Ipsilateral alterations in tryptophan hydroxylase activity in rat brain after hypothalamic 5,7-di-hydroxytryptamine lesion

The in vivo relationship between the amounts of tryptophan hydroxylase (TPH) protein and its intrinsic synthetic activity, measured by quantifying the amounts of α-[³H]methyl-5-hydroxytryptamine (α-[³H]M5-HT), is reported in cell body and terminal areas of intact and disturbed serotonergic neurons following a unilateral 5,7-dihydroxytryptamine (5,7-DHT) lesion of the dorsolateral hypothalamus. Five days after the lesion, the relationships between TPH and its synthetic product 5-HT were evaluated on adjacent brain sections in serotonergic cells bodies of the dorsal raphe nucleus (DRN) and nerve fibres of the medial forebrain bundle (MFB). On the side contralateral to the lesion, TPH and α-[³H]M5-HT levels in the intact hemi-DRN exhibited a caudo-rostral distribution and were positively and significantly correlated (P 0.001); the calculated TPH-specific activity was 0.76 nCi of α-[³H]M5-HT formed per U TPH. In the MFB, quantitative measurements of TPH and α-[³H]M5-HT showed no correlation between enzyme and product and no specific activity for TPH could be determined. On the side ipsilateral to the lesion, the density of TPH-immunoreactive fibers was drastically decreased in the dorsolateral hypothalamus where a significant reduction in TPH content (45.5% of control side,P < 0.001) was found. In the overall ipsilateral hemi-DRN, TPH and α-[³H]M5-HT levels, their correlation as well as TPH-specific activity were unaltered by the lesion but a significant increase in α-[³H]M5-HT and TPH contents was observed in the lateral wings of the DRN. The lesion also induced a significant increase in α-[³H]M5-HT and TPH levels (136% and 93.8%,P < 0.001, respectively) in the ipsilateral MFB, which resulted in a positive and significant correlation between these two markers and yielded a TPH-specific activity of 1.0 nCi of α-[³H]M5-HT formed per U TPH. TPH topological area was also significantly increased in the lateral aspect of the ipsilateral MFB 5 days post lesion. These results show that 5-HT synthesis in the intact DRN is proportional to and dependent on TPH activity while in the MFB, 5-HT accumulation appears unrelated to TPH content which is most likely in an inactive enzymatic form. Moreover, the data show that a local disruption of serotonergic terminals in the dorsolateral hypothalamus does not affect 5-HT synthesis in the overall ipsilateral DRN neurons but results in local activation of TPH within the serotonergic projection neurons and the ipsilateral MFB, as evidenced by active de novo synthesis of 5-HT. Altogether the results point to circumscribed activation of compensatory mechanisms in 5-HT synthesis after selective destruction of serotonergic terminals.     

Uptake of [H]serotonin in the abdominal ganglion ofAplysia california. Further studies on the morphological and biochemical basis of presynaptic facilitation

Sensitization of the gill-withdrawal reflex inAplysia california is mediated, in part, by a group of identified neurons, the L29 cells, which produce presynaptic facilitation of transmitter release from siphon sensory neurons. Physiological and pharmacological studies have provided indirect evidence that the L29 cells are serotonergic. In the present study we have used the specific uptake [³H]serotonin ([³H]5-HT) and electron-microscopic autoradiography in combination with horseradish peroxidase-labeling of identified neurons to characterize the fine structure ofAplysia serotonergic terminals and to examine more directly the transmitter biochemistry of the L29 neurons. Abdominal ganglia were incubated for 2 h in 10⁻⁶ M [³H]5-HT and thick and thin plastic sections examined with the light and electron microscope. L29 varicosities, identified by labeling with HRP, were found to accumulate [³H]5-HT. In addition, [³H]5-HT was localized to unidentified varicosities within the neuropil as well as to vesicle-filled terminals that formed axosomatic contacts in the cortical regions of the ganglion. The processes that accumulated [³H]5-HT contained conspicuous dense core vesicles identical in morphology to those previously described for L29. Some processes were found to make contact with HRP-labeled varicosities of sensory neurons. Comparison with results obtained from ganglia exposed to [³H]5-HT in the presence of either non-radioactive 5-HT or non-radioactive dopamine indicate that the iptake process is transmitter-specific. These studies provide additional evidence that the L29 cells are serotonergic and are consistent with the notion that aminergic neurons may be preferentially involved in modulatory synaptic actions.     

