Suppression of skin reactivity to purified protein derivative by hepatitis C virus among HTLV-I carriers in Japan

Gamma-aminobutyric acid (GABA)A receptors are the sites of action for many antiepileptic drugs such as benzodiazepines and barbiturates. We report the results of molecular cloning of the gamma1-subunit from seizure prone DBA/2J and resistant C57BL/6J inbred mice, and analyses of nucleotide sequences and expression of the gamma1-subunit messenger RNA (mRNA) in DBA/2 and C57BL/6 inbred mice. The mouse gamma1-subunit complementary DNA (cDNA) shares 98% similarity with that of the rat at the level of amino acid sequence. Northern blot hybridization indicates that the gamma1-subunit mRNA is expressed predominantly in areas other than the cerebral cortex and cerebellum and shows little change with postnatal development. No differences have been found for the subunit between DBA/2 and C57BL/6 mice either for nucleotide sequence or for level of expression of the subunit's mRNA in whole brain by Northern blots at 3 weeks of age.     

Reduction of GABA and glutamate transporter messenger RNAs in the severe-seizure genetically epilepsy-prone rat

The genetically epilepsy-prone rat is an animal model of inherited generalised tonic-clonic epilepsy that shows abnormal susceptibility to audiogenic seizures and a lowered threshold to a variety of seizure-inducing stimuli. Recent studies suggest a crucial role for glutamate and GABA transporters in epileptogenesis and seizure propagation. The present study examines the levels of expression of the messenger RNAs encoding the glial and neuronal glutamate transporters, GLT-1 and EAAC-1, and the neuronal GABA transporter, GAT-1, in paired male genetically epileptic-prone rats and Sprague Dawley control rats using the technique of in situ hybridization. In a parallel study, semiquantitative immunoblotting was used to assess GLT-1 and EAAC-1 protein levels in similarly paired animals. Animals were assessed for susceptibility to audiogenic seizures on six occasions, and killed seven days following the last audiogenic stimulus exposure. Rat brains were processed for in situ hybridization with radioactive 35S-labelled oligonucleotide probes (EAAC-1 and GAT-1), 35S-labelled riboprobes (GLT-1), and Fluorescein-labelled riboprobes (GLT-1 and GAT-1) or processed for immunoblotting using subtype-specific antibodies for GLT-1 and EAAC-1. Semiquantitative analyses were carried out on X-ray film autoradiograms in several brain regions for both in situ hybridization and immunoblotting studies. Reductions in GAT-1 messenger RNA were found in genetically epileptic-prone rats in all brain regions examined (-8 to -24% compared to control). Similar reductions in GLT-1 messenger RNA expression levels were seen in cortex, striatum, and CA1 (-8 to -12%) of genetically epileptic-prone rats; the largest reduction observed was in the inferior colliculus (-20%). There was a tendency for a reduced expression of EAAC-1 messenger RNA in most regions of the genetically epileptic-prone rat brain although this reached statistical significance only in the striatum (-12%). In contrast, no significant differences in GLT-1 and EAAC-1 protein between genetically epileptic-prone rats and control animals were observed in any region examined, although there was a tendency to follow the changes seen with the corresponding messenger RNAs. These results show differences in the messenger RNA expression levels of three crucial amino acid transporters. For the two glutamate transporters, GLT-1 and EAAC-1, differences in messenger RNA levels are not reflected or are only partially reflected in the expression of the corresponding proteins.     

The differential expression patterns of messenger RNAs encoding non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4) in the rat brain

The messenger RNA expression of non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4), considered alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid type, was investigated in rat brain by in situ hybridization histochemistry using oligonucleotide probes specific to each subunit sequence. GluR1-4 subunit messenger RNAs were expressed widely and abundantly throughout the CNS. However, the combination of expression pattern varied notably according to location. GluR2 messenger RNA was expressed most strongly and widely, with most areas except the Bergmann glia containing this messenger RNA. GluR4 messenger RNA was also present widely, although the expression level was low. However, we observed many areas which lacked or expressed very little GluR1 messenger RNA, such as some nuclei in the general motor system and auditory system. In addition, some nuclei in the hypothalamus and general somatosensory system lacked or expressed very little GluR3 messenger RNA. These results suggest that in the rat CNS non-N-methyl-D-aspartate receptors varied their composition according to the area where they were expressed, and that the combination pattern might be related to the functional role of neurons.     

