Stained Cell Bodies Research Articles

Selective alterations in the cellular distribution of apolipoprotein E immunoreactivity following transient cerebral ischaemia in the rat.

The aim of this study was to examine the cellular localization and alterations of apolipoprotein E (apoE) following a transient ischaemic insult using immunohistochemistry. Transient cerebral ischaemia was induced in Wistar rats by occlusion of both carotid arteries with hypotension followed by reperfusion for 4 h (n = 5), 24 h (n = 5) or 72 h (n = 6). In sham-operated animals (n = 9), the carotids were not occluded. In this model, ischaemia for 15 min results in selective neuronal damage in the caudate nucleus and neocortex (24 h after reperfusion) and the hippocampal CA1 pyramidal cells (72 h after reperfusion) while there is minimal damage in other areas such as the CA3 hippocampal region. In sham animals apoE immunoreactivity was confined to astrocytes and their processes. ApoE immunoreactivity was not altered at 4 h post-ischemic reperfusion. At 24 h reperfusion, intense apoE staining of the cytoplasm of astrocytes and neuropil within the caudate and neocortex was observed and at 72 h reperfusion apoE stained neuronal cell bodies within these regions. Within the CA1 region at 24 h reperfusion, there was increased immunoreactivity of the cytoplasm of astrocytes and the neuropil was more intensely stained compared with sham animals. At 72 h reperfusion, intense apoE staining of pyramidal cell bodies and dendrites was consistently observed in the CA1 region of the hippocampus. In contrast, at 72 h reperfusion, apoE staining of astrocytic processes was dramatically reduced in the CA1 region although GFAP staining indicated their preservation. The results demonstrate that following an ischaemic insult apoE is localized to degenerating neurons and their processes. This may indicate an inherent protective response of cells to injury. Alternatively, the results are consistent with the hypothesis that apoE is synthesized and released by astrocytes and taken up by neurons following injury.

Histochemical and immunohistochemical localisation of nitrergic neuronal and non-neuronal cells in the bursa of Fabricius of the chicken.

The bursa of Fabricius of 24 chickens, 2-4 weeks old, was investigated to determine the distribution of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) staining and nitric oxide synthase (NOS) antigen, by use of NADPH-d histochemistry and NOS immunohistochemistry, respectively. NADPH-d reaction product was localised in neuronal cell bodies and nerve fibres. The stained cell bodies were predominantly in the different bursal compartments; some occurred in the wall and between the lymphoid follicles, and some in the immediate vicinity of blood vessels. Stained nerve fibres travelled mostly with blood vessels, but many were also observed in the connective tissue of the bursa, independent of blood vessels, and some in contact with the lymphoid follicle and interfollicular epithelium. In addition to neuronal profiles, the interfollicular epithelium of the plicae, and the vascular endothelium were densely stained, and lymphocytes were moderately labelled. NOS immunoreactivity was found in neuron-like cells and fibres which were confirmed to be neuronal in adjacent sections stained with antibodies raised against neuron-specific enolase. It was also detected in interfollicular epithelium and lymphocytes but not in vascular endothelium. The distribution patterns of NADPH-d and NOS suggest that nitric oxide (NO) may play a role in the regulation of the secretory activity of interfollicular epithelium and the blood flow through the bursa.

Electrophysiology of porcine myenteric neurons revealed after vital staining of their cell bodies. A preliminary report

Due to practical limitations in visualizing and getting access to the ganglionic components of large mammals, electrophysiology of the enteric nervous system has been restricted mainly to small laboratory animals, more particularly the guinea-pig. The use of the vital dye 4-(4-diethylaminostyry1)-N-methylphyridinium iodide (4-Di-2-ASP), however, overcomes some of these difficulties. A 20-min incubation period with this dye, followed by a minimum period of 4 h in Krebs solution, suffices to stain the neuronal cell bodies, permitting selection of a neuron and positioning of the microelectrode for impalement and recording. The method has been applied to pig ileum and guinea-pig large and small bowel myenteric neurons. Impalements of untreated guinea-pig myenteric neurons were compared with those of 4-Di-2-ASP-pretreated ones. According to our preliminary data, the staining did not suppress the expression of apparently normal electrophysiological activity. Moreover, the procedure permitted impalement and recording of myenteric plexus neurons in pig ileal tissue with a rate of success equalling blind impalement on guinea-pig tissue. In contrast with formerly published results whereby staining of the neuronal cell bodies only occurred when the cells had been chemically damaged, our experiments suggest a possible correlation between fluorescence and cell viability.

