Serotonin projection patterns to the cochlear nucleus

The cochlear nucleus is well known as an obligatory relay center for primary auditory nerve fibers. Perhaps not so well known is the neural input to the cochlear nucleus from cells containing serotonin that reside near the midline in the midbrain raphe region. Although the specific locations of the main, if not sole, sources of serotonin within the dorsal cochlear nucleus subdivision are known to be the dorsal and median raphe nuclei, sources of serotonin located within other cochlear nucleus subdivisions are not currently known. Anterograde tract tracing was used to label fibers originating from the dorsal and median raphe nuclei while fluorescence immunohistochemistry was used to simultaneously label specific serotonin fibers in cat. Biotinylated dextran amine was injected into the dorsal and median raphe nuclei and was visualized with Texas Red, while serotonin was visualized with fluorescein. Thus, double-labeled fibers were unequivocally identified as serotoninergic and originating from one of the labeled neurons within the dorsal and median raphe nuclei. Double-labeled fiber segments, typically of fine caliber with oval varicosities, were observed in many areas of the cochlear nucleus. They were found in the molecular layer of the dorsal cochlear nucleus, in the small cell cap region, and in the granule cell and external regions of the cochlear nuclei, bilaterally, of all cats. However, the density of these double-labeled fiber segments varied considerably depending upon the exact region in which they were found. Fiber segments were most dense in the dorsal cochlear nucleus (especially in the molecular layer) and the large spherical cell area of the anteroventral cochlear nucleus; they were moderately dense in the small cell cap region; and fiber segments were least dense in the octopus and multipolar cell regions of the posteroventral cochlear nucleus. Because of the presence of labeled fiber segments in subdivisions of the cochlear nucleus other than the dorsal cochlear nucleus, we concluded that the serotoninergic projection pattern to the cochlear nucleus is divergent and non-specific. Double-labeled fiber segments were also present, but sparse, in the superior olive, localized mainly in periolivary regions; this indicated that the divergence of dorsal and median raphe neurons that extends throughout regions of the cochlear nucleus also extended well beyond the cochlear nucleus to include at least the superior olivary complex as well.     

Morphology of the cochlear nucleus of the normal and reeler mutant mouse

The morphology of the cochlear nuclei of normal and reeler mutant mice were studied in Nissl-stained sections. The cochlear nucleus in both mice is divisible into three parts: the anteroventral, posteroventral, and dorsal nuclei. Nine cell types can be recognized in the normal mouse. In the anteroventral nucleus spherical cells occupy the rostral pole. Globular cells are located caudally and extend to the interstitial region of the anteroventral nucleus. In the posteroventral nucleus multipolar cells are located rostrally and dark-staining cells occupy the caudal pole. Multipolar cells are also present in the anteroventral nucleus and in the deep region and molecular layer of the dorsal cochlear nucleus. The dorsal and lateral aspects of the ventral nuclei are covered by a granule cell layer. The dorsal nucleus consists of superficial molecular and pyramidal layers and a deep region. The deep region contains small and giant cells as well as multipolar cells. The pyramidal layer is made up of pyramidal cells, horizontal cells, and granule cells. Small cells are also present in the molecular layer and throughout the ventral nuclei. The dorsal cochlear nucleus of the reeler mutant mouse is disorganized and the molecular layer is reduced in thickness. The organization of the pyramidal layer is disrupted with granule cells superficial to pyramidal and horizontal cells. Cells which appear to be homologous to pyramidal cells are also present in the deep region of the dorsal nucleus. The total number of granule cells is reduced by an average of 42% over the whole nucleus and the reduction in granule cells is greatest in the granule cell cap covering the dorsal and lateral surface of the ventral cochlear nuclei. The cytoarchitecture of the ventral cochlear nucleus appears normal.     

Distribution and origin of noradrenergic and serotonergic fibers in the cochlear nucleus and inferior colliculus of the rat.

