Localization and quantification of 5-hydroxytryptophan and serotonin in the central nervous systems of Tritonia and Aplysia

Serotonin (5-hydroxytryptamine, 5-HT) plays a central role in several behaviors in marine molluscs and other species. In an effort to better understand the regulation of 5-HT synthesis, we used high performance liquid chromatography (HPLC) with electrochemical detection and immunohistochemistry to measure and map the distribution of the immediate precursor of 5-HT, 5-hydroxytryptophan (5-HTP), in two model opisthobranch molluscs, the nudibranch Tritonia diomedea and the anaspid Aplysia californica. HPLC measurements showed that 5-HTP is present at approximately the same level as the 5-HT metabolite, 5-hydroxyindolacetic acid (5-HIAA) but is more than 100 times lower in concentration than either 5-HT or dopamine in the same tissue. Specific 5-HTP immunoreactivity was colocalized with serotonin in both species. The overall intensity of 5-HTP immunoreactivity in individual ganglia agreed with HPLC measurements for those ganglia. The intensity of 5-HTP immunolabeling varied between cell types and was correlated with the intensity of 5-HT immunolabeling. In particular, differences in staining intensity were consistently seen among the three dorsal swim interneurons of the Tritonia swim central pattern generator circuit. Some nonserotonergic neurons also displayed low levels of 5-HTP immunolabeling that were above background levels. Together, these results support the notion that production of 5-HTP is a rate-limiting step in serotonin synthesis and suggest that there may be additional regulation that allows 5-HTP to accumulate to varying levels.     

Effects of castration and adrenalectomy on in vitro rates of tryptophan hydroxylation and levels of serotonin in microdissected brain nuclei of adult male rats

Rates of 5-hydroxytryptophan (5-HTP) synthesis and levels of serotonin (5-HT) were measured in microdissected brain nuclei following castration or adrenalectomy of adult male rats. Fourteen days following gonadectomy, 5-HTP synthesis decreased in the nucleus raphe dorsalis (DR) and nucleus centralis superior (NCS), while levels of 5-HT were unchanged in the 7 brain nuclei examined. Administration of testosterone to castrated rats not only did not reverse the castration-induced decrease in 5-HTP synthesis in the DR and NCS, but also decreased 5-HT synthesis in the nucleus amygdaloideus centralis (AGC) and the nucleus septalis lateralis (LS). Following administration of testosterone, 5-HT levels were unchanged. 10 days following bilateral adrenalectomy, 5-HTP synthesis increased in the NCS and the median eminence. Levels of 5-HT increased only in the median eminence. The increased 5-HTP synthesis and 5-HT levels following adrenalectomy were not reversed by corticosterone administration. In addition, these selective changes in 5-HT metabolism did not result from hormonal effects on the availability of tryptophan to the brain.We conclude that there are subsets of serotonergic neurons in rat central nervous system which respond uniquely to removal of the gonads and adrenals. Furthermore, the dissociation between serum and brain tryptophan concentrations and changes in rates of 5-HTP synthesis argue against tryptophan availability as being a primary determinant of 5-HT biosynthesis and for a direct endocrine central nervous system interaction with serotonergic neurons.     

Chronic SSRI Treatment Exacerbates Serotonin Deficiency in Humanized Tph2 Mutant Mice

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Changes in motor performance and rubral single unit activity in cats after microinjections of serotonin into the red nucleus area.

The serotonergic control exerted on the red nucleus (RN) was studied in unrestrained cats during the performance of a simple reaction time task which consisted of releasing a lever in response to an auditory go-signal. The effects of microinjections of serotonin-oxalate salt into the rubral area on the motor activity and on the firing of neurons recorded concomitantly in the red nucleus were investigated. Injections of serotonin (5-HT) (200-400 ng) into the red nucleus or its dorsal border induced subtle alterations in the conditioned motor performances but had no major effects on the spontaneous motor behavior. The changes in the conditioned motor output (an increase in the static force exerted on the lever and a speeding up of the lever release) are reminiscent of the facilitatory influence of serotonin on various motor reflexes previously reported. Changes in the neuronal activity were observed concomitantly with the effects on the motor output: 5-HT either enhanced or reduced the firing rate of the rubral neurons. These effects were apparently dependent on the discharge pattern of the neurons during the static motor activity. The results suggest that the serotonergic input to the red nucleus may participate in motor control by exerting a dual modulatory action on the activity of rubral neurons.     

