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Hybridization Histochemistry (hybridization + histochemistry)
Kinds of Hybridization Histochemistry Selected AbstractsGlutamatergic neurons are present in the rat ventral tegmental areaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2007Tsuyoshi Yamaguchi Abstract The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or ,-amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, we have proposed that additional cell types, such as those that express the neurotransmitter glutamate may also be present in this area. Thus, by using in situ hybridization histochemistry we investigated whether transcripts encoded by genes for the two vesicular glutamate transporters, VGluT1 or VGluT2, were expressed in the VTA. We found that VGluT2 mRNA but not VGluT1 mRNA is expressed in the VTA. Neurons expressing VGluT2 mRNA were differentially distributed throughout the rostro-caudal and medio-lateral aspects of the VTA, with the highest concentration detected in rostro-medial areas. Phenotypic characterization with double in situ hybridization of these neurons indicated that they rarely co,expressed mRNAs for tyrosine hydroxylase (TH, marker for DAergic neurons) or glutamic acid decarboxylase (GAD, marker for GABAergic neurons). Based on the results described here, we concluded that the VTA contains glutamatergic neurons that in their vast majority are clearly non-DAergic and non-GABAergic. [source] Dopaminergic regulation of orexin neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2005Michael Bubser Abstract Orexin/hypocretin neurons in the lateral hypothalamus and adjacent perifornical area (LH/PFA) innervate midbrain dopamine (DA) neurons that project to corticolimbic sites and subserve psychostimulant-induced locomotor activity. However, it is not known whether dopamine neurons in turn regulate the activity of orexin cells. We examined the ability of dopamine agonists to activate orexin neurons in the rat, as reflected by induction of Fos. The mixed dopamine agonist apomorphine increased Fos expression in orexin cells, with a greater effect on orexin neurons located medial to the fornix. Both the selective D1-like agonist, A-77636, and the D2-like agonist, quinpirole, also induced Fos in orexin cells, suggesting that stimulation of either receptor subtype is sufficient to activate orexin neurons. Consistent with this finding, combined SCH 23390 (D1 antagonist),haloperidol (D2 antagonist) pretreatment blocked apomorphine-induced activation of medial as well as lateral orexin neurons; in contrast, pretreatment with either the D1-like or D2-like antagonists alone did not attenuate apomorphine-induced activation of medial orexin cells. In situ hybridization histochemistry revealed that LH/PFA cells rarely express mRNAs encoding dopamine receptors, suggesting that orexin cells are transsynaptically activated by apomorphine. We therefore lesioned the nucleus accumbens, a site known to regulate orexin cells, but this treatment did not alter apomorphine-elicited activation of medial or lateral orexin neurons. Interestingly, apomorphine failed to activate orexin cells in isoflurane-anaesthetized animals. These data suggest that apomorphine-induced arousal but not accumbens-mediated hyperactivity is required for dopamine to transsynaptically activate orexin neurons. [source] Differential galanin receptor-1 and galanin expression by 5-HT neurons in dorsal raphé nucleus of rat and mouse: evidence for species-dependent modulation of serotonin transmissionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2003Jari A. Larm Abstract Galanin and galanin receptors are widely expressed by neurons in rat brain that either synthesize/release and/or are responsive to, classical transmitters such as ,-aminobutyric acid, acetylcholine, noradrenaline, histamine, dopamine and serotonin (5-hydroxytryptamine, 5-HT). The dorsal raphé nucleus (DRN) contains , 50% of the 5-HT neurons in the rat brain and a high percentage of these cells coexpress galanin and are responsive to exogenous galanin in vitro. However, the precise identity of the galanin receptor(s) present on these 5-HT neurons has not been previously established. Thus, the current study used a polyclonal antibody for the galanin receptor-1 (GalR1) to examine the possible expression of this receptor within the DRN of the rat and for comparative purposes also in the mouse. In the rat, intense GalR1-immunoreactivity (IR) was detected in a substantial population of 5-HT-immunoreactive neurons in the DRN, with prominent receptor immunostaining associated with soma and proximal dendrites. GalR1-IR was also observed in many cells within the adjacent median raphé nucleus. In mouse DRN, neurons exhibited similar levels and distribution of 5-HT-IR to that in the rat, but GalR1-IR was undetectable. Consistent with this, galanin and GalR1 mRNA were also undetectable in mouse DRN by in situ hybridization histochemistry, despite the detection of GalR1 mRNA (and GalR1-IR) in adjacent cells in the periaqueductal grey and other midbrain areas. 