Immunohistochemical localization of serotoninergic, enkephalinergic, and catecholaminergic cells in the brainstem and diencephalon of a cartilaginous fish, Hydrolagus colliei

We localized serotonin (5-HT), leu-enkephalin (LENK), and tyrosine hydroxylase (TH) immunoreactive cells in the brain of a holocephalian, Hydrolagus colliei, by use of antibodies made in rabbit and the peroxidase-antiperoxidase technique. Only three locations contained TH+ cells, the caudal myelencephalon, the locus coeruleus, and the diencephalon. Of these locations, the diencephalon contained the most cells and the locus coeruleus the least cells. The caudal TH+ myelencephalic cells formed a single large group that spanned both the dorsal and ventral portions of the brain (A1A2). The diencephalic TH+ cells were located in the posterior tuberculum, in the ventromedial and ventrolateral thalamic nuclei, and in the inferior lobe of the hypothalamus. Hydrolagus differed from mammals and the elasmobranchs, their sister group, in that no substantia nigra (A9), ventral tegmental area (A10), or A5 cell group was found. Distribution of LENK+ and 5-HT+ cells were similar to each other; the raphe nuclei contained most of the 5-HT+ and LENK+ cells. These 5-HT+ and LENK+ cells were found at all rostrocaudal levels of the myelencephalon. The nucleus reticularis magnocellularis, reticularis paragigantocellularis lateralis, the ventral met- and mesencephalon (B7 and B9 cell groups), the hypothalamus, and the pretectal area contained additional 5-HT+ and LENK+ cells. The solitary complex contained LENK+ cells but not but 5-HT+ cells. A dorsal raphe nucleus, which is the largest 5-HT+ cell group in mammals, was absent in Hydrolagus. A dorsal raphe nucleus is present in one galeomorph shark radiation but is absent in three radiations of batoids (rays, skates, and guitarfish). Thus even within cartilaginous fish, there are differences in the distribution of neurochemicals and possibly nuclei within their brains.     

Distribution of tyrosine hydroxylase, serotonin, and leu-enkephalin immunoreactive cells in the brainstem of a shark, Squalus acanthias.

The central nervous system location of neurochemicals that are widely distributed among extant animals may give us clues to changes that occurred in the brains of these animals during evolution. We have been studying the brains of cartilaginous fishes, a heterogeneous group whose central nervous system varies considerably. Squalus acanthias, the spiny dogfish shark, was chosen to represent the squalomorphs, a group of living sharks known to possess many primitive characters. The distribution of tyrosine hydroxylase (TH+), serotonin (5-HT+), and leu-enkephalin (LENK+) positive cells within the brainstem of Squalus was determined by use of antibodies to these substances. All the major raphe groups described for mammals were found in Squalus. The 5-HT+ cells in raphe nuclei were more uniformly distributed in Squalus than in Heterodontus, the horn shark. Other nuclei that were 5-HT+ and LENK+, and that have been identified in mammals, included reticularis paragigantocellularis lateralis, a B9 cell group, and reticularis magnocellularis. The postcommissural nucleus and pretectal area contained 5-HT+ and LENK+ cells. These cells have been described in a holocephalian, in teleosts, and in reptiles but not in other elasmobranchs or in mammals. Cells that were TH+ were located in prominent A1/A2, A6 (locus coeruleus), A9 (substantia nigra), and A10 (ventral tegmental area) cell groups, and in a very small A5 group. We conclude that the variation in chondrichthian brainstems exceeds that in mammals, and we suggest that this variation is related to life-style and the long evolutionary history of these fishes.     

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.     