Neuromodulation of the crab pyloric central pattern generator by serotonergic/cholinergic proprioceptive afferents

In the stomatogastric nervous system of the crab, Cancer borealis, a set of 4 serotonergic/cholinergic proprioceptive neurons, called gastropyloric receptor (GPR) cells, have effects on the pyloric motor pattern. In a semi-intact foregut preparation, the GPR cells are not activated by movements of the pyloric filter; instead they respond to the slower movements of the gastric mill (Katz et al., 1989). Thus, their activity is not synchronized to the pyloric motor pattern. However, when the GPR cells are stimulated in an in vitro preparation in a manner that resembles their normal firing pattern, they produce dramatic effects on the pyloric motor pattern. These effects include: (1) a prolonged increase in the pyloric cycle frequency, (2) a momentary pause in the motor pattern, (3) transient inhibition of some motor neurons, (4) strong excitation of other motor neurons, and (5) altered phase relationships of the different components of the motor pattern. These changes in the motor pattern are due to direct effects of the GPR cells on neurons in the pyloric central pattern generator (CPG). All of the cells in the pyloric circuit appear to receive GPR input. However, only 2 neurons receive detectable rapid nicotinic synaptic potentials. The other neurons receive only slower neuromodulatory input from GPR stimulation. The neuromodulatory effects include burst enhancement, plateau potential enhancement, excitation, and inhibition. These modulatory effects are largely mimicked by bath-applied serotonin (5-HT). Thus, primary sensory neurons can alter the production of motor patterns by a CPG through a phase-independent mechanism; these proprioceptors do not need to fire at a precise time in the cycle to be effective because their effects are mediated through the slower actions of the neuromodulator 5-HT.     

Inhibitory influence of GABA on central serotonergic transmission. Raphé nuclei as the neuroanatomical site of the GABAergic inhibition of cerebral serotonergic neurons

Acute injection of the gamma-aminobutyric acid (GABA) mimetics progabide, aminooxyacetic acid, gamma-acetylenic GABA and dipropylacetamide reduced 5-hydroxytryptophan (5-HTP) accumulation in serotonergic nerve terminal regions (prefrontal cortex, olfactory tubercle, septum, striatum, hypothalamus, hippocampus, substantia nigra, cerebellum and spinal cord) as well as in corresponding cell body areas (raphé dorsalis, medianus, pontis and magnus). This effect was antagonized by bicuculline. The inhibition of serotonin (5-HT) synthesis induced by a single progabide administration was accentuated on repeated treatment in the striatum, prefrontal cortex and cerebellum but was similar to that seen after acute treatment in the other areas. Local infusion of high concentrations of GABA or GABA mimetics into the striatum, septum or substantia nigra failed to modify 5-HTP accumulation in these areas. Cerebral hemitransection antagonized the ability of progabide (1200 mg/kg i.p.) to diminish 5-HTP accumulation in the striatum, hippocampus and prefrontal cortex. Intra-raphé dorsalis infusion of muscimol (0.1-100 ng) or GABA (1-100 micrograms) decreased 5-HT synthesis in the corresponding projection areas (e.g. striatum, substantia nigra, cortex) but not in the hippocampus or cerebellum. Conversely, intra-raphé medianus infusion of these drugs diminished 5-HTP accumulation in the corresponding projection areas (e.g. hippocampus, septum, cortex) but not in the striatum or cerebellum. Intra-raphé dorsalis or medianus injection of GABA antagonists (bicuculline, picrotoxinin, RU-5135) was without effect on cerebral 5-HT synthesis but antagonized the diminution of the amine synthesis observed in corresponding projection areas after intra-raphé dorsalis or medianus infusion of muscimol or GABA. These results suggest that GABA exerts an inhibitory (non-tonic) control over central serotonergic neurons which is mediated via GABA receptors located in the raphé nuclei.     

