Biology Department, The Open University, Milton Keynes, MK7 6AA, UK. Received 2 August .... locomotor response ('horizontal activity') [7.5 min injection: t( 16) = 3.22, P < 0.005; 15 ..... 89, 3830-3834. Orchinik, M., Murray, T. F. and Moore.
European Journal of Neuroscience, Vol. 8, pp. 794-800, I996
@ European Neuroscience Association
Novelty-related Rapid Locomotor Effects of Corticosterone in Rats Carmen Sandi, Cesar Venero and Carmen Guaza Psychobiology Research Group, Cajal Institute, CSIC, Avda Dr Arce 37, 28002 Madrid, Spain Keywords: glucocorticoids, exploration, corticosteroid receptor antagonists, rat
Abstract Glucocorticoids modulate brain function and behaviour through different mechanisms. Although classical effects are mediated through intracellular receptors that modulate gene transcription, recent evidence supports the existence of rapid, nongenomic steroid effects through the neuronal membrane. In this study, we explored possible rapid behavioural effects of corticosterone in the rat, which could provide a model to characterize further the mechanisms involved in rapid corticosteroid nongenomic actions. We found that a corticosterone injection, at doses (2.5 or 5 mg/kg) that mimic plasma concentrations produced by substantial stress, rapidly increases (within 7.5 min of its systemic administration) the locomotor response displayed by rats in a novel environment (activity cage). A lower dose of 1 mg/kg failed to induce this effect. In addition, corticosterone failed to increase locomotion when administered to rats that had been previously exposed to the activity cage. Corticosterone-induced increased locomotion in a novelty situation was not counteracted by either the intracerebroventricular administration of the protein synthesis inhibitor cycloheximide, or by the intracerebroventricular administration of specific antagonists for each type of intracellular corticosteroid receptor, i.e. RU28318, a mineralocorticoidreceptor antagonist and RU38486, a glucocorticoid receptor antagonist. Further studies supported the viability of the receptor antagonists to display an anti-corticosteroidaction interfering, as previously reported, with the behavioural swimming test. Therefore, the rapid actions of corticosterone in locomotor activity described here, which appear to be nongenomic, might provide a model for future research on the elucidation of the mechanisms involved in steroid-membrane interactions.
introduction Glucocorticoids, steroid hormones secreted by the adrenal glands in response to stressful situations, modulate brain function and behaviour. The classic mechanism of action involves binding to intracellular receptors, which subsequently bind to a glucocorticoid-responsive DNA element modulating gene transcription (Tsai and O’Malley, 1994). In the brain, genomic actions of corticosteroids are mediated by two types of receptors, mineralocorticoid and glucocorticoid receptors, which differ in their affinity to bind corticosterone, the naturally occurring glucocorticoid in the rat (McEwen ef al., 1986; de Kloet, 1991; Joels and de Kloet, 1994). However, cumulative evidence on the rapidity of certain steroid effects on neuronal excitability and behavioural modulation supports the existence of rapid, nongenomic, steroid effects mediated through an action on the neuronal membrane (Schumacher, 1990). Steroids may display a membrane action through different mechanisms, including the alteration of the membrane lipidic environment (Carlson et al., 1983), an interaction with other neurotransmitter receptors (Majewska et al., 1986; h i a et al., 1990) or the binding to specific steroid membrane receptors (Towle and Sze, 1983; Orchinik et al., 1991). A direct steroid action on the cell membrane represents a potentially relevant operating mechanism for the modulation of neuronal function and behaviour. During the last decade, specific
binding sites were reported for a number of steroids in synaptic membranes (Towle and Sze, 1983) and rapid steroid effects on neuronal excitability (Kasai and Yamashita, 1988; Saphier and Feldman, 1988; Hua and Chen, 1989). However, the physiological relevance of these findings was attenuated until more recent evidence accumulated for membrane-mediated behavioural effects. For instance, a rapid progesterone effect on oxytocin receptor binding in the hypothalamus of the female rat was related to the facilitation of lordosis behaviour (Schumacher et al., 1990). As for corticosteroids, Orchinik et al. (1991) characterized a corticosteroid receptor on neuronal membranes from the amphibian brain which appears to mediate rapid, stress-induced changes in male reproductive behaviour. However, rapid corticosteroid effects on behaviour have not been reported in mammals. In the present study, we aimed to explore possible rapid behavioural effects of corticosteroids which might provide a model to characterize further the mechanisms involved in steroid nongenomic actions. The exploratory response in a novel environment was considered as a possible candidate for a number of reasons. First, exposure to novelty increases plasma corticosterone levels (Dantzer and Mormkde, 1983). Moreover, the locomotor response displayed by rats in a novel environment is very sensitive to variations in the circulating levels
