Nuclear organization of orexinergic neurons in the hypothalamus of a lar gibbon and a chimpanzee

Employing orexin-A immunohistochemical staining we describe the nuclear parcellation of orexinergic neurons in the hypothalami of a lar gibbon and a chimpanzee. The clustering of orexinergic neurons within the hypothalamus and the terminal networks follow the patterns generally observed in other mammals, including laboratory rodents, strepsirrhine primates and humans. The orexinergic neurons were found within three distinct clusters in the ape hypothalamus, which include the main cluster, zona incerta cluster and optic tract cluster. In addition, the orexinergic neurons of the optic tract cluster appear to extend to a more rostral and medial location than observed in other species, being observed in the tuberal region in the anterior ventromedial aspect of the hypothalamus. While orexinergic terminal networks were observed throughout the brain, high density terminal networks were observed within the hypothalamus, medial and intralaminar nuclei of the dorsal thalamus, and within the serotonergic and noradrenergic regions of the midbrain and pons, which is typical for mammals. The expanded distribution of orexinergic neurons into the tuberal region of the ape hypothalamus, is a feature that needs to be investigated in other primate species, but appears to correlate with orexin gene expression in the same region of the human hypothalamus, but these neurons are not revealed with immunohistochemical staining in humans. Thus, it appears that apes have a broader distribution of orexinergic neurons compared to other primate species, but that the neurons within this extension of the optic tract cluster in humans, while expressing the orexin gene, do not produce the neuropeptide.     

Cellular location and major terminal networks of the orexinergic system in the brain of two megachiropterans

The present study describes the distribution of orexin-A immunoreactive neurons and their terminal networks in the brains of two species of megachiropterans. In general the organization of the orexinergic system in the mammalian brain is conserved across species, but as one of two groups of mammals that fly and have a high metabolic rate, it was of interest to determine whether there were any specific differences in the organization of this system in the megachiropterans. Orexinergic neurons were limited in distribution to the hypothalamus, and formed three distinct clusters, or nuclei, a main cluster with a perifornical location, a zona incerta cluster in the dorsolateral hypothalamus and an optic tract cluster in the ventrolateral hypothalamus. The nuclear parcellation of the orexinergic system in the megachiropterans is similar to that seen in many mammals, but differs from the microchiropterans where the optic tract cluster is absent. The terminal networks of the orexinergic neurons in the megachiropterans was similar to that seen in a range of mammalian species, with significant terminal networks being found in the hypothalamus, cholinergic pedunculopontine and laterodorsal tegemental nuclei, the noradrenergic locus coeruleus complex, all serotonergic nuclei, the paraventricular nuclei of the epithalamus and adjacent to the habenular nuclei. While the megachiropteran orexinergic system is typically mammalian in form, it does differ from that reported for microchiropterans, and thus provides an additional neural character arguing for independent evolution of these two chiropteran suborders.     

Nuclear organization of cholinergic,putative catecholaminergic and serotonergic nuclei in the brain of the eastern rock elephant shrew,Elephantulus myurus

The organization of the nuclear subdivisions of the cholinergic, putative catecholaminergic and serotonergic systems of the brain of the elephant shrew (Elephantulus myurus) were determined following immunohistochemistry for choline acetyltransferase, tyrosine hydroxylase and serotonin, respectively. This was done in order to determine if differences in the nuclear organization of these systems in comparison to other mammals were evident and how any noted differences may relate to specialized behaviours of the elephant shrew. The elephant shrew belongs to the order Macroscelidea, and forms part of the Afrotherian mammalian cohort. In general, the organization of the nuclei of these systems resembled that described in other mammalian species. The cholinergic system showed many features in common with that seen in the rock hyrax, rodents and primates; however, specific differences include: (1) cholinergic neurons were observed in the superior and inferior colliculi, as well as the cochlear nuclei; (2) cholinergic neurons were not observed in the anterior nuclei of the dorsal thalamus as seen in the rock hyrax; and (3) cholinergic parvocellular nerve cells forming subdivisions of the laterodorsal and pedunculopontine tegmental nuclei were not observed at the midbrain/pons interface as seen in the rock hyrax. The organization of the putative catecholaminergic system was very similar to that seen in the rock hyrax and rodents except for the lack of the rodent specific C3 nucleus, the dorsal division of the anterior hypothalamic group (A15d) and the compact division of the locus coeruleus (A6c). The nuclear organization of the serotonergic system was identical to that seen in all eutherian mammals studied to date. The additional cholinergic neurons found in the cochlear nucleus and colliculi may relate to a specific acoustic signalling system observed in elephant shrews expressed when the animals are under stress or detect a predator. These neurons may then function to increase attention to this type of acoustic signal termed foot drumming.     

