated by (a) an increased behavioural responsiveness to apomorphine, (b) a decreased ability of haloperidol to antagonize the apomorphine-elicited climbing behaviour, (c) a decreased rate of DA release reflected by a diminished striatal HVA level and (d) an increased ability of apomorphine to lower HVA level. This state of typical hypersensitivity appears as soon as the blockade of DA receptors is over (about 48 h after haloperidol administration) and disappears slowly with a half-life of about two days.On the other hand, administration of a single dose of apomorphine leads to a state of ‘behavioural facilitation’ also characterized by an increased behavioural responsiveness to apomorphine. Several arguments indicate that it differs from the state of typical hypersensitivity and that it might result from the hyposensitivity of DA ‘autoreceptors’: (a) it develops after stimulation of a class of DA receptors which respond to low doses of apomorphine or to weak agonists and is prevented by haloperidol; (b) it is characterized by a diminished effectiveness of low doses of apomorphine as regards both behavioural and biochemical actions and by an increased efficacy of haloperidol to enhance HVA level; (c) ‘behavioural facilitation’ and typical hypersensitivity elicited by haloperidol are additive; (d) ‘behavioural facilitation’ is no longer present after destruction of DA neurones by 6-hydroxydopamine. This change in sensitivity appears very rapidly (2 h after apomorphine administration) and disappears slowly with a half-life of about 7 days.Thus, the hyposensitivity of DA ‘autoreceptors’, responding to low doses of DA agonists, mediating an inhibition of DA release and, consequently, behavioural effects opposite to those resulting from the stimulation of postsynaptic receptors, may explain the ‘behavioural facilitation’.We suggest, in view of its rapid appearance and long-lasting occurrence, that this process represents a basic neurobiological mechanism accounting for long-term increases in synaptic efficacy.
Parkinson's disease is associated with degeneration of the dopaminergic component of the nigrostriatal pathway. However, the neurological symptoms of this disorder do not emerge until the degenerative process is almost complete. A comparable phenomenon can be observed in animal models of Parkinson's disease produced by the administration of the selective neurotoxin, 6-hydroxydopamine (6-OHDA). Studies using such models suggest that the extensive loss of dopaminergic neurons is compensated, in large part, by increased synthesis and release of dopamine (DA) from those DA neurons that remain, together with a reduced rate of DA inactivation. These findings may have important implications for the diagnosis and treatment of a variety of neurological and psychiatric diseases, as well as for our understanding of plasticity in monoaminergic systems.
The selective dopaminergic antagonist ligands [3H]SCH 23390 and [3H]sulpiride were used to reveal autoradiographically dopamine D1 and D2 receptors, respectively, in brain sections from rats which had received unilateral 6-hydroxydopamine (6-OHDA) injections destroying ascending nigrostriatal neurones. The binding of both ligands to striatal sections was first shown to be saturable, reversible and of high affinity and specificity [( 3H]SCH 23390: Bmax 2.16 pmol/mg protein, Kd 1.4 nM; [3H]sulpiride; Bmax 0.67 pmol/mg protein, Kd 10.7 nM). After unilateral stereotaxic 6-OHDA injections, rats rotated contralaterally when challenged with apomorphine (0.5 mg/kg), or specific D1 or D2 agonists, SKF 38393 (1.0-5.0 mg/kg) and LY 171555 (0.05-0.5 mg/kg), respectively. Loss of forebrain dopaminergic terminals was assessed autoradiographically using [3H]mazindol to label dopamine uptake sites. A loss of approximately 90-95% of uptake sites was reproducibly accompanied by an enhanced density of binding ipsilaterally for the D2 ligand, [3H]sulpiride, in all areas of the striatum, but most markedly in the lateral areas. An increase in the D2 binding site density was also seen in the ipsilateral nucleus accumbens and the olfactory tubercle. In contrast, in the same animals, the striatal D1 receptors were far less affected by dopaminergic denervation, with no consistent changes seen in the binding of [3H]SCH 23390. These results suggest that dopamine D2 receptors are more susceptible than D1 receptors to changes after dopaminergic denervation, which is expressed as an increase in the density of binding sites revealed here with [3H]sulpiride.