We examined the monoaminergic innervation of the rat cochlear nucleus (CN) and the inferior colliculus (IC) by using retrograde transport of the fluorescent dye Fluoro-Gold combined with immunohistochemistry. We used antisera against the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), and one against the transmitter serotonin (5-HT). Each substance revealed a distinct pattern of immunoreactive staining in the CN and the IC. In the CN, DBH-immunoreactive (-ir) fibers were present in all subnuclei. The molecular layer of the dorsal CN and the granular layer of the ventral CN, however, were largely devoid of DBH-ir fibers. In contrast, 5-HT-ir fibers were abundant in the molecular layer and the granular cell layer of the CN. In the dorsal CN and the postero- and anteroventral CN, however, this innervation was less dense and evenly distributed across subnuclei. In the IC, the DBH-ir fibers were slightly more numerous in layer 2 of the dorsal cortex than in other subnuclei, while the layer 1 of both the dorsal and the external cortex contained only a few fibers. In contrast, the 5-HT-ir fibers formed a dense network in both the dorsal and external cortices of the IC, while they were less abundant in the remaining subnuclei. PNMT-ir fibers were not found in any of the auditory brainstem nuclei. Following Fluoro-Gold injections into the CN or IC, retrogradely labeled DBH-ir neurons were found in the A6 noradrenergic cell group (locus coeruleus). The CN received additional projections from the A5 noradrenergic cell group, as well as sparse projections from the A4 and A7 cell groups. The serotonergic innervation of the CN and IC originated largely in the B7 serotonergic cell group (dorsal raphe nucleus). Serotonergic neurons in other groups of the raphe nuclei were only occasionally labeled. Our data indicate that both noradrenaline and serotonin may play a role in central auditory processing. Their differential distribution in the IC and CN subnuclei suggests that these transmitter systems might influence different functional circuits.     

Serotoninergic neurons in the mormyrid brain and their projection to the preelectromotor and primary electrosensory centers: immunohistochemical study

Serotonin-containing neurons in the brain of the weak-electric fish Gnathonemus petersii (mormyridae, teleostei) were studied with the aid of immunohistochemical labeling. Study of the central serotoninergic innervation was focused on the structures subserving the command of the electric organ and the first central relay of the electrosensory system. In the midline raphe nuclei, serotoninergic neurons formed a column that stretched from the ventral caudal medulla to the dorsal midbrain, ending caudal to the cerebellar peduncle. In the dorsal tegmentum, serotoninergic neurons were found bilaterally at the anterior margin of the decussation of the lateral lemniscus. Labeled neurons were also present bilaterally immediately anterior to the cerebellar peduncle and also in the pretectal region. In the hypothalamus, many serotoninergic neurons were in contact with the ventricular wall, and a few were present in the preoptic area. This distribution of serotoninergic cell bodies showed many similarities to that in other fish and higher vertebrates but lacked the lateral spread of the serotoninergic raphe system found in the midbrain tegmentum in mammals. Labeled fibers were found in both the preelectromotor medullary relay nucleus and the electromotor command nucleus. These serotoninergic projections were traced to the posterior raphe. Serotoninergic fibers also formed a dense network in the cortex and in the nucleus of the electrosensory lobe, both of which receive primary input from electroreceptors. These results suggest that serotonin may have a role in the modulation of the intrinsic, rhythmic electromotor command and in the gating of electrosensory input.     

Morphology and distribution of serotoninergic and oculomotor internuclear neurons in the cat midbrain

