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Expression and regulation of the immediate-early gene product Arc in the accessory olfactory bulb after mating in male rat
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Neuroscience Vol. 111, No. 2, pp. 251^258, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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EXPRESSION AND REGULATION OF THE IMMEDIATE-EARLY GENE PRODUCT ARC IN THE ACCESSORY OLFACTORY BULB AFTER MATING IN MALE RAT MASATO MATSUOKA,a * KANATO YAMAGATA,b HIROKO SUGIURA,b JUNKO YOSHIDA-MATSUOKA,a;c;d MASAO NORITAa and MASUMI ICHIKAWAc;d a
Division of Neurobiology and Anatomy, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Niigata 951-8510, Japan b
c
Department of Neuropharmacology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan
Department of Developmental Morphology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan d
CREST of the Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
AbstractöRecent studies of the accessory olfactory bulb have shown that the expression of immediate-early genes, e.g., c-fos, c-jun and egr-1, can be used as a marker of neuronal activity in response to pheromonal cues. In this study, we analyzed the expression pattern, in response to mating, of the novel immediate-early gene product Arc (an activityregulated cytoskeleton-associated protein). Arc is hypothesized to play a role in activity-dependent neuronal plasticity in the hippocampus. In a control group of male rats, only a small number of Arc-immunoreactive cells were observed in the accessory olfactory bulb. In a mating group, however, a marked increase in the number of Arc-immunoreactive cells was observed only in the granule cell layer of the accessory olfactory bulb. The increase in the number of Arc-immunoreactive cells after mating was similar to that observed for other immediate-early genes. However, for the mating group, the increase in Arc-positive cells was limited to the granule cell layer. Granule cells have been shown to exhibit a strong synaptic plasticity in response to pheromonal stimulation. From these ¢ndings we suggest that Arc plays an important role in neuronal plasticity in the accessory olfactory bulb. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: vomeronasal system, olfaction, pheromone, c-fos, immunocytochemistry.
which sni¡s and licks the secretion avidly (Macrides et al., 1974). The hypothalamus controls the reproductive system in accordance with the pheromonal information via the accessory olfactory bulb (AOB), the medial amygdala and the bed nucleus of the stria terminals (Scalia and Winans, 1975; Kevetter and Winans, 1981; Keverne, 1983; Ichikawa, 1988). The AOB is located at the posterocaudal aspect of the main olfactory bulb (MOB) and consists of simple neuronal circuits (Takami and Graziadei, 1991). A¡erent axons from sensory cells in the VNO terminate and form synaptic connections with dendrites of mitral/tufted cells in the glomerular layer of the AOB (Matsuoka et al., 1994; Rodriguez et al., 1999). Axons of mitral/tufted cells, whose cell bodies are located in the mitral/tufted cell layer (MTL), project to higher centers of the vomeronasal system (Mart|¤nezMarcos and Halpern, 1999). The mitral/tufted cell dendrites also form dendrodendritic synapses with granule cells, whose cell bodies exist in the granule cell layer (GRL) (Brennan et al., 1990; Matsuoka et al., 1998). It has been shown that the expression of immediateearly genes (IEGs) can be used as a marker for neuronal activity in response to chemosensory cues (Sagar et al., 1988; Morgan and Curran, 1991). After exposure to urine or soiled bedding of conspeci¢c animals of the opposite sex, the expression levels of the protein prod-
Pheromones are biomaterials detected by the vomeronasal organ (VNO) mainly and are involved in the regulation of various aspects of the mammalian reproductive function (Keverne, 1983; Meredith, 1983). In rodents, several biomaterials such as urine, £ank gland secretion, and vaginal secretion have dramatic e¡ects on reproductive physiology and behavior. For example, exposure of female mice to the urine of male mice results in an accelerated onset of puberty (Vandenbergh, 1969) and pregnancy block (Bruce, 1959). The vaginal secretion of the female hamster, in contrast, is a strong attractant to the male hamster (Johnston, 1974; Powers et al., 1979; Steel, 1984)
*Corresponding author. Tel. : +81-25-227-2055; fax: +81-25-2270753. E-mail address: [email protected] (M. Matsuoka). Abbreviations : AOB, accessory olfactory bulb; Arc, activity-regulated cytoskeleton-associated protein ; BSA, bovine serum albumin ; EDTA, ethylenediaminetetra-acetate; EGTA, ethylene glycol-bis (2-aminoethyl-ether)-N,N,NP,NP-tetraacetic acid ; GRL, granule cell layer; IEG, immediate-early gene; ir, immunoreactive; MOB, main olfactory bulb; MTL, mitral/tufted cell layer; PB, phosphate bu¡er ; PBS, phosphate-bu¡ered saline; PBST, PBS with 0.3% Triton X-100; SD, Sprague^Dawley; TH, tyrosine hydroxylase; VNO, vomeronasal organ. 251
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ucts of IEGs, e.g., c-fos and egr-1, increase in the VNO and the AOB and in higher centers of the vomeronasal system (Bakker et al., 1996; Bressler and Baum, 1996; Brennan et al., 1999; Dudley and Moss, 1999; Halem et al., 1999; Inamura et al., 1999; Matsuoka et al., 1999). Several studies have shown that mating increases the number of c-Fos-positive cells in the vomeronasal system, particularly in the AOB, of male and female rodents (Brennan et al., 1992; Fernandez-Fewell and Meredith, 1994; Greco et al., 1996). These previous studies of the AOB have shown that the expression of IEGs can be used as a marker of neuronal activity in response to pheromonal cues. However, almost all proteins encoded by these IEGs are actively transferred to the nucleus after synthesis, and their immunoreactivities are observed only in the nucleus. To understand the mechanism of discrimination and transmission of pheromonal information, it is necessary to ¢nd a novel marker responding to pheromonal cues. The protein product of the novel IEG, Arc (activity-regulated cytoskeleton-associated protein), is enriched in the brain and is rapidly regulated by neuronal activity (Lyford et al., 1995). Arc is hypothesized to play a role in the activity-dependent neuronal plasticity in the hippocampus (Steward et al., 1998). In addition, it was recently reported that Arc-immunoreactive (Arc-ir) cells increased on the dendrites in MOB glomeruli, which were activated by odor stimulation (Guthrie et al., 2000). In this study, we analyzed the expression pattern of Arc immunoreactivity in response to mating in the AOB of the adult male rat.