Pontomedullary distribution of 5-HT2A receptor-like protein in the rat

Serotonin (5-HT) exerts excitatory effects in many brainstem regions involved in autonomic, somatic, motor, and sensory functions, and in control of vigilance. To determine the potential role of 5-HT2A receptors in these effects, we immunohistochemically mapped the distribution of 5-HT2A receptor-like protein in the rat pontomedullary brainstem. Areas containing the densest labeling included the trigeminal, facial, hypoglossal, dorsal vagal motor nuclei, medullary linear nucleus, and the inferior olive. In the nucleus ambiguus, labeled cells were located in the areas containing pharyngeal and laryngeal motoneurons. Intensely labeled cells were loosely scattered in the reticular formation adjacent to the raphe magnus and obscurus nuclei, in the gigantocellular region, in the caudal pedunculopontine and laterodorsal tegmental nuclei, dorsomedial pontine reticular formation, and nucleus subcoeruleus. In the nucleus prepositus hypoglossi, all vestibular, abducens, cuneate, and lateral reticular nuclei, labeled neurons commingled with unlabeled ones. Few labeled neurons were located in the rostral and caudal ventrolateral medulla and parvicellular reticular formation. In the nucleus of the solitary tract, two patches of diffuse labeling not associated with cellular profiles were present: one in the medial, and the other in the interstitial subnucleus. Similar diffuse labeling was present in the lateral parabrachial region and the lateral rim of the caudal spinal trigeminal sensory nucleus. No labeled cells were found in the locus coeruleus, dorsal raphe, superior olive, or area postrema. The distinct pontomedullary distribution of 5-HT2A receptors, combined with the known arousal-dependent activity of serotonergic neurons, show that these receptors may mediate post- and presynaptic effects in the motor, selected somatic and visceral sensory, oculo-vestibulo-precerebellar, and sleep-related regions.     

Distribution of serotonin 2A, 2C and 3 receptor mRNA in spinal cord and medulla oblongata

It is known that 5-HT receptors have significant roles in nociceptive and motor functions. We have compared the cellular localization of the mRNAs encoding serotonin 5-HT(2A,) 5-HT(2C,) 5-HT(3) receptor subtypes within different levels of the rat spinal cord and medulla. In the spinal cord, 5-HT(2C) receptor mRNA is expressed at high levels in most of the gray matter, except for lamina II. In contrast, 5-HT(2A) receptor mRNA is expressed exclusively in lamina IX. 5-HT(3) receptor mRNA has a low level and diffuse pattern of expression increasing towards the ventral horn. In both gray and white matter, there is a characteristic presence of a few highly stained cells. For each subtype, the expression pattern is similar in all four levels of the spinal cord. In the medulla, 5-HT(2C) receptor mRNA is at high levels in many nuclei including the hypoglossal nucleus, the gigantocellular reticular nucleus alpha and the parvocellular reticular nucleus alpha, the spinal nucleus of the trigeminal tract, the facial, and the dorsal medullary reticular field. Moderate to low levels of expression are seen in the spinal vestibular nucleus, the vagus, the solitary nuclei and the raphe. 5-HT(2A) receptor is expressed at high levels in some nuclei such as the hypoglossal nucleus, the intercalate nucleus, the inferior olive and the lateral reticular nucleus. Moderate to low levels of expression are seen in the facial, the medial vestibular nuclei, the nucleus ambiguous, the vagus, and the gigantocellular reticular nucleus. 5-HT(3) receptor mRNA is present at low levels in most of the nuclei examined, with a few scattered strongly labeled cells. The results show a distinct distribution of the three subtypes of receptors supporting their physiological roles and will help to understand the mechanisms of nociception and motor function.     

Distribution of enkephalin and its relation to serotonin in cat and monkey spinal cord and brain stem.

