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We have demonstrated the coexistence of GABA-like and tyrosine hydroxylase-like immunoreactivities (GABA-LI and TH-LI, respectively) in the same neurons of the rat locus ceruleus (LC). The profiles of these cells were labeled by alternately immunostaining adjacent sections for GABA-LI or TH-LI by the avidin-biotin-peroxidase complex method or the peroxidase-anti-peroxidase method after perfusion (either Zamboni's fixative or PPG), and observation at light and electron microscopic levels. For light microscopy, pairs of adjacent sections of more than 590 (Zamboni's) and 260 (PPG), and for electron microscopy, 40 ultrathin sections cut from adjacent semithin plastic sections (Zamboni's), were examined. GABA-LI was found in 80% (1,309/1,642 in total) of small and medium-sized neurons, uniformly scattered throughout the LC. Observations unequivocally show that the majority of GABA-ergic neurons are also noradrenergic. Several neurons are neither noradrenergic nor GABA-ergic, while other noradrenergic neurons do not show GABA-LI. It is shown that astrocytes, but not oligodendrocytes, contain GABA. In situ hybridization using a probe DNA fragment of the glutamic acid decarboxylase (GAD) cDNA, amplified by the polymerase chain reaction, detected GAD mRNA signals in many neurons throughout the LC, supporting the presence of a GAD/GABA system in the LC. Multiple "classical" transmitters, including GABA, serotonin, and noradrenaline, coexist in many LC neurons and may contribute to its widely diverging projections throughout the entire CNS.
Anat Rec 1992 Dec
PMID:Immunocytochemical and in situ hybridization evidence for the coexistence of GABA and tyrosine hydroxylase in the rat locus ceruleus. 136 Jul 72

The present immunocytochemical study used an antiserum to tyrosine hydroxylase (TH), the first enzyme in the biosynthetic pathway of catecholamines, and revealed TH immunoreactivity in the ganglion cells and in the varicose nerve fibers of the cortex and medulla in the rat adrenal gland. TH immunoreactive nerve fibers in the cortex and medulla contained large and small granular vesicles, and also small clear vesicles. The immunoreactive nerve fibers were in close apposition to cortical cells in the cortex and in apposition to smooth muscle cells of blood vessels in both the cortex and medulla. Furthermore, TH immunoreactive nerve fibers were sometimes in close apposition to pericytes of blood vessels in the cortex and chromaffin cells in the medulla. The present results suggest that the catecholaminergic nerve fibers in the rat adrenal gland may be both intrinsic and extrinsic in origin.
Anat Rec 1991 Mar
PMID:Immunoelectron microscopic study of tyrosine hydroxylase immunoreactive nerve fibers and ganglion cells in the rat adrenal gland. 167 23

Spontaneous dwarf mice, in which both growth hormone (GH) and prolactin (PRL) are undetectable, are severely deficient in the PRL-inhibiting catecholamine dopamine (DA), as well as its synthetic enzyme, tyrosine hydroxylase (TH), in the basal hypothalamus (Phelps et al., Cell Tissue Res., 240:19-25, 1985; Phelps, Brain Res., 416:354-358, 1987). In contrast, transgenically constructed dwarf mice (Behringer et al., Genes Dev., 2:453-461, 1988) show complete ablation of pituitary GH cells, but PRL cells are retained at a level of approximately 10% of normal. In order to determine the feedback effect of this reduced, rather than absent, PRL on hypothalamic DA neurons, brains of transgenic dwarf mice were examined for catecholamine transmitters by histofluorescence, for the synthetic enzyme TH by immunocytochemistry, and for TH mRNA expression by in situ hybridization. DA histofluorescence in transgenic dwarfs was comparable to that of normal littermate mice in nonpituitary regulating areas (perikarya of zona incerta [A13] of hypothalamus and in midbrain substantia nigra area [A9]). Arcuate nucleus (A12) DA neurons that inhibit PRL secretion, however, showed dim to absent fluorescence in perikarya and in external median eminence terminals in dwarfs. There were reduced (P less than 0.05) numbers of A12 TH-immunoreactive neurons in transgenic dwarfs, to approximately 60% of those in normal mice. In contrast, TH-positive neurons in other hypothalamic areas (A13, A14) had average populations equivalent to those in normal mice. Quantification of TH mRNA abundance by in situ hybridization using both image analysis of hybridization over the arcuate nucleus, and grain counts per individual A12 cell in this nucleus, indicated that relative mRNA levels were the same in normal and transgenic dwarfs. The observations indicate that reduction in pituitary PRL is accompanied by defective expression in hypothalamic tuberoinfundibular neurons, which is severe at the DA neurotransmitter level, significant regarding observable TH immunoreactivity, and undetectable with regard to TH mRNA expression. Collectively, the findings suggest that posttranscriptional processes are involved with the mediation of PRL feedback upon hypothalamic neurons. Technically and quantitatively, the report presents the feasibility of simultaneous evaluation of transmitter histofluorescence, synthetic enzyme immunocytochemistry, and mRNA expression in individual animals.
Anat Rec 1991 Dec
PMID:Hypothalamic dopaminergic neurons in transgenic dwarf mice: histofluorescence, immunocytochemical, and in situ hybridization studies. 168 35

