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.     

Distribution of orexin-A immunoreactive neurons and their terminal networks in the brain of the rock hyrax, Procavia capensis

The present study describes the distribution of orexin-A immunoreactive neurons and terminal networks in relation to the previously described catecholaminergic, cholinergic and serotonergic systems within the brain of the rock hyrax, Procavia capensis. Adult female rock hyrax brains were sectioned and immunohistochemically stained with an antibody to orexin-A. The staining revealed that the neurons were mainly located within the hypothalamus as with other mammals. The orexinergic terminal network distribution also resembled the typical mammalian plan. High-density orexinergic terminal networks were located within regions of the diencephalon (e.g. paraventricular nuclei), midbrain (e.g. serotonergic nuclei) and pons (locus coeruleus), while medium density orexinergic terminal networks were evident in the telencephalic (e.g. basal forebrain), diencephalic (e.g. hypothalamus), midbrain (e.g. periaqueductal gray matter), pontine (e.g. serotonergic nuclei) and medullary regions (e.g. serotonergic and catecholaminergic nuclei). Although the distribution of the orexinergic terminal networks was typically mammalian, the rock hyrax did show one atypical feature, the presence of a high-density orexinergic terminal network within the anterodorsal nucleus of the dorsal thalamus (AD). The dense orexinergic innervation of the AD nucleus has only been reported previously in the Nile grass rat, Arvicanthis niloticus and Syrian hamster, Mesocricetus auratus, both diurnal mammals. It is possible that orexinergic innervation of the AD nucleus might be a unique feature associated with diurnal mammals. It was also noted that the dense orexinergic innervation of the AD nucleus coincided with previously identified cholinergic neurons and terminal networks in this particular nucleus of the rock hyrax brain. It is possible that this dense orexinergic innervation of the AD nucleus in the brain of the rock hyrax may act in concert with the cholinergic neurons and/or the cholinergic axonal terminals, which in turn may influence arousal states and motivational processing.     

Orexinergic bouton density is lower in the cerebral cortex of cetaceans compared to artiodactyls

The species of the cetacean and artiodactyl suborders, which constitute the order Cetartiodactyla, exhibit very different sleep phenomenology, with artiodactyls showing typical bihemispheric slow wave and REM sleep, while cetaceans show unihemispheric slow wave sleep and appear to lack REM sleep. The aim of this study was to determine whether cetaceans and artiodactyls have differently organized orexinergic arousal systems by examining the density of orexinergic innervation to the cerebral cortex, as this projection will be involved in various aspects of cortical arousal. This study provides a comparison of orexinergic bouton density in the cerebral cortex of twelve Cetartiodactyla species (ten artiodactyls and two cetaceans) by means of immunohistochemical staining and stereological analysis. It was found that the morphology of the axonal projections of the orexinergic system to the cerebral cortex was similar across all species, as the presence, size and proportion of large and small orexinergic boutons were similar. Despite this, orexinergic bouton density was lower in the cerebral cortex of the cetaceans studied compared to the artiodactyls studied, even when corrected for brain mass, neuron density, glial density and glial:neuron ratio. Results from correlational and principal component analyses indicate that glial density is a major determinant of the observed differences between artiodactyl and cetacean cortical orexinergic bouton density.     

Vasopressin- and neurophysin-immunoreactive neurons in the septal region, medial amygdala and locus coeruleus in colchicine-treated rats

The distribution and morphology of neurons containing vasopressin, oxytocin and their associated neurophysins were examined immunohistochemically in rats given intracerebroventricular injections of colchicine. Under these conditions, numerous neurons containing vasopressin and neurophysin were found in several brain areas in addition to those previously described in the hypothalamus. Individual parvocellular vasopressin neurons were scattered in the medial and lateral septum and vertical limb of the nucleus of the diagonal band, while a large number of such neurons were found throughout both the bed nucleus of the stria terminals and the dorsal portion of the medial amygdala. In addition a small cluster of parvocellular vasopressin neurons was present adjacent to the top of the third ventricle in the posterior dorsal hypothalamic area and a number of such neurons were found in the ventral locus coeruleus and sub coeruleus. The mean diameters of these parvocellular vasopressin neurons ranged from 16.6 to 19.8 micron in the different regions, in contrast to the 25.4 micron mean diameter of hypothalamic magnocellular vasopressin neurons, or the 13.7 micron mean diameter of parvocellular vasopressin neurons in the suprachiasmatic nucleus. No vasopressin neurons were found in other brain and spinal cord regions under the conditions used in this study, although all regions were examined. No oxytocin neurons other than those previously described in the hypothalamus and immediately contiguous regions were found. Measurement of the mean diameter of oxytocin neurons showed that neurons in the caudal paraventricular nucleus were clearly smaller (18.9 micron) than magnocellular oxytocin neurons (24.8 micron) in other parts of the hypothalamus. These parvocellular oxytocin neurons with experimentally documented central connections were similar in both size and appearance to the parvocellular vasopressin neurons seen after colchicine treatment, which are potential sources of certain central vasopressin pathways. These findings indicate that there are at least two types of oxytocin neurons in the hypothalamus and several types of vasopressin neurons in a variety of different areas in the brain, many of which are outside of the hypothalamus.     

