An immunocytochemical study of the development of central serotoninergic neurons in the chick embryo

The development of central serotoninergic neurons in the chick embryo has been investigated immunocytochemically by utilizing an antiserum to serotonin (5-HT). Immunoreactive neurons are first detected in the brainstem on embryonic day 4 (E4, stage 23), days earlier than 5-HT systems have been detected previously by biochemical techniques. The earliest 5-HT-containing cells at E4 appear rostral to the pontine flexure, yet by E5, 5-HT neuronal groups are observed throughout the brainstem from just caudal to the mesencephalic flexure to the cervical flexure. During this and subsequent phases of development, two distinct patterns of cellular migration seem to be involved in the formation of the various 5-HT neuronal groups. One pattern involves a ventral migration of 5-HT cells, which appears dependent upon the directional guidance of midline radial processes (formed by floor plate cells) that extend across the neuroepithelium. The other pattern involves a lateral migration of cells, followed by an aggregation and rearrangement of 5-HT neurons into distinct subgroups or clusters. Through these patterns of migration most components of the 5-HT neuronal system can be recognized as early as E12, with the mature organization of the 5-HT cell groups occurring by E17. One unexpected finding was the comparatively late appearance (between E9 and E12) of 5-HT neurons in the paraventricular organ of the hypothalamus. Thus, in comparison to the initial observation of the majority of brainstem 5-HT neurons at E4 to E5, the hypothalamic 5-HT cells appear after a delay of between 5 and 7 days. Such differences illustrate the fact that neurons sharing a common neurotransmitter phenotype do not necessarily share the same developmental timetable for the expression of that particular phenotype, or they may undergo neurogenesis during considerably different periods of embryogenesis.     

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.     

Postnatal development of the serotonin innervation of the hippocampus and dentate gyrus following raphe implants

Serotoninergic (5-HT) neurons derived from the embryonic raphe nuclear area (brainstem, embryonic days (E 16-18) were implanted into the entorhinal cortex of 6-day-old (P6) neonatal rat recipients which had received a fimbria lesion and entorhinal cortex ablation on P3. The hippocampus, dentate gyrus, and the raphe implant area were examined with 5-HT immunohistochemistry 7, 14, 21, 30, and 60 days after implantation. The pattern of 5-HT reinnervation was compared to that of normal and lesioned animals, and to previous studies in which rats received septal or striatal implants. In the hippocampus adjacent to the implant 5-HT-immunoreactive fibers were first observed by 7 days postimplantation and increased in density and in their septotemporal and dorsoventral extent with increasing time postimplantation. Moderately dense fiber networks were diffusely distributed in the hippocampus and dentate gyrus at 30 and 60 days postimplant. Little, if any, indication of lamination was present. Retrogradely labeled neurons (the majority of which contained 5-HT immunoreactivity) were observed in the raphe implant following injections of Fast Blue into the hippocampal formation. A few retrogradely labeled cells did not contain 5-HT, methionine-enkephalin (ME), or substance P (SP) immunoreactivity, although ME- and SP- immunoreactive neurons were observed in the implants. The lamination patterns and the increased density of 5-HT-immunoreactive fibers following a raphe implant into the entorhinal cortex clearly differ from the normal 5-HT pattern and from the patterns of lamination following a striatal or septal implant.     

Fetal raphe and hippocampal transplants into adult and aged C57BL/6N mice: A preliminary immunocytochemical study

Fetal mouse raphe and hippocampal tissue (from embryos with crown-rump length (CRL) 13–16 mm) was transplanted into adult and aged isogenetic mice to study the growth of serotonergic fibers between host and donor tissue. A specific antibody against serotonin (5-HT) was used to immunocytochemically visualize 5-HT containing cell bodies and fibers. Unilateral fetal transplants into the hippocampi of adult (4–6 mo.) or aged (24 mo.) mice matured and sent out processes which very densely innervated the transplant tissue itself and extended into the host hippocampus. The termination of these fibers was consistent with the known 5-HT-hippocampal lamination pattern in normal animals. Qualitative comparisons suggested that the density of outgrowth into adult hippocampus was greater than into aged hippocampus. Conversely, adult 5-HT neurons send sprouts into fetal hippocampal tissue transplanted into the lateral ventricle. Therefore, immunocytochemical procedures can be used to monitor outgrowth from the fetal tissue to the host and ingrowth from the adult host to the fetal tissue. Furthermore, the apparent normal 5-HT lamination pattern produced by fetal raphe axons in adult hippocampus is consistent with reports that neuronal transplantation is effective in reversing the anatomical and behavioral deficits produced by homotypic denervation of a terminal field.     

