Distribution of nerve growth factor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergic system--II. Brainstem, cerebellum and spinal cord

Multiple nuclei and fiber tracts in the adult rat brainstem and spinal cord were found to contain nerve growth factor receptor-related protein, as recognized by the monoclonal antibody 192-IgG. Both cholinergic and non-cholinergic sensory and motor regions demonstrated immunoreactive cell bodies and fibers. Nerve growth factor receptor-immunoreactive cells were seen in the mesencephalic nucleus of trigeminal nerve, superior colliculus, parabrachial, prepositus hypoglossal, raphe, dorsal and ventral cochlear, interstitial nucleus of the vestibular nerve, ambiguus and reticular nuclei, cerebellum and ventral spinal cord. Immunoreactive cells resembling neuroglia were distributed subpially along the superior colliculus. Intracerebroventricular injection of colchicine resulted in significantly increased nerve growth factor receptor immunoreactivity in all previously positive neurons and especially in certain neurons of the cochlear and ambiguus nuclei. It also resulted in the visualization of receptor immunoreactivity in certain neurons which were normally non-immunoreactive including cerebellar Purkinje cells, neurons of the central gray, locus coeruleus, facial, dorsal motor vagal and hypoglossal nuclei. In normal animals, nerve growth factor receptor-immunoreactive fibers and varicosities occurred in the trigeminal nerve nuclei, pontine, vestibular, parabrachial, facial, hypoglossal, dorsal motor vagal, solitary, gracile and cuneate nuclei and spinal cord. Although most fiber-like immunoreactive structures were probably axons and nerve terminals, neuroglial or extracellular localizations could not be excluded in some areas. For example, the medial nucleus of the inferior olive and most cerebellar nuclei contained diffuse non-fibrillar receptor immunoreactivity. The presence of nerve growth factor receptor-like immunoreactivity in cell bodies and fibers of several sensory and motor areas of the adult rat brainstem, cerebellum and spinal cord suggests multifocal actions of nerve growth factor or a nerve growth factor-like substance. Although the degree of overlap between nerve growth factor receptor- and choline acetyltransferase-containing regions in the brainstem is not as great as in the forebrain, our findings suggest a potential influence of nerve growth factor or nerve growth factor-like substances on cholinergic systems outside the forebrain. Furthermore, the disparities which occur imply that non-cholinergic nerve growth factor receptor-containing neurons of the brainstem, cerebellum and spinal cord may be affected by such trophic substances.     

Nerve growth factor receptors in rat spinal cord: an autoradiographic and immunohistochemical study

The distribution of nerve growth factor receptors in the lumbar spinal cord of the rat was studied with autoradiographic and immunohistochemical techniques: [125I]nerve growth factor and specific monoclonal antibody (Mab 192) against nerve growth factor receptor were used to localize nerve growth factor binding sites. The distributions of nerve growth factor binding sites with highest density within the superficial layers (laminae I and II) of the dorsal gray matter were virtually identical as demonstrated by these two ligands; this suggests that Mab 192 can be used as a specific probe to identify nerve growth factor receptors in rat nervous system. Nerve growth factor receptor binding sites, as demonstrated by autoradiography, were also found in longitudinal bundles of fibers running dorsolaterally in the lateral funiculus. However, no immunoreactivity was detected in these areas by immunohistochemistry. No specific binding was found in the dorsal horn when [125I]nerve growth factor was co-injected with unlabeled nerve growth factor or after incubation with nonspecific monoclonal antibody. Dorsal root section produced a complete loss of nerve growth factor-specific labeling pattern throughout laminae I-II of the spinal cord. This suggests that nerve growth factor receptors are localized on the nerve terminals of primary afferent fibers which synapse in the region of the spinal cord. The presence of nerve growth factor binding sites in the dorsal horn of the spinal cord is consistent with the possibility that nerve growth factor, or a nerve growth factor-like substance, derived from the central nervous system, may have a role in trophic support of dorsal root ganglion neurons.     

