inobutyric acid activity in the olfactory bulb of the rat during the sexual cycle and response to olfactory stimuli N. NAVARRO BECERRA AND N. I. MUNARO~ CdtesZra de F'arncologf~~ Facultad de Ciencias Me'dicas, U~iversidadMacisnsl de Cbrdoba, Cbrdobs 50m, Argentina
Can. J. Physiol. Pharmacol. 1992.70:922-925. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 12/13/14. For personal use only.
Received October 3, 1991 BECERRA, N. N., and MUNARQ,N. I. 1992. 7-Aminobutyric acid activity in the olfactory bulb of the rat during the sexual cycle and response to olfactory stimuli. Can. J. Physiol. Pharmcol. 70: 922-925. Gluhrnic acid decarboxylase activity in the main and accessory olfactory bulbs throughout the sexual cycle of the rat was studied. The effect of male pheromonal secretion on emyme activity during proestms and estrus day was also tested. The enzyme activity showed circadian rhythm during the estrous cycle. This rhythm was disrupted during diestms-2 afternoon in the main bulb and came back during proestms afternoon. A different pattern of emyme activity was present in the accessory bulb, since the circadian rhythm was altered during proestms morning, returning during estms afternoon. Male odor exposition did not change enzyme profile activity during proestms day and during estms morning in the main bulb. In contrast, in the accessory bulb the olfactory stimuli induced opposite changes to that found in rats from the vivarium during proestrus. Comparison of emyme activity in olfactory stimuli-deprived rats with that of pheromone-stimulated rats during proestrus showed that male odor exposure specifically affects accessory bulb emyme activity. It is concluded that the changes of the olfactory bulb GABAergic system during proestms and estms day, or that evoked by odor stimuli, demonstrate the discriminative response of h i s system between the accessory olfactory bulb and the main olfactory bulb. Key words: glutamic acid decarboxylase, pheromone, olfactory bulb, GABA, sexual cycle. BECERRA, N. N., et MWNARQ, N. 1. 1992. y-Aminobutyric acid activity in the olfactory bulb sf the rat during the sexual cycle and response to olfactory stimuli. Can. J. Physiol. Pharmacol. 7@ : 922-925. On a examin6 19activitCde 19acideglubmique dCcarboxylase dans les bulbes olfactifs primaire et accessoire au cours du cycle sexuel du rat. On a aussi examine 19effetde la dcrCtion phCromonale mile sur 19activitCemymatique au moment de l'oestms et du proestms. L'activitC emymatique a suivi un rythme circadien durant le cycle oestral. Dans le bulbe primaire, ce rythme a kt6 interrompu 19aprks-midiau moment du dioestms et est reappam l'aprks-midi au moment du proestms. Le bulble olfactif accessoire a present6 un profil d9activitCenzymatique diffkrent, le rythme circadien ayant Ct6 dtt5rC en matinee lors du proestms, pour revenir 2 la n o d e en aprks-midi de l'oestms. Une exposition a l'odeur m&len9apas modifik l9ac%ivitC du profil enzymtique du burbe primaire au jour du proestms et au matin de l'oestrus. Bans 1e bulbe secondaire, les stimuli olfactifs on8 induit des variations diamCtralement opposCes h celles obsem%lCeschez les rats du vivarium au moment du proestms. Une cornparaison de 19activitCemymatique de rats privks de stimuli olfactifs avec celle de rats stimulCs aux phCromones au moment de 190estmsa month6 que l'exposition l'odeur male affecte sp5cifiquement 19activit6enzymatique dam bulbe accessoire* On conclut que les variations du systkme GABAergique du bulbe olfactif au moment du proestms et de l'oestrus, ou ceIIes provoquCes par les stimuli olfactifs, illustrent la rkponse discriminatoire de ce systkme pour ce qui est des bulbes olfactifs primaire et secondaire. Mots ckks : acide glutamique dCcarboxylase, phCromone, bulbe olfactif, GABA, cycle sexuel. [Traduit par la ~Cdaction]
Both y-arninobutyrm'c acid (GABA) and glutarnic acid decarbsxylase (GAD), the enzyme required for GABA synthesis, have been localized in the olfactory bulb (Roberts and Frankel 1958; Riback et d. 1977). GABA has been implicated in inhibitory synapses s f the olfactory bulb cell layers such as the dendrodendritkc synapses of granule cells onto rnitral and tufted cells (Rall et al. 1966; Nicoll 1941). Furthermore, GAD imunoreactiviey has been localized in the periglornemlar and granule cells somata and dewdritic spines (Ribak et d. 19'97). However, relatively little is h o w n about biochemical mechanisms involving neuroendocrine pheromond-induced responses. In brain areas distinct from the olfactory bulb, evidence indicates the existence s f daily fluctuations in both GAABA levels and GAD activity (Casanueva et al. 1984; Munaro et d. 1991). ]In the rat hypsthdamus, GABA levels change throughout the estrous cycle (Alsatti et d. 1980) and a GAB activity circadian rhythm is present in the locus coemleus nucleus during the rat estrous cycle (Munaro et A. 1986) or in normal mde median eminence (Casanueva et al. 1984). The removal of the olfactory bulbs or specific olfactory stimuli are capable 'Author for correspondence. Printed in Canada 1 Imprim6 au Canada
of influencing the estrous cycle in rats and mice (Arm 4979; Whitten 1958). Furthermore, a mouse bulbectorny produces sugression of the estrous cycle (Whitten 1956). The effect of pheromones on the GAD olfactory bulb has reveded that the enzyme activity is able to change by olfactory stimuli (Munaro 1990). It was considered s f interest to evaluate GAD activity in the olfactory bulbs throughout the rat sexud cycle and the changes that pheromond sensory inputs can induce on enzyme activity at proestms and estms, the period of reproductive readiness of the female.