Kinectin distribution in chicken nervous system

Kinectin, a major kinesin receptor on endoplasmic reticulum, was visualized with anti-kinectin monoclonal antibodies (mAbs) in the adult chicken nervous system in comparison with kinesin immunostaining. Anti-kinectin mAbs punctately stained cell bodies and proximal dendrites of motor neurons in spinal cords. Axons of motor neurons were not stained with anti-kinectin mAbs, but stained heavily with anti-kinesin mAbs. This suggest that the kinesin receptor responsible for kinesin-driven anterograde fast axonal transport is different from kinectin. Anti-kinectin mAbs strongly stained neuronal cell bodies in spinal ganglion, nuclei in brainstem, cerebellar nuclei, striatum and cerebral cortex. Small neurons in cerebellar cortex and optic lobe showed relatively weak reaction, suggesting that the amount of kinectin correlates with the size of neuronal cell bodies.

Cellular localization of pan‐trk immunoreactivity and trkC mRNA in the enteric nervous system

The members of the trk family of tyrosine receptor kinases, trkA, trkB, and trkC, are the functional receptors for neurotrophins, a family of related neurotrophic factors. In this study, we investigated 1) the distribution of neurotrophin receptors in the developing and adult rat digestive tract with a pan-trk antibody that recognizes all known trks and 2) the cellular localization of trk-encoding mRNAs in the adult gut with single-stranded RNA probes specific for trkA, trkB, and trkC. In the developing myenteric plexus, trk immunoreactivity was present at embryonic day (ED) 14. Cells and fibers immunoreactive for trk could be visualized in the myenteric plexus at ED 16. At this age, dense staining was found in thick bundles of fibers in proximity to the myenteric plexus in the longitudinal muscle and in association with blood vessels in the mesentery. At ED 18, trk immunoreactivity was also seen in thin processes running from the myenteric plexus into the circular muscle, and in fibers and cells in intrapancreatic ganglia. By ED 20, immunoreactive staining was quite dense in both the myenteric and submucosal plexuses. At birth, virtually all enteric ganglia displayed strong trk immunoreactivity; the intensity of the staining at this age made it difficult to discern individual cells. During postnatal development, there was a decrease in cell body staining and an increase in the density of trk-containing fibers that became widely distributed to the gut wall and pancreas. The adult pattern of trk immunoreactivity was established between postnatal days 5 and 10. In adults, trk immunoreactivity was found in numerous enteric and intrapancreatic ganglion cells and in dense networks of fibers innervating all the layers of the gut, the pancreas, and vasculature. The trkC mRNA was expressed in adult enteric ganglion cells of both the myenteric and submucous plexus. By contrast, the trkA and trkB mRNAs could not be detected in enteric ganglia. All three trk mRNAs were expressed in dorsal root ganglia, which were used as positive controls. The density and wide distribution of trk immunoreactivity together with its persistence in adulthood support the concept that neurotrophins play a broad role in the digestive system from development through adult life, perhaps being involved in differentiation, phenotypic expression, and tissue maintenance. The presence of trkC mRNA in enteric neurons along with recent evidence that neurotrophin-3 plays a role in the development of the enteric nervous system suggest that trkC and neurotrophin-3 are a major neurotrophin system in the gastrointestinal tract. © 1996 Wiley-Liss, Inc.

Cellular localization of Pan-trk immunoreactivity and trkC mRNA in the enteric nervous system.