5-HT neuron activity in the DRN is primarily regulated via 5-HT1A autoreceptors, via inhibition of adenylate cyclase and activation of inward-rectifying K+ channels. Notably, the GalR1 receptor subtype signals via identical mechanisms and our findings establish that galanin modulates 5-HT neuron activity in the DRN of the rat via GalR1 (auto)receptors. However, these studies also identify important species differences in the relationship between midbrain galanin and 5-HT systems, which should prompt further investigations in relation to comparative human neurochemistry and which have implications for studies of animal models of relevant neurological conditions such as stress, anxiety and depression. [source] Cellular and behavioural effects of the adenosine A2a receptor antagonist KW-6002 in a rat model of l -DOPA-induced dyskinesiaJOURNAL OF NEUROCHEMISTRY, Issue 6 2003M. Lundblad Abstract We have examined the ability of KW-6002, an adenosine A2a antagonist, to modulate the dyskinetic effects of l -DOPA in 6-hydroxydopamine-lesioned rats. In animals rendered dyskinetic by a previous course of l -DOPA treatment, KW-6002 did not elicit any abnormal involuntary movements on its own, but failed to reduce the severity of dyskinesia when coadministered with l -DOPA. A second experiment was undertaken in order to study the effects of KW-6002 in l -DOPA-naive rats. Thirty-five animals were allotted to four groups to receive a 21-day treatment with: (i) KW-6002 (10 mg/kg/day); (ii) l -DOPA (6 mg/kg/day) i.p.; (iii) KW-6002 plus l -DOPA (same doses as above) or (iv) vehicle. Chronic treatment with KW-6002-only produced a significant relief of motor disability in the rotarod test in the absence of any abnormal involuntary movements. Combined treatment with l -DOPA and KW-6002 improved rotarod performance to a significantly higher degree than did each of the two drugs alone. However, this combined treatment induced dyskinesia to about the same degree as did l -DOPA alone. In situ hybridization histochemistry showed that KW-6002 treatment alone caused an approximately 20% reduction in the striatal levels of preproenkephalin mRNA, whereas neither the coadministration of KW-6002 and l -DOPA nor l -DOPA alone significantly altered the expression of this transcript in the dopamine-denervated striatum. Either alone or in combination with l -DOPA, KW-6002 did not have any modulatory effect on prodynorphin mRNA expression or FosB/,FosB-like immunoreactivity in the dopamine-denervated striatum. These results show that monotreatment with an adenosine A2a receptor antagonist can relieve motor disability without inducing behavioural and cellular signs of dyskinesia in rats with 6-hydroxydopamine lesions. Cotreatment with KW-6002 and l -DOPA potentiates the therapeutic effect but not the dyskinesiogenic potential of the latter drug. [source] Ageing and the Diurnal Expression of the mRNAs for Vasopressin and for the V1a and V1b Vasopressin Receptors in the Suprachiasmatic Nucleus of Male RatsJOURNAL OF NEUROENDOCRINOLOGY, Issue 6 2004T. Kalamatianos Abstract Changes in the function of neuropeptide synthesizing cells within the suprachiasmatic nucleus (SCN), the site of the predominant circadian pacemaker, may underlie the disturbance of rhythms observed during ageing. Arginine vasopressin (AVP) is synthesized by nearly one-third of SCN neurones in the rat. This peptide has predominantly excitatory actions within the SCN mediated by V1 -type receptors; the extent to which the V1a and/or V1b receptor subtypes are involved in SCN functions remains to be determined. The present study used isotopic in situ hybridization histochemistry to examine the effects of ageing on expression of mRNAs for AVP and V1a in the SCN and for V1b in the SCN and supraoptic nucleus (SON) of male rats kept under a 12 : 12 h light/dark cycle. Analysis of film autoradiographs from young adult (2,3-month-old; n = 40) or aged (19,20-month-old; n = 40) animals, at eight time points across the light/dark cycle, revealed an equivalent pattern and amplitude for the diurnal rhythm of AVP mRNA in the SCN of the young adult and aged groups. Both