Materials and metho& Attimls and procedures Adult female Wistar rats (200-250 g) were used. The animals were kept in a vivarium under artificial illumination (lights on from 05:06 to 19:W) and food and water were allowed ad libitum. The rats were housed in groups of five to six per cage. In the experiments designed to measure GAD activity throughout the sexud cycle, the vagina was examined and smears were taken daily. On different days sf the cycle, the rats were Elled by decapitation both at IB:W and 1790. The experimental groups were as follows.
BECERRA AND MUNARO
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PIG. 1. GAD activity measured at 11 and 17 h in the m i n olfactory bulb (MOB) (-) and accessory olfactory bulb (---) (AOB) throughout = 7, p < 0.8% ; Dl at 11 VS. the sexual cycle. Each point represents the mean of four to six rats f SEM. MOB: analysis of variance, F[1,431 1'7 h, g < 0.05; D, at 11 vs. 17 h, NS; P at 11 vs. 17 la, p < 0.05; E at 11 vs. 1%h, p < 0.05; Newman-Keuls t-test. AOB: analysis = 4 , p < 8.835;Dl at 11 vs. 17 h, NS; D2 at I1 VS. 17 h, p < 0.05; P at 11 vs. 17 h , p < 0.05; E at 11 vs. 17 h, NS; of variance, B;I Newman-Keuls f-test. MOB vs. AOB: P at 11 h, p < 0.05; P at 17 la, p < 0.05; E at 11 la, NS; E at 17 h, p < 0.01; paired f-test.
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(i) The first group of rats was maintained in a vivarium with normal conditions. (ii) Group 2 rats, during the day of proestrus or estrus, were subjected to the olfactory stimuli arising from soiled bedding from a male's cage. Female rats were placed in an isolated room and housed singly for 3 days. The males providing the olfactory stimuli were similarly housed in an isolated room, in individual cages containing wood shavings, During the morning or afternoon of the experimental day, the male was separated from his cage, which was covered with a wire mesh. The females were placed over this mesh and subjected to the olfactory signal arising from the soiled wood shavings. 'The females were covered with a clean plastic cage adapted at the top of the d e ' s cage and were left there for 2 h. Following the exposure perid, they were killed by decapitation. (iii) Control rats were kept under exactly the same conditions as group 2 but without being exposed to the olfactory stimulus of the vivarium or male pheromone. Tissue prepamfiora After decapitation, brains were immediately removed from the skull and the olfactory bulbs were dissected out and placed on ice. The main olfactory bulb (MOB) was separated from the accessory olfactory bulb (AOB) by an oblique cut (45') to eke longitudinal axis of the bulb. This cut was placed at 0.5 m to eke rostral pole sf the ABB. The anterior part contained the MOB and the caudal part eonh i n d the AdSB and the anterior olfactory nudeus, which was separated by a cut placed perpendicularly to eke oblique cut.
GAD esti~mation Enzyme activity (GAD activity) was estimated through the production of ""C0, according to the method of Albers and Brady (1959) with minor modifications. The samples were homogenized in 25 ranEba
phosphate buffer (pH 6.5) and incubated for 30 rnin at 37°C with equal volumes of substrate containing L-['"G]glutamic acid, pyridoxal phosphate, dithiothreitol, and potassium glutamate. The reaction was stopped by injecting 50 pL of 5 M H2S04. The tubes were returned to 37°C for 45 min and the evolved CO, was trapped by 50 p% of methy~kn~ethoHnium hydroxide in a test tube. The tubes were counted in a liquid scintillation apparatus. Blank values were boiled homogenates plus all reactants. GAD activity was expressed as nanomoles I4C8, formed per milligram protein per hour. Samples of the homogenate were taken for protein determination using the method of Lowry et al. (1951). Statistical a n d p i s The results were expressed as mean values f SEM. Analysis sf variance, Student's P-test,and paired &test were used when appropriate. A probability of error less than 0.05 was selected as the criterion for statistical significance.
Results GAD ac*ivi@in the The study of GAD activity during dl phases of the cycle? both in MOB and AOB at 11 and 17 h, revealed the existence of a circadian rhythm with a daily increase in GAD activity from 11 to 17 h, an herease that was more evident in MOB than in A8B. In MOB this rhythm was dtered from diestms-2 (D2) afternoon until Proestms (PB afterl~oon in which the enzyme activiby rose signifi~ant1-J' ( p < 0.05). On the contrary in the AOB, the circadian changes were disrupted during P morning,
CAN. B. PHYSIQL. PHARMACOL. VOL. 70, 1992
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Can. J. Physiol. Pharmacol. 1992.70:922-925. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 12/13/14. For personal use only.
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FIG.2. GAB activity measured at 1 1 and 17 h in MOB during P and E day in rats that were maintained in the vivarium (@), exposed to male pheromones (x), and odor deprived go). Each point represents the mean s f four to six rats SEM. *p < 64.01 versus rats exposed to olfaetsq stimuli (vivarium and male pheromones) at the corresponding day and hour (Student's P-test).
