Evidence for dual serotonergic projections to neocortex: axons from the dorsal and median raphe nuclei are differentially vulnerable to the neurotoxin p-chloroamphetamine (PCA)

Previous studies have shown that there are morphologically dissimilar serotonergic (5-HT) axon types in rat cerebral cortex which are differentially sensitive to the neurotoxic effects of certain psychotropic drugs: methylenedioxyamphetamines (MDA and MDMA) and p-chloroamphetamine (PCA) cause degeneration of fine 5-HT axon terminals in cortex, while sparing beaded axons. Moreover, a recent anterograde transport study suggests that fine and beaded 5-HT axons arise from the dorsal raphe (DR) and median raphe (MR) nuclei, respectively. These data led us to propose that the DR projection to neocortex is selectively vulnerable to the neurotoxic effects of PCA, while the MR projection is resistant; this hypothesis was tested in the present study by comparing retrograde axonal transport of the fluorescent tracer Fluoro-Gold in PCA-treated and control rats. Using this method, only axons that survive PCA treatment can take up and transport the injected label back to the cell bodies of origin, thus allowing us to determine which raphe-cortical projections remain intact after PCA. The results show that PCA administration produces a loss of fine 5-HT axon terminals in neocortex and a concomitant reduction in the number of retrogradely labeled neurons in the DR (77% decrease), when compared to controls. In contrast, beaded 5-HT axon terminals are spared and the number of labeled neurons in the MR remains unchanged after PCA. These results demonstrate that DR and MR projections to cortex are differentially vulnerable to PCA: fine axon terminals arise from neurons in the DR and are highly sensitive to the neurotoxic effects, whereas beaded axons from the MR are resistant. We therefore propose that there are two anatomically and functionally separate 5-HT projections to cortex having different (1) nuclei of origin, (2) axon morphology, (3) regional distributions, and (4) pharmacological properties. Since the mood-altering substances MDA, MDMA, and PCA act specifically upon 5-HT axon terminals from the dorsal raphe nucleus, DR neurons may be preferentially involved in the control of affective state.     

Dual serotoninergic projections to forebrain in the rat: morphologically distinct 5-HT axon terminals exhibit differential vulnerability to neurotoxic amphetamine derivatives.

The cerebral cortex of the rat and other mammals is innervated by two morphologically distinct classes of serotoninergic (5-HT) axon terminals: fine axons with minute varicosities and beaded axons characterized by large, spherical varicosities. Fine and beaded 5-HT axons exhibit different regional and laminar distributions in forebrain and arise from separate brainstem nuclei, the dorsal and median raphe nuclei, respectively. The present neuroanatomic study, based on immunocytochemical methods to visualize 5-HT axons, demonstrates that the two axon types differ markedly in their vulnerability to the neurotoxic amphetamine derivatives, methylenedioxyamphetamine (MDA), and p-chloroamphetamine (PCA). While both drugs cause extensive degeneration of fine 5-HT axons throughout forebrain, beaded 5-HT axons are consistently spared. Fine 5-HT axons, which richly innervate most regions of dorsal forebrain in control rats, are rarely seen 2 weeks after treatment with MDA or PCA; this loss of fine axons reflects a marked denervation that persists for months after drug administration. The serotoninergic axon terminals remaining after MDA or PCA administration are almost entirely of the beaded type and appear to be unaffected by both drugs. Over a wide range of doses (2.5-40 mg/kg PCA) and survival times (2 weeks to 2 months), these spared 5-HT axons with large, spherical varicosities cannot be distinguished from the normal, beaded 5-HT axons in control rats by morphologic criteria. Moreover, beaded 5-HT axons exhibit a highly characteristic regional distribution which is the same in control as in MDA- and PCA-treated rats: these axons innervate specific zones or layers within parietal and occipital cortex, hippocampus, cingulate cortex, entorhinal cortex, and the olfactory bulb, among other forebrain areas, and they form a dense plexus lining the ventricular system. Taken together, the results of this study demonstrate that fine 5-HT axons are highly vulnerable to the neurotoxic effects of the amphetamine derivatives MDA and PCA, while beaded 5-HT axons are markedly resistant. These findings are consistent with the hypothesis that there are two anatomically and functionally distinct sets of serotoninergic neurons projecting to forebrain. While both of these neuronal systems utilize 5-HT as a neurotransmitter, they differ in several features: 1) origin from separate nuclei in the brainstem (the dorsal and median raphe), 2) two types of morphologically distinct axon terminals, 3) markedly different distribution and innervation patterns in forebrain, and 4) dissimilar pharmacological properties. The results further suggest that psychotropic amphetamine derivatives have a selective action upon fine serotoninergic axons that arise from the dorsal raphe nucleus.     

