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.     

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)     

The origin of the vasopressinergic and oxytocinergic innervation of the rat brain with special reference to the lateral septum

The origin of the vasopressin-containing fibers in the rat lateral septum was studied by means of lesioning specific areas, in which vasopressin-containing cells are found, or by surgically separating the septum from the underlying structures. Following these procedures sections of the brain were stained immunocytochemically for the presence of vasopressin. In addition, retrograde labelling tracers were injected in the lateral septum.Lesioning of the paraventricular nucleus did not result in the disappearance of vasopressin fibers from the lateral septum, nor from the various other areas studied. It did, however, cause the disappearance of fibers from the nucleus of the solitary tract and the nucleus ambiguus. By contrast, after the same lesion practically the whole oxytocinergic innervation of the brain disappeared. Injection of tracers into the lateral septum revealed retrograde labeled cells, e.g. in the bed nucleus of the stria terminalis, but not in the paraventricular and supraoptic nucleus.Horizontal cuts under the lateral septum, intersecting the diagonal band of Broca, resulted in a dramatic decrease of the vasopressin fibers in the lateral septum, suggesting that the fibers enter the septum via this structure. Moreover, since the vasopressin fiber density was found to decrease drastically in the lateral septum after lesioning the bed nucleus of the stria terminalis, the vasopressin cells found in this area are probably the source of these fibers. Other areas where fibers were seen to decrease after lesions of the bed nucleus are the diagonal band of Broca, the area of the anterior amygdala, the lateral habenular nucleus, the periventricular gray, and the locus coeruleus.     

Noradrenergic innervation of the developing and mature septal area of the rat

The noradrenergic innervation of the developing and mature septal area of the rat was examined with light and electron microscopic immunocytochemistry using an antibody against dopamine-beta-hydroxylase. At birth, a small number of relatively thick noradrenergic fibers were found to innervate the lateral septum (mainly its intermediate part) and the nuclei of the vertical and horizontal limbs of the diagonal band of Broca. By postnatal day 7, a substantial increase in their density was observed. At this age some labeled fibers left the medial forebrain bundle and invaded the nucleus of the horizontal limb of the diagonal band. These fibers then ran in a ventrodorsal direction and innervated the nucleus of the vertical limb before entering the medial septum. Immunoreactive fibers were finer and more varicose than at birth. In the subsequent 2 weeks, the density of labeled fibers in the septal area was further increased. By postnatal day 21, the distribution pattern and density of the noradrenergic innervation appeared similar to the adult. In the adult, noradrenergic fibers exhibited more varicosities than in younger rats. Electron microscopic analysis revealed a low proportion (peaked at P7) of noradrenergic varicosities engaged in synaptic contacts throughout development. The overwhelming majority of these synapses were symmetrical, predominantly with small or medium-sized dendrites. The present findings provide the morphological basis for the functional interactions between noradrenergic afferents and neuronal elements in the septal area. The low proportion of synaptic contacts found in this study suggests that noradrenaline may exert its action in the septal area mainly through transmission by diffusion (volume transmission), as has been suggested for other areas of the developing and adult brain.     

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.     

Cholinergic and non-cholinergic forebrain projections to the interpenduncular nucleus

A combined fluorescent retrograde tracing and acetylcholinesterase (AChE) histochemical technique was used for the study of some forebrain projections to the interpeduncular nucleus (IPN). After injections of a fluorescent tracer into the IPN, the distribution of AChE-containing and of fluorescent retrogradely labeled neurons was simultaneously studied in the habenular nuclei, medial septum and diagonal band of Broca. In all these regions, the majority of retrogradely labeled neurons also contained AChE: neurons located in the habenular nuclei stained lightly or moderately for the enzyme, while neurons located in the diagonal band and medial septum displayed intense AChE staining and were classified as putatively cholinergic perikarya. In all regions, a minority of labeled neurons did not stain for AChE, and were identified as non-cholinergic neurons projecting to the IPN. The present study shows the existence of a biochemical heterogeneity in the habenulo-interpeduncular and telencephalo-interpeduncular pathways, and indicates that the latter contains putatively cholinergic as well as non-cholinergic fibers.     

