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benzodiazepine receptor complexes
Akuropharmacology, Vol. 36, No. 8, pp. 1071-1077, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 002%3908/97 $17.00 + 0.00
Pergamon PII: SOO2S-3908(97)00105-6
In Viva Administration of the 5-HTl* Receptor Agonist
8-OH-DPAT Interferes with Brain GABA*/ Benzodiazepine Receptor Complexes B. SGDERPALM, G. ANDERSSON, C. ENERBACK and J. A. ENGEL Institute of Physiology and Pharmacology, Department of Pharmacology, Medicinaregatan 7 S-413 90, Giiteborg, Sweden
Gateborg University,
(Accepted 2 May 1997)
Summary-In the present study the influence of in vivo administration, or in vitro addition, of the prototypic 5-HTi* receptor agonist 8-OH-DPAT on in vitro characteristics of GABAA/benzodiazepine receptor complexes was examined. In viva administration of 8OH-DPAT at a dose (32 &kg, S.C.- 10’) that has been reported to produce anxiolytic-like effects in the elevated plus-maze doubled the Kd for in vitro binding of 3Hflunitrazepam to rat cortical membranes (B,,, was unchanged) and enhanced GABA-stimulated (3, lo,30 and 100 PM) 36C1l influx in corticohippocampal synaptoneurosomes. In synaptoneurosomesfrom vehicle treated rats, diazepam (1, 3 and 10 /LM) potentiated GABA-stimulated (3 PM) 36C11 influx. No such effect was observed in tissue from 8-OH-DPAT treated rats, in which the GABA-stimulated (3 PM) 36C1l influx was similar to that caused by GABA + diazepam in tissue from vehicle treated rats. When added in vitro, S-OHDPAT failed to alter basal or GABA-stimulated 36C1-uptake. In vivo administration of a low “anxiolytic” dose of 8-OH-DPAT thus appears to interfere with GABAA/benzodiazepine receptor complexes, whereas in vitro application does not. The underlying mechanism remains to be elucidated but could involve in viilo release of positive modulators of GABAA/benzodiazepine receptor complexes, e.g. GABA, endozepines or neurosteroids. 0 1997 Elsevier Science Ltd.
Keywords-Anxiety, %OH-DPAT,5-HTi* receptor,GABAAlbenzodiazepinereceptorcomplex.
It is well established that decreased5-HT neurotransmission results in anxiolytic-like effects in various animal anxiety models. Thus extensive lesioning of brain 5-HT neurons by means of selective neurotoxins (5,6- and 5,7dihydroxytryptamine (DHT)) or inhibition of 5-HT synthesis with parachlorophenylalanine (PCPA) have been shown to produce anxiolytic-like effects both in classical punished conflict models and in models devoid of punishment, e.g. the elevated plus-maze and the social interaction test (Soubrie, 1986; Kahn et al., 1988; Siiderpalm, 1990). The non-benzodiazepine anxiolytics, e.g. buspirone (Buspar@), ipsapirone and gepirone, are believed to produce their anxiolytic effects by stimulating brain 5HTiA receptors (Traber and Glaser, 1987). These compounds, as well as the prototypic ~-HT~A receptor agonist 8-OH-DPAT (Hjorth et aZ., 1982), also produce anxiolytic-like effects in animal anxiety models, although
the effects are usually less pronounced than those obtained after the benzodiazepines (e.g. Engel et al., 1984; Eison et al., 1986). Some authors have suggested that the anxiolytic-like effects of these substancesare due to stimulation of 5-HT iA autoreceptors located on 5-HT cell-bodies in the raphe nuclei, leading to a decreaseof 5HT neuronal firing and release (e.g. Engel et al., 1984; Traber and Glaser, 1987; Carli and Samanin, 1988; Higgins et al., 1988; Sijderpalm et al., 1989; Schreiber and DeVry, 1993) whereas others have claimed that interaction with postsynaptic receptors accounts for this effect (cf. Schreiber and DeVry, 1993). According to the former view the net action of these compounds on 5-HT neurotransmission would be similar to that of extensive lesioning of 5-HT neurons, i.e. a decrease of 5-HT neurotransmission in neuronal circuits involved in the regulation of anxiety-related behaviors. Evidence was recently presented indicating that the anxiolytic-like effects observed after depletion of brain 5-HT by means of PCPA or 5,7-DHT pretreatment *To whom correspondence should be addressed. Tel: 46 31 involve an indirect activation of y-amino-butyric-acidA 773 34 20; Fax: 46 31 82 17 95; E-mail: bo.soderpalm@ (GABAA)/benzodiazepine receptor complexes located distally to 5-HT neurons, and that this activation may pharm.gu.se 1071
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B. Sijderpalmet al.