Correspondence fo: Dr Carmen Sandi. Biology Department, The Open University, Milton Keynes, MK7 6AA, UK Received 2 August 1995, revised 7 November 1995, accepted 24 November 1995
Rapid behavioural effects of corticosterone of corticosterone (McIntyre, 1976; Veldhuis et al., 1982; Veldhuis and de Kloet, 1983) and is influenced by previous stress experiences (West and Michael, 1988). Since changes in novelty-induced locomotor activity are frequently observed within a short period of exploration (i.e. 5-10 min) (McIntyre, 1976; Veldhuis et al., 1982; Sandi et al., 1992), we reasoned that a rapid effect of corticosterone on this behaviour would be implied, if novelty-induced elevations in plasma corticosterone were to be physiologically significant for this response. We now show that corticosterone rapidly increases, within 7.5 min of its injection, the locomotor response displayed by rats in a novel environment. In addition to its short onset, other criteria for considering nongenomic steroid actions, such as resistance to protein synthesis inhibition (Schumacher et al., 1990; Karst and Joels, 1991) and its occurrence even when the access to intracellular receptors is blocked (Orchinik et al., 1991; Sandi and Rose, 1994b), were fulfilled. Once we characterized this behavioural model for a rapid effect of corticosterone, the possible effector systems involved on the control of corticosterone-induced locomotion could be addressed. A preliminary brief description of some of these results has been published previously (Sandi and Guaza, 1994).
795
5 min test. The absence of hindleg movements was recorded as a measurement of immobility (cumulative recording). Treatments were administered intracerebroventricularly (i.c.v.) 5 min before the initial test.
Startle response In one experiment, startle responses were measured to test for the effectiveness of the protein synthesis inhibitor, cycloheximide, to alter a physiological measurement different from locomotor activity. Acoustic startle responses were measured in a stabilimeter cage (Responder Economy, Colombus, OH). It was constructed of Plexiglas and wire mesh and suspended within a steel frame between compression springs and an accelerometer. Cage movement resulting from a startle response was transduced by the accelerometer into a voltage that was proportional to the displacement velocity. The stabilimeter was housed in a sound-attenuating chamber with a background noise. The cage was located 10 cm from a high-frequency speaker that delivered the acoustic stimuli. After a 5 min acclimation period, during which time there was no stimulation, each rat received 10 startle stimuli (4000 Hz, 105 dB; each with a duration of 20 ms, including 0.4 ms rise-fall times) separated by a variable interval averaging 15 s.
Materials and methods Animals Adult male Wistar rats (250-300 g) from our in-house colony were used. They were housed 4-5 per cage in temperature (22 ? 2°C) and light (12 h light-dark cycle) controlled conditions and had free access to food and water. Testing always occurred between 1O:OO and 14:00 h. Animal care procedures were conducted in accordance with the guidelines set by the European Community Council Directives (86/609/EEC). Spontaneous locomotor activity A Digiscan Animal Activity Monitor System (activity cage), model RXYZCM TAO (Omnitech Electronics, Inc., Colombus, OH), was used to assess the activity of the animals in a novel environment. Briefly, the apparatus consists of a square area in which a plastic animal cage (40 X 40 X 35 cm) is placed. It contains two perpendicular arrays of 15 horizontal infrared beams and two vertical light screens (infrared). Each interruption of the beam generates an electric impulse counted by an internal electronic counter. Rats were tested in the activity cage for a 5 min session and different types of movement were precisely recorded. Data for the following variables of locomotor activity detected by the activity monitor were collected by an IBMcompatible computer system: (i) horizontal activity: the total number of beam interruptions in the horizontal sensor; (ii) vertical activity: the total number of beam interruptions in the vertical sensor; (iii) number of stereotypic movements: this parameter increased when the same beams were broken repeatedly within 1 s; (iv) margin time: the time (s) that the animal spent in the margins of the cage; and (v) average speed: the average speed of the animal’s movement in c d s .
