Immunohistological studies of masked indoleamine cells in the area postrema of the rat

Summary Masked indoleamine cells (MICS) in the area postrema and adjacent areas in the rat were immunohistochemically studied (the peroxidase-antiperoxidase method) using a serotonin antiserum. After pretreatment with nialamide (200–300 mg/kg), immunoreactive MICS could be observed. They were small cells (about 12 m in diameter) with several processes and were distributed in nearly all parts of the area postrema and also in the nucleus tructus solitarii. Following a single intraventricular injection of 75 g 5,6-dihydroxytryptamine, the immunoreactivity of these cells conspicuously decreased for several days. The submicroscopical structure of the cells was investigated using immunoelectron microscopy. Immunoreactive products were observed in the cytoplasm as particles with a diameter of 25–40 nm and high electron density, but these were not found in the nucleus or cell organelles.This work was supported by a grant (No. 57214028) from the Ministry of Education, Science and Culture, Japan     

Development of catecholaminergic neurons in the human medulla oblongata

Distribution and maturation of catecholaminergic (CA) neurons have been studied by tyrosine hydroxylase immunohistochemistry in the medulla oblongata of human fetuses aged 14.5-25 weeks of gestation. Already at 14.5 weeks, CA neurons were observed in two longitudinally oriented cell clusters, one located ventrolaterally in the area of the lateral reticular and ambiguous nuclei, the other one dorsomedially forming 4 groups related to the dorsal vagal nucleus, the commissural nucleus of the vagus, the nucleus of the tractus solitarius and the area postrema. CA neurons in the area postrema were often found close to blood vessels. Scattered intermediate CA neurons were seen between these two larger clusters. CA neurons still appeared immature exhibiting bipolar morphology with only one or two short stout processes, which hardly branched. At 21 weeks, CA neurons occupied essentially the same location, but had a more mature morphology. Though still bipolar in shape, they had thinner and much longer processes which frequently branched. Both in the ventrolateral and the dorsomedial cell clusters, these processes were frequently lying close to blood vessels. At 25 weeks, CA cells had matured into multipolar neurons with long thin processes forming fine fiber networks in the ventrolateral medulla as well as around and within the dorsal vagal and solitarius nuclei. Only at this stage, a distinct CA fiber tract was seen located in the region of the tractus solitarius. Our results indicate that CA neurons in the human medulla, which are presumably involved in the control of ventilation and blood pressure, though generated rather early during development, mature relatively late.     

Immunohistochemical localization of neuropeptide Y in the guinea pig medulla oblongata. Correlation with VIP and DBH

The immunohistochemical localization of neuropeptide Y (NPY) was correlated with those of dopamine-beta-hydroxylase (DBH) and vasoactive intestinal polypeptide (VIP) by mapping serial 7 micron paraffin sections at three levels of the guinea pig lower brainstem: a) area postrema, b) dorsal motor nucleus of the vagus, and c) nucleus prepositus of the hypoglossal nerve. Based on differences in transmitter expression, three populations of NPY-immunoreactive (IR) neurons were distinguished: NPY-IR catecholaminergic cells (NPY/CA), NPY-IR VIP-ergic cells (NPY/VIP), and NYP-IR cells which were not reactive to either DBH or VIP. Within these populations, size differences among neurons in characteristic locations allowed differentiation among the following subpopulations: NPY/CA neurons in the lateral reticular nucleus--magnocellular part (mean neuronal size 538 micron2) and parvocellular part (318 micron2)-, in the vagus-solitarius complex (433 micron2), and in the dorsal strip (348 micron2); NPY/VIP neurons in the vagus-solitarius complex (368 micron2) and in the nucleus ovalis (236 micron2). Apart from scattered NPY-IR cell bodies in the regions listed above, NPY-IR cell bodies in the lateral portion of the nucleus solitarius and in the caudal part of the spinal nucleus of the trigeminal nerve did not exhibit IR to either DBH or VIP. NPY-IR neurons in the area postrema occurred too infrequently for co-localization studies. The differential distribution of heterogeneous NPY-IR cell subpopulations may reflect the involvement of NPY in a variety of neuronal functions.     