Involvement of serotonergic system in the antidepressant-like effect of piperine

Piperine is a major alkaloid of black pepper (Piper nigrum Linn.) and long pepper (P. longum Linn.), and its antidepressant-like effect has been previously demonstrated. The purpose of this study was to explore the possible contribution of the serotonergic system in the antidepressant-like effect of piperine in mice. The results showed that piperine significantly reduced the immobility time in the forced swim test and tail suspension test in mice. The anti-immobility effect of piperine in the forced swim test and tail suspension test was completely abolished by pre-treating the mice with pCPA (an inhibitor of 5-HT synthesis). Piperine treatment also significantly potentiated the number of head-twitches of mice induced by 5-HTP (a metabolic precursor to 5-HT). In addition, the neurochemical assays showed that piperine produced a marked increase of 5-HT level in both the hippocampus and frontal cortex of mice. Taken together, these results clearly suggest that the antidepressant-like effect of piperine is mediated via the serotonergic system by enhancing 5-HT content in mouse brain.     

Influence of PCPA and MDMA (ecstasy) on physiology, development and behavior in Drosophila melanogaster

The effects of para-chlorophenylalanine (PCPA) and 3,4 methylenedioxy-methamphetamine (MDMA, 'ecstasy') were investigated in relation to development, behavior and physiology in larval Drosophila. PCPA blocks the synthesis of serotonin (5-HT) and MDMA is known to deplete 5-HT in mammalian neurons; thus these studies were conducted primarily to target the serotonergic system. Treatment with PCPA and MDMA delayed time to pupation and eclosion. The developmental rate was investigated with a survival analysis statistical approach that is unique for Drosophila studies. Locomotion and eating were reduced in animals exposed to MDMA or PCPA. Sensitivity to exogenously applied 5-HT on an evoked sensory-central nervous system (CNS)-motor circuit showed that the CNS is sensitive to 5-HT but that when depleted of 5-HT by PCPA a decreased sensitivity occurred. A diet with MDMA produced an enhanced response to exogenous 5-HT on the central circuit. Larvae eating MDMA from the first to third instar did not show a reduction in 5-HT within the CNS; however, eating PCPA reduced 5-HT as well as dopamine content as measured by high performance liquid chromatography from larval brains. As the heart serves as a good bioindex of 5-HT exposure, it was used in larvae fed PCPA and MDMA but no significant effects occurred with exogenous 5-HT. In summary, the action of these pharmacological compounds altered larval behaviors and development. PCPA treatment changed the sensitivity in the CNS to 5-HT, suggesting that 5-HT receptor regulation is modulated by neural activity of the serotonergic neurons. The actions of acute MDMA exposure suggest a 5-HT agonist action or possible dumping of 5-HT from neurons.     

Relationship between serotonin and the immune system in a teleost model

An immunomodulatory role for serotonin (5-HT) has been demonstrated in mammals and evidence for a similar role for 5-HT has recently emerged in fish. However, as limited studies are available, discrepancies often exist regarding the role of 5-HT in the teleost immune response. Therefore, studies were undertaken to help clarify this relationship. Lymphocyte proliferation and extracellular superoxide (O2.-) production were examined in cells from bluegill sunfish injected with either 5-HTP (the immediate precursor to 5-HT) or pCPA (an inhibitor of the rate-limiting enzyme in 5-HT synthesis), or, in vitro following exposure of immune cells to either 5-HT, the 5-HT(1A) receptor agonist, 8-OH-DPAT, or the receptor antagonist, NAN-190. Exposure of fish to 5-HTP increased whole brain 5-HT levels, while pCPA exposure decreased whole brain and splenic 5-HT. In vivo exposure of fish to pCPA depressed T- and B-lymphocyte proliferation; exposure to 5-HTP failed to alter either immune endpoint. In vitro exposure of bluegill splenocytes to 5-HT or 8-OH-DPAT inhibited lymphoproliferation; treatment with NAN-190 had no effect on immune function. Results suggest a link in bluegill between immune function and the serotonergic system. The disparity observed following in vivo- and in vitro-induced serotonergic alterations indicates the complexity of this neuro-immune relationship and emphasizes the need for further studies in this regard.     

