GTP-cyclohydrolase-I like immunoreactivity in rat brain

GTPCH-I immunoreactive structures in the rat brain were studied using a polyclonal antibody raised in the chick. General mapping was made using the avidin–biotin–peroxidase technique and compared with the distribution of tyrosine hydroxylase and serotonin immunoreactivities. Double immunofluorescence was performed in order to establish real intracellular colocalization. GTPCH-I immunoreactivity was generally found to be low. Immunostained neurons were present in all the serotonin cell groups. In catecholaminergic neurons, although tyrosine hydroxylase immunoreactivity was always very high, GTPCH-I immunoreactivity was extremely variable, from relatively strong (substantia nigra, ventral tegmental area) to low (locus coeruleus, caudal part of the hypothalamus), extremely low (rostral hypothalamus, ventral brainstem) or almost absent (dorsal brainstem, some hypothalamic nuclei). When feasible, double immunolabeling revealed that all the serotonin cells and most of the tyrosine hydroxylase cells were also expressing GTPCH-I. Our results argue in favor of a regulation of tyrosine hydroxylase activity by the intracellular synthesis of BH4.     

Dopamine transporter immunoreactivity in rat brain

The dopamine transporter (DAT) is a primary site for the action of cocaine in inducing euphoria. Its action is necessary for the selectivities of dopaminergic neurotoxins that provide the best current experimental models of Parkinson's disease. In the present report, rat ddpamine transporter-like immunoreactivity (iDAT) was assessed by immunohistochemistry using newly developed polyclonal antisera raised against conjugated peptides corresponding to sequences found in the dopainine transporter's carboxy- and amino-termini. Dense iDAT was observed in patterns consistent with neural processes and terminals in the striatum, nucleus accumbens, olfactory tubercle, nigrostriatal bundle, and lateral habenula. Perikarya in the substantia nigra pars compacts, were immunostained with moderate intensity using one of two immunohistochemical methods, while scattered ventral tegmental area perikarya were stained with somewhat less intensity. Immunoreactive neuronal processes with axonal and dendritic morphologies were stained in the substantia nigra and the paranigral and parabrachialis pigmentosus nuclei of the ventral tegmental area, while sparser processes were noted more medially in the ventral tegmental area. Neuronal processes were found in several laminae in the cingulate cortex, with notable fiber densities in the superficial aspects of lamina I and laminae II/III. The intensities of immunoreactivities in striatum and cerebral cortex were dramatically attenuated ipsilateral to nigrostriatal bundle 6-hydroxydopamine lesions. Specificity of immunostaining was supported by agreement of the results using sera directed against two distinct DAT segments, studies with preimmune and preadsorbed sera and studies of the extracted protein. These antisera identify and reveal details of the distribution of DAT immunoreactivity in rat brain and display ivariations in levels of DAT expression of likely functional significance. © 1995 Wiley-Liss, Inc.     

NGF receptor immunoreactivity in aged rat brain

The cellular distribution of nerve growth factor (NGF) receptor (NGFR) immunoreactivity in 3 cholinergic nuclei (medial septal nucleus, nucleus of the diagonal band and nucleus basalis magnocellularis) of the aged rat brain was compared to that of young-adult animals. In young-adult rats, NGFR immunoreactivity was strong in the neuronal body and in the whole dendritic tree. In aged animals, NGFR immunoreactivity was weak in both cell body and dendrites and was practically absent in the dendrite's distal portion. The loss of dendritic NGFR may play a critical role in the decline of neuronal function in the aging brain.     

Distribution of prosaposin-like immunoreactivity in rat brain

Prosaposin is the precursor for saposins A, B, C, and D, which are small lysosomal proteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. With a monospecific anti-saposin C antibody, which cross-reacts with prosaposin but not with saposin A, B, or D, the present immunoblot experiments showed that the rat brain expresses an unprocessed ～ 72 kDa protein (possibly prosaposin) and little saposin C. Regional analysis demonstrated that prosaposin is abundant in the brainstem, hypothalamus, cerebellum, striatum, and hippocampus, and less abundant in the cerebral cortex. Consistent with this finding, prosaposin-like immunoreactive neurons and fibers as revealed by immunohistochemistry were observed frequently in subcortical regions. The medial septum, diagonal bands, basal nucleus of Meynert, ventral striatum, medial habenular nucleus, and motor nuclei of cranial nerve had significant numbers of immunoreactive neurons. There were also nerve fibers with prosaposin-like immunoreactivity in several projection fields of the above nuclei. Other brain areas that contained prosaposin-like immunoreactive neurons and/or processes were: several brain nuclei (nucleus caudate putamen, globus pallidus, substantia nigra, red nucleus) constituting the so-called extrapyramidal system, reticular thalamic nucleus, entopeduncular nucleus, mammillary nuclei, auditory relay nuclei, cerebellum, sensory cranial nerve nuclei, and the reticular formation. The distribution pattern of prosaposin is apparently different from that of other neuroactive substances so far examined, and thus prosaposin may be involved in novel central events. © 1993 Wiley-Liss, Inc.     

