guanylate cyclase activity alters Fos expression by rat hypothalamic vasopressinergic neurons during acute glucose deprivation

Journal of Chemical Neuroanatomy 17 (1999) 13 – 19 www.elsevier.com/locate/jchemneu

Pharmacological manipulation of central nitric oxide/guanylate cyclase activity alters Fos expression by rat hypothalamic vasopressinergic neurons during acute glucose deprivation Karen P. Briski * Di6ision of Basic Pharmaceutical Sciences, College of Pharmacy, Northeast Louisiana Uni6ersity, Monroe, LA 71209, USA Received 19 August 1998; received in revised form 1 February 1999; accepted 13 June 1999

Abstract Neurohypophyseal secretion of arginine vasopressin is stimulated by decreased systemic glucose availability. Nitric oxide is produced by paraventricular and supraoptic magnocellular neurons, and is implicated in central mechanisms controlling plasma vasopressin and glucose levels. The current studies investigated the role of this neurotransmitter in glucoprivic induction of AP-1 transcriptional activity in hypothalamic vasopressinergic neurons by examining whether pharmacological manipulation of central nitric oxide/guanylate cyclase/cGMP signaling alters nuclear accumulation of Fos immunoreactivity in these cells. Adult male rats pretreated by intraventricular administration of saline exhibited extensive colabeling of vasopressinergic neurons in both brain sites for Fos following systemic injection of the glucose antimetabolite, 2-deoxy-D-glucose. Pretreatment with the nitric oxide donor, SIN1, resulted in decreased numbers of paraventricular and supraoptic Fos-positive vasopressinergic neurons during glucoprivation. In other animals, coadministration of SIN1 and the nitric-oxide sensitive guanylate cyclase inhibitor, ODQ, prior to the antimetabolite reversed these inhibitory effects of SIN1 on Fos expression by these cells. Intracerebral administration of ODQ alone did not significantly enhance expression of Fos by vasopressinergic neurons in either site. The present studies demonstrate that exogenous activation of the nitric oxide/guanylate cyclase/cGMP pathway in the brain inhibits nuclear accumulation of the AP-1 transcription factor, Fos, in vasopressinergic neurons during cellular glucopenia, and suggest that this neurotransmitter is critical for transactivational effects of glucoprivation on these neuropeptidergic neurons. © 1999 Elsevier Science B.V. All rights reserved. Keywords: 2-deoxy-D-glucose; Fos; Nitric oxide synthase; ODQ; Paraventricular nucleus; SIN1; Vasopressin

1. Introduction In the central nervous system (CNS), the soluble neurotransmitter, nitric oxide (NO), is generated from the amino acid, L-arginine, by catalytic action of nitric oxide synthase (NOS), a nicatinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase), under Ca2 + -dependent conditions (Bredt and Snyder, 1990). NO is implicated in the central regulation of hypothalamic arginine vasopressin (AVP) and oxytocin (OXY) secretion by reports that pharmacological inhibitors of NOS activity increase basal plasma levels of AVP and OXY (Kadekaro et al., 1997), facilitate deple* Tel.: +1-318-3423283; fax: + 1-318-3421606. E-mail address: [email protected] (K.P. Briski)

tion of both neuropeptides from the pars nervosa during hyperosmotic challenge (Kadowski et al., 1994), increase pituitary secretion of AVP during insulin-induced hypoglycemia (Chiodera et al., 1994), and elevate plasma OXY levels during water deprivation and mild hyperosmotic stimulation (Kadekaro et al., 1997). Evidence that magnocellular neurons in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei exhibit cytoplasmic NOS (Sagar and Ferriero, 1987; Arevalo et al., 1992; Miyagawa et al., 1994) suggests that NO may fulfill paracrine/autocrine regulatory functions within these neural structures. Recent findings that NO elevates hypothalamic tissue levels of the second messenger nucleotide, cGMP (Bhat et al., 1996) support a role for the guanylate cyclase/cGMP signal transduction pathway in intracellular mechanisms of NO action on target cells in this region of the brain.