Excitation of neurons in the canine area postrema by prostaglandins

The effects of prostaglandins on electrical activity of neurons in the canine area postrema were studied using the techniques of extracellular recording with iontophoresis. Excitatory responses were obtained upon application of prostaglandins A1, B1, B2, E1, F1 alpha, and F2 alpha in between 24 and 50% of the cells studied. The excitation was very similar in pattern to that observed to apomorphine, biogenic amines, and several neuropeptides in that it had a relatively long latency, low maimal frequency, and prolonged duration. Since the area postrema is known to play a central receptive role in initiating emesis to circulating toxins, these results suggest that prostaglandins may play a role in the initiation of some forms of emesis.     

Fine structure of the ependymal cells in the area postrema of the domestic fowl

Summary The ultrastructure of the ependymal cells in the area postrema of the domestic fowl was studied by scanning and transmission electron microscopy. The ependymal surface of the area postrema is covered with many furrows and ridges. These ridges consist of ependymal cells aggregated in a fan-like shape. The ependymal cell lacks clustered cilia, microvilli are few, and a long basal process extends through the parenchymal layer of the area postrema. Within the cytoplasm as well as in the basal process, a spherical body with a diameter ranging from 1.5 to 2 gmm is occasionally observed.This work was supported by a Scientific Research Grant, No. 144017, from the Ministry of Education of Japan to Professor M. YasudaThe authors are grateful to Drs. T. Fujioka and T. Watanabe for their valuable advice     

The ventricular surface of the area postrema and the adjacent area subpostrema in the rabbit brain]

The ependymal surface of the area postrema (rabitt) was examined by scanning and transmission electron microscopy. The flattened ependymal cells show few microvilli. Towards the central canal, the ependymal cells change gradually to a columnar shape; the number of microvilli increases concomitantly. The area postrema ependymal cell surface mostly bears a single cilia. In contrast, a region immediately adjacent to the area postrema, which has been named area subpostrema (Gwyn and Wolstencroft 1968), shows cilia arranged in bunches. These cilia are regularly covered with colloid -- like droplets. A period-acid-bisulfit-aldehydthionine method (Specht 1970) permits to identify these droplets with glyproteids.it has been suggested that the droplets might derive from the area subpostrema ependymal cells. Above the ependymal surface of the area postrema, a great number of fine unmyelinated neuronal processes and thicker processes are observed. Some of them show bulb-like endings. These terminals contain small vesicles, dense cored vesicles (400...800 A), and mitochondria which are mostly characterized by a single central prismatic tubule. The plasmalemma of some bulbs is in a synaptic contact with the apical plasmalemma of the ependyma, while other bulbs see to end freely in the ventricle. Some neuronal processes penetrate between ependymal cells of the area postrema into the ventricular lumen.     

Efferent projections of the area postrema demonstrated by autoradiography

The efferents connections of the area postrema (AP) have been studied autoradiographically following iontophoretic injections of 3H-glycine or 3H-leucine into the area postrema. Precise control of the diffusion of the labelled amino acids injected iontophorectically into the AP was made using the technique of sectioning with a cryostat. AP projects to a great number of structures. Projections to nucleus tractus solitarius (NFS), dorsal vagal nucleus, nucleus intercalatus, nucleus praepositus hypoglossi, nucleus hypoglossal, the mesencephalic nucleus of V nerve, locus coeruleus and superior and inferior colliculi are shown bilaterally. The density of the efferents was greatest to the NFS and the LC. Corelations are suggested with functional mechanisms of cardiovascular regulation.     

Cholecystokinin-octapeptide like immunoreactivity in the area postrema of the rat and cat

The distribution of cholecystokinin-8 (CCK-8)-like immunoreactivity in the area postrema of the rat and cat was visualized using the peroxidase, antiperoxidase technique. In the rat the greatest amount of immunostaining occurred in peripheral regions of the area postrema at intermediate and rostral levels. Caudally, scattered immunoreactivity predominated. After colchicine treatment, numerous immunoreactive somata were observed throughout the area postrema. The cat area postrema had a different and more complex pattern of immunostaining than the rat. Moderate to dense accumulations of immunostaining occurred in the ventromedial region of the area postrema bordering the solitary tract and dorsal vagal nuclei. The central region of the area postrema possessed scattered amounts of immunoreactivity at rostral levels. Following colchicine treatment, no visible CCK-8-like immunoreactive cell bodies were observed in the cat area postrema. Results of the present investigation provide morphological evidence for the role of CCK-8 in cardiovascular regulation and satiety. The difference in the distribution of CCK-8 in the rat and cat suggest a possible role in the emetic reflex.     

