Localization of nerve growth factor-like immunoreactivity in rat nervous tissue

The use of immunofluorescence with affinity-purified antibodies enabled cytological localization of nerve growth factor-like material in the rat. Immunoreactivity was observed along various nerve tracts of the foetal rat brain and spinal cord at day 15 of gestation. Longitudinal pathways in ventral and dorsal spinal cord, ventral lower brain stem, posterior commissure, retroflex fascicle and in the olfactory bulb were all positive. A weaker and more widely spread immunostaining was visible in many areas in the central nervous system. Cranial nerves were strongly immunoreactive. Neuronal perikarya in the retina and the olfactory mucosa as well as filae olfactoriae and the olfactory nerve all the way to the olfactory bulb were also positive. In sensory ganglia and peripheral nerves most immunoreactivity was confined to supporting tissues, probably including Schwann cells. In irides, the pattern of immunoreactivity was similar to that of the sensory and autonomic innervation. More intensively fluorescent material was found in regrowing nerve fibres in iris transplants. Our histochemical results suggest that nerve growth factor and/or a related protein is present in large amounts along nerve pathways in supportive tissues of the peripheral nervous system as well as in the central nervous system during early development.     

Isolation and amino acid sequence of two urotensin II peptides from Catostomus commersoni urophyses

Two peptides with urotensin II (UII = smooth muscle stimulating) activity have been isolated from urophyses of the sucker, Catostomus commersoni. The amino acid sequences of the two peptides, designated UIIA and UIIB, are as follows: UIIA, H-Gly-Ser-Gly-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH; UIIB, H-Gly-Ser-Asn-Thr-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. These peptides have the part sequence Cys-Phe-Trp-Lys-Tyr-Cys-Val in common with the UII peptide from Gillichthys mirabilis and the part-sequence Phe-Trp-Lys in common with somatostatin.     

Immunocytochemical localization of urotensin I neurons in the caudal neurosecretory system of the white sucker (Catostomus commersoni)

Summary The localization of urotensin I has been investigated in the caudal neurosecretory system of the white sucker (Catostomus commersoni). The peptide is present in all the cells of the system both large and small, in the large axons passing to the urophysis, and in fine beaded fibres not only within the urophysis but also in a fine plexus lateral to the large cells in the spinal cord proper. The possibility that the caudal neurosecretory system is not a functionally uniform system but rather a collection of dissimilar cells of different synaptic inputs with a common entity, urotensin I, is discussed. Moreover, the feasibility of a urotensin I feedback loop is described.Financial support for this investigation was provided in part by MRC (Canada). K.L. is MRC career investigator; K.L.W, was in receipt of an Alberta Heritage Foundation for Medical Research Fellowship. It is a pleasure to record the valuable technical assistance of Mrs. W. Ho and the dedicated assistance in the collection of the experimental animals by Mrs. Helen Wilson of Nanton, Alberta.     

Localization of corticotropin-releasing hormone (CRF)-like immunoreactivity in the central nervous system of the elasmobranch fish,Scyliorhinus canicula

Summary The occurrence and localization of immunoreactive corticotropin-releasing factor (CRF) in the brain and pituitary of the elasmobranch fish Scyliorhinus canicula, were studied by means of specific radioimmunoassay and immunohistochemistry using the indirect immunofluorescence method. Brain and pituitary extracts showed a good cross-reactivity with the ovine CRF antiserum, but serial dilutions of tissue samples did not completely parallel the standard curve. Relatively high concentrations of CRF-like material were found within the pituitary, diencephalon, and telencephalon. CRF-like immunoreactive perikarya were observed in the preoptic nucleus and in the nucleus lateralis tuberis. Numerous immunoreactive cells appeared to be of the CSF-contacting type. CRF-like immunopositive fibers were seen to run through the hypothalamus within the ventro-medial floor of the infundibular region. A dense plexus of immunoreactive nerve endings terminated in the median eminence and the neurointermediate lobe of the pituitary. These results indicate that a neurosecretory system containing CRF-like immunoreactivity exists in the brain of elasmobranchs, a group of vertebrates which has diverged early from the evolutionary line leading to mammals. In addition, our data support the notion that a CRF-like molecule is involved in the regulation of corticotropic and melanotropic cell activity in this primitive species of fish.     