Cholinergic and non-cholinergic forebrain projections to the interpeduncular nucleus

A combined fluorescent retrograde tracing and acetylcholinesterase (AChE) histochemical technique was used for the study of some forebrain projections to the interpeduncular nucleus (IPN). After injections of a fluorescent tracer into the IPN, the distribution of AChE-containing and of fluorescent retrogradely labeled neurons was simultaneously studied in the habenular nuclei, medial septum and diagonal band of Broca. In all these regions, the majority of retrogradely labeled neurons also contained AChE: neurons located in the habenular nuclei stained lightly or moderately for the enzyme, while neurons located in the diagonal band and medial septum displayed intense AChE staining and were classified as putatively cholinergic perikarya. In all regions, a minority of labeled neurons did not stain for AChE, and were identified as non-cholinergic neurons projecting to the IPN. The present study shows the existence of a biochemical heterogeneity in the habenulo-interpeduncular and telencephalo-interpeduncular pathways, and indicates that the latter contains putatively cholinergic as well as non-cholinergic fibers.     

Transplanted raphe and hippocampal fetal neurons do not displace afferent inputs to the dorsal hippocampus from serotonergic neurons in the median raphe nucleus of the rat

To determine if fetal transplants can substitute for or suppress intrinsic serotonergic (5-HT) innervation, we studied the relationship between transplanted and the endogenous raphe neurons projecting to the hippocampus. Fetal raphe transplants produced a 5-HT hyperinnervation of dorsal hippocampus in adult rats. Yet, transplants of fetal raphe tissue did not affect the number of median raphe nucleus (MRN) neurons, approximately 300, which retrogradely transported HRP from the hippocampus. This provides evidence that transplanted 5-HT neurons can co-exist with intrinsic 5-HT nerve terminals in the target area for at least one month. In the second part of this study, fetal hippocampal tissue was transplanted into the host hippocampus. Intrinsic 5-HT immunoreactive fibers innervated the transplanted fetal tissue. Nevertheless, the number of MRN neurons innervating the host tissue as revealed by HRP retrograde transport remained unchanged. Changes in the innervation pattern and 5-HT level in the dorsal hippocampus occur following transplantation of fetal tissue. These changes are discussed and suggest that both the target tissue and afferent neurons readjust to accommodate extrinsic transplanted tissue.     

A study of the reciprocal connections between the septum and the entorhinal area using anterograde and retrograde axonal transport methods in the rat brain

The reciprocal connections between the septum and the entorhinal area (EA) was studied in the rat brain using antero- and retrograde axonal transport methods. After injections of large volumes (2 × 100 nl) of horseradish peroxidase (HRP) conjugated to wheat-germ agglutinin (WGA) into the medial septum (MS) and the diagonal band of Broca (dbB), anterogradely transported HRP-WGA was found primarily in layers II and IV of the medial and lateral EA. Injections of HRP-WGA (50–100 nl) or fluorescent dyes (50–100 nl) into different parts of the retrohippocampal region resulted in labeling, by retrograde axonal transport, of cells in the MS and dbB, both ipsi- and contralateral to the injected hemisphere. The labeled cells were either small (long axis of soma: 10–15 μm), round, and oval, or medium (15–25 μm) to large (25–35 μm) of fusiform or multipolar shape. By using the method of retrograde fluorescent double labeling, the septal afferents to the EA were found to give off collaterals to other parts of the hippocampal region. A much smaller number of septal cells appeared to send bilateral projections to the EA of both hemispheres. Studies employing retrograde transport of HRP in combination with acetylcholinesterase (AChE) histochemistry on the same tissue section showed that, while a large number of cells projecting to the EA contain AChE, many projecting cells are devoid of AChE reaction products. These findings suggest that the septo-entorhinal projection consists of a cholinergic as well as a noncholinergic component. The entorhinal efferents to the septum were studied after injections of HRP-WGA into different parts of the retrohippocampal region. Labeled fibers could be traced through the fimbria to their terminal fields in the intermediate parts of the lateral septal nucleus and to the most lateral aspect of the vertical limb of the dbB. The cells giving rise to this projection were situated in layer IV of the medial and layers II through V of the lateral EA. Taken together, the present findings demonstrate a close anatomical relationship between the septum and the entorhinal area, in addition to the better known connections between the septum and the Ammon's horn.     