Neurotoxic effects of p-chloroamphetamine on the serotoninergic innervation of the trigeminal motor nucleus: a retrograde transport study

The rat forebrain receives projections from both dorsal and median raphe nuclei. It has recently been shown that serotoninergic axons arising from the dorsal raphe nucleus, but not those from the median raphe nucleus, degenerate following systemic administration of p-chloroamphetamine (PCA). The present study was conducted to determine (i) whether the motor nucleus of the trigeminal nerve is innervated by overlapping projections from multiple serotonin cell groups and (ii) whether a particular subset of serotoninergic axon terminals in the trigeminal motor nucleus are sensitive to the neurotoxic effects of PCA. Retrograde transport was used in combination with immunofluorescence to identify the serotonin-positive cells that project to the trigeminal motor nucleus both in control rats and in rats previously treated with PCA. In untreated rats, an average of 95 retrogradely labeled serotonin-positive neurons were found in the dorsal raphe nucleus, 135 in the nucleus raphe obscurus, 132 in the nucleus raphe pallidus and 63 in the ventrolateral medulla. After treatment with PCA, there was a marked decrease (-77%) in the number of retrogradely labeled serotoninergic neurons in the dorsal raphe nucleus, whereas the number of labeled neurons was unchanged in the raphe obscurus and raphe pallidus. These results demonstrate that PCA selectively lesions serotonin axon terminals arising from the dorsal raphe nucleus, while sparing projections from the raphe obscurus and raphe pallidus to the trigeminal motor nucleus. This conclusion is in agreement with previous findings that in the forebrain only axons from the dorsal raphe are vulnerable to PCA. The data provide further evidence that serotoninergic axons originating in the dorsal raphe nucleus differ from other serotoninergic axons in their pharmacological properties and that the dorsal raphe may contain a functionally unique subset of serotonin neurons.     

Immunocytochemical localization of serotonin in the rat dentate gyrus following raphe transplants

The ultrastructural features of the serotoninergic innervation of the rat dentate gyrus in normal adults and in animals receiving raphe nuclear area transplants was investigated using an antibody to serotonin (5-HT). Neonatal rats received a lesion of the fimbria-fornix and entorhinal cortex. Three days later, a portion of embryonic (E-16-18) raphe nuclear area was transplanted to the entorhinal cavity and the animals were allowed to survive for 60 days. Animals were processed for the immunocytochemical localization of 5-HT using the peroxidase-antiperoxidase method. Light microscopic observation showed that 5-HT-containing fibers from transplanted raphe neurons densely innervated the hilar and molecular zones of the dentate gyrus. Electron microscopic analysis showed that 5-HT immunoreactivity was contained only in axons and axon varicosities. There were no differences in the ultrastructural characteristics of axons and axon terminals between normal animals and those which had received raphe transplants. A mixture of both conventional synaptic junctions and non-synaptic axonal swellings were found in both groups.     

Reorganization of the area dentata serotoninergic plexus after lesions of the median raphe nucleus.