Low affinity nerve growth factor receptor binding in normal and Alzheimer's disease basal forebrain.

The binding characteristics of radiolabelled beta-nerve growth factor ([125I]NGF) have been determined on membrane preparations of basal forebrain from Alzheimer's disease (AD) brain and age-matched normal brains. [125I]NGF binds in a specific fashion indicative of a single receptor and is not displaced with microM concentrations of cytochrome c, insulin or epidermal growth factor (EGF). The mean dissociation constant (Kd) and the mean capacity (Bmax) of the NGF receptor were not significantly different between the 5 AD and 5 normal basal forebrain samples examined. Choline acetyltransferase (ChAT) activity was significantly reduced (P less than or equal to 0.001) in AD cerebral cortical samples compared with normal tissue.     

Regional development of muscarinic cholinergic binding sites in the prenatal rat brain.

The ontogeny of muscarinic cholinergic binding sites was studied in rat fetal central nervous system by in vitro autoradiographic techniques using [3H]N-methyl scopolamine as ligand (1 nM). Nonspecific binding was determined after the addition of 1 microM atropine. The main findings of this study are the early appearance of muscarinic cholinergic binding sites in fetal rat central nervous system before gestational day 14, their subsequent spread in a caudofrontal direction and the rapid change of patterns within individual brain regions. Muscarinic cholinergic sites are present shortly after cell birth, though the time-lag between cell generation and expression of muscarinic sites differs between neuronal cell populations. High receptor densities are noted in certain brainstem nuclei that are important for early fetal and neonatal behaviors.     

Nerve growth factor receptor is associated with cholinergic neurons of the basal forebrain but not the pontomesencephalon

Sequential immunohistochemical demonstration of nerve growth factor receptor and cholinergic acetyltransferase on the same tissue section in the rat revealed that approximately 92% of all cholinergic neurons in the basal forebrain possessed that receptor. Only 0.9% of the neurons demonstrating nerve growth factor receptor in the basal nuclear complex lacked the cholinergic synthetic enzyme, and a similarly small percentage of cholinergic cells, 7.1%, were choline acetyltransferase-positive but nerve growth factor receptor-negative. Affiliation of nerve growth factor receptor with structural entities morphologically indistinguishable from those demonstrating choline acetyltransferase on separate but corresponding tissue sections was also observed in the telencephalic fiber tracts and terminal fields of basal forebrain cholinergic neurons, including cholinergic puncta in the reticular nucleus of the thalamus. Nerve growth factor receptor was not found in association with choline acetyltransferase-positive somata of the pedunculopontine and laterodorsal tegmental nuclei, however, nor were fibers immunoreactive for nerve growth factor receptor observed originating from those cell bodies. These results suggest that nerve growth factor receptor, which is probably synthesized in cholinergic basal forebrain somata and transported throughout their dendritic and axonal arbors, has a physiologic role in those cells in the adult nervous system. This does not appear to be the case for phenotypically similar neurons of the pontomesencephalotegmental cholinergic complex.     

Localization of high-affinity and low-affinity nerve growth factor receptors in cultured rat basal forebrain

Previous work has indicated that nerve growth factor specifically and selectively increases choline acetyltransferase and acetylcholinesterase in organotypic cultures of rat basal forebrain-medial septal area. To determine whether these actions are potentially receptor-mediated, organotypic and dissociated basal forebrain-medial septal area cultures were examined. Two independent methods, [125I]nerve growth factor binding and immunocytochemistry with a monoclonal nerve growth factor receptor antibody (192-IgG), detected specific receptors. The nerve growth factor receptors were localized to two different cellular populations: flat, large, non-neuron-like cells, and small, round, process-bearing, neuron-like cells. Dissociation studies with [125I]nerve growth factor suggested that high-affinity receptors were localized to the neuron-like population, while only low-affinity receptors were localized to the non-neuron-like cells. We tentatively conclude that nerve growth factor may elicit cholinergic effects by directly binding to high-affinity receptors on neurons. To begin examining receptor regulation, cultures were exposed to exogenous, unlabeled nerve growth factor continuously for 10 days before binding studies were performed. Prior exposure to nerve growth factor did not alter binding characteristics of the receptor, using the present methods.     

