Neuroscience Research 99 (2015) 62–68
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Effects of a subconvulsive dose of kainic acid on the gene expressions of the arginine vasopressin, oxytocin and neuronal nitric oxide synthase in the rat hypothalamus Mitsuhiro Yoshimura a,1, Jun-ichi Ohkubo a,b,1, Hirofumi Hashimoto a, Takanori Matsuura a, Takashi Maruyama a , Tatsushi Onaka c , Hideaki Suzuki b , Yoichi Ueta a,∗ a
Department of Physiology and School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan Department of Otorhinolaryngology – Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan c Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University, Shimotsuke 329-0498, Japan b
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Article history: Received 20 February 2015 Received in revised form 27 April 2015 Accepted 10 May 2015 Available online 21 May 2015 Keywords: Arginine vasopressin Oxytocin Neuronal nitric oxide synthase Kainic acid Hypothalamus Rat
a b s t r a c t Arginine vasopressin (AVP) synthesis in the hypothalamo-neurohypophysial system (HNS) is upregulated by kainic acid (KA)-induced seizure in rats. However, it remains unknown whether a subconvulsive dose of KA affects the HNS. Here we examined the effects of subcutaneous (s.c.) administration of a low dose of KA (4 mg/kg) on the gene expressions of the AVP, oxytocin (OXT) and neuronal nitric oxide synthase (nNOS) in the supraoptic (SON) and paraventricular nuclei (PVN) of the rat hypothalamus, using in situ hybridization histochemistry. The expression of the AVP gene in the SON and PVN was judged to be up-regulated in KA-treated rats in comparison with saline-treated rats as controls. Next, the expression of the OXT gene was significantly increased in the SON at 6–24 h and in the PVN at 6 and 12 h after s.c. administration of KA. Finally, the expression of the nNOS gene was significantly increased in the SON and PVN at 3 and 6 h after s.c. administration of KA. These results suggest that up-regulation of the gene expressions of the AVP, OXT and nNOS in the rat hypothalamus may be differentially affected by peripheral administration of a subconvulsive dose of KA. © 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction The magnocellular neurosecretory cells (MNCs) in the hypothalamic supraoptic (SON) and the paraventricular nuclei (PVN) synthesize neurohypophysial hormones, arginine vasopressin (AVP) and oxytocin (OXT), project their axon terminals in the posterior pituitary and secrete AVP and OXT into the systemic circulation. The synthesis and secretion of AVP and OXT are modulated by various kinds of physiological and pathophysiological conditions, including the kainic acid (KA)-induced seizure (Iwanaga et al., 2011; Sun et al., 1996). KA is structurally related to the excitatory neurotransmitter glutamate, and binds strongly to the kainate subtype receptors as an agonist, result in excessive excitatory nerve and seizure (Collins et al., 1980). Systemic administration of KA is a good ∗ Corresponding author at: Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Tel.: +81 93 691 7420; fax: +81 93 692 1711. E-mail address: [email protected] (Y. Ueta). 1 These authors equally contributed to this work.
strategy to imitate the clinical and neuropathological features of temporal lobe epilepsy (Olney et al., 1974), and KA has been widely used in various kinds of seizure studies (Huang and Luijtelaar, 2012; Iwanaga et al., 2011; Ohno et al., 2012; Sakamoto et al., 2008; Turunc Bayrakdar et al., 2013). Commonly, 10–12 mg/kg of KA is systemically administered to induce seizure, which leading to massive excitotoxic damage of neuronal tissue (Doble, 1999; Iwanaga et al., 2011; Riljak et al., 2007). We demonstrated that AVP synthesis in the hypothalamo-neurohypophysial system (HNS) is up-regulated after subcutaneously (s.c.) administered 12 mg/kg kainic acid (KA)-induced seizure in rats (Iwanaga et al., 2011). On the other hand, several studies examined the effects of systemic administration of a subconvulsive or non-convulsive dose (5–6 mg/kg) of KA on the behavior in rats (Carbajal et al., 2004; Mikulecka et al., 1999; Riljak et al., 2014; Walls et al., 2014). However, up to our knowledge, there are no reports which refer to the hypothalamic neuropeptide fluctuation, in particular AVP and OXT, after systemic administration of a subconvulsive dose of KA. It has been demonstrated that systemic and central administrations of a subconvulsive dose of KA changes behavior such as locomotion,
http://dx.doi.org/10.1016/j.neures.2015.05.002 0168-0102/© 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
M. Yoshimura et al. / Neuroscience Research 99 (2015) 62–68
rearing, immobility and cognitive disturbances in rats (Arkhipov et al., 2008; Riljak et al., 2014). It is possible that a subconvulsive dose of KA could affect not only behavior but also HNS. In the present study, we investigated the effects on the gene expression of the AVP and OXT after s.c. administration of a subconvulsive dose of KA (4 mg/kg) in rats, using in situ hybridization histochemistry (ISH). The neuronal nitric oxide synthases (nNOS) are the family of enzymes that catalyze the conversion of l-arginine to l-citrullin and nitric oxide (NO). The nNOS is abundant besides the OXT producing neurons in the hypothalamus (Nylen et al., 2001; Yamamoto et al., 1997) and it has been considered that NO produced by nNOS is one of the synthetic accelerators of OXT as well as AVP (Aguila et al., 2011; Orlando et al., 2007). Hence, we examined the effects of subconvulsive dose of KA on the gene expression of the nNOS in the SON and PVN as well as AVP and OXT.