Serotonin-containing projections to the thalamus in the rat revealed by a horseradish peroxidase and peroxidase antiperoxidase double-staining technique

The retrograde transport of horseradish peroxidase (HRP) has been used in combination with peroxidase antiperoxidase (PAP) immunocytochemistry in order to investigate serotonin-containing projections to the thalamus of the rat. Sections were histochemically stained to reveal retrogradely transported HRP and then PAP immunostained using a monoclonal anti-serotonin (5-HT) antibody. Following HRP injections into the ventral thalamus, retrogradely labelled cells were observed in a number of sites in the brainstem and including areas known to be rich in 5-HT-containing neurons. At rostral levels of the dorsal raphe nucleus, retrogradely labelled cells were observed both on the midline and in a distinct lateral group extending diffusely into the periaqueductal gray (PAG). In both of these areas many 5-HT-immunoreactive HRP retrogradely labelled neurons were observed. However, except for the most rostral levels of the dorsal raphe nucleus, such double-labelled cells represented only a small proportion of the total population of 5-HT-immunoreactive neurons. In the lateral group, the retrograde labelling was mainly unilateral to the injection site but some contralateral labelling was also seen. At caudal levels of the dorsal raphe nucleus, retrogradely labelled cells were observed predominantly in the lateral group. At the level of the dorsolateral tegmental nucleus, few 5-HT of 5-HT/HRP labelled cells were observed in the lateral group, although HRP retrogradely labelled neurons were present. Double-stained cells were detected also in the medial raphe nucleus (corresponding to the B8 cell group according to the nomenclature of Dahlström and Fuxe¹³), among the fibres of the medial lemniscus (B9), and in nucleus raphe pontis (B5).     

Serotonin promotes region-specific glial influences on cultured serotonin and dopamine neurons.

To test the hypothesis that glia mediate interactions between embryonic serotonergic (5-HT) neurons and dopamine neurons, we studied the effects of 5-HT in co-cultures of E14 raphe neurons of mesencephalic dopamine neurons and radial glia/astrocytes derived from the same (homotypic) or opposite (heterotypic) brain region using a dose (10(-5) M) that would produce 5-HT uptake into glial cells as well as activate 5-HT receptors. Morphometric analysis of 5-HT and tyrosine hydroxylase (TH) immunoreactive neurons revealed regional differences in the effects of 5-HT (and nialamide) on survival, cell soma size, and dendrite-like neurite outgrowth in neuronal-glial co-cultures. In general, 5-HT had more significant effects on both types of monoamine neuron when they were cultured with mesencephalic glia (GSN). Stimulatory effects of 5-HT on growth of TH neurons in GSN cultures suggest that developing raphe axons, which reach the mesencephalon during the early differentiation of these neurons, may enhance the influence of local glial-derived trophic factors. Likewise, the promotion of 5-HT neuronal survival in these cultures suggests that glial factors in the mesencephalon may contribute to the support of 5-HT neurons in addition to the influences of raphe glia. The inhibitory effects of 5-HT on neurite outgrowth by raphe neurons in GSN co-cultures indicates enhanced sensitivity of these neurons to the inhibitory effects of 5-HT in the presence of mesencephalic glia. The region-specific effects of 5-HT and nialamide in glial co-cultures suggest that raphe and mesencephalic glia may express different capacities for 5-HT uptake, receptors, and/or monoamine oxidase (MAO) activities. These characteristics could be important for the specificity of growth-regulatory influences of glial cells on the development of brain monoamine neurons.     