Selective spread of neurotropic herpesviruses in the rat hippocampus

Transsynaptically spreading viruses are widely used for tracing neuronal circuits in both the central and peripheral nervous systems. However, viruses are capricious tools with selective spreading properties that can produce false-negative results. Using herpes simplex virus type 1 and two pseudorabies virus strains, we aimed at mapping quantitatively neuronal connections in the rat hippocampus. We found that none of the tested viruses infected CA3 pyramidal neurons across synapses following inoculation into the CA1 area. Combined injections of the viruses with the retrograde tracer cholera toxin B (CTB) resulted in CTB, but not virus labeling of CA3 pyramidal neurons. In contrast, other brain regions known to send inputs to the CA1 (the entorhinal cortex, medial septum and diagonal band of Broca, raphe nuclei) were transsynaptically infected. Our results indicate that Schaffer collaterals of CA3 pyramidal cells lack the appropriate cellular machinery for successful neurotropic herpesvirus infection. After injections of viruses into the dentate gyrus/CA3 area, we found labeling in commissurally projecting mossy cells and their afferent granule cells but not in contralateral CA3 pyramidal cells. Using this unique spreading property, we estimated that single mossy cells receive input from a compact cluster of 30-40 granule cells.     

The serotoninergic innervation of cerebral cortex: different classes of axon terminals arise from dorsal and median raphe nuclei

The objective of the present study was to characterize the morphology of serotoninergic axons in cerebral cortex of the rat and to determine whether dissimilar axon terminals arise from the dorsal vs. the median raphe nuclei. The anterograde tracer PHA-L was administered by iontophoresis into the dorsal (DR) and median (MR) raphe nuclei, and the morphologic features of the respective axonal projections from raphe to forebrain were analyzed. We have observed consistent structural differences between the axons from these two nuclei. Anterogradely labeled axons which arise from cells in the MR are characterized by large, spherical varicosities (type M axons) and by variations in axonal diameter. In contrast, DR fibers are very fine and typically have small, pleomorphic varicosities that are granular or fusiform in shape (type D axons). Similar features of serotonin (5-HT) axon morphology are also evident in 5-HT immunocytochemical preparations. In addition to structural differences, there is a differential topographic distribution of MR vs. DR fibers, with MR axons concentrated in particular areas of limbic cortex such as dentate gyrus, posterior cingulate, and entorhinal areas as well as in parietal cortex. Immunofluorescence with dual labels shows that over two-thirds of the raphe-cortical axons are serotoninergic. The dissimilarities in axon morphology indicate that individual raphe nuclei may form different patterns of synaptic organization. Based on the evidence that the dorsal and median raphe nuclei give rise to morphologically different axon terminals, we conclude that 5-HT axons in cortex may be subdivided into two distinct projections. This proposal is in accord with other, recent data showing that the two axon types have different pharmacologic properties and are likely to be functionally different.     