Swimming test The swimming test initially described by Porsolt et al. (1978) was used. Rats were individually placed in a narrow Plexiglas cylinder (diameter 18 cm, height 40 cm) containing 23 cm of water maintained at 25 ? 1°C. After 15 min in the water, they were removed and allowed to dry in a hot-air chamber for 30 min before being returned to their home cages. The animals were replaced in the cylinder 24 h later and the total duration of immobility was measured during a
Surge!Y In certain experiments, rats were cannulated i.c.v. Animals were anaesthetised with an i.p. injection of pentobarbital (Euta-Lender, Normon, Spain) dissolved in saline, at a dose of 40 mgkg. When narcosis became apparent, they were placed in a stereotaxic apparatus where its temperature was maintained by an electric underblanket. A unilateral craniotomy was performed following stereotaxic coordinates of Paxinos and Watson (1982) at 1.4 mm lateral to the midline and 0.8 mm anterior to the bregma. A sterile stainless guide cannula of 4 mm was inserted below the dura into the right lateral cerebral ventricle. Seven days were allowed for recovery after cannulation. After completion of the experiment, methylene blue dye was injected ( 10 pl) and the placement of the cannula was confirmed by observing the site and extent of staining. Drug administration Corticosterone (Sigma); RU283 18, the K salt of 3-(3-oxo-7-propyl17-hydroxy-androsta-4-one-17-yl)-propionicacid lactone, a specific mineralocorticoid receptor antagonist; RU38486 [ 17p-hydroxy-l lp(4-dimethylaminopheny1)-17-a-( 1-propyny1)-estra 4,9-dien-3-one], a specific glucocorticoid receptor antagonist (both provided by Roussel Uclaf, Paris, France); and cycloheximide, a protein synthesis inhibitor (Sigma), were used throughout the experiments. Corticosterone was dissolved in absolute ethanol and subsequently diluted in 0.9% saline to concentrations (1, 2.5, 5 or 10 mg/kg) that contained .-
**
5 3000
4 -
5 c
2000
0
4$ 0
1000
= Sal
corl
FIG. 3. Effects of an intraperitoneal injection of either vehicle (Sal) or corticosterone (Cort;5 mgkg) 15 min before testing in the horizontal activity displayed by rats in an environment to which they had been exposed the day before. Results are the mean t- SEM of 8-9 animals per group. * P < 0.05 versus corresponding saline group.