Nuclear organization of some immunohistochemically identifiable neural systems in two species of the Euarchontoglires: A Lagomorph,Lepus capensis,and a Scandentia,Tupaia belangeri

The present study describes the organization of the nuclei of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of two members of Euarchontoglires, Lepus capensis and Tupaia belangeri. The aim of the present study was to investigate the nuclear complement of these neural systems in comparison to previous studies on Euarchontoglires and generally with other mammalian species. Brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The majority of nuclei revealed in the current study were similar between the species investigated and to mammals generally, but certain differences in the nuclear complement highlight potential phylogenetic interrelationships within the Euarchontoglires and across mammals. In the northern tree shrew the nucleus of the trapezoid body contained neurons immunoreactive to the choline acetyltransferase antibody with some of these neurons extending into the lamellae within the superior olivary nuclear complex (SON). The cholinergic nature of the neurons of this nucleus, and the extension of cholinergic neurons into the SON, has not been noted in any mammal studied to date. In addition, cholinergic neurons forming the medullary tegmental field were also present in the northern tree shrew. Regarding the catecholaminergic system, the cape hare presented with the rodent specific rostral dorsal midline medullary nucleus (C3), and the northern tree shrew lacked both the ventral and dorsal divisions of the anterior hypothalamic group (A15v and A15d). Both species were lacking the primate/megachiropteran specific compact portion of the locus coeruleus complex (A6c). The nuclei of the serotonergic and orexinergic systems of both species were similar to those seen across most Eutherian mammals. Our results lend support to the monophyly of the Glires, and more broadly suggest that the megachiropterans are more closely related to the primates than are any other members of Euarchontoglires studied to date.     

Nuclear organization of cholinergic,catecholaminergic, serotonergic and orexinergic systems in the brain of the Tasmanian devil (Sarcophilus harrisii)

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brains of three male Tasmanian devils (Sarcophilus harrisii), which had a mean brain mass of 11.6 g. We found that the nuclei generally observed for these systems in other mammalian brains were present in the brain of the Tasmanian devil. Despite this, specific differences in the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems appear to carry a phylogenetic signal. In the cholinergic system, only the dorsal hypothalamic cholinergic nucleus could be observed, while an extra dorsal subdivision of the laterodorsal tegmental nucleus and cholinergic neurons within the gelatinous layer of the caudal spinal trigeminal nucleus were observed. Within the catecholaminergic system the A4 nucleus of the locus coeruleus complex was absent, as was the caudal ventrolateral serotonergic group of the serotonergic system. The organization of the orexinergic system was similar to that seen in many mammals previously studied. Overall, while showing strong similarities to the organization of these systems in other mammals, the specific differences observed in the Tasmanian devil reveal either order specific, or class specific, features of these systems. Further studies will reveal the extent of change in the nuclear organization of these systems in marsupials and how these potential changes may affect functionality.     