Stimulation of the rat dorsal raphe in vivo releases labeled serotonin from the parietal cortex

In vivo release of labeled serotonin ([³H]5-HT) from the parietal cortex was investigated by cortical cup technique and electrical stimulation of midbrain raphe in rats anesthetized with pentobarbital sodium. The spontaneous efflux of tritium from the parietal cortex preloaded with [³H]5-HT followed a multiphasic exponential course. After 120 min, the rate of efflux appeared to fit the single exponential function (slow phase). Imipramine (10⁻⁶−10⁻³ M) produced a dose-dependent increase in the spontaneous release. When pargyline in concentrations ranging from 10⁻⁴ to 10⁻³ M were added to the medium in the cup, the unchanged [³H]5-HT significantly increased in a dose-dependent manner and the slow declining coefficient of tritium efflux significantly decreased in the presence of 10⁻⁴ pargyline. Stimulation of the rostral two-thirds of the dorsal raphe and the lateral 5-HT bundle originating from the dorsal raphe significantly increased the release of [³H]5-HT and its metabolites while stimulation of the caudal one-third of the dorsal raphe did not produce a significant increase in the release of [³H]5-HT and its metabolites. Stimulation of the median raphe produced no or only a slight increase in the release of [³H]5-HT and its metabolites. These findings are a direct demonstration of the in vivo release of [³H]5-HT from the parietal cortex with stimulation of the dorsal raphe, particularly the rostral two-thirds of the nucleus and provide the neurochemical evidence for the dorsal raphe-cortical 5-HT pathway via the lateral 5-HT bundle.     

Retrograde axonal transport following injection of [H]serotonin in the olfactory bulb. I. Biochemical study

A retrograde axonal transport from the serotonergic nerve terminals in the olfactory bulb (OB) to their parent cell bodies in the midbrain raphe nuclei has been demonstrated after stereotaxic injection of [³H]5-HT into the OB of rats pretreated with a monoamine oxidase (MAO) inhibitor: at various time intervals thereafter (4–92 h) there was a preferential accumulation of radioactivity mainly in the raphe dorsalis nucleus (RDN). Maximal accumulation occurred at 24 h. Of this radioactivity, 30–50% was recovered as 5-HT. The accumulation was estimated to take place at two rates: a fast one (48 mm/day) and a slower one (16 mm/day). Under the same experimental conditions there was no clear evidence for a retrograde accumulation of [³H]norepinephrine in the RDN.A passive diffusion mechanism could be excluded since the diffusion of tracer towards the cerebrospinal fluid was prevented by prior mechanical obstruction of the olfactory diverticle of the lateral ventricle. Furthermore, colchicine strongly reduced (by 80%) the radioactive accumulation in the RDN.Destruction of serotonergic nerve terminals by 5,6-dihydroxytryptamine or inhibition of 5-HT uptake by fluoxetine decreased this retrograde accumulation whereas destruction of catecholaminergic nerve terminals by 6-hydroxydopamine was without effect. Pretreatment with reserpine decreased the amount of radioactivity transported to the RDN by 40%.In the absence of MAO inhibition pretreatment, animals still presented 35% of the tracer transported to the RDN. Intrabulbar injection of MAO inhibitor did not affect the accumulation rates when compared with animals which received the inhibitor by the intraperitoneal route.     