EXPERIMENTAL PROCEDURES
Animal treatment and tissue preparation Sexually inexperienced male Sprague^Dawley (SD) rats (Clea Japan, Japan), 7^8 weeks old, were used for immunoblot and immunocytochemical analyses. Animals were housed in standard laboratory cages (40U30U20 cm, one per cage) with food and water available ad libitum under 12-h reversed light cycles (lights on 8.00 h). The animals were randomly divided into two groups: (1) rats placed in contact with an estrous female in a cage with the soiled bedding of females which had not been cleaned for more than 24 h (mating group, n = 4 for immunoblot analysis and n = 6 for immunocytochemical analysis), and (2) rats exposed to clean bedding (control group, n = 4 for immunoblot analysis and n = 6 for immunocytochemical analysis). The estrous state of the females was monitored daily by taking vaginal smears. Mounting of all males in the mating group was observed. Successful mating was indicated by the presence of a vaginal plug. Males in the control group were placed individually in cages with clean bedding. Four hours after the males were transferred, the animals were killed by decapitation and their olfactory bulbs were removed from the skull for immunoblot analysis. For immunocytochemical analysis, the animals were deeply anesthetized by injection of sodium pentobarbital (5 mg/animal, i.p.), then perfused transcardially with saline followed by a ¢xative solution [4% paraformaldehyde in 0.1 M phosphate bu¡er (PB)]. The olfactory bulbs of the rats in each of the two groups were dissected out, post¢xed overnight at 4‡C in the same ¢xative solution, and used to study the pattern of neuronal activation in response to mating. Immunoblot analysis The AOB of each animal was homogenized with 5 vol. (v/w)
of 40 mM Tris/HCl bu¡er, pH 7.6, containing 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl £uoride, and 10 WM/ml protease inhibitors antipain, leupeptin and pepstatin A (bu¡er A). After centrifugation at 18 000Ug (Rav 6.5 cm) for 30 min at 4‡C, the supernatant was decanted. The precipitate was homogenized with 5 vol. of bu¡er A containing 0.1% Triton X-100. The precipitate fraction was separated by 10% polyacrylamide^sodium dodecyl sulfate gel electrophoresis and electrophoretically transferred to a polyvinylidene £uoride membrane. The transblot was preincubated with 5% nonfat dry skim milk in Tris-bu¡ered saline (TBS), pH 7.4, and then incubated overnight with polyclonal antibodies speci¢c to Arc (generated by Dr. K. Yamagata and coworkers; Lyford et al., 1995), c-Fos (rabbit polyclonal, Ab-5, Oncogene Science, MA, USA) and Gi2K (rabbit polyclonal, Wako, Japan). The membrane was washed with TBS containing 0.1% Triton X-100, and then incubated with a horseradish peroxidasecoupled antibody (diluted to 1:4000). Immunoreactive bands were detected using enhanced chemiluminescence reagents. Nerve transection To block the transmission of vomeronasal information on one side of the animal, we performed a left vomeronasal nerve transection. Four male SD rats, 7^8 weeks old, were anesthetized with sodium pentobarbital. The frontal bone covering the left olfactory bulb was removed, and a Te£on blade was used to cut the vomeronasal nerve ¢bers passing between the cribriform plate and the left olfactory bulb (Ichikawa et al., 1998). The vomeronasal nerves on the right side of the animal were left intact and served as a control. After a 40-day recovery period, the males were placed in contact with an estrous female in a cage with soiled bedding of females. Four hours after exposure, they were transferred for immunocytochemical study. Successful mating was indicated by the presence of a vaginal plug in the females. We investigated the e¡ect of nerve transection on the a¡erent and e¡erent projections to the AOB by immunocytochemical studies of the AOB using antibodies against N-CAM (mouse monoclonal, Sigma, MO, USA; diluted to 1:100), expressed in axon processes, and tyrosine hydroxylase (TH) (rabbit polyclonal, Chemicon, CA, USA; diluted to 1:250), a marker of dopaminergic ¢bers. All animal procedures used in this study were approved by the Institutional Animal Care Ethics Committee of the Tokyo Metropolitan Institute for Neuroscience. Immunocytochemistry The olfactory bulbs were immersed in 0.1 M PB containing 30% sucrose until they sank. Serial sagittal sections of the olfactory bulb (40-Wm thickness) were cut using a frozen microtome. Six sagittal sections (three from the right and three from the left) passing through the middle region of the AOB were selected from each animal for immunocytochemical analysis. After blocking the endogenous peroxidase activity by incubation with 3% hydrogen peroxide in methanol, the sections were rinsed in 50 mM phosphate-bu¡ered saline (PBS) containing 0.3% Triton X-100 (PBST, pH 7.4) for 45 min with three changes of the bu¡er, and then, nonspeci¢c binding components were blocked with a solution of 1% bovine serum albumin (BSA) in PBST (BSA^PBST) for 1 h at room temperature. Sections were incubated with a primary antibody for approximately 50 h at 4‡C. The primary antibodies were used in BSA^PBST at the following dilutions. The rabbit polyclonal anti-Arc antibody (Lyford et al., 1995) was diluted at 1:500 and the rabbit polyclonal anti-c-Fos antibody was diluted at 1:40 000. They were incubated further with biotinylated anti-rabbit IgG (7.5 Wg/ml; Vector Laboratories, Burlingame, CA, USA) in BSA^PBST for 1 h at room temperature, and then with the avidin^biotin complex (1:100; Amersham, Buckingham, UK) in BSA^PBST for 1 h at room temperature. Each step was followed by four washes with PBST for 15 min each. After the last wash, the sections were immersed in 0.175 M sodium acetate bu¡er, pH 7.4, for 30 min with two changes of the bu¡er, then incubated
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with the chromogen solution, which consisted of nickel chloride (0.25 mg/ml), 3,3P-diaminobenzidine tetrahydrochloride (0.2 mg/ ml) and 0.0025% hydrogen peroxide in the same bu¡er, for 10 min. The reaction was stopped by transferring the sections to sodium acetate bu¡er. Then, they were washed with 10 mM PBS for 20 min with three changes of the bu¡er. After washing with 10 mM PBS, the sections were mounted onto gelatin-coated glass slides.
induced in the AOB of the males of the mating group. In addition, we performed western blot analysis using a control protein from the experimental animals, and adopted the Gi2 antibody as the control protein. No induction was observed in Gi2 of the AOB of male rats after mating.
Quanti¢cation and data analyses
Localization of Arc-immunoreactivity in the AOB after mating
In this study, we observed the MTL and GRL in the AOB of SD male rats in light micrographs of 40-Wm-thick sagittal sections. Sections including large artifacts were excluded. We selected sections that had the largest glomerular regions of the AOB and quantitatively analyzed all MTL and GRL regions. The numbers of Arc-ir and c-Fos-ir cells in each of the two layers were determined and the total area of each layer was measured using an image analyzer (Zeiss KS300). Then, the numerical density of stained cells for each of the two layers was calculated and compared among the groups. Statistical analysis was performed using analysis of variance.