Mice receiving reserpine (1 mg/kg/day) during 5 days develop behavioral supersensitivity. To study the possible molecular correlates of these adaptive changes we compared binding parameters of D1 and D2 receptors and adenylate cyclase activity in striata from normal and reserpinized mice. Saturation curves using [3H]SCH 23390 showed no changes in maximum binding capacity (Bmax) or Kd of striatal D1 receptors taken from control or 5 days reserpine-treated mice. However, [3H]spiperone saturation curves showed a 31% increase in D2 receptors Bmax with no changes in Kd. Dopamine competition of [3H]SCH 23390 and [3H]spiperone binding in mouse striatum was also performed. Analysis of data by LIGAND showed that dopamine recognizes two subpopulations for D1 and for D2 receptors. The proportion of receptors in the high affinity state (D1high and D2high) were increased in reserpine-treated animals. The addition of 100 microM GTP produced a complete conversion of D1high and D2high receptors into their low-affinity states in striata from control and reserpinized mice. Five days of reserpine treatment increased basal adenylate cyclase activity of mouse striatum in the presence of Mn++ or Mg++ ions. Concentration curves with dopamine, NaF or forskolin revealed shifts to the left and higher maximum responses without changes in EC50 values in striata from reserpinized mice. Thus, a prolonged reserpine treatment produces marked changes in D1 and D2 receptors increasing the proportion of high affinity state subpopulations and the total Bmax of D2 receptors. Also, dopamine function may be enhanced through an increment of the catalytic component of striatal adenylate cyclase.
Profound depletion of forebrain dopamine by 6-hydroxydopamine in neonatal rats (day 3) was associated with up to 82% loss of D1 receptor sites labeled with [3H]SCH-23390 at day 21. Administration of the selective D1 agonist SKF-38393 (days 6-18) abolished the correlation between D1 receptor density and DA concentrations, even with greater than 99% depletion of DA. In intact control animals, there was an inverse correlation between spontaneous variation in levels of DA and D1 receptor site density in forebrain tissue (r = -0.79) which also was abolished by treatment with the D1 agonist. Thus, D1 receptor density may be regulated by reciprocal regulatory processes during normal development, but may fail to develop in the absence of an adequate level of stimulation.
Monoamine levels and the binding properties of [3H]SCH23390, a D1-specific ligand, and [3H]raclopride, a D2-specific ligand, were measured in the rostal and caudal neostriatum to investigate the fate of dopamine receptors following bilateral cerebroventricular injection of 6-hydroxydopamine in 3-day-old rats. After survival times of 15, 30 or 90 days, measurement of monoamine levels and of [3H]SCH23390 binding were also obtained from the cerebral cortex. At all three survival times, dopamine content was reduced by more than 90% of control values in both the rostral and caudal neostriatum; in cerebral cortex, the dopamine depletion was less profound (80%) and noticeable only after 1 and 3 months. In the rostral but not the caudal neostriatum, serotonin and 5-hydroxyindoleacetic acid concentrations were markedly increased at 1 and 3 months; cortical serotonin also was augmented at 3 months. There were no changes in neostriatal [3H]SCH23390 binding at any of the survival times, but a transient elevation occurred in the cortex at 1 month. In the rostral but not the caudal neostriatum, [3H]raclopride binding showed a slight elevation at 1 month and a further, highly significant increase at 3 months. As measured in individual rats, this increase in [3H]raclopride binding was linearly correlated with the increase in serotonin turnover (ratio of 5-hydroxyindoleacetic acid/serotonin). Such an up-regulation of D2 receptors, restricted to the rostral neostriatum which was also the site of a serotonin hyperinnervation, was probably indicative of a serotonin control on the expression of D2 receptors after dopamine denervation.
Autoradiographic experiments performed on rats with unilateral mesotelencephalic 6-hydroxydopamine (6-OHDA) injections revealed reduced binding of [3H]SCH23390 to D1 receptors in the striatum ipsilateral to the neurotoxin as well as increased binding of [3H]spiroperidol to D2 receptors in that hemisphere. These opposite influences of injury on the dopamine receptor subtypes occurred in rats sacrificed at 2 weeks or 11 months postoperatively, but neither change was evident at 4 days postoperatively. Equilibrium saturation analysis performed on rats sacrificed at 8 weeks postoperatively indicated that D1 and D2 receptor changes reflected altered Bmax values without KD modifications. Topographic analysis of the D1 decline by quantitative autoradiography revealed that the D1 decrease was greater in dorsal striatum than ventrally. Those striatal regions that showed greater declines in D1 density correspondingly had the greater losses of [3H]mazindol binding after the denervation, suggesting that the decline of D1 binding is a postsynaptic consequence of the reduced mesostriatal dopaminergic innervation. The findings indicate opposite influences of injury on D2 and D1 receptor levels and raise important questions concerning the mechanism by which 6-OHDA injection affects the D1 sites.
