Immunohistochemical Localization of Five Members of the KV1 Channel Subunits: Contrasting Subcellular Locations and Neuron-specific Co-localizations in Rat Brain

A large variety of potassium channels is involved in regulating integration and transmission of electrical signals in the nervous system. Different types of neurons, therefore, require specific patterns of potassium channel subunit expression and specific regulation of subunit coassembly into heteromultimeric channels, as well as subunit-specific sorting and segregation. This was investigated by studying in detail the expression of six different α-subunits of voltage-gated potassium channels in the rat hippocampus, cerebellum, olfactory bulb and spinal cord, combining in situ hybridization and immunocytochemistry. Specific polyclonal antibodies were prepared for five α-subunits (K_V1.1, K_V1.2, K_V1.3, K_V1.4, K_V1.6) of the Shaker-related subfamily of rat K_v channels, which encode delayed-rectifier type and rapidly inactivating A-type potassium channels. Their distribution was compared to that of an A-type potassium channel (K_V3.4), belonging to the Shaw-related subfamily of rat K_v channels. Our results show that these K_v channel α-subunits are differentially expressed in rat brain neurons. We did not observe in various neurons a stereotypical distribution of K_v channel α-subunits to dendritic and axonal compartments, but a complex differential subcellular subunit distribution. The different K_v channel subunits are targeted either to presynaptic or to postsynaptic domains, depending on neuronal cell type. Thus, distinct combinations of K_v1 α-subunits are co-localized in different neurons. The implications of these findings are that both differential expression and assembly as well as subcellular targeting of K_v channel α-subunits may contribute to K_v channel diversity and thereby to presynaptic and postsynaptic membrane excitability.     

Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse

Gamma-aminobutyric acid (GABA)ergic neurons in the central nervous system regulate the activity of other neurons and play a crucial role in information processing. To assist an advance in the research of GABAergic neurons, here we produced two lines of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse. The distribution pattern of GFP-positive somata was the same as that of the GAD67 in situ hybridization signal in the central nervous system. We encountered neither any apparent ectopic GFP expression in GAD67-negative cells nor any apparent lack of GFP expression in GAD67-positive neurons in the two GAD67-GFP knock-in mouse lines. The timing of GFP expression also paralleled that of GAD67 expression. Hence, we constructed a map of GFP distribution in the knock-in mouse brain. Moreover, we used the knock-in mice to investigate the colocalization of GFP with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in the frontal motor cortex. The proportion of GFP-positive cells among NeuN-positive cells (neocortical neurons) was approximately 19.5%. All the CR-, PV-, and SS-positive cells appeared positive for GFP. The CR-, PV, and SS-positive cells emitted GFP fluorescence at various intensities characteristics to them. The proportions of CR-, PV-, and SS-positive cells among GFP-positive cells were 13.9%, 40.1%, and 23.4%, respectively. Thus, the three subtypes of GABAergic neurons accounted for 77.4% of the GFP-positive cells. They accounted for 6.5% in layer I. In accord with unidentified GFP-positive cells, many medium-sized spherical somata emitting intense GFP fluorescence were observed in layer I.     

Neuropeptide Y Y1 receptor mRNA in rodent brain: distribution and colocalization with melanocortin-4 receptor