groups also displayed a significant diurnal rhythm in the expression of V1a receptor mRNA; however, the amplitude of this rhythm was reduced in the aged group, due to increased levels during the light phase and early part of night. Although the expression of V1b mRNA did not display a significant diurnal rhythm within the SCN or SON, persistently elevated levels for V1b mRNA were observed in the aged group at both sites. [source] Gene expression in peripheral arterial chemoreceptorsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2002Estelle B. Gauda Abstract The peripheral arterial chemoreceptors of the carotid body participate in the ventilatory responses to hypoxia and hypercapnia, the arousal responses to asphyxial apnea, and the acclimatization to high altitude. In response to an excitatory stimuli, glomus cells in the carotid body depolarize, their intracellular calcium levels rise, and neurotransmitters are released from them. Neurotransmitters then bind to autoreceptors on glomus cells and postsynaptic receptors on chemoafferents of the carotid sinus nerve. Binding to inhibitory or excitatory receptors on chemoafferents control the electrical activity of the carotid sinus nerve, which provides the input to respiratory-related brainstem nuclei. We and others have used gene expression in the carotid body as a tool to determine what neurotransmitters mediate the response of peripheral arterial chemoreceptors to excitatory stimuli, specifically hypoxia. Data from physiological studies support the involvement of numerous putative neurotransmitters in hypoxic chemosensitivity. This article reviews how in situ hybridization histochemistry and other cellular localization techniques confirm, refute, or expand what is known about the role of dopamine, norepinephrine, substance P, acetylcholine, adenosine, and ATP in chemotransmission. In spite of some species differences, review of the available data support that 1) dopamine and norepinephrine are synthesized and released from glomus cells in all species and play an inhibitory role in hypoxic chemosensitivity; 2) substance P and acetylcholine are not synthesized in glomus cells of most species but may be made and released from nerve fibers innervating the carotid body in essentially all species; 3) adenosine and ATP are ubiquitous molecules that most likely play an excitatory role in hypoxic chemosensitivity. Microsc. Res. Tech. 59:153,167, 2002. © 2002 Wiley-Liss, Inc. [source] Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibiansMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2001Laurent Yon Abstract Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide of the secretin/glucagon/vasoactive intestinal polypeptide superfamily, has been initially characterized in mammals in 1989 and, only 2 years later, its counterpart has been isolated in amphibians. A number of studies conducted in the frog Rana ridibunda have demonstrated that PACAP is widely distributed in the central nervous system (particularly in the hypothalamus and the median eminence) and in peripheral organs including the adrenal gland. The cDNAs encoding the PACAP precursor and 3 types of PACAP receptors have been cloned in amphibians and their distribution has been determined by in situ hybridization histochemistry. Ontogenetic studies have revealed that PACAP is expresssed early in the brain of tadpoles, soon after hatching. In the frog Rana ridibunda, PACAP exerts a large array of biological effects in the brain, pituitary, adrenal gland, and ovary, suggesting that, in amphibians as in mammals, PACAP may act as neurotrophic factor, a neurotransmitter and a neurohormone. Microsc. Res. Tech. 54:137,157, 2001. © 2001 Wiley-Liss, Inc. [source] Distribution of corticotropin-releasing hormone in the developing zebrafish brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 4 2007Gayathri Chandrasekar Abstract Corticotropin-releasing hormone (CRH) plays a central role in the physiological regulation of the hypothalamus-pituitary-adrenal/interrenal axis mediating endocrine, behavioral, autonomic, and immune responses to stress. Despite the wealth of knowledge about the physiological roles of CRH, the genetic mechanisms by which CRH neurons arise during development are poorly understood. As a first step toward analyzing the molecular and genetic pathways involved in CRH lineage specification, we describe the developmental distribution of CRH neurons in the embryonic zebrafish, a model organism for functional genomics and developmental biology. We searched available zebrafish expressed sequence tag (EST) databases for CRH-like