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with a significant decrease of GAD activity during P afternoon ( p < 0.05). (Fig. 1).
Efects of ok$kEctory stimuli on MOB- GAD activity during pr~estrusand estrus No changes in the values of enzyme activity were seen in rats exposed for 2 h to chemical cues from the male's soiled wood shavings when compared with the vdues observed in rats from the vivarium during P at 11 and 14 h and during estrus (E) morning. On the contrary, GAD activity showed a significant decrease in pheromone-stimulated rats during E afternoon ( p < 0.05). Rats deprived sf olfactory stimuli during P and E presented a similar pattern of enzyme activity but with lower values than that of olfactory-stimulated rats. Furthermore, these vdues paralleled those of the rats kept in the vivarium (Fig. 2). Efects of o@cko~"g,stimuki on AOB-GAD activity during proestrus and estrus In the rat AOB the presence of specific olfactory stimuli during P day at 11 and 17 h induced GAB activity changes opposite to those present in the AOB of rats kept in the vivarium. Male pheromone decreased enzyme activity during P morning ( p < 0.01), and in contrast, a marked increase ( p < 0.05) was present in the afternoon compared with rats from the vivarium. This was followed by a continuous decrease throughout the day of E. At the time studied during P and E, CAD activity values in rats that were odor stimulated paralleled and were always higher than that of odor-deprived rats (Fig. 3). Discussion The observations reported here demonstrate the participation of the rat olfactory bulb GABAergic system in the sexual cycle and GAD enzyme response to complex and specific olfactory stimuli. GAD activity and the subsequent synthesis of GABA could account for the inhibitory regulation involved
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FIG. 3. GAD activity measured at 11 and 1'7 h in AOB during P and E day in rats that were maintained in the vivarium ( O ) , expssed to male pheromones (x), and odor-deprived (a). Each point represents the mean s f four to six rats f SEM. Odor-deprived rats versus pheromone-stimulated rats: P at 11 1, NS; P at 17 h, p < 64.01; E at 11 h, p < (3.05; E at 1'7 h, NS; Student's P-test.Rats that were odor deprived versus odor vivarium rats: P at 1I h, p < 0.01; P at 17 h, NS: E at 11 h, p < 0.01; E at I7 h, p < 0.801;Student's e-test.
in the mentioned situations (Blindermann et al. 1980)' in addition to the process of transmission attenuation in olfactory bulb glomemlar neurons in response to olfactory inputs (Freeman 19'74). The existence of GAD activity circadian rhythm during the estrous cycle in the rat brain has been demonstrated (Munaro et al. 1986). En this work a different pattern of GAD activity diurnal variation between MOB and AOB was observed (Fig. 1). A daily increase of enzyme activity was found at 1'7 h in both parts of the bulb, which was disrupted during P at 17 h in AOB. The decrease of AOB enzyme activity during P afternoon may be due to high oestrsgen levels present at this moment (Freeman 1988) and agrees with the fact that in ovariectomized estrogen-treated rats AOB -GAB activity is lowered by the hormone action (B. N. Navarro and N. I. Munars, unpublished results). Estrogen-lowering effects of GAB activity in other brain regions have been observed (McGinnis et al. 1980). Ira contrast, at the same hour during P day a significant increase of GAD in MOB is present, which is greceded by low values from D2. This increase s f GAD in MOB is coincident with the peak values of the circadian rhythm found in the afternoon in both parts of the bulb. The mechanisms underlying the opposite kffects found during P in both parts s f olfactory bulb and the changes in MOB-GAD activity at D2 can not be established with the present data. Pheromone presence has been reported. to be impomnt in regulating md maintaining reproductive cycles (Clulow and Clarke 1968). In rodents, olfactory stimuli influence endocrine functions via the AOB, which projects to limbic streactures involved in the regulation of neuroendocrine and sexual functions (Powers and Winans 1975; Romers et al. 19963; Beltrarnino and Taleisnik 1983; Keverne 1983). The possibility of GAB activity changes under olfactory stimuli on stages
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BECEWM AND MUNARO
sf the cycle that are important for reproduction as P and E was tested. In this work, rat exposure to male pheromsnd secretion induced changes in cycBing GAB activity throughout P and E days. The profile of GABAergic activity was similar between MOB and AOB during E day when comparing rats maintained in the ambient complex odor of the rat vivarium and those exposed to mde pheromones (Figs. 2 - 3). This is not the case during P day when the pattern of GAD activity in AOB differs substantidly between both rat groups (Fig. 31, while no changes in MOB were observed (Fig. 2). This fact suggests a specific effect of mde pheromones in the AOB GABAergic activity. This contention is further supported by the effect of odor deprivation during P afternoon in AOB enzyme activity (Figs. 2 - 3). It is evident that the pheromone presence increases GAD-AOB activity at 1'7 Hn when compared with the values of the rats maintained in the vivarium. On the other hand, the complex &or of the vivarium specifically activates GAD during P morning in AOB. These two events, increase of GAD activity in AOB by complex odors or by mde pheromone, indicate the importance of the time of the day for the GABAergic response. In contrast, mde pheromone or the more complex odor of the rat vivarium both increase GABA-mediated inhibition similarly in MOB during B when compared with rats that were odor deprived (Fig. 2). These results indicate a discriminative induced response of the AOB GABA system to pheromone at P afternoon. The current results are in coincidence with previous reports (Munaro 1998) that indicate a specific CAD activity response to olfactory cues. Taken together, these findings led us to suggest that the specific cue activation of the GABAergic system in AOB represents a biochernicd mechanism important in modulating the olfactory input of mde pheromones. At E, MOB or AOB have a similar pattern of GAD activity either during the morning or afternoon for olfactory stimuli, but during E afternoon MOB -GAD activity in rats exposed to olfactory stimuli arising from a male reaches the levels sf rats odor deprived. This fact led us to presume a greater sensibility of the MOB to specific odor signals, particularly at this moment of the cycle. These results reveal that GAD activity in the olfactory bulb is influenced by internal and environmental factors (Kos&a et d. 1987), changing its response as a consequence of hormonal or olfactory stimulation.