The study of the deep pineal gland of the Mongolian gerbil and other neuronal tissue from the rat by means of confocal laser scanning microscopy (CLSM) is described. Opical serial sectioning was performed on thick (100-200 microns) sections of the deep pineal gland of the Mongolian gerbil stained immunohistochemically using antisera to S-antigen and tyrosine hydroxylase (TH). Both dual-stained and single-stained material was examined using the fluorochromes fluorescein isothiocyanate (FITC) and Texas Red. High resolution images were obtained showing that pinealocytes have 1-3 processes that extend primarily to other pinealocytes or presumptive pinealocytes. Pinealocytes are located within the deep pineal gland as well as adjacent to the posterior aspect of the medial habenular nuclei. Pinealocyte processes were not seen extending into the habenular nuclei, but rather ended within the deep pineal gland a significant distance from their perikarya. The TH-immunopositive fibers were distributed throughout the deep pineal gland, often forming "baskets" of fibers around pinealocytes rather than being associated primarily with blood vessels. Other uses of the confocal microscope are demonstrated on rat neural tissue reacted with peroxidase/diaminobenzidine (DAB) immunohistochemistry and FITC fluorescence immunohistochemistry (paraventricular nucleus) as well as Golgi-stained neuronal tissue (cerebral cortex). The HRP/DAB and Golgi-stained images were visualized using the reflected image mode of the confocal system.(ABSTRACT TRUNCATED AT 250 WORDS)
Anat Rec 1991 Dec
PMID:Application of confocal laser scanning microscopy to the deep pineal gland and other neural tissues. 168 36

We used immunocytochemical localization of tyrosine hydroxylase to trace the ontogenesis and anatomic distribution of sympathetic innervation in fetal, neonatal, and mature canine hearts. Sparse tyrosine hydroxylase-positive neural tissue first appeared in the atrium, including sinoatrial and atrioventricular nodes, and the ventricular epicardium at midgestation and progressively increased in extent to reach the adult pattern by 2 months following birth. Sympathetic innervation of the atrioventricular bundle occurred relatively later, with no nerve processes in the neonate but a mature pattern by 2 months. At each developmental stage the atria contained more tyrosine hydroxylase-positive neural tissue than the ventricles. Thus, sympathetic nerve processes appear in the developing canine heart earlier than was previously recognized. The time course of sympathetic innervation as defined by this anatomic study is in accord with electrophysiologic studies indicating progressive neonatal development of sympathetic effect which achieves maturity by 2 months of age.
Anat Rec 1990 Jan
PMID:Anatomic distribution of autonomic neural tissue in the developing dog heart: I. Sympathetic innervation. 196 14