Serotoninergic projections from the dorsal and median raphe nuclei to the area dentata of the hippocampal formation terminate mainly in the molecular layer and hilus, respectively. Consequently, a reduction in the density of the hilar serotoninergic plexus is seen by immunocytochemistry 2 weeks after lesions of the median raphe nucleus. Hippocampal serotonin concentration and serotonin high affinity uptake are also significantly reduced. Six weeks after lesion, surviving serotoninergic axons form a dense band in the inner molecular layer of the dorsal area dentata, a region that usually contains a sparse serotoninergic plexus. Moreover, serotoninergic fibers transverse the molecular layer and pass through the granule cell layer to reinnervate the hilus. Serotonin concentration and high affinity uptake have recovered to near normal levels by 6 weeks postlesion. Changes in the anatomical distribution of the area dentata serotoninergic plexus have not been reported in cases in which serotoninergic sprouting follows axotomy of serotoninergic projections. Thus direct lesions of serotoninergic neurons can produce a homotypic compensatory response that is qualitatively different from that generated by axotomy. The mechanistic basis for this reorganization is unclear, but the apparent extension of serotoninergic axon collaterals toward the hilus suggests that the denervated hilar neuropil is guiding reinnervation. Finally, anatomical evidence from animals studied 10 weeks postlesion suggests that the compensatory proliferation of serotoninergic axons observed 6 weeks after median raphe lesion is a transient event.     

Ultrastructural analysis of the innervation of TRH-immunoreactive neuronal elements located in the periventricular subdivision of the paraventricular nucleus of the rat hypothalamus

A combination of electron microscopic immunocytochemistry and autoradiography was employed to examine the synaptic organization of thyrotropin-releasing hormone (TRH) neurons in the periventricular subdivision of the paraventricular nucleus of the rat hypothalamus. TRH neurons were identified by immunocytochemistry. Selective uptake of tritiated serotonin (5-HT) was used to identify serotoninergic elements. TRH-immunoreactive axon terminals were found to be in synaptic contact with TRH-immunoreactive dendrites and with unlabeled dendritic branchlets. There were direct appositions between radiolabeled 5-HT terminals and TRH-immunoreactive dendrites, but differential synaptic contacts between 5-HT axonal elements and TRH neurons were not seen. TRH-immunopositive cell bodies and dendrites received a very intense innervation by unlabeled axon terminals or axonal varicosities showing morphologically defined synaptic junctions. These were mostly of the asymmetrical variety and different types could be distinguished. The findings substantiate the view that TRH neurons of the periventricular subvision of the paraventricular nucleus may be influenced by TRH axons, serotoninergic fibers and a large number of unidentified nerve terminals.     

An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat

The differential projections from the dorsal raphe and median raphe nuclei of the midbrain were autoradiographically traced in the rat brain after ³H-proline micro-injections. Six ascending fiber tracts were identified, the dorsal raphe nucleus being the sole source of four tracts and sharing one with the median raphe nucleus. The tracts can be classified as those lying within the medial forebrain bundle (dorsal raphe forebrain tract and the median raphe forebrain tract) and those lying entirely outside (dorsal raphe arcuate tract, dorsal raphe periventricular tract, dorsal raphe cortical tract, and raphe medial tract). The dorsal raphe forebrain tract lies in the ventrolateral aspect of the medial forebrain bundle (MFB) and projects mainly to lateral forebrain areas (e.g., basal ganglion, amygdala, and the pyriform cortex). The median raphe forebrain tract lies in the ventromedial aspect of the MFB and projects to medial forebrain areas (e.g., cingulate cortex, medial septum, and hippocampus). The dorsal raphe cortical tract lies ventrolaterally to the medial longitudinal fasciculus and projects to the caudate-putamen and the parieto-temporal cortex. The dorsal raphe periventricular tract lies immediately below the midbrain aqueduct and projects rostrally to the periventricular region of the thalamus and hypothalamus. The dorsal raphe arcuate tract curves laterally from the dorsal raphe nucleus to reach the ventrolateral edge of the midbrain and projects to ventrolateral geniculate body nuclei and the hypothalamic suprachiasmatic nuclei. Finally, the raphe medial tract receives fibers from both the median and dorsal raphe nuclei and runs ventrally between the fasciculus retroflexus and projects to the interpeduncular nucleus and the midline mammillary body. Further studies were done to test whether the fiber tracts travelling in the MFB contained 5-HT. Unilateral (left) injections of 5,7-dihydroxytryptamine (5 μgm/400 nl) 18 days before midbrain raphe microinjections of ³H-proline produced a reduction in the grain concentrations in all the ascending fibers within the MFB. Furthermore, pharmacological and behavioural evidence was obtained to show that the 5-HT system had been unilaterally damaged; these animals displayed preferential ipsilateral turning in a rotameter which was strongly reversed to contralateral turning after 5-hydroxytryptophan administration. The results show that DR and MR nuclei have numerous ascending projections whose axons contain the transmitter 5-HT. The results agree with the neuroanatomical distribution of the 5-HT system previously determined biochemically, histochemically, and neurophysiologically. The midbrain serotonin system seems to be organized by a series of fiber pathways. The fast transport rate in these fibers was found to be about 108 mm/day.     