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Recent studies show that circulating glucose and glucagon levels are regulated by AVP (Nagai et al., 1995), and that hypothalamic neuropeptide secretion is elevated in response to glucose substrate imbalance (Nussey et al., 1986; Engler et al., 1989; Caraty et al., 1990; Chiodera et al., 1992). Observations that intracerebroventricular (icv) administration of the NOS inhibitor, Nv-nitro-L-arginine methyl ester (L-NAME), increases plasma glucose levels (Uemura et al., 1997) suggest that NO may also be involved in central mechanisms governing blood glucose concentrations. Recent studies in our laboratory indicate that NOS-containing neurons in several hypothalamic structures are functionally responsive to pharmacological glucopenia, indicated by the transcriptional activation of cells in the PVN and SON exhibiting this cytoplasmic enzyme. The inducible immediate-early gene product, Fos, is expressed by neurons in both the PVN and SON following injection of the competitive glycolytic enzyme inhibitor, 2-deoxy-D-glucose (2DG), results that demonstrate the transactivational effects of decreased cellular glucose oxidation on these neurons (Ritter and Dinh, 1994; Briski, unpublished observations). The Fos protein is synthesized in response to plasma membrane receptor-mediated stimulation of cytoplasmic protein kinase C- and other kinase-signaling pathways, and forms heterodimeric complexes with jun gene family products that regulate gene transcription via AP-1 binding sites on DNA (Morgan and Curran, 1991). In the following experiments, we examined the role of central NO and guanylate cyclase/cGMP signaling in glucoprivic activation of the Fos stimulus-transcription cascade in AVP neurons in the PVN and SON by investigating the effects of icv administration of the NO donor, 3-morpholinosyndronimine (SIN1), in the presence of absence or the NO-sensitive guanylate cyclase inhibitor, ODQ, on nuclear accumulation of Fos-immunoreactivity (-ir) within these cells during pharmacological glucopenia.

2. Materials and methods

2.1. Animals Adult male Sprague – Dawley rats (220 – 240 g bw) were purchased from Simonsen Laboratories (Gilroy, CA, USA). The animals were housed 2/3 per cage under a 14 h light/10 h dark schedule (lights on at 05.00 h), and allowed free access to standard rat chow and water. Ten days after arrival, the rats were anesthetized by ip injection of ketamine:xylazine (0.1 ml/100 g bw, 100 mg ketamine:10 mg xylazine/ml; Henry Schein, Port Washington, NY, USA), and implanted with hand-held PE-20 polyethylene cannulas directed toward the left lateral cerebral ventricle. Seven days after

surgery, the accuracy of cannula placement was verified by assessment of drinking behavior induced by administration of 100 ng angiotensin II.

2.2. Experimental design At 09:00 h on the tenth day after surgery, groups of rats (n= 10/group) were injected icv with the following drugs in a 2.0 ul total volume: group 1: SIN1 [100 ug (Yokotani et al., 1997); Research Biochem., Natick, MA, USA; in saline]; group 2: ODQ [100 uM (Fedele et al., 1997); RBI; in propylene glycol (PG)]; group 3: SIN1 plus ODQ; in PG. Groups 4 and 5 were treated with saline or propylene glycol (PG) icv, respectively. Twenty minutes after these drug treatments, n=5 rats in each group were injected ip with 400 mg 2DG/kg [Sigma, St Louis, MO, USA (Ritter and Dinh, 1994)], while the remainder were injected with saline alone. Two hours after antimetabolite injection, the animals were anesthetized with sodium pentobarbital (75 mg/kg bw, ip), then perfused transcardially with 9.0% saline supplemented with 2.0% sodium nitrite, followed by 0.1 M phosphate buffer, pH 7.6, containing 4% paraformaldehyde and 0.2% picric acid. The brains were post-fixed overnight in fresh fixative, sunk in 25% sucrose, and cut into 25 mm serial sections on a Reichert–Jung freezing microtome. Tissue sections were cryoprotected in 30% sucrose prior to storage at − 20°C.