Neuronal organization of the avian paraventricular nucleus: intrinsic, afferent, and efferent connections

The heterogeneous paraventricular nucleus (PVN) of birds offers favorable conditions for the analysis of intrinsic, afferent, and efferent connections of neuroendocrine systems. Paraventricular neurons are successfully impregnated with the Golgi-technique. The findings indicate a direct influence of the cerebrospinal fluid (CSF) on the magnocellular neurons that, via their axon terminals in the neural lobe of the pituitary, are also exposed to the hemal milieu. The magnocellular neurons are intermingled with parvocellular elements which may represent local interneurons. A group of parvocellular nerve cells is identified as CSF-contacting neurons. This type of cell forms a basic morphologic component of the avian neuroendocrine apparatus. Immunocytochemical and ultrastructural studies further support the concept of neuronal interactions between parvocellular and magnocellular elements. Moreover, these findings speak in favor of the existence of recurrent collaterals of the magnocellular neurons. Nerve cells giving rise to afferent connections to the PVN are located in the limbic system and autonomic areas of the upper and lower brainstem. Further afferents may originate from the subfornical organ, the organon vasculosum laminae terminalis, the ventral tegmentum, and the area postrema. Via efferent projections, the PVN is connected to the nucleus accumbens, lateral septum, several hypothalamic nuclei, the neural lobe of the pituitary, the organon vasculosum laminae terminalis, the subfornical organ, the pineal organ, the area postrema, the lateral habenular complex, and various autonomic areas of the reticular formation in the upper and lower brainstem and the spinal cord. In conclusion, the PVN may be regarded as an integral component of the neuroendocrine apparatus reciprocally coupled to the limbic system, several circumventricular organs, and various autonomic centers of the brain.     

Mapping of alpha-melanocyte-stimulating hormone-like immunoreactivity in the cat brainstem

The distribution of alpha-melanocyte-stimulating hormone-like immunoreactive structures was studied in the brainstem of the cat using an indirect immunoperoxidase technique. Immunoreactivity was observed in several brainstem nuclei of the cat in which no immunoreactivity had been previously reported. Immunoreactive fibres were observed in the following; the inferior central nucleus; the pontine gray nuclei; the K?lliker-Fuse nucleus; the motor trigeminal nucleus, the anteroventral cochlear nucleus; the abducens nucleus; the retrofacial nucleus; the superior, lateral, inferior, and medial vestibular nuclei; the lateral nucleus of the superior olive; the external cuneate nucleus; the nucleus of the trapezoid body; the postpyramidal nucleus of the raphe; the medial accessory inferior olive; the dorsal accessory nucleus of the inferior olive; the nucleus ambiguus; the principal nucleus of the inferior olive; the preolivary nucleus; the nucleus ruber; the substantia nigra; and in the area postrema. Our results point to a more widespread distribution of alpha-melanocyte-stimulating hormone-like immunoreactive structures in the cat brainstem than that reported in previous studies carried out in the same region of the cat, rat and humans.     

Immunohistochemical localization of amylin in rat brainstem

Immunohistochemical studies were conducted on rat brainstem using a specific polyclonal antiserum against the COOH-terminal (25-37) of human amylin. Amylin-immunoreactive cell bodies were observed in the vestibular, cochlear, trapezoid, and inner cerebellar nuclei and in the mesencephalic nucleus of trigeminal nerve. Positive cell bodies were also found in lateral, gigantocellular and magnocellular reticular nuclei. Numerous amylin-immunoreactive nerve fibers were shown in the trigeminal spinal tract, in the solitary area and in the area postrema. Amylin-immunoreactive cell bodies were often surrounded by a network of tyrosine hydroxylase-immunoreactive nerve fibers. These results provide morphologic evidence that amylin may play a role in some discrete sensory functions.     