sequences and identified one EST that contained the complete zebrafish CRH open reading frame (ORF). The CRH precursor sequence contained a signal peptide, the CRH peptide, and a cryptic peptide with a conserved sequence motif. RT-PCR analysis showed crh expression in a wide range of adult tissues as well as during embryonic and larval stages. By whole-mount in situ hybridization histochemistry, discrete crh -expressing cell clusters were found in different parts of the embryonic zebrafish brain, including telencephalon, preoptic region, hypothalamus, posterior tuberculum, thalamus, epiphysis, midbrain tegmentum, and rostral hindbrain and in the neural retina. The localization of crh mRNA within the preoptic region is consistent with the central role of CRH in the teleost stress response through activation of the hypothalamic-pituitary-interrenal axis. The widespread distribution of CRH-synthesizing cells outside the preoptic region suggests additional functions of CRH in the embryonic zebrafish brain. J. Comp. Neurol. 505:337,351, 2007. © 2007 Wiley-Liss, Inc. [source] Localization of the mRNA encoding prolyl endopeptidase in the rat brain and pituitaryTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2004Gaelle Bellemère Abstract Prolyl endopeptidase (EC 3.4.21.26, PEP), a serine protease that hydrolyzes peptides at the carboxyl side of proline residues, is involved in the breakdown of several proline-containing neuropeptides and, thus, may contribute to the regulation of behavioral activities. In this study, the distribution of PEP mRNA was investigated in the central nervous system and pituitary of rat by means of quantitative reverse transcriptase-polymerase chain reaction analysis and in situ hybridization histochemistry. High densities of PEP transcripts were found in cerebellar Purkinje and granule cells, within most hypothalamic nuclei, in pyramidal neurons of the Ammon's horn, in granule cells of the dentate gyrus, and within the basolateral complex of the amygdala. Moderate levels of PEP mRNA were observed in layers 3,5 of the cerebral cortex, the anterior thalamic group, the septal region, the substantia nigra, the magnocellular neurons of the red nucleus, and the motor nuclei of the cranial nerves. Low concentrations of PEP mRNA were detected in the deep mesencephalic nuclei, the reticular formation, the pretectum, and the tectum. A high density of PEP mRNA was found in the intermediate and the anterior lobes of the pituitary, while the neural lobe was devoid of labeling. In several brain regions, the distribution pattern of PEP mRNA overlapped that of various neuropeptide receptors, suggesting that PEP is actually involved in the inactivation of regulatory neuropeptides. J. Comp. Neurol. 471:128,143, 2004. © 2004 Wiley-Liss, Inc. [source] Cell type- and region-specific expression of protein kinase C-substrate mRNAs in the cerebellum of the macaque monkeyTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2003Noriyuki Higo Abstract We performed nonradioactive in situ hybridization histochemistry in the monkey cerebellum to investigate the localization of protein kinase C-substrate (growth-associated protein-43 [GAP-43], myristoylated alanine-rich C-kinase substrate [MARCKS], and neurogranin) mRNAs. Hybridization signals for GAP-43 mRNA were observed in the molecular and granule cell layers of both infant and adult cerebellar cortices. Signals for MARCKS mRNA were observed in the molecular, Purkinje cell, and granule cell layers of both infant and adult cortices. Moreover, both GAP-43 and MARCKS mRNAs were expressed in the external granule cell layer of the infant cortex. In the adult cerebellar vermis, signals for both GAP-43 and MARCKS mRNAs were more intense in lobules I, IX, and X than in the remaining lobules. In the adult hemisphere, both mRNAs were more intense in the flocculus and the dorsal paraflocculus than in other lobules. Such lobule-specific expressions were not prominent in the infant cerebellar cortex. Signals for neurogranin, a postsynaptic substrate for protein kinase C, were weak or not detectable in any regions of either the infant or adult cerebellar cortex. The prominent signals for MARCKS mRNA were observed in the deep cerebellar nuclei, but signals for both GAP-43 and neurogranin mRNAs were weak or not detectable. The prominent signals for both GAP-43 and MARCKS mRNAs were observed in the inferior olive, but signals for neurogranin were weak or not detectable. The cell type- and region-specific expression of GAP-43 and MARCKS mRNAs in the cerebellum may be related to functional specialization regarding plasticity in each type of cell and each region of the cerebellum. J. Comp. Neurol. 