Distribution of orexinergic neurons and their terminal networks in the brains of two species of African mole rats

The distribution of orexinergic cell bodies and terminal networks within the brains of two species of African mole rat (Cape-dune mole rat--Bathyergus suillus and highveld mole rat--Cryptomys hottentotus) were identified using immunohistochemistry for orexin-A. The aim of the study was to investigate possible differences in the nuclear complement and terminal distribution of this system by comparing those of the mole rats to published studies of other rodents and mammals. The wild-caught mole rats used in this study live a subterranean lifestyle and are well known for their regressed visual system, which may lead to the prediction of differences in the distribution of the cell bodies and the terminal networks; however, we found that both species of mole rat displayed orexinergic nuclei limited to the hypothalamus in regions similar to those previously reported for other rodent and mammalian species. No immunoreactive neurons could be identified, in either species of mole rat within the anterior hypothalamic paraventricular nucleus, as has been reported for Murid rodents. The terminal networks, while remaining similar between the species, are more strongly expressed in the Cape-dune mole rat than in the highveld mole rat.     

Distribution of serotonin in the brain stem and spinal cord of the lizard Varanus exanthematicus: an immunohistochemical study

The distribution of serotonin-containing nerve cell bodies, fibers and terminals in the lizard Varanus exanthematicus was studied with the indirect immunofluorescence technique, using antibodies to serotonin. Most of the serotonin-containing cell bodies were found in the midline, in both of the raphe nuclei, i.e. the nuclei raphes superior and inferior. A considerable number of more laterally shifted serotonergic neurons was found particularly at three levels of the brain stem, viz. in the caudal mesencephalic tegmentum, at the isthmic level, and over a long distance in the medulla oblongata. These laterally situated serotonin-positive neurons were partly found within the confines of the substantia nigra, the nucleus reticularis superior and the lateral part of the nucleus reticularis medius and ventrolateral part of the nucleus reticularis inferior, respectively. No serotonergic cell bodies were found in the spinal cord. In the brain stem a dense serotonergic innervation was observed in all of the motor nuclei of the cranial nerves, in two layers of the tectum mesencephali, in the nucleus interpeduncularis pars ventralis, the nucleus profundus mesencephali pars rostralis, the periventricular grey, the nucleus parabrachialis, the vestibular nuclear complex, the nucleus descendens nervi trigemini, the nucleus raphes inferior, and parts of the nucleus tractus solitarii. Descending serotonergic pathways could be traced into the spinal cord via the dorsolateral, ventral and ventromedial funiculi, and were found to innervate mainly three parts of the spinal grey throughout the spinal cord, i.e. the dorsal part of the dorsal horn, the motoneuron area in the ventral horn, and the intermediate zone just lateral to the central canal. The results obtained in the present study suggest a close resemblance of the organization of the serotonergic system in reptiles and mammals, especially as to the serotonergic innervation of the spinal cord.     

A study of the organization of the locus coeruleus projections to the lateral geniculate nuclei in the albino rat

The lateral geniculate nuclei of the rat are known to receive an innervation from catecholamine-containing neurons. In the present study the origin, axonal projections and terminal distribution of this innervation was studied. The lateral geniculate nuclei contain a356 ± 20 ng norepinephrine/g and64 ± 7 ng dopamine/g tissue; the latter is within the range expected for dopamine as a precursor in a region innervated by a norepinephrine-containing terminal system. When separate norepinephrine-containing cell groups located at various brain stem levels are ablated or their axonal projections destroyed, only lesions in the locus coeruleus produce a significant decrease in the norepinephrine content of the lateral geniculate nuclei. Injections of horseradish peroxidase into the lateral geniculate nuclei result in retrograde transport of horseradish peroxidase only to the noradrenergic neurons of the locus coeruleus. The labelled neurons are pretent throughout the rostrocaudal and dorsoventral axes of both the ipsilateral (60%) and contralateral (40%) nucleus. Autoradiographic and fluorescence histo-chemical experiments indicate that axons that ascend from the locus coeruleus reach the lateral geniculate nuclei via the dorsal tegmental catecholamine-containing bundle and the medial forebrain bundle. These fibers enter the ventral lateral geniculate nucleus from the zona incerta and the dorsal lateral geniculate nucleus from the superior thalamic radiation, thalamic reticular nucleus, and lateral posterior nucleus. Contralateral fibers from the locus coeruleus cross in the posterior commissure, supraoptic and pontine decussations and join the ipsilateral projections to the lateral geniculate nuclei. The bilateral locus coeruleus innervation of the nuclei is comprised of a highly branched network of varicose axons. Neither the ipsilateral nor the contralateral projections appear to be topographically organized; instead, a single fiber may have collateral axons that branch throughout large areas of the nuclei. This innervation is moderately dense in the ventral, and very dense in the dorsal, lateral geniculate nucleus.The study indicates that both the dorsal and ventral lateral geniculate nuclei receive a diffuse catecholamine-containing innervation which arises solely from the norepinephrine-containing neurons of the locus coeruleus. The innervation of each lateral geniculate nucleus is bilateral, with noradrenergic neurons located throughout both the ipsilateral and the contralateral locus coeruleus contributing to ascending pathways that terminate as a diffuse, plexiform innervation interspersed among other afferents to the lateral geniculate nuclei. It is speculated that such a diffuse noradrenergic innervation might depress the spontaneous activity of neurons in the lateral geniculate nuclei, while preserving or enhancing their responsiveness to afferent optic stimulation.     

