A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

Developmental profile of glucocorticoid binding to synaptic plasma membrane from rat brain.

The plasma membranes of several mammalian tissues including the brain are known to have specific binding sites for glucocorticoids. The developmental changes in specific glucocorticoid binding to synaptic plasma membrane (SPM) from rat brain were determined at various postnatal ages, using [3H]triamcinolone acetonide (TA) as the steroid ligand. The specific binding of the labeled glucocorticoid to SPM during the first 2 postnatal weeks was only 40% of the adult level. An increase of the specific binding occurred after day 15, and this developmental rise of binding reached the adult level approximately by the end of the fourth week. Methodologically, these developmental data are detailed in the present article to include nonspecific binding as well as specific binding. Scatchard analysis indicates that the developmental rise of the specific glucocorticoid binding was due to an increase in the membrane binding sites. The ontogenetic increase of membrane binding sites during postnatal brain development provides additional evidence that these binding sites have physiological significance in brain function.

Changes in activity and mRNA for rat tryptophan hydroxylase and aromatic L-amino acid decarboxylase of brain serotonergic cell bodies and terminals following neonatal 5,7-dihydroxytryptamine.

In the present study, we examined time-dependent changes in activity, mRNA and immunoreactivity of the serotonin biosynthetic enzymes, tryptophan hydroxylase (TPH) and aromatic L-amino acid decarboxylase (AADC) in dorsal raphe nucleus (DRN), caudal brainstem and hypothalamus, following intracisternal injection of 5,7-dihydroxytryptamine (5,7-DHT) in neonatal rats. TPH activity in central serotonergic cell bodies and terminals was reduced to 20-30% of control levels at 1-8 weeks after neonatal, low-dose 5,7-DHT injection (24 micrograms free base). In contrast, AADC activity was either not changed or decreased to 40% of control levels, depending on the region. In situ hybridization and immunocytochemical staining indicated that 5,7-DHT caused a marked reduction in TPH and AADC message levels as well as the number of 5-HT and AADC-immunoreactive cells within the DRN as early as 1 week after 5,7-DHT. Even 15 weeks after drug administration recovery did not occur. This apparent neuronal loss was region-specific suggesting that some serotonergic neurons are more resistant to neonatal 5,7-DHT treatment than others. Taken together, these studies indicate that neonatal treatment with 5,7-DHT produces a marked and permanent (up to 15 weeks) reduction in the number of central serotonergic neurons.

Early post-natal administration of 5,7-dihydroxytryptamine destroys 5-HT neurons but does not affect spatial memory.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) may play an important role in learning and memory. It has also been suggested that 5-HT abnormalities may mediate some aspects of the cognitive disorders associated with Korsakoff syndrome and Alzheimer's Disease. The effect of intracisternally applied 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT) on learning and memory in rodents was evaluated. Three-day-old rat pups were treated with pargyline (40 mg/kg, i.p.) followed by 5,7-DHT (50 micrograms/pup) and returned to the dam for a month. At 75 days of age, rats were tested on a learning set problem in the Morris water maze for 5 days followed by 30 days of testing in a 12-arm radial maze with 8 of the 12 arms baited. In the Morris water maze, the latency to locate the hidden platform did not differ significantly for 5,7-DHT treated and control rats (F less than 1.0). Similarly, 5,7-DHT treated rats performed comparably to controls on the 12-arm radial maze (F less than 1.0). At 106 days of age the assay of tryptophan hydroxylase activity in the dorsal raphe nuclei and hippocampus showed marked reduction (86%, 78%, respectively) in 5,7-DHT treated animals compared to vehicle injected controls. Immunocytochemical analysis was consistent with the biochemical results. In 5,7-DHT treated animals there was severe loss of neurons that bind 5-HT antibody in the dorsal and medial raphe nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids stimulate transcription of the rat phenylethanolamine N-methyltransferase (PNMT) gene in vivo and in vitro.