Autoradiographic localization of binding sites for arginine vasopressin and atrial natriuretic peptide on astrocytes and neurons of cultured rat central nervous system.

The cellular localization of binding sites for [125I]arginine vasopressin and [125I]atrial natriuretic peptide was studied in explant cultures of rat spinal cord, brain stem and cerebellum by means of autoradiography. In brain stem cultures, especially in the nucleus of the solitary tract, a great number of neurons revealed binding sites for both peptides. In spinal cord cultures, many neurons of various sizes were labelled by [125I]arginine vasopressin, whereas only a small number of cells showed binding sites for [125I]atrial natriuretic peptide. Neurons in cerebellar cultures revealed little or no binding for the peptides. In addition to neurons, binding sites for [125I]arginine vasopressin and [125I]atrial natriuretic peptide were also observed on glial cells. Simultaneous staining of the cultures with glial fibrillary acidic protein has shown that the labelled cells were glial fibrillary acidic protein-positive and could therefore be identified as astrocytes. Labelling of the cells by [125I]arginine vasopressin and [125I]atrial natriuretic peptide was more intense in spinal cord and brain stem cultures than in cultures of cerebellum, providing evidence for a heterogeneity of astrocytes in different regions of the central nervous system. Binding of both [125I]arginine vasopressin and [125I]atrial natriuretic peptide to neurons and astrocytes could be competed by the unlabelled peptides, suggesting specific binding of the radioligands. Our autoradiographic studies provide good evidence that in addition to neurons, astrocytes also express receptors for arginine vasopressin and atrial natriuretic peptide.     

NGF treatment promotes development of basal forebrain tissue grafts in the anterior chamber of the eye

Summary The effects of nerve growth factor (NGF) on developing central cholinergic neurons were studied using intraocular grafts of rat fetal (E17) basal forebrain tissue. Prior to grafting, grafts were incubated in NGF or saline. Transplants were allowed to mature for six weeks, receiving weekly intraocular injections of NGF or saline. Measurements of NGF levels in oculo after one single injection showed that NGF slowly decreases in the anterior chamber fluid, and after one week, low but significant levels were still present in the eye. Following pretreatment with diisopropylfluorophosphate (DFP), the cholinergic neurons in the grafts were analyzed using three morphological markers: antibodies to cholineacetyltransferase (ChAT), antibodies to acetylcholinesterase (AChE Ab) and acetylcholinesterase histochemistry (AChE). The transplants grew well and became vascularized within the first week. The growth of the NGF-treated basal forebrain grafts was significantly enhanced as compared to the growth of the saline-treated grafts evaluated with repeated stereomicroscopical observations directly through the cornea of the etheranaesthetized hosts. The NGF-treated grafts contained almost twice as many cholinergic neurons seen with all the cholinergic markers used, as the salinetreated grafts. However, there was no difference in cholinergic cell density between the two groups. The morphology and size of an individual cholinergic neuron was similar in the two groups. The fiber density as evaluated with AChE-immunohistochemistry did not change after NGF-treatment. The DFP-treatment did not seem to affect the AChE-immunoreactivity since an extensive fiber network was found, whereas almost no fibers were seen using conventional AChE histochemistry. We have demonstrated that in oculo transplantation of basal forebrain is a useful model for examining in vivo effects of NGF on central cholinergic function. The marked volume increase of NGF-treated grafts and the unchanged density of cholinergic cells and terminals suggests, that NGF increases the survival of not only developing cholinergic neurons, but possibly other non-cholinergic neurons and non-neuronal cells as well. These results support the notion that NGF acts as a neurotrophic factor on cholinergic and possibly non-cholinergic cells in the central nervous system     

Data and hypotheses on the role of nerve growth factor and other neurotrophins in psychiatric disorders

Nerve growth factor (NGF) was discovered and characterized for its role on the growth, differentiation and maintenance of specific neurons of the peripheral nervous system. Subsequent studies revealed that NGF is synthesized and released within the central nervous system and exerts a trophic and functional role on basal forebrain cholinergic neurons; it is involved in a protective role following brain insults induced by an epileptic status, seizure, as well as surgical and chemical lesions.More recently our collaborative studies provided evidence that NGF is implicated in neurobehavioral response including cerebral alterations associated with psychiatric disorders. In this brief review, ongoing and emerging data are presented and discussed.     

Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1–Ch6)

Monoclonal antibodies to choline acetyltransferase and a histochemical method for the concurrent demonstration of acetylcholinesterase and horseradish peroxidase were used to investigate the organization of ascending cholinergic pathways in the central nervous system of the rat. The cortical mantle, the amygdaloid complex, the hippocampal formation, the olfactory bulb and the thalamic nuclei receive their cholinergic innervation principally, from cholinergic projection neurons of the basal forebrain and upper brainstem. On the basis of connectivity patterns, we subdivided these cholinergic neurons into six major sectors. The Chl and Ch2 sectors are contained within the medial septal nucleus and the vertical limb nucleus of the diagonal band, respectively. They provide the major cholinergic projections of the hippocampus. The Ch3 sector is contained mostly within the lateral portion of the horizontal limb nucleus of the diagonal band and provides the major cholinergic innervation to the olfactory bulb. The Ch4 sector includes cholinergic neurons in the nucleus basalis, and also within parts of the diagonal band nuclei. Neurons of the Ch4 sector provide the major cholinergic innervation of the cortical mantle and the amygdala. The Ch5–Ch6 sectors are contained mostly within the pedunculopontine nucleus of the pontomesencephalic reticular formation (Ch5) and within the laterodorsal tegmental gray of the periventricular area (Ch6). These sectors provide the major cholinergic innervation of the thalamus. The Ch5–Ch6 neurons also provide a minor component of the corticopetal cholinergic innervation.

These central cholinergic pathways have been implicated in a variety of behaviors and especially in memory function. It appears that the age-related changes of memory function as well as some of the behavioral disturbances seen in the dementia of Alzheimer's Disease may be related to pathological alterations along central cholinergic pathways.     

The D1 dopamine receptor in the rat brain: quantitative autoradiographic localization using an iodinated ligand

The distribution of dopamine D1 receptors in the rat, labeled with [125I]SCH 23982, was studied using a quantitative in-vitro light-microscopic autoradiographic method. The binding of [125I]SCH 23982 to slide-mounted tissue sections and membrane preparations of prefrontal cortex was saturable, specific and of high affinity. Scatchard analysis revealed a Kd of 1.15 +/- 0.47 nM and Bmax of 8.76 +/- 0.34 fmol/mg tissue in prefrontal cortex membranes and a Kd of 1.27 +/- 0.14 nM and Bmax of 67.6 +/- 3.75 fmol/mg tissue in slide-mounted tissue sections at the level of the striatum. [125I]SCH 23982 was found to predominantly label D1 receptors, but a small fraction of the binding was to serotonin receptors. D1 receptors were found throughout the forebrain and were concentrated in the substantia nigra pars reticulata, accumbens nucleus, caudate putamen, entopeduncular nucleus, olfactory tubercle and the major island of Calleja. [125I]SCH 23982 binding to serotonin receptors was concentrated in the cortices, dorsal raphe, central gray, anterior hypothalamic area and the molecular cell layer of the cerebellum. Knowledge of the distribution of D1 receptors may increase our understanding of the role of D1 receptors in central nervous system dopaminergic function. Furthermore, data on the potential sites of interaction of [125I]SCH 23982 with serotonin receptors may help to understand the complex physiology and pharmacology of the primarily D1 selective compound.     

Molecular cloning of rat trkC and distribution of cells expressing messenger RNAs for members of the trk family in the rat central nervous system.

