Fos expression in neurons immunoreactive for neuronal nitric oxide synthase in the rat paraventricular nucleus after intraperitoneal injection of interleukin-1β

Neuroscience Research 38 (2000) 321 – 324 www.elsevier.com/locate/neures

Rapid communication

Fos expression in neurons immunoreactive for neuronal nitric oxide synthase in the rat paraventricular nucleus after intraperitoneal injection of interleukin-1b Kazunari Todaka a, Yasushi Ishida a,*, Yuta Ishizuka a,b, Hiroyuki Hashiguchi a, Yoshio Mitsuyama a, Hiroshi Kannan b, Toshikazu Nishimori c a b

Department of Psychiatry, Miyazaki Medical College, Miyazaki 889 -1692, Japan Department of Physiology, Miyazaki Medical College, Miyazaki 889 -1692, Japan c Department of Biology, Miyazaki Medical College, Miyazaki 889 -1692, Japan Received 12 May 2000; accepted 29 June 2000

Abstract Double immunostaining for Fos and neuronal nitric oxide synthase (nNOS) was used to examine whether nNOS-immunoreactive neurons in the paraventricular hypothalamic nucleus (PVN) are activated to express Fos immunoreactivity by intraperitoneal injection of interleukin-1b (IL-1b) in the rat. Quantitative analysis revealed that some nNOS-positive PVN neurons are activated by IL-1b (4 mg/kg, i.p.) administration, but the majority of the IL-1b-activated PVN neurons do not express nNOS and are distributed mainly in the parvocellular part of the PVN. © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Interleukin-1b; Fos; Neuronal nitric oxide synthase; Paraventricular hypothalamic nucleus; Immunohistochemistry; Rat

Nitric oxide (NO) is recognized as an important messenger molecule in the central nervous system. It has been suggested that NO may modulate the release of corticotropin-releasing hormone (CRH), vasopressin, and oxytocin in the paraventricular hypothalamic nucleus (PVN, Costa et al., 1996), and that NO may modulate the stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis (Rivest and Rivier, 1994; Kishimoto et al., 1996). The NO synthetic enzyme nitric oxide synthase (NOS) has three major isoforms, neuronal NOS (nNOS), endothelial NOS, and inducible NOS (iNOS, Costa et al., 1996). The PVN, a constituent of the HPA axis, has been shown to express nNOS constitutively and intensely in the brain (Vincent and Kimura, 1992; Grossman et al., 1994). Interleukin-1 (IL-1), one of the key mediators of immunological and pathological responses to stress and * Corresponding author. Fax: +81-985-855475. E-mail address: [email protected] (Y. Ishida).

a variety of diseases developing through infectious and inflammatory processes (Dinarello, 1989), modulates the activity of the HPA axis (Chang et al., 1993; Brady et al., 1994; Ericsson et al., 1994; Rivest and Rivier, 1994; Rivest et al., 1992; Costa et al., 1996). In the present study, we examined whether nNOS-expressing PVN neurons are activated by peripherally administered IL-1b. We performed double immunohistochemistry for Fos and nNOS on rat tissue 2 h after intraperitoneal injection of IL-1b. We used male Wistar rats (Japan SLC) weighing 180–200 g. The rats were housed under a 12-h light/ dark cycle with free access to food and water. The experimental protocols used in this study were approved by the ethical committee on animal experimentation of the Miyazaki Medical College. Two hours after intraperitoneal injection of recombinant human IL-1b (4 mg/kg; Otsuka Pharmaceutical, Tokyo, Japan), the rats (n= 7) were anesthetized with pentobarbital (100 mg/kg, i.p.) and perfused transcardially at room

0168-0102/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 0 ) 0 0 1 6 6 - 8