involve release of an endogenous agonist for this receptor complex (Stiderpalm and Engel, 1989, 1991; Siiderpalm et aZ., 1992). Interestingly, a recent report indicates that the anxiolytic-like effects of some 5-HTrA receptor agonists may be counteracted by the benzodiazepine receptor antagonist flumazenil (Lopez-Rubalcava et al., 1992), further supporting the hypothesis that the anxiolytic-like effects of .5-HTI* receptor agonists may in fact involve an indirect activation of GABA*benzodiazepine receptor complexes (Siiderpalm et aI.. 1993). In the present study we have examined the effect of in viva administration of 8-OH-DPAT in a low dose (32 pg/kg, s.c.) that produces anxiolytic-like effects in the elevated plus-maze (Soderpalm et al., 1989) on in vitro binding of 3H-flunitrazepam to rat cortical membranes and on the in vitro function (36C1- flux) of GABA*benzodiazepine receptor complexes in corticohippocampal synaptoneurosomes, according to the method of Schwartz and co-workers (Schwartz et cd., 1984; Luu et al., 1987; Sijderpalm et aZ., 1992). The effect of in vitro addition of 8-OH-DPAT in the 36C1-flux model was also investigated. MATERIALS
AND METHODS
Animals
Male Sprague-Dawley rats (Alab, Sollentuna, Sweden) weighing 250-350 g were used. The animals were kept under regular light-dark conditions (light on at 5:00 a.m. and off at 7:00 p.m.) and at constant temperature (25°C) and humidity (65%) with free accessto food and water. An adaptation period of at least seven days to the animal maintenance facilities of the department was allowed prior to the start of the experiments. This study was approved by the Ethics Committee for Animal Experiments, Gbteborg, Sweden. In vitro 3H-junitrazepam binding. Brain tissue from rat cortex cerebri was homogenized in 20 volumes of 0.32 M sucrose and 5 mM HEPES (N-2hydroxyethylpiperazinyl-N-2 ethene sulfonic acid; pH 7.4) using a glass teflon homogenizer (8-10 strokes). The homogenate was centrifuged at 1OOOgfor 10 min and the pellet was discarded. The recovered supernatant was centrifuged at 48000g for 20 min and the recovered pellet was washed five times with cold buffer (50 mM NaKP04, 100 mM NaCl; pH 7.4). The final pellet was resuspended in the phosphate buffer to give a protein concentration of about 0.25 mg protein/ml. 3H-Flunitrazepam (74.8 Ci/mmol; New England Nuclear, MA, U.S.A.) binding was measured by incubating 0.4 ml aliquots of the membrane suspension at 0°C with various concentrations of 3H-flunitrazepam in a total volume of 0.5 ml. Non-specific binding was determined in the presence of 10 PM diazepam (generously supplied by Hoffmann-La Roche, Switzerland). After 75 min, aliquots of the incubation mixture were filtered over
Whatman GF/B filters and the filters were washed three times with 3 ml ice-cold buffer. The filters were placed into scintillation vials and dried overnight. Radioactivity of the samples was determined by conventional scintillation counting procedures and Scatchard plots were established from which B,, and Kd were determined. Preparation
of synaptoneurosomes