5-Hydroxytryptophan-induced cortisol response and CSF 5-HIAA in depressed patients

To determine if the enhanced cortisol response to oral administration of the serotonin (5-HT) precursor 5-hydroxytryptophan (5-HTP) that has been reported in unmedicated depressed and manic patients might be related to brain monoaminergic metabolism, the authors assessed correlations between 5-HTP-induced cortisol response and CSF in nine depressed patients. They found a significant negative correlation with CSF levels of 5-hydroxyindoleacetic acid, a 5-HT metabolite, but not with CSF levels of other monoamine metabolites. This finding is consistent with the hypothesis that low presynaptic brain serotonergic activity may be related to enhanced cortisol response to 5-HTP in depressed patients.     

Further studies on the activation of rat median raphe serotonergic neurons by inescapable sound stress

Previous studies, using a biochemical measure of serotonergic neuronal function, show that inescapable, randomly presented sound pulses activate serotonergic neurons in the rat median raphe but not dorsal raphe nucleus. The present study reveals that this activation also occurs in serotonin projection areas, in hippocampus, nucleus accumbens and cortex but not in caudate nucleus. The selectivity of this response is examined by comparing the response to sound stress with that produced by morphine, a treatment known to selectively activate dorsal raphe but not median raphe serotonergic neurons. Two approaches are used in Sprague-Dawley rat to measure the activation of serotonergic neurons: (1) determination ex vivo of accumulation of 5-hydroxytryptophan (5-HTP) in tissue from the dorsal and median raphe nuclei, hippocampus, cortex, caudate nucleus, and nucleus accumbens following in vivo inhibition of aromatic amino acid decarboxylase; and (2) measurement of extracellular serotonin levels in hippocampus, caudate nucleus, and nucleus accumbens. Sound stress increases 5-HTP accumulation in median raphe nucleus, hippocampus, cortex, and nucleus accumbens, but not dorsal raphe nucleus or caudate nucleus. Sound stress also enhances extracellular serotonin levels in hippocampus and nucleus accumbens, but not caudate nucleus. In contrast, the morphine treatment enhances 5-HTP accumulation in dorsal raphe nucleus, cortex and caudate nucleus, but not in median raphe nucleus, hippocampus or nucleus accumbens. Furthermore, it increases extracellular serotonin levels in only the caudate nucleus. The combined effects of sound stress and morphine on 5-HTP accumulation are identical to those obtained by each treatment individually. These findings provide further support for the presence of serotonergic neurons within the median raphe nucleus that have a unique response profile. These neurons may have an important role in responses or adaptations to stress.     

Alterations in alcohol consumption, withdrawal seizures, and monoamine transmission in rats treated with phentermine and 5-hydroxy-L-tryptophan

We have previously shown that coadministration of the dopamine (DA) agonist phentermine plus the serotonergic agonist fenfluramine suppresses alcohol intake and withdrawal seizures in rats. In the present study, phentermine and the serotonin (5-HT) precursor, 5-hydroxy-L-tryptophan (5-HTP), were administered alone, or in combination, to rats fed on a 6% alcohol-containing diet or an isocaloric control diet. Following a 9-h withdrawal period from the alcohol-containing diet, phentermine enhanced the effects of 5-HTP on both reduction of alcohol withdrawal seizures as well as changes in striatal serotonin. Food intake was monitored for 24 h after drug treatment, and neurochemical measures were examined at various time points. Phentermine alone reduced food intake in all diet conditions, but this anorectic effect was followed by hyperphagia in control rats. Phentermine plus 5-HTP reduced the consumption of the alcohol-containing diet, while its effects on consumption of control diets were mixed. In vivo microdialysis in rat nucleus accumbens revealed that phentermine increased extracellular DA, whereas 5-HTP caused marked elevations in extracellular 5-HT. Coadministration of phentermine and 5-HTP evoked simultaneous elevations in extracellular DA and 5-HT that mirrored the effects of each drug alone. Collectively, these findings show that coadministered phentermine plus 5-HTP is effective in reducing alcohol intake and suppressing alcohol withdrawal seizures. These therapeutic actions may be related to elevations in synaptic DA and 5-HT in critical brain regions.     