Evidence for substance P, serotonin and oxytocin input to medullary catecholamine neurons with diencephalic projections

Using the retrograde transport of horseradish peroxidase (HRP) in combination with two-color immunoperoxidase staining, boutons stained with antisera to substance P (SP), serotonin (5HT) and oxytocin (OX) have been observed in contiguity with neurons in the rostral and caudal medulla that showed immunoreactivity for phenylethanolamine N-methyl transferase (PNMT) and tyrosine hydroxylase (TH), respectively, and which were backfilled with HRP injected into the diencephalon. The juxtaposition of these immunostained structures indicates that SP, 5HT and OX released from fibers in the medulla may affect the activity of adrenergic and noradrenergic medullary neurons that project to the diencephalon. Moreover, the presence of 5HT- and OX-immunoreactive processes in contiguity with medullary CA cells that send fibers to the diencephalon indicates that the raphe nuclei and the paraventricular nucleus of the hypothalamus can directly influence ascending pathways that are known to innervate the hypothalamus and appear to effect changes in vasopressin release.     

Pharmacological characterization of medullary serotonin neurons

In the present study we investigated the characteristics of medullary raphe serotonergic neurons. Specifically, we sought to examine further the similarities between medullospinal 5-HT neurons and the more extensively studied neurons of the dorsal raphe. Intravenous administration of 5-methoxy-dimethyltryptamine (5-MeODMT) produced a dose-related inhibition of the firing of midline medullary 5-HT neurons. Microiontophoretically applied 5-MeODMT also inhibited medullary 5-HT neurons. The inhibitory potency of 5-MeODMT was nearly identical to that observed for dorsal raphe 5-HT neurons. Microiontophoretic or intravenous administration of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) did not alter the firing rate of medullary 5-HT neurons. Intravenous administration of the alpha 1-receptor antagonist prazosin resulted in an inhibition of the medullary 5-HT neuronal firing. The discharge of medullary 5-HT neurons increased during iontophoresis of norepinephrine. These data are discussed in relation to the identification and characterization of medullary 5-HT neurons. In addition, the data suggest that the firing rate of medullary 5-HT neurons is regulated in part by a tonic excitatory noradrenergic input.     

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.     

Analysis of the change in number of serotonergic neurons in the chick spinal cord during embryonic development

The existence of serotonin (5-HT)-containing neurons in the spinal cord of the chick embryo was examined by anti-5-HT immunocytochemistry. The first immunoreactive cells were observed in embryos at 7 days of incubation (E7) and were initially located within the floor plate of the early spinal cord. By E9, immunostained cells occurred throughout the length of the spinal cord and were frequently encountered in most transverse sections of the cord. When examined at later embryonic ages of E12, 17 and at hatching (E21 or 22), the 5-HT cells became progressively more difficult to find with the advancing age of the embryos. To determine if this population of spinal cord 5-HT neurons actually diminished during development, a detailed quantitative analysis was undertaken to estimate the number of 5-HT cells in the cord of chick embryos at different ages. The results of this investigation demonstrated that the size of the 5-HT neuronal population rose rapidly from E7 and plateaued (at approximately 3500 neurons) between E9 and E12. As anticipated, the number of 5-HT cells at E17 decreased at all cord levels. Surprisingly, however, the number of spinal cord 5-HT neurons at hatching increased (depending on the cord level) either back to, or above, the counts estimated for the earlier ages of E9 and E12. Therefore, cells expressing the 5-HT phenotype in the spinal cord of the chick embryo persist throughout the period of embryonic development, rather than appear transiently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Significant non-serotonergic raphe projection to the visual cortex of the rat. An immunohistochemical study combined with retrograde tracing.

The present study investigated the distribution of serotonergic and non-serotonergic raphe neurons with direct projections to the visual cortex. The study employed the WGA-apoHRP-Au retrograde transport technique combined with 5-HT immunohistochemical staining. Retrogradely labeled cells were observed in the dorsal raphe nucleus, the median raphe nucleus, and in the B9 and B6 cell groups. One notable finding was the great number of retrogradely labeled, non-5-HT immunoreactive cells. The average percentages of such cells in the various raphe regions were as follows: DR: 52% (n = 401); MR: 35% (n = 311); B9: 24% (n = 129); B6: 95% (n = 200). The present study demonstrated the presence of a significant proportion of non-serotonergic raphe region neurons projecting to the primary visual cortex in the rat. It is suggested that these neurons may complement the aminergic neurons as part of the ascending system which controls the functions of the visual cortex.     

Median raphe serotonergic innervation of medial septum/diagonal band of broca (MSDB) parvalbumin-containing neurons: possible involvement of the MSDB in the desynchronization of the hippocampal EEG.

