Immunocytochemical Localization of TASK-3 Channels in Rat Motor Neurons

Motor neurons are large cholinergic neurons located in the brain stem and spinal cord. In recent years, a functional role for TASK channels in cellular excitability and vulnerability to anesthetics of motor neurons has been described. Using a polyclonal monospecific antibody against the tandem pore domain K⁺ channel (K2P channel) TWIK-related acid-sensitive K⁺ channel (TASK-3), we analyzed the expression of the TASK-3 protein in motor systems of the rat CNS. Immunocytochemical staining showed strong TASK-3 expression in motor neurons of the facial, trigeminal, ambiguus, and hypoglossal nuclei. Oculomotor nuclei (including trochlear and abducens nucleus) were also strongly positive for TASK-3. The parasympathetic Edinger-Westphal nucleus and dorsal vagal nucleus showed significant, but weaker expression compared with somato- and branchiomotoric neurons. In addition, motor neurons in the anterior horn of the spinal cord were also strongly labeled for TASK-3 immunoreactivity. Based on morphological criteria, TASK-3 was found in the somatodendritic compartment of motor neurons. Cellular staining using methyl green and immunofluorescence double-labeling with anti-vesicular acetylcholine transporter (anti-vAChT) indicated ubiquitous TASK-3 expression in motor neurons, whereas in other brain regions TASK-3 showed a widespread but not ubiquitous expression. In situ hybridization using a TASK-3 specific riboprobe verified the expression of TASK-3 in motor neurons at the mRNA level.     

Distribution of Preproatrial Natriuretic Peptide mRNA in Rat Brain Detected by In Situ Hybridization of DNA Oligonucleotides: Enrichment in Hypothalamic and Limbic Regions

The expression and distribution of mRNA encoding preproatrial natriuretic peptide (ppANP) in rat brain has been investigated by in situ hybridization of two 35S-labeled synthetic DNA oligonucleotides, based on a cDNA clone sequence that encodes rat ppANP. The highest relative concentrations of ppANP mRNA were detected in the medial preoptic hypothalamic nucleus ("anteroventral/third ventricle region") and the medial habenula. Moderate concentrations of ppANP mRNA were observed in the CA1 pyramidal cells of the hippocampus, the endopiriform nucleus, the arcuate nucleus, the zona incerta, and cells of the pontine tegmental and peduculopontine nuclei. Several of these regions, including the habenula and the hypothalamic areas, have previously been reported to contain atrial natriuretic peptide (ANP)-like immunoreactivity, but the expression of ppANP mRNA in CA1 pyramidal cells suggests the occurrence of differential translation of ppANP mRNA into protein product in different brain regions, or the existence of different immunological forms of the peptide. The abundance of ppANP mRNA in brain was relatively low in comparison with that previously reported for many other mRNA species encoding other brain neuropeptides. These results demonstrate that ANP gene expression occurs in discrete neuronal populations of the CNS and that studies of the regulation of this expression should now be possible using quantitative in situ hybridization.     

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min.2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region.3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group.4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare.5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.     

Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain

Neural visinin-like proteins (VILIPs) are members of the neuronal subfamily of intracellular EF-hand calcium sensor proteins termed the NCS family, which are thought to play important roles in cellular signal transduction. While numerous studies suggest a wide but uneven distribution of these proteins in rat and chicken brain, their location in, and possible significance for, the human brain, remains to be established. We used specific polyclonal antisera to map the human brain for VILIP-1 and VILIP-3 immunoreactivities. VILIP-1 was detected in cortical pyramidal cells and interneurons, septal, subthalamic and hippocampal neurons (subfields CA1 and CA4 pyramidal cells and especially hilar interneurons) as well as in cerebellar Golgi, basket, granule, stellate and dentate nucleus neurons. Purkinje cells were free of immunoreaction. VILIP-3 was more restricted in its distribution. It was identified in cerebellar Purkinje cells and a subpopulation of granule neurons. Further, neurons belonging to different nuclei of the brain stem and multiple subcortical nerve cells stained for visinin-like protein 3. A weak immunoreaction appeared in cortical and hippocampal neurons. Intracellularly the immunoreactivity appeared in the perikarya, dendrites and some axons. Sometimes, immunostaining was found in the neuropil. Glia did not express visinin-like proteins. Our findings support, from a neuroanatomical viewpoint, the idea that these calcium sensor proteins may be of relevance for neuronal signalling in the human CNS.     

Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of Gbeta- and Ggamma2/3-subunits suggests that region-specific combinations of G-protein subunits mediate signal transduction in the central nervous system. The different subcellular distribution of Gbeta-subunits in cultivated neurons reflects that observed in tissue where Gbeta5 and Gbeta2 associate preferentially with the perikarya and the neuropil, respectively, and suggests an additional association of Gbeta2 with secretory vesicles.     