Serotoninergic fibers have been reported in both the abducens and facial nuclei of the cat. Furthermore, serotoninergic dorsal raphe and oculomotor internuclear neurons occupy similar locations in the periaqueductal gray overlying the oculomotor and trochlear motor nuclei. To resolve the issue of whether these two populations of neurons overlap, serotoninergic fibers were assayed in the abducens and facial nucleus; then the morphologies and distributions of identified serotoninergic neurons and oculomotor internuclear neurons were determined. Both the abducens and facial nuclei contained varicosities labelled with antibody to serotonin, but a much higher density of immunoreactive fibers was present in the latter, especially in its medial aspect. Distinct synaptic profiles labelled with antibodies to serotonin were observed in both nuclei. In both cases, terminal profiles contained numerous small, predominantly spheroidal, synaptic vesicles as well as a few, large, dense-core vesicles. These profiles made synaptic contacts onto dendritic and, in the facial nucleus, somatic profiles that occasionally displayed asymmetric, postsynaptic, membrane densifications. Following injection of horseradish peroxidase into either the abducens or facial nuclei, double-label immunohistochemical techniques demonstrated that the serotoninergic and oculomotor internuclear neurons form two distinct cell populations. The immunoreactive serotoninergic cells were distributed within the dorsal raphe nucleus, predominantly caudal to the retrogradely labelled oculomotor internuclear neurons. The latter were located in the oculomotor nucleus along its dorsal border and in the adjacent supraoculomotor area. Intracellular injection of horseradish peroxidase revealed that oculomotor internuclear neurons have multipolar somata with up to ten long, tapering dendrites that bifurcate approximately five times. Their dendritic fields were generally contained within the nucleus and adjacent supraoculomotor area. In contrast, putative serotoninergic neurons were often spindle-shaped and exhibited far fewer primary dendrites. Many of these long, narrow, sparsely branched dendrites crossed the midline and extended to the surface of the cerebral aqueduct. In the vicinity of the aqueduct they branched repeatedly to form a dendritic thicket. The axons of the intracellularly stained serotoninergic neurons emerged either from the somata or the end of a process with dendritic morphology, and in some cases they produced axon collaterals within the periaqueductal gray. Thus the oculomotor internuclear and serotoninergic populations differ in both distribution and morphology.(ABSTRACT TRUNCATED AT 400 WORDS)     

Topographical organization in the origin of serotoninergic projections to different regions of the cat cerebellar cortex

The distribution of serotonin immunoreactivity in the cat cerebellum was studied by using the indirect antibody peroxidase-antiperoxidase (PAP) technique. Furthermore, the origin of these chemically defined afferents was determined by combining the retrograde transport of horseradish peroxidase (HRP) with the PAP technique. In the cerebellar cortex, serotonin immunoreactivity is present in a plexus of beaded fibers that is confined almost exclusively to the granule and Purkinje cell layers; a few fibers are present in the molecular layer. Serotoninergic axons and varicosities have a dense and uniform distribution throughout all lobules of the cerebellum with the exception of lobule X where the fiber density is sparse. Serotonin cell bodies were not found within the cerebellar cortex. However, following pretreatment with pargyline and L-tryptophan, serotonin positive cell bodies were found in all deep cerebellar nuclei as well as the raphe and reticular nuclei in the brainstem. The present study demonstrates that the serotoninergic projection to the cat's cerebellum has some degree of topographical organization. Serotoninergic fibers in the anterior vermis (lobules I-V) were shown to arise from neurons located within the paramedian reticular nucleus, the lateral reticular nucleus, and the lateral tegmental field. Injections of HRP into either the posterior vermis (lobule VI-IX) or the paramedian lobule, labeled serotoninergic neurons exclusively in the lateral reticular nucleus. Lobus simplex, crus I and crus II (the hemisphere) receive a serotoninergic input from cells located in the lateral tegmental field, the peri-olivary reticular formation and the paramedian reticular nucleus. In no cases were neurons in the raphe double-labeled, although there were cells positive for HRP or serotonin alone. The data indicate that there is a topographical organization in the serotoninergic projection from the caudal brainstem to specific regions of the cat's cerebellar cortex. In addition to climbing and mossy fibers, this projection represents a third major source of cerebellar afferents based on its dense and widespread distribution as well as its morphological and chemical characteristics.     

