Autoradiographic localization of thyrotropin-releasing hormone receptors in the rat central nervous system

We employed quantitative autoradiography to examine the distribution of thyrotropin-releasing hormone (TRH) receptors in the rat CNS. The binding of [3H]3-methyl-histidine-TRH [( 3H]MeTRH) to TRH receptors in frozen rat brain sections was saturable, of a high affinity (Kd = 5 nM), and specific for TRH analogs. Autoradiograms of [3H]MeTRH binding showed highest concentrations of TRH receptors in the rhinencephalon, including accessory olfactory bulb, nuclei of the amygdala, and the ventral dentate gyrus and subiculum of the hippocampus. Moderate TRH receptor concentrations were found within the thalamus and hypothalamus, in most regions of the rhombencephalon, such as the cranial nerve nuclei, and in the substantia gelatinosa of the spinal cord. Neocortex and basal ganglia contained low densities of TRH receptors. This distribution correlates well with the sensitivity of brain regions to the known effects of TRH, and suggests that TRH receptors may mediate the actions of TRH in the rat CNS.     

Immunochemical and immunohistochemical localization of Bcl-x protein in the rat central nervous system

To explore the role of bcl-x in the regulation of cell death in the nervous system, we produced monoclonal antibodies against rat Bcl-x_L protein, the major product of the rat bcl-x gene that inhibits apoptosis, and defined its distribution in rat neural tissues by immunochemical and immunohistochemical means. Western blotting of tissue homogenates identified the Bcl-x protein as two bands with molecular weights of about 29 and 31 kDa. The level of Bcl-x expression in the nervous system was high, being comparable to that in the hematolymphoid system, and higher in the fetal than in the adult brain. Subcellular fractionation studies localized Bcl-x to various subcellular compartments. In tissue culture, Bcl-x was produced by all the cell types examined, including neurons, astrocytes, oligodendrocytes and microglial cells. Immunohistochemistry revealed that Bcl-x immunoreactivity was more intense in the gray than in the white matter. In the fetal cerebral cortex, labeling was mostly confined to the neuronal perikarya, whereas in the more mature brain, the neuropil of the gray matter, as well as the glial cells in the white matter, was also stained.     

Pituitary adenylate cyclase-activating peptide in the rat central nervous system: an immunohistochemical and in situ hybridization study

In the present study the localization of pituitary adenylate cyclase-activating peptide (PACAP)-expressing cell bodies and PACAP projections were mapped in the adult rat brain and spinal cord by using immunohistochemistry and in situ hybridization histochemistry. A widespread occurrence of PACAP-containing cell bodies was found, with the greatest accumulation in several hypothalamic nuclei and in several brainstem nuclei, especially the habenular nuclei, the pontine nucleus, the lateral parabrachial nucleus (LPB), and the vagal complex. PACAP was also present in cell bodies in the olfactory areas, in neocortical areas, in the hippocampus, in the vestibulo- and cochlear nuclei, in cell bodies of the intermediolateral cell column of the spinal cord and in Purkinje cells of the cerebellum, in the subfornical organ, and in the organum vasculosum of the lamina terminalis. An intense accumulation of PACAP-immunoreactive (-IR) nerve fibers was observed throughout the hypothalamus, in the amydaloid and extended amygdaloid complex, in the anterior and paraventricular thalamic nuclei, in the intergeniculate leaflet, in the pretectum, and in several brainstem nuclei, such as the parabrachial nucleus, the sensory trigeminal nucleus, and the nucleus of the solitary tract. PACAP-IR nerve fibers were also found in the area postrema, the posterior pituitary and the choroid plexus, and the dorsal and ventral horn of the spinal cord. The widespread distribution of PACAP in the brain and spinal cord suggests that PACAP is involved in the control of many autonomic and sensory functions as well as higher cortical processes.     