Stimulant-induced alterations in dopaminergic and serotonergic function in fetal raphe neurons

Methamphetamine and its structural analogues have been demonstrated to be neurotoxic to CNS dopamine (DA) and serotonin (5-HT) neurons both in vivo and in vitro. Our laboratory has been actively characterizing mesencephalic cultures and the effects of methamphetamine exposure on neurochemical and immunochemical indices. The purpose of the present studies was to extend our findings with mesencephalic cultures and compare them with methamphetamine-induced alterations in fetal raphe cultures that contain both DA and 5-HT cells. Methamphetamine (10 and 100 μM) was added to the cultures 24 h after plating and fresh daily thereafter. The effects of chronic methamphetamine exposure on [³H]-DA and [³H]-5-HT uptake were determined after 5 days of drug treatment. Additional cultures were immunochemically analyzed for the presence of DA- and 5-HT-containing cells and total neuronal density. Results indicate that repeated methamphetamine exposure decreased DA and 5-HT uptake. Furthermore, repeated exposure to the higher concentration of methamphetamine (100 μM) caused a significant reduction in the number of DA and 5-HT cells as well as reducing the total neuronal density. This would suggest that this higher concentration of methamphetamine results in generalized neurotoxicity. Exposure to 10 μM methamphetamine resulted in more specific effects on dopaminergic function. These findings indicate that repeated methamphetamine administration can induce similar alterations in both dopaminergic and serotonergic neurons in raphe cultures.     

Effect of nerve activity on the in vivo release of [H]serotonin continuously formed from l-[H]tryptophan in the caudate nucleus of the cat

A new isotopic approach has been developed to study the in vivo release of serotonin (5-HT). ‘Ence´phale isole´cats were implanted with a push-pull cannula in the ventrocaudal part of the head of the caudate nucleus to estimate the release of [³H]5-HT continuously synthesized froml-[³H]tryptophan. Both [³H]5-HT and [³H]tryptamine were found in superfusates. Resting steady state in the release of [³H]indoleamines was observed as soon as 20 min after the beginning of the superfusion withl-[³H]tryptophan; the levels of [³H]5-HT in superfusates were 2.5 times those of [³H]tryptamine and about 6 times the blank value. They were markedly enhanced in the presence of fluoxetine (5 × 10^-6M), a blocker of the 5-HT uptake process.A marked increase in the release of [³H]5-HT was seen during the local depolarization of 5-HT terminals with potassium chloride (60 mM) or batrachotoxin (10^-6M) or during the stimulation of 5-HT cell bodies in the nucleus raphe dorsalis withl-glutamic acid (5 × 10^-5M). These treatments did not enhance the efflux of [³H]tryptamine. The potassium-evoked release of [³H]5-HT was reduced by LSD (10^-5M). LSD added alone in the superfusing fluid was without effect. The batrachotoxin-evoked release of [³H]5-HT was inhibited in the presence of tetrodotoxin (9 × 10^-6M).The spontaneous release of [³H]5-HT and [³H]tryptamine was markedly reduced in the presence of a calcium-free medium containing cobalt (10 mM). A transient slight reduction in the spontaneous release of [³H]5-HT was observed in the presence of tetrodotoxin (9 × 10^-6M). The local cooling of 5-HT cell bodies with a cryoelectrode induced a slight reversible decrease in [³H]5-HT release. These last two treatments were without significant effect on [³H]tryptamine efflux in superfusates.     

The serotonin neurons in nucleus raphe dorsalis of adult rat: A light and electron microscope radioautographic study

The serotoninergic nerve cell body population of nucleus raphe dorsalis (RD) was identified by radioautography following cerebroventricular instillation of tritiated serotonin ([³H]5-HT) in adult rats pretreated with a monoamine oxidase inhibitor. Series of histological sections taken throughout the midbrain and upper pons exhibited a similar distribution and number of labeled nerve cell bodies in RD after prolonged administration ofeither 10^?5 or 10^?4 M [³H]5-HT or 10^?4 M [³H]5-HT and 10^?3 M nonradioactive noradrenaline. This allowed systematic mapping and quantification of serotoninergic nerve cell bodies at various levels of the RD. Their extrapolated total number averaged 11,500. Twice as many unreactive (nonserotoninergic) neurons were present within the same region. In electron microscope radioautographs, the labeled cells were usually larger (17.9 μm mean diameter) than their unlabeled congeners (13.1 μm), but stereological sampling of their perikarial organelle content failed to reveal any difference in cytoplasmic composition. Few [³H]5-HT-labeled axonal varicosities were observed in RD and none were found in close apposition or in synaptic junction with labeled nerve cell bodies, dendrites, or unreactive perikarya. A detailed statistical analysis of silver grain distribution in both labeled and “unlabeled” nerve cell bodies indicated that in the former, but not in the latter, dense bodies had a relatively high affinity for [³H]5-HT. Mitochondria and the cytoplasmic membrane were the only other organelles to show higher labeling indices in labeled than in unlabeled cells. Other sites of[³H]5-HT localization could be ascribed to artefactitious cross-linkage of the tracer by the fixative, since they had the same relative affinity in the two cell populations. These results provide new insights into the morphology and cytofunctional properties of the 5-HT neurons of rat RD.     