Cell bodies of origin of serotonin-immunoreactive afferents to the inferior olivary complex of the rat

Previous studies have used immunohistochemistry to localize serotonin to distinct olivary nuclei in several mammalian species. However, the location of the cell bodies of origin for the serotoninergic projection to the inferior olive in any of these species was unknown. In the present study, a paradigm which combines transport of horseradish peroxidase (HRP) and serotonin immunohistochemistry (PAP) was used to identify the cell bodies of origin of this afferent system to the inferior olivary complex of the rat. Cells which contain both retrogradely transported HRP and brown cytoplasmic staining indicating that they are serotoninergic cells that project to the inferior olivary complex are found exclusively in an area dorsal to the rostrolateral dorsal accessory olive within the nucleus reticularis paragigantocellularis. Neurons within this nucleus were also found to be a source of serotoninergic afferents to the cerebellum and spinal cord of the rat. This raises the possibility that individual serotonin-immunoreactive neurons within this nucleus may project to all 3 areas. Future studies will be designed to address this possibility. No double-labeled cells were observed within any of the raphe nuclei.     

Central innervation of the rat ependyma and subcommissural organ with special reference to ascending serotoninergic projections from the raphe nuclei

The subcommissural organ (SCO) and the cerebral ependyma receive serotoninergic innervation, but little is known about their origin in the raphe nuclei. Application of the retrograde tracer cholera toxin subunit B (ChB) in the third ventricle resulted in uptake in ependymal axons and backfilling of perikarya in the dorsomedian part of the dorsal raphe nucleus, immediately under the caudal aqueduct. By using dual staining with antisera against serotonin and ChB, a portion of the retrogradely labeled neurons was observed to co-store serotonin. Phaseolus vulgaris–leucoagglutinin (PHA-L) was injected into different raphe nuclei to fill the neurons in the same areas where the retrogradely labeled neurons were found. PHA-L injection in the midline of the dorsal raphe nucleus gave rise to ascending axonal processes in the mesencephalic central gray, from where they entered the periventricular strata and the third ventricular ependyma. In the cerebral ependyma, large numbers of positive fibers were consistently found in the ventral part of the lateral ventricles and in the dorsal part of the third ventricle. A large number of PHA-L-immunoreactive fibers were observed in the hypendymal layer of the lateral part of the SCO. Terminal fibers near the ependymal cells were also observed. In all cases, the PHA-L injections labeled innervating fibers both within the ependyma and in the SCO, whereas injections into the median raphe nucleus or in other raphe nuclei (i.e., the raphe pallidus and the raphe pontis) labeled fibers neither in the SCO nor in the ependyma. This study shows that a specific group of predominantly serotoninergic neurons innervates both the ependyma and the SCO and is probably involved in cerebrospinal fluid regulation. J. Comp. Neurol. 384:556–568, 1997. © 1997 Wiley-Liss, Inc.     

Auditory projections from the cochlear nucleus to pontine and mesencephalic reticular nuclei in the rat.

We investigated projections from the cochlear nucleus in the rat using the anterograde tracer Phaseolus vulgaris-leucoagglutinin. We focused on nuclei in the brainstem which are not considered to be part of the classical auditory pathway. In addition to labeling in auditory nuclei, we found presumed terminal fibers in 4 pontine and mesencephalic areas: (1) the pontine nucleus (PN), which receives bilateral projections from the antero- and posteroventral cochlear nuclei; (2) the ventrolateral tegmental nucleus (VLTg), which receives a contralateral projection from the rostral portion of the anteroventral cochlear nucleus; (3) the caudal pontine reticular nucleus (PnC), which receives bilateral input originating predominantly in the dorsal cochlear nucleus; and (4) the lateral paragigantocellular nucleus (LPGi), which receives projections from all subdivisions of the cochlear nuclei. In the VLTg and PnC, anterogradely labeled varicose axons were often found in close apposition to the primary dendrites and somata of large reticular neurons. Injections of the retrograde fluorescent tracer Fluoro-Gold into the VLTg demonstrated that the neurons of origin are mainly located contralaterally in the rostral anteroventral cochlear nucleus and in the cochlear root nucleus. The relevance of these auditory projections for short-latency audio-motor behaviors and acoustically elicited autonomic responses is discussed.     