Action sites of adrenomedullin in the rat brain: functional mapping by Fos expression.

The effects of intracerebroventricular (icv) administration of adrenomedullin (AM) and proadrenomedullin NH2-terminal 20 peptide (PAMP) on the expression of Fos in the central nervous system (CNS) were examined in conscious rats, using immunohistochemistry. Fos-like immunoreactivity (LI) was detected in various brain areas of the rats, including the supraoptic nucleus, the paraventricular nucleus, the locus coeruleus, the area postrema and the nucleus of the tractus solitarius 90 min after icv administration of AM. Few cells with Fos-LI were found in the CNS 90 min after icv administration of saline. Fos-LI was also detected in the various hypothalamic areas after icv administration of PAMP. These results suggest that centrally administered AM and PAMP may cause physiological responses through the activation of a neural network in the hypothalamus and the brainstem.     

Ultrastructure of the area postrema of the monkey, Macaca fascicularis

The area postrema of the monkey, Macaca fascicularis, were a pair of oval organs at the caudal end of the floor of fourth ventricle. Their ependymal lining was covered by well-developed microvilli with occasional overlying supraependymal cells. Two types of lining cells were present: pyramidad- and flattened cells. The pyramidal cell showed a long extending basal process resting on the underlying blood vessels. In transmission electron microscopy, the organ showed numerous fenestrated sinusoids characterized by a distinct perivascular space containing mast cells, macrophages and collagen fibrils. The parenchyma of the organ was composed of neurons and glial elements. Only one type of neuron ranging from 9.5 to 15 microns could be distinguished. The neurons contained an indented nucleus surrounded by organelle rich cytoplasm. The soma of the neuron was enclosed by glial element resembling astrocyte. The glial processes terminated on the blood vessel where they were "tunnelled" by a variable number of nerve fibres some of which gained a direct access to the external basal lamina of the perivascular space. Synapses in the neuropil predominantly of the axodendritic variety were observed. Axon terminals containing round agranular vesicles were seen to make synaptic contacts with the neuronal soma. No structural changes were observed in the area postrema following bilateral cervical vagotomy. However, degenerating axon terminals were observed in the subpostremal zone 7, 14 and 21 days after vagotomy suggesting a direct afferent projection into this region.     

Transneuronal labeling of neurons in the adult rat central nervous system following inoculation of pseudorabies virus into the colon

Transneuronal tracing with pseudorabies virus (PRV) was used to identify sites in the central nervous system involved in the neural control of colon function. PRV-immunoreactive (IR) cells were primarily localized to the caudal lumbosacral (L6-S1) and caudal thoracic-rostral lumbar (T13-L1) spinal segments with the distribution varying according to survival time (72-96 h). In the lumbosacral spinal cord at all time points examined, significantly (PА.005) greater numbers of PRV-IR cells were present in the region of the sacral parasympathetic nucleus (SPN) of the S1 spinal segment compared to that of the L6 segment. These studies also revealed morphologically distinct cell types with a differential distribution (probably interneurons and preganglionic parasympathetic neurons) in the region of the SPN in the L6-S1 spinal segments following colon inoculation. PRV-labeled neurons were located at various levels of the neuraxis and at many sites had a distribution similar to that following injection of virus to other urogenital organs. However, some unique sites in the dorsal motor nucleus of the vagus, nucleus of the solitary tract, nucleus ambiguus and area postrema were also identified. To determine if labeling in these caudal medullary sites was mediated by spinal or vagal pathways, the colon was inoculated with PRV in animals with a complete spinal cord (T8) transection (5-7 days prior). Following spinal transection, PRV-infected cells were detected in the same caudal medullary regions; however, labeling in other regions (e.g., Barrington's nucleus) was eliminated or significantly reduced. These studies have yielded several novel observations concerning the central neural control of colonic function: (1) the preganglionic efferent and primary afferent innervation of the colon arises primarily from the S1 spinal segment; (2) the distribution of PRV-infected neurons in the central nervous system following colon inoculation was similar to that following PRV inoculation of other urogenital organs; (3) Barrington's nucleus, which has been identified previously as the pontine micturition center, may have a role in colonic function; and (4) PRV infection in Barrington's nucleus following colon inoculation is mediated by bulbospinal pathways whereas labeling in caudal medullary regions is mediated, at least in part, by vagal pathways.     