The central neuropeptide Y (NPY) Y1 receptor (Y1-R) system has been implicated in feeding, endocrine, and autonomic regulation. In the present study, we systematically examined the brain distribution of Y1-R mRNA in rodents by using radioisotopic in situ hybridization histochemistry (ISHH) with a novel sensitive cRNA probe. Within the rat hypothalamus, Y1-R-specific hybridization was observed in the anteroventral periventricular, ventromedial preoptic, suprachiasmatic, paraventricular (PVH), dorsomedial, ventromedial, arcuate, and mamillary nuclei. In the rat, Y1-R mRNA expression was also seen in the subfornical organ, anterior hypothalamic area, dorsal hypothalamic area, and in the lateral hypothalamic area. In addition, Y1-R hybridization was evident in several extrahypothalamic forebrain and hindbrain sites involved in feeding and/or autonomic regulation in the rat. A similar distribution pattern of Y1-R mRNA was observed in the mouse brain. Moreover, by using a transgenic mouse line expressing green fluorescent protein under the control of the melanocortin-4 receptor (MC4-R) promoter, we observed Y1-R mRNA expression in MC4-R-positive cells in several brain sites such as the PVH and central nucleus of the amygdala. Additionally, dual-label ISHH demonstrated that hypophysiotropic PVH cells coexpress Y1-R and pro-thyrotropin-releasing hormone mRNAs in the rat. These observations are consistent with the proposed roles of the central NPY/Y1-R system in energy homeostasis.     

Distribution of Somatostatin Receptors 1, 2 and 3 mRNA in Rat Brain and Pituitary

In this study sequence-specific antisense oligonucleotide probes have been used to investigate the distribution of the mRNAs coding for the somatostatin receptor subtypes termed somatostatin receptor 1, somatostatin receptor 2 and somatostatin receptor 3 in the rat brain and pituitary using in situ hybridization techniques. The three receptor subtype mRNAs were found to be widely distributed in the brain with different patterns of expression, but with some overlap. Somatostatin receptor 1 mRNA was particularly concentrated in the cerebral and piriform cortex, magnocellular preoptic nucleus, hypothalamus, amygdala, hippocampus, and several nuclei of the brainstem. Somatostatin receptor 3 mRNA was very abundant in the cerebellum and pituitary (in contrast to somatostatin receptor 1), but it was also found in hippocampus, amygdala, hypothalamus and in motor nuclei of the brainstem. Somatostatin receptor 2 mRNA levels were very low relative to the other two mRNAs evaluated. Receptor 2 mRNA was observed in the anterior pituitary, and in the brain it was found in the medial habenular nucleus, claustrum, endopiriform nucleus, hippocampus, some amygdala nuclei, cerebral cortex and hypothalamus. None of the three somatostatin receptor mRNAs studied here was found in the caudate nucleus. Northern analysis revealed distinct sizes of mRNAs for each subtype, and displacement experiments showed that each probe sequence was subtype-specific.     

Postnatal expression of H1‐receptor mRNA in the rat brain: correlation to l‐histidine decarboxylase expression and local upregulation in limbic seizures

Histamine is implicated in the regulation of brain functions through three distinct receptors. Endogenous histamine in the brain is derived from mast cells and neurons, but the importance of these two pools during early postnatal development is still unknown. The expression of histamine H₁-receptor in the rat brain was examined using in situ hybridization during postnatal development and in adults. For comparison, the expression of l -histidine decarboxylase (HDC) in the two pools was revealed. H₁-receptor was evenly expressed throughout the brain on the first postnatal days, but resembled the adult, uneven pattern already on postnatal day 5 (P5). HDC was expressed in both mast cells and tuberomammillary neurons from birth until P5, after which the mast cell expression was no more detectable. In adult rat brain, high or moderate levels of H₁-receptor expression were found in the hippocampus, zona incerta, medial amygdaloid nucleus and reticular thalamic nucleus. In most areas of the adult brain the expression of H₁-receptor mRNA correlates well with binding data and histaminergic innervation. A notable exception is the hypothalamus, with high fibre density but moderate or low H₁-receptor expression. Systemic kainic acid administration induced increased expression of H₁-receptor mRNA in the caudate-putamen and dentate gyrus, whereas no change was seen in the hippocampal subfields CA1–CA3 or in the entorhinal cortex 6 h after kainic acid injections. This significant increase supports the concept that histaminergic transmission, through H₁-receptor, is involved in the regulation of seizure activity in the brain.     