2.3. Dual-antigen immunocytochemistry Every sixth section through the anterior hypothalamus (1 in 6 series) was washed stringently in 0.05 M Tris–buffered saline, pH 7.6 (TBS), then incubated for 30 min with normal goat serum (Vector rabbit Elite kit, PK-6101, Vector Laboratories, Burlingame, CA, USA) at room temperature. The tissues were then incubated for 48 h at 4°C, under gentle rotation, with a rabbit polyclonal antiserum against human Fos4 – 17 (1:100 000, Ab-5, Oncogene Sciences, Cambridge, MA, USA), diluted in TBS containing 0.05% Triton X-100 (TBS-Tx). The sections were subsequently incubated sequentially with biotinylated goat-anti rabbit second antibody and ABC reagent from rabbit Elite ABC kits, diluted per instructions with TBS, for 60 min each. Nuclear Fos antigenic sites were visualized by processing with TBS containing 0.014% 3,3%-diaminobenzidine hydrochloride (DAB), 0.01% hydrogen peroxide (H2O2), 0.4% nickel ammonium sulfate hexahydrate, and 0.23% sodium acetate trihydrate (Hrabovszky et al., 1995) for 2–5 min. After quenching of residual peroxidase with 0.5% H2O2 for 15 min, the sections were rinsed extensively in TBS, incubated for 30 min with normal goat serum, then exposed to rabbit polyclonal antisera against AVP (1:10 000, prod. no. 20069, lot no. 704156) [INCstar,

K.P. Briski / Journal of Chemical Neuroanatomy 17 (1999) 13–19

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Table 1 Effects of pharmacological manipulation of central NO/guanylate cyclase/cGMP signaling on expression of Fos-ir by hypothalamic vasopressinergic neurons in 2DG-treated male rats Neural structure Treatment groups

Paraventricular nucleus Post. Magnocell. Div. Posterior subnucleus Supraoptic nucleus

Vehicleicv+2DGip

SIN1icv+2DGip

ODQicv+2DGip

(SIN1+ODQ)icv+2DGip

AVP-ir a

AVP-+Fos-ir b

AVP-ir

AVP-+Fos-ir

AVP-ir

AVP-+Fos-ir

AVP-ir

AVP-+Fos-ir

290 9 31

226 9 24

279 9 30

529 4c

264 928

241 9 25

282 9 31

183 9 21

82 910

52 96

76 9 9

24 9 4c

92 913

59 9 7

86 9 10

27 9 4c

238 926

162918

217 9 23

659 8c

249 927

155 917

223 924

136 916

Mean numbers of AV-immunopositive neurons 9 SEM for n = 5 rats. Mean numbers of neurons colabeled for cytoplasmic AV- and nuclear Fos-ir9 SEM for n = 5 rats. c PB0.05, compared to double-labeled cells in the 2DG+vehicle group. a

b

Stillwater, MN], diluted in TBS, for 48 h at 4°C. The tissues were reacted sequentially with goat anti-rabbit second antibody and ABC reagent, for 60 min each. Cytoplasmic antigenic sites for AVP were labeled magenta by incubation for 3 – 5 min with reagents from Vector’s VIP substrate kit (PK-4600). The sections were rinsed sequentially in TBS and distilled water, mounted on gelatin-coated glass slides, then covered with Crystal Mount (Fisher Scientific, Pittsburg, PA) and dried on a slide warmer. The sections were examined and photographed with a Zeiss Axioskop brightfield microscope. Immunostaining controls included substitution of the primary antisera with normal rabbit serum, and sequential elimination of the biotinylated second antibody and ABC reagent from both staining series.

2.4. Statistics Mean counts of (1) total AVP neurons, e.g. Fos-negative plus Fos-positive, and (2) AVP neurons colabeled for Fos were obtained from 1 in 6 rostro-caudal series of sections through individual neural structures, and values for each structure were compared between 2DGtreated groups by one-way analysis of variance, followed by Duncan’s multiple range test.

3. Results Animals treated by systemic injection of the vehicle, saline, exhibited negligible immunolabeling of vasopressinergic neurons in both the posterior magnocellular (PVNpm; Fig. 1A) and caudal subnuclear division of the PVN. As shown in this figure, representative AVP-immunopositive neurons in the PVNpm of a saline-treated control exhibited a lack of demonstrable nuclear staining for Fos. The data in Table 1 show that injection of the glucose antimetabolite, 2DG, resulted in colabeling of AVP-positive neurons in both structures. In Fig. 1C and D, arrowheads depict Fos expression by vasopressinergic cells in the PVNpm and caudal subnucleus, respectively, while arrows indicate AVP-negative cells that are immunostained for this nuclear protein. As shown in Fig. 1B, Fos-positive AVP neurons in the drug-treated rats (arrowheads) were characterized by separate magenta and brown/black labeling, indicative of the distribution of AVP- and Fos-ir within cytoplasmic and nuclear cell compartments, respectively. Table 1 shows that mean numbers of AVP-immunopositive neurons did not significantly differ among groups pretreated with vehicle or various drugs prior to 2DG. Icv administration of the NO donor, SIN1, re-