Molecular cloning and characterization of Drosophila genes encoding small GTPases of the rab and rho families

We have isolated eight genes from Drosophila, small GTPases. They can be classified into three rab family genes (Drab2, Drab5, Drab11) and five rho family genes (Drac1a, Drac1b, Drac3, Dcdc42, DrhoA). While Drac3 is a novel type of rac gene, others are homologues of known mammalian genes for small GTPases. Northern blot analyses showed that all the genes are expressed throughout all developmental stages from embryo to adult. In situ hybridization to embryos revealed that Drab2, Drac1b, and Drac3 are highly expressed in the nervous system, in the trunk mesoderm, and in the cephalic mesoderm, respectively. Since hemocytes are derived from the cephalic mesoderm, we carried out double stainings using a hemocyte marker anti-peroxidasin antibody and Drac3 in situ hybridization. We found that Drac3 is expressed in hemocyte precursor cells. In the Drac3 deficiency embryos, the hemocyte precursor cells start to differentiate normally, but never develop into mature hemocytes, indicating that Drac3 is essential for their maturation. The DrhoA and Dcdc42 genes complemented S. cerevisiae rho1 and cdc42 mutations in the same manner as human rhoA and CDC42, respectively. These results suggest functional similarity between Drosophila and mammalian small GTPase genes.     

Expression of the CB1 and CB2 receptor messenger RNAs during embryonic development in the rat

We mapped the distribution of CB1 and CB2 receptor messenger RNAs in the developing rat to gain insight into how cannabinoids may affect embryogenesis. In situ hybridization histochemistry studies were done using riboprobes specific for CB1 or CB2 receptor messenger RNAs. We found that CB1 and CB2 receptor messenger RNAs are expressed in the placental cone and in the smooth muscle of the maternal uterus at the earliest gestational periods studied [from eight days of gestation (E8) through E12]. In the embryo, as early as E11, CB1 receptor messenger RNA is expressed in some cells of the neural tube and, at later embryological stages (from E15 to E21), in several distinct structures within the central nervous system. In addition, high levels of CB1 receptor messenger RNA were also found in areas of the peripheral nervous system such as the sympathetic and parasympathetic ganglia, in the retina and in the enteric ganglia of the gastrointestinal tract. In addition to neural structures, high levels of the CB1 receptor messenger RNA were also present in two endocrine organs, the thyroid gland and the adrenal gland. On the other hand, CB2 receptor messenger RNA is expressed exclusively in the liver of the embryo as early as E13. The region-specific expression of CB1 and CB2 receptor messenger RNAs suggests that these receptors have a functional role during embryogenesis.     

Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brain of children with AIDS

The neuropeptide Y Y2 receptor is one of six receptor subtypes mediating the multiform physiological actions of neuropeptide Y. The Y2 receptor has been demonstrated to be the most predominant receptor subtype in the human brain and appears to be involved in many neuropeptide Y actions, such as the regulation of locomotor activity, cardiovascular functions, memory processing, circadian rhythms and release of other neurotransmitters. We have recently demonstrated the widespread and abundant distribution of neuropeptide Y Y1 receptor messenger RNA in the human cerebral cortex (different laminar patterns within distinct cortical regions), hippocampal dentate gyrus and striatum. To assess a possible differential distribution of Y1 and Y2 receptor messenger RNAs, the regional expression of neuropeptide Y Y2 messenger RNA-containing cells in the human brain was analysed, in particular within the cerebral cortex and striatum. In situ hybridization experiments revealed the localization of the Y2 messenger RNA signal throughout all cortical regions, with the highest intensity per cell apparent in lamina IV, with the exception of the striate cortex, which showed an intense labelling primarily in layer VI. The striatum expressed low to undetectable levels of the Y2 receptor messenger RNA. The dentate gyrus and the CA2 region presented the highest hybridization signals, while a very weak Y2 messenger RNA expression was found in the CA1 region and subiculum. Positive Y2 messenger RNA hybridization signals were also detected in the lateral geniculate nucleus, amygdala, substantia nigra, hypothalamus, cerebellum and choroid plexus. These results demonstrate the widespread distribution of neuropeptide Y Y2 receptor messenger RNA in the human brain, with a pattern of expression distinct from the Y1 subtype, suggesting that these two receptor subtypes may mediate different neuropeptide Y functions in the human brain, mainly through actions on different neuronal systems.     

Increased expression of trkB and trkC messenger RNAs in the rat forebrain after focal mechanical injury

Tyrosine protein kinases trkA, trkB and trkC are signal transduction receptors for a family of neurotrophic factors known as the neurotrophins. Here we report on changes in the expression of messenger RNAs for trkA, trkB and trkC in the brain following an injury caused by insertion of a 30-gauge needle into adult rat hippocampus or neocortex. Quantitative in situ hybridization revealed no change in the level of trkA messenger RNAs in any brain region following this insult. In contrast, increased levels of trkB messenger RNA compared to untreated animals were seen in the granule cell layer of the dentate gyrus ipsilateral to the injury already 30 min after the injury. The increase reached maximal levels (four-fold) between 2 and 4 h, but returned to control levels 8 h after the injury. No change was seen in the contralateral dentate gyrus. The levels of trkC messenger RNA increased in the same brain regions as trkB messenger RNA, though with a delayed response, reaching a maximal increase of 3.3-fold 4 h after the injury. As for trkB messenger RNA, the level of trkC messenger RNA then tapered off and reached control levels 8 h after the injury. However, 4 h after the injury, a 1.7-fold increase of trkB and trkC messenger RNAs were seen in the ipsilateral piriform cortex. The increases of trkB and trkC messenger RNAs were confirmed using a nuclease protection assay. Increases of both trkB and trkC messenger RNAs were also seen in the piriform cortex, but not in the hippocampus, following needle insertion into the neocortex. Pretreatment of the animals with the non-competitive N-methyl-D-aspartate antagonist ketamine completely prevented the increases of trkB and trkC messenger RNAs, suggesting that the brain injury caused a release of glutamate with subsequent activation of N-methyl-D-aspartate receptors. In contrast, the anticonvulsive drug diazepam, the muscarinic antagonist atropine and the calcium-channel antagonist nimodipine had no effect on the increases of trkB and trkC messenger RNAs. Combined with previous data on the expression of neurotrophin messenger RNAs following similar injuries, our results support the hypothesis that increased levels of neurotrophins and their receptors could protect against neuronal damage following a brain insult.     

A single point mutation decreases picrotoxinin sensitivity of the human GABA receptor rho 1 subunit

The GABA receptor rho subunits are thought to form bicuculline-insensitive and picrotoxinin-sensitive GABAC receptors. We have investigated the role of the amino acid at position 309 in transmembrane segment M2 of the human rho 1 subunit as a determinant for picrotoxinin sensitivity. The mutant rho 1P309S was constructed by exchanging proline 309 for serine, the corresponding amino acid of the human rho 2 subunit. Whole-cell recordings from HEK-293 cells transfected with rho 1P309S cDNA revealed that the sensitivity of the rho 1P309S channels for picrotoxinin was four-fold lower than that of the wild type rho 1 subunit. The affinity of the mutant receptor for GABA was only slightly changed. These results provide direct evidence that the amino acid at position 309 is an important determinant for the picrotoxinin sensitivity of GABA receptors formed by the rho subunits.     