A clinicopathological and immunohistochemical study of central nervous system hemangiopericytomas

Hemangiopericytomas (HPC) of the central nervous system (CNS) are uncommon dural-based tumors that mimic meningiomas clinically and radiologically. Because there are few reports about these tumors from India, we aimed to study the clinico-pathological and immunohistochemical features of CNS HPC. During 2000 to 2008 all 23 patients diagnosed with HPC of CNS at our Institution were reviewed in the study (11 males and 12 females, mean age of 46 years). Clinical, radiological and histopathological features were reviewed. There were 14 patients with grade II and nine with grade III tumors. Immunohistochemistry with antibodies to epithelial membrane antigen (EMA), vimentin, S-100, CD34 and Ki-67 was done on routinely processed, paraffin-embedded sections of 20 tumors. All patients were EMA and S-100 negative, and vimentin positive. CD34 was positive in only five out of 20 patients. The mean Ki-67 labeling index was 4.25% in grade II tumors and 7.8% in grade III tumors. We concluded that HPC are distinct from meningiomas in morphology, immunohistochemistry and biologic behavior, although they resemble each other clinically and radiologically, HPCs need to be differentiated from meningiomas.     

Fatty acid amide hydrolase localization in the human central nervous system: an immunohistochemical study

Recent discoveries have opened new fields for research on the biochemistry and pharmacology of cannabinoids. Among them, and most importantly, are the characterization and molecular cloning of central and peripheral cannabinoid receptors as well as the isolation of the first putative endogenous ligands that bind to them, anandamide and 2-arachidonylglycerol. The enzyme that degrades these so-called "endocannabinoids" is an integral membrane protein, fatty acid amide hydrolase. Its distribution and biochemistry in rat brain suggest that it plays a critical role in the regulation of the endocannabinoid system. However, few data exist regarding its distribution and mechanism of action in human tissues. To that end, we have studied its cellular distribution in the human central nervous system by immunohistochemistry. Using an affinity-purified antibody, we report that fatty acid amide hydrolase is localized to specific and well-delimited cell populations, including cortical pyramidal neurons, subcortical white matter astrocytes, striatal and striatoefferent projecting neurons, hypothalamic and midbrain nuclei, granular and molecular layers of the cerebellum, Purkinje neurons, dentate cerebellar nucleus, inferior olivary nuclei and others. This distribution resembles that of the central cannabinoid receptors as well as that of the enzyme distribution in the rat brain. In summary, the cellular localization of the degradative enzyme of the endogenous cannabinoid ligands in human central nervous system reveals its presence on both neuronal and glial elements and shows a significant overlapping with that of central cannabinoid receptors, mainly in areas related with motor control, confirming the notion that the endocannabinoid system plays a critical role in the control of movement.     

Immunohistochemical localization of aspartoacylase in the rat central nervous system

Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cell-specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (approximately 37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotin-peroxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.     

中枢神经系统肠源性囊肿的形态及免疫组化研究

目的：观察中枢神经系统肠源性囊肿的组织形态及免疫组织化学特征。方法：使用病理组织学及免疫组织化学方法对３１例中枢神经系统不同部位的肠源性囊肿进行研究。结果：囊肿壁上皮可为单层或复层扁平、立方、柱状上皮；一些病例可见两种以上上皮组织成分，并相互移行；某些病例，在囊肿壁内可含有黑色素、软骨、平滑肌、粘液腺等多种组织成分。免疫组织化学染色ＥＭＡ均呈阳性；ＣＥＡ染色仅一例阴性，而此例ＮＳＥ阳性，故应诊断为神经上皮性囊肿；Ｓ－１００和ＣＫ染色结果不固定；Ｖｉ－ｍｅｎｔｉｎ和ＧＦＡＰ均呈阴性。结论：肠源性囊肿的组织形态和免疫组织化学特点可作为鉴别其他类型囊肿的依据。     

中枢神经系统神经节细胞胶质瘤组织学及免疫组织化学研究

目的：研究中枢神经系统神经节细胞胶质瘤的组织成分、组织发生、生物学特性及与临床的关系。方法：对２８例神经节细胞胶质瘤进行组织学及免疫组织化学观察。结果：神经细胞大小不等、形态多样、核大、核仁明显，９例可见双核；胶质细胞多为星形细胞，６例含有少枝胶质细胞成分，９例胶质细胞分化不良；血管增生扩张、血管周围淋巴细胞浸润、出血、坏死、囊性变常见。突触素２７例阳性，嗜酪粒蛋白Ａ２３例阳性，神经丝蛋白２５例阳性，神经特异性烯醇化酶、Ｓ－１００、胶质纤维酸性蛋白全部阳性。结论：肿瘤分化与年龄、病程无关；缺乏双核神经元不影响诊断；该肿瘤可能具有神经内分泌功能；本文对该肿瘤的组织发生进行了探讨。     

Immunocytological localization of pedal peptide in the central nervous system and periphery of Aplysia

