Immunocytochemical localization of GABA containing neurons in the ventrolateral medulla oblongata of the rat

Localization of gamma-aminobutyric acid (GABA) in the ventrolateral medulla oblongata of the rat was studied, using antisera directed against GABA molecule fixed to bovine serum albumin. Within the rostral portion of the ventrolateral medulla, GABA-like immunoreactive neurons were found in the lateral wing of the raphe magnus and in the region of the paragigantocellular reticular nucleus. In the caudal portion of the ventrolateral medulla, a lesser number of GABA-stained neurons were found in the region around the nucleus reticularis lateralis. GABA-like immunoreactive punctate structures were also found throughout the ventrolateral medulla. These results provide further evidence for the existence of GABAergic neurons in the ventrolateral medulla oblongata of the rat.     

Coexistence of catecholamine and methionine enkephalin-Arg6-Gly7-Leu8 in neurons of the rat ventrolateral medulla oblongata

Summary An overlapping distribution of catecholamine-containing cells and proenkephaline—A derived peptide-containing neurons have been identified in the rat medulla oblongata. However, it is not evident whether the coexistence of these bioactive substances occurs in the same neurons or not. Therefore, we examined the coexistence of catecholamine and methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), a proenkephaline—A derived peptide, using a combination of histofluorescence and peroxidase-anti-peroxidase (PAP) immunohistochemical (modified formaldehyde-glutalaldehyde (Faglu)) methods on the same tissue sections. We found one third of A1/C1 catecholamine fluorescent cells show MEAGL-like immunoreactivity.     

Nitroxidergic neurons in nuclei of rat and human medulla oblongata

Four types of neurons distinguished by NADPH-diaphorase reaction have been identified in seven nuclei of human and rat medulla oblongata. It was found that nitrogen oxide (NO)-positive neurons had similar distributions; neurons with high NO-synthase activity predominated in vasomotor nuclei and, in sensitive nuclei, most neurons had low NO-syntase activity.     

Effects of 5-HT3 receptor blockade on visceral nociceptive neurons in the ventrolateral reticular field of the rat medulla oblongata

The caudal ventrolateral reticular formation of the medulla oblongata is the first layer of visceral nociceptive processing. In experiments on rats, neuronal responses in this zone to nociceptive stimulation of the large intestine were examined and the effects of selective blockade of 5-HT3 receptors on these responses were assessed. By the character of responses to nociceptive colorectal stimulation (CRS), the recorded medullary neurons were divided into three groups—excited, inhibited and indifferent. Intravenous injection of 5-HT3 antagonist granisetron (1 and 2 mg/kg) as well as local application of this agent on the surface of the medulla oblongata (1.25 and 2.5 nmole) suppressed the background and evoked firing of CRS-excited reticular neurons in a dose-dependent manner but did not exert as pronounced influence on the cells inhibited by visceral nociceptive stimulation. Spike activity in the group of CRS-indifferent neurons under similar conditions was 5-HT3-independent. The results obtained provide evidence that 5-HT3 receptors mediate the facilitating effect of serotonin on supraspinal transmission of the abdominal nociceptive stimulus which, at least in part, is realized via selective activation of visceral medullary nociceptive neurons. A shutdown of this mechanism may underlie the analgesic effect of 5-HT3 antagonists in abdominal pain syndromes.     

Parvalbumin in respiratory neurons of the ventrolateral medulla of the adult rat

A column of parvalbumin immunoreactive neurons is closely associated with the location of respiratory neurons in the ventrolateral medulla of the rat. The majority (66%) of bulbospinal neurons in the medullary ventral respiratory column (VRC) that were retrogradely labeled by tracer injections in the phrenic nucleus were also positive for parvalbumin. In contrast, only 18.8% of VRC neurons retrogradely labeled after a tracer injection in the VRC, also expressed parvalbumin. The average cross-sectional area of VRC neurons retrogradely labeled after VRC injections was 193.8 μm² ± 6.6 SE. These were significantly smaller than VRC parvalbumin neurons (271.9 μm² ± 12.3 SE). Parvalbumin neurons were found in the Bötzinger Complex, the rostral ventral respiratory group (VRG), and the caudal VRG, areas which all contribute to the bulbospinal projection. In contrast, parvalbumin neurons were sparse or absent in the preBötzinger Complex and in the vicinity of the retrotrapezoid nucleus, areas that have few bulbospinal projections. Parvalbumin was rarely colocalized within Neurokinin-1 receptor positive (NK1R) VRC neurons, which are found in the preBötzinger complex and in the anteroventral part of the rostral VRG. Parvalbumin neurons in the Bötzinger Complex and rostral VRG help define the rostrocaudal extent of these regions. The absence of parvalbumin neurons from the intervening preBötzinger complex also helps establish the boundaries of this region. Regional boundaries described in this manner are in good agreement with earlier physiological and anatomical studies. Taken together, the distributions of parvalbumin, NK1R and bulbospinal neurons suggest that the rostral VRG may be subdivided into distinct, anterodorsal, anteroventral, and posterior subdivisions.     