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experiments in this study were carried out in accordance with the guidelines of the Physiological Society of Japan under the control of the Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health, Japan. 2.2. Experimental procedure Rats were s.c. administered 0.9% saline (0.5 mL/rat) as control (CTR) or KA (4 mg/kg). KA was purchased from Nacalai Tesque Inc. (Kyoto, Japan). KA was dissolved in saline and adjusted to pH 7.0. Three hours (h), 6 h, 12 h, 24 h, 48 h and 1 week after s.c. administration of saline or KA, they were decapitated without being anesthetized (n = 6–7 each). After decapitation, brains were immediately removed, placed on dry ice, and stored at −80 ◦ C in a deep freezer until use in ISH analysis. 2.3. Definition for a subconvulsive dose of KA
2. Materials and methods 2.1. Animals Adult male Wistar rats (170–190 g) were used for all the experiments. The animals were housed in standard plastic cages at 23–25 ◦ C on a 12 h light (07.00–19.00 h)–12 h dark cycle. All
High dose of s.c. administration of KA (12 mg/kg) induced convulsion according to our previous studies (Iwanaga et al., 2011; Ohno et al., 2012). The defined seizure scale (Racine, 1972) which were rated in our previous studies exhibited around 3–4 at 3 h, the peak of the seizures, after s.c. administration of KA. In the present study every 30 min until 3 h after s.c. administration of KA (4 mg/kg)
Fig. 1. AVP mRNA in the SON and PVN. (A) Representative microphotographs obtained from emulsion dipped sections that indicate AVP mRNA in the SON (a and c) and PVN (b and d). At 6 h after administration of saline (CTR) (a and b) or KA (c and d) in emulsion-dipped slides. Each part encircled by broken red line indicates the location used for the analysis. Scale bars, 200 m. (B) Quantification of AVP signals identified in the SON. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7. (C) Quantification of AVP signals identified in the PVN. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7.
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and at each time point of decapitation, at least two investigators observed whether seizures were developed or not by rating the seizure scale (Racine, 1972). On the other hand, the gene expression of the c-fos is one of the most valuable markers to detect neuronal activation (Yoshimura et al., 2013b). Previous studies have revealed that c-fos was up-regulated in the amygdala or the hippocampus by KA (10–15 mg/kg) induced seizure (Pereno et al., 2011; Riba-Bosch and Perez-Clausell, 2004). Thus, we examined whether the gene expression of the c-fos was observed in the hippocampus or not after s.c. administration of KA (4 mg/kg) by ISH. 2.4. ISH for AVP, hnAVP, OXT and nNOS ISH was performed on frozen 12-m-thick coronal brain cryostat sections cut at −20 ◦ C, thawed, and mounted onto gelatin/chrome alum-coated slides that were kept at −80 ◦ C until use. The SON and the PVN were localized by referring to the atlas. The detail of ISH procedures has been described in previously (Ueta et al., 1995). A 35 S 3 -end-labeled deoxyoligonucleotide complementary to transcripts encoding AVP (AVP probe sequence 5 -CAG CTC CCG GGC TGG CCC GTC CAG CT-3 ), hnAVP (hnAVP probe sequence 5 -GCA CTG TCA GCA GCC CTG AAC GGA CCA CAG TGG TAC-3 ), OXT (OXT probe sequence 5 -CTC GGA GAA GGC AGA CTC AGG GTC GCA GGC-3 ) and nNOS (nNOS probe sequence 5 GCC TTG GGC ATG CTG AGG GCC ATT ACC CAG ACC TGT GAC
TCT GTC-3 ) were used for the hybridization. The specificity of these probes has been authenticated by previous studies (Iwanaga et al., 2011; Ueta et al., 1995; Yoshimura et al., 2013a). The probe was 3 -end-labeled using terminal deoxynucleotidyl transferase and [35 S] dATP. The ISH protocol has been previously described in detail. Hybridization was carried out overnight at 37 ◦ C in 45 L of hybridization buffer under a Nescofilm coverslip (Bando Kagaku, Osaka, Japan). A total of 1 × 105 c.p.m./slide was used for AVP and OXT transcripts and 1 × 106 c.p.m./slide for hnAVP and nNOS transcripts. After hybridization, sections were washed four times with SSC (150 mM NaCl and 15 mM sodium citrate) for 1 h at 55 ◦ C and for an additional 1 h with two changes of SSC at room temperature. Hybridized sections were exposed to autoradiography film (Hyperfilm, Amersham, Bucks, UK) for 6 h for OXT and AVP, and for 7 days for hnAVP and nNOS. The images were analyzed by computerized densitometry using an MCID imaging analyzer (Imaging Research Inc., Ontario, Canada). The mean optical densities (ODs) of the autoradiographs were measured by comparison with simultaneously exposed 14 C-labeled microscale samples and represented in arbitrary units, with the mean OD obtained from control rats set to 100. The results were blinded and analyzed randomly in duplicate by at least two researchers in order to avoid bias. Slides hybridized with the probes were dipped in a nuclear emulsion (K-5; Ilford, Cheshire, UK) and exposed for further 3 days for AVP and OXT, and for 3 weeks for hnAVP and nNOS.
Fig. 2. AVP hnRNA in the SON and PVN. (A) Representative microphotographs obtained from emulsion dipped sections that indicate AVP hnRNA in the SON (a and c) and PVN (b and d). At 6 h after administration of saline (CTR) (a and b) or KA (d and e) in emulsion-dipped slides. Each part encircled by broken red line indicates the location used for the analysis. Scale bars, 200 m. (B) Quantification of hnAVP signals identified in the SON. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7. (C) Quantification of hnAVP signals identified in the PVN. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7.