Tyrosine protein kinases trk, trkB and trkC are signal-transducing receptors for the neurotrophins nerve growth factor, brain-derived nerve growth factor, neurotrophin-3 and neurotrophin-4. Here we report on the isolation of cDNA fragments encoding a part of rat trk and trkB proteins, respectively, and characterization of a full-length cDNA clone encoding rat trkC. Cells expressing mRNAs for the different members of the trk family were identified in the rat central nervous system by in situ hybridization using oligonucleotide probes designed from the isolated cDNA sequences and complementary to mRNA sequences coding for the extracellular region of the receptors. The expression of trk mRNA was found to be restricted to neurons of the basal forebrain, caudate-putamen with features of cholinergic cells and to magnocellular neurons of several brainstem nuclei. In contrast, cells expressing trkB and trkC mRNAs were widely distributed in the brain. Areas expressing high levels of trkB or trkC mRNAs included olfactory formations, neocortex, hippocampus, thalamic and hypothalamic nuclei, brainstem nuclei, cerebellum and spinal cord motoneurons. A similar distribution for trkB and trkC mRNAs was shown in most areas but each probe specific for these mRNAs also provided distinct labeling patterns in different subregions, layers and cells. Comparison between our data and previous analyses of cells expressing mRNAs for neurotrophins and the low-affinity nerve growth factor receptor suggests that different modes of action and different combinations of receptors mediate biological responses to neurotrophins in the adult rat brain.     

Neurotrophic effect of brain-derived neurotrophic factor on basal forebrain cholinergic neurons in culture from postnatal rats.

We examined the effect of brain-derived neurotrophic factor (BDNF) on cholinergic neurons in culture from postnatal rat basal forebrain by assay of choline acetyltransferase (ChAT) activity and cytochemical staining for acetylcholinesterase (AChE). BDNF was found to increase the ChAT activities but failed to promote the survival of AChE-positive neurons in cultures from neonatal (P3) rats, suggesting that its main role is cholinergic differentiation. In contrast, an enhancement of the survival of AChE-positive neurons and of ChAT activity was observed in cultures from P15-16 rats, suggesting that BDNF's main action is the maintenance of cholinergic neurons. Our results indicate a similarity between BDNF and nerve growth factor effects on the responses of cholinergic neurons of postnatal rat basal forebrain in culture.     

Differential distribution of somatostatin receptor subtypes in rat brain revealed by newly developed somatostatin analogs.

Somatostatin receptor subtypes were labeled with the somatostatin analogs [125I]CGP 23996 and [125I]MK 678 and the distribution of these receptors in rat brain was investigated using quantitative autoradiographic techniques. [125I]CGP 23996 and [125I]MK 678 specifically label different populations of somatostatin receptors in rat brain. In a number of brain regions striking differences in the distribution of the somatostatin receptor subtypes labeled by each peptide were observed. High levels of binding sites for both [125I]CGP 23996 and [125I]MK 678 were present in the cerebral cortex, CA1 region and subiculum of the hippocampus. In contrast, high levels of [125I]MK 678 binding were found in the dentate gyrus of the hippocampus while few [125I]CGP 23996 binding sites were observed in this brain region. [125I]CGP 23996 binding was detected in the central region of the interpeduncular nucleus whereas the dorsal and lateral subnuclei of this brain area expressed mainly somatostatin receptors with high affinity for MK 678. The locus coeruleus and regions of the superior colliculus and hypothalamus selectively express [125I]MK 678-sensitive somatostatin receptors. Furthermore, limbic structures such as the lateral septum, the nucleus accumbens and ventromedial striatum had much higher levels of [125I]MK 678 binding sites than [125I]CGP 23996 binding sites. Differences in the expression of the somatostatin receptor subtypes were also detected in the substantia nigra. [125I]CGP 23996 binding was present in the pars reticulata but not the pars compacta whereas the reverse distribution for [125I]MK 678 binding sites was observed. The differential distribution of [125I]CGP 23996 and [125I]MK 678 binding sites in rat brain supports the hypothesis that these peptides selectively label different somatostatin receptor subtypes in the central nervous system.     

The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures

Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.     