The distribution of enkephalin (ENK)-like immunoreactivity (LI) in spinal cord and medulla oblongata of cat and gray monkey (Macaca fascicularis) was studied by use of immunofluorescence and peroxidase antiperoxidase (PAP) techniques. Possible coexistence between ENK- and 5-hydroxytryptamine (5-HT)-LI was also analyzed with double labeling immunofluorescence. Furthermore, in situ hybridization was used to demonstrate cell bodies in the brain stem expressing mRNA encoding for ENK. ENK-immunoreactive (IR) axonal varicosities and fibers were demonstrated throughout the spinal cord gray matter, with the highest density in the superficial dorsal horn, the area around the central canal, the intermediolateral cell column, the sacral parasympathetic nucleus, and in Onuf's nucleus. In the monkey ventral horn, ENK-IR varicose fibers could in some cases be demonstrated in very close apposition to cell bodies. A low degree of co-localization between ENK- and 5-HT-LI was seen in the spinal cord of both species. Still, fibers containing both compounds could as a rule be demonstrated in every section studied. The highest degree of coexistence was encountered in the motor nucleus of the ventral horn. Six weeks after a low thoracic spinal cord transection a decreased staining for ENK-LI was demonstrated in the ventral horn motor nucleus, whereas other parts of the spinal cord appeared unaffected. In the brain stem of cats after colchicine treatment, ENK-LI was found in a majority of the 5-HT-IR cell bodies in the raphe nuclei (nucleus raphe magnus, pallidus and obscurus) and in the lateral reticular nucleus (rostroventrolateral reticular nucleus). In cat not pretreated with colchicine, a few weakly stained ENK-IR cell bodies could be found in the midline raphe nuclei and in the lateral reticular nucleus with the PAP technique. In the monkey brain stem without colchicine treatment, using the PAP technique, heavily stained ENK-IR cell bodies could be seen in the lateral reticular nucleus whereas, as in the cat, only a few, weakly stained ENK-IR cell bodies could be seen in the midline raphe nuclei. Using in situ hybridization technique, ENK mRNA expressing cells were demonstrated in the lateral reticular nucleus while no convincing mRNA signal could be found over cell bodies in the raphe nuclei. It is concluded that part of the ENKergic innervation of the cord in both species derives from supraspinal or suprasegmental levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monoamine cell distribution in the cat brain stem. A fluorescence histochemical study with quantification of indolaminergic and locus coeruleus cell groups

The distribution of monoaminergic cell bodies in the brainstem of the cat has been examined with Falck-Hillarp fluorescence histochemical technique. Quantitative determinations indicate that the cat brainstem contains about 60,300 indolaminergic (IA) cells. The majority of these (about 46,700, or 77.5%) are located within raphe nuclei. The largest number is contained within nucleus raphe dorsalis (RD), accounting for around 24,300 IA cells, while raphe pallidus (RP) holds about 8,000, raphe centralis superior (RCS) 7,400, raphe magnus (RM) 2,400, raphe obscurus (RO) 2,300, linearis intermedius (LI) 2,100, and the raphe pontis (RPo) only some 280 IA cells. The IA cells represent, however, only part of the neuronal population of raphe nuclei, which, in addition, hold varying numbers of other medium-sized and small-sized neurons. Thus, quantifications in Nissl-stained material indicate that the IA cells make up about 70% of the medium-sized cells in RD, 50% in RP, 35% in RCS and RO, 25% in LI, 15% in RM, and only 10% in RPo. The substantial numbers of small-sized perikarya observed in all raphe nuclei may represent interneurons. Significant numbers of IA cells were consistently located outside the raphe nuclei at all brainstem levels. In all, these amounted to approximately 13,600, or 22.5% of the total number of IA cells. Thus, IA cells occurred in the myelinated bundles, and sometimes in reticular formation, bordering the raphe nuclei; in the ventral brainstem forming a lateral extension from the ventral raphe (RP, RM, RPo, RCS, and LI) to the position of the rubrospinal bundle; in the periventricular gray and subjacent tegmentum of dorsal pons and caudal mesencephalon; in the locus coeruleus (LC) complex; around the motor trigeminal nucleus; caudal to the red nucleus; and in the interpeduncular and interfascicular nuclei. The wide distribution of IA cells leads to a considerable mixing with catecholaminergic (CA) cell groups. Our observations on CA cell distribution are essentially in accordance with previous reports. Quantifications indicate that the LC complex contains about 9,150 CA cells, unilaterally. A previously unnoticed group of scattered CA cells was found in relation to the vestibular nuclei and extending dorsally toward the deep cerebellar nuclei.     