Hypothalamic and other connections with dorsal CA2 area of the mouse hippocampus

The CA2 area is an important, although relatively unexplored, component of the hippocampus. We used various tracers to provide a comprehensive analysis of CA2 connections in C57BL/6J mice. Using various adeno‐associated viruses that express fluorescent proteins, we found a vasopressinergic projection from the paraventricular nuclei of the hypothalamus (PVN) to the CA2 as well as a projection from pyramidal neurons of the CA2 to the supramammillary nuclei. These projections were confirmed by retrograde tracing. As expected, we observed CA2 afferent projections from neurons in ipsilateral entorhinal cortical layer II as well as from bilateral dorsal CA2 and CA3 using retrograde tracers. Additionally, we saw CA2 neuronal input from bilateral medial septal nuclei, vertical and horizontal limbs of the nucleus of diagonal band of Broca, supramammillary nuclei (SUM), and median raphe nucleus. Dorsal CA2 injections of adeno‐associated virus expressing green fluorescent protein revealed axonal projections primarily to dorsal CA1, CA2, and CA3 bilaterally. No projection was detected to the entorhinal cortex from the dorsal CA2. These results are consistent with recent observations that the dorsal CA2 forms disynaptic connections with the entorhinal cortex to influence dynamic memory processing. Mouse dorsal CA2 neurons send bilateral projections to the medial and lateral septal nuclei, vertical and horizontal limbs of the diagonal band of Broca, and SUM. Novel connections from the PVN and to the SUM suggest important regulatory roles for CA2 in mediating social and emotional input for memory processing. J. Comp. Neurol. 521:1844–1866, 2013. © 2012 Wiley Periodicals, Inc.     

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

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

Basal forebrain inputs to the interpeduncular nucleus in the rat studied with the Phaseolus vulgaris-leucoagglutinin tracing method

The connections between the basal forebrain and the interpeduncular nucleus (IP) were studied in the rat using the anterograde transport of Phaseolus vulgaris-leucoagglutinin (PHA-L). PHA-L was injected in the septum-diagonal band complex, the preoptic area, the substantia innominata, the globus pallidus, and the ventral pallidum. In a number of cases sections of the IP were double-immunostained for PHA-L and serotonin. Only following PHA-L injections in the medial septal nucleus and the nucleus of the vertical limb of the diagonal band, were substantial terminations found throughout all subnuclei of IP. Following injections in the medial and lateral preoptic area labeling was confined to the caudal part of IP. This finding suggests that the area in the basal forebrain that contributes to these projections is smaller than has been indicated by previous retrograde tracing studies. Labeled fibers reach the IP predominantly via the medial forebrain bundle. Only a very small number of fibers reaches the ventral mesencephalon, and possibly the IP, via the stria medullaris and the fasciculus retroflexus. The highest density of terminations was seen in the apical subnucleus. The apical subnucleus contains serotonin-immunoreactive neurons, in the direct vicinity of which varicosities on the labeled fibers were seen. This finding suggests innervation of the serotonergic neurons by fibers from the medial septum and preoptic area.     

Basal forebrain inputs to the interpeduncular nucleus in the rat studied with the Phaseolus vulgaris-leucoagglutinin tracing method

The connections between the basal forebrain and the interpeduncular nucleus (IP) were studied in the rat using the anterograde transport of Phaseolus vulgaris-leucoagglutinin (PHA-L). PHA-L was injected in the septum-diagonal band complex, the preoptic area, the substantia innominata, the globus pallidusand the ventral pallidum. In a number of cases sections of the IP were double-immunostained for PHA-L and serotonin. Only following PHA-L injections in the medial septal nucleus and the nucleus of the vertical limb of the diagonal band, were substantial terminations found throughout all subnuclei of IP. Following injections in the medial and lateral preoptic area labeling was confined to the caudal part of IP. This finding suggests that the area in the basal forebrain that contributes to these projections is smaller than has been indicated by previous retrograde tracing studies. Labeled fibers reach the IP predominantly via the medial forebrain bundle. Only a very small number of fibers reaches the ventral mesencephalonand possibly the IP, via the stria medullaris and the fasciculus retroflexus. The highest density of terminations was seen in the apical subnucleus. The apical subnucleus contains serotonin-immunoreactive neurons, in the direct vicinity of which varicosities on the labeled fibers were seen. This finding suggests innervation of the serotonergic neurons by fibers from the medial septum and preoptic area.     