Co-localization of the immunoreactivities of corticotropin-releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni

Summary Using the label-fracture technique, an ultrastructural comparison was made of the number and distribution of wheat germ agglutinin (WGA)-binding sites between human normal and sickle red blood cells. The WGA was adsorbed to colloidal gold, and quantitative analysis of the electron micrographs revealed that more binding sites were present on the sickle erythrocytes than on the normal erythrocytes. Moreover, the sites were more clustered on the sickle red cells than on the normal red cells. Use of another lectin, Bandieraea simplicifolia-II, revealed that it did not bind to normal or sickle red cells. Because of the affinity of the WGA for sialic acid residues, it is probable that the WGA is binding to a transmembrane sialoglycoprotein, glycophorin A. The conformation and/or distribution of the glycophorin A molecules may be altered by the sickle hemoglobin that binds to the red cell membrane. Hence, as detected by WGA, new surface receptors, which could play a role in the adhesion of sickle cells to endothelium may be exposed.     

Distribution and localization of pro-brain-derived neurotrophic factor-like immunoreactivity in the peripheral and central nervous system of the adult rat

The precursors for neurotrophins are proteolytically cleaved to form biologically active mature molecules which activate their receptors p75NTR and trks. A recent study showed that the precursor for nerve growth factor (NGF) can bind to p75NTR with a high affinity and induces apoptosis of neurons in vitro. Mutation in Val66Met of brain-derived neurotrophic factor (BDNF) results in reduction in hippocampal function in learning and in the dysfunction of intracellular BDNF sorting and secretion. To examine the functions of pro-neurotrophins in vivo, it is essential to know where they are expressed in the nervous system. In the present study, we have raised and characterized rabbit polyclonal antibodies against a peptide coding for the precursor region of the BDNF gene. The antibody specifically recognizes the precursor for BDNF by western blot. With the affinity purified precursor antibody, we have mapped the distribution and localization of the precursor for BDNF. The results showed that, like mature BDNF, pro-BDNF is localized to nerve terminals in the superficial layers of dorsal horn, trigeminal nuclei, nuclei tractus solitarius, amygdaloid complex, hippocampus, hypothalamus and some peripheral tissues. These results suggest that pro-BDNF, like mature BDNF, is anterogradely transported to nerve terminals and may have important functions in synaptic transmission in the spinal cord and brain.     

Localization of CRF immunoreactivity in the central nervous system of three vertebrate and one insect species

Summary By use of a specific antiserum against synthetic ovine corticotropin-releasing factor (CRF) in the peroxidase-antiperoxidase (PAP) immunocytochemical procedure (Vandesande and Dierickx 1976), CRF-like antigenic determinants were demonstrated in the central nervous system of a human fetus, the Wistar rat, the frog Rana ridibunda, and the American cockroach Periplaneta americana. The immunoreactive CRF-producing cells occur mainly in the nucleus paraventricularis of the rat, while in Rana ridibunda these cells occur in the nucleus praeopticus. Immunoreactive CRF-containing fibres were also visualized. Very clear CRF-immunoreactive products were observed in the brain as well as the corpora cardiaca (CC) and corpora allata (CA) of the cockroach Periplaneta americana. ACTH-immunoreactivity was also demonstrated in the brain-CC-CA complex of this insect. Double immunohistochemical staining (Vandesande 1983) also revealed that both the CRFand ACTH-like substances occur in different neurosecretory neurons and nerve fibres. These results suggest that the antigenic determinants of CRF are very similar in vertebrates and insects bespeaking their very long evolutionary history.     

Glutamate-like immunoreactivity in the leech central nervous system

Summary Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.     