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.     

Co-expression of vasopressin and androgen-binding protein in the rat hypothalamus

In previous studies we have observed the expression of androgen binding protein (ABP) in the rat hypothalamo-neurohypophysial system. With immunocytochemical double staining we found partial co-localization with oxytocin. In the present study we used antibodies to the anti-diuretic hormone arginine vasopressin (AVP) for co-localization with ABP in the rat hypothalamus. Both antigens were seen in the magnocellular paraventricular and supraoptic nuclei. Dense fiber networks with varicosities containing both AVP and ABP immunoreactivity were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed also co-existence in the parvocellular portion of the paraventricular nucleus and in the suprachiasmatic nucleus. ABP immunoreactive neurons in the preoptic region were devoid of AVP staining, AVP neurons in the bed nucleus of the stria terminalis stained only occasionally for ABP. We conclude that both the magnocellular and the parvocellular hypothalamic vasopressin systems are capable of expressing the steroid binding globulin, which is probably subject to axonal transport, along with the peptide hormone. Intrahypothalamic expression of ABP may be among the mechanisms necessary for rapid actions of steroids on hypothalamic neuroendocrine systems.     

Effect of adrenalectomy on miniature inhibitory postsynaptic currents in the paraventricular nucleus of the hypothalamus

Within the rat paraventricular nucleus of the hypothalamus two types of neurons have been distinguished based on morphological appearance, i.e., parvocellular and magnocellular neurons. The parvocellular neurons play a key role in regulating the activity of the hypothalamo-pituitary-adrenal axis, which is activated, e.g., after stress exposure. These neurons receive humoral negative feedback via the adrenal hormone corticosterone but also neuronal inhibitory input, either directly or transsynaptically relayed via GABAergic interneurons. In the present study we examined to what extent the neuronal GABAergic input is influenced by the humoral signal. To this end, miniature inhibitory postsynaptic currents (mIPSCs) were recorded in parvo- and magnocellular neurons of adrenalectomized rats, which lack corticosterone, and in sham-operated controls. Under visual control neurons in coronal slices containing the paraventricular nucleus were designated as putative parvocellular or magnocellular neurons: the former were located in the medial part of the nucleus and displayed a small fusiform soma; the latter were mostly located in the lateral part and were recognized by their large round soma. Compared with putative magnocellular neurons, parvocellular neurons generally exhibited a lower membrane capacitance, lower mIPSC frequency, and smaller mIPSC amplitude. Following adrenalectomy, the mIPSC frequency was significantly enhanced in parvo- but not magnocellular neurons. Other properties of the cells were not affected. In a second series of experiments we examined whether the increase in mIPSC frequency was due to the absence of corticosterone or caused by other effects related to adrenalectomy. The data support the former explanation since implantation of a corticosterone releasing pellet after adrenalectomy fully prevented the change in mIPSC frequency. We conclude that, in the absence of humoral negative feedback, local GABAergic input of parvocellular neurons in the paraventricular nucleus is enhanced. This may provide a compensatory mechanism necessary for maintaining controllable network activity.     