Serotonergic projections and serotonin receptor expression in the reticular nucleus of the thalamus in the rat

The reticular nucleus (RT) of the thalamus, a thin sheet of GABAergic neurons located between the external medullary lamina and the internal capsule of the thalamus, has functionally distinct afferent and efferent connections with thalamic nuclei, the neocortex, the basal forebrain and the brainstem. RT is critically positioned to rhythmically pace thalamocortical networks leading to the generation of spindle activity during the early phases of sleep and during absence (spike-wave) seizures. Serotonin, acting on 5-HT(1A) receptors on parvalbumin-containing cells of RT, has been implicated in this rhythmicity. However, the precise source(s) of 5-HT afferents to the RT remains to be determined. In the present study, we injected the retrograde tracer, Fluorogold, into dorsal and ventral regions of RT to determine the origins of raphe input to RT. We further characterized the distribution of 5-HT fibers to RT by using immunohistochemistry for 5-HT and for the 5HT transporter (SERT) detection. Finally, we described the presence of the two major postsynaptic 5-HT receptors in RT, 5-HT(1A) and 5-HT(2A) receptors. Our results show that the dorsal raphe nucleus and the supralemniscal nucleus (B9) of the midbrain are the principal sources of raphe projections to RT. In addition, serotonergic fibers (5-HT and SERT positive) were richly distributed throughout RT, and 5-HT(1A) and 5-HT(2A) receptors were highly expressed on RT neurons and dendrites. These findings suggest a significant 5-HT modulatory influence on GABAergic neurons of RT in the control of rhythmical (or spindle) activity in thalamocortical systems directly associated with sleep and possibly with absence seizures.     

Origins of serotonergic projections to the spinal cord in rat: An immunocytochemical-retrograde transport study

The origins of the serotonergic projections to the spinal cord in the rat were determined by employing the retrograde cell marker HRP coupled with the unlabeled antibody, peroxidase-antiperoxidase immunocytochemical method of Sternberger. Large numbers of stained neurons (> 70%) in the medullary raphe nuclear complex were found to contain both HRP retrogradely transported from the spinal cord and positive 5-HT staining. These serotonergic cell groups, including the nucleus raphe obscurus, raphe pallidus, raphe magnus, and the ventral parts of the reticular formation, project to all spinal cord levels. In addition, some neurons contained HRP granules, but were unstained for 5-HT, suggesting that they may contain other non-serotonergic neurotransmitters. More rostrally in the midbrain reticular formation, many 5-HT neurons were found to have projections exclusively to the cervical spinal cord. These findings indicate that the descending serotonin inputs to the spinal cord originate not only from the serotonergic neurons located in the medullary raphe complex, but also from other new sources located in the central gray and reticular formation of the midbrain.     

The efferent connections of the nucleus raphe centralis superior in the rat as revealed by radioautography.

By chronically implanting a glass micropipette filled with tritiated leucine in the raphe centralis superior of the rat, the projection of this nucleus was traced by radioautography. The majority of the ascending projections were located within the ventral tegmental area and, further rostrally, the median forebrain bundle. Along the course of this bundle numerous fibers branched successively into the mammillary peduncle, the fasciculus retroflexus, the stria medullaris, the fornix and the cingulum. The most significant projections included the ones to the interpeduncular nucleus, the mammillary bodies, the habenular nuclei and the hippocampus. No projections were detected in the striatum, the cortex piriformis or the amygdala. Descending projections diffused to the pontine reticular formation and central gray through the medial and the dorsal longitudinal bundles. In addition widespread projections were also seen in nuclei located near the raphe centralis superior: raphe nuclei, dorsal and ventral tegmental nuclei.     

