Protein components of the blood–brain barrier (BBB) in the brainstem area postrema–nucleus tractus solitarius region

The blood–brain barrier (BBB) prevents entry of circulating substances into the brain. The circumventricular organs (CVOs) lack a BBB and have a direct communication with the circulation blood. One of the CVOs, the area postrema (AP), which has a close relationship with the nucleus of the tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMX), plays a role in controlling the entry of blood-borne substances to neurons of the brainstem. To clarify the cellular localization of protein components of the BBB in the brainstem AP-NTS region, we used antisera to—(1) Tight junctions: claudin-5 and zona occludens-1 (ZO-1). (2) Endothelial cells: (a) all endothelial cells—rat endothelial cell antigen-1 (RECA-1) and (b) endothelial cells at BBB—endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR). (3) Basal lamina—laminin. (4) Vascular smooth muscle cells—smooth muscle actin (SMA). (5) Pericytes—chondroitin sulfate proteoglycan (NG2). (6) Glial cells: (a) astrocytes—glial fibrillary acidic protein (GFAP), (b) tanycytes—dopamine- and cAMP-regulated phosphoprotein of 32 kDA (DARPP-32), and (c) microglia—CD11b. Neuronal cell bodies in the NTS were visualized by antisera to neuropeptide Y (NPY) and α-melanocyte-stimulating hormone (α-MSH), two peptides regulating energy balance. This study provides a detailed analysis of the cellular localization of BBB proteins in the AP and NTS and shows the existence of vessels in the dorsomedial aspect of the NTS that lack immunoreactivity for the BBB markers EBA and TfR. Such vessels may represent a route of entry for circulating substances to neurons in the NTS that inter alia regulate energy balance.     

A second look at the barriers of the medial basal hypothalamus

The cell bodies of hypothalamic secretory neurons are localized in areas protected by the blood-brain barrier (BBB), whereas their axon terminals are localized in the median eminence, which lacks a BBB. This implies a complex barrier system, allowing neurons of the central nervous system to secrete into the blood stream without making the BBB leaky. In the present study, three experimental protocols were applied to clarify certain relevant aspects of the barriers operating in the medial basal hypothalamus of the rat. We established that the milieu of the arcuate nucleus is exposed to both the ventricular and the subarachnoidal cerebrospinal fluid (CSF).The median eminence milieu, the perivascular space of the portal vessels, and the subarachnoid space appear to be in open communication; also, beta2-tanycytes establish an efficient barrier between the median eminence milieu and the ventricular CSF. Similarly, beta1-tanycytes establish a lateral barrier, separating the intercellular space of the median eminence from that of the arcuate nucleus. We also found that the glucose transporter I (GLUT I), a BBB marker, is localized throughout the whole plasma membrane of beta1-tanycytes, but is missing from beta2-tanycytes. Expression of GLUT I by tanycytes progressively develops during the first postnatal weeks; while the degree of damage of the arcuate nucleus by administration of monosodium glutamate, at different postnatal intervals, parallels that of the GLUT I immunoreactivity of beta1-tanycytes. An explanation is offered for the selective destruction of the arcuate neurons by the parenteral administration of monosodium glutamate to infant rats.     

Dendritic arborization and axon trajectory of neurons in the hypothalamic arcuate nucleus of the rat

Summary Dendritic arborization patterns of neurons in the hypothalamic arcuate nucleus and the course of their axons outside the median eminence were studied using Golgi material, electron microscopic detection of degenerated axon terminals following surgical interference, and horseradish peroxidase technique.Two kinds of neurons can be distinguished in the arcuate nucleus: small fusiform and somewhat larger polygonal cells. Fusiform neurons having two, sparsely arborizing stem dendrites are localized mainly in the medial and dorsal parts of the nucleus. A variant of the fusiform neurons (pear shaped cells) has only one dendritic stem, and the axon originates at the other pole of the neuron. These latter cells are found along the ependymal wall of the third ventricle. Polygonal neurons which occupy the ventral and lateral portions of the nucleus have 4–5 repeatedly branching stem dendrites. In addition to the well known course of the axons of the arcuate neurons towards the median eminence, there are axons of both fusiform and polygonal cells which leave the nucleus in lateral or dorsolateral direction. Axons having this course frequently issue a collateral branch along the first 100–150 m of their trajectory.A knife-cut through the arcuate nucleus or along its lateral border resulted in degeneration of axon terminals in the area between the arcuate and ventromedial nuclei, and in a ventromedial sector of the ventromedial nucleus itself. A cut located in the area between the arcuate and ventromedial nuclei caused degenerated axon terminals over a large part of the ventromedial nucleus with a considerable increase in the ventromedial portion.Labeled fusiform and polygonal neurons can be seen in the arcuate nucleus following the injection of horseradish peroxidase into the lateral hypothalamus.The results suggest unequivocally that the neurons in the arcuate nucleus have efferent connections not only towards the median eminence but also to the hypothalamic ventromedial nucleus and lateral hypothalamic area.     