Immunocytology using antisera raised to conjugated pedal peptide (Pep) was used to localize the peptide in the CNS and periphery of Aplysia. A total of over 200 neurons in the CNS exhibited Pep-like immunoreactivity. As expected from results presented in the previous paper, immunoreactive neurons were heavily concentrated in the pedal ganglia, primarily in a broad ribbon comprised of about 60 large contiguous neurons on the dorsal side of each ganglion. Smaller and less numerous immunoreactive neurons were found in the other ganglia. A number of neurons primarily located in the abdominal ganglia had dense networks of immunoreactive varicose fibers surrounding their cell bodies. Many immunoreactive axons were observed in peripheral nerves, particularly those nerves leaving the pedal ganglia. Analyses of sections of body wall indicated that Pep-like immunoreactivity was localized to a series of varicose axons that appeared to be associated with vascular spaces, muscle fibers, and other large cells. These axons likely arise from pedal ganglion nerves that were shown to transport large amounts of 35S-labeled Pep to the periphery. These results suggest that Pep is a transmitter-like neuropeptide that is likely to have a number of important physiological actions in Aplysia.     

Evidence for proctolin-like substances in the central nervous system of the leech Hirudo medicinalis

The distribution of proctolin-like immunoreactive (PLI) cells was mapped in the central nervous system of the leech Hirudo medicinalis. In segmental ganglia, PLI cells can be divided into two groups: cells that stain repeatedly in every successive ganglion, and cells that stain only in specific segmental ganglia. The number of PLI cells, therefore, ranged from eight to 20 cells per ganglion. One bilateral pair of PLI cells (cells PLI-1) was further characterized morphologically by Lucifer yellow and horseradish peroxidase cell injections. Cell PLI-1 conforms in soma position, morphology, and physiological properties with cell 101, which has been previously classified as an inhibitory motoneuron to the flattener muscles. A locust bioassay (O'Shea and Adams, Science 213:567-569, 1981) was used to detect the presence of proctolin-like bioactivity. Extracts of leech ganglia when applied to the extensor tibialis muscle of the locust leg induced a proctolin-like response similar to the responses induced by proctolin standards. This work extends the finding of proctolin-like substances to the annelid phylum.     

Autoradiographic localization and characterization of atrial natriuretic peptide binding sites in the rat central nervous system and adrenal gland

Atrial natriuretic peptides (ANP) have recently been identified in both heart and CNS. These peptides possess potent natriuretic, diuretic, and vasorelaxant activities, and are all apparently derived from a single prohormone. Specific ANP binding sites have been characterized in the adrenal zona glomerulosa and kidney cortex, and one study reported ANP binding sites in the CNS. However, a detailed examination of the localization of ANP binding sites throughout the brain has not been reported. In this study, quantitative autoradiography was employed to examine the distribution of ANP receptors in the rat CNS. The binding of (3-125I-iodotyrosyl28) rat ANP-28 to binding sites in the rat CNS was saturable, specific for ANP-related peptides, and displayed high affinity (Kd = 600 pM). When the relative concentrations of ANP binding sites were determined throughout the rat brain, the highest levels of ANP binding were localized to the circumventricular organs, including the area postrema and subfornical organ, and the olfactory apparatus. Moderate levels of ANP binding sites were present throughout the midbrain and brain stem, while low levels were found in the forebrain, diencephalon, basal ganglia, cortex, and cerebellum. The presence of ANP binding sites in the subfornical organ and the area postrema, regions considered to be outside the blood-brain barrier, suggests that peripheral ANP levels may regulate some aspects of CNS control of salt and water balance. The possible functions of ANP binding sites in other regions of the rat brain are not known, but, like many other peptides, ANP may act as a neurotransmitter or neuromodulator at these loci.     

5-hydroxytryptamine in the central nervous system and sexual receptivity of female rhesus monkeys.