The members of the trk family of tyrosine receptor kinases, trkA, trkB, and trkC, are the functional receptors for neurotrophins, a family of related neurotrophic factors. In this study, we investigated 1) the distribution of neurotrophin receptors in the developing and adult rat digestive tract with a pan-trk antibody that recognizes all known trks and 2) the cellular localization of trk-encoding mRNAs in the adult gut with single-stranded RNA probes specific for trkA, trkB, and trkC. In the developing myenteric plexus, trk immunoreactivity was present at embryonic day (ED) 14. Cells and fibers immunoreactive for trk could be visualized in the myenteric plexus at ED 16. At this age, dense staining was found in thick bundles of fibers in proximity to the myenteric plexus in the longitudinal muscle and in association with blood vessels in the mesentery. At ED 18, trk immunoreactivity was also seen in thin processes running from the myenteric plexus into the circular muscle, and in fibers and cells in intrapancreatic ganglia. By ED 20, immunoreactive staining was quite dense in both the myenteric and submucosal plexuses. At birth, virtually all enteric ganglia displayed strong trk immunoreactivity; the intensity of the staining at this age made it difficult to discern individual cells. During postnatal development, there was a decrease in cell body staining and an increase in the density of trk-containing fibers that became widely distributed to the gut wall and pancreas. The adult pattern of trk immunoreactivity was established between postnatal days 5 and 10. In adults, trk immunoreactivity was found in numerous enteric and intrapancreatic ganglion cells and in dense networks of fibers innervating all the layers of the gut, the pancreas, and vasculature. The trkC mRNA was expressed in adult enteric ganglion cells of both the myenteric and submucous plexus. By contrast, the trkA and trkB mRNAs could not be detected in enteric ganglia. All three trk mRNAs were expressed in dorsal root ganglia, which were used as positive controls. The density and wide distribution of trk immunoreactivity together with its persistence in adulthood support the concept that neurotrophins play a broad role in the digestive system from development through adult life, perhaps being involved in differentiation, phenotypic expression, and tissue maintenance. The presence of trkC mRNA in enteric neurons along with recent evidence that neurotrophin-3 plays a role in the development of the enteric nervous system suggest that trkC and neurotrophin-3 are a major neurotrophin system in the gastrointestinal tract.

Enhanced synaptophysin immunoreactivity in rat hippocampal culture by 5-HT1A agonist, S100b, and corticosteroid receptor agonists

Serotonin (5-HT) has been shown to modulate brain maturation during development and adult plasticity. This effect in the whole animal may be due to activation of 5-HT1A receptors and a corresponding increases in S100b and corticosterone. Synaptophysin, an integral protein of the synaptic vesicle membrane that correlates with synaptic density and neurotransmitter release, is reduced by depletion of 5-HT in the cortex and hippocampus of the adult rat. Injections of a 5-HT1A agonist or dexamethasone can reverse the loss of synaptophysin immunoreactivity (IR). In this study we used morphometric analysis of synaptophysin-IR to study the effects of the 5-HT1A agonist, ipsapirone, and the neuronal extension factor, S100b on hippocampal neurons grown in a serum and steroid free media. Both compounds increased the synaptophysin-IR at doses previously established to be highly specific. Ipsapirone (10(-9)M) was more effective on neuronal cell bodies staining and S100b (10 ng/ml) was more effective in increasing the number of synaptophysin-IR varicosities on neuronal processes. In addition both types of corticosteroid receptor agonists, at previously established specific doses, Ru28362 (10(-8) M) and aldosterone (10(-9) M) produced smaller increases compared to control groups in both the cell body staining and the number of varicosities. The effect of these differentiating factors on the expression of synaptophysin-IR suggests multiple regulation sites for producing and maintaining pre-synaptic elements in the brain.

Ultrastructural Localization of a Voltage‐gated K+ Channel a Subunit (Kv1.2) in the Rat Cerebellum