In situ hybridization of corticotropin-releasing factor-encoding messenger RNA in the hypothalamus of the white sucker,Catostomus commersoni

Summary In situ hybridization procedure with a ³²P-labelled synthetic oligonucleotide probe was used to detect corticotropin-releasing factor-encoding messenger RNA (CRF mRNA) in the hypothalamus of the white sucker, Catostomus commersoni. Adjacent sections were immunostained by a sucker CRF-specific antiserum. CRF mRNA-containing neurons were identified by autoradiography in the magnocellular and parvocellular subdivisions of the preoptic nucleus (PON). Many of these neurons were also immunostained by sucker antiserum, showing the same distribution patterns. These results confirm the presence of CRF mRNA and CRF peptide in the white sucker hypothalamus and support the view that the magnocellular and parvocellular neurons of the PON may be involved in the control of adrenocorticotropic hormone secretion from the pituitary in the white sucker.     

Relationship between urotensin II- and somatostatin-immunoreactive spinal cord neurons of Catostomus commersoni and Oncorhynchus kisutch (Teleostei)

Summary This immunocytochemical study describes the presence of separate immunoreactive (IR)-urotensin II (UII) and IR-somatostatin (SOM) systems in the spinal cord of two species of teleost fish. Both systems are arranged in a close spatial interrelationship in which IR-SOM fibres apparently innervate cerebrospinal fluid (CSF)-contacting IR-UII neurons. Specimens of Oncorhynchus kisutch also display CSF-contacting IR-SOM neurons located in the lateral ependymal walls of the central canal, in addition to CSF-contacting IR-UII neurons located ventrally. It is suggested that, in this species, CSF-contacting IR-SOM and IR-UII neurons perceive different stimuli from the CSF and are integrated in such a way that one peptidergic system may modulate the function of the other.     

Localization of growth hormone-releasing factor-like immunoreactivity in the hypothalamo-hypophysial system of some teleost species

Summary An antiserum to growth hormone-releasing factor (GRF) 1-44 was applied on brain and pituitary sections of nine teleost species. Immunoreactive (ir) perikarya were demonstrated in parvo- and magnocellular portions of the preoptic nucleus (PON) and occasionally in the nucleus lateralis tuberis. The two tracts originating in the PON ran ventro-laterally toward the optic chiasm and then caudally in the basal hypothalamus. In the pars distalis (PD) of the eel, carp, goldfish and salmonids, GRF-ir fibers did not enter the rostral PD and few fibers passed close to somatotropes. In.Myoxocephalus andMugil, a variable number of ir-fibers passed close to cells of the rostral and proximal PD. In the neurointermediate lobe, GRF-ir fibers were located exclusively in the neural tissue of the eel and trout. In goldfish, carp andMyoxocephalus, GRF-ir fibers entered the intermediate lobe. This antiserum also labeled corticotrops and, to a lesser extent, melanotrops in the pituitary of cyprinids. A variable number of perikarya contained both GRF and vasotocin in the PON of the eel. In all teleost species studied so far, the distribution patterns of GRF are different, and the function of the various adenohypophysial cell types appears to be differently modulated, according to the variable distribution of GRF in the pituitary.     

Glutamate-like immunoreactivity in the leech central nervous system.

Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.     

Localization of cholecystokinin-like immunoreactivity in the central nervous system of Triatoma infestans (Insecta: Heteroptera)

The distribution of cholecystokinin-like immunoreactivity was studied in the central nervous system of the heteropteran insect Triatoma infestans using high-sensitivity immunocytochemistry. In the protocerebrum, CCK-IR somata were observed in the anteromedial, anterolateral and posterior cell-body layers. The neuropils displayed different densities of immunoreactive neurites. Few immunoreactive somata were found in the optic lobe in both the medial and lateral soma rinds, as well as in the proximal optic lobe. Immunoreactive fibers were present in the medulla and lobula neuropils. The sensory deutocerebrum contained a higher number of immunopositive perikarya than the antennal mechanosensory and motor center. The antennal lobe glomeruli displayed a moderate density of immunoreactive fibers. With regard to the subesophageal ganglion, numerous CCK-IR somata were found close to the root of the mandibular nerve; others were present in the soma rind of the remaining neuromeres. CCK-IR perikarya were present in both thoracic ganglia, with the abdominal neuromeres containing the highest number of positive somata. The neuropils of both ganglia showed moderate densities of immunopositive processes. The distribution of CCK-LI in somata and neuropils of central nervous system of T. infestans is widespread suggesting that a CCK-like peptide may act mainly as a neuromodulator in the integration of information from distinct sensory receptors.     