This work was supported by a grant from the Consejo de Investigacisnes Cientificas y Tecnologicas de la Provincia de Cbrdoba, Argentina. The authors thank Mr. A. RsHdan for his technical assistance. Albers, N., and Brady, 8. D. 1959. The distribution of glutamate decarboxylase in the nervous system of the rhesus monkey. J. Biol. Chern. 234: 926-928. Alsatti, M., Ciesielskici,L., Kempe, K., et al. 1986). Involvement of serotonin and y-aminobutyric acid in the timing s f estrous receptivity in the cycling female rat. Psychonearroendocrinology, 5: 319-328. Arsn, C. 1979. Mechanisms of control of the reproductive function by olfactory stimuli in female m m a l s . Physiol. Rev. 59: 229284.
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Beltramino, C., and Taleisnik, S. 1983. Release of LH in the female rat by olfactory stimuli. Neurmndocrhology, 36: 53 -58. Blindermnn, J. M., De Fendis, F. V., Maitre, M., et al. 1980. G l u ~ m a t decarboxylase e activity in brain regions of differentiallyhoused mice: a difference in the olfactory bulb. Ewperientia, 36: 853-854. Casanueva, H., Apud, J. A*,Masotto, C., et al. 1984. Daily fluctuations in the activity of the tuberoinhndibular GABAergic system and plasma prolactin levels. Neuroendocrinology , 39: 367 -370. Clulsw, F. B . , and Clarke, 9. R. 1968. Pregnancy block in Mcrotus agrestts and induced ovulator. Nature (London), 219: 51 1 . Freeman, M. E. 1988. The ovarian cycle sf the rat. In The physiology of reproduction. Edited by E. Knobil et al. Raven Press, New York. pp. 1893- 1928. Freeman, W. J. 1974. Relation of glomemlar neursnal activity to glomemlar transmission attenuation, Brain Res. 65: 9 '1 - 107. Keveme, E. B. 1983. Pheromonal influences on the endocrine regulation of reproduction. Trends Neurosc. 6: 381 -384, Kosaka, T., Kosaka, K., Kiyoshi, H., et ak. 1987. Differentid effect of functional olfactory deprivation on the GABAergic and catecho8aminergic traits in the rat main olfactory bulb. Brain Wes. 413: 1 97 -203. Lowry, 8.H.,Rosenbrough, N. J., Farr, A. L., and Randall, W. J. 195 1. Protein measurement with the Folin phenol reagent. 9. Biol. Chern. 193: 265 -275. McGinnis, M. Y., Gordon, 9. H., and @orski, W. A. 1980. Time course and localization of the effects of estrogen on glutamic acid decarboxylase activity. 9, Neurochem. 34: 785 -792. Munaro, N . I. 1990. Maternal behavior: glubrnic acid decarboxylase activity in the olfactory bulb of the rat. Phamacol. Biochem. Behav. 36: $1-84. Munaro, N. I., Dotti, C . , and Taleisnik, S. 1986. Glubibmic acid decarboxylase activity in the hypothalamus of the rat: effect of estrogen. In GABA and endocrine hnction. Edited by C. Racagni and A. 8.Donoso. Raven Press, New York. pp. 201 -207. Muanaro, N.I., Morello, H., and Taleisnik, S. 1991. Glartamdc acid decarboxylase activity of the preoptic area and hypothalamus is influenced by the serotonergic system. 9. Neurochem. 57: 13021306. Nicoll, R. A. 1971. Pharmacological evidence for GABA as the transmitter in granule cell inhibition in the olfactory bulb. Brain Res, 35: 137 - 149. Powers, J. B., and Winans, S. S. 1975. Vomeronasal organ: critical role in mediating sexual behavior on the male hamster. Science (Washington, D.C.), 187: 961 -963. Rall, W., Sheferd, G. M . , Reese, T o S . , and Brightman, M. W . 1966. Dendrodendrjltic synaptic pathway for inhibition in the olfactory bulb. Exp. Neurol. 14: 44 -56. Riback, C. E., Vaughn, 9. E., S a b , K., et al. 1977. Glutamate decarboxylase localization in neurons of the olfactory bulb. Brain Res. 126: 1-18. Roberts, E., and Frankel, S . 1950. Aminobntyric acid in brain: its formation from glutamic acid. J. Biol. Chem. 187: 55 -63. R o m r s , P. R.,Beltramino, C. A., and Caner, H. F. 1990. Participation of the olfactory system in the control to approach behavior of the female rat to the male. Physiol. Behav. 47: 685 -698. Whitten, W. K. 1956. The effect of removal of olfactory bulb on the gonads of mice. J. Endocrinol. 14: 160- 163. Whitten, W. K. 1958. Modification of the sestrous cycle of the mouse by external stimuli associated with the male changes in the oestrous cycle determined by vaginal smears. J. ~ndocrinol.17: 307-313.