The ascending serotonergic system in the hamster: comparison with projections of the dorsal and median raphe nuclei

The ascending serotonergic projections are derived largely from the midbrain median and dorsal raphe nuclei, and contribute to the regulation of many behavioral and physiological systems. Serotonergic innervation of the hamster circadian system has been shown to be substantially different from earlier results obtained with other methods and species. The present study was conducted to determine whether similar differences are observed in other brain regions.Ascending projections from the hamster dorsal or median raphe were identified using an anterograde tracer, Phaseolus vulgaris leucoagglutinin, injected by iontophoresis into each nucleus. Brains were processed for tracer immunoreactivity, and drawings were made of the median raphe and dorsal raphe efferent projection patterns. The efferents were also compared to the distribution of normal serotonergic innervation of the hamster midbrain and forebrain. The results show widespread, overlapping projection patterns from both the median and dorsal raphe, with innervation generally greater from the dorsal raphe. In several brain regions, including parts of the pretectum, lateral geniculate and basal forebrain, nuclei are innervated by the dorsal, but not the median, raphe. The hypothalamic suprachiasmatic nucleus is the only site innervated exclusively by the median and not by the dorsal raphe. The pattern of normal serotonin fiber and terminal distribution is generally more robust than would be inferred from the anterograde tracer material. However, there is good qualitative similarity between the two sets of data. The oculomotor nucleus and the medial habenula are unusual to the extent that each has a moderately dense serotonin terminal plexus, although neither receives innervation from the median or dorsal raphe. In contrast, the centrolateral thalamic nucleus and lateral habenula have little serotonergic innervation, but receive substantial other neural input from the raphe nuclei. The normal serotonergic innervation of the hamster brain is similar to that in the rat, although there are exceptions. The anterograde tracing of ascending median or dorsal raphe projections reveals a high, but imperfect, degree of correspondence with the serotonin innervation data, and with data from rats derived from immunohistochemical and autoradiographic tract-tracing techniques.     

Origin of projections from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus in the rat: A combined retrograde and anterograde tracing study