Complementary and overlapping expression of Y1, Y2 and Y5 receptors in the developing and adult mouse nervous system

Neuropeptide Y, a 36 amino acid peptide, mediates its biological effects by activating the Y1, Y2, Y5 and Y6 receptors, which are also receptors for the structurally related peptide YY. Different classes of receptors have been suggested to be involved in different neuropeptide Y functions. In this report, we have characterized the developmental regulation and compared the cellular localization of these receptors in the developing and in the adult central and peripheral nervous systems of the mouse. RNase protection assays revealed that Y1, Y2 and Y5 messenger RNAs were expressed very early in spinal cord, brain, cerebellum and dorsal root ganglion development and were often down-regulated at times corresponding to their acquirement of the adult function in neurotransmission. In situ hybridization of the adult brain showed that Y1 was widely expressed, Y2 displayed a more restricted pattern, Y5 was expressed at very low levels and only in a few brain nuclei and Y6 was not expressed. Virtually all areas containing neurons positive for Y5 also expressed Y1, whereas many Y1-positive cells clearly did not express Y5. In contrast, Y2 was not expressed by the neurons expressing Y1 or Y5. These findings suggest that neuropeptide Y signaling in the brain could be mediated by simultaneous Y1 and Y5 activation. Similar results were also obtained in peripheral sensory neurons. Furthermore, our results suggest that neuropeptide Y/peptide YY receptors play an important role in nervous system development and that selective receptor combinations are responsible for signaling the different effects of neuropeptide Y in the peripheral and central nervous systems.     

Reduction in enkephalin and substance P messenger RNA in the striatum of early grade Huntington's disease: a detailed cellular in situ hybridization study

The expression of enkephalin and substance P messenger RNAs was examined in the caudate-putamen of human post mortem tissue from control and Huntington's disease tissue using in situ hybridization techniques and human specific enkephalin and substance P [35S] oligonucleotides. Macroscopic and microscopic quantification of enkephalin and substance P gene expression was carried out using computer-assisted image analysis. Tissue was collected from six control cases with no sign of neurological disease and six Huntington's disease cases ranging from grades 0 to 3 as determined by neuropathological evaluation. The clinical and pathological diagnosis of Huntington's disease was confirmed unequivocally by genetic analysis of the CAG repeat length in both copies of IT15, the Huntington's disease gene. A marked reduction in both enkephalin and substance P messenger RNAs was detected in all regions of the caudate nucleus and putamen in Huntington's disease grades 2/3 when compared to controls; in the dorsal caudate few enkephalin or substance P messenger RNA-positive cells were detected. For the early grade (0/1) Huntington's disease cases, a heterogeneous reduction in both enkephalin and substance P messenger RNAs were noted; for enkephalin messenger RNA the striatal autoradiograms displayed a conspicuous patchy appearance. Detailed cellular analysis of the dorsal caudate revealed a striking reduction in the number of enkephalin and substance P messenger RNA-positive cells detected and in the intensity of hybridization signal/cell. These data suggest that both the "indirect" GABA/enkephalin and "direct" GABA/substance P pathways are perturbed very early in the course of the disease and that these early changes in chemical signalling may possibly underlie the onset of clinical symptoms.     

Distribution of somatostatin receptor messenger RNAs in the rat gastrointestinal tract

BACKGROUND & AIMS: The gastrointestinal (GI) tract is a major source and target of somatostatin (SRIF). Recently, five pharmacologically different SRIF receptors (sst1-5) were cloned. The cellular and tissue distribution of the sst1-5 messenger RNAs (mRNAs) were studied in the rat GI tract using in situ hybridization histochemistry (ISHH). METHODS: Two sets of (35)S-uridine triphosphate (UTP)-labeled antisense and sense riboprobes were prepared for each sst. ISHH was conducted on frozen tissue samples from rat stomach, duodenum, jejunum, ileum, colon, and pancreas. RESULTS: mRNAs of all five sst-s are widely expressed in the rat GI tract. The distribution pattern for each sst mRNA was identical with both antisense probes. No specific signal was found with any of the sense probes. Each layer of the different parts of the gut expressed mRNAs of multiple sst subtypes. All organs expressed sst3 mRNA very intensely. The lowest levels of mRNA expression for all five subtypes within the GI tract were found in the pancreas. CONCLUSIONS: The widespread expression of sst mRNAs suggests a significant role for SRIF in the regulation of GI function.     