Fig. 1. Expression of the nuclear transcription factor, Fos, by PVN vasopressinergic neurons during acute glucose deprivation. Treatments are indicated in the top right-hand corners of panels A–D. Arrows in panel A depict scattered colabeled AVP neurons following saline injection. Arrowheads in panels 1B, C and depict colabeled vasopressinergic neurons in 2DG-injected rats; arrows in the same panels show Fos-positive nuclei of nonvasopressinergic neurons. The scale bars in panels A, C, and D equal 80 mm, while the bar in panel B equals 40 mm. Fig. 2. Effects of pharmacological manipulation of central NO/guanylate cyclase/cGMP signaling on Fos expression by PVN vasopressinergic neurons during acute glucose deprivation. Treatments are indicated in the top right-hand corners of panels A-D. Arrows in panel A show minimal Fos expression by nonvasopressinergic neurons in the PVNpm following icv administration of SIN1; in this figure, scattered AVP-positive neurons exhibit faint nuclear staining for Fos. In panels C and D, arrows indicate dual-labeled vasopressinergic neurons, while arrowheads depict Fos-positive nonvasopressinergic neurons following 2DG injection of SIN1 plus ODQ vs. SIN1-pretreated rats, respectively. The scale bars equal 80 mm.

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Figs 1 and 2.

K.P. Briski / Journal of Chemical Neuroanatomy 17 (1999) 13–19

sulted in negligible nuclear immunostaining within both the PVNpm (Fig. 2B) and PVN caudal subnucleus for Fos. The data in Table 1 indicate that in each structure, mean numbers of vasopressinergic neurons expressing Fos during acute glucose deprivation were decreased by SIN1 pretreatment, compared to animals pretreated with vehicle alone. Representative Fos-positive AVP neurons in the PVNpm of a SIN1 pretreated, 2DG-injected animal are indicated by arrows in Fig. 2D; while these cells were morphologically similar to dual-labeled neurons in this brain site in animals pretreated with vehicle prior to 2DG, their numbers were comparatively diminished. In the same figure, arrowheads depict nonvasopressinergic neurons (arrowheads) in the PVNpm that expressed Fos in response to acute glucose deprivation. Icv administration of the NO-sensitive guanylate cyclase inhibitor, ODQ, did not elicit Fos expression in either the PVNpm or caudal subnucleus, while Table 1 reveals that pretreatment with ODQ did not enhance mean numbers of Fos-positive AVP neurons in either structure in the glucoprivic rats. Icv administration of the combination of SIN1 and ODQ resulted in faint immunostaining of a small proportion of AVP neurons in the PVNpm for Fos (Fig. 2A), but no labeling of vasopressinergic neurons in the caudal subnucleus. The results in Table 1 indicate that mean numbers of dual-labeled vasopressinergic neurons in the PVNpm were not different between animals pretreated with SIN1 plus ODQ versus those pretreated with vehicle only, whereas numbers of Fos-positive AVP neurons in the caudal subnucleus of animals given the drug combination remained significantly less than those counted in the vehicle-pretreated controls. Fig. 2C shows colabeling of vasopressinergic (arrows) and nonvasopressinergic neurons (arrowheads) n the PVNpm for nuclear Fos-ir following 2DG injection of animals pretreated with SIN1 plus ODQ. Similar findings were observed in the SON. The data in Table 1 indicate that mean numbers of AVP-immunopositive neurons in the SON were not significantly modified by the various pretreatments, relative to the vehicle-pretreated controls. Animals pretreated with the NO donor, SIN1, exhibited a reduction in mean numbers of dual-labeled vasopressinergic neurons in this brain site versus the vehicle-pretreated rats following injection of 2DG, whereas these numbers did not differ between the groups pretreated with the SIN1 and ODQ combination or with vehicle alone. This table also shows that pretreatment with ODQ did not result in greater numbers of Fos-positive AVP neurons in the SON following injection of 2DG. Fig. 3A depicts colabeling of several SON AVP neurons for Fos (arrow) during glucoprivation, while Fig. 3B shows that pretreatment with SIN1-attenuated expression of this nuclear protein in these cells. Fig. 3C shows that pretreatment with SIN1 plus ODQ-enhanced immunostaining of vasopressinergic neurons for Fos following injection of 2DG.