Basal and stimulated extracellular serotonin concentration in the brain of rats with altered serotonin uptake

We examined the relationship between the density of serotonergic (5-hydroxytryptamine [5-HT]) uptake sites and extracellular 5-HT concentration in the rat brain using microdialysis with two different models, lesions with 5,7-dihydroxytryptamine (50 μg in the dorsal raphe nucleus (DRN) 15 days before) and sublines of rats genetically selected displaying extreme values of platelet 5-HT uptake. Compared to controls, lesioned rats had a reduced cortical concentration of 5-hydroxyindoles (45%), unchanged basal extracellular 5-HT in the DRN and ventral hippocampus (VHPC), and reduced basal 5-hydroxyindoleacetic acid (5-HIAA) concentrations (46%, DRN; 22%, VHPC). Yet the perfusion of 100 mmol/L KCl or 1 μmol/L citalopram elevated dialysate 5-HT significantly more in the DRN and VHPC of controls. In genetically selected rats, platelet 5-HT content and uptake were highly correlated (r² = 0.9145). Baseline dialysate 5-HT (VHPC) was not different between high and low 5-HT rats and from normal Wistar rats. However, KCl or citalopram perfusion increased dialysate 5-HT significantly more in high 5-HT than in low 5-HT rats, and the former displayed a greater in vivo tissue 5-HT recovery. Significant but small differences in the same direction were noted in [³H]citalopram binding in several brain areas, as measured autoradiographically. Thus, basal extracellular 5-HT (but not 5-HIAA) concentrations are largely independent on the density of serotonergic innervation and associated changes in uptake sites. However, marked differences emerge during axonal depolarization or reuptake blockade. The significance of these findings for the treatment of mood disorders in patients with neurological disorders is discussed. Synapse 28:313–321, 1998. © 1998 Wiley-Liss, Inc.     

Serotonergic retinopetal projections from the dorsal raphe nucleus in the mouse demonstrated by combined [H] 5-HT retrograde tracing and immunolabeling of endogenous 5-HT

The present study demonstrated a direct serotonergic retinopetal projection in the mouse stemming from the lateral portion of the dorsal raphe nucleus bilaterally. A double-labeling technique was employed combining: (1) radioautography and retrograde axonal tracing following intraocular injection of [³H] 5-HT and (2) immunocytochemical identification of endogenous 5-HT. Radiolabeled neurons were only observed within the dorsal raphe nucleus and were always double-labeled with the 5-HT antibody. The radiolabeling appeared to be specific resulting from the retrograde transport of a radioactive 5-HT derivative product following uptake of the neurotransmitter by intraretinal terminals.     

Raphe serotonergic neurons projecting to the olfactory bulb contain galanin or somatostatin but not neurotensin

Retrograde axonal transport with [3H]5-HT has been developed as a specific tracing technique to identify serotonergic projections. This method, in combination with immunocytochemistry, offers considerable advantage of specificity and sensitivity to study pathways of multitransmitter-containing neurons. In this work, we studied the presence of galanin, somatostatin, and neurotensin in serotonergic neurons of dorsal and median raphe, which project to the olfactory bulbs. After [3H]5-HT injections into the rat olfactory bulbs, double galanin-immunoreactive and [3H]5-HT radiolabelled cells were located in the dorsal, lateral, and ventral region of dorsal raphe, but they were never seen in the median raphe. In the dorsal raphe, galanin-radiolabelled neurons represented 28% of the total number of radiolabelled cells. Double somatostatin-immunoreactive and radiolabelled neurons were located in the dorsal and median raphe. In the dorsal raphe, double somatostatin-radiolabelled neurons represented only 11% of the radiolabelled cells and they were mainly located ventral to the aqueduct. In the median raphe, 15% of radiolabelled cells were also immunopositive for somatostatin. In contrast, neurotensin-immunoreactive cells in the dorsal and median raphe were distributed among [3H]5-HT radiolabelled neurons but they were never radiolabelled. Our results show subpopulations of serotonergic raphe-olfactory bulb projection neurons containing either galanin or somatostatin, but not neurotensin.     