Calcium-binding proteins in the cerebellar cortex of the bottlenose dolphin and harbour porpoise

Studying the distribution of Ca²⁺-binding proteins allows one to discover specific neuron chemotypes involved in the regulation of the activity of various neural elements. While extensive data exist on Ca²⁺-binding proteins in the nervous system, in particular, in the cerebellar cortex of terrestrial mammals, the localization of these proteins in the cerebellar cortex of marine mammals has not been studied. We studied the localization of calretinin, calbindin, and parvalbumin immunoreactivity in the cerebellar cortex of the bottlenose dolphin Tursiops truncates and harbour porpoise Phocoena phocoena. In both species, most Purkinje cells were calbindin-immunoreactive, while calretinin and parvalbumin were expressed in a small portion of Purkinje cells. In addition, calretinin-immunoreactive unipolar brush and granule cells and calbindin- and parvalbumin-immunoreactive basket, stellate, and Golgi cells were observed. Calretinin-immunoreactive corticopetal (mossy and climbing) fibers were found. Based on the length of the primary dendrite, short-, middle-, and long-dendrite unipolar brush cells could be distinguished. The validity of this classification was supported using cluster analysis suggesting the presence of several natural types of these cells. The distribution of Ca²⁺-binding proteins in the cerebellar cortex of the cetaceans studied was generally similar to that reported for terrestrial mammals, suggesting that this trait is evolutionarily conservative in mammals.     

The influence of salt loading on vasopressin gene expression in magno- and parvocellular hypothalamic neurons: an immunocytochemical and in situ hybridization analysis

Arginine vasopressin peptide and messenger RNA expression were examined at the cellular level in the magnocellular and parvocellular neurons in the rat paraventricular nucleus after dehydration and rehydration, employing immunocytochemistry and in situ hybridization histochemistry on the same tissue sections. Most magnocellular vasopressinergic neurons of control animals expressed both vasopressin-like immunoreactivity and messenger RNA. However, neurons negative for vasopressin-like immunoreactivity but expressing messenger RNA were also detected, and their number increased during dehydration. In contrast, almost all of the parvocellular vasopressinergic neurons of dehydrated animals expressed vasopressin messenger RNA alone, with continued increase in their number after rehydration, despite return of the number of magnocellular vasopressinergic neurons to the control level. Vasopressin messenger RNA and corticotropin releasing factor-like immunoreactivity were co-localized in the same parvocellular neurons, and vasopressin-immunoreactive nerve terminals were detected in the external zone of the median eminence. These findings suggest that magno- and parvocellular vasopressinergic neurons are differentially activated during dehydration/rehydration. Osmotic stimuli activate all magnocellular vasopressinergic neurons, but the effect is not simultaneous in all of these neurons. Parvocellular vasopressinergic neurons are also activated by the stress of dehydration which effect appears to last longer than in the magnocellular system.     

Differential expression of calcium-binding proteins in the red nucleus of the developing and adult human brain

The adult human red nucleus consists of two parts: (1) the parvocellular part, which is clearly separated from (2) the magnocellular part. The latter and its rubrospinal projection is known to be rudimentary in the adult human brain. Information concerning the fetal or neonatal features of the red nucleus is sparse. This study is aimed at providing a detailed account of the distribution of three calcium-binding proteins: calretinin (CR), calbindin (CB), and parvalbumin (PV), which are known to be expressed in distinct neuronal populations. Special attention has been paid to transient phenomena. CB was the most abundant protein in the magnocellular part in fetal and perinatal brains; immunoreactive (ir) neurons appeared numerous and densely packed. In the adult only few and widely spaced ir nerve cells were present. CR-expression largely corresponds to that of CB, except that fewer neurons were immunolabelled. In double- labellings the majority of neurons expressed both CB and CR; a moderate number of nerve cells solely expressing CR was present in the magnocellular part. PV-ir fibers and a moderate number of small cells were observed in the fetal, perinatal as well as the adult parvocellular part. A few PV-ir neurons were seen in the magnocellular part of the fetal and perinatal brains. Our results indicated that: (1) the magnocellular and parvocellular parts of the red nucleus were well-demarcated portions from fetal life onwards, thus a dominance of the parvocellular part over the magnocellular occurred during development; (2) the magnocellular part was more prominent in the fetal period than in adulthood; (3) neurons in the red nucleus were heterogeneous with respect to the immunoreactivities towards the three calcium-binding proteins examined; (4) the transient prominence of the magnocellular part might be a substrate for a specific transitory pattern of motor behaviour. Accepted: 7 September 2000     

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.     