Transiently increased colocalization of vesicular glutamate transporters 1 and 2 at single axon terminals during postnatal development of mouse neocortex: a quantitative analysis with correlation coefficient

Vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 show complementary distribution in neocortex; VGLUT1 is expressed mainly in axon terminals of neocortical neurons, whereas VGLUT2 is located chiefly in thalamocortical axon terminals. However, we recently reported a frequent colocalization of VGLUT1 and VGLUT2 at a subset of axon terminals in postnatal developing neocortex. We here quantified the frequency of colocalization between VGLUT1 and VGLUT2 immunoreactivities at single axon terminals by using the correlation coefficient (CC) as an indicator in order to determine the time course and spatial extent of the colocalization during postnatal development of mouse neocortex. The colocalization was more frequent in the primary somatosensory (S1) area than in both the primary visual (V1) and the motor areas; of area S1 cortical layers, colocalization was most evident in layer IV barrels at postnatal day (P) 7 and in adulthood. CC in layer IV showed a peak at P7 in area S1, and at P10 in area V1 though the latter peak was much smaller than the former. These results suggest that thalamocortical axon terminals contained not only VGLUT2 but also VGLUT1, especially at P7-10. Double fluorescence in situ hybridization confirmed coexpression of VGLUT1 and VGLUT2 mRNAs at P7 in the somatosensory thalamic nuclei and later in the thalamic dorsal lateral geniculate nucleus. As VGLUT1 is often used in axon terminals that show synaptic plasticity in adult brain, the present findings suggest that VGLUT1 is used in thalamocortical axons transiently during the postnatal period when plasticity is required.     

Innexins in the lobster stomatogastric nervous system: cloning, phylogenetic analysis, developmental changes and expression within adult identified dye and electrically coupled neurons

Gap junctions play a key role in the operation of neuronal networks by enabling direct electrical and metabolic communication between neurons. Suitable models to investigate their role in network operation and plasticity are invertebrate motor networks, which are built of comparatively few identified neurons, and can be examined throughout development; an excellent example is the lobster stomatogastric nervous system. In invertebrates, gap junctions are formed by proteins that belong to the innexin family. Here, we report the first molecular characterization of two crustacean innexins: the lobster Homarus gammarus innexin 1 (Hg-inx1) and 2 (Hg-inx2). Phylogenetic analysis reveals that innexin gene duplication occurred within the arthropod clade before the separation of insect and crustacean lineages. Using in situ hybridization, we find that each innexin is expressed within the adult and developing lobster stomatogastric nervous system and undergoes a marked down-regulation throughout development within the stomatogastric ganglion (STG).The number of innexin expressing neurons is significantly higher in the embryo than in the adult. By combining in situ hybridization, dye and electrical coupling experiments on identified neurons, we demonstrate that adult neurons that express at least one innexin are dye and electrically coupled with at least one other STG neuron. Finally, two STG neurons display no detectable amount of either innexin mRNAs but may express weak electrical coupling with other STG neurons, suggesting the existence of other forms of innexins. Altogether, we provide evidence that innexins are expressed within small neuronal networks built of dye and electrically coupled neurons and may be developmentally regulated.     

Distribution of tuberoinfundibular peptide of 39 residues and its receptor, parathyroid hormone 2 receptor, in the mouse brain