Differential origin of brainstem serotoninergic projections to the midbrain periaqueductal gray and superior colliculus of the rat

Previous studies have shown that both the midbrain periaqueductal gray (PAG) and the superior colliculus receive a significant serotoninergic (5-HT) innervation. In the present study the origins of these 5-HT projections to the rodent PAG and superior colliculus were analyzed by using a combined immunohistochemical-retrograde transport technique. Thirteen brainstem regions were found to contain double-labelled 5-HT-like immunoreactive neurons following HRP injections into the PAG while only four brainstem nuclei contained double-labelled neurons following superior collicular injections. After HRP deposits into the ventral PAG, the largest percentage of double-labelled neurons was identified in nucleus raphe magnus, pars alpha of the nucleus gigantocellularis, and the paragigantocellular nucleus. The dorsal PAG, on the other hand, received the largest percentage of its 5-HT projections from nuclei raphe dorsalis, raphe obscurus, raphe pontis, and raphe medianis. The 5-HT input to the superior colliculus was found to arise exclusively from nuclei raphe dorsalis, raphe medianis, and raphe pontis and from the contralateral periaqueductal gray. Raphe nuclei were found to contribute serotoninergic projections to both the PAG and the superior colliculus while reticular nuclei contributed 5-HT projections only to the PAG. Injections of the fluorescent retrograde tracers true blue and nuclear yellow were then made into the PAG and superior colliculus to ascertain if neurons located in raphe nuclei that projected to both structures provided axon collaterals to both areas. Generally, less than 10% of raphe neurons projecting to the superior colliculus were identified as providing axon collaterals to the PAG. The present results demonstrate major quantitative and qualitative differences in the origin of 5-HT projections to the ventral PAG and superior colliculus. The origin of 5-HT input to the dorsal PAG, on the other hand, showed many similarities to the origin of 5-HT innervation of the superior colliculus. These data also indicate that approximately 35% of raphe neurons provide nonserotoninergic projections to the PAG and superior colliculus.     

Association of serotonin-like immunoreactive axons with nociceptive projection neurons in the caudal spinal trigeminal nucleus of the rat