Lateral evaginations from the third ventricle into the rat mediobasal hypothalamus: an amplification of the ventricular route.

In this work we report the existence of several evaginations extending out of the third ventricle within the mediobasal hypothalamus of the rat. In coronal sections, these evaginations appear as very narrow canaliculi integrating a canalicular system, which increases the contact surface between the ventricular lining and the nervous tissue. Consequently these evaginations enlarge the ventricular route for the transport of active principles present in the cerebrospinal fluid, such as (neuro)hormones and neurotransmitters. The mediobasal hypothalamus includes the arcuate nucleus and the median eminence (both involved in neuroendocrine mechanisms and in the regulation of pituitary function). A possible implication of our finding is that the neuroactive substance-containing ventricular cerebrospinal fluid may reach the intercellular spaces of the surrounding neuropil of the arcuate nucleus. According to literature these substances cross the ependyma of the lateral wall of the infundibular recess of the third ventricle. We suggest that such substances might also pass through the ependymal lining of the canalicular system, which displays the same ultrastructural characteristics as the rest of the ependyma of the lateral wall of the third ventricle. Therefore, the arcuate neurons may be influenced not only by synaptic inputs (afferent fibers) but also by non-synaptic diffusion neurotransmission (by means of neuroactive substances present in the cerebrospinal fluid).     

Intrahypothalamic connections: An electron microscopic study in the rat

Summary Terminal degeneration within the hypothalamus was studied by electron microscopy 1 or 2 days (1) after carefully placed microlesions in the arcuate, anterior periventricular, ventromedial, premammillary and posterior hypothalamic nuclei and (2) after microlesions placed in the hypothalamus deafferented 3 weeks earlier.In the median eminence terminal degeneration was found after each of these lesions. Projections from the ventromedial nucleus reach the arcuate, suprachiasmatic, and anterior periventricular nuclei.Projections from the arcuate nucleus terminate in the medial preoptic, anterior periventricular, and ventromedial nuclei.After lesioning the premammillary nuclei degeneration was found in the supraoptic, arcuate, anterior hypothalamic and ventromedial nuclei.     

Preprotachykinin gene expression in the mediobasal hypothalamus of estrogen-treated and ovariectomized control rats

Summary We have determined the localization of preprotachykinin (PPT) mRNA-containing neurons in the mediobasal hypothalamus of the rat. PPT mRNA-containing neurons are present in the ventromedial nucleus (with a concentration in the ventrolateral aspect (VL-VM)), the dorsomedial nucleus, the lateral hypothalamus and the arcuate nucleus. This distribution is consistent with the findings of immunocytochemical studies of substance P-immunoreactive neurons in the hypothalamus. We have also examined whether PPT gene expression is regulated by estrogen in the VL-VM by comparing the levels of PPT mRNA in the VL-VM of ovariectomized rats and ovariectomized, estrogen-replaced rats. Both in situ hybridization and slot blot hybridization analysis revealed no changes in PPT mRNA content in the VL-VM following estrogen treatment. These results suggest that estrogen does not regulate lordosis behavior by affecting PPT gene expression in VL-VM neurons.     