Can. J. Physiol. Pharmacol. 1992.70:922-925. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 12/13/14. For personal use only.
Received October 3, 1991 BECERRA, N. N., and MUNARQ,N. I. 1992. 7-Aminobutyric acid activity in the olfactory bulb of the rat during the sexual cycle and response to olfactory stimuli. Can. J. Physiol. Pharmcol. 70: 922-925. Gluhrnic acid decarboxylase activity in the main and accessory olfactory bulbs throughout the sexual cycle of the rat was studied. The effect of male pheromonal secretion on emyme activity during proestms and estrus day was also tested. The enzyme activity showed circadian rhythm during the estrous cycle. This rhythm was disrupted during diestms-2 afternoon in the main bulb and came back during proestms afternoon. A different pattern of emyme activity was present in the accessory bulb, since the circadian rhythm was altered during proestms morning, returning during estms afternoon. Male odor exposition did not change enzyme profile activity during proestms day and during estms morning in the main bulb. In contrast, in the accessory bulb the olfactory stimuli induced opposite changes to that found in rats from the vivarium during proestrus. Comparison of emyme activity in olfactory stimuli-deprived rats with that of pheromone-stimulated rats during proestrus showed that male odor exposure specifically affects accessory bulb emyme activity. It is concluded that the changes of the olfactory bulb GABAergic system during proestms and estms day, or that evoked by odor stimuli, demonstrate the discriminative response of h i s system between the accessory olfactory bulb and the main olfactory bulb. Key words: glutamic acid decarboxylase, pheromone, olfactory bulb, GABA, sexual cycle. BECERRA, N. N., et MWNARQ, N. 1. 1992. y-Aminobutyric acid activity in the olfactory bulb sf the rat during the sexual cycle and response to olfactory stimuli. Can. J. Physiol. Pharmacol. 7@ : 922-925. On a examin6 19activitCde 19acideglubmique dCcarboxylase dans les bulbes olfactifs primaire et accessoire au cours du cycle sexuel du rat. On a aussi examine 19effetde la dcrCtion phCromonale mile sur 19activitCemymatique au moment de l'oestms et du proestms. L'activitC emymatique a suivi un rythme circadien durant le cycle oestral. Dans le bulbe primaire, ce rythme a kt6 interrompu 19aprks-midiau moment du dioestms et est reappam l'aprks-midi au moment du proestms. Le bulble olfactif accessoire a present6 un profil d9activitCenzymatique diffkrent, le rythme circadien ayant Ct6 dtt5rC en matinee lors du proestms, pour revenir 2 la n o d e en aprks-midi de l'oestms. Une exposition a l'odeur m&len9apas modifik l9ac%ivitC du profil enzymtique du burbe primaire au jour du proestms et au matin de l'oestrus. Bans 1e bulbe secondaire, les stimuli olfactifs on8 induit des variations diamCtralement opposCes h celles obsem%lCeschez les rats du vivarium au moment du proestms. Une cornparaison de 19activitCemymatique de rats privks de stimuli olfactifs avec celle de rats stimulCs aux phCromones au moment de 190estmsa month6 que l'exposition l'odeur male affecte sp5cifiquement 19activit6enzymatique dam bulbe accessoire* On conclut que les variations du systkme GABAergique du bulbe olfactif au moment du proestms et de l'oestrus, ou ceIIes provoquCes par les stimuli olfactifs, illustrent la rkponse discriminatoire de ce systkme pour ce qui est des bulbes olfactifs primaire et secondaire. Mots ckks : acide glutamique dCcarboxylase, phCromone, bulbe olfactif, GABA, cycle sexuel. [Traduit par la ~Cdaction]
Both y-arninobutyrm'c acid (GABA) and glutarnic acid decarbsxylase (GAD), the enzyme required for GABA synthesis, have been localized in the olfactory bulb (Roberts and Frankel 1958; Riback et d. 1977). GABA has been implicated in inhibitory synapses s f the olfactory bulb cell layers such as the dendrodendritkc synapses of granule cells onto rnitral and tufted cells (Rall et al. 1966; Nicoll 1941). Furthermore, GAD imunoreactiviey has been localized in the periglornemlar and granule cells somata and dewdritic spines (Ribak et d. 19'97). However, relatively little is h o w n about biochemical mechanisms involving neuroendocrine pheromond-induced responses. In brain areas distinct from the olfactory bulb, evidence indicates the existence s f daily fluctuations in both GAABA levels and GAD activity (Casanueva et al. 1984; Munaro et d. 1991). ]In the rat hypsthdamus, GABA levels change throughout the estrous cycle (Alsatti et d. 1980) and a GAB activity circadian rhythm is present in the locus coemleus nucleus during the rat estrous cycle (Munaro et A. 1986) or in normal mde median eminence (Casanueva et al. 1984). The removal of the olfactory bulbs or specific olfactory stimuli are capable 'Author for correspondence. Printed in Canada 1 Imprim6 au Canada
of influencing the estrous cycle in rats and mice (Arm 4979; Whitten 1958). Furthermore, a mouse bulbectorny produces sugression of the estrous cycle (Whitten 1956). The effect of pheromones on the GAD olfactory bulb has reveded that the enzyme activity is able to change by olfactory stimuli (Munaro 1990). It was considered s f interest to evaluate GAD activity in the olfactory bulbs throughout the rat sexud cycle and the changes that pheromond sensory inputs can induce on enzyme activity at proestms and estms, the period of reproductive readiness of the female.