A number of neuronal functions governed by the hypothalamic paraventricular nucleus are influenced by serotonin, and it is generally believed that the moderate density of serotonin-immunoreactive fibres and terminals within the paraventricular nucleus originates from the midbrain dorsal and median raphe nuclei. To further evaluate the intricate anatomy of projections from brain stem raphe nuclei of the rat, a combination of retrograde and anterograde tracing experiments were conducted to determine the medullary raphe nuclei projection to the paraventricular nucleus. Rhodamine-labelled latex microspheres, Cholera toxin subunit B and FluoroGold we used as retrograde tracers. Intracerebroventricular injections into the third ventricle of all retrograde tracers labelled a distinct population of neurons in the dorsal raphe situated in the subependymal stratum adjacent to the cerebral aqueduct indicating that these cells take up the tracer from the cerebrospinal fluid. Very few retrogradely labelled neurons were seen in the median raphe after i.c.v. administration of the tracers. Retrograde tracers delivered into the medial part of the paraventricular nucleus labelled no further cells in the midbrain dorsal and median raphe nuclei, whereas a substantial number of retrogradely labelled cells emerged in the pontine raphe magnus. However, when the retrograde tracers were delivered into the lateral part of the paraventricular nucleus, avoiding leakage of the tracer into the ventricle, very few labelled neurons were seen in the dorsal and median raphe, whereas the prominent labelling of raphe magnus neurons persisted. The anatomical organization of nerve fibres terminating in the area of paraventricular nucleus originating from midbrain raphe nuclei was studied in a series of anterograde tracing experiments using the plant lectin Phaseolus vulgaris leucoagglutinin. Injections delivered into the dorsal raphe or median raphe labelled but a few fibres in the paraventricular nucleus proper. A high number of fine calibered nerve fibres overlying the ependyma adjacent to the paraventricular nucleus was, however, seen after the injections into the subependymal rostral part of the dorsal raphe. Injections delivered into the raphe magnus gave rise to a dense plexus of terminating fibres in the parvicellular parts of the paraventricular nucleus and moderately innervated the posterior magnocellular part of the paraventricular nucleus as well as the magnocellular supraoptic nucleus. Concomitant visualization of serotonin-immunoreactive neurons and retrograde FluoroGold-tracing from the paraventricular nucleus revealed that none of the serotonergic neurons of the raphe magnus projects to this nucleus, while a few of the neurons putatively projecting to the paraventricular nucleus from the median raphe are serotonergic.

The current observations suggest that the raphe magnus constitute by far the largest raphe input to the paraventricular nucleus and strongly questions the earlier held view that most raphe fibres innervating the paraventricular nucleus are derived from the midbrain dorsal and median raphe. However, the source of serotonergic innervation of the paraventricular nucleus remains elusive.     

Selective anterograde tracing of the individual serotonergic and nonserotonergic components of the dorsal raphe nucleus projection to the vestibular nuclei

It is well known that the dorsal raphe nucleus (DRN) sends serotonergic and nonserotonergic projections to target regions in the brain stem and forebrain, including the vestibular nuclei. Although retrograde tracing studies have reported consistently that there are differences in the relative innervation of different target regions by serotonergic and nonserotonergic DRN neurons, the relative termination patterns of these two projections have not been compared using anterograde tracing methods. The object of the present investigation was to trace anterogradely the individual serotonergic and nonserotonergic components of the projection from DRN to the vestibular nuclei in rats. To trace nonserotonergic DRN projections, animals were pretreated with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), and then, after 7 days, the anterograde tracer biotinylated dextran amine (BDA) was iontophoretically injected into the DRN. In animals treated with 5,7-DHT, nonserotonergic BDA-labeled fibers were found to descend exclusively within the ventricular plexus and to terminate predominantly within the periventricular aspect of the vestibular nuclei. Serotonergic DRN projections were traced by injecting 5,7-DHT directly into DRN, and amino-cupric-silver staining was used to visualize the resulting pattern of terminal degeneration. Eighteen hours after microinjection of 5,7-DHT into the DRN, fine-caliber degenerating serotonergic terminals were found within the region of the medial vestibular nucleus (MVN) that borders the fourth ventricle, and a mixture of fine- and heavier-caliber degenerating serotonergic terminals was located further laterally within the vestibular nuclear complex. These findings indicate that fine-caliber projections from serotonergic and nonserotonergic DRN neurons primarily innervate the periventricular regions of MVN, whereas heavier-caliber projections from serotonergic DRN neurons innervate terminal fields located in more lateral regions of the vestibular nuclei. Thus, serotonergic and nonserotonergic DRN axons target distinct but partially overlapping terminal fields within the vestibular nuclear complex, raising the possibility that these two DRN projection systems are organized in a manner that permits regionally-specialized regulation of processing within the vestibular nuclei.     