322

K. Todaka et al. / Neuroscience Research 38 (2000) 321–324

temperature with saline, followed by 4% para formaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Control animals (n=7) were injected intraperitoneally with the same volume of saline. The brains were removed immediately, postfixed in the same fixative at 4°C for 1 h, soaked in 10% sucrose in PB for 1 h and then in 30% sucrose in PB at 4°C overnight. The brains were cut into frontal 40 mm-thick sections on a freezing microtome. Immunohistochemistry for Fos was performed using the streptavidin – biotin system (Histofine SAB-PO(R) kit; Nichirei, Tokyo, Japan, Ishida et al., 1998). After incubation in 10% normal goat serum for 20 min, the sections were incubated at 4°C overnight with rabbit polyclonal antibody that recognizes c-Fos (diluted 1:5000; Santa Cruz Biotechnology, Santa Cruz, CA). The sections were thoroughly rinsed in 0.1 M phosphate-buffered saline (PBS; pH 7.4), incubated in biotinylated goat anti-rabbit IgG at room temperature for 45 min, rinsed again in PBS, and further incubated with a streptavidin-peroxidase complex at room temperature for 15 min. After several rinses in PBS and pretreatment with 0.25% cobalt chloride to intensify visualization of immunostaining, the reaction products were visualized with 0.01% diaminobenzidine tetrahydrochloride (DAB) and 0.0003% hydrogen peroxide.

After Fos immunostaining, nNOS expression was examined using the avidin–biotin–peroxidase system (Vectastain Elite kit; Vector, Burlingame, CA). After incubation in 10% normal horse serum for 60 min, the sections were incubated at 4°C overnight with a mouse monoclonal antibody against nNOS (1:3000; Sigma, St. Louis, MO). The sections were thoroughly rinsed with PBS and incubated with a secondary biotinylated horse anti-mouse IgG at room temperature for 60 min. After several additional rinses in PBS, the sections were incubated with an avidin–peroxidase complex at room temperature for 30 min. The reaction products were visualized with 0.03% DAB and 0.0009% hydrogen peroxide. The cross-reactivity was immunohistochemically tested in the sections by omitting either of the two primary antibodies, and the test showed no crossreactivity between anti-c-Fos antibody and antinNOS antibody used in the double labeling immunohistochemistry. The sections were mounted on gelatincoated glass slides, air-dried, dehydrated, cover slipped, and observed at the microscope under bright-field illumination. To estimate the degree of colocalization of Fos and nNOS in cells of the PVN, we counted the number of Fos-positive cells in the PVN labeled or not labeled

Fig. 1. Photomicrographs of frontal sections through the PVN, showing Fos/nNOS-immunopositive cells (A, B, C) and Nissl-stained (cresyl violet) cells (D) in the PVN 2 h after the intraperitoneal injection of saline (A, D) or of IL-1b (4 mg/kg, B, C). Arrowheads indicate nNOS-/Fos+cells (black). Black arrows indicate nNOS+ /Fos-cells (brown). White arrows indicate double-labeled (nNOS +/Fos + ) cells, 3 V, third ventricle. Scale bars, 100 mm (in A, B, D); 50 mm (in C).

K. Todaka et al. / Neuroscience Research 38 (2000) 321–324

Fig. 2. The histograms illustrate the mean number of Fos-positive (single- and double-labeled) cells (A), nNOS-positive (single- and double-labeled) cells (B), double-labeled (nNOS + /Fos+ ) cells (C), and the percentage of Fos-positive cells expressing nNOS (D) in the PVN 2 h after intraperitoneal injection of saline for the control (n =7) or of IL-1b (4 mg/kg, n= 7). Results are mean 9 S.E.M. *P B 0.05, **PB0.01 compared with each corresponding value of the control (saline injection) group.

with nNOS using a 10 × microscope objective. We also determined the number of cells immunopositive for nNOS labeled and not labeled with Fos. Thereafter, the numbers obtained from two or three sections per animal were averaged. Sections from the experimental (IL-1b injection) and control (saline injection) animals were matched for rostro – caudal level (plate 25 of the atlas of Paxinos and Watson, 1997). We calculated the percentage of Fos-positive cells expressing nNOS for each group. Data were analyzed with the Mann – Whitney U-test; P values B0.05 were regarded as statistically significant. A marked induction of Fos protein was observed in the PVN following IL-1b administration. The Fos-positive cells were distributed mainly in the parvocellular part of the PVN (Fig. 1B). In contrast, nNOS-positive cells were distributed mainly in the magnocellular part of the PVN in both the saline-injected control animals and in the animals injected with IL-1b (Fig. 1A and B). At the quantitative analysis, the number of Fos-positive [single labeled (nNOS −/Fos +) and double labeled (nNOS +/Fos + )] cells was found to significantly increased (PB 0.01) in the IL-1b-treated animals (Fig. 2A). The number of nNOS-positive [single labeled (nNOS+/Fos−) and double labeled (nNOS+/Fos+ )] cells did not change instead in the PVN after IL-1b administration (Fig. 2B). The number of double labeled (nNOS +/Fos + ) cells increased (P B0.05, Fig. 2C),