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Fig. 3. Effects of pharmacological manipulation of central NO/guanylate cyclase/cGMP signaling on Fos expression by SON vasopressinergic neurons during acute glucose deprivation. Treatments are indicated in the top right-hand corners of panels A – C. Arrows in panels A and C depict Fos expression by AVP-positive neurons in vehicle versus SIN1 plus ODQ-pretreated rats. Panel B shows that pretreatment with SIN1 alone decreased immunolabeling of vasopressinergic neurons for Fos following injection of 2DG. The scale bars equal 80 mm.

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4. Discussion The current studies provide evidence that pharmacological manipulation of central NO/guanylate cyclase/ cGMP activity alters glucoprivic induction of Fos expression by vasopressinergic neurons in the hypothalamic PVN and SON. The results show that animals pretreated by icv delivery of the NO donor, SIN1, exhibited decreased nuclear immunostaining of AVP neurons in both neural structures for Fos following injection of 2DG, whereas coadministration of SIN1 and the NO-sensitive guanylyl cyclase inhibitor, ODQ, prior to antimetabolite injection reversed inhibitory effects of SIN1 on numbers of cells in the PVNpm and SON that were colabeled for the AVP and Fos anti-gens. These data reveal that exogenous activation of NO/guanylate cyclase/cGMP signaling within the CNS inhibits Fosmediated AP-1 transcriptional regulatory activity in AVP neurons in these both hypothalamic structures during glucose substrate imbalance. Neuroanatomical evidence for NOS expression by magnocellular neurons in the PVN and SON (Sagar and Ferriero, 1987; Arevalo et al., 1992; Miyagawa et al., 1994), together with pharmacological evidence implicating this neurotransmitter in the regulation of circulating AVP and OXY concentrations (Chiodera et al., 1994; Kadowski et al., 1994; Kadekaro et al., 1997), suggest that NO may influence neuropeptidergic neurosecretion through autocrine and/or paracrine mechanisms within these hypothalamic structures. The present findings that icv administration of SIN-1 decreased Fos expression by AVP neurons in both loci suggest that cellular substrates for neuromodulatory actions of NO are accessible from the cerebroventricular system. While our studies did not determine whether the posterior magnocellular and subnuclear divisions of the PVN are accessible to SIN1 following intracerebral administration of the dose utilized above, further experiments are planned to investigate whether infusion of pharmacological agents capable of altering NO/cGMP signaling into either subregion of the PVN alters the functional status of local AVP neurons. In contrast to the PVN, which extends across the periventricular and medial zones of the hypothalamus, the SON is located far lateral from the third ventricle, presumably outside the diffusion range of this structure, and is thus inaccessible to the various drugs administered into the cerebroventricular system. The current findings that pretreatment with SIN1 decreased mean numbers of local AVP neurons that expressed Fos-ir during glucoprivation suggest that NO is capable of influencing the functional status of these cells via indirect mechanisms, involving afferent stimuli relayed to this structure from the periventricular CNS. Earlier studies involving human subjects reported that peripheral administration of the NOS inhibitor, Nv-nitro-L-arginine methyl ester (L-NAME), enhances hypo-