1. Phenylethanolamine N-methyltransferase (PNMT) is regulated by glucocorticoid hormones. This study investigates the ability of glucocorticoids to modulate transcription of the rat PNMT gene in vivo and in vitro. 2. In the adrenal glands of hypophysectomized (HPX'd) rats, the synthetic glucocorticoid dexamethasone (DEX) stimulates production of PNMT mRNA. Quantitative hybridization reveals that the levels of PNMT mRNA increase approximately threefold in total and poly(A)+RNA after 4 days of DEX treatment of HPX'd rats, a level which is maximal for this treatment. 3. ACTH, the hormonal stimulus of glucocorticoid biosynthesis in the adrenal cortex, enhances PNMT mRNA production to levels comparable to that achieved with DEX in this system. The steroid responsiveness of PNMT message production is specific for glucocorticoids. DEX also increases PNMT mRNA in the brain stem, although the magnitude and speed of response are lower than observed in the adrenal gland. 4. Additional confirmation of the inductive ability of glucocorticoids is demonstrated by the increase in PNMT immunoprecipitated following translation in vitro of adrenal RNAs from DEX-treated rats. Furthermore, the PNMT mRNA signal obtained by in situ hybridization histochemistry in adrenal sections and in primary cultures of dispersed rat adrenal medullae reveals that DEX effects on PNMT mRNA can be elicited both in vivo and in vitro. 5. Specifically, glucocorticoids exert their effects on expression of PNMT mRNA by elevating the rate of PNMT gene transcription: a 2.3-fold increase in PNMT transcription persists for 18 hr following DEX treatment of HPX'd rats. In summary, this study establishes that glucocorticoids directly and rapidly stimulate transcription of the rat PNMT gene.

Vesicular and cytoplasmic localization of neurotensin-like immunoreactivity (NTLI) in neurons postsynaptic to terminals containing NTLI and/or tyrosine hydroxylase in the rat central nucleus of the amygdala.

Neurotensin and catecholamines in the central nucleus of the amygdala (CNA) have both been implicated in the integration of autonomic responses to stress. We examined whether there might be a cellular substrate for interactions involving these putative neurotransmitters in the CNA. Sections of acrolein-fixed rat brain were processed either (1) for the ultrastructural localization of a rat antiserum against neurotensin using the peroxidase-antiperoxidase (PAP) method, or (2) for the dual localization of rat neurotensin antiserum and rabbit antiserum against the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), using the PAP method and immunoautoradiography. The rat polyclonal antiserum against neurotensin was shown in immunoblots to recognize neuromedin N and Lys-Arg-neurotensin (LANT-6) in addition to neurotensin. In single and dual labeling studies, the neurotensin-like immunoreactivity (NTLI) was detected in perikarya and processes. The NTLI was localized predominantly to dense core vesicles in one group of perikarya and dendrites, while a second group had labeling both in dense core vesicles and more diffusely throughout the cytoplasm. Terminals also showed NTLI, particularly in association with dense core vesicles. The labeled terminals formed primarily symmetric junctions with both cell bodies and dendrites. In the dual labeling study, perikarya contained only NTLI while terminals contained TH and/or NTLI. Terminals containing TH or NTLI separately innervated cell bodies and dendrites displaying NTLI, and formed separate or convergent inputs onto unlabeled neuronal targets. Terminals colocalizing both TH and NTLI formed junctions only on unlabeled dendrites. These findings show that in the rat CNA two populations of neurons differ with respect to their distribution of NTLI, and that the output from neurons containing NTLI is modulated by direct synaptic input from terminals containing neurotensin and/or catecholamines. Release of neurotensin and catecholamines, most likely dopamine, from the same or separate terminals on common targets in the CNA may account for certain similarities in their stress-related functions.

Ultrastructural localization of neurotensin-like immunoreactivity within dense core vesicles in perikarya, but not terminals, colocalizing tyrosine hydroxylase in the rat ventral tegmental area.