RESULTS
Immunoblot analysis To investigate whether the Arc protein is induced in the AOB after mating, the anti-Arc antibody was used for western blot analysis. Consequently, single protein bands with an apparent molecular weight of 55 kDa were identi¢ed by western blot analysis of AOB of male rats. The Arc protein was observed in the AOB of control group, but it was strongly induced in that of the mating group (Fig. 1). The c-Fos protein was also
We used immunocytochemistry to investigate the localization of Arc in the AOB after mating. The AOB includes ¢ve layers, namely, the vomeronasal nerve layer, the glomerular layer, the MTL, the olfactory tract layer and the GRL. There are numerous neurons present in the glomerular layer, the MTL and the GRL. In rats of the control group, only a few Arc-ir cells were observed throughout these three layers of the AOB. In contrast, in rats of the mating group, a marked increase in the number of Arc-ir cells was observed within the GRL. A high density of Arc-ir cells was observed along the anterior^ posterior axis (Fig. 2). No Arc-ir cells were observed in the glomerular layer. Arc-ir cells were observed at a low density in the basal region of the MTL, near the boundaries of the olfactory tract layer, and no Arc-ir cells were observed in the middle region of the MTL. In addition, all Arc-ir cells in the MTL appeared to be smaller than the mitral/tufted cells. The intracellular staining pattern of Arc, whose expression was observed in cell bodies and dendrite trees, di¡ered from those of other products of IEGs (Fig. 2E). In this study, we performed a quantitative analysis of Arc-ir cells in the MTL and the GRL of the AOB. Results of the quantitative analysis were con-
Fig. 1. Expression of Arc, c-Fos and Gi2 in the AOB of male SD rats after mating. Arc and c-Fos proteins were induced after mating with a female SD rat in the AOB of the male SD rats. In particular, induction of the Arc protein after mating was intense. No induction was observed in Gi2 of the AOB of male rats after mating. C, control ; M, mating.
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Fig. 2. Light photomicrographs showing Arc-ir cells in the AOB of adult male SD rats after mating. The 40-Wm-thick sagittal sections were stained with an antibody against Arc. Rostral of the AOB is right side and caudal is left. (A) AOB of adult male SD rats after exposure to clean bedding (control group). (B) Higher magni¢cation of the MTL and the GRL of (A). (C) AOB of adult male SD rats after mating (mating group). (D) Higher magni¢cation of the MTL and the GRL of (C). (E) High-power magni¢cation of granule cells in the AOB of an adult SD male rat after mating. Arc-ir is observed in a granule cell dendrite (arrows). Scale bars = 250 Wm (A, C); 100 Wm (B, D); 50 Wm (E).
sistent with immunocytochemical observations. Data are shown in Fig. 5. Thus, after mating, the expression of the Arc protein is concentrated in the granule cells in the AOB of SD male rats.
antibody (Fig. 3). After mating, many Arc-ir cells were observed in the GRL on the AOB of the left side, but on the nerve-transected side (right side), no Arc-ir cells were observed (Fig. 3).
E¡ect of vomeronasal nerve transection on Arc-immunoreactivity
Expression pattern of Fos-immunoreactivity in the AOB after mating
We investigated the e¡ect of vomeronasal stimulation on Arc-ir labeling in the AOB by vomeronasal nerve transection. Forty days after surgery, immunocytochemical observation using the N-CAM antibody con¢rmed that the vomeronasal nerve had degenerated and did not recover (Fig. 3). However, the animals (n = 4) still accomplished mating, because they had an intact vomeronasal and olfactory system on the left side and a recovered olfactory system on the right side. In addition, it was con¢rmed that the e¡erent dopaminergic projections to the AOB were not a¡ected by nerve transection as determined by immunocytochemical observation using the TH
To compare the expression patterns between Arc and Fos immunoreactivites, we performed immunocytochemical staining using the c-Fos antibody. There were numerous Fos-ir cells in the glomerular layer, the MTL and the GRL of the AOB after mating, and their expression is localized to the nuclei. However, even in the control group, some Fos-ir cells were observed in all three layers of the AOB (Fig. 4). In addition, we performed a quantitative analysis of Fos-ir cells in the MTL and the GRL of the AOB. Results of the quantitative analysis were consistent with immunocytochemical observations. Data are shown in Fig. 5. Thus, the expression pattern of
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Fig. 3. Light micrographs showing Arc- (A, D), N-CAM- (B, E) and TH- (C, F) immunoreactivity in the AOB of treated male rats (nerve transection) after mating. The 40-Wm-thick sagittal sections were stained with each antibody. Rostral of the AOB is right side and caudal is left. (A^C) AOB on the intact side of adult male SD rats after mating. (D^F) AOB on the vomeronasal nerve-transected side of same animal. Scale bars = 250 Wm (A, B, D, E); 100 Wm (C, F). VNL: vomeronasal nerve layer; GL: glomerular layer; OTL: olfactory tract layer.
the c-Fos-protein immunoreactivity after mating was similar to that of the Arc-protein immunoreactivity, but the localization in the AOB and the intracellular expression was extremely di¡erent between the two proteins.
DISCUSSION
Localization of Arc protein in the AOB after mating The vomeronasal system has an important role in the mammalian reproductive function (Wysocki, 1979; Halpern, 1987). The AOB is the ¢rst relay station in the vomeronasal system. The aim of the present study was to investigate the changes of the novel IEG product, Arc, in the AOB of male rats after mating. Firstly, we performed western blot analysis of AOB of male rats. A single protein band with an apparent molecular weight of 55 kDa was identi¢ed, and induction of the protein in the AOB of male SD rats was observed after mating
(Fig. 1). The size of this protein is consistent with the reported molecular size of the Arc protein in the rat cortex and hippocampus (Lyford et al., 1995). Therefore, it is likely that this single band represents the Arc protein. We examined the localization of the Arc protein in the AOB of male rats after mating. The number of Arc-ir cells in the AOB of adult male rats increased markedly after mating; a similar increase was observed in the other IEG, c-fos. However, the increase in the number of Arcir cells was localized to the GRL and di¡ered from that of Fos-ir cells that were expressed in all layers of the AOB. An increase in the number of Arc-ir cells was also detected in the MTL of the AOB. However, Arc-ir cells were concentrated in the outer layer of the MTL near the boundaries of the olfactory tract layer, and appeared to be smaller than the mitral/tufted cells. Hence, it is possible that Arc-ir cells in the MTL are granule cells. Thus, the results in the present study suggest that Arc is speci¢cally expressed in granule cells in the AOB after mating. It is very interesting that the Arc protein is expressed in one subpopulation of granule cells
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Fig. 4. Light photomicrographs showing Fos-ir cells in the AOB of adult male SD rats after mating. Rostral of the AOB is right side and caudal is left. (A) AOB of adult male SD rats after exposure to clean bedding (control group). (B) Higher magni¢cation of the MTL and the GRL of (A). (C) AOB of adult male SD rats after mating (mating group). (D) Higher magni¢cation of the MTL and the GRL of (C). Scale bars = 250 Wm (A, C); 100 Wm (B, D).