Effects of corticosteroid receptor antagonists on the cotticosterone-induced behavioural changes in a novel environment The short onset of the observed behavioural changes induced by corticosterone, as well as their rapid reversibility, are compatible with a nongenomic effect mediated through a membrane action. However, we addressed the possibility that the corticosterone effects were mediated by the classic intracellular corticosteroid receptors. The specific mineralocorticoid, RU283 18 (500 @at) and glucocorticoid, RU38486 (500 @rat), receptor antagonists were used. In order to ensure brain corticosteroid blockade, the antagonist dose selected was at least five times greater than doses previously proved to be effective to impair memory for stressful types of learning (de Kloet et al., 1988; Oitzl and de Kloet, 1992) and to alter effectively neuroendocrine and cardiovascular regulation (Ratka et al., 1989; van den Berg et al., 1990), in times ranging from 15 to 45 min post-injection. Four groups of animals received two simultaneous injections, one i.c.v. and one i.p., 15 min before being exposed to the activity cage. For the i.c.v. injection, two groups received saline and either of the 2 remaining groups received one of the receptor antagonists, RU28318 or RU38486. For the i.p. injection, one group on the saline i.c.v. condition was injected with saline (Sal-Sal) and the other three groups with corticosterone (Sal-Cort, RU283 18-Cort and RU38486-Cort). The results corresponding to horizontal activity are shown in Figure 4. An ANOVA revealed a significant treatment effect [F(3,12) = 9.91, P < 0.0021. Again, the Sal-Cort group increased locomotor response compared with controls (P < 0.01). Treatment with either corticosteroid antagonist failed to influence the corticosterone effect since neither the RU28318-Cort nor the RU38486-Cort group differed from Sal-Cort group (as.), but showed significantly higher values than controls (P < 0.01 and P < 0.05 respectively). Effects of corticosteroid receptor antagonists on behavioural retention in the swimming test Given that administration of the corticosteroid receptor antagonists failed to alter the behavioural effects of corticosterone in the novel environment, we aimed to test the efficacy of the antagonists under our experimental conditions through an independent measurement. The swimming test, initially described by Porsolt et al. (1978). was selected since behavioural retention has been shown to be affected
o
FIG.4. Horizontal activity displayed in a novel environment by rats submitted to two simultaneous injections (one i.c.v. and one i.p.) 15 min prior to testing. For the i.c.v. injection, rats received either saline, the mineralocorticoid receptor antagonist RU283 18 (500 &rat) or the glucocorticoid receptor antagonist RU38486 (500 &at). For the i.p. injection, they received either a vehicle (Sal) or a corticosterone injection (Cort;5 mgkg). Results are mean t- SEM of four animals per group. * P < 0.05 and **P < 0.01 versus corresponding Sal-Sal group.
TABLE 1. Effects of i.c.v. administration of the mineralocorticoid receptor antagonist RU28318 or the glucocorticoid receptor antagonist RU38486 on retention of the immobility acquired during the forced swimming test Treatment (ng)
n
Immobility (8)
Saline, vehicle RU28318, 100 RU28318, 500 RU38486, 100 RU38486.500
9 I 7 7 8
43.9 c 43.4 Z 37.6 ? 11.5 t18.7 ?
3.64 6.67 5.84 3.55* 2.33*
Treatments were given 5 min pre-training and their effects assessed during a 5 min test performed 24 h post-training. Results are the mean 5 SEM, *P < 0.01 versus saline group.
by corticosteroid receptor blockade (Veldhuis et al., 1985; de Kloet ef al., 1988). In particular, de Kloet ef al. (1988) showed that i.c.v. administration of RU38486, at doses ranging 10-100 ng/rat, dose dependently impaired the retention of acquired immobility, whereas RU28318 failed to influence retention at doses ranging from 1 to lo00 @rat. In order to replicate the de Kloet et al. (1988) study, all experimental groups in our study received an i.c.v. injection 5 min prior the initial 15 min test and retention of acquired immobility was evaluated on a 5 min test 24 h later. For each antagonist, the doses used were 500 @rat, as in the activity cage study (see above), and 100 @at, selected on the basis of de Kloet et al. (1988) data. None of the antagonist doses affected the level of immobility during the 15 min immediately after injection (n.s.). Table 1 shows the results on retention of acquired immobility 24 h after the initial forced swimming procedure. The ANOVA of the data yielded a significant treatment effect [F(1,33) = 10.97, P < 0.00011. Post-hoc Tukey analyses indicated that RU38486, at both doses tested (100 and 500 ng), significantly impaired the retention of acquired immobility (P < 0.01), whereas RU28318, at both doses used, did not interfere with retention (n.s.).