Within the rat ventral tegmental area (VTA), the parabrachial pigmentosus and paranigral subdivisions are known to differ in their functional responses to injected neurotensin. These subdivisions also vary in their connections with other brain regions and in their number of neurotensin-containing perikarya as seen by light microscopy. In both subdivisions, there may be intracellular as well as synaptic relations between dopamine and neurotensin. Dopaminergic neurons are known to be physiologically activated by neurotensin (NT) and may also contain this peptide. To characterize further the cellular relationships in each subdivision, we examined the ultrastructural immunocytochemical localization of a rat antiserum against NT and a rabbit antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in single sections. The NT antiserum was raised against the entire peptide sequence. Immunoblots showed that the antiserum recognized the original antigen as well as the related peptides neuromedin N and lysine 8- arginine 9- neurotensin 10-13 (LANT-6). In both the parabrachial pigmentosus and paranigral subdivisions, neurotensin-like immunoreactivity (NTLI) was localized predominantly in the large (80-100 nm) dense core vesicles using the peroxidase anti-peroxidase (PAP) method. In tissue labeled for NT by the PAP method and for TH by immunoautoradiography, serial section analysis revealed that all perikarya containing NTLI (n = 19) were also TH-positive. Three times as many perikarya colocalized NTLI and TH in the parabrachial pigmentosus subdivision (n = 15) as in the paranigral subdivision (n = 4). Occasionally, a perikaryon containing TH and NTLI could be found in direct apposition to a TH-labeled perikaryon without glial separation. In contrast to perikarya and dendrites, terminals showing NTLI (38 in parabrachial pigmentosus, 29 in paranigral) lacked detectable TH labeling. Of the terminals containing NTLI whose synaptic junctions could be identified, 48% were symmetric and 10% were asymmetric. The targets of these terminals included perikarya and dendrites lacking detectable immunoreactivity (69% in parabrachial pigmentosus, 55% in paranigral), immunolabeled for TH (26% in parabrachial pigmentosus, 38% in paranigral) or containing both NTLI and TH (5% in parabrachial pigmentosus, 7% in paranigral). Single terminals containing NTLI sometimes contacted more than one neuronal target, some of which were apposed to each other without glial separation. TH-labeled terminals synapsed onto double-labeled perikarya in the paranigral subdivision, but were not observed to do so in the parabrachial pigmentosus subdivision.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuronal-glial interactions: quantitation of astrocytic influences on development of catecholamine neurons.

Effects of astroglia on the morphological and biochemical differentiation of catecholamine neurons from embryonic rat mesencephalon were studied in vitro, and compared to results obtained with fibroblasts. Neurite outgrowth and complexity were measured using computer-assisted morphometry on tyrosine hydroxylase immunoreactive neurons growing on preformed monolayers of astrocytes or fibroblasts. The morphological differentiation of these neurons was stimulated by the presence of astrocytes, and this effect was evident in various cellular compartments, including the size of the cell soma, length of neurites and neuritic segments, and the numbers of these segments. Tyrosine hydroxylase activity was measured biochemically in these cultures and was also found to be stimulated by the presence of astroglial monolayers. The implication of these results for the understanding of specific neuron-glial interactions during embryonic brain development is discussed.

Serotonergic innervation of the rat caudate following a neonatal 6-hydroxydopamine lesion: an anatomical, biochemical and pharmacological study.

6-Hydroxydopamine (6-OHDA) treatment of neonatal rats resulted in a dose-related loss of striatal dopamine (DA). These reductions corresponded closely with the loss of tyrosine hydroxylase-containing terminals at this brain site. Striatal serotonin (5-HT) concentration increased only after DA was maximally depleted by the highest dose of 6-OHDA. Quantitative immunohistochemistry revealed that the increased 5-HT content after neonatal 6-OHDA lesioning was due to a proliferation of 5-HT nerve terminals. The density of immunoreactive 5-HT-containing terminals appeared to increase more than did the 5-HT content. The present study examined whether 5-HT hyperinnervation was playing a role in behavioral responses induced by D1-DA agonists and antagonists in neonatally lesioned rats, because reports have suggested that these drugs may interact with 5-HT receptors. However, SCH-23390, the D1-DA antagonist (0.3 mg/kg), did not alter behavioral responses to 5-HTP and SKF-38393 (3 mg/kg), a D1-DA agonist did not produce any signs of activating 5-HT receptors in 5,7-DHT-lesioned rats. These data indicate that these compounds affecting D1-DA receptors do not have a significant effect on 5-HT function at doses which have maximal effects on D1-DA receptor function. Pretreatment with the 5-HT antagonist methysergide did not produce a change in apomorphine-induced locomotion and did not antagonize the self-mutilation or the other behaviors produced by L-DOPA or SKF-38393 in neonatally lesioned rats, suggesting that 5-HT hyperinnervation is not responsible for these drug-induced changes in neonatal 6-OHDA-lesioned rats.