Materials and metho& Attimls and procedures Adult female Wistar rats (200-250 g) were used. The animals were kept in a vivarium under artificial illumination (lights on from 05:06 to 19:W) and food and water were allowed ad libitum. The rats were housed in groups of five to six per cage. In the experiments designed to measure GAD activity throughout the sexud cycle, the vagina was examined and smears were taken daily. On different days sf the cycle, the rats were Elled by decapitation both at IB:W and 1790. The experimental groups were as follows.
BECERRA AND MUNARO
Can. J. Physiol. Pharmacol. 1992.70:922-925. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 12/13/14. For personal use only.
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PIG. 1. GAD activity measured at 11 and 17 h in the m i n olfactory bulb (MOB) (-) and accessory olfactory bulb (---) (AOB) throughout = 7, p < 0.8% ; Dl at 11 VS. the sexual cycle. Each point represents the mean of four to six rats f SEM. MOB: analysis of variance, F[1,431 1'7 h, g < 0.05; D, at 11 vs. 17 h, NS; P at 11 vs. 17 la, p < 0.05; E at 11 vs. 1%h, p < 0.05; Newman-Keuls t-test. AOB: analysis = 4 , p < 8.835;Dl at 11 vs. 17 h, NS; D2 at I1 VS. 17 h, p < 0.05; P at 11 vs. 17 h , p < 0.05; E at 11 vs. 17 h, NS; of variance, B;I Newman-Keuls f-test. MOB vs. AOB: P at 11 h, p < 0.05; P at 17 la, p < 0.05; E at 11 la, NS; E at 17 h, p < 0.01; paired f-test.
,,
(i) The first group of rats was maintained in a vivarium with normal conditions. (ii) Group 2 rats, during the day of proestrus or estrus, were subjected to the olfactory stimuli arising from soiled bedding from a male's cage. Female rats were placed in an isolated room and housed singly for 3 days. The males providing the olfactory stimuli were similarly housed in an isolated room, in individual cages containing wood shavings, During the morning or afternoon of the experimental day, the male was separated from his cage, which was covered with a wire mesh. The females were placed over this mesh and subjected to the olfactory signal arising from the soiled wood shavings. 'The females were covered with a clean plastic cage adapted at the top of the d e ' s cage and were left there for 2 h. Following the exposure perid, they were killed by decapitation. (iii) Control rats were kept under exactly the same conditions as group 2 but without being exposed to the olfactory stimulus of the vivarium or male pheromone. Tissue prepamfiora After decapitation, brains were immediately removed from the skull and the olfactory bulbs were dissected out and placed on ice. The main olfactory bulb (MOB) was separated from the accessory olfactory bulb (AOB) by an oblique cut (45') to eke longitudinal axis of the bulb. This cut was placed at 0.5 m to eke rostral pole sf the ABB. The anterior part contained the MOB and the caudal part eonh i n d the AdSB and the anterior olfactory nudeus, which was separated by a cut placed perpendicularly to eke oblique cut.
GAD esti~mation Enzyme activity (GAD activity) was estimated through the production of ""C0, according to the method of Albers and Brady (1959) with minor modifications. The samples were homogenized in 25 ranEba
phosphate buffer (pH 6.5) and incubated for 30 rnin at 37°C with equal volumes of substrate containing L-['"G]glutamic acid, pyridoxal phosphate, dithiothreitol, and potassium glutamate. The reaction was stopped by injecting 50 pL of 5 M H2S04. The tubes were returned to 37°C for 45 min and the evolved CO, was trapped by 50 p% of methy~kn~ethoHnium hydroxide in a test tube. The tubes were counted in a liquid scintillation apparatus. Blank values were boiled homogenates plus all reactants. GAD activity was expressed as nanomoles I4C8, formed per milligram protein per hour. Samples of the homogenate were taken for protein determination using the method of Lowry et al. (1951). Statistical a n d p i s The results were expressed as mean values f SEM. Analysis sf variance, Student's P-test,and paired &test were used when appropriate. A probability of error less than 0.05 was selected as the criterion for statistical significance.
Results GAD ac*ivi@in the The study of GAD activity during dl phases of the cycle? both in MOB and AOB at 11 and 17 h, revealed the existence of a circadian rhythm with a daily increase in GAD activity from 11 to 17 h, an herease that was more evident in MOB than in A8B. In MOB this rhythm was dtered from diestms-2 (D2) afternoon until Proestms (PB afterl~oon in which the enzyme activiby rose signifi~ant1-J' ( p < 0.05). On the contrary in the AOB, the circadian changes were disrupted during P morning,
CAN. B. PHYSIQL. PHARMACOL. VOL. 70, 1992
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Can. J. Physiol. Pharmacol. 1992.70:922-925. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 12/13/14. For personal use only.
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FIG.2. GAB activity measured at 1 1 and 17 h in MOB during P and E day in rats that were maintained in the vivarium (@), exposed to male pheromones (x), and odor deprived go). Each point represents the mean s f four to six rats SEM. *p < 64.01 versus rats exposed to olfaetsq stimuli (vivarium and male pheromones) at the corresponding day and hour (Student's P-test).
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with a significant decrease of GAD activity during P afternoon ( p < 0.05). (Fig. 1).