Neurotransmitters,neuropeptides and binding sites in the rat mediobasal hypothalamus: effects of monosodium glutamate (MSG) lesions

Summary Indirect immunofluorescence histochemistry and receptor autoradiography were used to study the localization of transmitter-/peptidecontaining neurons and peptide binding sites in the mediobasal hypothalamus in normal rats and in rats treated neonatally with repeated doses of the neurotoxin monosodium-glutamate (MSG). In the arcuate nucleus, the results showed a virtually complete loss of cell bodies containing immunoreactivity for growth hormone-releasing factor (GRF), galanin (GAL), dynorphin (DYN), enkephalin (ENK), corticotropin-like intermediate peptide (CLIP), neuropeptide Y (NPY), and neuropeptide K (NPK). Tyrosine hydroxylase(TH)-, glutamic acid decarboxylase(GAD)-, neurotensin(NT)- and somatostatin(SOM)-immunoreactive (IR) cells were, however, always detected in the ventrally dislocated, dorsomedial division of the arcuate nucleus. In the median eminence, marked decreases in numbers of GAD-, NT-, GAL-, GRF-, DYN-and ENK-IR fibers were observed. The numbers of TH-, SOM-and NPY-IR fibers were in contrast not or only affected to a very small extent, as revealed with the immunofluorescence technique. Biochemical analysis showed a tendency for MSG to reduce dopamine levels in the median eminence of female rats, whereas no effect was observed in male rats. Autoradiographic studies showed high to moderate NT binding sites, including strong binding over presumably dorsomedial dopamine cells. In MSG-treated rats, there was a marked reduction in GAL binding in the ventromedial nucleus. The findings implicate that most neurons in the ventrolateral and ventromedial arcuate nucleus are sensitive to the toxic effects of MSG, whereas a subpopulation of cells in the dorsomedial division of the arcuate nucleus, including dopamine neurons, are not susceptible to MSG-neurotoxicity. The results indicate, moreover that the very dense TH-IR fiber network in the median eminence predominantly arises from the dorsomedial TH-IR arcuate cells, whereas the GAD-, NT-, GAL-, GRF-and DYN-IR fibers in the median eminence to a large extent arise from the ventrolateral arcuate nucleus. Some ENK-and NPK-positive cells in the arcuate nucleus seem to project to the lateral palisade zone of the median eminence, but most of the ENK-IR fibers in the median eminence, located in the medial palisade zone, seem to primarily originate from an area(s) located outside the arcuate nucleus, presumably the paraventricular nucleus. The NPY-positive fibers in the median eminence contain to a large extent immunoreactive dopamine -hydroxylase (DBH), and do not arise from the ventromedial arcuate nucleus. SOM-IR cells in the dorsal periventricular arcuate nucleus do not send major projections to the median eminence. The present findings thus show that MSG treatment represents a valuable tool to clarify the organization of chemically identified neuron populations in the arcuate nucleus-median eminence complex and provide further information for understanding the neuroendocrine effects of neonatal MSG treatment.     

Influence of amygdala stimulation on the activity of identified tuberoinfundibular neurones in the rat hypothalamus.

1. Extracellular action potentials were recorded from 1246 neurones in the mediobasal hypothalamus of pentobarbitone or urethane anaesthetized male rats. Antidromic invasion from the surface of the median eminence identified 165 cells, located in the arcuate and ventromedial nuclei and the periventricular area, as tuberoinfundibular neurones. The majority (65%) of these cells displayed no spontaneous activity. 2. Latencies for antidromic invasion from median eminence ranged from 0-5 to 14-0 msec (mean 4-3 +/- 2-9 msec, S.D.). Conduction velocities for axons of tuberoinfundibular neurones were under 1-0 m/sec, and were slowest (under 0-2 m/sec) for those tuberoinfundibular neurones located in the arcuate nucleus. 3. Single 1 HZ stimulation of amygdala evoked short latency (mean 18-8 +/- 7-0 msec; n = 30) excitation of tuberoinfundibular neurones in the ventromedial nucleus. Stria terminalis stimulation evoked similar responses at a shorter latency (mean 10-2 +/- 3-5 msec; n = 12) from other ventromedial tuberoinfundibular neurones. Three of these neurones were also excited by amygdala stimulation at comparably longer latencies. In spontaneously active tuberoinfundibular cells, the initial excitation was followed by a decrease in excitability lasting 70-150 msec. Tuberoinfundibular neurones soldom followed orthodromic activation at frequencies beyond 30 HZ. 4. An initial decrease in activity at latencies of 18-40 msec (mean 29-2 +/- 10-2 msec) characterized the amygdala evoked responses from nine tuberoinfundibular neurones. A similar response from one other tuberoinfundibular neurone followed stria terminalis stimulation at a latency of 11 msec. Most of these tuberoinfundibular neurones were located in the dorsal part of the ventromedial nucleus. 5. Two ventromedial tuberoinfundibular neurones also displayed antidromic invasion from the amygdala; interaction studies suggested an axon collateral pathway that originated close to the origin of the axon. 6. Tuberoinfundibular neurones unresponsive to amygdala stimulation were usually located in the arcuate nucleus or periventricular area. 7. These results provide electrophysiological evidence for a direct influence of the amygdala on the activity of tuberoinfundibular neurones in the ventromedial hypothalamic nucleus. There are also data to indicate that some ventromedial tuberoinfundibular neurones have axon collaterals that return to the amygdala. These reciprocal connexions between the amygdala and ventromedial tuberoinfundibular neurones may indicate neural circuits important for extrahypothalamic modulation of adenohypophyseal secretion.     