Atrial natriuretic peptide (ANP) in relation to blood pressure: a study in middle-aged men with normal and elevated blood pressure.

In order to investigate the potential role of atrial natriuretic peptide (ANP) in mild to moderate essential hypertension, a study was conducted in groups of normotensive and hypertensive middle-aged men born in 1926 and 1927. Venous plasma concentrations of immunoreactive ANP (irANP) were studied in relation to measurements of cardiac structure and function, urinary electrolytes as well as some cardiovascular hormones. Plasma irANP did not differ between normotensive controls (31 +/- 14 pmol l-1) and borderline or untreated hypertensive patients. However, irANP concentrations were slightly but significantly (P less than 0.05) lower in the borderline (26 +/- 8 pmol l-1) compared to the untreated established hypertensives (35 +/- 14 pmol l-1). No relationships were found between irANP and blood pressure, indices of left ventricular structure and function or hormone parameters in subgroups or the whole study group. Our data do not support the view that plasma irANP is increased in uncomplicated essential hypertension, since our groups of borderline or established hypertensive middle-aged men without major cardiac involvement did not differ in irANP concentrations compared to normotensive controls. Thus, during the development or in the early stages of essential hypertension, ANP secretion does not seem to be abnormal.

Clonidine-specific antisera recognize an endogenous clonidine-displacing substance in brain.

An endogenous substance in brain, clonidine-displacing substance, binds to the same receptor populations as clonidine and is biologically active. Since receptor binding sites can be modeled by using specific antiligand antibodies, we tested the hypothesis that polyclonal antibodies raised in rat and rabbit against the clonidine analog p-aminoclonidine coupled to hemocyanin would recognize compounds structurally related to clonidine, including clonidine-displacing substance. Binding to anti-p-aminoclonidine antibodies was examined by using a competitive radioimmunoassay with tritiated p-aminoclonidine as the radioligand. Central vasodepressor agents that, like clonidine, are known to bind with high affinity to both imidazole sites and alpha 2-adrenergic receptors in brain inhibited radioligand binding to anti-p-aminoclonidine antibodies. All of these agents contain imidazol(in)e and phenyl ring moieties as part of their chemical structures (e.g., oxymetazoline); a number of other compounds without one or both of these rings failed to cross-react with the antisera. Clonidine-displacing substance, partially purified from bovine brain, also inhibited specific radioligand binding to anti-p-aminoclonidine antibodies. The inhibition was dose dependent and high affinity (IC50, 4 Units). The endogenous substance had no effect on the apparent affinity of the antibodies for the radioligand, but blocked a specific number of binding sites. Immunoprecipitation experiments showed that authentic clonidine-displacing substance, that which displaces tritiated p-aminoclonidine binding to membrane receptors, is recognized by anti-p-aminoclonidine antibodies. We conclude that a unique subset of structural determinants required for ligand interaction with both imidazole and alpha 2-adrenergic receptors is critical for binding to anti-p-aminoclonidine antibodies, and that since clonidine-displacing substance is recognized by highly clonidine-specific antisera, it may also contain these determinants within its structure, namely the imidazol(in)e and phenyl ring systems.

Tryptophan hydroxylase activity and 5-hydroxytryptamine-immunoreactive cells in the superior cervical ganglion of hydrocortisone-treated neonatal rats.

Early postnatal glucocorticoid injections led to a large increase in the number of 5-hydroxytryptamine(5-HT)-immunoreactive small cells in the rat superior cervical ganglion. Tryptophan hydroxylase (TPH) activity in ganglia from animals treated with glucocorticoids was not significantly different from saline-injected controls. Both 5-HT immunoreactivity and TPH activity were decreased in ganglia from animals treated with glucocorticoid and the TPH inhibitor parachlorophenylalanine, but not in animals treated with 5-HT uptake inhibitor fluoxetine. These results suggest that 5-HT is synthesized in the small intensely fluorescent (SIF) cells.