3H]-serotonin retrograde labelling in serotonergic fibers

Autoradiography, following retrograde axonal transport with [3H]-serotonin (10(-4) M) was used to identify, selectively, the serotonergic neurons of medullary raphe that innervate the spinal cord. In combination with this technique, immunocytochemical detection of endogenous serotonin showed some raphe magnus cell bodies immunostained by 5-HT antibodies and [3H]5-HT radiolabelled. Serotonergic fibers of lateral and dorsolateral funiculus of ligated spinal cord were also characterized following retrograde axonal transport with 10(-4) M [3H]-serotonin. At the ultrastructural level, mainly myelinated, although unmyelinated labelled fibers were also identified as swollen axons. Clusters of large membranous organelles, predominantly mitochondria, some dense lamellar bodies, multivesicular bodies and lysosomes accumulated on the distal segment of the ligation. Autoradiographic reaction was always observed to be very intense and silver grains overlapped mainly mitochondria (41%) and clusters of membranous organelles (37%). However, smooth endoplasmic reticulum seemed to be devoid of silver grains. From our results, serotonergic myelinated spinal fibers were labelled after uptake-retrograde axonal transport with [3H]5-HT. Mitochondria and membrane organelles could convey tritiated compounds derived from [3H]-serotonin uptake towards the serotonergic cell bodies.     

Uptake of [3H]serotonin in the abdominal ganglion of Aplysia californica. Further studies on the morphological and biochemical basis of presynaptic facilitation

Sensitization of the gill-withdrawal reflex in Aplysia california is mediated, in part, by a group of identified neurons, the L29 cells, which produce presynaptic facilitation of transmitter release from siphon sensory neurons. Physiological and pharmacological studies have provided indirect evidence that the L29 cells are serotonergic. In the present study we have used the specific uptake [3H]serotonin ([3H]5-HT) and electron-microscopic autoradiography in combination with horseradish peroxidase-labeling of identified neurons to characterize the fine structure of Aplysia serotonergic terminals and to examine more directly the transmitter biochemistry of the L29 neurons. Abdominal ganglia were incubated for 2 h in 10(-6) M [3H]5-HT and thick and thin plastic sections examined with the light and electron microscope. L29 varicosities, identified by labeling with HRP, were found to accumulate [3H]5-HT. In addition, [3H]-5-HT was localized to unidentified varicosities within the neuropil as well as to vesicle-filled terminals that formed axosomatic contacts in the cortical regions of the ganglion. The processes that accumulated [3H]5-HT contained conspicuous dense core vesicles identical in morphology to those previously described for L29. Some processes were found to make contact with HRP-labeled varicosities of sensory neurons. Comparison with results obtained from ganglia exposed to [3H]5-HT in the presence of either non-radioactive 5-HT or non-radioactive dopamine indicate that the uptake process is transmitter-specific. These studies provide additional evidence that the L29 cells are serotonergic and are consistent with the notion that aminergic neurons may be preferentially involved in modulatory synaptic actions.     

Retrograde axonal transport following injection of [H]-serotonin into the olfactory bulb. II. Radioautographic study