Mapping of somatostatin-28 (1-12) in the alpaca diencephalon

Using an immunocytochemical technique, we report for the first time the distribution of immunoreactive cell bodies and fibers containing somatostatin-28 (1-12) in the alpaca diencephalon. Somatostatin-28 (1-12)-immunoreactive cell bodies were only observed in the hypothalamus (lateral hypothalamic area, arcuate nucleus and ventromedial hypothalamic nucleus). However, immunoreactive fibers were widely distributed throughout the thalamus and hypothalamus. A high density of such fibers was observed in the central medial thalamic nucleus, laterodorsal thalamic nucleus, lateral habenular nucleus, mediodorsal thalamic nucleus, paraventricular thalamic nucleus, reuniens thalamic nucleus, rhomboid thalamic nucleus, subparafascicular thalamic nucleus, anterior hypothalamic area, arcuate nucleus, dorsal hypothalamic area, around the fornix, lateral hypothalamic area, lateral mammilary nucleus, posterior hypothalamic nucleus, paraventricular hypothalamic nucleus, suprachiasmatic nucleus, supraoptic hypothalamic nucleus, and in the ventromedial hypothalamic nucleus. The widespread distribution of somatostatin-28 (1-12) in the thalamus and hypothalamus of the alpaca suggests that the neuropeptide could be involved in many physiological actions.     

The arcuate nucleus as a circumventricular organ in the mouse

The present study searched for morphological correlates of the permeability of the ventromedial arcuate nucleus of the mouse to blood-borne proteins. First, we determined that highly permeable microvessels are detected in the ventromedial arcuate nucleus using a rat monoclonal antibody to a mouse-specific endothelial phenotype (clone MECA32) recently recognized as a marker of endothelial fenestral diaphragms and previously shown to label circumventricular organs. Second, in the mild conditions of tissue fixation mandatory for use of MECA32, we observed that after a rapid vascular flush with saline, endogenous immunoglobulins are especially retained in circumventricular organs and ventromedial arcuate nucleus. The ventromedial arcuate nucleus thus shares features in common with classical circumventricular organs.     

Presence of calbindin and lack of parvalbumin in progesterone receptor-containing neurons of the monkey mediobasal hypothalamus.

All of the progesterone receptor-containing cells of the monkey hypothalamus are GABAergic. The aim of this study was to further characterize these GABAergic progesterone receptor-containing neurons based on their calbindin or parvalbumin content. These calcium-binding proteins are characteristic markers of different populations of GABAergic neurons in the central nervous system. Double-immunolabeling for progesterone receptor and either calbindin or parvalbumin was performed on hypothalamic Vibratome sections of estrogen primed African green monkeys (Cercopithecus aethiops). Progesterone receptor-containing calbindin-immunoreactive neurons were observed in the ventromedial and periventricular areas of the hypothalamus. Forty-one per cent of the progesterone receptor-containing cells in this area were calbindin immunopositive. No double-immunolabeled neurons could be detected in the infundibular (arcuate) nucleus. In tissue double-immunolabeled for progesterone receptor and parvalbumin, none of the progesterone receptor-containing neurons exhibited immunoreactivity for parvalbumin. Electron microscopic double-immunostaining for progesterone receptor and calbindin confirmed the light microscopic results. Furthermore, a large number of asymmetric synaptic contacts were observed on the calbindin-immunoreactive neurons. These observations demonstrate that progesterone receptor-containing cells in the monkey mediobasal hypothalamus consist of at least two different types of GABA neurons, and indicate that progesterone receptor-containing calbindin cells may be postsynaptic targets of excitatory fibers.     