Substance P in the ventrolateral medulla oblongata regulates ventilatory responses.

Local injection of substance P (SP) into the ventral portion of the nucleus gigantocellularis, nucleus reticularis lateralis, and nucleus retrofacialis of the ventrolateral medulla oblongata (VLM) or direct application on the ventral surface of the medulla oblongata caused marked stimulation of tidal volume (VT) and/or minute ventilation (VE). The ventilatory response to hypoxia was significantly blunted after SP in the VLM but not in the dorsal medulla oblongata (DM) (nucleus tractus solitarius). The SP antagonist [D-Pro2,D-Trp7,9]SP almost completely inhibited this response when applied locally to a wide area of the superficial layer of the VLM but not of the DM. Unilateral or bilateral application of 0.3-1.5 nmol of the SP antagonist in the VLM (corpus trapezoideum and the caudal region extending from the rootlets of the nucleus hypoglossus to the first cervical segment) markedly attenuated the response to a 5% CO2 inhalation. The inhibition of the CO2 response was seen after [D-Pro2,D-Trp7,9]SP in the rostral areas of the medulla oblongata corresponding to the corpus trapezoideum and the caudal region extending from the rootlets of the nucleus hypoglossus to the first cervical segment of the cervical cord. Electric somatosensory-induced ventilatory stimulation could be depressed by approximately 70% by [D-Pro2,D-Trp7,9]SP locally applied on the surface of the VLM. We conclude that SP is involved in the hypoxic, hypercapnic, and somatosensory ventilatory responses in the rat. However, these respiratory reflexes are mediated via different neuronal pools in the medulla oblongata, mainly the VLM.     

Immunocytochemical localization of somatostatin-containing neurons in the rat hypothalamus

Summary The rat hypothalamus was studied at the light microscopic level with the use of single and double immunocytochemical staining methods. It was shown that the rat supraoptic and paraventricular hypothalamic nuclei, and their accessory neurosecretory nuclei, do not contain magnocellular somatostatin neurons. The distribution of the hypothalamic parvocellular somatostatin cells is described. The parvocellular component of the rat hypothalamic paraventricular nucleus is, at least partly, composed of somatostatin cells: they form a fairly well circumscribed periventricular cell mass. The rat suprachiasmatic nuclei contain separate somatostatin neurons and vasopressin neurons. Scattered somatostatin cells are present in the entire arcuate nucleus. In addition to the periventricular somatostatin cells located in the preopticanterior hypothalamic area and in the arcuate nucleus, the rat hypothalamus also contains numerous scattered somatostatin cells located distant from the third ventricle.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Cytochemical localization of alkaline phosphatase in the ependyma of the rat medulla oblongata

Summary The ependyma of the IVth ventricle and the central canal of the rat medulla oblongata was investigated using the cytochemical technique for alkaline phosphatase (AlPase) which revealed two types of ependymal cells in the medulla. The central canal type of the ependymal cell occupying the dorsal part of the central canal in the lower medulla exhibited intense AlPase activity with light microscopy. These cells had reaction products in all plasma membranes, including the microvilli and the cilia at the luminal cell surface. Some cells appeared to be tanycytes, since the process reached the basement membrane of the parenchymal blood vessel. The ventricular type of ependymal cells, which form the floor of the IVth ventricle and the central canal, contained no reaction products in any structure of the luminal cell surface.The possible relationship between the cerebrospinal fluid and the nervous tissues through the ependymal linings is discussed.     