Selective labeling of spiral ganglion and granule cells with D-aspartate in the auditory system of cat and guinea pig

The present study sought to locate putative glutamatergic or aspartatergic pathways in the auditory system of cats and guinea pigs. We injected 0.06 to 3 mM D-[3H] aspartate (D-Asp) in the cochlear nucleus before preparation for light microscopic autoradiography. At short survival times (15 and 40 min) there was heavy labeling of astrocytic somata. Labeling patterns typical of cochlear nerve endings decorated neurons in the cochlear nucleus, e.g., cell bodies and dendritic trunks of octopus cells. Labeling patterns consistent with retrograde axonal transport by the parallel fibers of granule cells appeared in the molecular layer of the dorsal cochlear nucleus and in the external granular layer. Retrograde labeling of the cochlear nerve root fibers also occurred. Consistent with these results are companion biochemical findings on the rapidly dissected cochlear nuclei of guinea pigs. The dorsal, anteroventral, and posteroventral cochlear nuclei, each, evinced uptake of D-Asp. Subsequently, electrical stimulation of each nucleus released a portion of the accumulated amino acid. Most of this release probably came from synaptic endings. Another group of experiments compared autoradiographic localization of 0.06 to 3 mM D-Asp to that of horseradish peroxidase (HRP) 6 hr to 2 d after injections in the cochlear nucleus. Astroglial cell bodies were no longer labeled by D-Asp, but spiral ganglion cell bodies in the cochlea and granule cell bodies in the cochlear nucleus were. Perikarya of the periolivary and ventral cochlear nuclei projecting to the dorsal cochlear nucleus were labeled by HRP and not by D-Asp. Thus, comparisons with the HRP findings indicate that D-Asp labeling resulted from a selective retrograde transport. There was no evidence for a selective anterograde axonal transport. The present observations support the hypothesis that cochlear nerve fibers and granule cells may use L-glutamate and/or L-aspartate as a transmitter in the cochlear 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.     

The origins, course and distribution of the dorsal and intermediate acoustic striae in the rhesus monkey

Attempts were made to produce discrete stereotactic lesions in the dorsal cochlear nucleus (Dc) in a series of rhesus monkeys. Anterograde degeneration was studied using Nauta and Fink-Heimer techniques. Following lesions confined to the caudal pole of Dc, a few degenerated fibers projected to the contralateral lateral superior olivary nucleus via the dorsal acoustic stria. Degeneration was distributed to the contralateral ventral and to a lesser extent dorsal nuclei of the lateral lemniscus. The contralateral central nucleus of the inferior colliculus was a major site of termination of second order auditory fibers; a small number of degenerated fibers ascended to this nucleus ipsilaterally, and a few crossed in its commissure. A few degenerated fibers also were present in the contralateral magnocellular division of the medial geniculate body. When Dc was damaged at levels which overlie the posteroventral cochlear nucleus (Pv), or after lesions involving both subdivisions, degeneration was seen in the intermediate as well as in the dorsal acoustic stria. Fibers arising in Pv coursed through Dc and were interrupted by lesions in the latter. In addition to degeneration described above, fibers projected to the ipsilateral lateral superior olivary nucleus, contralateral medial trapezoid nucleus, and bilaterally to dorsal retroolivary and to preolivary cell groups. No degeneration was seen in the trapezoid body. Lesions involving the posteroventral and anteroventral (Av) cochlear nuclei produced degeneration in the intermediate acoustic stria and in the trapezoid body, but not in the dorsal stria. Degeneration in the intermediate acoustic stria was similar to that present after lesions affecting Dc and Pv. Thus, since Av does not project crossed fibers to this structure, these findings indicate that Pv sends some fibers to the contralateral as well as ipsilateral lateral superior olivary nucleus.     