Tuberoinfundibular peptide of 39 residues (TIP39) was identified as a potent parathyroid hormone 2 receptor (PTH2R) agonist. Existing anatomical data also support the suggestion that TIP39 is the PTH2R's endogenous ligand, but a comprehensive comparison of TIP39 and PTH2R distributions has not been performed. In the present study, we compared the distributions of TIP39 and PTH2R on adjacent mouse brain sections. In addition, we determined the locations of PTH2R-expressing cell bodies by in situ hybridization histochemistry and by labeling beta-galactosidase driven by the PTH2R promoter in knockin mice. An excellent correlation was found between the distributions of TIP39-containing fibers and PTH2R-containing cell bodies and fibers throughout the brain. TIP39 and the PTH2R are abundant in medial prefrontal, insular, and ectorhinal cortices, the lateral septal nucleus, the bed nucleus of the stria terminalis, the fundus striati, the amygdala, the ventral subiculum, the hypothalamus, midline and intralaminar thalamic nuclei, the medial geniculate body, the periaqueductal gray, the ventral tegmental area, the superior and inferior colliculi, the parabrachial nuclei, the locus coeruleus, subcoeruleus and periolivary areas, and the nucleus of the solitary tract. Furthermore, even the subregional distribution of TIP39- and PTH2R-immunoreactive fibers in these regions showed remarkable similarities, providing anatomical evidence that TIP39 may act on the PTH2R. Based on these observations and on previous pharmacological data, we propose that TIP39 is an endogenous ligand of the PTH2R and that they form a neuromodulator system, which is optimally positioned to regulate limbic, endocrine, and auditory brain functions. Published 2007 Wiley-Liss, Inc.     

Expression and Subcellular Localization of p42IP4/ Centaurin-α, a Brain-specific, High-affinity Receptor for lnositol 1,3,4,5-Tetrakisphosphate and Phosphatidylinositol 3,4,5-Trisphosphate in Rat Brain

Recently emerging evidence suggests important roles for inositol polyphosphates and inositol phospholipids in neuronal Ca²⁺ signalling, membrane vesicle trafficking and cytoskeletal rearrangement. A prerequisite for a detailed physiological characterization of the signalling of both potential second messengers inositol-(1,3,4,5)-tetrakisphosphate (InsP₄) and phosphatidylinositol-3,4,5-trisphosphate (PtdlnsP₃) in the nervous system is the precise cellular localization of their receptors. Based on the cDNA sequence of a recently cloned brain-specific receptor with high affinity for both InsP₄ and PtdlnsP₃ (InsP₄–PtdlnsP₃R), p42^IP4/centaurin-α, we localized the mRNA and the protein in rat brain. In situ hybridization revealed a widespread expression of the InsP₄–PtdlnsP₃R with prominent labelling in cerebellum, hippocampus, cortex and thalamus, which moreover is developmentally regulated. Using peptide-specific antibodies, the immunoreactivity was localized in the adult brain in the vast majority of neuronal cell types and probably also in some glial cells. Prominent immunoreactivity was found in axonal processes and in cell types characterized by extensive neurites. In the hypothalamus a subpopulation of parvocellular neurons in the peri- and paraventricular nuclei was most heavily labelled. This was confined by strong immunoreactivity in the lamina externa of the median eminence in close proximity to portal plexus blood vessels. Electron microscopy revealed that the InsP₄-PtdlnsP₃R was frequently associated with presynaptic vesicular structures. Further studies should identify the role of the InsP₄-PtdlnsP₃R in cellular neural processes.     

Distribution of type 1 cannabinoid receptor-expressing neurons in the septal-hypothalamic region of the mouse: colocalization with GABAergic and glutamatergic markers

Type 1 cannabinoid receptor (CB1) is the principal mediator of retrograde endocannabinoid signaling in the brain. In this study, we addressed the topographic distribution and amino acid neurotransmitter phenotype of endocannabinoid-sensitive hypothalamic neurons in mice. The in situ hybridization detection of CB1 mRNA revealed high levels of expression in the medial septum (MS) and the diagonal band of Broca (DBB), moderate levels in the preoptic area and the hypothalamic lateroanterior (LA), paraventricular (Pa), ventromedial (VMH), lateral mammillary (LM), and ventral premammillary (PMV) nuclei, and low levels in many other hypothalamic regions including the suprachiasmatic (SCh) and arcuate (Arc) nuclei. This regional distribution pattern was compared with location of γ-aminobutyric acid (GABA)ergic and glutamatergic cell groups, as identified by the expression of glutamic acid decarboxylase 65 (GAD65) and type 2 vesicular glutamate transporter (VGLUT2) mRNAs, respectively. The MS, DBB, and preoptic area showed overlaps between GABAergic and CB1-expressing neurons, whereas hypothalamic sites with moderate CB1 signals, including the LA, Pa, VMH, LM, and PMV, were dominated by glutamatergic neurons. Low CB1 mRNA levels were also present in other glutamatergic and GABAergic regions. Dual-label in situ hybridization experiments confirmed the cellular co-expression of CB1 with both glutamatergic and GABAergic markers. In this report we provide a detailed anatomical map of hypothalamic glutamatergic and GABAergic systems whose neurotransmitter release is controlled by retrograde endocannabinoid signaling from hypothalamic and extrahypothalamic target neurons. This neuroanatomical information contributes to an understanding of the role that the endocannabinoid system plays in the regulation of endocrine and metabolic functions.     