Activation of median raphe serotonergic neurons results in the desynchronization of hippocampal electroencephalographic (EEG) activity. This could be a direct effect, because serotonin (5-HT) fibers terminate on a specific population of hippocampal interneurons. On the other hand, it could be an indirect action through the medial septum/diagonal band of Broca (MSDB) pacemaker cells, because, in addition to previously described inhibitory effects, excitatory actions of 5-HT have been demonstrated on MSDB gamma-aminobutyric acid (GABA)-containing neurons through 5-HT2A receptors. Electron microscopic double immunostaining for Phaseolus vulgaris-leucoagglutinin (PHA-L) injected into the median raphe (MR) and parvalbumin, choline acetyltransferase, or calretinin as well as double immunostaining for 5-HT and parvalbumin, and colocalization for parvalbumin and 5-HT2A receptors were done in rats. The results demonstrated that: 1) MR axons form perisomatic and peridendritic baskets and asymmetric synaptic contacts on MSDB parvalbumin neurons; 2) these fibers do not terminate on septal cholinergic and calretinin neurons; 3) 5-HT fibers form synapses identical to those formed by PHA-L-immunolabeled axons with parvalbumin neurons; and 4) MSDB parvalbumin cells contain 5-HT2A receptors. These observations indicate that 5-HT has a dual action on the activity of hippocampal principal cells: 1) an inhibition of the input sector by activation of hippocampal GABA neurons that terminate exclusively on apical dendrites of pyramidal cells, and 2) a disinhibition of the output sector of principal neurons. MSDB parvalbumin-containing GABAergic neurons specifically innervate hippocampal basket and chandelier cells. Thus, 5-HT-elicited activation of MSDB GABAergic neurons will result in a powerful inhibition of these GABA neurons.     

Pre- and post-synaptic effects of the 5-HT3 agonist 2-Methyl-5-HT on the 5-HT system in the rat brain

Microiontophoretic applications of 5-HT and of the 5-HT₃ agonist 2-methyl-5-HT produced a current-dependent suppression of firing activity of both hippocampal (CA₁ and CA₃) and cortical neurons in anesthetized rats. Concomitant microiontophoretic applications of the 5-HT₃ antagonists BRL 46470A and S-zacopride, as well as their intravenous injection, did not antagonize the inhibitory effect of 5-HT and 2-methyl-5-HT. In contrast, the 5-HT_1A antagonist BMY 7378, applied by microiontophoresis or administered intravenously, significantly reduced the inhibitory action of 5-HT and 2-methyl-5-HT. The firing activity of dorsal raphe 5-HT neurons was also reduced by 5-HT, 2-methyl-5-HT and the 5-HT_1A agonist 8-OH-DPAT applied by microiontophoresis. While BRL 46470A (0.1 and 1 mg/kg, i.v.) did not antagonize the inhibitory effect of the three 5-HT agonists on 5-HT neuronal firing activity, only that of 8-OH-DPAT was attenuated by the 5-HT_1A antagonist (+) WAY 100135. R-zacopride significantly reduced the duration of suppression of firing activity of CA₃ pyramidal neurons induced by the electrical stimulation of the ascending 5-HT pathway, and this reducing effect was prevented by the three 5-HT₃/5-HT₄ antagonists renzapride, S-zacopride and tropisetron, but not by BRL 46470A. Finally, in in vitro superfusion experiments, both BRL 46470A and S-zacopride antagonized the enhancing action of 2-methyld-HT on the electrically-evoked release of [³H]-5-HT in both rat frontal cortex and hippocampus slices. These findings suggest that, in vivo, the suppressant effect of 2-methyl-5-HT on the firing activity of dorsal hippocampus pyramidal, somatosensory cortical, and dorsal raphe 5-HT neurons is not mediated by 5-HT₃ receptors, but rather by 5-HT_1A receptors. The attenuating effect of R-zacopride on the effectiveness of the stimulation of the ascending 5-HT pathway is not mediated by 5-HT₃ receptors. In contrast, in vitro, the enhancing action of 2-methyl-5-HT on the electrically-evoked release of [³H]5-HT in both frontal cortex and hippocampus slices is mediated by 5-HT₃ receptors. © 1995 Wiley-Liss, Inc.     