Organization and number of orexinergic neurons in the hypothalamus of two species of Cetartiodactyla: a comparison of giraffe (Giraffa camelopardalis) and harbour porpoise (Phocoena phocoena)

The present study describes the organization of the orexinergic (hypocretinergic) neurons in the hypothalamus of the giraffe and harbour porpoise--two members of the mammalian Order Cetartiodactyla which is comprised of the even-toed ungulates and the cetaceans as they share a monophyletic ancestry. Diencephalons from two sub-adult male giraffes and two adult male harbour porpoises were coronally sectioned and immunohistochemically stained for orexin-A. The staining revealed that the orexinergic neurons could be readily divided into two distinct neuronal types based on somal volume, area and length, these being the parvocellular and magnocellular orexin-A immunopositive (OxA+) groups. The magnocellular group could be further subdivided, on topological grounds, into three distinct clusters--a main cluster in the perifornical and lateral hypothalamus, a cluster associated with the zona incerta and a cluster associated with the optic tract. The parvocellular neurons were found in the medial hypothalamus, but could not be subdivided, rather they form a topologically amorphous cluster. The parvocellular cluster appears to be unique to the Cetartiodactyla as these neurons have not been described in other mammals to date, while the magnocellular nuclei appear to be homologous to similar nuclei described in other mammals. The overall size of both the parvocellular and magnocellular neurons (based on somal volume, area and length) were larger in the giraffe than the harbour porpoise, but the harbour porpoise had a higher number of both parvocellular and magnocellular orexinergic neurons than the giraffe despite both having a similar brain mass. The higher number of both parvocellular and magnocellular orexinergic neurons in the harbour porpoise may relate to the unusual sleep mechanisms in the cetaceans.     

The cholinergic innervation of normal and transplanted superior colliculus in the rat: an immunohistochemical study

The distribution of choline acetyltransferase was determined in normal and transplanted rat superior colliculus with choline acetyltransferase immunohistochemistry. This distribution was compared to the pattern of histochemically detected acetylcholinesterase activity. To determine cholinergic input to the superficial superior colliculus, double labelling experiments combining retrograde tracing methods and choline acetyltransferase immunohistochemistry were carried out. No choline acetyltransferase-containing neurons were observed in the rat superior colliculus. A dense network of choline acetyltransferase-immunoreactive fibres and terminals was seen in the intermediate layers of the normal superior colliculus. The distribution was patchy and very similar to the pattern of acetylcholinesterase activity. Occasional fibres and terminals were seen in the deep tectal laminae. The superficial layers contained a low number of choline acetyltransferase-stained fibres and terminals but a comparatively high level of acetylcholinesterase activity. Following a unilateral injection of a tracer into the superficial superior colliculus, retrogradely labelled choline acetyltransferase-immunoreactive neurons were found in the dorsal and ventral subnuclei of the ipsilateral parabigeminal nucleus. As in the normal superior colliculus, choline acetyltransferase-positive neurons were not found in tectal transplants. However, choline acetyltransferase-immunoreactive fibres and terminals were seen in grafts but only in those which had extensive connections with the host midbrain. The pattern of staining most closely resembled that seen in the intermediate layers of the normal superior colliculus. The similar arrangement of choline acetyltransferase and acetylcholinesterase activity in the intermediate layers of normal rat superior colliculus provides further evidence for cholinergic innervation to these layers, probably originating in the dorsal and pedunculopontine tegmental nuclei. The data from the double labelling experiments indicate that the choline acetyltransferase-immunoreactive terminals observed in the superficial layers represent the terminal field of an ipsilateral cholinergic projection from the parabigeminal nucleus. Tectal grafts receive cholinergic innervation from the host. The evidence suggests that much of this input derives from the cholinergic nuclei in the brainstem tegmentum which normally project to the intermediate tectal layers.     