Distribution and Subcellular Localization of High-Molecular-Weight Microtubule-Associated Protein-2 Expressing Exon 8 in Brain and Spinal Cord

Abstract: The expression of high-molecular-weight (HMW) microtubule-associated protein-2 (MAP-2) expressing exon 8 (MAP-2+8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP-2+8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP-2+8 comigrated with MAP-2a. In human adult brain, HMW MAP-2+8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium-sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultra-structural level, HMW MAP-2+8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP-2+8. The presence of HMW MAP-2+8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.     

Norepinephrine-induced expression of cytokines in isolated biventricular working rat hearts

Whereas ATP consumption increases with neural activity and is buffered by phosphocreatine (PCr), it is not known whether PCr synthesis by ubiquitous mitochondrial creatine kinase (uMtCK) supports energy metabolism in all neurons. To explore the possibility that uMtCK expression in neurons is modulated by activity and during development, we used immunocytochemistry to detect uMtCK-containing mitochondria. In the adult brain, subsets of neurons including layer Va pyramidal cells, most thalamic nuclei, cerebellar Purkinje cells, olfactory mitral cells and hippocampal interneurons strongly express uMtCK. uMtCK is transiently expressed by a larger group of neurons at birth. Neurons in all cortical layers express uMtCK at birth (P0), but uMtCK is restricted to layer Va by P12. uMtCK is detected in cerebellar Purkinje cells at birth, but localization to dendrites is only observed after P5 and is maximal on P14. Hippocampal CA1 and CA3 pyramidal neurons contain uMtCK-positive mitochondria at birth, but this pattern becomes progressively restricted to interneurons. Seizures induced uMtCK expression in cortical layers II–III and CA1 pyramidal neurons. In the cortex, but not in CA1, blockade of seizures prevented the induction of uMtCK. These findings support the concept that uMtCK expression in neurons is (1) developmentally regulated in post-natal life, (2) constitutively restricted in the adult brain, and (3) regulated by activity in the cortex and hippocampus. This implies that mitochondrial synthesis of PCr is restricted to those neurons that express uMtCK and may contribute to protect these cells during periods of increased energy demands.     

A Brain-Specific Protein p25 Is Localized and Associated with Oligodendrocytes, Neuropil, and Fiber-Like Structures of the CA3 Hippocampal Region in the Rat Brain

Abstract: Developmental expression and cellular localization of a novel brain-specific 25-kDa protein (p25), a substrate of tau protein kinase II, were investigated in the rat brain using polyclonal antibodies raised against peptides synthesized based on the p25 amino acid sequence. By western immunoblotting, p25 was found to be expressed only slightly in the embryonic period; the expression increased from 11 days up to 5 weeks of age, and continued to increase gradually until 1–2 years of age. Immunohistochemistry revealed distinct staining of glial cells in most regions of the central nervous system in the adult rat brain. These positively immunostained cells were especially abundant in the white matter, such as the corpus callosum, cingulum, external capsule, and internal capsule. The glial cells were identified as oligodendrocytes, and the nuclei of the cells remained unstained. Whereas the neuropil in most parts of the brain was immunostained less intensely than glias, the neuropil in the first and second layers of the cerebral cortex and the dentate gyrus was relatively strongly stained. Fiber-like structures were also stained in the CA3 region of hippocampus.     

Interaction of neuronal NOS and catecholamines in regulation of expression of proteins of apoptosis by vasopressinergic hypothalamic neurons

The work studied vasopressinergic neurons of hypothalamic supraoptic and paravenricular nuclei of the wild type mice and the neuronal nitric oxide synthase (nNOS) gene knockouted mice at a decrease of the brain catecholamine (CA) level caused by administration of the blocker of activity of tyrosine hydroxylase alpha-methyl-paratyrosine (alpha-MPT) and at the CA level decrease on the background of functional activity of the vasopressinergic neurons caused by dehydration of animals. There were analyzed changes in the number of neurons in both magnocellular hypothalamic nuclei expressing proapoptotic proteins caspase-8 and caspase-9, p53, and antiapoptotic protein Bcl-2. The disturbance of the CA-ergic innervation was shown to be a strong damaging factor leading to apoptosis of neurons regardless of the presence of nNOS in the cells. However, at disturbance of the CA-ergic innervation due to the 5-day mouse dehydration, no death of neurons by apoptosis was revealed. Thus, it is possible that functional activation prevents the hypothalamic vasopressinergic neurons from death at a decrease of the CA level in brain. The main difference of the nNOS gene knockouts is the absence of activation of the Bcl-2 expression under all used actions. This confirms our suggestion about interaction of CA and NO in triggering of expression of the antiapoptotic protein Bcl-2.     