DSIP-like immunoreactivity in the developing rat brain

Delta sleep-inducing peptide (DSIP) is a naturally occurring nonapeptide that has been reported to affect sleep. The concentration of DSIP-like material was measured by a highly specific radioimmunoassay in brain tissue from developing rats. DSIP-like immunoreactivity was present in fetal brain at increased levels shortly before birth. In one experiment, a significant elevation in the concentration of the peptide occurred at postnatal day 20, but in a second, similar, study the increase occurred later. No marked differences in the levels of DSIP-like immunoreactivity were found in the brains of rats injected 2 months earlier with DSIP or among 10 parts of brains obtained at postnatal day 20. Regardless of the function of DSIP, it is available to the rat throughout early development.     

Immunohistochemical studies of FMRF-amide-like immunoreactivity in rat brain

The anatomical distribution of neuronal perikarya and nerve fibres containing FMRF-amide-like immunoreactivity in the brain, spinal cord and pituitary of the rat has been studied by immunohistochemistry. In animals pretreated with colchicine, the highest concentration of nerve cell bodies occurred in hypothalamic nuclei. Cells were also present in the cortex, striatum, septum, thalamus and in the brainstem. Beaded nerve fibres were abundant in the septum, nucleus of the striae terminalis, hypothalamus, medial regions of the thalamus, the parabrachial nucleus, the ventrolateral medulla, the substantia gelatinosa of the spinal trigeminal nucleus and the dorsal horn of the spinal cord. Fibres were also present in the cortex, striatum, amygdala, pons, ventral spinal cord and the neural lobe of the pituitary. The localization was specific in that preabsorbtion of the antisera with FMRF-amide, but not structurally related molecules such as Met-Enk-Arg6Phe7, APP or BPP, completely abolished the localization. The mammalian counterparts of FMRF-amide may have a neurotransmitter or neuromodulatory role.     

Abdominal surgery induces Fos immunoreactivity in the rat brain

Previous neuropharmacological studies indicate that brain peptides are involved in mediating gastric stasis induced by abdominal surgery. Central pathways activated by abdominal surgery were investigated in the rat by using Fos protein as a marker of neuronal activation. Abdominal surgery (laparotomy alone or combined with cecal manipulation) was performed under brief enflurance anesthesia (7–8 minutes), and 1 hour later rats were killed and brains processed for Fos immunoreactivity. Double labeling with Fos and arginine vasopressin, oxytocin, or tyrosine hydroxylase antibodies was also performed. Abdominal surgery induced Fos staining in the nucleus tractus solitarii, paraventricular and supraoptic nuclei of the hypothalamus, locus coeruleus, and ventrolateral medulla. After abdominal surgery, 18–25% of vasopressin and 18–33% of oxytocin-labeled cells were found to be Fos positive in the paraventricular nucleus and 15% of activated cells in the nucleus tractus solitarii were positive for tyrosine hydroxylase immunoreactivity. Enflurane alone induced c-fos expression in the same brain area; however, the number of Fos-positive cells and double-labeled cells were decreased two-to fivefold and three- to eightfold, respectively, compared with the abdominal surgery groups. These data show that abdominal surgery induced activation of specific hypothalamic, pontine, and medullary neurons. These findings may have implications for the understanding of central mechanisms involved in mediating gastric ileus following abdominal surgery. © 1994 Wiley-Liss, Inc.     