Cotransmitters: differential effects of serotonin (5-HT)-depleting drugs on levels of 5-HT and TRH and their receptors in rat brain and spinal cord.

Following codepletion of endogenous serotonin (5-HT, greater than 90%) and thyrotropin-releasing hormone (TRH, 66%) by neonatal treatment with the serotonergic neurotoxin, 5,7-dihydroxytryptamine (DHT), a 33% (n = 12, P less than 0.01) increase in specific TRH receptor binding was observed in adult rat spinal cord (SC) homogenates. A 20-21% increase in TRH receptors was also observed in the medulla/pons (MP) (n = 12, P less than 0.05) and midbrain (MB) (n = 12, P less than 0.02), but no changes were detected in 6 rostral brain regions. The depletion of 5-HT after DHT-treatment was also accompanied by a 34-42% increase in 5-HT1 binding in the SC, MP and MB. Eadie-Hofstee analysis revealed that the changes in TRH receptor levels observed after DHT-lesions were due to an increase in receptor number rather than any significant changes in receptor affinity. Chronic treatment of adult rats with the 5-HT-depleting drugs, p-chlorophenylalanine (PCPA) and reserpine, produced a 90-97% decrease in 5-HT in the SC, MP and MB and elevated 5-HT1 binding above controls in these tissues. However, neither drug treatment caused any significant alterations in the levels of TRH or its receptors in any of these tissues. In conclusion, these results have provided further support for the coexistence of 5-HT and TRH in the MP and SC and revealed possible new areas of such colocalization in the MB. Furthermore, these data have demonstrated that only DHT-treatment, as apposed to PCPA or reserpine, can produce long-lasting codepletion of 5-HT and TRH with simultaneous compensatory up-regulation of their receptor systems in the SC and other caudal tissues.

Haemodynamics and plasma ANP (atrial natriuretic peptide) after acute blood volume expansion in normotensive and spontaneously hypertensive rats.

Atrial natriuretic peptide (ANP) was measured in plasma during acute volume load in conscious, spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. During basal conditions immunoreactive ANP were similar in the SHR (630 +/- 56 pmoles l-1) and the WKY (657 +/- 114 pmoles l-1) groups. An acute 10% and 20% whole blood volume expansion resulted in a linear increase in immunoreactive plasma ANP in the WKY. In the SHR the increase in plasma ANP was attenuated during the 20% volume load. During the 10% and 20% volume load central venous pressure (CVP), central blood volume (CBV) and cardiac output increased relatively more in the SHR compared with the WKY group. In contrast, the increase in peripheral blood volume (PBV) and decrease in heart rate (HR) was attenuated in the SH rats. In the SHR group there was a shift of the ANP vs. CVP and ANP vs. CBV curves to the right compared with the WKY. We conclude that acute volume loading is a potent stimulus for ANP release in WKY as well as SHR. However, in the SHR, ANP release was blunted in spite of the increased centralization of the volume load in this rat strain. Thus, the decreased responsiveness of the ANP hormonal system may contribute to the development and maintenance of hypertension in this genetic form of hypertension.

Gamma-aminobutyric acid in the medial rat nucleus accumbens: ultrastructural localization in neurons receiving monosynaptic input from catecholaminergic afferents.