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Fig. 3. OXT mRNA in the SON and PVN. (A) Representative microphotographs obtained from emulsion dipped sections that indicate OXT mRNA in the SON (a and c) and PVN (b and d). At 6 h after administration of saline (CTR) (a and b) or KA (d and e) in emulsion-dipped slides. Each part encircled by broken red line indicates the location used for the analysis. Scale bars, 200 m. (B) Quantification of OXT signals identified in the SON. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7. (C) Quantification of OXT signals identified in the PVN. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7.
2.5. Data analysis The results obtained from the ISH are presented as means ± standard errors of the means (SEMs). The results of the gene expression analysis are shown as percentage of each CTR group. All data were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni-type adjustment for multiple comparisons (Origin Pro version 8.5J, Lightstone, Tokyo, Japan). Differences or correlations were considered significant when P-values were less than 0.05. 3. Results 3.1. Definition for a subconvulsive dose of KA Until 3 h after s.c. administration of KA, KA-treated rats did not develop seizure, thus, seizure scale was judged as 0. At each time point (3 h, 6 h, 12 h, 24 h, 48 h and 1 week) of decapitation, seizure was not observed in all KA-treated rats (data not shown). ISH analysis revealed that the gene expression of the c-fos was not induced in the cerebral cortex nor in the hippocampus 3 h after s.c. administration of KA (4 mg/kg), however, the c-fos expression was robustly induced in the SON and the PVN 30 min after s.c. administration of KA (4 mg/kg) (data not shown). All KAtreated rats used in the experiment were judged as a subconvulsive
condition because of seizure scale 0 and no induction of the c-fos gene expression in the cerebral cortex and the hippocampus in those rats. In addition, as a positive control, we observed abundant induction of the c-fos gene expression in the cortex and hippocampus 3 h after s.c. administration of high dose of KA (12 mg/kg) (data not shown). 3.2. AVP mRNA in the SON and PVN Fig. 1A showed the representative signals for AVP mRNA obtained from autoradiographs. AVP mRNA levels in the SON (Fig. 1B) and PVN (Fig. 1C) did not differ between CTR and KA at each time point. 3.3. AVP hnRNA in the SON and PVN Fig. 2A showed the representative signals for AVP hnRNA obtained from autoradiographs. Our previous study confirmed that the hnAVP changed rapidly after various kinds of stimuli (Iwanaga et al., 2011; Nomura et al., 1999) and the signals for the hnAVP tended to cluster around the nucleus of individual MNCs (Fig. 2A). The enlarged microphotographic observation was similar with previous our study (Kawasaki et al., 2005). The hnAVP levels were significantly increased at 3 h, 6 h, 12 h and 24 h after s.c.
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Fig. 4. The nNOS mRNA in the SON and PVN. (A) Representative microphotographs obtained from emulsion dipped sections that indicate nNOS mRNA in the SON (a and c) and PVN (b and d) in emulsion-dipped slides. At 3 h after administration of saline (CTR) (a and b) or KA (d and e). Each part encircled by broken red line indicates the location used for the analysis. Scale bars, 200 m. (B) Quantification of nNOS signals identified in the SON. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7. (C) Quantification of nNOS signals identified in the PVN. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7.
administration of KA in the SON (Fig. 2Ac and B) and in the PVN (Fig. 2Ad and C) compared to CTR (Fig. 2Aa, Ab, B and C). 3.4. OXT mRNA in the SON and PVN Fig. 3A showed the representative signals for OXT mRNA obtained from autoradiographs. OXT mRNA levels were significantly increased at 6 h, 12 h and 24 h after s.c. administration of KA in the SON (Fig. 3Ac and B) and at 6 h and 12 h in the PVN (Fig. 3Ad and C) compared to CTR (Fig. 3Aa, Ab, B and C). 3.5. The nNOS mRNA in the SON and PVN Fig. 4A showed the representative signals for nNOS mRNA obtained from autoradiographs. The nNOS mRNA levels were significantly increased at 3 h and 6 h after s.c. administration of KA in the SON (Fig. 4Ac and B) and the PVN (Fig. 4Ad and C) compared to CTR (Fig. 4Aa, Ab, B and C). 4. Discussion The present study demonstrated that the gene expressions of the AVP, the OXT and the nNOS in the SON and the PVN were significantly up-regulated after s.c. administration of a subconvulsive dose (4 mg/kg) of KA compared to CTR. AVP hnRNA levels in the
SON and PVN were significantly increased, though AVP mRNA did not change. OXT mRNA levels were significantly increased at the peak around 6–12 h after s.c. administration of KA. The nNOS mRNA levels were significantly increased transiently within 6 h after s.c. administration of KA. These results suggest that the hypothalamic peptide synthesis may be differentially affected even by systemic administration of a subconvulsive dose of KA. Although AVP mRNA levels in the SON and the PVN had no significant differences between CTR and KA at each time point, AVP hnRNA levels were significantly increased after s.c. administration of KA. In addition, the up-regulation of AVP hnRNA was observed not only in the magnocellular division but also in the parvocellular division. It is possible that AVP neurons were co-localized with corticotrophin releasing hormone (CRH) producing neurons, resulting in induction of the stress response (Herman et al., 1994). In the present study, the increased ratio of hypothalamic AVP hnRNA levels in KA (4 mg/kg)-treated rats were 1.5 folds in the SON and 2.5 folds in the PVN vs. CTR. Our previous study used high dose of KA (12 mg/kg) sowed that the increased ratio of the hypothalamic AVP hnRNA levels were 2.0 folds in the SON and 4.5 folds in the PVN vs. CTR (Iwanaga et al., 2011). One possible explanation about those differences in the SON and the PVN is that seizure induced by a high dose of KA may be involved in the up-regulation of the AVP gene expressions in the SON and the PVN. Another possible explanation is that the differences may be caused by pharmacological effects