Detailed mapping of serotonin 5-HT1B and 5-HT1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

The similar pharmacology of the 5-HT1B and 5-HT1D receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [35S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [3H]alniditan). The anatomical patterns of 5-HT1B and 5-HT1D receptor messenger RNA were quite different. While 5-HT1B receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT1D receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT(1B/1D) binding sites (combined) obtained with [3H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT1B receptor labelling in the presence of ketanserin under conditions to occlude 5-HT1D receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT1B and 5-HT1D receptors.     

Galanin messenger RNA during postnatal development of the rat brain: expression patterns in Purkinje cells differentiate anterior and posterior lobes of cerebellum

Following our initial mapping of preprogalanin messenger RNA in adult brain and its presence in a subpopulation of cerebellar Purkinje neurons [Ryan M. C. and Gundlach A. C. (1996) Neuroscience 70, 709-728], the present study examined the ontogenic expression of preprogalanin messenger RNA in the postnatal rat brain focussing on the Purkinje cells of the cerebellar cortex. Using in situ hybridization histochemistry, preprogalanin messenger RNA was detected in the developing forebrain and hindbrain from postnatal day 4 to day 60 (adult). On postnatal day 4 very light hybridization signal (labelling) was observed in cells of a number of nuclei including the central amygdaloid nucleus, the medial preoptic area, paraventricular nucleus and dorsomedial hypothalamic nucleus of the forebrain while lightly-labelled cells were detected in neurons of the nucleus of the solitary tract and locus coeruleus of the hindbrain. Hybridization signal was not apparent in other nuclei until later, with positively-labelled neurons first apparent in the dorsal cochlear nucleus at postnatal day 21. The abundance of preprogalanin messenger RNA-positive neurons and the intensity of the hybridization signal increased, in most regions, until postnatal day 28 when labelling resembled that of the mature rat. Preprogalanin messenger RNA was first detected in the cerebellum on postnatal day 10 only in Purkinje cells of lobule 10 of the posterior vermis and increased in distribution throughout Purkinje cell layers of the entire cerebellar cortex by postnatal day 13. The intensity of hybridization signal in Purkinje cells varied between lobules, with Purkinje cells in lobule 10 displaying a moderate to heavy degree of labelling, while lobules 6-9 and the more posterior lobules of the hemisphere including crus 2 of the ansiform lobule, the paramedian lobule and the copula pyramis, displayed only light labelling. The intensity of labelling in the anterior vermis and the remaining lobules of the hemisphere including crus 1 of the ansiform lobule, the simple lobule, the paraflocculus and the flocculus, was homogeneously weak. By postnatal day 21, Purkinje cell labelling reached maximum intensity in all lobules. Regional differences were still apparent, however, with labelling in the posterior vermis and hemisphere ranging from moderate to heavy, with only light to moderate labelling detected in the anterior vermis. The intensity of labelling in the posterior vermis and most lobules of the hemisphere was similar from postnatal day 21 to adulthood, while, in the anterior vermis, crus 1 of the ansiform lobule and the simple lobule, the intensity of hybridization decreased slightly by postnatal day 28 and was completely absent in Purkinje cells of the adult rat. Differential expression of preprogalanin messenger RNA in Purkinje cells of the developing rat cerebellum and transient expression in certain lobules suggests that galanin gene products may have a role in both the developing and mature rat brain and that galanin gene expression may represent a useful marker for differentiating the anterior and posterior cerebellar lobes.