Mice homozygous for the autosomal recessive gene weaver (wv) exhibit a regionally specific depletion of forebrain dopamine (DA). DA is reduced approximately 70% in the dorsal striatum of homozygotes (wv/wv) relative to heterozygous (+/wv) controls while DA content in ventral striatum is relatively unchanged. The goal of the present study was to determine the regional effects of the weaver mutation on striatal DA receptors and DA uptake sites using quantitative autoradiography. Catecholamine histofluorescence was used to examine midbrain DA-containing cell bodies. Compared to behaviorally normal (+/-) littermates, the binding of [3H]spiroperidol to D2 sites was significantly increased in the dorsal but not ventral striatum of wv/wv mice. Binding of the D1 ligand, [3H]SCH23390, was significantly decreased throughout the striatum of wv/wv mice. The binding of [3H]mazindol to DA uptake sites was dramatically reduced in all wv/wv striatal regions except the ventrolateral portion. Compared to +/- littermates, wv/wv mice had far fewer fluorescent cell bodies in the substantia nigra and a less pronounced reduction of ventral tegmental area fluorescent somata. These findings support the hypothesis that heterogeneities exist in the genetic control of the mesotelencephalic DA system. The results underscore the usefulness of the weaver mouse in the study of mesostriatal sub-systems, receptor regulation, and potentially as a model of human neuropathologies that affect distinct populations of cells in the mesotelencephalic system.
Dopamine-sensitive adenylate cyclase and 3H-SCH 23390 binding parameters were measured in the rat substantia nigra and striatum 15 days after the injection of 6-hydroxydopamine into the medial forebrain bundle. The activity of nigral dopamine-sensitive adenylate cyclase and the binding of 3H-SCH 23390 to rat nigral D-1 dopamine receptors were markedly decreased after the lesion. On the contrary, 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway enhanced both adenylate cyclase activity and the density of 3H-SCH 23390 binding sites in striatal membrane preparations. The changes in 3H-SCH 23390 binding found in both nigral and striatal membrane preparations were associated with changes in the total number of binding sites with no modifications in their apparent affinity. The results indicate that: within the substantia nigra a fraction (30%) of D-1 dopamine receptors coupled to the adenylate cyclase is located on cell bodies and/or dendrites of dopaminergic neurons; striatal D-1 dopamine receptors are tonically innervated by nigrostriatal afferent fibers.
The effects of 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 38393) (D1 agonist) on the motor behaviour of mice rendered hypokinetic with reserpine, were studied in the absence and presence of additional treatment with N-n-propyl-N-phenylethyl-p(3-hydroxyphenyl)ethylamine hydrochloride (RU 24213), lisuride (D2 agonists) or apomorphine (mixed D1/D2 agonist). Three hours after reserpine (5 mg/kg) stimulating dopamine D2 receptors evoked slow, ponderous walking and head-down sniffing. SKF 38393 (1.5-15 mg/kg) had no direct effect of its own, but greatly amplified the D2 response, giving more fluent locomotion, rearing and grooming. The facilitatory action of SKF 38393 was inhibited by the D1 antagonist (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin l -7-ol (SCH 23390) (0.05 mg/kg), whereas D2-mediated responses were sensitive both to SCH 23390 and the D2 antagonist metoclopramide (0.5 mg/kg). Mice treated with reserpine for 24 h became more sensitive to the motor stimulant actions of all four agonists. SKF 38393 now promoted rapid locomotion, rearing and grooming directly. The effects of D2 stimulation were weak by comparison and often antagonistic (not synergistic) with those of the D1 agonist. Both sets of agonists were now attenuated only by their respective antagonists. Reserpine caused pronounced falls in the concentrations of dopamine, 5-hydroxytryptamine and noradrenaline in the striatum, olfactory tubercle and cerebral cortex, with correspondingly elevated metabolite levels. These results indicate that D1 and D2 agonists at doses that are relatively ineffective at stimulating behaviour when given in isolation 3 h after reserpine, interact when given together to partially restore locomotion, rearing and grooming. This interaction is not apparent 24 h post-reserpine, a time at which D1 and D2 agonists produce significant effects of their own.
Diurnal cycles for 8 ligand receptor pairs and choline acetyltransferase (ChAT) activity in 3 brain regions differed markedly in two rat lines, both of Wistar origin. Statistically significant differences between diurnal cycles in the two rat lines were found in the following parameters: 24 h means in 6 of 11 measurements, magnitude of cycle amplitudes, and phase position in 6 of 11 measurements, up to complete reversal of the acrophases in the case of ChAT activity in hippocampus. The importance of these findings--such major differences in two closely related rat lines--is obvious in any attempt to compare receptor-binding studies per se between laboratories using the same strain but not line, in studies of receptor rhythm characteristics, and in particular, for analysing the effects of brain-reactive drugs. While there are some reports on strain-dependency of cyclic functions, we are not aware that line-dependency has previously been described.