Spiny calretinin-immunoreactive neurons in the hilus and CA3 region of the rat hippocampus: Local axon circuits,synaptic connections,and glutamic acid decarboxylase 65/67 mRNA expression

We have used the Golgi method and Golgi electron microscopic techniques to analyze the axonal arborization and efferent connections of spiny calretinin-immunoreactive neurons in the CA3 region and hilus of the rat hippocampal formation. In the hilus, the axons of spiny calretinin-immunoreactive neurons sent out numerous collaterals that arborized in the hilar region and the molecular layer. In the CA3 region, these axons extended mainly to the stratum radiatum and pyramidal layer but also to the stratum oriens and stratum lacunosum-moleculare. Axonal varicosities were distributed widely throughout the axonal collaterals. Electron microscopic studies revealed that the axon terminals of spiny calretinin-immunoreactive neurons established synaptic contacts mainly with dendritic shafts. We next analyzed the expression of glutamic acid decarboxylase (GAD65/67) mRNAs in spiny nonpyramidal neurons that were identified by calretinin immunoreactivity. We found that spiny calretinin-positive neurons in the CA3 region and hilus of the rat hippocampal formation expressed the two isoforms of GAD: GAD65 and GAD67 mRNAs. These findings show that the spiny calretinin-immunoreactive neurons of hippocampus give rise to local axonal arborizations, suggesting that they are inhibitory. J. Comp. Neurol. 404:438–448, 1999. © 1999 Wiley-Liss, Inc.     

Perivascular T cells are infected with HTLV-I in the spinal cord lesions with HTLV-I-associated myelopathy/tropical spastic paraparesis: double staining of immunohistochemistry and polymerase chain reaction in situ hybridization

HTLV-I-infected cells play an important role in pathogenesis HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Our previous studies of quantitative polymerase chain reaction (PCR) and in situ PCR suggested that T cells infiltrating in the spinal cord lesion were infected with HTLV-I. To elucidate the localization of HTLV-I proviral DNA directly, we performed double staining using immunohistochemistry and PCR in situ hybridization (PCR-ISH). Fresh frozen sections of the spinal cord from four HAM patients taken at autopsy were first immunostained with antibodies to pan T cells (UCHL-1), macrophages (KP-1) and helper/inducer T cells (OPD4). Then PCR-ISH was carried out with specific primers and probe for the HTLV-I pX region. UCHL-1-positive cells were noted around perivascular areas and, to some extent, in the parenchyma. Of the UCHL-1-positive cells, 9.4% (case 1), 9.6% (case 2), 1.1% (case 3) and 6.7% (case 4) became positive in HTLV-I PCR-ISH. UCHL-1-negative cells were HTLV-I PCR-ISH negative and almost all KP-1-positive cells were HTLV-I negative. HTLV-I was localized to OPD4-positive cells in examined lesions of cases 2 and 4. These data are a direct demonstration of HTLV-I proviral DNA localizing to infiltrated T cells in HAM/ TSP spinal cord lesions. Received: 1 December 1997 / Revised, accepted: 20 March 1998