Recent electrophysiological studies of neurons of the myenteric plexus of the corpus of the guinea pig stomach have revealed that slow synaptic events are extremely rare. In contrast, they are commonly encountered in similar investigations of myenteric ganglia of the guinea pig small intestine. The current immunocytochemical analysis of the myenteric plexus and innervation of the muscularis externa of the corpus of the guinea pig stomach was undertaken in order to determine whether putative neurotransmitters capable of mediating slow synaptic events are present in gastric ganglia. A major difference between the small intestine and the stomach was found in the innervation of the musculature. Whereas the longitudinal muscle layer of the small intestine contains very few nerve fibers and is innervated mainly at its interface with the myenteric plexus, the longitudinal muscle of the corpus of the stomach contained as many varicose substance P (SP)-, vasocative intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive axons as the circular muscle layer. These putative neurotransmitters were also present in the ganglia of the myenteric plexus, where varicose SP-, VIP-, and NPY-immunoreactive fibers encircled nonimmunoreactive neurons. Varicose 5-hydroxytryptamine (5-HT)-immunoreactive terminal axons were essentially limited to the myenteric plexus and were found both in ganglia and in interganglionic connectives, where they were particularly numerous; 5-HT-immunoreactive neurons appeared to be more abundant in the stomach than in the small intestine. Tyrosine hydroxylase (TH)- and calcitonin-gene-related-peptide (CGRP)-immunoreactive axons were also more common in the myenteric plexus than in the musculature, but of these, only the TH-immunoreactive neurites tended, like those of the other putative transmitters, to encircle neurons in myenteric ganglia. Evidence was obtained that, as in the small intestine, at least some of the SP-, VIP-, NPY-, and 5-HT-immunoreactive fibers in the stomach are derived from intrinsic gastric myenteric neurons. In contrast, unlike the small intestine, gastric myenteric ganglia appeared to lack intrinsic CGRP-immunoreactive neurons; therefore, the CGRP-immunoreactive gastric axons are probably of extrinsic origin.(ABSTRACT TRUNCATED AT 400 WORDS)
Anat Rec 1989 Jul
PMID:Immunocytochemical analysis of potential neurotransmitters present in the myenteric plexus and muscular layers of the corpus of the guinea pig stomach. 247 50

The present work examines the ability of prolactin to enter the CNS of the rat and effect its feedback stimulation of dopamine release prior to the appearance of prolactin receptors in choroid plexus (i.e., 10 days postnatal). An inhibitor of tyrosine hydroxylase was used to allow the assessment of dopamine turnover separate from synthesis and transport of the amine. Chronic but not acute hyperprolactinemia resulted in increased dopamine release relative to vehicle-treated controls, as shown by diminished fluorescence intensity in the median eminence. These results indicate that activation of the prolactin short-loop feedback system occurs by 10 days postnatal, prior to the appearance of prolactin receptors at the choroid plexus.
Anat Rec 1987 Jan
PMID:Prolactin causes increased turnover of dopamine in 10-day-old rat median eminence. 345 64

Recent physiological evidence indicates that vagal postganglionic control of left ventricular contractility is mediated by neurons found in a ventricular epicardial fat pad ganglion. In the dog this region has been referred to as the cranial medial ventricular (CMV) ganglion [J.L. Ardell, Structure and function of mammalian intrinsic cardiac neurons, in: J.A. Armour, J.L. Ardell (Eds.). Neurocardiology, Oxford Univ. Press, New York, 1994, pp. 95-114; B.X. Yuan, J.L. Ardell, D.A. Hopkins, A.M. Losier, J.A. Armour, Gross and microscopic anatomy of the canine intrinsic cardiac nervous system, Anat. Rec., 239 (1994) 75-87]. Since activation of the vagal neuronal input to the CMV ganglion reduces left ventricular contractility without influencing cardiac rate or AV conduction, this ganglion contains a functionally selective pool of negative inotropic parasympathetic postganglionic neurons. In the present report we have defined the light microscopic distribution of preganglionic negative inotropic neurons in the CNS which are retrogradely labeled from the CMV ganglion. Some tissues were also processed for the simultaneous immunocytochemical visualization of tyrosine hydroxylase (TH: a marker for catecholaminergic neurons) and examined with both light microscopic and electron microscopic methods. Histochemically visualized neurons were observed in a long slender column in the ventrolateral nucleus ambiguus (NA-VL). The greatest number of retrogradely labeled neurons were observed just rostral to the level of the area postrema. TH perikarya and dendrites were commonly observed interspersed with vagal motoneurons in the NA-VL. TH nerve terminals formed axo-dendritic synapses upon negative inotropic vagal motoneurons, however the origin of these terminals remains to be determined. We conclude that synaptic interactions exist which would permit the parasympathetic preganglionic vagal control of left ventricular contractility to be modulated monosynaptically by catecholaminergic afferents to the NA-VL.
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PMID:Neural control of left ventricular contractility in the dog heart: synaptic interactions of negative inotropic vagal preganglionic neurons in the nucleus ambiguus with tyrosine hydroxylase immunoreactive terminals. 974 80