A highly specific monoclonal antibody and pre-embedding immunocytochemistry were employed to examine the distribution of the K+ channel, alpha subunit K(V)1.2 in the rat cerebellum. At the light microscopic level, the heaviest immunoreactivity was seen in the basket cell pinceau at the base of Purkinje cells, with lighter staining of basket and Golgi cell bodies and a punctate pattern in the granule cell and molecular layers. Electron microscopy was performed to identify the ultrastructural location of K(V)1.2 alpha subunit in these labelled structures. This revealed that the labelling of the pinceau was confined to the preterminal axonal plexus, the area immediately around the Purkinje axon initial segment being relatively devoid of staining. Basket cell parent axons were not immunostained, but gave rise to heavily stained fine processes. Immunoreactivity was also seen in myelinated axons in the granule cell layer and in the medial cerebellar nucleus, the staining being most concentrated at the juxtaparanodal regions of the axons. An unusual pattern of staining was seen in some mossy fibre terminals, with staining restricted to fine protuberances of mossy fibre glomeruli. Structures contacted by these protuberances included adjoining glial processes. Immunostaining was absent from Purkinje cell bodies, dendrites, their axon initial segments and their terminals in the medial cerebellar nucleus. In this study, the alpha subunit K(V)1.2 was localized to a number of different cell types in the cerebellum. Each neuronal type displays a distinct subcellular distribution of the subunit.

Transient decrease of acetylcholinesterase in ventral horn neurons caudal to a low thoracic spinal cord hemisection in the adult rat

Light microscopic enzyme: histochemistry was employed to study the alterations of acetylcholinesterase (AChE) within lumbosacral ventral horn neurons at survival times of 1, 4, 7, 14, 28, 60, and 90 days after low thoracic spinal cord hemisection in adult rats. The intensity of histochemical staining was quantified using densitometric techniques. Virtually all ventral horn neurons of sham-operated and unoperated animals, which served as controls, displayed intense AChE staining. Hemisection of the spinal cord induced a transient, ipsilateral decrease of AChE staining in most neuronal cell bodies and in the neuropil of lamina IX at all segmental levels caudal to the lesion. Quantitative analysis of representative segments revealed a reduction of AChE in the ventral horn during a postoperative (p.o.) period of 1 to 28 days followed by a phase of recovery over the next two months. AChE activity still remained slightly reduced, even at 90 days p.o. The transient decrease in ACNE is a well-known metabolic response of axotomized motoneurons. However, the observed changes of AChE reactivity in intact motoneurons ipsilateral and caudal to the hemisection are presumably induced by the interruption of supraspinal descending pathways. These metabolic changes may functionally affect the whole motor unit and be involved in the disturbances of motor function following spinal cord injury.

Distribution ofα1A, α1B andα1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.

Changes of MAP2 phosphorylation during brain development.

The microtubule-associated protein MAP2 is essential for development of early neuronal morphology and maintenance of adult neuronal morphology. Several splice variants exist, MAP2a-d, with a lack of MAP2a in cat brain. MAP2 is widely used as a neuronal marker. In this study we compared five monoclonal antibodies (MAbs) against MAP2. They show differences in the immunocytochemical distribution of MAP2 isoforms during development of the visual cortex and cerebellum of the cat. Local and temporal differences were seen with MAb AP18, an antibody directed against a phosphorylation-dependent epitope near the N-terminal end. In large pyramidal dendrites in visual cortex, the AP18 epitope remained in parts immunoreactive after treatment with alkaline phosphatase. Three MAbs, AP14, MT-01, and MT-02, recognized the central region of the MAP2b molecule, which is not present in MAP2c and 2d, and reacted with phosphorylation-independent epitopes. During the first postnatal week the immunostaining in cerebellum differed between antibodies in that some cellular elements in external and internal granular layers and Purkinje cells were stained to various degrees, whereas at later stages staining patterns were similar. At early stages, antibody MT-02 stained cell bodies and dendrites in cerebral cortex and cerebellum. With progressing maturation, immunoreactivity became restricted to distal parts of apical dendrites of pyramidal cells and was absent from perikarya and finer proximal dendrites in cortex. MT-02 did not stain MAP2 in cerebellum of adult animals. This study demonstrates that the immunocytochemical detection of MAP2 depends on modifications such as phosphorylation and conformational changes of the molecule, and that MAP2 staining patterns differ between MAbs. Phosphorylation and specific conformations in the molecule may be essential for modulating function and molecular stability of MAP2, and monoclonal antibodies against such sites may provide tools for studying the functional role of modifications.

Development of calretinin-immunoreactive unipolar brush-like cells and an afferent pathway to the embryonic and early postnatal mouse cerebellum.