Distribution of nicotinic cholinergic receptors in the rat tel- and diencephalon: a quantitative receptor autoradiographical study using [3H]-acetylcholine, [alpha-125I]bungarotoxin and [3H]nicotine

The topographical distribution of [alpha-125I]bungarotoxin [125I]BTX, [3H]nicotine ([3H]Nic), [3H]acetylcholine ([3H]ACh) (in the presence of atropine) binding in rat tel- and diencephalon was investigated using a quantitative receptor autoradiographical technique. With the [3H]ACh and [3H]Nic radioligands, a strong labelling was observed in various thalamic nuclei, including the medial habenula, a moderate labelling in different areas of the cortex cerebri, the nucleus caudatus putamen, the nucleus accumbens and tuberculum olfactorium and a uniform weak labelling in the hypothalamus. When the binding data for [3H]Nic were plotted against binding data for [3H]ACh in various brain nuclei, a significant correlation was obtained. Considering [125I]BTX, the strongest labelling was observed in the lateral mammillary nucleus and the hilus gyrus dentatus of the hippocampal formation. A weak labelling occurred in areas such as the nucleus causatus putamen, the thalamus and the cerebral cortex. No significant correlation was therefore obtained between the degree of [125I]BTX binding in various brain nuclei and the degree of binding observed with [3H]Nic or [3H]ACh. The present results underline the view that the high-affinity [3H]Nic and [3H]ACh binding sites label the same cholinergic nicotinic receptor binding site, while [125I]BTX labels another subpopulation of nicotinic cholinergic receptors, predominantly found in discrete areas of the hypothalamus and the limbic cortex.     

Quantitative light microscopic autoradiographic localization of cholinergic muscarinic receptors in the human brain: forebrain

The distribution of muscarinic cholinergic receptors in the human forebrain and cerebellum was studied in detail by quantitative autoradiography using N-[3H]methylscopolamine as a ligand. Only postmortem tissue from patients free of neurological diseases was used in this study. The highest densities of muscarinic cholinergic receptors were found in the striatum, olfactory tubercle and tuberal nuclei of the hypothalamus. Intermediate to high densities were observed in the amygdala, hippocampal formation and cerebral cortex. In the thalamus muscarinic cholinergic receptors were heterogeneously distributed, with densities ranging from very low to intermediate or high. N-[3H]Methylscopolamine binding was low in the hypothalamus, globus pallidus and basal forebrain nuclei, and very low in the cerebellum and white matter tracts. The localization of the putative muscarinic cholinergic receptors subtypes M1 and M2 was analysed in parallel using carbachol and pirenzepine at a single concentration to partially inhibit N-[3H]methylscopolamine binding. Mixed populations of both subtypes were found in all regions. M1 sites were largely predominant in the basal ganglia, amygdala and hippocampus, and constituted the majority of muscarinic cholinergic receptors in the cerebral cortex. M2 sites were preferentially localized in the diencephalon, basal forebrain and cerebellum. In some areas such as the striatum and substantia innominata there was a tendency to lower densities of muscarinic cholinergic receptors with increasing age. In general, we observed a slight decrease in M2 sites in elderly cases. Muscarinic cholinergic receptor concentrations seemed to be reduced following longer postmortem periods. The distribution of acetylcholinesterase was also studied using histochemical methods, and compared with the localization of muscarinic cholinergic receptors and other cholinergic markers. The correlation between the presence of muscarinic cholinergic receptors and the involvement of cholinergic mechanisms in the function of specific brain areas is discussed. Their implication in neurological diseases is also reviewed.     

Autoradiographic localization of [125I]-endothelin-1 binding sites in rat brain

Autoradiograms of [125I]-endothelin (ET) binding in the rat brain demonstrated that the receptors for endothelin are localized mainly in the brainstem, basal ganglia, and cerebellum. Among the many other nuclei in these regions, there also appeared nuclei which are considered to play important roles in the central nervous regulation of the cardiovascular system: they include the nuclei of the anteroventral third ventricle area, the supraoptic nucleus, and the subfornical organ, for example. From these findings, we suggest that ET-1 or its analogous peptide(s) may act as a neuropeptide regulating central nervous functions, including cardiovascular functions.     