Synaptoneurosomes were prepared essentially according to the method of Hollingsworth et aZ. (1985). Rats were decapitated and their brains rapidly taken out and placed in ice-cold preparation buffer (PB = NaCl 118.5 mM, KC1 4.7 mM, MgS04 1.18 mM, CaCll 2.5 mM, HEPES (2-(4-(2-Hydroxyethyl)-l-piperazinyl)ethansulfonic acid) 20 mM and Tris-Base 9mM). The brains were then placed on a chilled Petri dish on a filter paper soaked with PB. The cerebral cortices and hippocampi were dissected free and the white matter and the dura and pia mater were removed. Thereafter the cortices and hippocampi from each single animal were pooled and placed in ice-cold PB. The tissue was then decanted over a filter paper, weighed and homogenized in 7 ml of PB in a glass-to-glass homogenizer (five strokes). The homogenate was gravity filtered through three layers of nylon-filter (160 PM) in a Sweenex filter holder, and the glass-to-glass homogenizer was rinsed with 30 ml of PB. The resultant material was filtered once more over a 10 pm Millipore filter. The filtrate was centrifuged at 1OOOgfor 15 min and the supematant was decanted. The pellet was gently resuspended in 2 ml of PB in a glassteflon-homogenizer, and was thereafter diluted with 30 ml of PB and centrifuged once more at 1OOOgfor 15 min. The supematant was again decanted and the remaining pellet was diluted with PB, to yield a final protein concentration of approximately 10 mg/ml. The resulting material after a very similar cortical preparation has been described in detail and is considered to mainly be made up of sealed neuronal membranes, often arranged in a “snowman”-like fashion (“synaptoneurosomes”), with a very limited number of other “cells” (a “cell-free” preparation) (Schwartz et al., 1984; Luu et al., 1987). In the present study we added hippocampus to the preparation, since (1) besides being implicated in anxiety, this brain region shows a high density of GABAJbenzodiazepine receptors, and (2) this procedure increases the tissue yield from each animal. 36Cl- uptake in synaptoneurosomes
Assay tubes containing 300 ~1 assay-buffer (PB with pH 7.4 at 30°C were prewarmed in a water-bath (30”(Z), before addition of 100 ~1 of the synaptoneurosomal suspension previously described. The suspension was left to incubate for 20 min before the addition of 0.5 &i of 36C1- The mixture was rapidly vortexed and the flux of 36C1-‘terminated 15 set later by the addition of 5 ml icecold PB containing 100 PM picrotoxin. The mixture was then immediately vacuum-filtered (Schleicher and Scheull filters, GF31, 24 mm) and the tube and the filter
8-OH-DPAT and GABAAibenzodiazepinereceptors Table 1. Effect of in vivo administration of 8-OH-DPAT (32 /*g/kg s.c., 10 min prior to decapitation) on the in vitro binding characteristics for 3H-flunitrazepam to cortical membranes. Shown arc the means f SEM of five separate experiments. Statistics: Student’s t-test.
Vehicle 8-OH-DPAT
&, (pmokd
Kd
2.05 * 0.2 n.s. 2.51 * 0.3
138.4 f 22 p = 0.026 233.9 T 21
(nM)
was rinsed twice more with 5 ml of the picrotoxincontaining buffer. The filters were placed into scintillation vials and 4 ml of sqintillation fluid (Beckmann Ready-Safe) were added. Radioactivity was counted over night (DPM), using conventional liquid scintillation techniques. Except when indicated data are expressed as percent stimulation of baseline 36C1- accumulation (15 set in the absence of GABA) of the respective animal. In all experiments GABA was added simultaneously with 36C1-. Protein was determined by the method of Lowry et aE.(1951).