The accessory olfactory bulb (AOB) is a primary center of the vomeronasal system. In the dog, the position and morphology of the AOB remained vague for a long time. Recently, the morphological characteristics of the dog AOB were demonstrated by means of lectin-histochemical, histological, and immunohistochemical staining, although the distribution of each kind of neuron, especially granule cells, remains controversial in the dog AOB. In the present study, we examined the distribution of neuronal elements in the dog AOB by means of immunohistochemical and enzyme-histochemical staining. Horizontal paraffin or frozen sections of the dog AOB were immunostained with antisera against protein gene product 9.5 (PGP 9.5), brain nitric oxide synthase (NOS), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), substance P (SP), and vasoactive intestinal polypeptide (VIP) by avidin-biotin peroxidase complex method. In addition, frozen sections were stained enzyme-histochemically for NADPH-diaphorase. In the dog AOB, vomeronasal nerve fibers, glomeruli, and mitral/tufted cells were PGP 9.5-immunopositive. Mitral/tufted cells were observed in the glomerular layer (GL) and the neuronal cell layer (NCL). In the NCL, a small number of NOS-, GAD-, and SP-immunopositive and NADPH-diaphorase positive granule cells were observed. In the GL, GAD-, TH-, and VIP-immunopositive periglomerular cells were observed. In the GL and the NCL, TH-, and VIP-immunopositive short axon cells were also observed. In addition to these neurons, TH- and SP-immunopositive afferent fibers were observed in the GL and the NCL. We could distinctly demonstrate the distribution of neuronal elements in the dog AOB. Since only a small number of granule cells were present in the dog AOB, the dog AOB did not display such a well-developed GCL as observed in the other mammals.
Anat Rec 1998 11
PMID:Immunohistochemical and enzyme-histochemical study on the accessory olfactory bulb of the dog. 981 Dec 17

In the chick heart, sympathetic innervation is derived from the sympathetic neural crest (trunk neural crest arising from somite level 10-20). Since the trunk neural crest gives rise to sympathetic ganglia of their corresponding level, it suggests that the sympathetic neural crest develops into cervical ganglia 4-14. We therefore tested the hypothesis that, in addition to the first thoracic ganglia, the cervical ganglia might contribute to cardiac innervation as well. Putative sympathetic nerve connections between the cervical ganglia and the heart were demonstrated using the differentiation markers tyrosine hydroxylase and HNK-1. In addition, heterospecific transplantation (quail to chick) of the cardiac and trunk neural crest was used to study the relation between the sympathetic neural crest and the cervical ganglia. Quail cells were visualized using the quail nuclear antibody QCPN. The results by immunohistochemical study show that the superior and the middle cervical ganglia and possibly the carotid paraganglia contribute to the carotid nerve. This nerve subsequently joins the nodose ganglion of the vagal nerve via which it contributes to nerve fibers in cardiac vagal branches entering the arterial and venous pole of the heart. In addition, the carotid nerve contributes to nerve fibers connected to putative baro- and chemoreceptors in and near the wall of pharyngeal arch arteries suggesting a role of the superior and middle cervical ganglia and the paraganglia of the carotid plexus in sensory afferent innervation. The lower cervical ganglia 13 and 14 contribute predominantly to nerve branches entering the venous pole via the anterior cardinal veins. We did not observe a thoracic contribution. Heterospecific transplantation shows that the cervical ganglia 4-14 as well as the carotid paraganglia are derived from the sympathetic neural crest. The cardiac neural crest does not contribute to the neurons of the cervical ganglia. We conclude that the cervical ganglia contribute to cardiac innervation which explains the contribution of the sympathetic neural crest to the innervation of the chick heart.
Anat Rec 1999 08 01
PMID:Contribution of the cervical sympathetic ganglia to the innervation of the pharyngeal arch arteries and the heart in the chick embryo. 1040 14


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