Aminergic modulation of graded synaptic transmission in the lobster stomatogastric ganglion

Graded chemical synaptic transmission is important for establishing the motor patterns produced by the pyloric central pattern generator (CPG) circuit of the lobster stomatogastric ganglion (Raper, 1979; Anderson and Barker, 1981; Graubard et al., 1983). We examined the modulatory effects of the amines dopamine (DA), serotonin (5-HT), and octopamine (Oct) on graded synaptic transmission at all the central chemical synapses made by the pyloric dilator (PD) neuron onto its follower cells, using synaptic input-output curves measured from cell somata. DA strongly reduced the graded synaptic strength at all the PD synapses. DA reduction of chemical synaptic strength from PD onto the inferior cardiac (IC) neuron could change the sign of synaptic interaction between these 2 cells from inhibitory to excitatory by uncovering a weak electrical connection. 5-HT had weaker and more variable effects, reducing graded synaptic strength from the PD onto the lateral pyloric and pyloric neurons and enhancing the weak synapse from the PD to the IC cell. Oct strongly enhanced the graded synaptic strength at all the PD central synapses. Oct enhancement of graded synaptic strength between the PD and IC cells could also change the sign of the interaction: weak, excitatory electrical coupling, which was sometimes dominant before Oct, was masked by the enhanced chemical inhibitory interaction during Oct application. Measurements of electrical coupling between 2 PD cells and between 2 postsynaptic cells suggest that Oct does not change the input resistance of these cells and may act directly at the PD synapses. The effects of DA and 5-HT are most easily explained by their general reductions in pre- and postsynaptic input resistance. DA, 5-HT, and Oct each produce a distinct pyloric motor pattern (Flamm and Harris-Warrick, 1986a). These amine-induced motor patterns may be explained by the unique actions of each amine on the intrinsic membrane properties of different pyloric CPG neurons (Flamm and Harris-Warrick, 1986b) and by modulation of graded synaptic transmission between the pyloric neurons.     

Serotonin, via HTR2 receptors, excites neurons in a cortical-like premotor nucleus necessary for song learning and production

Serotonin (5-HT) is a neuromodulator that is important for neural development, learning and memory, mood, and perception. Dysfunction of the serotonin system is central to depression and other clinically important mood disorders and has been linked with learning deficits. In mammals, 5-HT release from the raphe nuclei in the brainstem can modulate the functional properties of cortical neurons, influencing sensory and motor processing. Birds also have serotonergic neurons in the dorsal raphe, suggesting that 5-HT plays similar roles in sensory and motor processing, perhaps modulating brain circuitry underlying birdsong. To investigate this possibility, we measured the effects of 5-HT on spontaneous firing of projection neurons in the premotor robust nucleus of the arcopallium in brain slices from male zebra finches. These neurons are thought be akin to cortical layer V pyramidal neurons. 5-HT dramatically and reversibly enhanced the endogenous firing of RA neurons. Using pharmacological agonists and antagonists in vitro, we determined this action is mediated via HTR2 receptors, which we verified are expressed by in situ hybridization. Finally, focal administration of the serotonin selective reuptake inhibitor fluvoxamine revealed that endogenous 5-HT is sufficient to mediate this effect in vivo. These findings reveal a modulatory action of serotonin on the physiology of the song system circuitry and suggest a novel role of serotonin in regulating song production and/or learning; further understanding of the role of 5-HT in this system may help illuminate the complex role of this neuromodulator in social interactions and motor plasticity in humans.     

The Expression of Brain-Derived Neurotrophic Factor and Tryptophan Hydroxylase in the Dorsal Raphe Nucleus during Repeated Stress

The mechanisms of development of stress-induced depression symptoms may include interaction between the elements of the serotonergic (5-HT) system and brain-derived neurotrophic factor (BDNF) at the level of the cell bodies of 5-HT neurons. In order to evaluate this situation, we used immunofluorescent staining of BDNF protein and the enzyme of serotonin synthesis tryptophan hydroxylase (TPH) in the dorsal raphe nucleus (DRN) of adult male rats that were subjected daily to a 15-min forced swim stress for 1, 2, 7, or 14 days. The expression of the BDNF protein was substantially decreased in the DRN after 7- or 14-day stress, whereas the expression of the TPH protein significantly increased after the 14-day stress and did not differ from the control after the shorter duration of repeated stress. The absence of correlation between some indices indicates their independence from each other at the level of the cell bodies of 5-HT neurons.     