Galanin-like immunoreactivity is present in human substantia innominata and in senile plaques in Alzheimer's disease

Galanin immunoreactivity has been shown to be present in cholinergic magnocellular basal forebrain neurons in both rat and monkey brain. In the present study galanin immunoreactivity in human substantia innominata was found in both the magnocellular neurons of the nucleus basalis of Meynert and in a dorsomedial group of intensely immunoreactive parvocellular neurons. Scattered galanin-immunoreactive fibers, but no immunoreactive neurons, were found in normal human cerebral cortex. Galanin-immunoreactive neurites were found in a small number of senile plaques in the hippocampus and temporal isocortex of Alzheimer's disease patients. The existence of galanin immunoreactivity in human magnocellular basal forebrain neurons which are depleted in Alzheimer's disease suggests that galanin may be similarly affected.     

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.     

GABA-immunoreactive neurons in the mediodorsal nucleus of the monkey thalamus

The mediodorsal nucleus of the thalamus is an important component of the brain's circuitry for memory 9,20 and yet surprisingly little is known of its intrinsic organization. In the present study we have examined the distribution, spatial relationships and morphology of gamma-aminobutyric acid (GABA)-containing cells within the magnocellular and parvocellular subdivisions of the mediodorsal nucleus. These subdivisions have anatomically and functionally distinct connections with the orbital (limbic) and dorsolateral (association) areas of the prefrontal cortex, and accordingly, we investigated whether there were corresponding differences in their local circuit organization. Our findings show that round or ovoid GABA-immunoreactive neurons were abundant in both subdivisions. However, these neurons were larger in diameter in the magnocellular aspect (mean diameter = 10.4 +/- 0.1 micron) than in the parvocellular moiety (mean diameter = 9.9 +/- 0.1 micron) and the intensity of reactivity was also greater for the magnocellular neurons. Comparison of the densities of GABA-immunoreactive neurons revealed not only a greater density of neurons in the parvocellular division but also that the proportion of all neurons which were GABA-containing was greater in this region. These differences in the morphology and density of inhibitory local circuit neurons may contribute to the functional duality of prefrontal cortex innervated by these thalamo-cortical pathways. Certain qualitative features were common to both subdivisions; thus GABA-immunoreactive neurons were found in small clusters throughout the magnocellular and parvocellular divisions of the mediodorsal nucleus and, in addition, regions with few or no GABA-immunoreactive cells were often surrounded by strings of GABA-containing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of neuron number in the ageing mouse paraventricular nucleus.

The number of neurons in the paraventricular nucleus was estimated in brains from male mice aged 6, 25, 28 and 31 months. There was no significant variation with age in neuron number (mean 2715). At all ages most neurons were parvocellular neurons and the neurosecretory magnocellular neurons made up only 10% of the total. There was no significant variation in neuronal nuclear diameter (mean 9.4 microns) with age. The nuclear diameter of both parvocellular and magnocellular neurons was identical. The difference in size between the two types of neurons was due to the difference in volume of their perikarya.     

Influence of aging on the neurochemical organization of the rat paraventricular nucleus

The paraventricular nucleus (PVH) of the hypothalamus is a key region for the integration of the autonomic and neuroendocrine mechanisms. This integration becomes less reliable with age. Some critical functions, such as eating and drinking, body-temperature regulation, autonomic and endocrine responses which regulate the cardiovascular system seem to be particularly affected by the aging-related processes. In this paper, we analysed by means of immunocytochemistry the neurochemical organization of the magnocellular and parvocellular component of the PVH in old male rats. The main results concerning the neurohormones and the carrier proteins are the following: a significant decrease in the number of the oxytocin- (OXY) like immunoreactive neurons of the medial and lateral parvocellular nuclei; a decrease in the vasopressin- (VAS) like immunoreactive neurons of the medial and lateral parvocellular nuclei and also of the medial magnocellular nucleus; a decrease in the neurophysin- (NRP) like immunoreactive neurons of the lateral parvocellular nucleus. We also found a decrease in the mean area of magnocellular OXY- and VAS-like immunoreactive neurons, a decrease in the extension of the dendritic tree sampled in the medial part of the nucleus; a decrease in the number of varicosities along the neurons coming from the PVH, and in the density of axons in the median eminence and in the vagal complex. The NRP-like immunoreactive structures in the substantia gelatinosa of the spinal cord of old rats were also decreased in respect to younger adult animals. Among the neuropeptides investigated (corticotropin-releasing factor, leu-enkephalin, somatostatin, cholecystokinin and neurotensin) we found a decrease in the leu-enkephalin-like immunoreactive neurons of the dorsal and medial parvocellular nuclei. Our data support--from a morphological point of view--the existence of involution processes in the neurochemical organization of the PVH during aging.