Radioautography was used to study the intraneuronal distribution of [³H]-serotonin (5-HT) and/or its derivatives selectively taken up by the olfactory bulb (OB) serotonergic terminals and subsequently transported to their parent cell bodies in the midbrain raphe nuclei. This was done 24 h after injection of [³H]5-HT into the main OB of rats either pretreated or not with monoamine oxidase (MAO) inhibitor. A prior mechanical obstruction of the rostral ventricular cavities prevented diffusion of the tracer towards cerebrospinal fluid.Heavily labelled nerve cell bodies were found mainly in the ipsilateral raphe dorsalis nucleus (RDN) and to a lesser extent in the raphe centralis nucleus. The radio-autographic reaction often extended to dendritic processes while sparing the nucleus. A diffuse reaction was also observed but limited to the raphe area. The supraependymal 5-HT fibers were found to be free of labelling.Neither local destruction of catecholaminergic terminals with 6-OHDA, nor absence of MAO inhibition, impaired this radioautographic pattern, while destruction of serotonergic terminals with 5,6-dihydroxytryptamine in OB resulted in the disappearance of labelled axonal varicosities and neurons in the OB and the RDN respectively.At the electron microscope level, labelled cell bodies in the RDN were medium-sized (12–15 μm). Silver grains were localized mainly on mitochondria and, to a lesser extent, on lysosomes and endoplasmic reticulum but spared the nucleus and the nucleolus. Silver grains were also found near the nuclear membran and outside the neuronal membrane. The observation of heavy metal impregnated thich sections confirmed the preferential localization of silver grains on mitochondria with or without inhibition of MAO.These resuls could account for the subcellular compartments involved in the retrograde axonal transport of [³H]-5-HT and its subsequent degradation and/or dendritic release.     

Retrograde axonal transport following injection of [3H]serotonin in the olfactory bulb. I. Biochemical study

A retrograde axonal transport from the serotonergic nerve terminals in the olfactory bulb (OB) to their parent cell bodies in the midbrain raphe nuclei has been demonstrated after stereotaxic injection of [2H]5-HT into the OB of rats pretreated with a monoamine oxidase (MAO) inhibitor: at various time intervals thereafter (4-92 h) there was a preferential accumulation of radioactivity mainly in the raphe dorsalis nucleus (RDN). Maximal accumulation occurred at 24 h. Of this radioactivity, 30-50% was recovered as 5-HT. The accumulation was estimated to take place at two rates: a fast one (48 mm/day) and a slower one (16 mm/day). Under the same experimental conditions there was no clear evidence for a retrograde accumulation of [3H]norepinephrine in the RDN. A passive diffusion mechanism could be excluded since the diffuson of tracer towards the cerebrospinal fluid was prevented by prior mechanical obstruction of the olfactory diverticle of the lateral ventricle. Furthermore, colchicine strongly reduced (by 80%) the radioactive accumulatin in the RDN. Destruction of serotonergic nerve terminals by 5,6-dihydroxytryptamine or inhibiton of 5-HT uptake by fluoxetine decreased this retrograde accumulation whereas destruction of catecholaminergic nerve terminals by 6-hydroxydopamine was without effect. Pretreatment with reserpine decreased the amount of radioactivity transported to the RDN by 40%. In the absence of MAO inhibition pretreatment, animals still presentd 35% of the tracer transported to the RDN. Intrabulbar injection of MAO inhibitor did not affect the accumulation rates when compared with animals which received the inhibitor by the intraperitoneal route. In conclusion, the retrograde axonal transport following [3H]5-HT injection in the serotonergic RDN-OB system occurs via an active process which depends on a colchicine-sensitive mechanism and is partially linked to a reserp ine-sensitive structure. During its transport, the amine seems to be relatively protected from metabolic inactivation.     

Tracing specific transmitter pathways in the rat CNS: combination of [H]serotonin retrograde labelling with immunocytochemical detection of endogenous transmitters

Selective retrograde labelling with [³H]serotonin ([³H]5-HT) can be used to identify serotonergic cell bodies after specific [³H]5-HT uptake by the corresponding nerve terminals. In the present study, we demonstrate that autoradiography of this [³H]5-HT radiolabelling can be combined with immunocytochemical detection of endogenous serotonin, GABA or substance P on the same tissue section. The midbrain raphe serotonergic projections to the olfactory bulb and the spinal projections of medullary serotonergic nuclei were investigated. The specificity of retrograde labelling with [³H]5-HT was confirmed by immunoreactivity of the radiolabelled cells for serotonin, using an antiserum specific for formaldehyde-fixed serotonin. After spinal injections of [³H]5-HT, many retrogradely labelled cells in the medullary raphe were immunopositive for substance P, and a few for GABA. These results are in agreement with the available information on the co-existence of putative transmitters in the spinal projections of caudal raphe neurons. Therefore, autoradiography of [³H]5-HT retrograde labelling combined with immunocytochemistry offers a possibility to test the specificity of transmitter-selective retrograde labelling, to identify transmitter-defined neuronal interactions and to investigate the projection fields of multitransmitter containing neurons.     