in the AOB after mating. In the AOB, the functional roles of the Arc protein have been uncertain. In the other parts of the CNS, the expression of Arc mRNA was induced by electroconvulsive shock (ECS) in the cerebral cortex and the hippocampus (Lyford et al., 1995; Steward et al., 1998). In these areas, the Arc protein is hypothesized to play roles in the activity-dependent neuronal plasticity and modi¢cation of the dendrite structure for interaction with the neuronal cytoskeleton (Lyford et al., 1995). Similarly, it is possible that the Arc protein plays an important role in neuronal plasticity in the AOB. Further studies using morphological, physiological, or molecular strategies may be helpful in con¢rming the functional roles of the Arc protein in the AOB. E¡ect of vomeronasal nerve transection on Arc expression The AOB receives both a¡erent and e¡erent inputs. Axons of vomeronasal sensory cells terminate in the glomeruli of the AOB (Raisman, 1985). In addition, it has been demonstrated that noradrenergic nerve ¢bers from the locus coeruleus terminate in the GRL and the MTL (Shipley et al., 1985). Naturally, during mating, animals are exposed to pheromones of their partner. Moreover, when a male and female mate, the brain noradrenergic system is activated, thereby increasing the noradrenaline level in the AOB (Keverne and de la Riva, 1982; Rosser and Keverne, 1985). To con¢rm which input strongly a¡ects the increase in the number of Arc-ir cells in the AOB, we performed vomeronasal nerve transection to selectively block the vomeronasal a¡erent input. Forty days after transection, axon projections of the olfactory
nerve to the MOB recovered, but projections of the vomeronasal nerve to the AOB did not recover, which is consistent with results of a previous report (Ichikawa, 1999). In addition, immunocytochemical observation using the TH antibody con¢rmed that e¡erent dopaminergic projections to the AOB were not a¡ected by nerve transection. Consequently, on the nerve-transected side, Arc-ir cells disappeared completely after mating. A previous study revealed that removal of the VNO eliminated the increase in c-Fos-protein expression levels in the granule cells induced by exposure to soiled bedding or mating (Fernandez-Fewell and Meredith, 1994; Rajendren and Moss, 1994). Thus, the results of the present study agree with those of previous studies, i.e., the increase in the Arc expression level in the AOB depended strongly on pheromonal stimulation, the absence of which eliminated the increase in the expression level of Arc or other IEGs. However, this evidence cannot exclude the e¡ect of activation of noradrenergic ¢bers on Arc expression in the AOB. Further experiments are necessary to con¢rm these observations. Comparative study between Arc and c-Fos proteins One of the main objectives of the present study was to analyze di¡erences in the intracellular localization and expression pattern in the AOB between the Arc protein and proteins encoded by other IEGs. We adopted the c-Fos protein encoded by a typical IEG, c-fos, for comparison, because there have been several published reports on c-Fos in both the main olfactory and the vomeronasal system. Several di¡erences in the intracellular localization and expression pattern between Arc and
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Fig. 5. Expression pattern of Arc and Fos in the AOB of male rats after mating (mean number of stained nuclei/10 000 Wm2 þ S.E.M.) #P 6 0.01 statistically signi¢cant di¡erence from the control group (analysis of variance).
c-Fos proteins have been demonstrated in this study. (1) As stated above, the Arc-protein expression was localized in the GRL of the AOB, while c-Fos-protein expression was not. (2) The Arc protein was not expressed in the control group, but the c-Fos protein was expressed in both the control and mating groups. (3) In contrast to c-Fos, Arc is not restricted to the nucleus; instead, it appears to be present in the cytoplasm of the granule cell body and in dendritic processes. This observation is very important and makes Arc an excellent marker of neuronal activity after mating. The c-Fos protein forms heterodimers with proteins encoded by the jun family, which are transferred actively to the nucleus. In contrast, the Arc protein has a modest degree of homology with the cytoskeletal protein K-spectrin (Lyford et al., 1995). Hence, it is hypothesized that in the dentate gyrus, newly produced Arc mRNA by ECS is transferred to rapidly activated postsynaptic processes, and continuously synthesized proteins play an important role in activity-dependent synaptic modi¢cation involving an interaction with neurocytoskeleton molecules (Steward et al., 1998). Thus, in the extranucleus, the Arc protein is hypothesized to be a modulator involved in synaptic plasticity. In contrast, it is known that there is a £exible synaptic plasticity in the granule cells of the AOB. It has been reported that pheromonal memory
formation during mating was accomplished by the postsynaptic modi¢cation of excitatory synapses between mitral/tufted and granule cells, that is, the modi¢cation of postsynaptic density in the granule cell dendritic spines (Matsuoka et al., 1997). However, not all synapses in the granule cells of the AOB need to be modi¢ed during memory formation; probably, only a part of the subpopulation of these synapses related to the partner’s pheromones should be modi¢ed. If the Arc protein plays an important role related to the synaptic plasticity in the dendritic spines of the granule cells, it should be possible to observe activated spines using Arc immunoreactivity. Thus, the application of Arc is a useful and accurate marker of activated synapses in response to pheromonal stimulation, which is related to memory formation during mating. AcknowledgementsöWe wish to thank Dr. R.M. Costanzo for helpful discussions and assistance. This work was supported in part by CREST of JST (M.M. and M.I.), a Grant-in-Aid for Scienti¢c Research (13760192) from the Japan Society for the Promotion of Science and the Ministry of Education, Science, Sports and Culture of Japan (M.M.), grants from the Japan Society for the Promotion of Science and the Ministry of Education, Science, Sports and Culture of Japan (K.Y.) and a Grant-in-Aid for Scienti¢c Research (C) from Japan Society for the Promotion Science (H.S.).