Neuroendocrine responses to serotonergic agents in alcoholics.

Previous studies have suggested a possible deficit in serotonergic function in alcoholism. In order to further assess the serotonergic system in alcoholism, the plasma cortisol and prolactin (PRL) responses following 6-chloro-2-[1-piperazinyl]pyrazine (MK-212), a direct-acting serotonin2 (5-HT2)/5-HT1c receptor agonist, L-5-hydroxytryptophan (L-5-HTP), a precursor of 5-HT, and placebo were compared in male alcoholics and normal controls. The increase in plasma cortisol following L-5-HTP was significantly lower in the alcoholic subjects compared with the normal controls. The plasma PRL, but not the plasma cortisol response, following MK-212 was also significantly lower in the alcoholics. L-5-HTP had no significant effect on plasma PRL levels in either group. The basal plasma cortisol and PRL concentrations of the alcoholics and normal controls were not significantly different. These data are consistent with previous reports of a serotonergic abnormality in alcoholism.     

In vivo and in vitro studies on the effect of the serotoninergic system on luteinizing hormone and luteinizing hormone-releasing hormone secretion in prepubertal and peripubertal female rats

The present investigations were designed to assess the effect of the serotoninergic system on luteinizing hormone (LH) and LH-releasing hormone (LH-RH) secretion in female rats aged 14 and 30 days. The administration of 5-hydroxytryptophan (5-HTP; 75 mg/kg i.p.) increased hypothalamic serotonin (5-HT) concentrations in both age groups, and did not affect hypothalamic norepinephrine (NE) concentrations or release. Serum LH levels were raised by 5-HTP in 14-day-old, but not in 30-day-old rats. Basal and KCl- (28 mM) stimulated LH-RH release by incubated hypothalamic fragments was significantly enhanced when 5-HTP was injected previously to 14-day-old animals. In 30-day-old rats, 5-HTP treatment did not modify basal LH-RH release, and decreased the KCl-stimulated LH-RH output. Similarly, the addition of 5-HT (10(-7) M) to superfused hypothalamic fragments enhanced basal LH-RH release in 14-day-old rats and blocked the increment in LH-RH release evoked by KCl in 30-day-old rats. The present results show that in 14-day-old female rats, the serotoninergic system (activated in vivo by 5-HTP treatment, or in vitro by 5-HT addition) exerts a stimulatory effect on LH-RH, and thus, on LH release. On the contrary, in 30-day-old animals, stimulated LH-RH secretion was inhibited by 5-HT. Apparently, the hypothalamic NE system is not implicated in this response. The participation of this changing effect of 5-HT on LH-RH/LH release at the onset of puberty is postulated.     

Cellular effects of swim stress in the dorsal raphe nucleus

Swim stress regulates forebrain 5-hydroxytryptamine (5-HT) release in a complex manner and its effects are initiated in the serotonergic dorsal raphe nucleus (DRN). The purpose of this study was to examine the effects of swim stress on the physiology of DRN neurons in conjunction with 5-HT immunohistochemistry. Basic membrane properties, 5-HT(1A) and 5-HT(1B) receptor-mediated responses and glutamatergic excitatory postsynaptic currents (EPSCs) were measured using whole-cell patch clamp techniques. Rats were forced to swim for 15min and 24h later DRN brain slices were prepared for electrophysiology. Swim stress altered the resting membrane potential, input resistance and action potential duration of DRN neurons in a neurochemical-specific manner. Swim stress selectively elevated glutamate EPSC frequency in 5-HT DRN neurons. Swim stress non-selectively reduced EPSC amplitude in all DRN cells. Swim stress elevated the 5-HT(1B) receptor-mediated inhibition of glutamatergic synaptic activity that selectively targeted 5-HT cells. Non-5-HT DRN neurons appeared to be particularly responsive to the effects of a milder handling stress. Handling elevated EPSC frequency, reduced EPSC decay time and enhanced a 5-HT(1B) receptor-mediated inhibition of mEPSC frequency selectively in non-5-HT DRN cells. These results indicate that swim stress has both direct, i.e., changes in membrane characteristics, and indirect effects, i.e., via glutamatergic afferents, on DRN neurons. These results also indicate that there are distinct local glutamatergic afferents to neurochemically specific populations of DRN neurons, and furthermore that these distinct afferents are differentially regulated by swim stress. These cellular changes may contribute to the complex effects of swim stress on 5-HT neurotransmission and/or the behavioral changes underlying the forced swimming test model of depression.     