323

while the percentage of Fos-positive cells expressing nNOS decreased significantly in the PVN after IL-1b administration (PB 0.05, Fig. 2D). In agreement with a previous report (Ericsson et al., 1994), the present results confirmed a marked induction of Fos protein in the PVN, mainly in the parvocellular part of the nucleus, after intraperitoneal administration of IL-1b. The present findings indicate that neurons in the parvocellular part of the PVN can be activated not only by the central administration of IL-1b (Chang et al., 1993; Rivest and Rivier, 1994; Rivest et al., 1992) but also by systemic administration of this cytokine. Although NADPH-diaphorase histochemistry has been reported to reveal the distribution of NOS in the brain (Woodside and Amir, 1996; Harada et al., 1999), discrepancies between NADPH-diaphorase-positive and NOS-positive structures have been found (Tracey et al., 1993). Therefore, we used NOS immunohistochemistry in the present study. The previous studies investigating stress-induced alterations in nNOS expression also reported that nNOS mRNA levels within the PVN increased 6 h after immobilization stress (Kishimoto et al., 1996), and that nNOS expression increased 2–3 h following LPS administration (Lee et al., 1995; Harada et al., 1999). In the present study, quantitative analysis of nNOS protein was performed by counting the number of nNOS-immunopositive cells at 2 h following IL-1b administration. Therefore, our results cannot be directly compared with those of the abovementioned previous reports. The levels of nNOS protein within the PVN could change after IL-1b administration, and the enzyme could be induced in the PVN by the treatment in neurons that are nNOS-immunonegative in basal conditions. Monitoring nNOS expression over a longer period of time, by means of Western blotting, following IL-1b treatment may address this issue. In the present study, we showed that the number of double-labeled (nNOS+ /Fos+ ) cells significantly increased, while the percentage of Fos-positive cells expressing nNOS significantly decreased in the PVN 2 h after IL-1b administration. Taken together with the marked induction of Fos, such decrease in the percentage of activated cells reflects the finding that a majority of Fos-positive neurons are not immunopositive for nNOS in the PVN 2 h after the IL-1b injection. Although these double-labeled cells represented only a small proportion of the total population of activated PVN cells, some nNOS-positive cells appeared to be activated by the IL-1b injection in the present study. The stimulatory effects of immune signals, represented by LPS and IL-1b, on CRH and vasopressin release, exerted via activation of the cyclo-oxygenase pathway and production of prostaglandin E2, may be counteracted by the increased formation of NOS and NO (Costa et al., 1996). Such putative mechanisms are

324

K. Todaka et al. / Neuroscience Research 38 (2000) 321–324

supported by the evidence that peripheral administration of LPS induces a simultaneous increase in nNOS and iNOS levels (Lee et al., 1995; Harada et al., 1999) and that peripheral administration of IL-1b induces NO release (estimated by extracellular levels of NO− 2 and NO− 3 ) in the PVN (Ishizuka et al., 1998). Thus, the present results suggest that the intraperitoneal injection of IL-1b stimulates some nNOS-positive cells in the PVN and may lead to the increased formation of NOS and NO as an agent counteracting the HPA response to immune activation. The Fos-positive and also nNOS-negative neurons may be represented, at least in part, by CRHor vasopressin-positive neurons upon which NO may act as a paracrine regulator of CRH or vasopressin upon HPA axis activation.

Acknowledgements Recombinant human IL-1b was a gift from Otsuka Pharmaceutical Company, Japan.