大鼠运动病敏感性性别差异与相关脑区AVP及其V1b受体表达的关系

目的：检测不同性别大鼠旋转刺激后脑内相关区域精氨酸加压素（AVP）含量及V1b受体表达的变化,探讨AVP及受体参与运动病的可能机制。方法：给予SD大鼠30 min绕水平轴的旋转刺激,然后采用放免法检测相关脑区AVP含量,并通过荧光免疫组化方法测定相应脑区V1b受体的表达情况。结果：①在雌性大鼠,旋转刺激组各脑区AVP含量无显著性改变;对于雄性大鼠,对照组各检测脑区AVP含量高于雌性,旋转刺激组小脑、延髓内AVP含量的变化无显著性意义,但前脑、间脑、脑桥内AVP含量较对照组明显降低（P〈0.05）。②雌性大鼠视上核AVP的V1b受体表达阳性神经元数量旋转刺激组显著低于对照组（P〈0.05）,而前庭核、最后区V1b受体表达阳性神经元数量明显多于对照组（P〈0.05）;在雄性大鼠,旋转刺激组视上核与前庭核V1b受体表达阳性神经元数量无显著性改变,而最后区V1b受体表达阳性神经元数量有所增加（P〈0.05）,但增加幅度没有雌性大鼠明显。结论：前脑、间脑、脑桥内AVP含量与前庭核和最后区V1b受体表达及对旋转刺激反应的差异可能与运动病敏感性性别差异有关,并且前庭核、最后区可能是AVP-V1受体拮抗剂抗运动病作用的靶点。     

Plasma hormone levels and central c-Fos expression in ferrets after systemic administration of cholecystokinin

Posterior pituitary hormone secretion and central neural expression of the immediate-early gene product c-Fos was examined in adult ferrets after intravenous administration of CCK octapeptide. Pharmacological doses of CCK (1, 5, 10, or 50 microg/kg) did not induce emesis, but elicited behavioral signs of nausea and dose-related increases in plasma vasopressin (AVP) levels without significant increases in plasma oxytocin (OT) levels. CCK activated neuronal c-Fos expression in several brain stem viscerosensory regions, including a dose-related activation of neurons in the dorsal vagal complex (DVC). Activated brain stem neurons included catecholaminergic and glucagon-like peptide-1-positive cells in the DVC and ventrolateral medulla. In the forebrain, activated neurons were prevalent in the paraventricular and supraoptic nuclei of the hypothalamus and also were observed in the central nucleus of the amygdala and bed nucleus of the stria terminalis. Activated hypothalamic neurons included cells that were immunoreactive for AVP, OT, and corticotropin-releasing factor. Comparable patterns of brain stem and forebrain c-Fos activation were observed in ferrets after intraperitoneal injection of lithium chloride (LiCl; 86 mg/kg), a classic emetic agent. However, LiCl activated more neurons in the area postrema and fewer neurons in the nucleus of the solitary tract compared with CCK. Together with results from previous studies in rodents, our findings support the view that nauseogenic treatments activate similar central neural circuits in emetic and nonemetic species, despite differences in treatment-induced emesis and pituitary hormone secretion.     

Neuronal activation in the hypothalamus and brainstem during feeding in rats

We trained rats to a regime of scheduled feeding, in which food was available for only 2 hr each day. After 10 days, rats were euthanized at defined times relative to food availability, and their brains were analyzed to map Fos expression in neuronal populations to test the hypothesis that some populations are activated by hunger whereas others are activated by satiety signals. Fos expression accompanied feeding in several hypothalamic and brainstem nuclei. Food ingestion was critical for Fos expression in noradrenergic and non-noradrenergic cells in the nucleus tractus solitarii and area postrema and in the supraoptic nucleus, as well as in melanocortin-containing cells of the arcuate nucleus. However, anticipation of food alone activated other neurons in the arcuate nucleus and in the lateral and ventromedial hypothalamus, including orexin neurons. Thus orexigenic populations are strongly and rapidly activated at the onset of food presentation, followed rapidly by activity in anorexigenic populations when food is ingested.     