Effect of cycloheximide on the corticostemne-induced behavioural changes in a novel environment We then checked the possibility that the corticosterone effect might be mediated through an effect on protein synthesis mechanisms. Four
798 Rapid behavioural effects of corticosterone corticosterone (Sandi et al., 1996). Rats received an i.c.v. injection of either saline or a cycloheximide dose (300 pg/20 plhat) intermediate to the doses used in the locomotor activity experiments. After 50 min, they were tested for acoustic startle responses. The results showed that cycloheximide induced a significant reduction of the startle response: startle impulse Log, mean ? SEM, Sal = 5.64 ? 0.41 (n = 6); Cxm = 4.45 ? 0.21; t = 2.85, df = 10, P < 0.02, indicating that the protein synthesis inhibitor was effectively interfering with this physiological response.
Discussion FIG. 5. Horizontal activity displayed in a novel environment by rats submitted to two subsequent injections, one i.c.v. 60 min pre-testing, and one i.p. 15 min pre-testing. For the i.c.v. injection, rats received either saline (Sal), or the protein synthesis inhibitor cycloheximide (Cxm; 200 pg/rat). For the i.p. injection, they received either vehicle (Sal) or a corticosterone injection (5 mgkg). Results are mean 2 SEM of 7-9 animals per group. *P < 0.05 corresponding Sal-Sal group. Similar results were obtained with a cycloheximide dose of 400 @rat (see Results section).
groups of rats were injected at two separate times before testing in the activity cage, following a 2 X 2 design. After 60 min pre-testing, rats received an i.c.v. injection either with saline or the protein synthesis inhibitor cycloheximide (Cxm, 200 pg/rat). This dose of cycloheximide was selected based on previous studies showing that an equivalent concentration was effective to prevent corticosterone effects, mediated by intracellular corticosteroid receptors, on electrophysiological properties of neural membranes (Karst and Joels, 1991). At 15 min pre-testing, half of the animals in each condition at the first injection were i.p. injected with saline (Sal-Sal and Cxm-Sal groups), and the other half with corticosterone (Sal-Cort and CxmCort groups). As shown in Figure 5, the different groups displayed different levels of locomotor activity [F(3,26) = 3.02, P < 0.051. As previously, corticosterone-treated animals exhibited increased levels of horizontal activity (P < 0.05) as compared with controls. Pretreatment with cycloheximide failed to alter significantly the corticosterone effect, since the Cxm-Cort group did not differ from the Sal-Cort group (n.s.), but showed significantly higher values than controls (P < 0.05). No significant differences in locomotor activity were observed between Cxm-Sal and Sal-Sal groups (n.s.). In order to ensure the blockade of protein synthesis, we performed a further experiment using a higher dose of cycloheximide (400 pg/20 pl/rat) reported to block effectively in vivo protein synthesis-dependent behaviour in rats when administered i.c.v. (Fleming et al., 1990). The results showed a similar pattern, with the protein synthesis inhibitor failing to counteract the behavioural effect of the steroid (horizontal activity counts, mean 5 SEM: Sal-Sal: 1472 ? 165; Sal-Cort: 2268 2 139; Cxm-Sal: 1285 ? 155; Cxm-Cort: 2181 ? 152). However, higher doses of cycloheximide could not be used since pilot experiments indicated that doses 3500 pg/rat themselves reduced the locomotor activity displayed by rats in the activity cage. This cycloheximide-induced alteration in locomotor activity is in agreement with previous data obtained in mice exposed to an open field (Squire et aL, 1970). Given that cycloheximide administration was not effective to prevent corticosterone-induced exploratory changes in a novel environment, we tested the efficacy of this protein synthesisinhibitor to influence other physiological parameters to ensure its effectiveness under our experimental conditions. The acoustic startle response was selected since it has proved to be sensitive to circulating