Neurons containing gamma-aminobutyric acid (GABA) in the medial portion of the adult rat nucleus accumbens were characterized with respect to their ultrastructure, sites of termination, and catecholaminergic input. Antisera against GABA-conjugates and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), were localized within single sections by means of peroxidase-antiperoxidase (PAP) and immunoautoradiographic labeling methods. Peroxidase reaction product indicating GABA-like immunoreactivity (GABA-LI) was seen in medium-size (15-20 microns) perikarya containing either round and unindented or invaginated nuclear membranes. The cells with invaginated nuclei were few in number and usually exhibited more intense peroxidase reaction product in sections collected at the same distance from the surface of the tissue. Reaction product for GABA was also detected in proximal (1.5-3.0 microns) dendrites, axons, and terminals. Terminals with GABA-LI formed symmetric junctions on perikarya, proximal dendrites, and dendritic spines of neurons that usually lacked detectable immunoreactivity. Many of the GABAergic terminals also were apposed directly to other unlabeled terminals and to terminals exhibiting either peroxidase labeling for GABA or immunoautoradiographic labeling for TH. Many of the unlabeled terminals associated with the GABAergic axons formed asymmetric junctions on dendritic spines. From 138 TH-labeled, principally dopaminergic terminals that were examined in the medial nucleus accumbens, 4% were associated with the somata of GABAergic neurons and another 14% formed symmetric junctions with proximal dendrite showing GABA-LI. The remaining TH-immuno-reactive terminals either lacked recognizable densities or formed symmetric synapses on unlabeled dendrites and spines. A few of the unlabeled dendrites, as well as those containing GABA-LI, received symmetric synapses from both catecholaminergic and GABAergic terminals. We conclude that in the medial portion of the rat nucleus accumbens, GABA is localized to two morphologically distinct types of neurons, one or both of which receive monosynaptic input from catecholaminergic afferents, and that GABAergic terminals form symmetric synapses on other principally non-GABAergic neurons. The results also support earlier physiological evidence showing that GABA may modulate the output of other GABAergic and non-GABAergic neurons through presynaptic associations.

Distribution of glucagonlike peptide I (GLP-I), glucagon, and glicentin in the rat brain: an immunocytochemical study.

Although glucagonlike immunoreactants (GLIs) are present in the central nervous system of several mammalian species, their structural relationship with pancreatic proglucagon is not defined, and their precise anatomical distribution has not been studied extensively. To obtain further information about the structure and biological significance of brain GLIs, the anatomical distribution of three different antigenic determinants of pancreatic proglucagon--glucagonlike peptide I (GLP-I), glucagon, and glicentin--was mapped in the brain of colchicine-treated rats by immunocytochemistry using the avidin-biotin-peroxidase method. Neuronal cell bodies immunoreactive with antisera specific for GLP-I, glucagon, and glicentin were found only in the caudal medulla oblongata. Within the caudal medulla immunostained cell bodies were found at levels from approximately 0.55 mm rostral to the obex to 0.45 mm caudal to the obex, and were located within the nucleus of the solitary tract (NTS) and the dorsal (MdD) and ventral (MdV) parts of the medullary reticular nucleus. The NTS contained three times more immunoreactive cell bodies than the MdD and MdV, and these cell bodies were located in the midline, medial, and lateral subnuclei of the caudal third of the NTS. Immunostaining of the same cell bodies in paired adjacent sections incubated with GLP-I and glucagon antisera or glucagon and glicentin antisera provided evidence for coexistence of the three antigens within the same neurons of the NTS. Nerve fibers and terminals immunoreactive with GLP-I, glucagon, and glicentin antisera were widely distributed throughout the rat brain and there was no discernible difference in the distribution of fibers and terminals immunoreactive with each of the three antisera. The highest densities of immunostained fibers and terminals were observed in the hypothalamus, thalamus, and septal regions, and the lowest in the cortex and hindbrain. The localization of neuronal cell bodies containing GLP-I, glucagon, and glicentin within the NTS and the MdD and MdV, and the extensive distribution of immunoreactive fibers and terminals throughout the rat brain suggest a role for these peptides in the integration of autonomic as well as central nervous system functions.

Differential afferent regulation of dopaminergic and GABAergic neurons in the mouse main olfactory bulb.

Peripheral deafferentation of the mouse main olfactory bulb following intranasal irrigation with ZnSO4 produced profound decreases in tyrosine hydroxylase activity and immunoreactivity in intrinsic dopamine neurons normally localized to the juxtaglomerular region of the bulb. In contrast, only modest alterations in GABA-immunoreactivity and glutamic acid decarboxylase (GAD) activity were observed in the same region. In fact, when GAD activity was expressed per mg tissue, a reflection of enzyme concentration, no changes in activity were observed 3 weeks postlesion and only relatively modest decreases in specific activity were found following long survival times (4 months). When the data were expressed per bulb, as an indication of the total amount of enzyme present, GAD activity and bulb weight exhibited similar reductions. Olfactory marker protein levels, determined as an indication of the completeness of the deafferentation, were at or below the limits of detection in all lesioned mice. These data indicate that afferent regulation of transmitter expression in the juxtaglomerular neurons of the olfactory system is phenotype specific.