Interaction of neuronal NOS and catecholamines in regulation of expression of proteins of apoptosis by vasopressinergic hypothalamic neurons

The work deals with studies on vasopressinergic neurons of hypothalamic supraoptic and paravenricular nuclei in the wild type mice and the neuronal nitric oxide synthase (nNOS) in the gene knockouted mice at a decrease of the brain catecholamine (CA) level caused by administration of the blocker of activity of tyrosine hydroxylase α-methyl-paratyrosine (α-MPT) and at the CA level decrease on the background of functional activity of the vasopressinergic neurons caused by dehydration of animals. There were analyzed changes in the number of neurons in the magnocellular hypothalamic nuclei expressing proapoptotic proteins caspase-8 and caspase-9, p53, and antiapoptotic protein Bcl-2. Disturbance of the CAergic innervation was shown to be a strong damaging factor leading to apoptosis of neurons regardless of the presence of nNOS in the cells. However, at disturbance of the CAergic innervation due to the 5-day mouse dehydration, no death of neurons by apoptosis was revealed. Thus, it is possible that functional activation prevents the hypothalamic vasopressinergic neurons from death at a decrease of the CA level in brain. The main difference of the nNOS gene knockouts is the absence of activation of the Bcl-2 expression under all used actions. This confirms our suggestion about interaction of CA and NO in triggering of expression of the antiapoptotic protein Bcl-2.     

Immunocytochemical Localization of TASK-3 (K<Subscript>2P</Subscript>9.1) Channels in Monoaminergic and Cholinergic Neurons

Monoaminergic and cholinergic systems are important regulators of cortical and subcortical systems, and a variety of vegetative functions are controlled by the respective neurotransmitters. Neuronal excitability and transmitter release of these neurons are strongly regulated by their potassium conductances carried by Kir and K_2P channels. Here we describe the generation and characterization of a polyclonal monospecific antibody against rat TASK-3, a major brain K_2P channel. After removal of cross-reactivities and affinity purification the antibody was characterized by ELISA, immunocytochemistry of TASK-3 transfected cells, and Western blots indicating that the antibody only detects TASK-3 protein, but not its paralogs TASK-1 and TASK-5. Western blot analysis of brain membrane fractions showed a single band around 45 kD, close to the predicted molecular weight of the TASK-3 protein. In addition, specific immunolabeling using the anti-TASK-3 antibody in Western blot analysis and immunocytochemistry was blocked in a concentration dependent manner by its cognate antigen only. Immunocytochemical analysis of rat brain revealed strong expression of TASK-3 channels in serotoninergic neurons of the dorsal and median raphe, noradrenergic neurons of the locus coeruleus, histaminergic neurons of the tuberomammillary nucleus and in the cholinergic neurons of the basal nucleus of Meynert. Immunofluorescence double-labeling experiments with appropriate marker enzymes confirmed the expression of TASK-3 in cholinergic, serotoninergic, and noradrenergic neurons. In the dopaminergic system strong TASK-3 expression was found in the ventral tegmental area, whereas TASK-3 immunoreactivity in the substantia nigra compacta was only weak. All immunocytochemical results were supported by in situ hybridization using TASK-3 specific riboprobes.     

微管相关蛋白tau在不同年龄大鼠海马内的定位分布特点

目的 检测微管相关蛋白tau和Ser396/404位点磷酸化tau在胚胎期大鼠和出生后直至成熟期大鼠海马内的表达及变化规律,浅析与神经细胞分裂和分化的关系.方法 用免疫组织化学SABC法显示孕18d、出生后1d、1w、2w、2m的大鼠脑冠状切面总tau（R134d）、Ser396/404位点磷酸化tau（PHF-1）的表达.结果 ①胚胎期大鼠海马CA区的锥体细胞数量明显多于出生后,随着脑的发育,锥体细胞层的神经细胞数量逐渐下降;②孕18d大鼠海马内有丰富的总tau和PHF-1 tau的表达,并且海马内各区的阳性物质表达均匀,生后1d和1w两种物质表达仍然很强,阳性产物主要分布在海马CA1和CA2区以锥体细胞轴突为主要成分的始层,在锥体细胞树突集中的分子层和胞核内也有少量分布,而在生后2w和2m大鼠海马内各区均未见密集的阳性产物表达.结论 不同年龄大鼠海马内总tau和PHF-1 tau的表达和分布变化可能与神经系统发育过程中细胞的分裂和分化有关.     