This study shows that corticosterone, at doses that mimic plasma concentrations produced by substantial stress (Stein-Behrens et al., 1994), induces rapid changes (within 7.5-15 min) in the behavioural pattern displayed by rats in a novel environment, as evidenced by an increase in locomotor activity and stereotypy. This effect is novelty related since the steroid failed to modify the exploratory pattern in rats that had been previously exposed to the experimental situation. The rapid corticosterone effects are of relatively short duration (not manifested at 60 min after steroid administration), when compared with previously reported effects of corticosterone on behavioural reactivity (Oitzl et al., 1994) or learning and memory processes (Sandi and Rose, 1994a). which were visible between one and several hours after the corticosteroid injection. The rapid onset of these effects is consistent with an action through the neuronal membrane (Schumacher, 1990), rather than a genomic action mediated by intracellular steroid receptors (de Kloet, 1991; Ioels and de Kloet, 1994). This possibility is supported by two additional observations. First, central administration of selective antagonists for the two types of intracellular corticosteroid receptors-mineralocorticoid and glucocorticoid receptors-failed to counteract the increase in locomotor activity induced by corticosterone. The viability of the antagonists effectively to display an anti-corticosteroid action under our experimental conditions was supported by additional experiments using the swimming test. As previously reported (de Kloet et al., 1988), the glucocorticoid receptor antagonist RU38486 interfered with subsequent retention of acquired immobility, whereas the mineralocorticoid receptor antagonist failed to influence the subsequent behavioural pattern in the swimming test. Second, central administration of an inhibitor of protein synthesis, cycloheximide, was also not effective in preventing this behavioural action of corticosterone. Therefore, for the first time, these results present evidence for a behavioural effect of corticosterone in mammals that appears not to be mediated by the classic mechanism involving the actions of intracellular receptors on RNA-dependent protein synthesis, but rather by a rapid process through the cell-surface membrane. The only nongenomic behavioural action of corticosterone previously reported was in amphibians-the rapid suppression of male reproductive behaviour occumng within 8 min of i.p. injection, an effect that, in addition, was elegantly related to specific, high-affinity receptors in synaptic membranes (Orchinik et al., 1991). The involvement of glucocorticoid hormones in the locomotor reactivity to novelty was previously demonstrated in situations of corticosterone removal by adrenalectomy. Using experimental conditions similar to our study, chronic adrenalectomized rats showed reduced exploratory activity (McIntyre, 1976; Veldhuis er al., 1982) which was specifically restored by a corticosterone injection (Veldhuis et al., 1982; Veldhuis and de Kloet, 1983). Therefore, the absence of corticosterone results in the opposite effect (reduced locomotion) of that observed in this study after the acute increase, above basal values, of corticosterone levels (increased locomotion). In contrast
Rapid behavioural effects of corticosterone 799 with our results, previous studies reported that corticosterone administration to either adrenalectomized (Veldhuis et al., 1982; Veldhuis and de Kloet, 1983; Oitzl et al., 1994) or sham-operated animals (Veldhuis et al., 1982) failed to increase ambulation above control values. There might be two reasons for such a discrepancy. First, the doses employed were lower than the corticosterone dose we used. Secondly, in those studies steroid injections were given 60 min before testing, and our own time-dependent data indicate that increased locomotion observed at 7.5 or 15 min post-injection had reversibly disappeared if rats were tested 60 min post-injection. Therefore, the locomotor activity displayed by rats in a novel environment might depend upon tonic glucocorticoid control through intracellular receptors (although recent data present some discrepancy; Oitzl et al., 1994). However, in situations involving elevated circulating levels of corticosterone, such as stress, the end of the light period under a light-dark cycle or treatment with exogenous corticosterone, increased locomotion might be mediated by a rapid, nongenomic action of corticosterone exerted through a membrane action. A main aim of this study was to provide a model to investigate the mechanisms which underlaid this rapid behavioural action of corticosterone. Studies on the control of locomotion highlight the striatum and its complex network of connections with other structures, such as cortex, substantia nigra and thalamus, as a key brain region involved in this function (Graybiel, 1990; Carlsson, 1993; Angulo and McEwen, 1994). In particular, striatal dopaminergic transmission has largely been implicated on this behaviour. There is evidence supporting a striatal dopaminergic mediation