Efects of ok$kEctory stimuli on MOB- GAD activity during pr~estrusand estrus No changes in the values of enzyme activity were seen in rats exposed for 2 h to chemical cues from the male's soiled wood shavings when compared with the vdues observed in rats from the vivarium during P at 11 and 14 h and during estrus (E) morning. On the contrary, GAD activity showed a significant decrease in pheromone-stimulated rats during E afternoon ( p < 0.05). Rats deprived sf olfactory stimuli during P and E presented a similar pattern of enzyme activity but with lower values than that of olfactory-stimulated rats. Furthermore, these vdues paralleled those of the rats kept in the vivarium (Fig. 2). Efects of o@cko~"g,stimuki on AOB-GAD activity during proestrus and estrus In the rat AOB the presence of specific olfactory stimuli during P day at 11 and 17 h induced GAB activity changes opposite to those present in the AOB of rats kept in the vivarium. Male pheromone decreased enzyme activity during P morning ( p < 0.01), and in contrast, a marked increase ( p < 0.05) was present in the afternoon compared with rats from the vivarium. This was followed by a continuous decrease throughout the day of E. At the time studied during P and E, CAD activity values in rats that were odor stimulated paralleled and were always higher than that of odor-deprived rats (Fig. 3). Discussion The observations reported here demonstrate the participation of the rat olfactory bulb GABAergic system in the sexual cycle and GAD enzyme response to complex and specific olfactory stimuli. GAD activity and the subsequent synthesis of GABA could account for the inhibitory regulation involved
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FIG. 3. GAD activity measured at 11 and 1'7 h in AOB during P and E day in rats that were maintained in the vivarium ( O ) , expssed to male pheromones (x), and odor-deprived (a). Each point represents the mean s f four to six rats f SEM. Odor-deprived rats versus pheromone-stimulated rats: P at 11 1, NS; P at 17 h, p < 64.01; E at 11 h, p < (3.05; E at 1'7 h, NS; Student's P-test.Rats that were odor deprived versus odor vivarium rats: P at 1I h, p < 0.01; P at 17 h, NS: E at 11 h, p < 0.01; E at I7 h, p < 0.801;Student's e-test.
in the mentioned situations (Blindermann et al. 1980)' in addition to the process of transmission attenuation in olfactory bulb glomemlar neurons in response to olfactory inputs (Freeman 19'74). The existence of GAD activity circadian rhythm during the estrous cycle in the rat brain has been demonstrated (Munaro et al. 1986). En this work a different pattern of GAD activity diurnal variation between MOB and AOB was observed (Fig. 1). A daily increase of enzyme activity was found at 1'7 h in both parts of the bulb, which was disrupted during P at 17 h in AOB. The decrease of AOB enzyme activity during P afternoon may be due to high oestrsgen levels present at this moment (Freeman 1988) and agrees with the fact that in ovariectomized estrogen-treated rats AOB -GAB activity is lowered by the hormone action (B. N. Navarro and N. I. Munars, unpublished results). Estrogen-lowering effects of GAB activity in other brain regions have been observed (McGinnis et al. 1980). Ira contrast, at the same hour during P day a significant increase of GAD in MOB is present, which is greceded by low values from D2. This increase s f GAD in MOB is coincident with the peak values of the circadian rhythm found in the afternoon in both parts of the bulb. The mechanisms underlying the opposite kffects found during P in both parts s f olfactory bulb and the changes in MOB-GAD activity at D2 can not be established with the present data. Pheromone presence has been reported. to be impomnt in regulating md maintaining reproductive cycles (Clulow and Clarke 1968). In rodents, olfactory stimuli influence endocrine functions via the AOB, which projects to limbic streactures involved in the regulation of neuroendocrine and sexual functions (Powers and Winans 1975; Romers et al. 19963; Beltrarnino and Taleisnik 1983; Keverne 1983). The possibility of GAB activity changes under olfactory stimuli on stages
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BECEWM AND MUNARO
sf the cycle that are important for reproduction as P and E was tested. In this work, rat exposure to male pheromsnd secretion induced changes in cycBing GAB activity throughout P and E days. The profile of GABAergic activity was similar between MOB and AOB during E day when comparing rats maintained in the ambient complex odor of the rat vivarium and those exposed to mde pheromones (Figs. 2 - 3). This is not the case during P day when the pattern of GAD activity in AOB differs substantidly between both rat groups (Fig. 31, while no changes in MOB were observed (Fig. 2). This fact suggests a specific effect of mde pheromones in the AOB GABAergic activity. This contention is further supported by the effect of odor deprivation during P afternoon in AOB enzyme activity (Figs. 2 - 3). It is evident that the pheromone presence increases GAD-AOB activity at 1'7 Hn when compared with the values of the rats maintained in the vivarium. On the other hand, the complex &or of the vivarium specifically activates GAD during P morning in AOB. These two events, increase of GAD activity in AOB by complex odors or by mde pheromone, indicate the importance of the time of the day for the GABAergic response. In contrast, mde pheromone or the more complex odor of the rat vivarium both increase GABA-mediated inhibition similarly in MOB during B when compared with rats that were odor deprived (Fig. 2). These results indicate a discriminative induced response of the AOB GABA system to pheromone at P afternoon. The current results are in coincidence with previous reports (Munaro 1998) that indicate a specific CAD activity response to olfactory cues. Taken together, these findings led us to suggest that the specific cue activation of the GABAergic system in AOB represents a biochernicd mechanism important in modulating the olfactory input of mde pheromones. At E, MOB or AOB have a similar pattern of GAD activity either during the morning or afternoon for olfactory stimuli, but during E afternoon MOB -GAD activity in rats exposed to olfactory stimuli arising from a male reaches the levels sf rats odor deprived. This fact led us to presume a greater sensibility of the MOB to specific odor signals, particularly at this moment of the cycle. These results reveal that GAD activity in the olfactory bulb is influenced by internal and environmental factors (Kos&a et d. 1987), changing its response as a consequence of hormonal or olfactory stimulation.