Role of drug efflux transporters in the brain for drug disposition and treatment of brain diseases

The blood-brain barrier (BBB) serves as a protective mechanism for the brain by preventing entry of potentially harmful substances from free access to the central nervous system (CNS). Tight junctions present between the brain microvessel endothelial cells form a diffusion barrier, which selectively excludes most blood-borne substances from entering the brain. Astrocytic end-feet tightly ensheath the vessel wall and appear to be critical for the induction and maintenance of the barrier properties of the brain capillary endothelial cells. Because of these properties, the BBB only allows entry of lipophilic compounds with low molecular weights by passive diffusion. However, many lipophilic drugs show negligible brain uptake. They are substrates for drug efflux transporters such as P-glycoprotein (Pgp), multidrug resistance proteins (MRPs) or organic anion transporting polypeptides (OATPs) that are expressed at brain capillary endothelial cells and/or astrocytic end-feet and are key elements of the molecular machinery that confers the special permeability properties to the BBB. The combined action of these carrier systems results in rapid efflux of xenobiotics from the CNS. The objective of this review is to summarize transporter characteristics (cellular localization, specificity, regulation, and potential inhibition) for drug efflux transport systems identified in the BBB and blood-cerebrospinal fluid (CSF) barrier. A variety of experimental approaches available to ascertain or predict the impact of efflux transport on brain access of therapeutic drugs also are described and critically discussed. The potential impact of efflux transport on the pharmacodynamics of agents acting in the CNS is illustrated. Furthermore, the current knowledge about drug efflux transporters as a major determinant of multidrug resistance of brain diseases such as epilepsy is reviewed. Finally, we summarize strategies for modulating or by-passing drug efflux transporters at the BBB as novel therapeutic approaches to drug-resistant brain diseases.     

Precursor-protein convertase 1 gene expression in the mouse hypothalamus: differential regulation by ob gene mutation,energy deficit and administration of leptin,and coexpression with prepro-orexin

The expression of precursor-protein convertase (PC)1, PC2 and paired basic amino acid cleaving enzyme four mRNA was studied by in situ hybridisation in regions of the hypothalamus involved in energy regulation in relation to obese (ob) gene mutation and energy deficit. PC1 gene was differentially expressed in hypothalamic nuclei of mice from different genetic backgrounds or energetic status, whereas no differences in expression were observed for either the PC2 or paired basic amino acid cleaving enzyme four genes. In obese ob/ob mice, PC1 mRNA levels were increased in the paraventricular nucleus, decreased in the lateral hypothalamus and unchanged in the ventromedial nucleus and arcuate nucleus relative to lean controls. In response to intraperitoneal injection of murine leptin, PC1 mRNA levels in obese ob/ob mice decreased in the arcuate nucleus, increased in the lateral hypothalamus and were unchanged in both the paraventricular nucleus and ventromedial nucleus. In mice deprived of food for 24 h, PC1 mRNA levels were reduced in the ventromedial nucleus, increased in the lateral hypothalamus and unchanged in the paraventricular nucleus and arcuate nucleus relative to ad libitum-fed controls. Overall, whilst the data show effects related to leptin and energetic status, they do not support a strong and consistent link between PC1 gene expression and energy balance. This suggests that if PC1 is important to the control of energy balance then protein expression and activity, rather than gene expression may be the more critical parameters of regulation. The relationship between PC1 and candidate energy balance-related genes in the lateral hypothalamus was investigated by dual in situ hybridisation. PC1 mRNA was localised in prepro-orexin mRNA expressing neurons in the lateral hypothalamus, which suggests a functional relationship.     