References Brady, L.S., Lynn, A.B., Herkenham, M., Gottesfeld, Z., 1994. Systemic interleukin-1 induces early and late patterns of c-fos mRNA expression in brain. J. Neurosci. 14, 4951–4964. Chang, S.L., Ren, T., Zadina, J.E., 1993. Interleukin-1 activation of FOS proto-oncogene protein in the rat hypothalamus. Brain Res. 617, 123 – 130. Costa, A., Poma, A., Navarra, P., Forsling, M.L., Grossman, A., 1996. Gaseous transmitters as new agents in neuroendocrine regulation. J. Endocrinol. 149, 199–207. Dinarello, C.A., 1989. Interleukin-1 and its biologically related cytokines. Adv. Immunol. 44, 153–205. Ericsson, A., Kova´cs, K.J., Sawchenko, P.E., 1994. A functional anatomical analysis of central pathways subserving the effects of interleukin-1 on stress related neuroendocrine neurons. J. Neurosci. 14, 897 – 913.

.

Grossman, A.B., Rossmanith, W.G., Kabigting, E.B., Cadd, G., Clifton, D., Steiner, R.A., 1994. The distribution of hypothalamic nitric oxide synthase mRNA in relation to gonadotrophin-releasing hormone neurons. J. Endocrinol. 140, R5 – R8. Harada, S., Imaki, T., Chikada, N., Naruse, M., Demura, H., 1999. Distinct distribution and time-course changes in neuronal nitric oxide synthase and inducible NOS in the paraventricular nucleus following lipopolysaccharide injection. Brain Res. 821, 322– 332. Ishida, Y., Todaka, K., Kuwahara, I., Ishizuka, Y., Hashiguchi, H., Nishimori, T., Mitsuyama, Y., 1998. Methamphetamine induces Fos expression in the striatum and the substantia nigra pars reticulata in a rat model of Parkinson’s disease. Brain Res. 809, 107 – 114. Ishizuka, Y., Ishida, Y., Jin, Q.-H., Shimokawa, A., Saita, M., Kato, K., Kunitake, T., Hanamori, T., Mitsuyama, Y., Kannan, H., 1998. Abdominal vagotomy attenuates interleukin-1b-induced nitric oxide release in the paraventricular nucleus region in conscious rats. Brain Res. 789, 157 – 161. Kishimoto, J., Tsuchiya, T., Emson, P.C., Nakayama, Y., 1996. Immobilization-induced stress activates neuronal nitric oxide synthase (nNOS) mRNA and protein in hypothalamic-pituitaryadrenal axis in rats. Brain Res. 720, 159 – 171. Lee, S., Barbanel, G., Rivier, C., 1995. Systemic endotoxin increases steady-state gene expression of hypothalamic nitric oxide synthase: comparison with corticotropin-releasing factor and vasopressin gene transcripts. Brain Res. 705, 136 – 148. Paxinos, G., Watson, C., 1997. The Rat Brain in Stereotaxic Coordinates, Compact, third ed. Academic Press, San Diego. Rivest, S., Rivier, C., 1994. Stress and interleukin-1b-induced activation of c-fos, NGFI-B and CRF gene expression in the hypothalamic PVN: comparison between Sprague – Dawley, Fisher-344 and Lewis rats. J. Neuroendocrinol. 6, 101 – 117. Rivest, S., Torres, G., Rivier, C., 1992. Differential effects of central and peripheral injection of interleukin-1b on brain c-fos expression and neuroendocrine functions. Brain Res. 587, 13 –23. Tracey, W.R., Nakane, M., Pollock, J.S., Forstermann, U., 1993. Nitric oxide synthases in neuronal cells, macrophages and endothelium are NADPH diaphorases, but represent only a fraction of total cellular NADPH diaphorase activity. Biochem. Biophys. Res. Commun. 195, 1035 – 1040. Vincent, S.R., Kimura, H., 1992. Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46, 755 – 784. Woodside, B., Amir, S., 1996. Reproductive state changes NADPHdiaphorase staining in the paraventricular and supraoptic nuclei of female rats. Brain Res. 739, 339 – 342.