Lipocortin 1 immunoreactivity identifies microglia in adult rat brain

Immunohistochemical localization of two Ca⁺⁺-binding proteins, Lipocortin 1 (LC1) and S100-β, demonstrates two distinct classes of primitive glia in the floor plate of rat embryos. With proper fixation (formalin-lysine-periodate-acetic acid), dendritic glia in the CNS of adult rats also apparently stain for either LC1 or S100-β in the ratio of 1:3. In order to further distinguish and identify these two glial classes, we have examined their population density, topography, and responses to localized neuron death. Neurons of the ipsilateral thalamus undergo apoptosis following cortical ablation; the contralateral thalamus serves as control. By eight days post-lesion, the number of LC1 cells in the ipsilateral thalamus has increased >4-fold, the increase comprising primarily activated phagocytes adjacent to degenerating neurons. The S100-β glia in the same region are virtual- ly indistinguishable from control; but background staining (apparently representing extracellular S100-β) is increased. Thus, the responses of dendritic LC1 glia resemble those previously described for microglia and are quite different from the astrocytes identified by S100-β immunoreactivity. Both dendritic and activated forms of LC1 glia stain with the microglial marker, Griffonia simplicifolia iso-lectin B₄. However, before the correspondence of LC1 glia and microglia can be confirmed, two anomalies require resolution: (1) the LC1 glia are greater in number and more evenly distributed than microglia marked with other methods; (2) the dendritic LC1 glia apparently are progeny of primitive glia that form the midline raphe of the embryonic floor plate. The participation of LC1 glia in the removal of CNS debris supports the hypothesis that LC1 plays anti-inflammatory and/or immunosuppressive roles in phagocytes. © 1993 Wiley-Liss, Inc.     

Glial immunoreactivity for metallothionein in the rat brain.

A series of frozen and vibratome coronal sections of the rat brain were examined by immunocytochemistry for the presence of a cysteine-rich metal binding protein, metallothionein (MT). Astrocytes throughout the brain and brainstem stained positively for MT; neurons and oligodendroglia were unstained. Ependymal cells and tanycyte processes in the hypothalamus were also immunoreactive, along with a narrow zone of immunopositivity along the margins of the area postrema. Gomori-positive astrocytes in the hypothalamus, identifiable by toluidine blue staining, metal-containing cytoplasmic granules, represented a subset of MT-positive astrocytes that may be involved in reactions to blood-borne metal compounds that penetrate into circumventricular organs of the brain.     

Diazepam and buspirone alter neuropeptide Y-like immunoreactivity in rat brain

Neuropeptide Y-like immunoreactivity (NPY-LI) was investigated in naIve Sprague-Dawley rats subjected to acute, subchronic (7 days) or chronic (21 days) intraperitoneal treatment with diazepam (1 or 3 mg/kg once daily) or buspirone (1.5 or 5 mg/kg twice daily). NPY-LI was determined by radioimmunoassay in the amygdala, nucleus accumbens, hypothalamus and frontal cortex 24 h after the last dose of the drugs. Amygdala NPY-LI decreased after acute diazepam (3 mg/kg) or buspirone (1.5 mg/kg) and increased after subchronic treatment with both doses of diazepam and after chronic buspirone (1.5 mg/kg) treatment. Both diazepam and buspirone given in subchronic and chronic doses decreased NPY-LI levels in the nucleus accumbens. Hypothalamic NPY-LI changed only after chronic treatment: it decreased after diazepam and increased after buspirone (5 mg/kg). NPY-LI content in the frontal cortex decreased after subchronic diazepam (3 mg/kg) treatment and slightly increased after buspirone. The study has shown that both diazepam and buspirone affect NPY-LI levels in rats. These results suggest that the NPY system in the amygdala and nucleus accumbens is implicated in the anxiolytic effects of the drugs studied.     

Distribution and immunohistochemical characterization of torsinA immunoreactivity in rat brain

A mutation of the DYT1 gene on chromosome 9q34 has recently been identified as the cause of one form of autosomal-dominantly inherited dystonia. TorsinA, the protein product of this gene, has homology with the family of heat shock proteins, and is found in many peripheral tissues and brain regions. We used a polyclonal antibody to torsinA, developed in our laboratory, to systematically examine the regional distribution of torsinA in rat brain. We find that neurons in all examined structures are immunoreactive for this protein. There is intense immunoreactivity in most neuronal nuclei, with slightly less labeling of cytoplasm and proximal processes. Terminals also are labeled, especially in striatum, neocortex and hippocampus. Double-labeling fluorescence immunohistochemistry using antibodies to neurotransmitters and other neurochemical markers demonstrated that the majority of neurons of all studied neurochemical types are immunoreactive for torsinA. Our findings indicate that torsinA is widely distributed in the central nervous system implicating additional, localized factors, perhaps within the basal ganglia, in the development of dystonia. Many other proteins have a similar widespread distribution, including some which have been implicated in other movement disorders and neurodegenerative processes, such as parkin, -synuclein, ubiquitin and huntingtin. The distribution of torsinA in rat brain as demonstrated by immunohistochemistry contrasts with the results of in situ hybridization studies of torsinA mRNA in human postmortem brain in which a more limited distribution was found.     