News from the brain: the GPR124 orphan receptor directs brain-specific angiogenesis

During development, microvessels acquire specialized functions to meet the requirements of different tissues and organs. The vasculature of the brain constitutes one of the best examples of an organ-specific and highly specialized microvasculature, in which the endothelial cells that line blood vessels form an active permeability barrier and transport system called the blood-brain barrier (BBB); little is known, however, about the molecular mechanisms that instruct endothelial cells toward a BBB phenotype. Now Kuhnert et al. reveal that the orphan heterotrimeric GTP-binding protein-coupled receptor GPR124/TEM5 acts as an organ-specific regulator of brain angiogenesis, required for normal endothelial cell sprouting, migration, and expression of the BBB marker Glut-1 in the forebrain and neural tube. These findings add to our knowledge of brain vascularization and may open up possibilities for new therapeutic regimes to treat several diseases, including stroke, brain tumors, and vascular malformations.     

GABAergic and catecholaminergic innervation of mediobasal hypothalamic beta-endorphin cells projecting to the medial preoptic area.

In the absence of cellular estrogen receptors or proven direct estrogen action in the rat, it is assumed that estrogen indirectly regulates the secretory activity of the preoptic area luteinizing hormone-releasing hormone-producing cells. We have previously shown that pro-opiomelanocortin neurons in the arcuate nucleus of the rat send axons rostrally to connect with luteinizing hormone-releasing hormone neurons of the preoptic area. An experiment combining retrograde tracing and double-immunostaining was used to test the hypothesis that rat GABAergic and/or catecholaminergic neurons can influence luteinizing hormone-releasing hormone-producing cells via mediobasal hypothalamic beta-endorphin neurons. The retrograde tracer horseradish peroxidase was injected into the medial preoptic area; two days later, arcuate nucleus Vibratome sections were double-immunostained for beta-endorphin and glutamate decarboxylase or tyrosine hydroxylase. Light and electron microscopic analysis of these triple-labeled sections demonstrated that a population of beta-endorphin-immunoreactive neurons concentrated in the ventromedial arcuate nucleus contain retrogradely transported horseradish peroxidase granules and form synaptic contacts with glutamate decarboxylase- and tyrosine hydroxylase-immunoreactive axon terminals. The present data suggest that arcuate nucleus GABA and catecholamine fibers may influence luteinizing hormone-releasing hormone-containing neurons via projective pro-opiomelanocortin cells.     

Estradiol target sites immunoreactive for beta-endorphin in the arcuate nucleus of rat and mouse hypothalamus

Combined autoradiography and immunocytochemistry was performed to examine estradiol-concentrating neurons of the arcuate nucleus in the hypothalamus of ovariectomized rats and mice for beta-endorphin-like immunoreactivity. A high number of [3H]estradiol-labeled cells occurred in the arcuate nucleus of both species. Neurons immunoreactive for beta-endorphin could be visualized throughout the arcuate nucleus as well as ventrolaterally outside of this nucleus. Colocalization of [3H] estradiol nuclear labeling and beta-endorphin-like cytoplasmic immunoreactivity could be found scattered throughout the arcuate nucleus, most frequently in the ventromedial part, although most of the [3H]estradiol target neurons did not react with the antibodies. The results suggest that a subpopulation of beta-endorphin neurons is directly addressed by estradiol and that the arcuate nucleus contains different groups of estradiol receptive neurosecretory cells.     

毁损弓状核对动脉粥样硬化形成的影响

本实验分为四组：（1）单纯毁损弓状核组,（2）毁损弓状核＋动脉粥样硬比诱发剂组,（3）单纯给予动脉粥样硬化诱发剂组和（4）对照组,包括腹腔注射生理盐水及皮下注射谷氨酸单钠等2组。在通过电镜观察主动脉壁变化的同时探讨下丘脑弓状核对动脉粥样硬化形成的影响。结果发现：单纯毁损弓状核组血管内皮细胞明显变性,细胞核肿胀,内皮下层增厚并出现空泡,可见有平滑肌细胞向内皮下层迁移,形成明显的动脉粥样硬化早期病变;毁损弓状核＋动脉粥样硬化诱发剂组和单纯用动脉粥样硬化诱发剂组,血管壁的变化基本与单纯毁损弓状核组相同,都有内皮细胞变性,细胞核肿胀,内皮下层增厚和出现空泡等变化,与单纯毁损弓状核组相比未发现有明显不同的变化;对照组未发生任何病理变化。本研究提示下丘脑弓状核对动脉粥样硬化的形成可能有直接调控作用。     