Mast cells in the human brain

Mast cells, as adjudged by the metachromatic staining of their cytoplasmic granules, were found in 79% of the 97 humans brains studied. They were most numerous and most consistently present in the infundibulum, pineal organ, area postrema and choroid plexuses. They were also numerous in the leptomeninges surrounmding the pineal organ and infundibulum. Occasional mast cells were also seen within the supraoptic crest, the subfornical organ, the ventricles and the leptomeninges at sites other than over the infundibulum and pineal organ. They were not detectable elsewhere in the brain or spinal cord. In the infundibulum, pineal organ, area postrema and telencephalic choroid plexuses mast cells were most numerous in young individuals (i.e., 0-19 years of age); thereafter, their numbers progressively decreased with aging. Elsewhere mast cell numbers remained about the same with aging. Except in the area postrema where mast cells were more numerous and more consistently present in males, sex-related differences in mast cell number or distribution were not detected. No differences in either the abundance, the distribution or the percentage of individuals possessing mast cells at any of these sites were apparent between 'normative' brains, lesioned brains ('stroke', lobotomy, etc.) or those from individuals with either congenital or acquired encephalopathies.     

Ghrelin acts on the dorsal vagal complex to stimulate pancreatic protein secretion

Ghrelin receptors are present in the central nervous system. We hypothesized that ghrelin released from the stomach acts as an endocrine substance and stimulates brain stem vagovagal circuitry to evoke pancreatic secretion. In an in vivo anesthetized rat model, an intravenous infusion of ghrelin at doses of 5, 10, and 25 nmol increased pancreatic protein secretion from a basal level of 125 +/- 6 to 186 +/- 8, 295 +/- 12, and 356 +/- 11 mg/h, respectively. Pretreatment with atropine or hexamethonium or an acute vagotomy, but not a perivagal application of capsaicin, completely abolished pancreatic protein secretion responses to ghrelin. In conscious rats, an intravenous infusion of ghrelin at a dose of 10 nmol resulted in a 2.2-fold increase in pancreatic protein secretion over basal volume. Selective ablation of the area postrema abolished pancreatic protein secretion stimulated by intravenous infusion of ghrelin but did not alter the increase in pancreatic protein secretion evoked by diversion of bile-pancreatic juice. Immunohistochemical staining showed a marked increase in the number of c-Fos-expressing neurons in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus after an intravenous infusion of ghrelin in sham-lesioned rats; selective ablation of the area postrema eliminated this increase. In conclusion, ghrelin stimulates pancreatic secretion via a vagal cholinergic efferent pathway. Circulating ghrelin gains access to the brain stem vagovagal circuitry via the area postrema, which represents the primary target on which peripheral ghrelin may act as an endocrine substance to stimulate pancreatic secretion.     

Quantitative distribution of angiotensin II binding sites in rat brain by autoradiography

Angiotensin II binding sites were localized and quantified in individual brain nuclei from single rats by incubation of tissue sections with 1 nM 125I-[Sar1]-angiotensin II, [3H]-Ultrofilm autoradiography, computerized microdensitometry and comparison with 125I-standards. High angiotensin II binding was present in the circumventricular organs (organon vasculosum laminae terminalis, organon subfornicalis and area postrema), in selected hypothalamic nuclei (nuclei suprachiasmatis, periventricularis and paraventricularis) and in the nucleus tractus olfactorii lateralis, the nucleus preopticus medianus, the dorsal motor nucleus of the vagus and the nucleus tractus solitarii. High affinity (KA from 0.3 to 1.5 X 10(9) M-1) angiotensin II binding sites were demonstrated in the organon subfornicalis, the nucleus tractus solitarii and the area postrema after incubation of consecutive sections from single rat brains with 125I-[Sar1]-angiotensin II in concentrations from 100 pM to 5 nM. These results demonstrate and characterize brain binding sites for angiotensin II of variable high affinity binding both inside and outside the blood-brain barrier.