Brainstem origin of serotonin- and enkephalin-immunoreactive afferents to the opossum's cerebellum

Previous studies have described the distribution of serotonin- and enkephalin-immunoreactive elements in the posterior lobe vermis of the opossum's cerebellum. In the present study we have used a double labeling paradigm which combines the retrograde transport of horseradish peroxidase (HRP) with serotonin and enkephalin immunohistochemistry to determine the brainstem origin of serotoninergic and enkephalinergic neurons that project to the opossum's cerebellar cortex. Subsequent to HRP injections into the posterior lobe vermis, widespread areas of the medulla and pons were found to contain retrogradely labeled neurons. Serotonin-immunoreactive somata are present primarily in the raphe nuclei and the adjacent reticular formation. Enkephalinergic neurons were numerous in the raphe nuclei, medial accessory olive, gigantocellular reticular formation, locus coeruleus, and the nucleus of the trapezoid body. However, serotoninergic neurons that project to the cerebellum were located only in the medullary pyramids and the reticular formation adjacent to the raphe. Double-labeled enkephalinergic neurons were located 1) within the medullary pyramids, 2) throughout the extent of the caudal medial accessory olive, 3) in the rostral subnucleus a of the medial accessory olive, 4) in the nucleus reticularis gigantocellularis pars ventralis, 5) in the nucleus reticularis lateralis, and 6) in the nucleus reticularis ventralis lateral to the inferior olivary complex. These results indicate that although neurons containing serotonin and enkephalin immunoreactivity may be present in some of the same pontine and medullary nuclei, those serotoninergic and enkephalinergic neurons that project to the cerebellum are present primarily in restricted and spatially separate regions of the caudal medulla.     

Distribution of neuropeptide Y-like immunoreactive cell bodies and fibers in the brain stem of the cat

By using intratissue injections of colchicine and an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibers containing neuropeptide Y-like immunoreactivity in the brain stem of the cat. The densest clusters of immunoreactive perikarya were observed in the following nuclei: anteroventral cochlear, lateral reticular (internal and external divisions), dorsal tegmental, inferior colliculus and dorsal nucleus of the lateral lemniscus. By contrast, the nuclei abducens, the nucleus of the trapezoid body, preolivary, interpeduncularis, infratrigeminal, gigantocellular tegmental field, coeruleus and dorsal motor nucleus of the vagus had the lowest density. Finally, a moderate density of neuropeptide Y-like immunoreactive cell bodies was found in the nuclei: lateral tegmental field, laminar spinal trigeminal, praepositus hypoglossi, superior colliculus, lateral vestibular and motor trigeminal. In addition, a mapping of the neuropeptide Y-like immunoreactive fibers was carried out. Thus, the densest network of immunoreactive fibers was observed in the laminar spinal trigeminal nucleus. The nuclei periaqueductal gray, inferior central, praepositus hypoglossi, postpyramidal raphe, dorsal raphe, incertus and medial vestibular contained a moderate density of immunoreactive fibers, whereas the nuclei interpeduncularis, inferior colliculus, superior central, gracile, retrorubral, K?lliker-Fuse, dorsal tegmental, ambiguus and alaminar spinal trigeminal had the lowest density of neuropeptide Y-like immunoreactive fibers. The anatomical location of neuropeptide Y-like immunoreactivity suggests that the peptide could play an important role in several physiological functions, e.g., those involved in cardiovascular, auditory, motor, visual, nociceptive and somatosensory mechanisms.     