Serotoninergic projections to the spinal dorsal horn are implicated in the modulation of nociceptive transmission. However, morphological evidence indicating that serotoninergic projection fibers make synapses on nociceptive neurons in the medullary dorsal horn is still meager. Thus, we examined whether axonal varicosities with serotonin (5-HT)-like immunoreactivity (5-HT-LI) might make synapses on nociceptive projection neurons in the caudal spinal trigeminal nucleus (Vc) of the rat. Projection neurons were retrogradely labeled with tetramethylrhodamine-dextran amine (TMR-DA) or wheat germ agglutinin-horseradish peroxidase (WGA-HRP) that was injected into the parabrachial or thalamic region. Vc neurons in which c-fos protein-like immunoreactivity (Fos-LI) was induced by subcutaneous injection of formalin into the lip were considered nociceptive. Vc neurons in direct contact with axonal varicosities that bind isolectin I-B4 were also considered nociceptive. Triple labeling for 5-HT, TMR-DA, and Fos as well as that for 5-HT, TMR-DA, and I-B4 were done by using the immunofluorescence and fluorescence histochemical techniques. Confocal laser-scanning microscopy revealed that axonal varicosities with 5-HT-LI were in close apposition to TMR-DA-labeled neurons showing Fos-LI in lamina I and the outer part of lamina II (lamina IIo), and that both axonal varicosities with 5-HT-LI and those binding I-B4 were in close apposition to single neuronal profiles labeled with TMR-DA. The presumed nociceptive neuronal profiles in close apposition to axon terminals with 5-HT-LI were mainly those of laminae I and II neurons as well as dendrites of lamina III neurons. Electron microscopy confirmed that axon terminals with 5-HT-LI and those with I-B4 binding activity in laminae I and II made synapses on somatic and dendritic profiles that were labeled with WGA-HRP. The results indicate that serotoninergic neurons project directly on nociceptive projection neurons in the Vc. J. Comp. Neurol. 384:127-141, 1997. © 1997 Wiley-Liss, Inc.     

Methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) cause selective ablation of serotonergic axon terminals in forebrain: immunocytochemical evidence for neurotoxicity

The psychotropic amphetamine derivatives 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) have been used for recreational and therapeutic purposes in man. In rats, these drugs cause large reductions in brain levels of serotonin (5-HT). This study employs immunocytochemistry to characterize the neurotoxic effects of these compounds upon monoaminergic neurons in the rat brain. Two weeks after systemic administration of MDA or MDMA (20 mg/kg, s.c., twice daily for 4 d), there is profound loss of serotonergic (5-HT) axons throughout the forebrain; catecholamine axons are completely spared. Regional differences in drug toxicity are exemplified by partial sparing of 5-HT axons in hippocampus, lateral hypothalamus, basal forebrain, and in some areas of neocortex. The terminals of 5-HT axons are selectively ablated, while axons of passage and raphe cell bodies are spared. Thickened preterminal fibers exhibit increased staining due to damming-up of neurotransmitter and other axonal constituents. Fine 5-HT axon terminals are extremely vulnerable to these drugs, whereas terminal-like axons with large varicosities survive, raising the possibility that some 5-HT axons may be resistant to the neurotoxic effects. At short survivals, visualization of greatly swollen, fragmented 5-HT axons provides anatomic evidence for degeneration of 5-HT projections. The results establish that MDA and MDMA produce structural damage to 5-HT axon terminals followed by lasting denervation of the forebrain. Both drugs have similar effects, but MDA produces a greater reduction of 5-HT axons than does MDMA at the same dosage. The selective degeneration of 5-HT axons indicates that these drugs may serve as experimental tools to analyze the organization and function of 5-HT projections. Caution should be exercised until further studies determine whether these compounds may be hazardous in man.     

Organization of raphe-cortical projections in rat: A quantitative retrograde study

Retrograde transport of a fluorescent dye was employed to study the projections from raphe nuclei to neocortex in the rat. The spatial distributions of labeled raphe cells were analyzed quantitatively to determine whether the nuclei are topographically organized with respect to different cortical targets. The dorsal raphe nucleus (DRN), exclusive of the lateral wing regions, has a predominantly (3:1) ipsilateral projection with decreasing numbers of cells projecting to frontal, parietal, and occipital cortex. Overlapping cell groups within the DRN project differentially to these three cortical areas: DRN cells innervating frontal cortex extend more rostrally and laterally than those to either parietal or occipital cortex. The medium raphe and B9 projections are bilaterally symmetric, with equal cell numbers projecting to frontal, parietal, and occipital cortex. The rostro-caudal distributions of cells that project to disparate cortical areas differ in B9 but not in MR. The percentage of cortically projecting cells that are serotonergic is 80% for the DRN, 60% in the MR and 33% in the B9 cell group. The dorsal raphe nucleus and the B9 cell group are organized heterogeneously, and overlapping sets of neurons project differentially upon particular areas of neocortex. In contrast, the median raphe nucleus projects uniformly upon the neocortex and does not exhibit topographic organization. The three rostral raphe nuclei (DR, MR and B9) are each organized according to different rules with regard to their efferent projections to cortex. The differential organization of the raphe nuclei suggests that groups of cells within these three raphe nuclei are likely to innervate different combinations of cortical targets and thus to have different functional effects.     