Brainstem projections to midline and intralaminar thalamic nuclei of the rat

The projections from the brainstem to the midline and intralaminar thalamic nuclei were examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin beta -subunit (CTb) were made in each of the intralaminar nuclei of the dorsal thalamus: the lateral parafascicular, medial parafascicular, central lateral, paracentral, oval paracentral, and central medial nuclei; in the midline thalamic nuclei-the paraventricular, intermediodorsal, mediodorsal, paratenial, rhomboid, reuniens, and submedius nuclei; and, in the anteroventral, parvicellular part of the ventral posterior, and caudal ventral medial nuclei. The retrograde cell body labeling pattern within the brainstem nuclei was then analyzed. Nearly every thalamic site received a projection from the deep mesencephalic reticular, pedunculopontine tegmental, dorsal raphe, median raphe, laterodorsal tegmental, and locus coeruleus nuclei. Most intralaminar thalamic sites were also innervated by unique combinations of medullary and pontine reticular formation nuclei such as the subnucleus reticularis dorsalis, gigantocellular, dorsal paragigantocellular, lateral, parvicellular, caudal pontine, ventral pontine, and oral pontine reticular nuclei; the dorsomedial tegmental, subpeduncular tegmental, and ventral tegmental areas; and, the central tegmental field. In addition, most intralaminar injections resulted in retrograde cell body labeling in the substantia nigra, nucleus Darkschewitsch, interstitial nucleus of Cajal, and cuneiform nucleus. Details concerning the pathways from the spinal trigeminal, nucleus tractus solitarius, raphe magnus, raphe pallidus, and the rostral and caudal linear raphe nuclei to subsets of midline and intralaminar thalamic sites are discussed in the text. The discussion focuses on brainstem-thalamic pathways that are likely involved in arousal, somatosensory, and visceral functions.     

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.     

Changes in serotonergic neurons in the brain of pyrithiamine-induced acute thiamine-deficient mice

We examined changes in 5-hydroxytriptamine (5-HT, serotonin) neurons in pyrithiamine-induced thiamine deficiency in mice immunohistochemically. Extensive decreases in the densities of 5-HT-immunoreactive fibers were detected in the lateral septal nucleus, the thalamus, the medial mammillary nucleus, the dorsal and the median raphe nuclei, the raphe obscurus nucleus, the tegmental area, the cerebellum and the vestibular nucleus, though only a small decrease was detected in the inferior colliculus. Most remarkably, degenerative winding fibers were detected between the deep mesencephalic nucleus and the ventral tegmental area. Increases in intensity of 5-HT immunoreactivity in the dorsal raphe nucleus and decreases in the number of 5-HT-immunoreactive cell bodies in the dorsal and the median raphe nuclei were detected. These results demonstrated the differential vulnerability of 5-HT neurons in thiamine-deficient mice. This is the first report to demonstrate changes in 5-HT neurons immunohistochemically throughout the brain of pyrithiamine-induced thiamine deficient mouse. Received: 15 January 1999 / Revised 22 March 1999 / Accepted: 24 March 1999     

The distribution and origin of serotonin-containing fibers in the septal area: a combined immunohistochemical and fluorescent retrograde tracing study in the rat

The distribution of 5-hydroxytryptamine (serotonin, 5-HT)-containing nerve fibers and terminals in the septal area of the rat was studied by using immunohistochemistry with specific antibodies to 5-HT in combination with fluorescent retrograde tracing methods. The 5-HT innervation of the septum is heterogeneous with regard to both the morphology of individual processes and the density of distribution in different parts of the septum. Three major classes of 5-HT like immunoreactive processes can be distinguished: 1) thin, convoluted fibers with small, round or elongated varicosities; 2) thick and relatively straight fibers with few varicosities; and 3) pericellular plexuses with large varicosities in close association with perikarya in the lateral septum. Three areas of the septum receive a prominent innervation by 5-HT processes: the diagonal band of Broca, the ventral part of the lateral septum, and an area bordering the medial edge of the islands of Calleja (insula magna). Whereas the two latter areas contain dense terminal networks, the diagonal band of Broca is occupied primarily by 5-HT fibers en route to other parts of the septum. Intraseptal injections of HRP or fluorescent dyes (granular blue, propidium iodide) resulted in retrograde labeling of neuronal cell bodies in several nuclei of the brainstem which are known to contain 5-HT neurons: the dorsal raphe, the median raphe, the nucleus reticularis tegmenti pontis, the raphe pontis, and the raphe magnus. Where fluorescent retrograde tracing was performed with 5-HT immunohistochemistry on the same tissue section, a prominent 5-HT containing pathway and a non-5-HT-containing pathway from the raphe nuclei to the septum were revealed. Finally, double retrograde fluorescent labeling after injections of granular blue or propidium iodide into the septum and entorhinal area respectively of the same rat revealed extensive branching of the raphe efferents. Thus, individual raphe neurons may simultaneously connect with septum and the entorhinal area, two structures essential for normal hippocampal function.     