Early development of serotonin-containing neurons and pathways as seen in wholemount preparations of the fetal rat brain

The early development of serotonin-containing neurons was studied in wholemounts of the fetal rat brain (E12-E18). The wholemounts were treated immunocytochemically according to an immunoperoxidase technique to reveal a panorama of developing serotoninergic neurons. Serotoninergic neurons were localized to two discrete groups or clusters within the brainstem. Serotonin-containing neurons were identified first at E12 forming a rostral cluster of cells just caudal to the mesencephalic flexure. The more caudal cluster of cells first appeared at E14 in the medulla. During the period from E12 to E18, the immunoreactive cells increased in number and acquired a more complex dendritic tree while migrating to their permanent position. At E16, cells of the rostral group exhibited remarkably uniform mediolateral orientation. The rostral group of immunoreactive neurons gave rise to almost all ascending fibers, whereas the caudal group gave rise to the majority of descending fibers. Growing serotoninergic fibers were tipped by prominent growth cones, which were strongly immunoreactive. The fibers demonstrated prominent orientational selectivity with an almost total separation into ascending and descending bundles. Some of the ascending immunoreactive fibers displayed acute changes in their direction of growth, suggesting that the orientation of serotininergic fibers is mediated by directional cues that are specific to particular populations of serotoninergic fibers. Serotoninergic axons within the medial forebrain bundle were demonstrated particularly well and their ascent and rate of growth toward the forebrain could be easily followed. Immunoreactive fibers entered the telencephalon at E17 two portals, one along the lateral border of the hypothalamus and one rostrally, adjacent to the olfactory tubercle. In wholemounts at E18, fibers arising from this latter location could be followed as two distinct bands within the pallium; a basal band located ventrolaterally, adjacent to the lateral olfactory tract, and a dorsal band located at the medial edge of the telencephalon. Both fascicles were directed toward the occipital pole and contained unbranched fibers. At E18, serotoninergic axons arising from these two loosely organized fascicles covered most of the frontal telencephalon. The results of the present study indicate that wholemounts of embryonic brain can provide novel spaciotemporal data on the development of neuro-transmitter systems and may in the future prove to be useful experimental preparations in developmental neurobiology.     

Pre- and post-natal ontogeny of serotonergic projections to the rat spinal cord

The development of 5-hydroxytryptamine (5-HT) innervation in the spinal cord was studied from embryonic day 14 (E14) to adulthood. Sprague-Dawley rats were fixed by perfusion with 5% glutaraldehyde in cacodylate-sodium metabisulfite buffer, and vibratome sections were processed for immunocytochemistry with a 5-HT antiserum. For electron microscopy, the sections were flat-embedded in araldite, and thin sectioning was performed. 5-HT neurons caudally directed from raphe nuclei invade the spinal cord at E14 and reach the caudalmost levels by E16-E17. In longitudinal sections, axons are seen by E15, at cervical and upper thoracic levels, to invade the presumptive gray matter from the anterior and lateral funiculi. The invasion process occurred either by sharp angulation of the axon or by branching of a collateral. By E16, at thoracic level the anterior horn and the intermediolateral columns are profusely innervated by very thin, varicose fibers; synapses are seen at E17 and E18 using EM. 5-HT immunoreactive boutons are involved here. After birth, 5-HT innervation of these two areas evolves progressively from a diffuse network to a more restricted pattern, especially at the thoracic level for the intermediolateral column and at cervical and lumbar levels for the anterior horn. The adult pattern is reached by postnatal day 21 (P21). The growth of axons toward the dorsal horn becomes noticeable by E19 at all spinal levels, when fibers invade the neck of the horn from the lateral funiculus, and innervation proceeds diffusely until P5. At P7, thin fibers course dorsally and laterally along the border of the gray matter and ramify profusely in layers I and II. The adult pattern is also reached in the dorsal horn by P21. These results are discussed in relation to the postnatal maturation of motor and sensory circuits and to the development of transplanted raphe neurons in the rat spinal cord.     