Localization of NADPH diaphorase activity in monoaminergic neurons of the rat brain

Nitric oxide has recently been implicated as a neurotransmitter, and may modulate synaptic transmission, cerebral blood flow, and neurotoxicity. NADPH diaphorase histochemistry has been shown to be a reliable marker for nitric oxide synthase, the enzyme that synthesizes nitric oxide, in the nervous system. Because monoaminergic neurons frequently contain co-transmitters, we examined whether these cells also exhibit NADPH diaphorase activity. Frozen sections from postnatal and adult rat brains were stained for NADPH diaphorase activity and either serotonin-like immunoreactivity or tyrosine hydroxylase-like immunoreactivity. Numerous neurons in the mesopontine serotoninergic cell groups (including the caudal linear, dorsal, median, supralemniscal, and pontine raphe nuclei) contained both serotonin-like immunoreactivity and NADPH diaphorase activity. Within the dorsal raphe nucleus, approximately 70% of the serotoninergic neurons in the medial subnuclei displayed NADPH diaphorase activity, while less than 10% of the serotoninergic neurons in the lateral subnuclei were doubly labeled. Retrograde labeling with fluorescent microspheres indicated that many raphe-cortical neurons contained NADPH diaphorase activity. No NADPH diaphorase activity was detected in serotoninergic neurons in the medullary nuclei (including the raphe magnus, raphe pallidum, and raphe obscurus). Only a small proportion of tyrosine hydroxylase-like immunoreactive neurons in the periaqueductal gray, rostral linear nucleus, and rostrtrodorsal ventral tegmental area contained NADPH diaphorase activity. Tyrosine hydroxylase-like immunoreactive neurons in the substantia nigra, locus coeruleus, hypothalamus, olfactory bulb, and dorsal raphe nucleus did not contain detectable NADPH diaphorase activity. The observation that many mesopontine (but not medullary) serotoninergic neurons contain NADPH diaphorase activity suggests that these neurons may release both serotonin and nitric oxide. © Wiley-Liss, Inc.     

Projections from the sacculus to the cochlear nuclei in the Mongolian gerbil

The projections of the saccule, an otolith end organ, to the cochlear nuclei were studied using both transganglionic transport and intracellular injection techniques. Labeled fibers and terminals were observed in the anterior and posterior portions of the ventral cochlear nucleus and the dorsal cochlear nucleus. Most terminals were present in the granule cell domain, especially in the subpeduncular corner between the anteroventral cochlear nucleus and the floccular peduncle of the cerebellum. It has been hypothesized that the cochlea in mammals may have developed phylogenetically from the saccule. The projections from the saccule to the cochlear nuclei were investigated in a mammalian species, the Mongolian gerbil, in an attempt to obtain initial information supporting or refuting this hypothesis. The presence of an otolith end organ projection to the cochlear nuclei in rodents should encourage comparative studies in additional aspects of the evolution of the auditory system.     

Raphe nuclei in three cartilaginous fishes,Hydrolagus colliei,Heterodontus francisci,and Squalus acanthias

The vertebrate reticular formation, containing over 30 nuclei in mammals, is a core brainstem area with a long evolutionary history. However, not all reticular nuclei are equally old. Nuclei that are widespread among the vertebrate classes are probably ones that evolved early. We describe raphe nuclei in the reticular formation of three cartilaginous fishes that diverged from a common ancestor over 350 million years ago. These fishes are Hydrolagus colliei, a holocephalan, Squalus acanthias, a small-brained shark, and Heterodontus francisci, a large-brained shark. Nuclear identification was based on immunohistochemical localization of serotonin and leu-enkephalin, on brainstem location, and on cytoarchitectonics. Raphe nuclei are clustered in inferior and superior cell groups, but within these groups individual nuclei can be identified: raphe pallidus, raphe obscurus, and raphe magnus in the inferior group and raphe pontis, raphe dorsalis, raphe centralis superior, and raphe linearis in the superior group. Hydrolagus lacked a dorsal raphe, nucleus, but the nucleus was present in the sharks. The majority of immunoreactive cells are found in the superior group, especially in raphe centralis superior, but immunoreactive cells are present from spinal cord to caudal mesencephalon. The distribution and cytoarchitectonics of serotoninergic and enkephalinergic cells are Similar to each other, but raphe nuclei contain fewer enkephalinergic than serotoninergic cells. The cytoarchitectonics of immunoreactive raphe cells in cartilaginous fishes are remarkably Similar to those described for raphe nuclei in mammals; however, the lack of a raphe dorsalis in Hydrolagus indicates that either it evolved later than the other raphe nuclei or it was lost in holocephalan fishes. © 1995 Wiley-Liss, Inc.     