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(a low and high dose of KA). It is judged that AVP gene expression in the hypothalamus was up-regulated in a subconvulsive dose of KA-treated rats even if AVP mRNA remained unchanged because AVP hnRNA has a very short half-life and the change reflects gene transcription much better than AVP mRNA (Arima et al., 1999). In this study, it is judged that AVP synthesis was up-regulated even if AVP mRNA remained unchanged. As OXT mRNA was significantly increased both in the SON and the PVN in KA-treated rats, the expression of the OXT gene was upregulated by systemic administration of a subconvulsive dose of KA. Effects of low dose of KA on the expression of the OXT gene seemed to be stronger than that of the AVP gene in the SON and PVN. Our previous study showed that KA-induced inward currents in the OXT neurons were significantly larger than those in the AVP neurons, using whole cell patch-clamp recording in in vitro brain slice preparation (Ohkubo et al., 2014). In this in vitro experiments, MNCs isolated from the SON were identified to be AVP and OXT neurons by visible fluorescent proteins (Ohkubo et al., 2014) in transgenic rats lines (Katoh et al., 2011; Ueta et al., 2005). It is hypothesized that OXT neurons are more highly affected by peripherally administered KA than AVP neurons even in in vivo state. The nNOS gene expression is marked increased in the SON and the PVN when OXT and AVP release is enhanced by osmotic stimulation (Srisawat et al., 2004), parturition (Popeski et al., 1999), nociceptive stimulation (Kurose et al., 2001), and stress (Hatakeyama et al., 1996; Orlando et al., 2007). NO produced by nNOS in the SON and the PVN is considered as one of the potent stimulators to elicit AVP and OXT release (Aguila et al., 2011; Orlando et al., 2007). According to the previous study (Xiao et al., 2005), it may be possible that NO distributes more to OXT release than to AVP release, although it is difficult to define the distribution of NO from our experimental condition. The expressions of the nNOS gene in the SON and PVN were significantly increased after s.c. administration of a subconvulsive dose of KA in rats. These increases of nNOS mRNA levels were relatively transient rather than those of the AVP hnRNA and OXT mRNA levels. It is possible that NO produced by nNOS may regulate the gene expressions of the AVP and OXT and secretion of these peptides after systemic administration of KA. It may be also interesting to use a nNOS inhibitor, such as 3-bromo-7-nitoroindazole, in combination with KA to examine whether the expression of the AVP or OXT would be down-regulated. These points should be clarified by further study. The physiological relevance of upregulation of AVP and OXT gene expression in response to a sub-convulsive dose of KA remains unclear. According to the previous studies, AVP has proconvulsive effects (Croiset and De Wied, 1997; Richmond, 2003) and OXT has anticonvulsive effects (Clynen et al., 2014; Erbas et al., 2013). It is possible that the primarily up-regulation of AVP may be the cause of seizure and OXT may be up-regulated to prevent seizure by s.c. administration of a subconvulsive dose of KA. The physiological meanings of this difference should be clarified by further study, in particular, in in vivo study. In conclusion, we revealed that the gene expressions of the AVP, OXT and nNOS were up-regulated even with a subconvulsive dose of KA administration in rats. Our study may bring new insight into the pharmacological effects of a systemic administration of a subconvulsive dose of KA on the hypothalamic neuropeptides.
Author contribution Experimental design: M.Y., J.O., H.S. and Y.U. Carry out experiment: M.Y., J.O., H.H., T.M., T.M. and T.O. Data analysis: M.Y. and J.O. Interpretation of the data: M.Y., J.O., and Y.U. Prepare the draft: M.Y. Prepare the figure: M.Y. Final approval: Y.U.
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Acknowledgements We thank Ms. Kanako Shoguchi (University of Occupational and Environmental Health, Kitakyushu, Japan) for her technical assistance. This study was supported by a Grant-in-Aid for Scientific Research (B), No. 25293055 to Y.U. from the Japan Society for the Promotion of Science (JSPS) and by a Grant-in-Aid for Scientific Research on Innovative Area, No. 25113721 to Y.U. from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. References Aguila, F.A., Oliveira-Pelegrin, G.R., Yao, S.T., Murphy, D., Rocha, M.J., 2011. Anteroventral third ventricle (AV3V) lesion affects hypothalamic neuronal nitric oxide synthase (nNOS) expression following water deprivation. Brain Res. Bull. 86, 239–245. Arima, H., Kondo, K., Kakiya, S., Nagasaki, H., Yokoi, H., Yambe, Y., Murase, T., Iwasaki, Y., Oiso, Y., 1999. Rapid and sensitive vasopressin heteronuclear RNA responses to changes in plasma osmolality. J. Neuroendocrinol. 