Cathepsin B immunoreactivity is widely distributed in the rat brain

The cellular localization and regional distribution of cathepsin B within rat CNS was revealed by immunohistochemistry using a monospecific antiserum. Cathepsin B protein was found to be widely but unevenly distributed throughout rat brain. Neurons were always cathepsin B immunoreactive. Glial elements were only occasionally immunostained. The distribution of the enzyme resembles largely that of cathepsin D.     

Presynaptic NMDA receptor subunit immunoreactivity in GABAergic terminals in rat brain

N-methyl-D-aspartate (NMDA) receptors are commonly found post-synaptically; they mediate fast excitatory neurotransmission in the central nervous system. In this study, we provide immunocytochemical data supporting the existence of presynaptic NMDA receptors in GABAergic terminals using polyclonal antisera raised against the C-terminus of the NMDAR1 subunit. At the light microscope level, rich plexuses of NMDAR1-positive varicose fibers were found in various nuclei in the basal forebrain (bed nucleus of stria terminalis, septum, parastrial nucleus, vascular organ of the lamina terminalis), thalamus (paraventricular nucleus, midline nuclei), and hypothalamus (parvocellular paraventricular nucleus, arcuate nucleus, preoptic nucleus, suprachiasmatic nucleus). In the brainstem, labeled fibers were much less abundant and were confined to the ventral tegmental area, periaqueductal gray, parabrachial nucleus, and locus coeruleus. At the electron microscope level, NMDAR1-immunoreactive terminals examined in the bed nucleus of stria terminalis, parvocellular paraventricular hypothalamic nucleus, and arcuate nucleus formed symmetric synapses, contained darkly stained large dense-core vesicles, and displayed gamma-aminobutyric acid (GABA) immunoreactivity. Terminals with similar ultrastructural features were found in the paraventricular thalamic nucleus. These findings demonstrate the existence of NMDAR1 subunit immunoreactivity in subsets of GABAergic terminals, which raises questions about the potential roles and mechanisms of activation of presynaptic NMDA heteroreceptors in the rat central nervous system. The pattern of distribution and ultrastructural features of these boutons suggest that they may arise from local GABAergic projections interconnecting a group of brain structures mediating stress responses and/or other endocrine, autonomic, and limbic functions.     

Distribution of NPY Y5-like immunoreactivity in the rat brain

The pharmacology and brain mRNA distribution of the neuropeptide Y (NPY) rat Y5 (rY5) receptor has led to the hypothesis that this receptor might mediate the hypothalamic feeding response to NPY in addition to many other physiologic functions. However, through the use of autoradiographic techniques, only very low levels of Y5-like immunoreactive (Y5-ir) binding are detected in the rat brain. To localize the Y5 protein in the rat brain, polyclonal antibodies were raised to the carboxyl terminus of the rY5 receptor. The resulting antisera were affinity purified and characterized by specific binding to HEK293 cells that had been stably transfected with the rY5 receptor. Utilizing immunohistochemical techniques, we found a discrete pattern of Y5-ir in the rat brain. In initial studies, very low levels of Y5-ir were detected, and TSA amplification was required to visualize the staining. Areas with the highest levels of expression in clude the piriform cortex, supraoptic nucleus, and hippocampus. Areas with moderate levels of expression include the lateral septum, amygdala, arcuate nucleus, paraventricular hypothalamic nucleus, locus coeruleus, and cerebellum. With several exceptions, this pattern of distribution is consistent with earlier reports of rY5 mRNA and receptor protein expression.     

Distribution of metabotropic glutamate receptor mGluR5 immunoreactivity in rat brain

The receptor mGluR5 is a metabotropic glutamate receptor with messenger RNA abundantly present throughout cortex, hippocampus, and caudate/putamen that is also coupled to phosphatidyl inositide hydrolysis and calcium mobilization. In this study, the distribution of mGluR5 was examined in rat brain by immunocytochemistry. The antibody utilized is highly specific and does not cross react with the most closely related other metabotropic glutamate receptor, as determined by Western blot analysis of nonneuronal cells transfected with metabotropic receptor coding sequences. The receptor mGluR5 is widely expressed with the highest density in olfactory bulb, caudate/putamen, lateral septum, cortex, and hippocampus, as confirmed with both immunocytochemistry and Western blot analysis. Electron microscopic studies in hippocampus and cortex indicate that the labeling is mostly on membranes of dendritic spines and shafts. Light and electron microscopic evidence indicates that some mGluR5 immunoreactivity is located in presynaptic axon terminals, suggesting that mGluR5 may function as a presynaptic receptor.