In the developing mouse hindbrain, immunoreactivity for calretinin, a calcium-binding protein, was first observed at embryonic day 10, and was localized to neuronal cell bodies in the reticular formation. By embryonic day 12, fibers emanated rostrally from the calretinin-immunoreactive neurons, extended dorsally and then caudally in the uncinate fasciculus to reach the developing cerebellar plate. These fibers crossed the cerebellar midline and were distributed to the contralateral side of the cerebellum. The number and intensity of staining of cell bodies in the reticular formation was reduced in postnatal mice. After postnatal day 1, it was no longer possible to discern the calretinin-immunoreactive fiber bundle in the brainstem, although fibers were still visible at the level of the uncinate fasciculus and in the cerebellum. We also observed intensely calretinin-immunoreactive, smaller cells in the cerebellum (embryonic day 14) and dorsal cochlear nuclei (embryonic day 18), most of which we believe are destined to become the "unipolar brush", (also known as "pale" or "monodendritic") cells observed in the adult mammalian brain. An immature form of these cells exists in the developing mouse cerebellum. Thus, using calretinin antiserum as a marker, an afferent neuronal system was described which projects to the cerebellar primordium. It is suggested that the calretinin-containing hook bundle is an afferent projection which provides a feed-forward neuronal system to the cerebellum which, in turn, projects afferent fibers to the calretinin-containing and other cells of the reticular formation.

CNS distribution and overexpression of neurofilament light proteins (NF-L) in mice transgenic for the human NF-L: Aberrant accumulation in thalamic perikarya

Light microscopic immunocytochemistry with monoclonal antibodies recognizing both murine and human light neurofilament proteins (mNF-L and hNF-L) or hNF-L only was used to examine the distribution of NF-L in the CNS of adult mice, normal or transgenic for the human gene. In normal mice, major fiber bundles were immunoreactive to the first antibody, with few exceptions such as the internal capsule, anterior commissure, and corpus callosum. Strong immunoreactivity was also present in the perikarya of motoneurons in the spinal cord and brainstem, as well as in other brainstem nuclei. Faint cell body staining was visible in layers II, III, and V of the parietal cortex and layers V and VI of the retrosplenial cingulate cortex. In transgenic mice, all forebrain as well as brainstem fiber tracts were intensely immunoreactive to both antibodies. Cell body staining was more intense than in normal mouse and involved additional forebrain and brainstem regions, including extended areas of cerebral cortex. Abnormal cell body labeling was particularly striking in several thalamic nuclei, where numerous darkly stained perikarya were considerably enlarged by accumulated immunoreactive material and exhibited eccentric and fragmented nuclei. At the electron microscopic level, these perikarya were filled with disarrayed filaments displacing all other organelles against the cytoplasmic membrane. Such aberrant accumulation of NF-L was presumably the result of an overexpression in selective subpopulations of CNS neurons. It was compatible with prolonged survival of the animal and could provide a new experimental model of neurodegenerative disease.

Peptidergic innervation of leg muscles of the cockroach, Periplaneta americana (L.), and a possible role in modulation of muscle contraction.

FMRFamide-related peptides of insects are particularly important because of their possible function as neurohormones and neuromodulators on a wide variety of tissues. Part of this study was an investigation of the immunofluorescent staining of motor nerves which arise in the metathoracic ganglion, examined in wholemount using an antiserum that recognizes extended -RFamide peptides (generally recognized to be of the FMRFamide family). This antiserum revealed immunochemical staining of numerous cell bodies in the metathoracic ganglion and of axons in peripheral nerve 5, a large nerve which contains both motor and sensory fibres. Axons staining positive for FMRFamide-related peptides were traced in nerve 5 as far as the femur-tibia joint, and into the first (sensory-motor) and third (motor only) ramus of nerve 5. Reverse-phase HPLC with radioimmunoassay revealed a peak of FMRFamide-related peptide activity in nerve 5 that was coincident with a peak found when thoracic ganglia were processed in the same fashion. A physiological assay was devised to test the ability of various non-native peptides to alter the characteristics of contraction of skeletal muscles of the legs. Using neurally evoked contractions of coxal depressor muscles of the metathoracic leg it was determined that several non-native peptides could potentiate muscle contractions. The results of this study suggest that muscles of the legs receive innervation by identifiable, FMRFamide-related peptide-containing neurons and that the release of peptide(s) at the muscle may be yet another method of modulating the mechanics of muscle contraction.