11, 337–341. 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Turunc Bayrakdar, E., Bojnik, E., Armagan, G., Kanit, L., Benyhe, S., Borsodi, A., Yalcin, A., 2013. Kainic acid-induced seizure activity alters the mRNA expression and G-protein activation of the opioid/nociceptin receptors in the rat brain cortex. Epilepsy Res. 105, 13–19. Ueta, Y., Fujihara, H., Serino, R., Dayanithi, G., Ozawa, H., Matsuda, K., Kawata, M., Yamada, J., Ueno, S., Fukuda, A., Murphy, D., 2005. Transgenic expression of enhanced green fluorescent protein enables direct visualization for physiological studies of vasopressin neurons and isolated nerve terminals of the rat. Endocrinology 146, 406–413. Ueta, Y., Levy, A., Chowdrey, H.S., Lightman, S.L., 1995. Water deprivation in the rat induces nitric oxide synthase (NOS) gene expression in the hypothalamic paraventricular and supraoptic nuclei. Neurosci. Res. 23, 317–319. Walls, A.B., Eyjolfsson, E.M., Schousboe, A., Sonnewald, U., Waagepetersen, H.S., 2014. A subconvulsive dose of kainate selectively compromises astrocytic metabolism in the mouse brain in vivo. J. Cereb. Blood Flow Metab. 34, 1340–1346. Xiao, M., Ding, J., Wu, L., Han, Q., Wang, H., Zuo, G., Hu, G., 2005. The distribution of neural nitric oxide synthase-positive cerebrospinal fluid-contacting neurons in the third ventricular wall of male rats and coexistence with vasopressin or oxytocin. Brain Res. 1038, 150–162. Yamamoto, Y., Ueta, Y., Nomura, M., Serino, R., Kabashima, N., Shibuya, I., Yamashita, H., 1997. Upregulation of neuronal NOS mRNA in the PVN and SON of inherited diabetes insipidus rats. Neuroreport 8, 3907–3911. Yoshimura, M., Matsuura, T., Ohkubo, J., Ohno, M., Maruyama, T., Ishikura, T., Hashimoto, H., Kakuma, T., Yoshimatsu, H., Terawaki, K., Uezono, Y., Ueta, Y., 2013a. The gene expression of the hypothalamic feedingregulating peptides in cisplatin-induced anorexic rats. Peptides 46, 13–19. Yoshimura, M., Ohkubo, J., Katoh, A., Ohno, M., Ishikura, T., Kakuma, T., Yoshimatsu, H., Murphy, D., Ueta, Y., 2013b. A c-fos-monomeric red fluorescent protein 1 fusion transgene is differentially expressed in rat forebrain and brainstem after chronic dehydration and rehydration. J. Neuroendocrinol. 25, 478–487.
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Effects of a subconvulsive dose of kainic acid on the gene expressions of the arginine vasopressin, oxytocin and neuronal nitric oxide synthase in the rat hypothalamus Mitsuhiro Yoshimura a,1, Jun-ichi Ohkubo a,b,1, Hirofumi Hashimoto a, Takanori Matsuura a, Takashi Maruyama a , Tatsushi Onaka c , Hideaki Suzuki b , Yoichi Ueta a,∗ a
Department of Physiology and School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan Department of Otorhinolaryngology – Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan c Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University, Shimotsuke 329-0498, Japan b
a r t i c l e
i n f o
Article history: Received 20 February 2015 Received in revised form 27 April 2015 Accepted 10 May 2015 Available online 21 May 2015 Keywords: Arginine vasopressin Oxytocin Neuronal nitric oxide synthase Kainic acid Hypothalamus Rat
a b s t r a c t Arginine vasopressin (AVP) synthesis in the hypothalamo-neurohypophysial system (HNS) is upregulated by kainic acid (KA)-induced seizure in rats. However, it remains unknown whether a subconvulsive dose of KA affects the HNS. Here we examined the effects of subcutaneous (s.c.) administration of a low dose of KA (4 mg/kg) on the gene expressions of the AVP, oxytocin (OXT) and neuronal nitric oxide synthase (nNOS) in the supraoptic (SON) and paraventricular nuclei (PVN) of the rat hypothalamus, using in situ hybridization histochemistry. The expression of the AVP gene in the SON and PVN was judged to be up-regulated in KA-treated rats in comparison with saline-treated rats as controls. Next, the expression of the OXT gene was significantly increased in the SON at 6–24 h and in the PVN at 6 and 12 h after s.c. administration of KA. Finally, the expression of the nNOS gene was significantly increased in the SON and PVN at 3 and 6 h after s.c. administration of KA. These results suggest that up-regulation of the gene expressions of the AVP, OXT and nNOS in the rat hypothalamus may be differentially affected by peripheral administration of a subconvulsive dose of KA. © 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction The magnocellular neurosecretory cells (MNCs) in the hypothalamic supraoptic (SON) and the paraventricular nuclei (PVN) synthesize neurohypophysial hormones, arginine vasopressin (AVP) and oxytocin (OXT), project their axon terminals in the posterior pituitary and secrete AVP and OXT into the systemic circulation. The synthesis and secretion of AVP and OXT are modulated by various kinds of physiological and pathophysiological conditions, including the kainic acid (KA)-induced seizure (Iwanaga et al., 2011; Sun et al., 1996). KA is structurally related to the excitatory neurotransmitter glutamate, and binds strongly to the kainate subtype receptors as an agonist, result in excessive excitatory nerve and seizure (Collins et al., 1980). Systemic administration of KA is a good ∗ Corresponding author at: Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Tel.: +81 93 691 7420; fax: +81 93 692 1711. E-mail address: [email protected] (Y. Ueta). 1 These authors equally contributed to this work.