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Neurol. 197, 81^98. Lyford, G.L., Yamagata, K., Kaufmann, W.E., Barnes, C.A., Sanders, L.K., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Lanahan, A.A., Worley, P.F., 1995. Arc, a growth factor and activity-regulated gene, encoded a novel cytoskeleton-associated protein that is enriched in neuronal dendrites. Neuron 14, 433^445. Macrides, F., Bartke, A., Fernandez, F., D’Angelo, W., 1974. E¡ects of exposure to vaginal odor and receptive females on plasma testosterone in the male hamster. Neuroendocrinology 15, 355^364. Mart|¤nez-Marcos, A., Halpern, M., 1999. Di¡erential centrifugal a¡erents to the anterior and posterior accessory olfactory bulb. NeuroReport 10, 2011^2015. Matsuoka, M., Kaba, H., Mori, Y., Ichikawa, M., 1997. Synaptic plasticity in olfactory memory formation in female mice. NeuroReport 8, 2501^ 2504. Matsuoka, M., Mori, Y., Hoshino, K., Ichikawa, M., 1994. Social environment a¡ects synaptic structure in the glomerulus of the accessory olfactory bulb of the hamster. Neurosci. Res. 19, 187^193. Matsuoka, M., Mori, Y., Ichikawa, M., 1998. Morphological changes of synapses induced by urinary stimulation in the hamster accessory olfactory bulb. Synapse 28, 160^166. Matsuoka, M., Yokosuka, M., Mori, Y., Ichikawa, M., 1999. Speci¢c expression pattern of Fos in the accessory olfactory bulb of male mice after exposure to soiled bedding of females. Neurosci. Res. 35, 189^195. Meredith, M., 1983. Sensory physiology of pheromone communication. In: Vandenbergh, J.G. (Ed.), Pheromones and Reproduction in Mammals. Academic, New York, pp. 199^252. Morgan, J.I., Curran, T., 1991. Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu. Rev. Neurosci. 14, 421^451. Powers, J.B., Fields, R.B., Winans, S.S., 1979. Olfactory and vomeronasal system participation in male hamsters’ attraction to female vaginal secretions. Physiol. Behav. 22, 77^84. Raisman, G., 1985. Specialized neuroglial arrangement may explain the capacity of vomeronasal axons to reinnervate central neurons. Neuroscience 14, 237^254. Rajendren, G.V., Moss, R.L., 1994. Vomeronasal organ-mediated induction of fos in the central accessory olfactory pathways in repetitively mated female rats. Brain Res. Bull. 34, 53^59. Rodriguez, I., Feinstein, P., Mombaerts, P., 1999. Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system. Cell 97, 199^208. Rosser, A.E., Keverne, E.B., 1985. The importance of central noradrenergic neurones in the formation of an olfactory memory in the prevention of pregnancy block. Neuroscience 15, 1141^1147. Sagar, S.M., Sharp, F.R., Curran, T., 1988. Expression of c-fos protein in brain: metabolic mapping at the cellular level. Science 240, 1328^1331. Scalia, F., Winans, S.S., 1975. The di¡erential projections of the olfactory bulb and accessory olfactory bulb in mammals. J. Comp. Neurol. 161, 31^55. Shipley, M.T., Halloran, F.J., de la Torre, J., 1985. Surprisingly rich projection from locus coeruleus to the olfactory bulb in the rat. Brain Res. 329, 294^299. Steel, E., 1984. E¡ect of the odour of vaginal secretion on non-copulatory behaviour of male hamsters (Mesocricetus auratus). Anim. Behav. 32, 597^608. Steward, O., Wallace, C.S., Lyford, G.L., Worley, P.F., 1998. Synaptic activation causes the mRNA for the IEG Arc to localize selectively near activated postsynaptic sites on dendrites. Neuron 21, 741^751. Takami, S., Graziadei, P.P., 1991. Light microscopic Golgi study of mitral/tufted cells in the accessory olfactory bulb of the adult rat. J. Comp. Neurol. 311, 65^83. Vandenbergh, J.G., 1969. Male odor accelerates female sexual maturation in mice. Endocrinology 84, 658^660. Wysocki, C.J., 1979. Neurobehavioral evidence for the involvement of the vomeronasal system in mammalian reproduction. Neurosci. Biobehav. Rev. 3, 301^341. (Accepted 13 December 2001)
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Neuroscience Vol. 111, No. 2, pp. 251^258, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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EXPRESSION AND REGULATION OF THE IMMEDIATE-EARLY GENE PRODUCT ARC IN THE ACCESSORY OLFACTORY BULB AFTER MATING IN MALE RAT MASATO MATSUOKA,a * KANATO YAMAGATA,b HIROKO SUGIURA,b JUNKO YOSHIDA-MATSUOKA,a;c;d MASAO NORITAa and MASUMI ICHIKAWAc;d a
Division of Neurobiology and Anatomy, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Niigata 951-8510, Japan b
c
Department of Neuropharmacology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan
Department of Developmental Morphology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan d
CREST of the Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
AbstractöRecent studies of the accessory olfactory bulb have shown that the expression of immediate-early genes, e.g., c-fos, c-jun and egr-1, can be used as a marker of neuronal activity in response to pheromonal cues. In this study, we analyzed the expression pattern, in response to mating, of the novel immediate-early gene product Arc (an activityregulated cytoskeleton-associated protein). Arc is hypothesized to play a role in activity-dependent neuronal plasticity in the hippocampus. In a control group of male rats, only a small number of Arc-immunoreactive cells were observed in the accessory olfactory bulb. In a mating group, however, a marked increase in the number of Arc-immunoreactive cells was observed only in the granule cell layer of the accessory olfactory bulb. The increase in the number of Arc-immunoreactive cells after mating was similar to that observed for other immediate-early genes. However, for the mating group, the increase in Arc-positive cells was limited to the granule cell layer. Granule cells have been shown to exhibit a strong synaptic plasticity in response to pheromonal stimulation. From these ¢ndings we suggest that Arc plays an important role in neuronal plasticity in the accessory olfactory bulb. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: vomeronasal system, olfaction, pheromone, c-fos, immunocytochemistry.
which sni¡s and licks the secretion avidly (Macrides et al., 1974). The hypothalamus controls the reproductive system in accordance with the pheromonal information via the accessory olfactory bulb (AOB), the medial amygdala and the bed nucleus of the stria terminals (Scalia and Winans, 1975; Kevetter and Winans, 1981; Keverne, 1983; Ichikawa, 1988). The AOB is located at the posterocaudal aspect of the main olfactory bulb (MOB) and consists of simple neuronal circuits (Takami and Graziadei, 1991). A¡erent axons from sensory cells in the VNO terminate and form synaptic connections with dendrites of mitral/tufted cells in the glomerular layer of the AOB (Matsuoka et al., 1994; Rodriguez et al., 1999). Axons of mitral/tufted cells, whose cell bodies are located in the mitral/tufted cell layer (MTL), project to higher centers of the vomeronasal system (Mart|¤nezMarcos and Halpern, 1999). The mitral/tufted cell dendrites also form dendrodendritic synapses with granule cells, whose cell bodies exist in the granule cell layer (GRL) (Brennan et al., 1990; Matsuoka et al., 1998). It has been shown that the expression of immediateearly genes (IEGs) can be used as a marker for neuronal activity in response to chemosensory cues (Sagar et al., 1988; Morgan and Curran, 1991). After exposure to urine or soiled bedding of conspeci¢c animals of the opposite sex, the expression levels of the protein prod-
Pheromones are biomaterials detected by the vomeronasal organ (VNO) mainly and are involved in the regulation of various aspects of the mammalian reproductive function (Keverne, 1983; Meredith, 1983). In rodents, several biomaterials such as urine, £ank gland secretion, and vaginal secretion have dramatic e¡ects on reproductive physiology and behavior. For example, exposure of female mice to the urine of male mice results in an accelerated onset of puberty (Vandenbergh, 1969) and pregnancy block (Bruce, 1959). The vaginal secretion of the female hamster, in contrast, is a strong attractant to the male hamster (Johnston, 1974; Powers et al., 1979; Steel, 1984)
*Corresponding author. Tel. : +81-25-227-2055; fax: +81-25-2270753. E-mail address: [email protected] (M. Matsuoka). Abbreviations : AOB, accessory olfactory bulb; Arc, activity-regulated cytoskeleton-associated protein ; BSA, bovine serum albumin ; EDTA, ethylenediaminetetra-acetate; EGTA, ethylene glycol-bis (2-aminoethyl-ether)-N,N,NP,NP-tetraacetic acid ; GRL, granule cell layer; IEG, immediate-early gene; ir, immunoreactive; MOB, main olfactory bulb; MTL, mitral/tufted cell layer; PB, phosphate bu¡er ; PBS, phosphate-bu¡ered saline; PBST, PBS with 0.3% Triton X-100; SD, Sprague^Dawley; TH, tyrosine hydroxylase; VNO, vomeronasal organ. 251
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ucts of IEGs, e.g., c-fos and egr-1, increase in the VNO and the AOB and in higher centers of the vomeronasal system (Bakker et al., 1996; Bressler and Baum, 1996; Brennan et al., 1999; Dudley and Moss, 1999; Halem et al., 1999; Inamura et al., 1999; Matsuoka et al., 1999). Several studies have shown that mating increases the number of c-Fos-positive cells in the vomeronasal system, particularly in the AOB, of male and female rodents (Brennan et al., 1992; Fernandez-Fewell and Meredith, 1994; Greco et al., 1996). These previous studies of the AOB have shown that the expression of IEGs can be used as a marker of neuronal activity in response to pheromonal cues. However, almost all proteins encoded by these IEGs are actively transferred to the nucleus after synthesis, and their immunoreactivities are observed only in the nucleus. To understand the mechanism of discrimination and transmission of pheromonal information, it is necessary to ¢nd a novel marker responding to pheromonal cues. The protein product of the novel IEG, Arc (activity-regulated cytoskeleton-associated protein), is enriched in the brain and is rapidly regulated by neuronal activity (Lyford et al., 1995). Arc is hypothesized to play a role in the activity-dependent neuronal plasticity in the hippocampus (Steward et al., 1998). In addition, it was recently reported that Arc-immunoreactive (Arc-ir) cells increased on the dendrites in MOB glomeruli, which were activated by odor stimulation (Guthrie et al., 2000). In this study, we analyzed the expression pattern of Arc immunoreactivity in response to mating in the AOB of the adult male rat.