Organization of cholinergic,catecholaminergic, serotonergic and orexinergic nuclei in three strepsirrhine primates: Galago demidoff,Perodicticus potto and Lemur catta

The nuclear organization of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of three species of strepsirrhine primates is presented. We aimed to investigate the nuclear complement of these neural systems in comparison to those of simian primates, megachiropterans and other mammalian species. The brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The nuclei identified were identical among the strepsirrhine species investigated and identical to previous reports in simian primates. Moreover, a general similarity to other mammals was found, but specific differences in the nuclear complement highlighted potential phylogenetic interrelationships. The central feature of interest was the structure of the locus coeruleus complex in the primates, where a central compactly packed core (A6c) of tyrosine hydroxylase immunopositive neurons was surrounded by a shell of less densely packed (A6d) tyrosine hydroxylase immunopositive neurons. This combination of compact and diffuse divisions of the locus coeruleus complex is only found in primates and megachiropterans of all the mammalian species studied to date. This neural character, along with variances in a range of other neural characters, supports the phylogenetic grouping of primates with megachiropterans as a sister group.     

Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1–Ch6)

Monoclonal antibodies to choline acetyltransferase and a histochemical method for the concurrent demonstration of acetylcholinesterase and horseradish peroxidase were used to investigate the organization of ascending cholinergic pathways in the central nervous system of the rat. The cortical mantle, the amygdaloid complex, the hippocampal formation, the olfactory bulb and the thalamic nuclei receive their cholinergic innervation principally, from cholinergic projection neurons of the basal forebrain and upper brainstem. On the basis of connectivity patterns, we subdivided these cholinergic neurons into six major sectors. The Chl and Ch2 sectors are contained within the medial septal nucleus and the vertical limb nucleus of the diagonal band, respectively. They provide the major cholinergic projections of the hippocampus. The Ch3 sector is contained mostly within the lateral portion of the horizontal limb nucleus of the diagonal band and provides the major cholinergic innervation to the olfactory bulb. The Ch4 sector includes cholinergic neurons in the nucleus basalis, and also within parts of the diagonal band nuclei. Neurons of the Ch4 sector provide the major cholinergic innervation of the cortical mantle and the amygdala. The Ch5–Ch6 sectors are contained mostly within the pedunculopontine nucleus of the pontomesencephalic reticular formation (Ch5) and within the laterodorsal tegmental gray of the periventricular area (Ch6). These sectors provide the major cholinergic innervation of the thalamus. The Ch5–Ch6 neurons also provide a minor component of the corticopetal cholinergic innervation.

These central cholinergic pathways have been implicated in a variety of behaviors and especially in memory function. It appears that the age-related changes of memory function as well as some of the behavioral disturbances seen in the dementia of Alzheimer's Disease may be related to pathological alterations along central cholinergic pathways.     

Organization of cholinergic,putative catecholaminergic and serotonergic nuclei in the diencephalon,mibrain and pons of sub-adult male giraffes

The current study describes the nuclear organization and neuronal morphology of the cholinergic, putative catecholaminergic and serotonergic systems within the diencephalon, midbrain and pons of the giraffe using immunohistochemistry for choline acetyltransferase, tyrosine hydroxylase and serotonin. The giraffe has a unique phenotype (the long neck), a large brain (over 500 g) and is a non-domesticated animal, while previous studies examining the brains of other Artiodactyls have all been undertaken on domesticated animals. The aim of the present study was to investigate possible differences in the nuclear organization and neuronal morphology of the above-mentioned systems compared to that seen in other Artiodactyls and mammals. The nuclear organization of all three systems within the giraffe brain was similar to that of other Artiodactyls. Some features of interest were noted for the giraffe and in comparison to other mammals studied. The cholinergic neuronal somata of the laterodorsal tegmental nucleus were slightly larger than those of the pedunculopontine tegmental nucleus, a feature not described in other mammals. The putative catecholaminergic system of the giraffe appeared to lack an A15 dorsal nucleus, which is commonly seen in other mammals but absent in the Artiodactyls, had a large and expanded substantia nigra pars reticulata (A9 ventral), a small diffuse portion of the locus coerueleus (A6d), an expansive subcoeruleus (A7sc and A7d), and lacked the A4 nucleus of the locus coeruleus complex. The nuclear organization of the serotonergic system of the giraffe was identical to that seen in all other eutherian mammals studied to date. These observations in the giraffe demonstrate that despite significant changes in life history, phenotype, brain size and time of divergence, species within the same order show the same nuclear organization of the systems investigated.     