strategy to imitate the clinical and neuropathological features of temporal lobe epilepsy (Olney et al., 1974), and KA has been widely used in various kinds of seizure studies (Huang and Luijtelaar, 2012; Iwanaga et al., 2011; Ohno et al., 2012; Sakamoto et al., 2008; Turunc Bayrakdar et al., 2013). Commonly, 10–12 mg/kg of KA is systemically administered to induce seizure, which leading to massive excitotoxic damage of neuronal tissue (Doble, 1999; Iwanaga et al., 2011; Riljak et al., 2007). We demonstrated that AVP synthesis in the hypothalamo-neurohypophysial system (HNS) is up-regulated after subcutaneously (s.c.) administered 12 mg/kg kainic acid (KA)-induced seizure in rats (Iwanaga et al., 2011). On the other hand, several studies examined the effects of systemic administration of a subconvulsive or non-convulsive dose (5–6 mg/kg) of KA on the behavior in rats (Carbajal et al., 2004; Mikulecka et al., 1999; Riljak et al., 2014; Walls et al., 2014). However, up to our knowledge, there are no reports which refer to the hypothalamic neuropeptide fluctuation, in particular AVP and OXT, after systemic administration of a subconvulsive dose of KA. It has been demonstrated that systemic and central administrations of a subconvulsive dose of KA changes behavior such as locomotion,
http://dx.doi.org/10.1016/j.neures.2015.05.002 0168-0102/© 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
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rearing, immobility and cognitive disturbances in rats (Arkhipov et al., 2008; Riljak et al., 2014). It is possible that a subconvulsive dose of KA could affect not only behavior but also HNS. In the present study, we investigated the effects on the gene expression of the AVP and OXT after s.c. administration of a subconvulsive dose of KA (4 mg/kg) in rats, using in situ hybridization histochemistry (ISH). The neuronal nitric oxide synthases (nNOS) are the family of enzymes that catalyze the conversion of l-arginine to l-citrullin and nitric oxide (NO). The nNOS is abundant besides the OXT producing neurons in the hypothalamus (Nylen et al., 2001; Yamamoto et al., 1997) and it has been considered that NO produced by nNOS is one of the synthetic accelerators of OXT as well as AVP (Aguila et al., 2011; Orlando et al., 2007). Hence, we examined the effects of subconvulsive dose of KA on the gene expression of the nNOS in the SON and PVN as well as AVP and OXT.
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experiments in this study were carried out in accordance with the guidelines of the Physiological Society of Japan under the control of the Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health, Japan. 2.2. Experimental procedure Rats were s.c. administered 0.9% saline (0.5 mL/rat) as control (CTR) or KA (4 mg/kg). KA was purchased from Nacalai Tesque Inc. (Kyoto, Japan). KA was dissolved in saline and adjusted to pH 7.0. Three hours (h), 6 h, 12 h, 24 h, 48 h and 1 week after s.c. administration of saline or KA, they were decapitated without being anesthetized (n = 6–7 each). After decapitation, brains were immediately removed, placed on dry ice, and stored at −80 ◦ C in a deep freezer until use in ISH analysis. 2.3. Definition for a subconvulsive dose of KA
2. Materials and methods 2.1. Animals Adult male Wistar rats (170–190 g) were used for all the experiments. The animals were housed in standard plastic cages at 23–25 ◦ C on a 12 h light (07.00–19.00 h)–12 h dark cycle. All
High dose of s.c. administration of KA (12 mg/kg) induced convulsion according to our previous studies (Iwanaga et al., 2011; Ohno et al., 2012). The defined seizure scale (Racine, 1972) which were rated in our previous studies exhibited around 3–4 at 3 h, the peak of the seizures, after s.c. administration of KA. In the present study every 30 min until 3 h after s.c. administration of KA (4 mg/kg)
Fig. 1. AVP mRNA in the SON and PVN. (A) Representative microphotographs obtained from emulsion dipped sections that indicate AVP mRNA in the SON (a and c) and PVN (b and d). At 6 h after administration of saline (CTR) (a and b) or KA (c and d) in emulsion-dipped slides. Each part encircled by broken red line indicates the location used for the analysis. Scale bars, 200 m. (B) Quantification of AVP signals identified in the SON. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7. (C) Quantification of AVP signals identified in the PVN. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7.
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and at each time point of decapitation, at least two investigators observed whether seizures were developed or not by rating the seizure scale (Racine, 1972). On the other hand, the gene expression of the c-fos is one of the most valuable markers to detect neuronal activation (Yoshimura et al., 2013b). Previous studies have revealed that c-fos was up-regulated in the amygdala or the hippocampus by KA (10–15 mg/kg) induced seizure (Pereno et al., 2011; Riba-Bosch and Perez-Clausell, 2004). Thus, we examined whether the gene expression of the c-fos was observed in the hippocampus or not after s.c. administration of KA (4 mg/kg) by ISH. 2.4. ISH for AVP, hnAVP, OXT and nNOS ISH was performed on frozen 12-m-thick coronal brain cryostat sections cut at −20 ◦ C, thawed, and mounted onto gelatin/chrome alum-coated slides that were kept at −80 ◦ C until use. The SON and the PVN were localized by referring to the atlas. The detail of ISH procedures has been described in previously (Ueta et al., 1995). A 35 S 3 -end-labeled deoxyoligonucleotide complementary to transcripts encoding AVP (AVP probe sequence 5 -CAG CTC CCG GGC TGG CCC GTC CAG CT-3 ), hnAVP (hnAVP probe sequence 5 -GCA CTG TCA GCA GCC CTG AAC GGA CCA CAG TGG TAC-3 ), OXT (OXT probe sequence 5 -CTC GGA GAA GGC AGA CTC AGG GTC GCA GGC-3 ) and nNOS (nNOS probe sequence 5 GCC TTG GGC ATG CTG AGG GCC ATT ACC CAG ACC TGT GAC
TCT GTC-3 ) were used for the hybridization. The specificity of these probes has been authenticated by previous studies (Iwanaga et al., 2011; Ueta et al., 1995; Yoshimura et al., 2013a). The probe was 3 -end-labeled using terminal deoxynucleotidyl transferase and [35 S] dATP. The ISH protocol has been previously described in detail. Hybridization was carried out overnight at 37 ◦ C in 45 L of hybridization buffer under a Nescofilm coverslip (Bando Kagaku, Osaka, Japan). A total of 1 × 105 c.p.m./slide was used for AVP and OXT transcripts and 1 × 106 c.p.m./slide for hnAVP and nNOS transcripts. After hybridization, sections were washed four times with SSC (150 mM NaCl and 15 mM sodium citrate) for 1 h at 55 ◦ C and for an additional 1 h with two changes of SSC at room temperature. Hybridized sections were exposed to autoradiography film (Hyperfilm, Amersham, Bucks, UK) for 6 h for OXT and AVP, and for 7 days for hnAVP and nNOS. The images were analyzed by computerized densitometry using an MCID imaging analyzer (Imaging Research Inc., Ontario, Canada). The mean optical densities (ODs) of the autoradiographs were measured by comparison with simultaneously exposed 14 C-labeled microscale samples and represented in arbitrary units, with the mean OD obtained from control rats set to 100. The results were blinded and analyzed randomly in duplicate by at least two researchers in order to avoid bias. Slides hybridized with the probes were dipped in a nuclear emulsion (K-5; Ilford, Cheshire, UK) and exposed for further 3 days for AVP and OXT, and for 3 weeks for hnAVP and nNOS.