The role of 5-hydroxytryptamine (5-HT) in the control of sexual receptivity in female rhesus monkeys has been studied in 24 adult females paired with 6 adult males. p-Chlorophenylalanine (PCPA, 75 mg/kg or 100 mg/kg, every fourth day), a selective inhibitor of 5-HT, was found to reverse unreceptivity induced by adrenalectomy in ovariectomised, oestrogen-treated females. PCPA-treated females presented more frequently and initiated more sexual behaviour, or else they refused fewer of the male's attempts to mount. These effects were in turn reversed by 5-hydroxytryptophan (5-HTP, 20 mg/kg every second day), when this was given to PCPA-treated animals. In addition, 5-HTP given alone to ovariectomised oestrogen-treated females reduced their receptivity. Parallel biochemical experiments showed that PCPA in the doses used lowered the levels of 5-HT in the brain as measured by the levels of 5-hydroxyindole-3-acetic acid (5-HIAA) in the CSF, and that these were restored by 5-HTP. Both oestradiol benzoate (15 mug/day for 10 days) and testosterone propionate (250 mug/day or 400 mug/day for 10 days) lowered the turn-over rates of 5-HT in the brain (as measured by the probenecid test) in ovariectomised female monkeys. These effects of oestradiol on turnover were antagonised by progesterone (15 mg/day for 10 days, given with oestradiol). A substance other than an adrenal androgen has thus been found to reverse the effects of adrenalectomy on sexual receptivity in female monkeys. It is therefore possible that androgens regulate receptivity in female monkeys by modifying the activity of 5-HT-containing neural systems.     

Differential subcellular localization of the 5-HT3-As receptor subunit in the rat central nervous system

Following the cloning and sequencing of the A subunit of the 5-HT3 receptor, two alternatively spliced isoforms, 5-HT3-AS and 5-HT3-AL, have been identified. In order to analyse the distribution of the receptor, a polyclonal antibody has been produced against the short form which is the most abundant in the central nervous system [Doucet et al. (2000) Neuroscience 95, 881-892]. As expected from the recognition of functional 5-HT3 receptors, immunostaining by this anti-5-HT3-R-AS antibody matched the distribution of the high-affinity 5-HT3 binding sites in the rat brain and spinal cord. 5-HT3-AS-like immunoreactivity was detected at low levels in the limbic system, particularly in the amygdala and the hippocampus, and in the frontal, piriform and entorhinal cortices. High levels of immunoreactivity were found in the brainstem, mainly in the nucleus tractus solitarius and the nucleus of the spinal tract of the trigeminal nerve, and in the dorsal horn of the spinal cord. At the ultrastructural level, immunostaining was generally found associated with axons and nerve terminals (70-80%) except in the hippocampus, where labelled dendrites were more abundant (56%). This preferential localization on nerve endings is consistent with the well-documented physiological role of 5-HT3 receptors in the control of neurotransmitter release. However, the different distribution in the hippocampus raises the question of whether differential addressing mechanisms exist for preferentially targeting 5-HT3 receptors to postsynaptic dendritic sites as compared to presynaptic nerve endings, depending on the nature of the neurons bearing these receptors.     

Immunohistochemical localization of phospholipase D1 in rat central nervous system

Phospholipase D (PLD) is one of the intracellular signal transduction enzymes and plays an important role in a variety of cellular functions. We investigated the distribution of PLD isozyme, PLD1 in the rat brain and spinal cord using an immunological approach. Western blot analysis showed the presence of PLD1 protein in all tissues studied, with significantly higher levels in the brainstem and spinal cord, which was correlated with the results obtained from PLD activity assay. Prominent and specific signals of PLD1 were observed in many functionally diverse brain areas, including the olfactory bulb, medial septum-diagonal band complex, cerebral cortex, brainstem, cerebellum, and spinal cord. In the brainstem, the red nucleus, substantia nigra, interpeduncular nucleus, cranial motor nuclei (trigeminal motor, abducent, facial, and hypoglossal), sensory cranial nerve nuclei (spinal trigeminal, vestibular, and cochlear), as well as nuclei of the reticular formation, all showed intense immunoreactivity. Purkinje cells and deep cerebellar nuclei of the cerebellum were also labeled intensely. However, no significant labeling was found in the thalamus, epithalamus, and basal ganglia. Although many of the PLD1 immunoreactive cells were neurons, PLD1 was also expressed in glial cells such as presumed astrocytes and tanycytes. These findings suggest that PLD1 may play an important role in the central nervous system of the adult rat.     

Immunohistochemical localization of ORL-1 in the central nervous system of the rat

A novel member of the opioid receptor family (ORL-1) has been cloned from a variety of vertebrates. ORL-1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N-terminus of ORL-1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II–V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1–CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL-1 was observed in telencephalic areas such as caudate-putamen. In the cerebellum, ORL-1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL-1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL-1 receptor in the rat CNS. © 1996 Wiley-Liss, Inc.