The origin of serotoninergic afferents to the cat's cerebellar nuclei

In the cat, serotoninergic (5HT) axons and terminals form a dense plexus that is present throughout the granule cell and Purkinje cell layers of the cerebellar cortex and all of the cerebellar nuclei. The intent of the present study was to identify the source of 5HT fibers in the cerebellar nuclei. The medial, interposed, and lateral cerebellar nuclei were selectively injected with either rhodamine or fluorescein-labeled latex microspheres that were retrogradely transported to brainstem neurons. Transverse sections of the brainstem were processed with a primary antibody to 5HT and secondary antibody tagged with either rhodamine or fluorescein. The location of neurons containing both serotonin-like immunoreactivity and retrogradely transported microspheres was plotted. All three of the cerebellar nuclei receive 5HT afferents from the nucleus locus coeruleus, the dorsal raphe nucleus, and the dorsal tegmental nucleus. In addition, the medial nucleus receives projections from the superior central nucleus, the nucleus raphe obscurus, the nucleus raphe magnus, and the periolivary reticular formation. The interposed nuclei receive additional projections from the nucleus raphe magnus, whereas the lateral nucleus receives additional projections from the superior central nucleus. In conclusion, the 5HT projections to the cerebellar nuclei do not appear to be collaterals of those projecting to the cortex (Kerr and Bishop, J Comp Neurol 304:502–515, 1991). These findings suggest that, although the cortex and nuclei are anatomically and physiologically related, they do not process all information in parallel. © Wiley-Liss, Inc.     

Projections of the median raphe nucleus in the rat

No previous report in any species has examined comprehensively the projections of the median raphe (MR) nucleus with modern tracing techniques. The present report represents an in depth analysis of the projections of MR by use of the anterograde anatomical tracer Phaseolus vulgaris-leucoagglutinin. MR fibers descend along the midline within the brainstem and mainly ascend within the medial forebrain bundle in the forebrain. MR fibers distribute densely to the following brainstem/forebrain sites: caudal raphe nuclei, laterodorsal tegmental nucleus, dorsal raphe nucleus, interpeduncular nucleus, medial mammillary body, supramammillary nucleus, posterior nucleus and perifornical region of the hypothalamus, midline and intralaminar nuclei of thalamus, dopamine-containing cell region of medial zona incerta, lateral habenula, horizontal and vertical limbs of the diagonal band nuclei, medial septum, and hippocampal formation. Virtually all of these structures lie on or close to the midline, indicating that the MR represents a midline/para-midline system of projections. Overall, MR projections to the cortex are light. MR projects moderately to the perirhinal, entorhinal and frontal cortices, but sparingly to remaining regions of cortex. A comparison of MR with dorsal raphe (DR) projections (Vertes RP. 1991. J Comp Neurol 313:643-668) shows that these two major serotonin-containing cell groups of the midbrain distribute to essentially nonoverlapping regions of the forebrain; that is, the MR and DR project to complementary sites in the forebrain. A direct role for the MR in the desynchronization of the electroencephalographic activity of the hippocampus and its possible consequences for memory-associated functions of the hippocampus is discussed.     