Corticotropin releasing factor-like immunoreactivity in sensory ganglia and capsaicin sensitive neurons of the rat central nervous system: colocalization with other neuropeptides

Immunohistochemistry and radioimmunoassay (RIA) revealed that corticotropin releasing factor (CRF)-like immunoreactivity was found to be colocalized with substance P (SP)-, somatostatin (SST)- and leu-enkephalin (LENK)-like immunoreactivity in the dorsal root- and trigeminal ganglia, the dorsal horn of the spinal cord (laminae I and II), the substantia gelatinosa, and at the lateral border of the spinal nucleus and in the tractus spinalis of the trigeminal nerve. These peptides were also located in fast blue labeled cells of the trigeminal ganglion following injection of the dye into the spinal trigeminal area. This indicates that there are possible sensory projections of these peptides into the spinal trigeminal area. Capsaicin treatment of neonatal rats resulted in a marked decrease in the density of CRF-, SP-, VIP- and CCK-containing neurons in the above mentioned hindbrain areas, whereas SST- and LENK-immunoreactivity were not changed. RIA revealed that, compared to controls, CRF, SP and VIP concentrations in these areas were decreased in rats pretreated with capsaicin, while SST levels were increased; CCK and LENK levels were unchanged. It is concluded that the primary afferent neurons of the nucleus and tractus spinalis of the trigeminal nerve are richly endowed with a number of peptides some of which are sensitive to capsaicin action. The close anatomical proximity of these peptide containing neurons suggests the possibility of a coexistance of one or more of these substances.     

Sauvagine-like and corticotropin-releasing factor-like immunoreactivity in the brain of the bullfrog (Rana catesbeiana)

Summary Immunocytochemical methods were used to investigate the occurrence and distribution of sauvagine, corticotropin-releasing factor-, or urotensin I-like immunoreactivities (SVG-ir, CRF-ir, UI-ir, respectively) in the bullfrog (Rana catesbeiana) brain, using specific antisera raised against non-conjugated SVG, ovine CRF, rat/human CRF, and UI. In the hypothalamus, SVG-ir was found in the magnocellular perikarya, in the dorsal and ventral regions of the preoptic nucleus, and in the hypothalamo-hypophyseal projections to the external zone as well as the internal zone of the median eminence, to pars nervosa, and in fibres running from the pars nervosa to the pars intermedia of the pituitary. In contrast, CRF-ir was found only in parvocellular perikarya, mainly localized in the rostro-ventral part of the preoptic nucleus, with fine processes protruding through the ependyma of the third ventricle, fibre projections terminating in the anterior preoptic area and in the neuropil of the periventricular gray, and a caudal projection to the external zone of the median eminence. No CRF-ir staining was seen in the pars nervosa and pars intermedia. The use of UI-specific antisera failed to give a positive response in the frog brain. It is concluded that, in the frog brain, two anatomically different CRF-like (or SVG-like) systems co-exist, comparable to the reported co-existence of UI-ir and CRF-ir neuronal systems in fish brain.     

Peptide YY-like immunoreactivity in the central nervous system of the rat

The concentration of peptide YY (PYY)-like immunoreactivity in rat brain and spinal cord was determined by radioimmunoassay. The highest concentrations were found in the cervical spinal cord (18.1 +/- 1.3 ng/g, mean +/- S.E.M.) and in the medulla oblongata (16.3 +/- 1.5 ng/g). Lower amounts were found in the pons and in the hypothalamus. Chromatographic analysis of the PYY-like immunoreactivity from various regions of the brain revealed 95% of the immunoreactive material to be indistinguishable from synthetic porcine PYY. PYY-immunoreactive nerve cell bodies could be demonstrated by immunocytochemistry in the medulla oblongata of colchicine-treated rats, the largest group of cells being found in the midline area between and partly in the raphe pontis and obscurus nuclei. Another large group of immunoreactive cells was detected more laterally in the medial parts of the gigantocellular reticular nucleus. A few cells, finally, were seen in the dorsal parts of the medulla, including the nucleus of the solitary tract. Varicose nerve fibers displaying PYY immunoreactivity were observed in many parts of the hypothalamus, pons, medulla and spinal cord.     