of corticosterone effects in locomotion. Thus, stress and corticosterone treatments have been reported to increase striatal dopaminergic activity (Mittleman et al., 1992; Keefe el al., 1993) as well as to facilitate dopamine-mediated locomotor effects of psychostimulants and opioids (Deroche et al., 1992; Marinelli et al., 1994). Glutamatergic transmission seems to play a key role in this picture, since stress rapidly (between 10 and 20 min) increases the release of excitatory amino acids in the striatum (Moghaddam, 1993), an effect that in the hippocampus is attenuated by adrenalectomy (Lowy et al., 1993). Glutamate and related compounds also facilitate dopamine release from striatum very rapidly (Krebs et al., 1991; Morari et al., 1993; Jin and Fredholm, 1994). Nevertheless, an effect through other brain areas with a putative role in the control of locomotion, such as the mesencephalic locomotor region (Coles et al., 1989) and neurotransmitter systems, cannot be discarded. One question that remains to be addressed is the type of steroidmembrane interaction that might mediate the corticosterone-induced rapid effect on exploratory behaviour reported here. Even though, given their high degree of lipophilicity, steroids might disrupt the properties of the neuronal membrane, recent data favour a more specific action through membrane receptors (Schumacher, 1990). They may either be specific corticosteroid receptors (Towle and Sze, 1983; Orchinik et al., 1991) or receptors for other neurotransmitters, such as the GABAA receptor complex. Evidence for the latter possibility has been reported for other steroids in the mammalian brain (Majewska et al., 1986; Lan et al., 1990; Puia et al., 1990). In particular, corticosterone shows mixed agonistichntagonistiic effects on GABAA function ( h i a et al., 1990). At high doses, corticosterone has been shown to depress GABAergic postsynaptic inhibition in the hippocampus and neocortex (Zeise et al., 1992). If that were the case in the striaturn, it could also explain our results on corticosteroneinduced locomotion. Thus, a corticosterone antagonistic action on striatal GABAergic interneurons might remove the tonic inhibitory GABAergic modulation on dopamine efflux with the subsequent increase of dopamine release. As for the specific corticosteroid
membrane receptors, evidence in mammals comes from radioligand binding (Towle and Sze, 1983) and electrophysiological techniques (Kasai and Yamashita, 1988; Saphier and Feldman, 1988; Hua and Chen, 1989; Chen et al., 1991). However, the characterization of these receptors has only been demonstrated to be functionally relevant in amphibian neuronal membranes (Orchinik et al., 1991). These receptors mediate rapid corticosterone actions through guanine nucleotide-binding proteins (G proteins) (Orchinik et al., 1992). We have obtained preliminary evidence for the involvement of the gas molecule, nitric oxide, in the rapid corticosterone effects on locomotion reported here (Sandi and Guaza, 1994; Sandi et al., 1996). Interestingly, in peripheral physiological systems, nitric oxide production has been shown both to be activated by G proteins (Murthy et al., 1993) and to activate these guanine proteins (Lander et al., 1993). In summary, we present evidence here for a model of rapid, nongenomic behavioural action of corticosterone which might be exerted through the neuronal membrane. Provided with this model, further studies will be conducted to try to understand the kind of steroid-membrane interactions involved, as well as the possible neurochemical chain effecting corticosterone actions. Current work is being undertaken to test some of the hypotheses presented above in the possible role of glutamatergic and GABAergic systems.
Acknowledgements We thank Roussel Uclaf (Paris, France) for providing RU28318 and RU38486. We also thank Prof. E.R de Kloet, Dr S. del Cerro, and two anonymous referees for helpful advice, Dr J. Taylor for critical reading of the manuscript and C. Garcia for technical assistance. This work was supported by a grant from the DGICYT (92/014), Spain, and is part of a collaborative project under the European Neuroscience Programme on the Neural Mechanisms of Learning and Memory.
Abbreviations ANOVA Cort Cxm GABA i.c.v. RU283 18 RU38486 Sal
analysis of variance corticosterone cycloheximide gamma-aminobutyric acid intracerebroventricular K salt of 3-(3-oxo-7-propyl-17-hydroxy-androsta-4-one-17y1)-propionic acid lactone 17P-hydroxy-l l P-(4-dimethylaminophenyl)-17-a-(1 propynyl)-estra-4.9-dien-3-one saline
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