Localization of quinolinic acid metabolizing enzymes in the rat brain. Immunohistochemical studies using antibodies to 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase

Specific antibodies raised in rabbits against 3-hydroxyanthranilic acid oxygenase (EC 1.13.11.6) and quinolinic acid phosphoribosyltransferase (EC 1.13.11.6) and quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) were used in immunohistochemical studies to map the cellular localization of the quinolinic acid metabolizing enzymes in the adult male rat brain. 3-Hydroxyanthranilic acid oxygenase immunoreactivity was found to be present in glial cells of presumed astroglial identity, as judged by co-localization with glial fibrillary acidic protein. 3-Hydroxyanthranilic acid oxygenase-immunoreactive glial cells were present in all brain regions and within major fiber tracts. The density of 3-hydroxyanthranilic acid oxygenase-immunoreactive glial cells as well as the intensity of staining of these cells differed among brain regions. In general, telencephalic acid diencephalic areas harbored a larger number of 3-hydroxyanthranilic acid oxygenase-positive cells than did mesencephalic regions. In the former regions the caudate nucleus, septum, nucleus accumbens, neocortex and hippocampus were particularly enriched in 3-hydroxyanthranilic acid oxygenase-immunoreactive cells. In the thalamus, regional differences were noted with regard to the intensity of staining among glial cells with high densities of 3-hydroxyanthranilic acid oxygenase cells in the anteroventral, reticular and ventromedial nuclei. In the inferior and superior colliculi, numerous 3-hydroxyanthranilic acid oxygenase-positive glial cells were found in all layers. In the hypothalamus, 3-hydroxyanthranilic acid oxygenase-immunoreactive glial cells were encountered in the zona incerta, the lateral hypothalamic area, the caudal preoptic region and in the dorsomedial nucleus. In the mesencephalon, the substantia nigra contained numerous, moderately stained cells. At caudal levels of the brain-stem, a relatively large number of cells was detected in the nucleus of the solitary tract, the pontine nucleus and in the fascial nerve nucleus, while other nuclei, such as the reticular formation and the area postrema were relatively poor in 3-hydroxyanthranilic acid oxygenase-immunoreactive cells. In addition to staining of glial cells, neuronal cell bodies containing 3-hydroxyanthranilic acid oxygenase immunoreactivity were detected in the main and in the accessory olfactory bulb, as well as in the ventromedial nucleus of the hypothalamus. Quinolinic acid phosphoribosyltransferase immunoreactivity was observed within glial cells and in association with neuronal cell bodies. Some, but not all, quinolinic acid phosphoribosyltransferase positive glial cells contained glial fibrillary acidic protein (K?hl     

Distribution of cholinergic neurons and fibers in the hypothalamus of the rat using choline acetyltransferase as a marker

The distribution of choline-acetyltransferase-like immunoreactive structures in the rat hypothalamus and preoptic area was examined by using avidin-biotin immunocytochemistry. We found that the hypothalamus is richly innervated by the cholinergic neuron system. Sites containing cholinergic neurons of varying density were: medial and lateral preoptic areas, septohypothalamic nucleus, median preoptic area, lateral hypothalamus including the perifornical area, anterior hypothalamic nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus, dorsal and ventral premammilary nuclei, neuropil mediodorsal to the anterior hypothalamic nucleus, neuropil ventral to the anterior hypothalamic nucleus and ventromedial hypothalamic nucleus, neuropil between lateral hypothalamus and ventromedial hypothalamus, and neuropil between dorsal premammilary nucleus and posterior hypothalamic nucleus. There were also many varicose and non-varicose fibers in the preoptic area and hypothalamus. Two kinds of varicose fibers, one with strong immunoreactivity and the other with weak immunoreactivity, were seen. Non-varicose fibers were also detected in the optic chiasma and habenulo-interpeduncular tract. These fibers were passing fibers.