467:135,149, 2003. © 2003 Wiley-Liss, Inc. [source] Characterization of CART neurons in the rat and human hypothalamusTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 1 2001Carol F. Elias Cocaine- and amphetamine-regulated transcript (CART) is a recently described neuropeptide widely expressed in the rat brain. CART mRNA and peptides are found in hypothalamic sites such as the paraventricular nucleus (PVH), the supraoptic nucleus (SON), the lateral hypothalamic area (LHA), the dorsomedial nucleus of the hypothalamus (DMH), the arcuate nucleus (Arc), the periventricular nucleus (Pe), and the ventral premammillary nucleus (PMV). Intracerebroventricular administration of recombinant CART peptide decreases food intake and CART mRNA levels in the Arc are regulated by leptin. Leptin administration induces Fos expression in hypothalamic CART neurons in the PVH, the DMH, the Arc, and the PMV. In the current study, we used double label in situ hybridization histochemistry to investigate the potential direct action of leptin on hypothalamic CART neurons and to define the chemical identity of the hypothalamic CART neurons in the rat brain. We found that CART neurons in the Arc, DMH, and PMV express long form leptin-receptor mRNA, and the suppressor of cytokine signaling-3 (SOCS-3) mRNA after an acute dose of intravenous leptin. We also found that CART neurons in the parvicellular PVH, in the DMH and in the posterior Pe coexpress thyrotropin-releasing hormone (TRH) mRNA. CART neurons in the magnocellular PVH and in the SON coexpress dynorphin (DYN), and CART cell bodies in the LHA and in the posterior Pe coexpress melanin-concentrating hormone (MCH) and glutamic acid decarboxylase (GAD-67) mRNA. In the Arc, a few CART neurons coexpress neurotensin (NT) mRNA. In addition, we examined the distribution of CART immunoreactivity in the human hypothalamus. We found CART cell bodies in the PVH, in the SON, in the LHA, in the Arc (infundibular nucleus) and in the DMH. We also observed CART fibers throughout the hypothalamus, in the bed nucleus of the stria terminalis, and in the amygdala. Our results indicate that leptin directly acts on CART neurons in distinct nuclei of the rat hypothalamus. Furthermore, hypothalamic CART neurons coexpress neuropeptides involved in energy homeostasis, including MCH, TRH, DYN, and NT. The distribution of CART cell bodies and fibers in the human hypothalamus indicates that CART may also play a role in the regulation of energy homeostasis in humans. J. Comp. Neurol. 432:1,19, 2001. © 2001 Wiley-Liss, Inc. [source] Decreased GAD65 mRNA levels in select subpopulations of neurons in the cerebellar dentate nuclei in autism: an in situ hybridization studyAUTISM RESEARCH, Issue 1 2009Jane Yip Abstract The laterally positioned dentate nuclei lie in a key position in the cerebellum to receive input from Purkinje cells in the lateral cerebellar hemisphere participating in both motor and cognitive functions. Although neuropathology of the four cerebellar nuclei using Nissl staining has been qualitatively reported in children and adults with autism, surprisingly the dentate nuclei appeared less affected despite reported reductions in Purkinje cells in the posterolateral cerebellar hemisphere. To determine any underlying abnormalities in the critically important GABAergic system, the rate-limiting GABA synthesizing enzyme, glutamic acid decarboxylase (GAD) type 65 was measured via in situ hybridization histochemistry in dentate somata. GAD65 mRNA labeling revealed two distinct subpopulations of neurons in adult control and autism postmortem brains: small-sized cells (about 10,12,µm in diameter, presumed interneurons) and larger-sized neurons (about 18,20,µm in diameter, likely feedback to inferior olivary neurons). A mean 51% reduction in GAD65 mRNA levels was found in the larger labeled cells in the autistic group compared with the control group (P=0.009; independent t -test) but not in the smaller cell subpopulation. This suggests a disturbance in the intrinsic cerebellar circuitry in the autism group potentially interfering with the synchronous firing of inferior olivary neurons, and the timing of Purkinje cell firing and inputs to the dentate nuclei. Disturbances in critical neural substrates within these key circuits could disrupt afferents to motor and/or cognitive cerebral association areas in the autistic brain likely contributing to the marked behavioral consequences characteristic of autism. 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