5-HT2A receptor antagonist M100907 reduces serotonin synthesis: an autoradiographic study

The effects of the administration of the serotonin (5-HT)_2A antagonist, M100907, on 5-HT synthesis rates, were evaluated using the α-[¹⁴C]methyl-l-tryptophan (α-MTrp) autoradiographic method. In the treatment study, M100907 (10 mg/kg) was injected intraperitoneally 30 min before the α-MTrp injection (30 μCi over 2 min). A single dose of M100907 caused a significant decrease in the synthesis in the anterior olfactory nucleus, accumbens nucleus, frontal cortex, sensory-motor cortex, cingulate cortex, medial caudate-putamen, dorsal thalamus, substantia nigra, inferior collicus, raphe magnus nucleus, superior olive, and raphe pallidus nucleus.These data suggest that the terminal 5-HT_2A receptors are involved in the regulation of 5-HT synthesis in the entire brain. Further, 5-HT synthesis is likely regulated by the 5-HT_2A antagonistic property of M100907 in the cortices, anterior olfactory nucleus, caudate putamen, and nucleus accumbens.     

Synthesis of serotonin by neurons of the myenteric plexus in situ and in organotypic tissue culture.

Previous studies have suggested that serotonin is a neurotransmitter in the mammalian myenteric plexus. The present study was done to test this hypothesis by determining whether neurons of the myenteric plexus synthesize 5-HT from L-trypophan. Isolated strips of longitudinal muscle with attached myenteric plexus took up L-tryptophan by a saturable transport mechanism K_m 4.7 × 10^?5M; V_max 6.7 nmoles/g/min). These strips also synthesized tritiated serotonin ([³H]5-HT) and [³H]5-hydroxy-indoleacetic acid from L-[³H]tryptophan. This synthesis of [³H]5-HT was antagonized by prior treatment of guinea pigs with p-chlorophenylalanine. The drug treatment did not interfere with tryptophan uptake as did inclusion of p-chlorophenylalanine with L-[³H]tryptophan in vitro and thus probably decreased synthesis of [³H]5-HT by inhibiting tryptophan hydroxylase. Organotypic tissue cultures of intestine, lacking mucosa, argentaffin cells or mast cells also converted L-[³H]tryptophan to [³H]5-HT and [³H]5-hydroxyindoleacetic acid. Examination of the myenteric plexus from chemically sympathectomized (6-hydrpxydopamine) animals given L-tryptophan and a monoamine oxidase inhibitor for formaldehyde-induced histofluorescence revealed a diffuse yellow fluorophore in the myenteric plexus with the spectral characteristics of 5-HT. Pretreatment with p-chorophenylalanine prevented development of the diffuse fluorescence induced by L-tryptophan but revealed clusters of fluorescent neuronal cell bodies. The fluorescence of these cell bodies also had the spectral characteristics of the 5-HT fluorophore. Organotypic tissue cultures of intestine, grown for 3 weeks, and then dried and exposed to gaseous formaldehyde contained groups of fluorescent neuronal cell bodies. Their fluorescence was enhanced by adding L-tryptophan and an inhibitor of monoamine oxidase to the culture medium. This fluorescence also had the spectral characteristics of 5-HT. We conclude that the myenteric plexus synthesizes 5-HT from L-tryptophan. The responsible neurons survive in culture and are therefore intrinsic to the gut itself. These observations support the hypothesis that 5-HT is a transmitter of neurons in the mammalian myenteric plexus.     