Connections of the parahippocampal cortex in the cat. IV. Subcortical efferents

The present report deals with the projections from the entorhinal and perirhinal cortices to subcortical forebrain structures and the brainstem in the cat. By using anterograde and retrograde tracing techniques, it could be demonstrated that the entire mediolateral extent of the parahippocampal cortex issues prominent projections to the dorsal and ventral striatum, the amygdala, and the claustrum. In addition, the entorhinal cortex sends projections to the septum and the diagonal band of Broca. Only the perirhinal cortex gives rise to a weak projection to the dorsolateral periaquaductal gray and the ventral pontine region. The major proportion of the subcortical projections originates in the perirhinal cortex and the lateral entorhinal cortex, whereas the medial entorhinal cortex has a much sparser output and sends no fibers to the amygdala. The subcortical projections from both the entorhinal cortex and the perirhinal cortex arise mostly from their deep layers. It was further found that these projections are topographically organized along the mediolateral axis of the parahippocampal cortex. This mediolateral axis is related to a ventrolateral to dorsomedial axis in the septum, a mediolateral axis in the amygdala and the ventral striatum, and a ventrodorsal coordinate in the dorsal striatum and the claustrum. A further topography was observed in the projections from the perirhinal cortex to the lateral amygdaloid nucleus. A rostrocaudal axis in the perirhinal cortex corresponds to a mediolateral axis in the lateral amygdaloid nucleus. The present observations are compared with data concerning the connectivity of the parahippocampal cortex with the hippocampal formation and other cortical structures. It is suggested that the parahippocampal cortex in the cat may be conceptualized as an interface between the hippocampal formation and several subcortical structures in the realm of the limbic and motor systems.     

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.     

Cytoarchitecture and efferent projections of the nucleus incertus of the rat

The nucleus incertus is located caudal to the dorsal raphe and medial to the dorsal tegmentum. It is composed of a pars compacta and a pars dissipata and contains acetylcholinesterase, glutamic acid decarboxylase, and cholecystokinin-positive somata. In the present study, anterograde tracer injections in the nucleus incertus resulted in terminal-like labeling in the perirhinal cortex and the dorsal endopyriform nucleus, the hippocampus, the medial septum diagonal band complex, lateral and triangular septum medial amygdala, the intralaminar thalamic nuclei, and the lateral habenula. The hypothalamus contained dense plexuses of fibers in the medial forebrain bundle that spread in nearly all nuclei. Labeling in the suprachiasmatic nucleus filled specifically the ventral half. In the midbrain, labeled fibers were observed in the interpeduncular nuclei, ventral tegmental area, periaqueductal gray, superior colliculus, pericentral inferior colliculus, pretectal area, the raphe nuclei, and the nucleus reticularis pontis oralis. Retrograde tracer injections were made in areas reached by anterogradely labeled fibers including the medial prefrontal cortex, hippocampus, amygdala, habenula, nucleus reuniens, superior colliculus, periaqueductal gray, and interpeduncular nuclei. All these injections gave rise to retrograde labeling in the nucleus incertus but not in the dorsal tegmental nucleus. These data led us to conclude that there is a system of ascending projections arising from the nucleus incertus to the median raphe, mammillary complex, hypothalamus, lateral habenula, nucleus reuniens, amygdala, entorhinal cortex, medial septum, and hippocampus. Many of the targets of the nucleus incertus were involved in arousal mechanisms including the synchronization and desynchronization of the theta rhythm.     