Three-dimensional organization of the cytoplasmic neuroglobin-immunopositive structures in the rat medulla oblongata neurons

Neuroglobin is an iron-containing protein, most abundant in the vertebrate nervous system. Since neuroglobin is able to bind oxygen reversibly owing to the heme prosthetic group, it was believed that its function is an intercellular transport of oxygen in the nervous system and accumulation of oxygen for energy supply of cells in hypoxic conditions. In this work, a three-dimensional reconstruction of the neuroglobin distribution in large neurons of the rat medulla oblongata was carried out by means of immunocytochemistry and confocal laser microscopy. Positive neuroglobin immunocytochemical reaction was observed mainly in the perinuclear areas of large nerve cells exhibiting a discrete staining of the cytoplasm. Examination under the microscope at a high magnification revealed some neuroglobin-immunopositive granules, ring-like objects 1–2 μm in diameter, as well as linear and branched structures in neuronal cytoplasm and, occasionally, in the proximal segments of neuronal processes. Three-dimensional reconstruction of the neuroglobin-immunopositive structures showed that they mainly have the form of continuous lines and curves interlaced in some sites, about 1.0–1.5 μm thick, forming a complex network in the cytoplasm. The neuroglobin-immunopositive complexes found for the first time in neuronal cytoplasm are not identical to any known cytoplasmic compartments of nerve cells, but the diameter of their elements, as well as the shape and location suggest a possible link of neuroglobin with a mitochondrial network.     

Age-related changes in nitroxidergic neurons in some nuclei of rat medulla oblongata

The paper presents a comparative study of NO neurons in the solitary tract nucleus, giant-cell, and lateral reticular nuclei in rats at 4, 7, 10, 14, 30, 45, and 60 days old and 3, 6, 12, 18, 24 months old. We determine the active quantitative and qualitative changes that occur in NO-positive neurons in the studied nuclei during the course of postnatal development. A low level of enzyme activity is observed on the first day; it reaches a peak level around the first-third month, then slowly declines. The size and number of nitroxidergic neurons increases, while the relative cell density decreases until the third month of life. We reveal local differences in the ontogenetic development of NO neurons in the studied nuclei. Solitary tract neurons have the highest rate of development, while NO neurons of old animals undergo early and extreme changes as compared to other studied nuclei of rat medulla oblongata.     

Glycine immunoreactive neurons in the medulla oblongata in cats

Using a highly specific antiserum to Glycine and a very sensitive immunohistochemical technique with streptavidin-HRP, we visualized for the first time a considerable number of glycine immunoreactive cell bodies and fibers in the cat medulla oblongata. These results suggest that glycine may play an essential role in nearly all the physiological functions involving the medulla oblongata, including the muscular atonia occurring during paradoxical sleep.     

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.     

Sex differences in angiotensin signaling in bulbospinal neurons in the rat rostral ventrolateral medulla

Sex differences may play a significant role in determining the risk of hypertension. Bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are involved in the tonic regulation of arterial pressure and participate in the central mechanisms of hypertension. Angiotensin II (ANG II) acting on angiotensin type 1 (AT(1)) receptors in RVLM neurons is implicated in the development of hypertension by activating NADPH oxidase and producing reactive oxygen species (ROS). Therefore, we analyzed RVLM bulbospinal neurons to determine whether there are sex differences in: 1) immunolabeling for AT(1) receptors and the key NADPH oxidase subunit p47 using dual-label immunoelectron microscopy, and 2) the effects of ANG II on ROS production and Ca(2+) currents using, respectively, hydroethidine fluoromicrography and patch-clamping. In tyrosine hydroxylase-positive RVLM neurons, female rats displayed significantly more AT(1) receptor immunoreactivity and less p47 immunoreactivity than male rats (P < 0.05). Although ANG II (100 nM) induced comparable ROS production in dissociated RVLM bulbospinal neurons of female and male rats (P > 0.05), an effect mediated by AT(1) receptors and NADPH oxidase, it triggered significantly larger dihydropyridine-sensitive long-lasting (L-type) Ca(2+) currents in female RVLM neurons (P < 0.05). These observations suggest that an increase in AT(1) receptors in female RVLM neurons is counterbalanced by a reduction in p47 levels, such that ANG II-induced ROS production does not differ between females and males. Since the Ca(2+) current activator Bay K 8644 induced larger Ca(2+) currents in females than in male RVLM neurons, increased ANG II-induced L-type Ca(2+) currents in females may result from sex differences in calcium channel densities or dynamics.     

Changes in renal efferent activity and autonomic response to GABA applied to structures of the ventrolateral surface of the cat medulla oblongata

Effects of injecting 1 µl 1·10^–5–5·10^–4 M gamma-aminobutyric acid (GABA) into the neuronal structures of the chemically sensitive area of the bulbar ventrolateral surface and clearly-defined borders of this area with GABA-sensitive cells were investigated in cats anesthetized by a mixture of chloralose and urethane. It was found that GABA, interacting with bicuculline-sensitive GABA_a receptors, exercises an inhibitory effect on the activity of neuronal structures of the ventrolateral surface involved in governing blood pressure, heart rate, and vascular tonus.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 327–334, May–June, 1987.     