Correspondence between 5-HT2 receptors and serotonergic axons in rat neocortex

The anatomic relationship between serotonergic (5-HT) axons and 5-HT2 receptors in the rat forebrain was determined by a combined analysis of transmitter immunocytochemistry and receptor autoradiography. High densities of 5-HT2 receptors, localized by the ligand N1-methyl-2-125I-LSD (125I-MIL), are found in neocortex and striatum; these regions also receive a dense serotonergic innervation. Regional variations in the density of 5-HT2 receptors and 5-HT axons correspond closely in most, but not all, areas of the forebrain. In somatosensory cortex (SI), the laminar distribution of 5-HT2 receptors closely matches that of 5-HT axons: in particular, a dense band of 5-HT2 receptors in layer Va of SI is in precise register with a dense plexus of fine 5-HT axons. We have also observed a close spatial relationship between 5-HT2 receptors and fine axons in other areas of the forebrain, suggesting that 5-HT2 receptors may be selectively linked to a particular type of 5-HT axon terminal. Since fine axons of this type have been reported to arise from the dorsal raphe nucleus, it appears likely that 5-HT2 receptors may mediate the effects of dorsal but not median raphe projections.     

Effect of the selective lesion of serotoninergic neurons on the regional distribution of 5-HT1A receptor mRNA in the rat brain.

The effects of the selective lesion of serotoninergic neurons by an intra-raphe administration of 5,7-dihydroxytryptamine on the 5-HT1A receptor protein and the 5-HT1A receptor mRNA were examined in various regions of the rat brain using specific antibodies and an antisense riboprobe, respectively. Twenty one days after the treatment, the 5-HT1A receptor protein was no longer detected within the dorsal raphe nucleus but was still present in the hippocampus and entorhinal cortex. Quantitative in situ hybridization showed an 85% decrease in the levels of 5-HT1A receptor mRNA within the dorsal raphe nucleus, but no significant change in the hippocampus, interpeduncular nucleus and entorhinal cortex of 5,7-dihydroxytryptamine-treated rats. These data demonstrate that 5-HT1A receptors are synthesized by serotoninergic neurons in the dorsal raphe nucleus, and by neurons located postsynaptically with regard to serotoninergic projections in other areas. The unchanged levels of 5-HT1A receptor mRNA in the hippocampus, interpeduncular nucleus and entorhinal cortex three weeks after the extensive lesion of serotoninergic neurons are consistent with the absence of 5-HT1A receptor up regulation already reported under this condition.     

Serotonergic afferents to the rat olfactory bulb: I. Origins and laminar specificity of serotonergic inputs in the adult rat

This is a study of the chemoanatomical organization of the projection from the raphe nuclei to the main olfactory bulb in the rat. A heavy projection from the dorsal and median raphe nuclei to the main olfactory bulb was shown by both retrograde and anterograde tracing techniques. Following injections of 1% wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the main olfactory bulb, up to 1300 neurons were retrogradely labeled in the dorsal and median raphe nuclei, a much larger number than has been suggested by previous studies. By combining 5-HT immunofluorescence with True blue retrograde fluorescent labeling, it was determined that the vast majority of raphe neurons that project to the olfactory bulb contain serotonin. Injections of WGA-HRP into dorsal and/or median raphe produced dense anterograde labeling in the glomeruli, while fewer labeled fibers were observed in the external plexiform layer, internal plexiform layer, and granule cell layer. In contrast to the number of other centrifugal afferents to the bulb, a significant contingent of fibers from the raphe nuclei enters the main olfactory bulb (MOB) from outside in, i.e., via the olfactory nerve layer. Serotonergic fibers in MOB were visualized by immunocytochemistry, and the distribution of specific 5-HT fibers closely matched the distribution of anterograde terminal label resulting from injections of WGA-HRP in the raphe nuclei. The serotonergic fibers have a specific laminar distribution and morphology in MOB. Thus, the density of the serotonergic innervation to the glomerular layer is 2-3 times that of any other layer in MOB. In addition to their preferential innervation of the glomeruli, glomerular and infraglomerular serotonergic fibers are morphologically different. Serotonergic fibers located in the glomerular layer are generally thick (0.25-0.60 micron) compared to the thinner (0.25-0.35 micron) fibers that predominate in infraglomerular layers. Another difference is that the glomerular fibers often contain varicosities that are greater than 1 micron in diameter, while varicosities along infraglomerular fibers are usually barely larger than the axonal diameter. Finally, glomerular fibers are much more intensely stained than infraglomerular fibers. Electrolytic lesions of the dorsal and median raphe caused a total depletion of serotonin fiber staining in the bulb, demonstrating that the raphe nuclei are the sole source of the serotonergic input to the main olfactory bulb. Thus, it has been demonstrated that serotonergic neurons in dorsal and median raphe project very heavily to glomeruli and less heavily to other layers in the main olfactory bulb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct projections from serotonergic neurons in the dorsal and median raphe nuclei of midbrain to the suprachiasmatic nucleus in Tupaia belangeri chinensis