Differential effect of orexins (hypocretins) on serotonin release in the dorsal and median raphe nuclei of freely behaving rats

Orexin (hypocretin)-containing neurons in the perifornical hypothalamus project to widespread regions of the brain, including the dorsal and median raphe nuclei [Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS (1998) Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996-10015; Wang QP, Koyama Y, Guan JL, Takahashi K, Kayama Y, Shioda S (2005) The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus. Regul Pept 126:35-42]. Orexin-A or orexin-B was infused by reverse microdialysis into the dorsal raphe nucleus or median raphe nucleus of freely behaving rats, and extracellular serotonin was simultaneously collected by microdialysis and analyzed by high-performance liquid chromatography. We have found that orexin-A produced a dose-dependent increase of serotonin in the dorsal raphe nucleus, but not in the median raphe nucleus. However, orexin-B elicited a small but significant effect in both the dorsal raphe nucleus and median raphe nucleus. Orexins may have regionally selective effects on serotonin release in the CNS, implying a unique interaction between orexins and serotonin in the regulation of activities including sleep-wakefulness.     

Projections of cholinergic and non-cholinergic neurons of the brainstem core to relay and associational thalamic nuclei in the cat and macaque monkey

The projections of brainstem core neurons to relay and associational thalamic nuclei were studied in the cat and macaque monkey by combining the retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase with choline acetyltransferase immunohistochemistry. All major sensory (medial geniculate, lateral geniculate, ventrobasal), motor (ventroanterior, ventrolateral, ventromedial), associational (mediodorsal, pulvinar, lateral posterior) and limbic (anteromedial, anteroventral) thalamic nuclei of the cat were found to receive projections from cholinergic neurons located in the peribrachial area of the pedunculopontine nucleus and in the laterodorsal tegmental nucleus as well as from non-cholinergic neurons in the rostral (perirubral) part of the central tegmental mesencephalic field. Specific relay nuclei receive less than 10% of their brainstem afferents from non-cholinergic neurons located at rostral midbrain levels and receive 85-96% of their brainstem innervation from a region at midbrain-pontine junction where the cholinergic peribrachial area and laterodorsal tegmental nucleus are maximally developed. Of the total number of horseradish peroxidase-positive brainstem neurons seen after injections in various specific relay nuclei, the double-labeled (horseradish peroxidase + choline acetyltransferase) neurons represent approximately 70-85%. Three to eight times more numerous horseradish peroxidase-labeled brainstem cells were found after injections in associational (mediodorsal and pulvinar-lateral posterior complex) and diffusely cortically-projecting (ventromedial) thalamic nuclei of cat than after injections in specific relay nuclei. The striking retrograde cell labeling observed after injections in nuclei with associative functions and widespread cortical projections was due to massive afferentation from non-cholinergic parts of the midbrain and pontine reticular formation, on both ipsi- and contralateral sides. After wheat germ agglutinin-horseradish peroxidase injections in the associative pulvinar-lateral posterior complex and mediodorsal nucleus of Macaca sylvana, 45-50% of horseradish peroxidase-positive brainstem peribrachial neurons were also choline acetyltransferase-positive. While cells in the medial part of the cholinergic peribrachial area were found to project especially towards the pulvinar-lateral posterior nuclear complex in monkey, the retrograde cell labeling seen after the mediodorsal injection was mostly confined to the lateral part of both dorsal and ventral aspects of the peribrachial area.(ABSTRACT TRUNCATED AT 400 WORDS)