Corpuscles of Stannius and stanniocalcin-like immunoreactivity in the white sucker (Catostomus commersoni): evidence for a new cell-type

The distribution of stanniocalcin immunoreactivity was examined in the corpuscles of Stannius of the white sucker (Catostomus commersoni) by using a chum salmon stanniocalcin antiserum, Western blotting, and light and electron microscopy. The white sucker possesses at least two stanniocalcin-immunoreactive corpuscles in the most posterior portions of the kidneys. Immunocytochemistry and ultrastructure revealed two cell-types in the corpuscle parenchyma, only one of which was immunoreactive. The nonimmunoreactive cells contained dense-cored vesicles and long processes that extended between the immunoreactive cells and terminated at perivascular spaces. When corpuscle extracts were subjected to electrophoresis and Western blotting, three nonreduced stanniocalcin-like immunoreactive bands (approximately 56, 61, and 64 kDa) were observed. However, in the presence of a reductant, a diffuse band migrating in the range of 28 to 32 kDa was noted. The results of this study on the white sucker demonstrate the presence of a dimeric stanniocalcin-like molecule and present evidence of a previously uncharacterized cell-type in the corpuscles of Stannius.     

Characterization and distribution of FMRFamide immunoreactivity in the rat central nervous system

FMRFamide immunoreactive material (irFMRFamide) was studied in rat brain and gastrointestinal tract. Highest irFMRFamide concentrations were found in tissues of the gastrointestinal tract and, in the brain, highest concentrations were found in the hippocampus, midbrain, brainstem and hypothalamus. High pressure liquid chromatographic characterization of irFMRFamide demonstrated that the immunoreactive material in brain, pancreas and duodenum was different from molluscan FMRFamide but it was also distinct from any known neuropeptide.     

Quantitative distribution of galanin-like immunoreactivity in the rat central nervous system

Using an antiserum directed against synthetic galanin (GAL) a sensitive radioimmunoassay was developed. The antiserum interaction with GAL was characterized by displacement curve characteristics and high performance liquid chromatography. Besides the main GAL-immunoreactive peak several small peaks with GAL-like immunoreactivity were observed. No cross-reactivity of the GAL-antiserum with several other peptides was observed. GAL-like immunoreactivity was measured in 37 microdissected areas of the rat central nervous system. High concentrations (greater than 2000 fmol/mg protein) were observed in the amygdaloid nuclei, the septum, globus pallidus, bed nuclei of the stria terminalis, all hypothalamic nuclei, the superior colliculus, locus coeruleus, the nucleus of the solitary tract and the neurointermediate lobe of the pituitary. Moderate concentrations (1000-2000 fmol/mg protein) were observed in the hippocampus, the nucleus accumbens and nucleus of the diagonal tract, the caudate-putamen, the central gray, the nucleus, tract and substantia gelatinosa of the spinal trigeminal nerve. The results generally correlate with those previously published by immunocytochemistry. The widespread distribution of GAL-like immunoreactivity in the rat central nervous system suggests an involvement of GAL in a variety of brain functions.     

Histamine immunoreactivity in the central nervous system of the spider Cupiennius salei

In this study, immunohistochemistry on Vibracut sections is used to demonstrate anti-histamine immunoreactivity in the brain of the spider, Cupiennius salei (Keys.) (Ctenidae). We describe a system of histamine-immunoreactive neurons within the central nervous system that consists of six omnisegmental neurons. These histamine-immunoreactive neurons form two subgroups: a dorsal system with two cells per hemisphere and a ventral system with only one cell per hemisphere. The cells have extended arborizations in the motor and sensory areas of all neuromeres in the suboesophageal ganglionic mass. We have also found histamine immunoreactivity in the photoreceptors of C. salei and suggest that histamine is a neurotransmitter of photoreceptors in all arthropods, since it is also known to occur in the photoreceptors of the other main arthropod taxa (Merostomata, Crustacea, and Insecta).