Cellular Localization of Enzymatically Active Thrombin in Intact Human Tissues by Hirudin Binding

Cellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inhibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

Histochemical localization of nitric oxide neurons in the hypothalamus: association with gonadotropin-releasing hormone neurons and co-localization with N-methyl-D-aspartate receptors.

The neurotransmitter glutamate plays an important role in the control of gonadotropin-releasing hormone (GnRH) secretion. Recent evidence suggests that the novel transmitter nitric oxide may also play a role in controlling GnRH release and may be an important mediator of glutamate effects. To explore the role of nitric oxide in these events, the present study determined the distribution of the enzyme which catalyzes nitric oxide production, nitric oxide synthase (NOS) in the hypothalamus, its association with GnRH neurons, and whether NOS neurons contain NMDA receptors. NOS was localized by staining hypothalamic sections from female rats for NADPH-diaphorase activity. Specific antibodies for GnRH and the NMDAR1 receptor subunit were used for double-staining to determine NOS association with GnRH neurons and the presence of NMDA R1 receptor subunits in hypothalamic NOS neurons. The studies showed intense NOS cell body and fiber staining in the organum vasculosum of the lamina terminalis (OVLT) where numerous GnRH cell bodies are located. Other major GnRH cell body sites such as the median preoptic nucleus (MPN) and medial preoptic area (MPOA) displayed moderate staining of NOS cell bodies and fibers. Intense NOS staining was also observed in the median eminence, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus of the hypothalamus. While no GnRH neurons were found to double stain for NOS in the hypothalamus, GnRH neurons were frequently surrounded by NOS neurons in the OVLT, MPN and MPOA with potential contacts between NOS and GnRH neurons in these areas. In addition, there was significant overlap of GnRH and NOS fibers in the lateral portion of the internal zone of the median eminence where GnRH fibers and terminals converge. Double-staining studies for NADPH-diaphorase and NMDA R1 receptor subunit showed that many NOS neurons in the OVLT, MPOA, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus co-localize the NMDA R1 receptor subunit. Localization of NMDA R1 receptor subunit immunoreactivity in B-NOS neurons in the hypothalamus was further confirmed by using combined immunohistochemistry-in situ hybridization. Finally, the functional importance of this co-localization was shown by the finding that central administration of a nitric oxide synthase inhibitor blocked the ability of NMDA to induce LH secretion. Taken as a whole, these studies provide evidence which support a role for nitric oxide as an important regulator of GnRH neurons in the female. They also suggest that hypothalamic NOS neurons are targets for glutamate regulation as evidenced by co-localization of the NMDA R1 receptor subunit.

How does thermotherapy effectively work on benign prostatic hyperplasia--an experimental study

Isometric contractile force of rabbit prostatic tissue in response to electric field stimulation (EFS), KCl, and phenylephrine were measured at incubation temperature of 37 degrees C, before and after thermal exposure to 42 degrees C, 45 degrees C, 48 degrees C and 50 degrees C for 30 minutes. The contractile force in response to EFS decreased after thermal exposure above 45 degrees C, and the contractile force in response to KCL or phenylephrine decreased after thermal exposure above 48 degrees C. All the contractile response abolished after thermal exposure to 50 degrees C. The results indicate that the nerve is more hear-sensitive than the smooth muscle in the prostate. Histological examination revealed shrinkage of cell body and dark staining of nuclear chromatin of the smooth muscle cells after thermal exposure above 48 degrees C. The same histological change of the smooth muscle as well as degenerative change of the nerve cells was observed on the prostate 3-7 months after clinical thermotherapy. From these results, it is suggested that clinical effect of thermotherapy is brought about from both neural and muscular damage of the prostate. Since the least temperature to cause an irreversible tissue damage ranges from 48 degrees C through 50 degrees C, we believe it is ideal to heat the prostate around 50 degrees C to obtain a good clinical effect of thermotherapy on benign prostatic hyperplasia as a minimum invasive treatment.