Osteocalcin- and Osteopontin-Containing Neurons in the Rat Hind Brain

Immunohistochemistry for osteocalcin (OC) and osteopontin (OPN) was performed to know their distributions in the hind brain of adult rats. OC- and OPN-immunoreactivity (-ir) were detected in neuronal cell bodies, including perikarya and proximal dendrites and the neuropil. In the cranial nerve motor nuclei, numerous OC- and OPN-immunoreactive (-ir) neurons were detected. The neuropil in the cranial motor nuclei mostly showed strong OC- and OPN-staining intensity. The cranial nerve sensory nuclei and other relay and modulating structures in the lower brain stem also contained various numbers of OC- and OPN-ir neurons. The staining intensities in the neuropil were varied among these regions. In the cerebellar cortex, Purkinje cells and granule cells showed OPN-ir but not OC-ir. However, OC- and OPN-ir neurons were abundantly distributed throughout the cerebellar nuclei. The neuropil in the cerebellar nuclei showed moderate OC-ir and strong OPN-ir staining intensities. These findings indicate that the distribution patterns of OC- and OPN-ir neurons were similar in many structures within the hind brain. OC may play a role in modulating neuroprotective function of OPN.     

Cloning,characterization and expression of two alternatively splicing isoforms of Ca2+/calmodulin-dependent protein kinase I gamma in the rat brain

Ca2+/calmodulin-dependent protein kinase I (CaMKI), originally identified as a protein kinase phosphorylating synapsin I, has been shown to constitute a family of closely related isoforms (alpha, beta and gamma). Here, we have isolated and determined the complete primary structures of two alternatively splicing isoforms of CaMKI termed CaMKI gamma 1 and -gamma 2. CaMKI gamma 1 and -gamma 2 contain an identical N-terminal catalytic domain with different C-terminal regions due to the deletion of the 425-bp nucleotide sequence of CaMKI gamma 1 in CaMKI gamma 2. In vitro kinase assay has demonstrated the marked enhancement of the Ca2+/CaM-dependent activity of CaMKI gamma 1 by the preincubation with Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), but no significant activation of CaMKI gamma 2. Northern blot analysis has demonstrated the predominant expression of CaMKI gamma in the brain. RT-PCR analysis has revealed similar expression patterns between CaMKI gamma 1 and CaMKI gamma 2 in various brain regions. In situ hybridization analysis has demonstrated that CaMKI gamma mRNA is expressed in a distinct pattern from other isoforms of CaMKI with predominant expression in some restricted brain regions such as the olfactory bulb, hippocampal pyramidal cell layer of CA3, central amygdaloid nuclei, ventromedial hypothalamic nucleus and pineal gland. In the primary hippocampal neurons and NG108-15 cells, transfected CaMKI gamma 1 and -gamma 2 are localized primarily in the cytoplasm and neurites but not in the nucleus. These findings suggest that both isoforms of CaMKI gamma may be involved in Ca2+ signal transduction in the cytoplasmic compartment of certain neuronal population.     

Cellular expression of MAP 2 kinase in rat brain

The cellular localization of microtubule-associated protein (MAP) 2 kinase mRNA in rat brain was examined by in situ hybridization histochemistry using a synthetic oligonucleotide probe. MAP 2 kinase was expressed in both neuronal and non-neuronal cells. ‘Areas of high density of mRNA label by the MAP 2 kinase probe appeared to be associated with high cellular packing density. Thus, MAP 2 kinase expression was particularly high in regions such as the locus coeruleus, the piriform cortex, the dentate gyrus granule cell layer, pyramidal cells of the hippocampus, the mitral cells of the olfactory bulb, and the large motor neurons of the V and VII nerves. This apparent ubiquitous distribution suggests an important role of MAP 2 kinase in the cellular functions in most cells of the adult brain.     

A histochemical study of the regional distribution in the rat brain of enzymatic activity hydrolyzing glucose- and 2-deoxyglucose-6-phosphate

A modified Wachstein-Meisel lead salt method using glucose-6-phosphate or 2-deoxyglucose-6-phosphate as substrates was employed at the light microscopic level to map the rat brain for glucose-6-phosphatase (G-6-Pase). As has been described, most of the activity of the enzyme resided in neuronal cell bodies and dendritic stems. No differences were found between the results obtained with the two substrates. Two categories of brain structures with heavy and with moderate staining could be distinguished while the majority of brain regions contained only barely discernible neurons. Structures displaying very high enzyme activity included nuclei of cranial nerves, nuclei of the reticular formation, Purkinje cells, and some parts of the limbic system, e.g., CA 3 and CA 4 pyramidal fields of the hippocampus. It is pointed out that accurate biochemical determinations of G-6-Pase activity will critically depend on painstaking microdissection of nuclei and cell layers. The histochemical results may be pertinent to the interpretation of the 2-deoxyglucose method for assessment of regional glucose utilization rates in brain. The present observations make it unlikely that regional variations in G-6-Pase activity account for differences in uptake and retention of radioactivity from (1-14C)glucose and (14C)2-deoxyglucose reported previously by our group.