glycemic patterns of AVP secretion (Chiodera et al., 1994). In the current studies, experimental inhibition of central cGMP production by icv delivery of ODQ did not result in further increases in numbers of Fos-ir-positive AVP neurons in the PVN or SON of glucoprivic rats. Since we did not quantify immunodemonstrable Fos or total AP-1 binding in individual labeled cells, the possibility that mean nuclear levels of this protein were modified by intracerebral delivery of the guanylate cyclase inhibitor cannot be discounted. Alternatively, our data may indicate that pharmacological decreases in central NO/cGMP signaling have only minimal amplifying effects on glucoprivic induction of the Fos stimulus transcription cascade in AVP neurons. Although Tassorelli and Joseph (1995) previously reported that systemic administration of the organic nitrate and NO donor, nitroglycerin, resulted in immunolabeling of NADPH-diaphorase-containing neurons in the PVN and SON for Fos, our results showed that nuclear labeling for this protein was negligible in both structures following icv delivery of a single dose of another NO donor, SIN1. These disparate results could reflect different pharmacological effects caused by systemic versus intracerebral drug administration, differential potency of the two NO donors, and methodological factors such as choice of primary antiserum used to detect Fos. The present studies showed that pretreatment with SIN1 significantly decreased glucoprivic induction of Fos expression within each brain region examined, but that coadministration of ODQ plus SIN-1 effectively reversed this drug-induced inhibition of neuronal transactivation in the PVNpm and SON, not the caudal subnucleus. These observations suggest that in the latter site, regulatory effects of NO on vasopressinergic nuclear accumulation of Fos-ir during glucoprivation may be mediated by cGMP-independent mechanisms. In summary, the present studies demonstrate that intraventricular administration of exogenous NO decreases mean numbers of hypothalamic AVP neurons that express immunoreactivity for the AP-1 transcription factor, Fos, in response to glucoprivation. The data also provide evidence that guanylate cyclase/cGMP signaling may be critical for modulatory effects of NO on genomic regulatory activity in these cells during glucose substrate imbalance. The current results thus support the view that NO may function as a negative neuromodulator of vasopressinergic neuronal function during this metabolic challenge. References Arevalo, R., Sanchez, F., Alonso, J.R., Carretero, J., Vasquez, R., Aijon, J., 1992. NADPH-diaphorase activity in the hypothalamic magnocellular neurosecretory nuclei of the rat. Brain Res. Bull. 28, 599 – 603.

K.P. Briski / Journal of Chemical Neuroanatomy 17 (1999) 13–19 Bhat, G., Mahesh, V.B., Aguan, K., Brann, D.W., 1996. Evidence that brain nitric oxide synthase is the major nitric oxide synthase isoform in the hypothalamus of the adult female rat and that nitric oxide potently regulates hypothalamic cGMP levels. Neuroendocrinology 64, 93–102. Bredt, D.S., Snyder, S.H., 1990. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc. Natl. Acad. Sci. 87, 682 – 685. Caraty, A., Grino, M., Locatelli, A., Guillaume, V., Bourdouresque, F., Conte-Devoix, B., et al., 1990. Insulin-induced hypoglycemia stimulates corticotropin-releasing factor and arginine vasopressin secretion into hypophyseal portal blood of conscious, unrestrained rams. J. Clin. Invest. 85, 1716–1721. Chiodera, P., Volpi, R., Capretti, L., Speroni, G., Marcato, A., Rossi, G., et al., 1992. Hypoglycemia-induced arginine vasopressin and oxytocin release is mediated by glucoreceptors located inside the blood-brain barrier. Neuroendocrinology 55, 655 – 659. Chiodera, P., Volpi, R., Coiro, V., 1994. Inhibitory control of nitric oxide on the arginine-vasopressin and oxytocin response to hypoglycemia in normal men. NeuroReport 5, 1822–1824. Engler, D., Pham, T., Fullerton, M.J., Ooi, G., Funder, J.W., Clarke, I.J., 1989. Studies of the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophyseal-portal circulation of the conscious sheep. Neuroendocrinology 49, 367–381. Fedele, E., Conti, A., Raiteri, M., 1997. The glutamate receptor/NO/ cyclic GMP pathway in the hippocampus of freely moving rats: modulation by cyclothiazide, interaction with GABA and the behavioral consequences. Neuropharmacology 36, 1393–1403. Hrabovszky, E.., Vrontakis, M.E., Petersen, S.L., 1995. Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats. J. Histo. Cyto. 43, 363–370. Kadekaro, M., Liu, H., Terrell, M.L., Gestl, S., Bui, V., SummyLong, J.Y., 1997. Role of NO on vasopressin and oxytocin release and blood pressure responses during osmotic stimulation in rats. Am. J. Physiol. 273, R1024–1030. Kadowski, K., Kishimoto, J., Leng, G., Emson, P.C., 1994. Up-regu-

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