EXPERIMENTAL PROCEDURES
Animal treatment and tissue preparation Sexually inexperienced male Sprague^Dawley (SD) rats (Clea Japan, Japan), 7^8 weeks old, were used for immunoblot and immunocytochemical analyses. Animals were housed in standard laboratory cages (40U30U20 cm, one per cage) with food and water available ad libitum under 12-h reversed light cycles (lights on 8.00 h). The animals were randomly divided into two groups: (1) rats placed in contact with an estrous female in a cage with the soiled bedding of females which had not been cleaned for more than 24 h (mating group, n = 4 for immunoblot analysis and n = 6 for immunocytochemical analysis), and (2) rats exposed to clean bedding (control group, n = 4 for immunoblot analysis and n = 6 for immunocytochemical analysis). The estrous state of the females was monitored daily by taking vaginal smears. Mounting of all males in the mating group was observed. Successful mating was indicated by the presence of a vaginal plug. Males in the control group were placed individually in cages with clean bedding. Four hours after the males were transferred, the animals were killed by decapitation and their olfactory bulbs were removed from the skull for immunoblot analysis. For immunocytochemical analysis, the animals were deeply anesthetized by injection of sodium pentobarbital (5 mg/animal, i.p.), then perfused transcardially with saline followed by a ¢xative solution [4% paraformaldehyde in 0.1 M phosphate bu¡er (PB)]. The olfactory bulbs of the rats in each of the two groups were dissected out, post¢xed overnight at 4‡C in the same ¢xative solution, and used to study the pattern of neuronal activation in response to mating. Immunoblot analysis The AOB of each animal was homogenized with 5 vol. (v/w)
of 40 mM Tris/HCl bu¡er, pH 7.6, containing 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl £uoride, and 10 WM/ml protease inhibitors antipain, leupeptin and pepstatin A (bu¡er A). After centrifugation at 18 000Ug (Rav 6.5 cm) for 30 min at 4‡C, the supernatant was decanted. The precipitate was homogenized with 5 vol. of bu¡er A containing 0.1% Triton X-100. The precipitate fraction was separated by 10% polyacrylamide^sodium dodecyl sulfate gel electrophoresis and electrophoretically transferred to a polyvinylidene £uoride membrane. The transblot was preincubated with 5% nonfat dry skim milk in Tris-bu¡ered saline (TBS), pH 7.4, and then incubated overnight with polyclonal antibodies speci¢c to Arc (generated by Dr. K. Yamagata and coworkers; Lyford et al., 1995), c-Fos (rabbit polyclonal, Ab-5, Oncogene Science, MA, USA) and Gi2K (rabbit polyclonal, Wako, Japan). The membrane was washed with TBS containing 0.1% Triton X-100, and then incubated with a horseradish peroxidasecoupled antibody (diluted to 1:4000). Immunoreactive bands were detected using enhanced chemiluminescence reagents. Nerve transection To block the transmission of vomeronasal information on one side of the animal, we performed a left vomeronasal nerve transection. Four male SD rats, 7^8 weeks old, were anesthetized with sodium pentobarbital. The frontal bone covering the left olfactory bulb was removed, and a Te£on blade was used to cut the vomeronasal nerve ¢bers passing between the cribriform plate and the left olfactory bulb (Ichikawa et al., 1998). The vomeronasal nerves on the right side of the animal were left intact and served as a control. After a 40-day recovery period, the males were placed in contact with an estrous female in a cage with soiled bedding of females. Four hours after exposure, they were transferred for immunocytochemical study. Successful mating was indicated by the presence of a vaginal plug in the females. We investigated the e¡ect of nerve transection on the a¡erent and e¡erent projections to the AOB by immunocytochemical studies of the AOB using antibodies against N-CAM (mouse monoclonal, Sigma, MO, USA; diluted to 1:100), expressed in axon processes, and tyrosine hydroxylase (TH) (rabbit polyclonal, Chemicon, CA, USA; diluted to 1:250), a marker of dopaminergic ¢bers. All animal procedures used in this study were approved by the Institutional Animal Care Ethics Committee of the Tokyo Metropolitan Institute for Neuroscience. Immunocytochemistry The olfactory bulbs were immersed in 0.1 M PB containing 30% sucrose until they sank. Serial sagittal sections of the olfactory bulb (40-Wm thickness) were cut using a frozen microtome. Six sagittal sections (three from the right and three from the left) passing through the middle region of the AOB were selected from each animal for immunocytochemical analysis. After blocking the endogenous peroxidase activity by incubation with 3% hydrogen peroxide in methanol, the sections were rinsed in 50 mM phosphate-bu¡ered saline (PBS) containing 0.3% Triton X-100 (PBST, pH 7.4) for 45 min with three changes of the bu¡er, and then, nonspeci¢c binding components were blocked with a solution of 1% bovine serum albumin (BSA) in PBST (BSA^PBST) for 1 h at room temperature. Sections were incubated with a primary antibody for approximately 50 h at 4‡C. The primary antibodies were used in BSA^PBST at the following dilutions. The rabbit polyclonal anti-Arc antibody (Lyford et al., 1995) was diluted at 1:500 and the rabbit polyclonal anti-c-Fos antibody was diluted at 1:40 000. They were incubated further with biotinylated anti-rabbit IgG (7.5 Wg/ml; Vector Laboratories, Burlingame, CA, USA) in BSA^PBST for 1 h at room temperature, and then with the avidin^biotin complex (1:100; Amersham, Buckingham, UK) in BSA^PBST for 1 h at room temperature. Each step was followed by four washes with PBST for 15 min each. After the last wash, the sections were immersed in 0.175 M sodium acetate bu¡er, pH 7.4, for 30 min with two changes of the bu¡er, then incubated
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with the chromogen solution, which consisted of nickel chloride (0.25 mg/ml), 3,3P-diaminobenzidine tetrahydrochloride (0.2 mg/ ml) and 0.0025% hydrogen peroxide in the same bu¡er, for 10 min. The reaction was stopped by transferring the sections to sodium acetate bu¡er. Then, they were washed with 10 mM PBS for 20 min with three changes of the bu¡er. After washing with 10 mM PBS, the sections were mounted onto gelatin-coated glass slides.
induced in the AOB of the males of the mating group. In addition, we performed western blot analysis using a control protein from the experimental animals, and adopted the Gi2 antibody as the control protein. No induction was observed in Gi2 of the AOB of male rats after mating.