The existence of FGFR1-5-HT1A receptor heterocomplexes in midbrain 5-HT neurons of the rat: relevance for neuroplasticity

The ascending midbrain 5-HT neurons to the forebrain may be dysregulated in depression and have a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by coimmunoprecipitation and colocation of the FGFR1 and 5-HT1A immunoreactivities in the midbrain raphe cells, evidence for the existence of FGFR1-5-HT1A receptor heterocomplexes in the dorsal and median raphe nuclei of the Sprague Dawley rat as well as in the rat medullary raphe RN33B cells has been obtained. Especially after combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA clusters was found in the RN33B cells. Similar results were reached with the FRET technique in HEK293T cells, where TM-V of the 5HT1A receptor was found to be part of the receptor interface. The combined treatment with FGF-2 and the 5-HT1A agonist also synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and the RN33B cells as well as their differentiation, as seen from development of the increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TM-V but not by TM-II. Together, the results indicate that the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A receptor heterocomplex in the midbrain raphe 5-HT nerve cells appear to have a trophic role in the central 5-HT neuron systems in addition to playing a key role in reducing the firing of these neurons.     

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.     

Distribution,quantification, and morphological characteristics of serotonin-immunoreactive cells of the supralemniscal nucleus (B9) and pontomesencephalic reticular formation in the rat

In their initial report on the rat, Dahlstrom and Fuxe ([1964] Acta Physiol. Scand. 62:1–55) identified nine brainstem serotonin-containing cell groups, which they termed B1–B9. B9 has received considerably less attention than other serotonergic nuclei (B1–B8) due in part to the fact that its precise location and extent have not been well documented in subprimates. B9 (supralemniscal nucleus; SLN) has been viewed as a minor serotonergic cell group. In addition, 5-hydroxytryptamine (5-HT)-containing cells have been shown to be only sparsely distributed throughout the pontomesencephalic reticular formation (PMRF). By using 5-HT immunohistochemical techniques, we examined the distribution and morphological characteristics of SLN and PMRF 5-HT neurons of the pontomesencephalic tegmentum. We showed that 5-HT cells of both the SLN and the PMRF extend rostrocaudally from the rostral midbrain to the midpons. 5-HT SLN cells are located within or dorsal to the medial lemniscus (ML); those of the PMRF are widely distributed throughout the PMRF. The mean numbers of 5-HT containing cells in the SLN, PMRF, dorsal raphe, and median raphe nuclei were 4,571, 1,948, 15,191, and 4,114, respectively. The SLN (B9) contains more 5-HT neurons than any serotonergic group other than the dorsal raphe nucleus. The dendrites of both SLN and PMRF 5-HT cells are primarily oriented mediolaterally and generally extend for long distances (75–300 μm), running perpendicular to the fibers of the ML (SLN) or, to those coursing through the brainstem (PMRF). The present anatomical delineation of SLN and PMRF shows that they are major 5-HT-containing cell groups in the rat and provides the foundation for the further examination of their properties and functions. J. Comp. Neurol. 378:411–424, 1997. © 1997 Wiley-Liss, Inc.     

Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio).

The histaminergic system and its relationships to the other aminergic transmitter systems in the brain of the zebrafish were studied by using confocal microscopy and immunohistochemistry on brain whole-mounts and sections. All monoaminergic systems displayed extensive, widespread fiber systems that innervated all major brain areas, often in a complementary manner. The ventrocaudal hypothalamus contained all monoamine neurons except noradrenaline cells. Histamine (HA), tyrosine hydroxylase (TH), and serotonin (5-HT) -containing neurons were all found around the posterior recess (PR) of the caudal hypothalamus. TH- and 5-HT-containing neurons were found in the periventricular cell layer of PR, whereas the HA-containing neurons were in the surrounding cell layer as a distinct boundary. Histaminergic neurons, which send widespread ascending and descending fibers, were all confined to the ventrocaudal hypothalamus. Histaminergic neurons were medium in size (approximately 12 microm) with varicose ascending and descending ipsilateral and contralateral fiber projections. Histamine was stored in vesicles in two types of neurons and fibers. A close relationship between HA fibers and serotonergic raphe neurons and noradrenergic locus coeruleus neurons was evident. Putative synaptic contacts were occasionally detected between HA and TH or 5-HT neurons. These results indicate that reciprocal contacts between monoaminergic systems are abundant and complex. The results also provide evidence of homologies to mammalian systems and allow identification of several previously uncharacterized systems in zebrafish mutants.