High-performance liquid chromatography with electrochemical detection was used to measure the concentrations of 3,4-dihydroxyphenylethylamine (dopamine), 5-hydroxytryptamine (5-HT), p-hydroxyphenylethanolamine (octopamine), alpha-methyl-p-tyrosine, and tryptophan in the cerebral ganglia of cockroaches (Periplaneta americana) after peripheral administration of alpha-methyl-p-tyrosine and alpha-methyltryptophan. In addition, the levels of dopamine, 5-HT, octopamine, alpha-methyl-p-tyrosine, and tryptophan were determined after injection of alpha-methyl-p-tyrosine, 6-hydroxydopamine, or 5,7-dihydroxytryptamine directly into the cerebral ganglia by means of microinjection needles. Peripheral administration of alpha-methyl-p-tyrosine (400-1,600 micrograms/insect) caused a reduction in dopamine and 5-HT concentrations in cockroach cerebral ganglia, although the reduction in dopamine concentrations was more pronounced. Peripheral injections of alpha-methyl-p-tyrosine also reduced octopamine levels in the cerebral ganglia. Peripheral injection of alpha-methyltryptophan (400-1,600 micrograms/insect) caused a marked reduction in 5-HT and tryptophan concentrations in cockroach cerebral ganglia without altering dopamine or octopamine concentrations. Central injections of alpha-methyl-p-tyrosine (80 micrograms/insect) reduced dopamine concentrations in the cerebral ganglia. However, neither 6-hydroxydopamine (20 micrograms/insect) nor 5,7-dihydroxytryptamine (20 micrograms/insect) caused reductions in amine levels when applied near or directly into the cerebral ganglia. The results suggest that specific lesions of aminergic neurons in insects by either 6-hydroxydopamine or 5,7-dihydroxytryptamine are impractical. The specific, long-lasting depletion of 5-HT by alpha-methyltryptophan suggests that this chemical may be useful in elucidating the functions of 5-HT in insects.
Two classes of compounds have been investigated as inhibitors of purified beef adrenal tyrosine hydroxylase. Among the aromatic amino acids tyrosine analogues were found to be most potent, particularly those having an α-methyl or 3-halogen substitution. Two normal metabolites, mono- and diiodo-tyrosine, were found to be very effective inhibitors. Inhibition by the amino acid analogues was shown to be competitive with the substrate. Catechols were also inhibitory, particularly 3,4-dihydroxy-phenylpropylacetamide (compound H 22/54). Inhibition by the latter was reversed by cofactor (tetrahydrofolate or DMPH4) but not by tyrosine.
Rats given unilateral injections of 6-OHDA along the course of the mesotelencephalic dopaminergic projection show impairments in contralateral sensorimotor functions from which they often recover. Such rats also display an enhanced sensitivity to DA receptor stimulants, e.g. apomorphine, as revealed by contralateral turning, and an increased binding of neuroleptic compounds (e.g. [3H]spiroperidol) to the denervated striatum. This research examines the relationship of these receptor changes to both behavioral supersensitivity and recovery of sensorimotor functions by quantifying the time course of each phenomenon after injury. The supersensitivity to apomorphine and the behavioral recovery developed with a similar time course after injury, being evident within 1.5-3 days and reaching nearly maximal levels by 2 weeks postoperatively. A significant increase in in vivo [3H]spiroperidol binding to the denervated striatum occurred by 4 days postoperatively, and the magnitude of this change increased linearly during the first postoperative month. In contrast, the in vitro binding of this ligand to membranes of the denervated striatum was not increased until 3 weeks after the lesion. The results suggest that a proliferation of DA receptors may contribute to the pharmacological supersensitivity and the recovery of function, and that these early receptor changes may be revealed with greater sensitivity using in vivo binding techniques.
The discovery that various states of sleep, rest, wakefulness and arousal in man can be correlated with specific forms of the electroencephalogram1 has led to intensive studies of these states, mostly in mammals2-5. Today it is generally accepted that circadian sleep-wakefulness cycles occur in mammals and birds2,3,6. Behavioural observations on sleep in moths have also been published7; many other invertebrates demonstrate rest/activity cycles8. Circadian sensitivity fluctuations in both central9 and peripheral10-15 components of the visual system of various nocturnal arthropod species have been demonstrated. We now report that long-term, extracellular, single-unit recordings from optomotor interneurones in the optic lobes of forager honey bees reveal an oscillation in their sensitivity to moving visual stimuli16, 17. The oscillation displays properties typical of a circadian rhythm6, 18. The sensitivity of the neurons is higher during the subjective day than during the subjective night. The locomotor activity of individual, fixed walking forager bees shows a similar circadian oscillation and is also higher during the subjective day. Visual and mechanical stimuli can act directly on the interneurones and restore their sensitivity during times of reduced neuronal responsiveness. A comparison with results available for mammals makes it likely that the neuronal phenomena presented here are correlates of the bee's circadian sleep-wakefulness rhythm.