This study investigated the direct serotonergic projections to the suprachiasmatic nucleus (SCN) from the dorsal and median raphe nuclei (DR/MR) of the midbrain in Tupaia belangeri chinensis (TBC) by combined application of retrograde horseradish peroxidase (HRP) tract tracing, immunohistochemistry, and electron microscope techniques. The results provide evidence for the direct projections to the SCN from serotonergic neurons distributed predominantly in the MR (mainly in its lateral portion) and to a lesser degree in the DR (in its ventrolateral portion) more caudally in the midbrain, and the existence of abundant symmetrical and asymmetrical synaptic connections between the serotonergic terminals and the postsynaptic elements in the SCN TBC. The results also revealed that almost all DR neurons projecting to the SCN contained serotonin, whereas about one-half of MR neurons projecting to the SCN were immunoreactive for serotonin.     

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.     

Serotonin1B receptors: from protein to physiological function and behavior

The serotonin (5-HT)1B receptor is expressed in the central nervous system (CNS) of rodents and its homologous 5-HT1D beta receptor is expressed in human. These receptors are distributed in both serotonergic and non-serotonergic neurons, where they act as auto- or heteroreceptors, respectively. Studies from ours and other laboratories have shown that 5-HT1B receptors are densely expressed in the ventral pallidum, globus pallidus, substantia nigra and dorsal subiculum and moderately expressed in the cerebral cortex, the molecular layer of the hippocampus, the entopeduncular nucleus, the superficial gray layer of the superior colliculus, the caudate putamen and the deep nuclei of the cerebellum. At the ultrastructural level, 5-HT1B receptors were found distributed in axons and axon terminals and these receptors are located on the plasma membrane of unmyelinated axon terminals and in the cytoplasm close to the plasmalemma. The terminal localization of the 5-HT1B receptors in CNS suggests that there is a signal responsible for the protein transport toward the nerve terminals. Studies from ours and other groups using lesion, radioligand binding sites, viral transfection and anterograde methods have shown that 5-HT1B receptors are located at the nerve terminals of different pathways. The 5-HT1B receptors act as terminal receptors and are involved in regulation of the release of various neurotransmitters, including 5-HT itself. The regulation of gamma-aminobutyric acid release by 5-HT1B receptors has been found in projections: from caudate putamen to the globus pallidus or substantia nigra, from nucleus accumbens to the ventral tegmentum area, and from purkinje neurons to the deep nuclei of the cerebellum. The control of glutamate release by 5-HT1B receptors has been found in projections from hippocampus to the dorsal subiculum and of N-acetyl-aspartyl-glutamate release from retinal ganglion cells to the superficial gray layer of the superior colliculus. The control of 5-HT release by 5-HT1B receptors was shown in projections arising from the raphe nuclei to fore- and midbrain regions. Multiple evidences suggest that 5-HT1B receptors are implicated in several physiological functions, behavior and psychiatric diseases including migraine, locomotor activity, drug abuse reinforcement, migraine, aggressive behavior, depression and anxiety states.