Origin of the neurotensinergic innervation of the rat basal forebrain studied by retrograde transport of cholera toxin

Basal forebrain cholinergic neurons project to the hippocampus and cerebral cortex where they play an important role in cortical activation, attention, and memory. These neurons have been shown to express functional neurotensin receptors and to receive a dense neurotensinergic innervation. In the present study, we investigated the origin of this innervation by combining retrograde transport of cholera toxin with immunohistochemical detection of neurotensin. After injection of cholera toxin in the anterior substantia innominata and diagonal band of Broca, retrogradely labelled cells were widely distributed throughout forebrain limbic structures. Only a small proportion of these cells, located (by decreasing order of frequency) in the lateral septum, medial preoptic area, rostral hypothalamus, nucleus accumbens, and rostral basal forebrain, were dually labelled for neurotensin. After injection of cholera toxin in the posterior substantia innominata and magnocellular preoptic nucleus, retrogradely labelled cells were detected throughout the limbic forebrain and ponto-mesencephalic tegmentum. Here again, only a small proportion of these cells, located (by decreasing order of frequency) in the nucleus accumbens, lateral septum, rostral basal forebrain, hypothalamus, bed nucleus of the stria terminalis, supramammilliary nucleus, ventral tegmental area, and raphe complex co-localized neurotensin. In view of the burst generating properties of neurotensin on basal forebrain cholinergic neurons, our results suggest that neurotensin projections may be part of the septo-hippocampo-septal loop regulating hippocampal theta activity. More caudally, neurotensin axons originating from the lateral hypothalamus and pontomesencephalic tegmentum may contribute to the contingent of ascending reticular formation fibers involved in the regulation of the sleep-wake cycle. J. Comp. Neurol. 391:30–41, 1998. © 1998 Wiley-Liss, Inc.     

Olfactory relationships of the telencephalon and diencephalon in the rabbit. III. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations.

The axoplasmic retrograde transport of horseradish peroxidase (HRP) from axon terminals to their parent cell bodies and histochemical fluorescence microscopy have been used to study the ipsilateral centrifugal fibers to the olfactory bulbs and anterior olfactory nucleus in the rabbit. Focal injections of peroxidase were placed unilaterally into the main or accessory olfactory bulb or into the anterior olfactory nucleus. In animals with injected HRP confined within the main bulb, perikarya retrogradely labeled with the protein in the ipsilateral forebrain were observed in the anterior prepyriform cortex horizontal limb of the nucleus of the diagonal band, and far lateral preoptic and rostral lateral hypothalamic areas. Brain stem cell groups that contained HRP-positive somata include the locus coeruleus and midbrain dorsal raphe nucleus. Except for the prepyriform cortex, the basal forebrain structures with labeled perikarya correlate well with locations of cell bodies containing acetylcholinesterase and choline acetyltransferase. These somata may represent a cholinergic afferent system to the main olfactory bulb. Peroxidase-labeled cell bodies in the locus coeruleus and midbrain raphe are indicative of noradrenergic and serotonergic innervations respectively of the olfactory bulb. In rabbits in which peroxidase was injected or diffused into the accessory olfactory bulb and anterior alfactory nucleus, HRP-positive somata were identified in the prepyriform cortex bilaterally, the horizontal limb of the diagonal band nucleus, lateral hypothalamic region, nucleus of the lateral olfactory tract, corticomedial complex of the amygdala, mitral and tufted cell layers of the ipsilateral main olfactory bulb, locus coeruleus, and the midbrain raphe. Evidence for centrifugal fibers to the accessory olfactory bulb from the corticomedial complex of the amygdala, locus coeruleus, and possibly the nucleus of the lateral olfactory tract and midbrain raphe is discussed. A similar distribution of labeled perikarya in the forebrain and brain stem was seen in rats in which peroxidase injected into the main olfactory bulb had spread into the accessory bulb and anterior olfactory nucleus. Histochemical fluorescence microscopy of the main and accessory olfactory bulbs in the rabbit and rat revealed fine caliber, green fluorescent fibers and varicosities predominantly in the granule cell layer and less so among cells in the glomerular layer. In sections through the root of the main olfactory bulb, a similar fluorescence was seen in the deep half of the plexiform layer of the pars externa of the anterior alfactory nucleus. These fluorescent fibers likely represent the noradrenergic innervation of the olfactory bulbar and retrobulbar formations. A fluorescent yellow hue was observed in the glomerular layer of the main bulb and may signify a serotonergic innervation of this lamina...