This work was supported by a grant from the Consejo de Investigacisnes Cientificas y Tecnologicas de la Provincia de Cbrdoba, Argentina. The authors thank Mr. A. RsHdan for his technical assistance. Albers, N., and Brady, 8. D. 1959. The distribution of glutamate decarboxylase in the nervous system of the rhesus monkey. J. Biol. Chern. 234: 926-928. Alsatti, M., Ciesielskici,L., Kempe, K., et al. 1986). Involvement of serotonin and y-aminobutyric acid in the timing s f estrous receptivity in the cycling female rat. Psychonearroendocrinology, 5: 319-328. Arsn, C. 1979. Mechanisms of control of the reproductive function by olfactory stimuli in female m m a l s . Physiol. Rev. 59: 229284.
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Beltramino, C., and Taleisnik, S. 1983. Release of LH in the female rat by olfactory stimuli. Neurmndocrhology, 36: 53 -58. Blindermnn, J. M., De Fendis, F. V., Maitre, M., et al. 1980. G l u ~ m a t decarboxylase e activity in brain regions of differentiallyhoused mice: a difference in the olfactory bulb. Ewperientia, 36: 853-854. Casanueva, H., Apud, J. A*,Masotto, C., et al. 1984. Daily fluctuations in the activity of the tuberoinhndibular GABAergic system and plasma prolactin levels. Neuroendocrinology , 39: 367 -370. Clulsw, F. B . , and Clarke, 9. R. 1968. Pregnancy block in Mcrotus agrestts and induced ovulator. Nature (London), 219: 51 1 . Freeman, M. E. 1988. The ovarian cycle sf the rat. In The physiology of reproduction. Edited by E. Knobil et al. Raven Press, New York. pp. 1893- 1928. Freeman, W. J. 1974. Relation of glomemlar neursnal activity to glomemlar transmission attenuation, Brain Res. 65: 9 '1 - 107. Keveme, E. B. 1983. Pheromonal influences on the endocrine regulation of reproduction. Trends Neurosc. 6: 381 -384, Kosaka, T., Kosaka, K., Kiyoshi, H., et ak. 1987. Differentid effect of functional olfactory deprivation on the GABAergic and catecho8aminergic traits in the rat main olfactory bulb. Brain Wes. 413: 1 97 -203. Lowry, 8.H.,Rosenbrough, N. J., Farr, A. L., and Randall, W. J. 195 1. Protein measurement with the Folin phenol reagent. 9. Biol. Chern. 193: 265 -275. McGinnis, M. Y., Gordon, 9. H., and @orski, W. A. 1980. Time course and localization of the effects of estrogen on glutamic acid decarboxylase activity. 9, Neurochem. 34: 785 -792. Munaro, N . I. 1990. Maternal behavior: glubrnic acid decarboxylase activity in the olfactory bulb of the rat. Phamacol. Biochem. Behav. 36: $1-84. Munaro, N. I., Dotti, C . , and Taleisnik, S. 1986. Glubibmic acid decarboxylase activity in the hypothalamus of the rat: effect of estrogen. In GABA and endocrine hnction. Edited by C. Racagni and A. 8.Donoso. Raven Press, New York. pp. 201 -207. Muanaro, N.I., Morello, H., and Taleisnik, S. 1991. Glartamdc acid decarboxylase activity of the preoptic area and hypothalamus is influenced by the serotonergic system. 9. Neurochem. 57: 13021306. Nicoll, R. A. 1971. Pharmacological evidence for GABA as the transmitter in granule cell inhibition in the olfactory bulb. Brain Res, 35: 137 - 149. Powers, J. B., and Winans, S. S. 1975. Vomeronasal organ: critical role in mediating sexual behavior on the male hamster. Science (Washington, D.C.), 187: 961 -963. Rall, W., Sheferd, G. M . , Reese, T o S . , and Brightman, M. W . 1966. Dendrodendrjltic synaptic pathway for inhibition in the olfactory bulb. Exp. Neurol. 14: 44 -56. Riback, C. E., Vaughn, 9. E., S a b , K., et al. 1977. Glutamate decarboxylase localization in neurons of the olfactory bulb. Brain Res. 126: 1-18. Roberts, E., and Frankel, S . 1950. Aminobntyric acid in brain: its formation from glutamic acid. J. Biol. Chem. 187: 55 -63. R o m r s , P. R.,Beltramino, C. A., and Caner, H. F. 1990. Participation of the olfactory system in the control to approach behavior of the female rat to the male. Physiol. Behav. 47: 685 -698. Whitten, W. K. 1956. The effect of removal of olfactory bulb on the gonads of mice. J. Endocrinol. 14: 160- 163. Whitten, W. K. 1958. Modification of the sestrous cycle of the mouse by external stimuli associated with the male changes in the oestrous cycle determined by vaginal smears. J. ~ndocrinol.17: 307-313.