Pathways connecting the right and left cochlear nuclei

Connections between the right and left cochlear nuclei were studied with retrograde and anterograde axonal transport techniques. Large, multipolar neurons in the anterior and posterior divisions of the anteroventral cochlear nucleus and in the posteroventral cochlear nucleus project to the ventral and dorsal cochlear nuclei on the opposite side. In addition, giant cells in the deep layers of the dorsal cochlear nucleus project to the contralateral posteroventral cochlear nucleus and possibly also to the contralateral dorsal cochlear nucleus. The pattern of terminal distribution of the crossed connections was determined by using the anterograde axonal transport of horseradish peroxidase-labelled wheat germ lectin. Although no part of the cochlear nuclear complex is completely free of anterograde label, the densest labelling is found in the anterior division of the anteroventral cochlear nucleus, throughout the posteroventral cochlear nucleus (where it is closely associated with cell bodies), and in the fusiform and superficial layers of the dorsal cochlear nucleus. These direct synaptic connections from one cochlear nucleus to the other could play a significant role in processes that depend on binaural interactions within the central nervous system.     

Extent of colocalization of serotonin and GABA in the neurons of the rat raphe nuclei

Previous investigations of the distribution of neurons containing both serotonin and GABA in the brainstem raphe nuclei have yielded discrepant results amongst different authors. This study attempted to clarify the distribution as well as the proportions of raphe and other brainstem neurons that contain both neurotransmitters. All the nine serotonergic cell groups known to be present in the brainstem were examined with an indirect immunofluorescence method using antibodies against serotonin and glutamic acid decarboxylase in colchicine-treated rats. Sections were incubated either simultaneously or sequentially for the two immunolabels. Brainstem neurons that were labelled for both markers were generally infrequent. Of all the serotonin cell groups in the brainstem, the nucleus raphe magnus contained the most double-labelled cells (a mean of 3.6% of a total of 625–1155 serotonin-immunoreactive cells counted in this nucleus), followed by the nucleus raphe obscurus (1.5% of a total of 220–550 serotonin-immunoreactive neurons counted). The dorsal, median and pontine raphe nuclei as well as the supralemniscal nucleus (the B9 group) contained very few double-labelled cells, which comprised a mean of 0.1–0.7% of all serotonin-immunoreactive cells in each of these nuclei. No double labelled cells were present in the caudal linear raphe nucleus or the nucleus raphe pallidus, nor in the B4 group. These results suggest that only a very small percentage of serotonergic neurons in the medullary raphe nuclei (raphe magnus and raphe obscurus) also contain GABA, whereas such cells are virtually absent in the midbrain raphe nuclei or in the non-raphe serotonergic cell groups in the brainstem.     

The hypothalamic paraventricular and lateral parabrachial nuclei receive collaterals from raphe nucleus neurons: a combined double retrograde and immunocytochemical study.

Retrograde tracer injections of fluorescein- and rhodamine-labelled latex microspheres centered in the parvicellular zone of the hypothalamic paraventricular nucleus and pontine lateral parabrachial nucleus revealed that 36% of the labelled neurons in the dorsal raphe nucleus send collaterals to both structures. These cells were organized in a well-distinguishable cluster within the dorsal raphe nucleus. By combining retrograde tracing with immunocytochemistry, it was found that less than 8% of the double-labelled cells stained positively for serotonin. Of the remaining raphe nuclei that were examined, only the median raphe nucleus contributed a minor nonserotoninergic projection to the paraventricular or lateral parabrachial nuclei. Few of the retrogradely labelled cells in the median raphe nucleus contained both tracers. These results suggest that nonserotoninergic and serotoninergic neurons in the dorsal raphe nucleus, via collateral branching, may simultaneously influence the activity of two central nervous system nuclei involved in autonomic control.