Extent of colocalization of serotonin and GABA in neurons of the ventral medulla oblongata in rat

The colocalization of serotonin (5-hydroxytryptamine; 5-HT) and γ-aminobutyric acid (GABA) in the ventral aspect of the rat medulla oblongata was studied using antibodies directed against 5-HT and GABA. Although 5-HT- and GABA-immunoreactive cell bodies were observed over the entire rostral-caudal extent of the ventral medulla, the colocalization of these two classical neurotransmitters in single cells was, for the most part, limited to a region that corresponds anatomically to nucleus raphe magnus/nucleus paragigantocellularis. Schematic drawings showing the distribution of 5-HT/GABA cell bodies in the ventral medulla are provided.     

GABA-synthesizing neurons in the medulla: their relationship to serotonin-containing and spinally projecting neurons in the rat.

GABA-synthesizing neurons were identified in the medulla of the rat by peroxidase-antiperoxidase (PAP) immunohistochemistry for glutamic acid decarboxylase (GAD). Using diaminobenzidine (DAB) either alone or intensified with silver, a relatively large number of GAD-immunoreactive neurons were evident within the reticular formation, raphe nuclei and vestibular nuclei. In all these areas, profuse GAD-immunoreactive varicosities appeared to contact the soma and dendrites of both non-GABA and GABA neurons. These observations suggest that GABA neurons may act as interneurons or local projection neurons within the medulla and accordingly exert a potent inhibitory and/or disinhibitory control on bulbar projection neurons. Within the ventral reticular formation (pars alpha and ventralis of the gigantocellular reticular field) and raphe magnus, large numbers of prominent GAD-immunoreactive neurons resembled in size and morphology and overlapped in distribution the serotonin-immunoreactive neurons of the same regions. However, by sequential double immunostaining utilizing DAB as a chromogen for serotonin (5-HT) and benzidine dihydrochloride (BDHC) for GAD, it was found that GAD-containing neurons were distinct from 5-HT-containing neurons. Following injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the upper cervical spinal cord and combined processing for WGA-HRP (using tetramethylbenzidine [TMB] with cobalt) and immunohistochemistry (with DAB), a contingent of spinally projecting neurons were found to contain GAD. The GAD-immunoreactive reticulo- and raphe-spinal neurons were most frequent within the pars alpha and ventralis of the gigantocellular reticular fields and the raphe magnus, where they were approximately equal in number to the coexistent, but distinct 5-HT spinally projecting neurons. GABA neurons of the medulla may thus contribute directly to the bulbar inhibitory influence upon spinal sensory and motor systems.     

Comparison of alpha2 nicotinic acetylcholine receptor subunit mRNA expression in the central nervous system of rats and mice

The nicotinic acetylcholine receptor (nAChR) alpha2 subunit was the first neuronal nAChR to be cloned. However, data for the distribution of alpha2 mRNA in the rodent exists in only a few studies. Therefore, we investigated the expression of alpha2 mRNA in the rat and mouse central nervous systems using nonradioactive in situ hybridization histochemistry. We detected strong hybridization signals in cell bodies located in the internal plexiform layer of the olfactory bulb, the interpeduncular nucleus of the midbrain, the ventral and dorsal tegmental nuclei, the median raphe nucleus of the pons, the ventral part of the medullary reticular nucleus, the ventral horn in the spinal cord of both rats and mice, and in a few Purkinje cells of rats, but not of mice. Cells that moderately express alpha2 mRNA were localized to the cerebral cortex layers V and VI, the subiculum, the oriens layer of CA1, the medial septum, the diagonal band complex, the substantia innominata, and the amygdala of both animals. They were also located in a few midbrain nuclei of rats, whereas in mice they were either few or absent in these areas. However, in the upper medulla oblongata alpha2 mRNA was expressed in several large neurons of the gigantocellular reticular nucleus and the raphe magnus nucleus of mice, but not of rats. The data obtained show that a similar pattern of alpha2 mRNA expression exists in both rats and mice, with the exception of a few regions, and provide the basis for cellular level analysis.     