A specific antiserum recognizes clonidine-displacing substance: implications for the structure of the brain's own clonidine.

A polyclonal antiserum was raised in rabbit against the clonidine analog p-aminoclonidine (PAC) coupled to hemocyanin. The antiserum (anti-PAC3) exhibited high affinity for unconjugated [3H]PAC (Kd 0.32 +/- 0.07 nM) in a rapid-filtration radioimmunoassay. Competition experiments showed that PAC, clonidine, and naphazoline cross-reacted with the anti-PAC3 antiserum, whereas a number of other structurally related compounds did not. An endogenous clonidine-displacing substance (CDS) partially purified from bovine brain also inhibited specific [3H]PAC binding to anti-PAC3 in a dose-dependent manner. Thus, (a) anti-PAC3 antiserum is specific for clonidine and closely related compounds, and (b) CDS may structurally resemble clonidine since it is recognized by this highly specific antiserum.

Identification of somatomedin/insulin-like growth factor immunoreactive cells in the human fetus.

Somatomedins/insulin-like growth factors (Sm/IGFs) are present in blood and in extracts from multiple tissues of the human fetus and induce the proliferation of cultured human fetal cells. To identify the cellular location of immunoreactive Sm/IGF in human fetal tissues, we have performed immunocytochemistry in tissues from prostaglandin-induced human fetal abortuses of 12 to 20 wk in gestation. Every tissue studied except the cerebral cortex contains Sm/IGF immunoreactive cells. Cells staining positively include hepatocytes, hepatic hemopoietic cells, columnar epithelia of the pulmonary airways, intestine and kidney tubules, adrenal cortical cells, dermal cells, skeletal and cardiac muscle fibers, and pancreatic islet and acinar cells. Immunostaining was specific for Sm/IGFs, but because of the cross-reactivity of the antibodies it was not possible to determine whether the immunoreactivity represented Sm-C/IGF I, IGF II, or both. Liver contained the greatest proportion of immunoreactive cells, while the thymus and spleen had only a few immunostained cells. With the possible exception of dermal and some adrenal cortical cells, the immunoreactive cells do not appear to be the primary sites of Sm/IGF synthesis, because parallel in situ hybridization histochemical studies using Sm/IGF oligodeoxyribonucleotide probes show that Sm/IGF mRNAs are localized predominantly to fibroblasts and mesenchymal cells. Therefore the immunoreactive cells identified in this study may define sites of action of Sm/IGFs.

Glutamate-positive neurons in the somatic sensory cortex of rats and monkeys.

The morphology and laminar distribution of neurons labeled with an antiserum prepared against glutamic acid (Glu) conjugated to keyhole limpet hemocyanin have been studied in the somatic sensory cortex of rats and monkeys. In both species, the vast majority of immunostained neurons are pyramidal; some nonpyramidal neurons are also present. Positive neurons are observed in all cortical layers, although variations are found in the percentage of Glu-positive neurons in the different layers. In rats they are most numerous in layer V (36%), followed by layer II (33%), layer III (32%), and layer VI (29%). In layer IV, 13% of all neurons are positive. Immunoreactive neurons are very sparse in layer I. In monkeys, Glu-positive neurons represent 51% of all neurons in layer V, 49% in layer III, 40% in layers II and VI, and 19% in layer IV. No differences are evident in the laminar distribution of Glu-positive neurons among cytoarchitectonic areas 3a, 3b, 1, and 2. As in rats, Glu-positive neurons are very sparse in layer I. Since Glu and GABA metabolisms are closely related, double-labeling experiments were performed in which thin, adjacent paraffin sections were stained alternately with the anti-Glu serum and with an anti-GABA serum. The 2 populations are almost completely segregated, even though a small fraction of neurons (less than 5%) are labeled by the antisera against both antigens.(ABSTRACT TRUNCATED AT 250 WORDS)