Transforming growth factor beta isoforms in the adult rat central and peripheral nervous system.

The distribution of transforming growth factor-beta isoforms 1, 2 and 3 and transforming growth factor-beta 2 and 3 mRNAs in adult rat central and peripheral nervous system was examined using Northern blotting and isoform specific antibodies for immunocytochemistry. Transforming growth factor-beta 2 and 3 mRNA were present in all brain areas including cerebral cortex, hippocampus, striatum, cerebellum and brainstem. In sciatic nerve, transforming growth factor-beta 3 mRNA was highly expressed, but transforming growth factor-beta 2 mRNA was not detectable. Transforming growth factor-beta 1-like immunoreactivity was confined to meninges and choroid plexus in the brain and connective tissue in peripheral ganglia and nerves. Transforming growth factor-beta 2 and 3 immunoreactivity entirely overlapped and, in general, were found in large multipolar neurons. Highest densities of immunoreactive neuronal perikarya were present in spinal cord and brainstem motor nuclei, hypothalamus, amygdaloid complex, hippocampus and cerebral cortical layers II, III and V. Most thalamic nuclei, superior colliculi, periaqueductal gray and striatum were almost devoid of transforming growth factor-beta 2- and 3-immunoreactive neurons. Fibrous astrocytes in white matter areas were intensely immunostained. Most dorsal root ganglionic neurons, their satellite cells and Schwann cells in peripheral nerves were also labeled. Transforming growth factor-beta 2- and 3-immunoreactive neurons were localized in brain regions that have been shown to contain neurons synthesizing and/or storing basic fibroblast growth factor suggesting possible opposing or synergistic effects of these peptide growth factors. However, the precise functions of local synthesis and storage of the transforming growth factor-beta isoforms in the nervous system are as yet unknown.     

Autoradiographic mapping of [mono[125I]iodo-Tyr10, MetO17]vasoactive intestinal peptide binding sites in the rat brain

The distribution of vasoactive intestinal peptide binding sites in the rat brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) previously shown to maintain in the central nervous system the full biological activity of native vasoactive intestinal peptide was used for this study. In initial kinetic and pharmacological experiments the binding of M-[125I]vasoactive intestinal peptide to slide-mounted sections was shown to be time-dependent, saturable and reversible. Association of M-[125I]VIP specific binding was maximal within 90-120 minutes. Specific binding, corresponding to approximately 50% of total binding was saturable, of high affinity (Kd of 76.6 pM) and low capacity (fmol/mg prot range). Dissociation of M-[125I]VIP was maximal at 10 minutes. Unlabeled vasoactive intestinal peptide and the two structurally related peptides "peptide-histidine-isoleucine" (PHI) and secretin competed in a concentration-dependent manner for sites labeled by M-[125I]vasoactive intestinal peptide with the following rank order of potencies: vasoactive intestinal peptide greater than PHI greater than secretin. Vasoactive intestinal peptide receptors, as revealed by quantitative autoradiography, are present at various levels of the neuraxis. High densities were observed in olfactory bulb, cerebral cortex (highest in layers I, II, IV and VI), dentate gyrus, subiculum, various thalamic and hypothalamic nuclei, superior colliculus, locus coeruleus, area postrema, subependymal layer and pineal gland. Intermediate densities were found in the amygdala, nucleus accumbens, caudate-putamen, septum, bed nucleus of the stria terminalis, CA1 to CA4 fields of the hippocampus and central gray. No specific binding of M-[125I]vasoactive intestinal peptide was observed in white matter tracts such as corpus callosum, anterior commissure, medial forebrain bundle and fornix. The mapping of M-[125I]vasoactive intestinal peptide binding sites as revealed by autoradiography on slide-mounted sections indicates an association, although not exclusive, of vasoactive intestinal peptide receptors with brain regions involved in the processing of specific sensory inputs.