Serotoninergic and noradrenergic projections to the ventral posterolateral nucleus of the monkey thalamus

In the present study, serotoninergic and noradrenergic varicosities were identified in the ventral posterolateral nucleus of the macaque monkey. Monoaminergic neurons projecting to the ventral posterolateral nucleus of the thalamus were identified by using retrograde labeling with horseradish peroxidase combined with immunocytochemical staining for serotonin or dopamine-beta-hydroxylase. The midbrain nucleus raphe dorsalis was the major site of origin for neurons providing a serotoninergic projection to the ventral posterolateral nucleus. A few retrogradely labeled serotonin-containing neurons were also observed in the central superior and the raphe pontis nuclei. Noradrenergic cells with projections to the thalamus were primarily located in the nucleus locus coeruleus with some projection neurons in the nucleus subcoeruleus, and the A5 catecholamine cell group of the pons.     

Autoradiographic analysis of ascending projections from the pontine and mesencephalic reticular formation and the median raphe nucleus in the rat

Ascending projections from the medial pontine reticular formation, the mesencephalic reticular formation, and the median raphe nucleus were examined using the autoradiographic technique. The majority of the ascending fibers labeled after injections of [3H]-leucine into the nucleus pontis caudalis (RPC) course through the brainstem within the tracts of Forel (tractus fasciculorum tegmenti of Forel) and directly ventral to them. At the caudal diencephalon, Forel's bundle divides into dorsal and ventral components bound primarily for the dorsal thalamus and the subthalamus, respectively. RPC fibers project to several regions involved in oculomotor/visual functions. These include the abducens nucleus, the intermediate gray layer of the superior colliculus (SCi), the anterior pretectal nucleus (APN), the ventral lateral geniculate nucleus (LGNv), and regions of the central gray directly bordering the oculomotor nucleus, the interstitial nucleus of Cajal, and the nucleus of Darkschewitsch. Few, if any, fibers from RPC (or from nucleus pontis oralis-RPO) terminate within the oculomotor nucleus proper. Other sites receiving heavy projections from the RPC include adjacent regions of the pontomesencephalic reticular formation (RF), the parafascicular (PF) and central lateral (CL) nuclei of the thalamus and the fields of Forel/zona incerta (FF-ZI). RPO fibers also ascend predominantly in Forel's bundle. Other ascending tracts for these fibers are the medial longitudinal fasciculus and the central tegmental tract (CTT). RPO fibers distribute significantly to the same structures of the oculomotor/visual system receiving projections from RPC. The RPO projections to the SCi and the APN are particularly pronounced. RPO fibers terminate heavily in several nuclei located ventrally within the rostral midbrain/caudal diencephalon. These include major dopamine-containing cell groups (the retrorubral nucleus, the ventral tegmental area, and the substantia nigra-pars compacta) as well as the interpeduncular nucleus, the lateral mammillary nucleus, and the supramammillary nucleus. Other prominent targets for RPO fibers include the mesencephalic RF, specific regions of the central gray, the PF, the CL, the paracentral and central medial nuclei of the thalamus, and the FF/ZI. The major bundle of the ascending fibers labeled after injections of the mesencephalic reticular formation (MRF) travels within the CTT in a position just lateral to the central gray, but a significant number of labeled axons also course in Forel's bundle.(ABSTRACT TRUNCATED AT 400 WORDS)