Age and differentiation-related differences in neuron-specific tubulin immunostaining of olfactory sensory neurons

Olfactory sensory neurons (OSNs) are unusual mammalian neurons because they are produced continually throughout adult life and because their production is upregulated after injury. Because OSNs also have an unusual immunological profile and do not bind the commonly used antibody markers for neurons, we sought new antibody markers for studies of OSN regeneration. In this report, we characterize the staining patterns of antibodies to the Class III β, neuron-specific, tubulin (NST) in rat olfactory tissue sections, to determine if these antibodies specifically label OSNs. In tissue sections from newborn rats, monoclonal antibodies to NST labeled cell bodies and processes of both immature (olfactory marker protein, OMP, -negative) and mature (OMP-positive) OSNs. In tissue sections from adult rats, immature OSNs showed both cell body and dendrite staining with anti-NST, while mature OSNs showed little or no cell body staining. Mature OSNs appeared to have both axonal and dendritic anti-NST staining. Axonal staining was suggested by the complete labeling of the olfactory nerve bundles and the nerve fiber layer of the olfactory bulb. The extent of labeling was judged by comparison with anti-OMP staining. Mature OSN dendritic staining was suggested because a much higher number of dendrites were anti-NST stained in the epithelium than cell bodies. These changes suggest both age and differentiation-related changes in subcellular distribution of NST in OSNs. NST antibodies are thus good markers for all OSNs in the newborn rat, but selective markers for immature OSNs and mature OSN processes in the adult rat. NST antibodies may also be useful probes for βIII tubulin function in neurons.

Postnatal development of [D-Ala2]deltorphin-I-like immunoreactive structures in the rat brain.

[D-Ala2]deltorphin-I, a highly selective ligand for delta opioid receptors, is a heptapeptide originally purified from frog skin. Previous immunohistochemical studies indicate that [D-Ala2]deltorphin-I-like molecule(s) may be present in adult rat brain, including specific neuronal cells and fibers partially overlapping with the mesocortical and nigrostriatal dopaminergic systems. Here, we examined the developmental aspect of such immunoreactive brain structures in early postnatal rats. In newborn to 21-day-old rats, positive staining in the brain occurred mainly in subpopulations of neurons and occasionally in tanycytes. On postnatal day 0, neuronal cell bodies containing [D-Ala2]deltorphin-I-like immunoreactivity were found in various brain regions, including the olfactory tubercle, ventral pallidum, hippocampus, ventral tegmental area, pars compacta of the substantia nigra, supramammillary nucleus, and dorsal raphe nucleus. Immunoreactive nerve fibers were observed in the main and accessory olfactory bulbs, olfactory tubercle, prelimbic area, anterior cingulate cortex, neostriatum, accumbens, lateral septal nucleus, lateral habenular nucleus, and superior colliculus. As pups grew, positive staining of cell bodies decreased gradually in both density and intensity, and those in the olfactory tubercle and ventral pallidum were no longer visible on postnatal day 14. On postnatal day 21, positive cells were found only in the ventral midbrain, including the pars compacta of the substantia nigra, ventral tegmental area, A8 region, and supramammillary nucleus. Positive fibers also decreased in density with age except in the accessory olfactory bulb, olfactory tubercle, prelimbic area, and anterior cingulate cortex.

Toluene inhibits synaptic transmission without causing gross morphological disturbances

The effects of toluene on synaptic transmission and neuronal morphology were investigated using guinea pig hippocampal slices. Population spikes (PS) were elicited in granule cell layer by stimulation of the perforant path and antidromic potentials (AP) were evoked in the same locii by stimulation of mossy fibers. Toluene at a concentration of 1000 μg/ml completely depressed post-synaptic responses within 15 min but increased the AP to 140% of its original value. The PS recovered completely when washout was begun 10 and 20 min after onset of depression, but exhibited only partial recovery following a 40 min depression. There were no evident changes in staining of axons or cell bodies after toluene treatment. These results indicate that toluene at high concentrations (1000 μg/ml) inhibits synaptic transmission selectively and with longer exposures causes lasting physiological effects unaccompanied by gross morphological changes.