Effect of moxonidine on putative sympathetic neurons in the rostral ventrolateral medulla of the rat

We used an intracellular recording technique in vitro to investigate the effects of moxonidine on neurons in the rostral ventrolateral medulla (RVLM) with electrophysiological properties similar to premotor sympathetic neurons in vivo. These neurons were classified as firing regularly and irregularly, according to previous reports. Moxonidine is a sympathoinhibitory and antihypertensive agent that is thought to be a ligand of alpha(2)-adrenergic receptors and imidazoline type-1 receptors in the RVLM. Moxonidine (2-10 microM) was applied to the perfusate on 4 irregularly firing neurons, and 2 regularly firing neurons. Moxonidine (2 microM) produced only minor depolarization in 2 of these neurons. However, on 4 tested neurons, moxonidine (10 microM) elicited a profound inhibitory effect with hyperpolarization (near -20 mV); these neurons practically ceased firing. These changes were partially reversible. The results would indicate that neurons in the RVLM, recorded in vitro and with similar electrophysiological characteristics to a group of neurons previously identified in vivo in the same bulbar region as barosensitive premotor sympathetic neurons, can be modulated by imidazoline-derivative adrenergic agonists. These results could help to understand the hypotensive effects of moxonidine.     

Hypoxic excitation in neurons cultured from the rostral ventrolateral medulla of the neonatal rat.

Neurons within cardiorespiratory regions of the rostral ventrolateral medulla (RVLM) have been shown to be excited by local hypoxia. To determine the electrophysiological properties of these excitatory responses to hypoxia, we developed a primary dissociated cell culture system to examine the intrinsic response of RVLM neurons to hypoxia. Neonatal rat neurons plated on medullary astrocyte monolayers were studied using the whole cell perforated patch-clamp technique. Sodium cyanide (NaCN, 0.5-10 mM) was used, and membrane potential (V(m)), firing frequency, and input resistance were examined. In 11 of 19 neurons, NaCN produced a V(m) depolarization, an increase in firing frequency, and a decrease in input resistance, suggesting the opening of a cation channel. The hypoxic depolarization had a linear dose response and was dependent on baseline V(m), with a greater response at more hyperpolarized V(m). In 8 of 19 neurons, NaCN produced a V(m) hyperpolarization, decrease in firing frequency, and variable changes in input resistance. The V(m) hyperpolarization exhibited an all-or-none dose response and was independent of baseline V(m). These differential responses to NaCN were retained after synaptic blockade with low Ca(2+)-high Mg(2+) or TTX. Thus hypoxic excitation 1) is maintained in cell culture, 2) is an intrinsic response, and 3) is likely due to the increase in a cation current. These hypoxia-excited neurons are likely candidates to function as central oxygen sensors.     

Immunocytochemical localization of soluble protein in the adrenal medulla

Synopsis An indirect immunocytochemical technique (Nakane, 1970) was employed to localize the soluble proteins purified from lysates of catecholamine (CA)-containing vesicles of the bovine adrenal medulla. Antiserum to the proteins, produced in rabbits, was used for incubation of sections of bovine adrenal tissue prepared by fixation in glutaraldehyde and embedding in serum albumin (McLean & Singer, 1970). The site of the antigen-antibody complex was visualized by incubating the sections with anti-rabbit -globulin (goat) conjugated to peroxidase, followed by the deposition of electronopaque reaction product generated by the enzyme. The reaction product, also visible at the level of the light microscope, appeared to have a distribution similar to that of the CA-storage vesicles. Electron microscopic examination revealed that nearly all the reaction product was deposited over the electron-opaque core of the vesicles. The intravesicular localization is consistent with the proposal that these proteins exist primarily in the CA-containing granular vesicle and may function to stabilize the CA-storage complex.     

Immunohistochemical localization of neuropeptide Y in the guinea pig medulla oblongata

Summary The immunohistochemical localization of neuropeptide Y (NPY) was correlated with those of dopamine--hydroxylase (DBH) and vasoactive intestinal polypeptide (VIP) by mapping serial 7 m paraffin sections at three levels of the guina 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 NPY-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 m²) and parvocellular part (318 m²)-, in the vagus-solitarius complex (433 m²), and in the dorsal strip (348 m²); NPY/VIP neurons in the vagus-solitarius complex (368 m²) and in the nucleus ovalis (236 m²). 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.Supported by the Deutsche Forschungsgemeinschaft, grant He 919/6-1