Fig. 2. AVP hnRNA in the SON and PVN. (A) Representative microphotographs obtained from emulsion dipped sections that indicate AVP hnRNA in the SON (a and c) and PVN (b and d). At 6 h after administration of saline (CTR) (a and b) or KA (d and e) in emulsion-dipped slides. Each part encircled by broken red line indicates the location used for the analysis. Scale bars, 200 m. (B) Quantification of hnAVP signals identified in the SON. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7. (C) Quantification of hnAVP signals identified in the PVN. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7.
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Fig. 3. OXT mRNA in the SON and PVN. (A) Representative microphotographs obtained from emulsion dipped sections that indicate OXT mRNA in the SON (a and c) and PVN (b and d). At 6 h after administration of saline (CTR) (a and b) or KA (d and e) in emulsion-dipped slides. Each part encircled by broken red line indicates the location used for the analysis. Scale bars, 200 m. (B) Quantification of OXT signals identified in the SON. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7. (C) Quantification of OXT signals identified in the PVN. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7.
2.5. Data analysis The results obtained from the ISH are presented as means ± standard errors of the means (SEMs). The results of the gene expression analysis are shown as percentage of each CTR group. All data were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni-type adjustment for multiple comparisons (Origin Pro version 8.5J, Lightstone, Tokyo, Japan). Differences or correlations were considered significant when P-values were less than 0.05. 3. Results 3.1. Definition for a subconvulsive dose of KA Until 3 h after s.c. administration of KA, KA-treated rats did not develop seizure, thus, seizure scale was judged as 0. At each time point (3 h, 6 h, 12 h, 24 h, 48 h and 1 week) of decapitation, seizure was not observed in all KA-treated rats (data not shown). ISH analysis revealed that the gene expression of the c-fos was not induced in the cerebral cortex nor in the hippocampus 3 h after s.c. administration of KA (4 mg/kg), however, the c-fos expression was robustly induced in the SON and the PVN 30 min after s.c. administration of KA (4 mg/kg) (data not shown). All KAtreated rats used in the experiment were judged as a subconvulsive
condition because of seizure scale 0 and no induction of the c-fos gene expression in the cerebral cortex and the hippocampus in those rats. In addition, as a positive control, we observed abundant induction of the c-fos gene expression in the cortex and hippocampus 3 h after s.c. administration of high dose of KA (12 mg/kg) (data not shown). 3.2. AVP mRNA in the SON and PVN Fig. 1A showed the representative signals for AVP mRNA obtained from autoradiographs. AVP mRNA levels in the SON (Fig. 1B) and PVN (Fig. 1C) did not differ between CTR and KA at each time point. 3.3. AVP hnRNA in the SON and PVN Fig. 2A showed the representative signals for AVP hnRNA obtained from autoradiographs. Our previous study confirmed that the hnAVP changed rapidly after various kinds of stimuli (Iwanaga et al., 2011; Nomura et al., 1999) and the signals for the hnAVP tended to cluster around the nucleus of individual MNCs (Fig. 2A). The enlarged microphotographic observation was similar with previous our study (Kawasaki et al., 2005). The hnAVP levels were significantly increased at 3 h, 6 h, 12 h and 24 h after s.c.
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Fig. 4. The nNOS mRNA in the SON and PVN. (A) Representative microphotographs obtained from emulsion dipped sections that indicate nNOS mRNA in the SON (a and c) and PVN (b and d) in emulsion-dipped slides. At 3 h after administration of saline (CTR) (a and b) or KA (d and e). Each part encircled by broken red line indicates the location used for the analysis. Scale bars, 200 m. (B) Quantification of nNOS signals identified in the SON. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7. (C) Quantification of nNOS signals identified in the PVN. *P < 0.05 vs. CTR; **P < 0.01 vs. CTR. Signals for the each CTR group were set at 100%. Data are presented as means ± SEMs. CTR, n = 6; KA, n = 6–7.