Autoradiographic quantification of serotonin1A receptors in rat brain following antidepressant drug treatment

There is growing evidence that the serotonergic (5-HT) system is involved in the pathogenesis and treatment of major depression. The 5-HT receptor subtype involved in the enhancing effect of antidepressant treatments, however, has not been identified. The present study was undertaken to quantify 5-HT1A sites in the rat brain by autoradiography and membrane binding, using the selective ligand [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT), following long-term antidepressant treatment. Following a 21-day treatment with amitriptyline (10 mg/kg/day), there was a significant increase of [3H]8-OH-DPAT binding measured by autoradiography in the dorsal hippocampus, but there was no change in the nucleus raphe dorsalis; whole brain membrane binding revealed an increase in the number of binding sites, with no change in the affinity for [3H]8-OH-DPAT. Conversely, fluoxetine (10 mg/kg/day), a selective blocker of 5-HT reuptake, and gepirone (10 mg/kg/day), a 5-HT1A agonist, both administered for 21 days, significantly reduced [3H]8-OH-DPAT binding measured by autoradiography in the nucleus raphe dorsalis without altering hippocampal binding sites. The control active treatment with diazepam (2 mg/kg/day) did not alter [3H]8-OH-DPAT binding in the hippocampus or in the nucleus raphe dorsalis. All groups were compared to a 21-day vehicle-treated control group. These results are fully consistent with previous electrophysiological and behavioral studies and suggest that alterations of 5-HT1A receptors might underlie the enhancement of 5-HT neurotransmission by antidepressant treatments.     

Effects of antipsychotic drugs on dopamine and serotonin contents and metabolites, dopamine and serotonin transporters, and serotonin1A receptors

Summary. The effects of neuroleptics have been attributed to dopamine (DA) receptor blockade; however, other neurotransmitters, in particular serotonin (5-HT), have also been implicated. In this study, we examined the effects of clozapine and haloperidol on the distribution of DA and 5-HT transporters, on endogenous DA, 5-HT and their major metabolites, and on 5-HT_1A receptors. Adult male Sprague-Dawley rats were treated with either haloperidol (1 mg/kg/day, i.p.), clozapine (20 mg/kg/day, i.p.) or saline for 21 days, and following 3 days of withdrawal, the brains were removed. Tissue levels of DA and 5-HT and their metabolites were measured by high-performance liquid chromatography in 16 brain regions, while quantitative autoradiography with [¹²⁵I]RTI-121, [³H]citalopram and [³H]8-OH-DPAT was employed to label DA transporters, 5-HT transporters and 5-HT_1A receptors, respectively. After haloperidol, densities of 5-HT transporters were increased in the dorsal insular cortex and in the ventral half of caudal neostriatum, while 5-HT_1A receptors augmented in cingulate cortex but decreased in the entorhinal area. After clozapine, [³H]citalopram labelling was increased in ventral hippocampus, ventral caudal neostriatum and nucleus raphe dorsalis, but decreased in medio-dorsal and latero-dorsal neostriatum as well as in substantia nigra. Binding of [³H]8-OH-DPAT following clozapine was decreased in frontal, parietal, temporal and entorhinal cortices but increased in the CA3 division of Ammon's horn. The changes in 5-HT transporters in nucleus raphe dorsalis and substantia nigra, as well as the 5-HT_1A receptor down-regulations caused by clozapine but not by haloperidol, may explain effects obtained with clozapine and other atypical neuroleptics. There were no modifications in densities of DA transporters, nor of tissue DA levels, after the chronic neuroleptic treatments. The results are in line with previous suggestions that a certain degree of tolerance to neuroleptics develops, in spite of profound D₁ and D₂ receptor changes that persist during the entire chronic treatment with these psychotropic agents. Received September 2, 1997; accepted July 9, 1998     

differential expression of serotonin and [met]enkephalin-Arg-Gly-Leu in neurons of the rat brain stem

The hypothesis that serotonin (5-HT) and [Met]enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL) coexist in the rat brain stem raphe nuclei was tested by a technique combining histofluorescence with immunocytochemistry, after treatment with colchicine and nialamide. In midbrain and pons serotonergic cell groups (B5-B9), no coexistence of 5-HT and MEAGL was detected. In serotonergic cell groups of the medulla oblongata (B1-B3), only 0.3–1.5% of 5-HT-fuorescent cells were MEAGL-immunoreactive. These findings suggest that putative 5-HT and MEAGL are mostly expressed in different populations of neurons in the rat raphe nuclei.