Quanti¢cation and data analyses
Localization of Arc-immunoreactivity in the AOB after mating
In this study, we observed the MTL and GRL in the AOB of SD male rats in light micrographs of 40-Wm-thick sagittal sections. Sections including large artifacts were excluded. We selected sections that had the largest glomerular regions of the AOB and quantitatively analyzed all MTL and GRL regions. The numbers of Arc-ir and c-Fos-ir cells in each of the two layers were determined and the total area of each layer was measured using an image analyzer (Zeiss KS300). Then, the numerical density of stained cells for each of the two layers was calculated and compared among the groups. Statistical analysis was performed using analysis of variance.
RESULTS
Immunoblot analysis To investigate whether the Arc protein is induced in the AOB after mating, the anti-Arc antibody was used for western blot analysis. Consequently, single protein bands with an apparent molecular weight of 55 kDa were identi¢ed by western blot analysis of AOB of male rats. The Arc protein was observed in the AOB of control group, but it was strongly induced in that of the mating group (Fig. 1). The c-Fos protein was also
We used immunocytochemistry to investigate the localization of Arc in the AOB after mating. The AOB includes ¢ve layers, namely, the vomeronasal nerve layer, the glomerular layer, the MTL, the olfactory tract layer and the GRL. There are numerous neurons present in the glomerular layer, the MTL and the GRL. In rats of the control group, only a few Arc-ir cells were observed throughout these three layers of the AOB. In contrast, in rats of the mating group, a marked increase in the number of Arc-ir cells was observed within the GRL. A high density of Arc-ir cells was observed along the anterior^ posterior axis (Fig. 2). No Arc-ir cells were observed in the glomerular layer. Arc-ir cells were observed at a low density in the basal region of the MTL, near the boundaries of the olfactory tract layer, and no Arc-ir cells were observed in the middle region of the MTL. In addition, all Arc-ir cells in the MTL appeared to be smaller than the mitral/tufted cells. The intracellular staining pattern of Arc, whose expression was observed in cell bodies and dendrite trees, di¡ered from those of other products of IEGs (Fig. 2E). In this study, we performed a quantitative analysis of Arc-ir cells in the MTL and the GRL of the AOB. Results of the quantitative analysis were con-
Fig. 1. Expression of Arc, c-Fos and Gi2 in the AOB of male SD rats after mating. Arc and c-Fos proteins were induced after mating with a female SD rat in the AOB of the male SD rats. In particular, induction of the Arc protein after mating was intense. No induction was observed in Gi2 of the AOB of male rats after mating. C, control ; M, mating.
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Fig. 2. Light photomicrographs showing Arc-ir cells in the AOB of adult male SD rats after mating. The 40-Wm-thick sagittal sections were stained with an antibody against Arc. Rostral of the AOB is right side and caudal is left. (A) AOB of adult male SD rats after exposure to clean bedding (control group). (B) Higher magni¢cation of the MTL and the GRL of (A). (C) AOB of adult male SD rats after mating (mating group). (D) Higher magni¢cation of the MTL and the GRL of (C). (E) High-power magni¢cation of granule cells in the AOB of an adult SD male rat after mating. Arc-ir is observed in a granule cell dendrite (arrows). Scale bars = 250 Wm (A, C); 100 Wm (B, D); 50 Wm (E).
sistent with immunocytochemical observations. Data are shown in Fig. 5. Thus, after mating, the expression of the Arc protein is concentrated in the granule cells in the AOB of SD male rats.
antibody (Fig. 3). After mating, many Arc-ir cells were observed in the GRL on the AOB of the left side, but on the nerve-transected side (right side), no Arc-ir cells were observed (Fig. 3).
E¡ect of vomeronasal nerve transection on Arc-immunoreactivity
Expression pattern of Fos-immunoreactivity in the AOB after mating
We investigated the e¡ect of vomeronasal stimulation on Arc-ir labeling in the AOB by vomeronasal nerve transection. Forty days after surgery, immunocytochemical observation using the N-CAM antibody con¢rmed that the vomeronasal nerve had degenerated and did not recover (Fig. 3). However, the animals (n = 4) still accomplished mating, because they had an intact vomeronasal and olfactory system on the left side and a recovered olfactory system on the right side. In addition, it was con¢rmed that the e¡erent dopaminergic projections to the AOB were not a¡ected by nerve transection as determined by immunocytochemical observation using the TH
To compare the expression patterns between Arc and Fos immunoreactivites, we performed immunocytochemical staining using the c-Fos antibody. There were numerous Fos-ir cells in the glomerular layer, the MTL and the GRL of the AOB after mating, and their expression is localized to the nuclei. However, even in the control group, some Fos-ir cells were observed in all three layers of the AOB (Fig. 4). In addition, we performed a quantitative analysis of Fos-ir cells in the MTL and the GRL of the AOB. Results of the quantitative analysis were consistent with immunocytochemical observations. Data are shown in Fig. 5. Thus, the expression pattern of
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Fig. 3. Light micrographs showing Arc- (A, D), N-CAM- (B, E) and TH- (C, F) immunoreactivity in the AOB of treated male rats (nerve transection) after mating. The 40-Wm-thick sagittal sections were stained with each antibody. Rostral of the AOB is right side and caudal is left. (A^C) AOB on the intact side of adult male SD rats after mating. (D^F) AOB on the vomeronasal nerve-transected side of same animal. Scale bars = 250 Wm (A, B, D, E); 100 Wm (C, F). VNL: vomeronasal nerve layer; GL: glomerular layer; OTL: olfactory tract layer.
the c-Fos-protein immunoreactivity after mating was similar to that of the Arc-protein immunoreactivity, but the localization in the AOB and the intracellular expression was extremely di¡erent between the two proteins.