administration of KA in the SON (Fig. 2Ac and B) and in the PVN (Fig. 2Ad and C) compared to CTR (Fig. 2Aa, Ab, B and C). 3.4. OXT mRNA in the SON and PVN Fig. 3A showed the representative signals for OXT mRNA obtained from autoradiographs. OXT mRNA levels were significantly increased at 6 h, 12 h and 24 h after s.c. administration of KA in the SON (Fig. 3Ac and B) and at 6 h and 12 h in the PVN (Fig. 3Ad and C) compared to CTR (Fig. 3Aa, Ab, B and C). 3.5. The nNOS mRNA in the SON and PVN Fig. 4A showed the representative signals for nNOS mRNA obtained from autoradiographs. The nNOS mRNA levels were significantly increased at 3 h and 6 h after s.c. administration of KA in the SON (Fig. 4Ac and B) and the PVN (Fig. 4Ad and C) compared to CTR (Fig. 4Aa, Ab, B and C). 4. Discussion The present study demonstrated that the gene expressions of the AVP, the OXT and the nNOS in the SON and the PVN were significantly up-regulated after s.c. administration of a subconvulsive dose (4 mg/kg) of KA compared to CTR. AVP hnRNA levels in the
SON and PVN were significantly increased, though AVP mRNA did not change. OXT mRNA levels were significantly increased at the peak around 6–12 h after s.c. administration of KA. The nNOS mRNA levels were significantly increased transiently within 6 h after s.c. administration of KA. These results suggest that the hypothalamic peptide synthesis may be differentially affected even by systemic administration of a subconvulsive dose of KA. Although AVP mRNA levels in the SON and the PVN had no significant differences between CTR and KA at each time point, AVP hnRNA levels were significantly increased after s.c. administration of KA. In addition, the up-regulation of AVP hnRNA was observed not only in the magnocellular division but also in the parvocellular division. It is possible that AVP neurons were co-localized with corticotrophin releasing hormone (CRH) producing neurons, resulting in induction of the stress response (Herman et al., 1994). In the present study, the increased ratio of hypothalamic AVP hnRNA levels in KA (4 mg/kg)-treated rats were 1.5 folds in the SON and 2.5 folds in the PVN vs. CTR. Our previous study used high dose of KA (12 mg/kg) sowed that the increased ratio of the hypothalamic AVP hnRNA levels were 2.0 folds in the SON and 4.5 folds in the PVN vs. CTR (Iwanaga et al., 2011). One possible explanation about those differences in the SON and the PVN is that seizure induced by a high dose of KA may be involved in the up-regulation of the AVP gene expressions in the SON and the PVN. Another possible explanation is that the differences may be caused by pharmacological effects
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(a low and high dose of KA). It is judged that AVP gene expression in the hypothalamus was up-regulated in a subconvulsive dose of KA-treated rats even if AVP mRNA remained unchanged because AVP hnRNA has a very short half-life and the change reflects gene transcription much better than AVP mRNA (Arima et al., 1999). In this study, it is judged that AVP synthesis was up-regulated even if AVP mRNA remained unchanged. As OXT mRNA was significantly increased both in the SON and the PVN in KA-treated rats, the expression of the OXT gene was upregulated by systemic administration of a subconvulsive dose of KA. Effects of low dose of KA on the expression of the OXT gene seemed to be stronger than that of the AVP gene in the SON and PVN. Our previous study showed that KA-induced inward currents in the OXT neurons were significantly larger than those in the AVP neurons, using whole cell patch-clamp recording in in vitro brain slice preparation (Ohkubo et al., 2014). In this in vitro experiments, MNCs isolated from the SON were identified to be AVP and OXT neurons by visible fluorescent proteins (Ohkubo et al., 2014) in transgenic rats lines (Katoh et al., 2011; Ueta et al., 2005). It is hypothesized that OXT neurons are more highly affected by peripherally administered KA than AVP neurons even in in vivo state. The nNOS gene expression is marked increased in the SON and the PVN when OXT and AVP release is enhanced by osmotic stimulation (Srisawat et al., 2004), parturition (Popeski et al., 1999), nociceptive stimulation (Kurose et al., 2001), and stress (Hatakeyama et al., 1996; Orlando et al., 2007). NO produced by nNOS in the SON and the PVN is considered as one of the potent stimulators to elicit AVP and OXT release (Aguila et al., 2011; Orlando et al., 2007). According to the previous study (Xiao et al., 2005), it may be possible that NO distributes more to OXT release than to AVP release, although it is difficult to define the distribution of NO from our experimental condition. The expressions of the nNOS gene in the SON and PVN were significantly increased after s.c. administration of a subconvulsive dose of KA in rats. These increases of nNOS mRNA levels were relatively transient rather than those of the AVP hnRNA and OXT mRNA levels. It is possible that NO produced by nNOS may regulate the gene expressions of the AVP and OXT and secretion of these peptides after systemic administration of KA. It may be also interesting to use a nNOS inhibitor, such as 3-bromo-7-nitoroindazole, in combination with KA to examine whether the expression of the AVP or OXT would be down-regulated. These points should be clarified by further study. The physiological relevance of upregulation of AVP and OXT gene expression in response to a sub-convulsive dose of KA remains unclear. According to the previous studies, AVP has proconvulsive effects (Croiset and De Wied, 1997; Richmond, 2003) and OXT has anticonvulsive effects (Clynen et al., 2014; Erbas et al., 2013). It is possible that the primarily up-regulation of AVP may be the cause of seizure and OXT may be up-regulated to prevent seizure by s.c. administration of a subconvulsive dose of KA. The physiological meanings of this difference should be clarified by further study, in particular, in in vivo study. In conclusion, we revealed that the gene expressions of the AVP, OXT and nNOS were up-regulated even with a subconvulsive dose of KA administration in rats. Our study may bring new insight into the pharmacological effects of a systemic administration of a subconvulsive dose of KA on the hypothalamic neuropeptides.
Author contribution Experimental design: M.Y., J.O., H.S. and Y.U. Carry out experiment: M.Y., J.O., H.H., T.M., T.M. and T.O. Data analysis: M.Y. and J.O. Interpretation of the data: M.Y., J.O., and Y.U. Prepare the draft: M.Y. Prepare the figure: M.Y. Final approval: Y.U.
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