Biosynthesis of dopamine and serotonin in the rat brain after repeated cocaine injections: A microdissection mapping study

The purpose of the present study was to examine the effects of chronic cocaine on dopamine (DA) and serotonin (5-HT) synthesis in several rat brain regions implicated in drug reinforcement. Male rats were treated twice daily with cocaine (15 mg/kg, ip) or saline for 1 week. After 42 hr of abstinence, rats were challenged with either cocaine (15 mg/kg, ip) or saline, followed by the aromatic L-amino acid decarboxylase inhibitor 3-hydroxybenzylhydrazine (NSD-1015; 100 mg/kg, ip). Animals were decapitated 30 min after NSD-1015 and discrete brain regions were microdissected from 300 μm frozen sections. Postmortem tissue levels of 3, 4-dihydroxyphenylalanine (DOPA) and 5-hyroxytryptophan (5-HTP) were quantified by HPLC with electrochemical detection and used to estimate biosynthesis of DA and 5-HT, respectively. In chronic saline-treated rats, cocaine dramatically suppressed DA and 5-HT synthesis in all forebrain regions examined, including: medial prefrontal cortex, nucleus accumbens, caudate nucleus, olfactory tubercle, and basolateral amygdala. The degree of inhibition ranged from 35-65% and was more pronounced in 5-HT neurons compared to DA neurons in the same tissue sample. In general, chronic cocaine did not significantly alter basal levels of DOPA or 5-HTP; a notable exception was lateral hypothalamus, where chronic cocaine reduced basal DA synthesis to 75% of control. After repeated cocaine injections, the synthesisiinhibiting effect of a challenge injection of cocaine was attenuated in many brain areas. These data suggest that whereas acute cocaine decreases DA and 5-HT synthesis in forebrain, chronic cocaine is not neurotoxic to DA and 5-HT neurons. In addition, the mechanism(s) mediating cocaine-induced suppression of monoamine synthesis may become desensitized by chronic exposure to the drug. Published 1993 Wiley-Liss, Inc.     

Influence of inhibitory and excitatory inputs on serotonin efflux differs in the dorsal and median raphe nuclei

The dorsal (DRN) and median raphe nuclei (MRN) are two major sources of serotonergic projections to forebrain that are involved in regulation of behavioral state and motor activity, and implicated in affective disorders such as depression and schizophrenia. To investigate afferent influences on serotonergic neurons, this study compared the role of endogenous GABA and glutamate in the DRN and MRN using microdialysis and measurement of locomotor activity in freely behaving rats. Local infusion of the GABA(A) receptor antagonist bicuculline increased serotonin (5-HT) efflux in the DRN but not the MRN. In contrast, infusion of glutamate receptor antagonists produced larger decreases in 5-HT efflux in the MRN compared with the DRN. Moreover, glutamate receptor antagonists attenuated the increase in 5-HT efflux produced by GABA receptor blockade in the DRN. Thus, the disinhibitory effect of GABA blockers could be ascribed in part to an enhanced influence of glutamate. Measurements of locomotor activity indicate that changes in 5-HT were not simply correlated with behavioral activity induced by drug infusion. In summary, the role of inhibitory and excitatory afferents was strikingly different in the DRN and MRN. GABA afferents were the predominant tonic influence on serotonergic neurons in the DRN. In contrast, glutamatergic but not GABAergic afferents had a strong tonic influence on serotonergic neurons in the MRN.