DISCUSSION
Localization of Arc protein in the AOB after mating The vomeronasal system has an important role in the mammalian reproductive function (Wysocki, 1979; Halpern, 1987). The AOB is the ¢rst relay station in the vomeronasal system. The aim of the present study was to investigate the changes of the novel IEG product, Arc, in the AOB of male rats after mating. Firstly, we performed western blot analysis of AOB of male rats. A single protein band with an apparent molecular weight of 55 kDa was identi¢ed, and induction of the protein in the AOB of male SD rats was observed after mating
(Fig. 1). The size of this protein is consistent with the reported molecular size of the Arc protein in the rat cortex and hippocampus (Lyford et al., 1995). Therefore, it is likely that this single band represents the Arc protein. We examined the localization of the Arc protein in the AOB of male rats after mating. The number of Arc-ir cells in the AOB of adult male rats increased markedly after mating; a similar increase was observed in the other IEG, c-fos. However, the increase in the number of Arcir cells was localized to the GRL and di¡ered from that of Fos-ir cells that were expressed in all layers of the AOB. An increase in the number of Arc-ir cells was also detected in the MTL of the AOB. However, Arc-ir cells were concentrated in the outer layer of the MTL near the boundaries of the olfactory tract layer, and appeared to be smaller than the mitral/tufted cells. Hence, it is possible that Arc-ir cells in the MTL are granule cells. Thus, the results in the present study suggest that Arc is speci¢cally expressed in granule cells in the AOB after mating. It is very interesting that the Arc protein is expressed in one subpopulation of granule cells
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Fig. 4. Light photomicrographs showing Fos-ir cells in the AOB of adult male SD rats after mating. Rostral of the AOB is right side and caudal is left. (A) AOB of adult male SD rats after exposure to clean bedding (control group). (B) Higher magni¢cation of the MTL and the GRL of (A). (C) AOB of adult male SD rats after mating (mating group). (D) Higher magni¢cation of the MTL and the GRL of (C). Scale bars = 250 Wm (A, C); 100 Wm (B, D).
in the AOB after mating. In the AOB, the functional roles of the Arc protein have been uncertain. In the other parts of the CNS, the expression of Arc mRNA was induced by electroconvulsive shock (ECS) in the cerebral cortex and the hippocampus (Lyford et al., 1995; Steward et al., 1998). In these areas, the Arc protein is hypothesized to play roles in the activity-dependent neuronal plasticity and modi¢cation of the dendrite structure for interaction with the neuronal cytoskeleton (Lyford et al., 1995). Similarly, it is possible that the Arc protein plays an important role in neuronal plasticity in the AOB. Further studies using morphological, physiological, or molecular strategies may be helpful in con¢rming the functional roles of the Arc protein in the AOB. E¡ect of vomeronasal nerve transection on Arc expression The AOB receives both a¡erent and e¡erent inputs. Axons of vomeronasal sensory cells terminate in the glomeruli of the AOB (Raisman, 1985). In addition, it has been demonstrated that noradrenergic nerve ¢bers from the locus coeruleus terminate in the GRL and the MTL (Shipley et al., 1985). Naturally, during mating, animals are exposed to pheromones of their partner. Moreover, when a male and female mate, the brain noradrenergic system is activated, thereby increasing the noradrenaline level in the AOB (Keverne and de la Riva, 1982; Rosser and Keverne, 1985). To con¢rm which input strongly a¡ects the increase in the number of Arc-ir cells in the AOB, we performed vomeronasal nerve transection to selectively block the vomeronasal a¡erent input. Forty days after transection, axon projections of the olfactory
nerve to the MOB recovered, but projections of the vomeronasal nerve to the AOB did not recover, which is consistent with results of a previous report (Ichikawa, 1999). In addition, immunocytochemical observation using the TH antibody con¢rmed that e¡erent dopaminergic projections to the AOB were not a¡ected by nerve transection. Consequently, on the nerve-transected side, Arc-ir cells disappeared completely after mating. A previous study revealed that removal of the VNO eliminated the increase in c-Fos-protein expression levels in the granule cells induced by exposure to soiled bedding or mating (Fernandez-Fewell and Meredith, 1994; Rajendren and Moss, 1994). Thus, the results of the present study agree with those of previous studies, i.e., the increase in the Arc expression level in the AOB depended strongly on pheromonal stimulation, the absence of which eliminated the increase in the expression level of Arc or other IEGs. However, this evidence cannot exclude the e¡ect of activation of noradrenergic ¢bers on Arc expression in the AOB. Further experiments are necessary to con¢rm these observations. Comparative study between Arc and c-Fos proteins One of the main objectives of the present study was to analyze di¡erences in the intracellular localization and expression pattern in the AOB between the Arc protein and proteins encoded by other IEGs. We adopted the c-Fos protein encoded by a typical IEG, c-fos, for comparison, because there have been several published reports on c-Fos in both the main olfactory and the vomeronasal system. Several di¡erences in the intracellular localization and expression pattern between Arc and
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Fig. 5. Expression pattern of Arc and Fos in the AOB of male rats after mating (mean number of stained nuclei/10 000 Wm2 þ S.E.M.) #P 6 0.01 statistically signi¢cant di¡erence from the control group (analysis of variance).
c-Fos proteins have been demonstrated in this study. (1) As stated above, the Arc-protein expression was localized in the GRL of the AOB, while c-Fos-protein expression was not. (2) The Arc protein was not expressed in the control group, but the c-Fos protein was expressed in both the control and mating groups. (3) In contrast to c-Fos, Arc is not restricted to the nucleus; instead, it appears to be present in the cytoplasm of the granule cell body and in dendritic processes. This observation is very important and makes Arc an excellent marker of neuronal activity after mating. The c-Fos protein forms heterodimers with proteins encoded by the jun family, which are transferred actively to the nucleus. In contrast, the Arc protein has a modest degree of homology with the cytoskeletal protein K-spectrin (Lyford et al., 1995). Hence, it is hypothesized that in the dentate gyrus, newly produced Arc mRNA by ECS is transferred to rapidly activated postsynaptic processes, and continuously synthesized proteins play an important role in activity-dependent synaptic modi¢cation involving an interaction with neurocytoskeleton molecules (Steward et al., 1998). Thus, in the extranucleus, the Arc protein is hypothesized to be a modulator involved in synaptic plasticity. In contrast, it is known that there is a £exible synaptic plasticity in the granule cells of the AOB. It has been reported that pheromonal memory
formation during mating was accomplished by the postsynaptic modi¢cation of excitatory synapses between mitral/tufted and granule cells, that is, the modi¢cation of postsynaptic density in the granule cell dendritic spines (Matsuoka et al., 1997). However, not all synapses in the granule cells of the AOB need to be modi¢ed during memory formation; probably, only a part of the subpopulation of these synapses related to the partner’s pheromones should be modi¢ed. If the Arc protein plays an important role related to the synaptic plasticity in the dendritic spines of the granule cells, it should be possible to observe activated spines using Arc immunoreactivity. Thus, the application of Arc is a useful and accurate marker of activated synapses in response to pheromonal stimulation, which is related to memory formation during mating. AcknowledgementsöWe wish to thank Dr. R.M. Costanzo for helpful discussions and assistance. This work was supported in part by CREST of JST (M.M. and M.I.), a Grant-in-Aid for Scienti¢c Research (13760192) from the Japan Society for the Promotion of Science and the Ministry of Education, Science, Sports and Culture of Japan (M.M.), grants from the Japan Society for the Promotion of Science and the Ministry of Education, Science, Sports and Culture of Japan (K.Y.) and a Grant-in-Aid for Scienti¢c Research (C) from Japan Society for the Promotion Science (H.S.).
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