RESULTS
In uivo administration of S-OH-DPAT (32 pg/kg, s.c., 10 min prior to decapitation) significantly increased (Student’s t-test, p = 0.026) the dissociation constant (I&) for 3H-flunitrazepam. binding to cortical membranes
250
r
200
-
(Table 1). There was no significant change of B,, (Student’s t-test, ~~0.05). The same dose of 8-OH-DPAT injected in viuo increased GABA-stimulated 36C1-flux into corticohippocampal synaptoneurosomes at all concentrations of GABA tested, whereas in vitro addition of the drug (1 PM) did not influence the GABA-response (Fig. 1). A two factor repeated measures ANOVA on seven pooled in vivo experiments revealed significant treatment (8OH-DPAT versus vehicle; F( 1,52) = 4.99, p = 0.0298) and concentration (GABA 3, 10, 30, 100pM; F(3,52) = 183.74, p c 0.0001) effects, but no significant interaction (F(3,52) = 0.46, p = 0.713). For three pooled in vitro experiments the ANOVA showed a significant concentration effect (F(1,12) = 678.7, p < 0.0001) but a non-significant treatment effect (F(2,12) = 0.40, p = 0.5623) and no interaction (F(2,12) = 0.384, p = 0.6932). There was no statistically significant difference between the basal accumulation of 36C1- in synaptoneurosomes obtained from vehicle- or 8-OH-DPAT-treated animals (Student’s t-test, p >0.05), and in vitro addition of various concentrations of 8-OH-DPAT did not influence the basal accumulation of 36C1- in synaptoneurosomes (paired t-tests versus [0] nM, p > 0.05; Table 2). As illustrated in Fig. 2, addition of various concentrations of diazepam to the test-tubes enhanced (paired ttest, p c 0.01 or p < 0.05) GABA-induced (3 PM) 36C1flux into synaptoneurosomes obtained from vehicletreated animals, whereas no such enhancement was
250 -
-eveh - 0 - 8-OH-DPAT (in vitro)
200
-eveh - 0 - 8-OH-DPAT (in vivo)
A
-
E: .g $ 150 u e:
E zz 150 ‘F m 25 2 F: .F 100 i.i I8 50
1073
5 f
100
-
Y
-
50 -
o-
o-5.5
-4.5 -5 log[GABA]M
-4
I
-5.5
I
I
-4.5 -5 log[GABA]M
I
-4
after in vitro Fig. 1. Effect of 8-OH-DPAT on basal36C1-influx in rat corticohippocampalsynaptoneurosomes addition (left; 1 PM) or in viva administration(right; 32 pg/kg s.c., 10 min proir to decapitation).Shownare the results of representative experiments performed in triplicate and repeated at least three times with similar results.
B. SGderpalmet al.
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Table 2. Lack of effect of S-OH-DPAT on basal 36C1Finflux in rat corticohippocampal synaptoneurosomes after in viva administration (32 pg/kg s.c., 10 min prior to decapitation) or in virro addition (various concentrations). In viva treatment (n = 6)
36C1- influx (DPIWmg protein)
Vehicle S-OH-DPAT (32 &kg
350.6 k 13.1 341.4 f 14.0
s.c., - 10’)
In vitro addition &OH-DPAT]
nM (n = 3)
0 1 10 100 1000 10000
observed in synaptoneurosomes from 8-OH-DPATtreated rats. As in Fig. 1, 36C1- influx after GABA 3 PM was, however, greater in synaptoneurosomes from 8-OH-DPAT-treated rats (Student’s t-test, p c 0.05; Fig. 2). DISCUSSION
The present study has demonstrated alterations of the
36Cl- influx (percentage stimulation) 0f 7.3 * 10.0 ) 13.7 k 14.7 & 7.0 +
8.6 4.7 6.2 5.8 12.3 4.5
specific binding), reported enhancement of 3H-flunitrazepam and 3H-Ro-15-1788 binding, respectively. Whether this enhancement was due to alterations of & or Bmaxis, however, not clear. Using an autoradiographic approach Gobbi et al. (1991) reported a decrease of benzodiazepine receptor binding in the substantia nigra after acute drug treatment and an enhancement in the same brain region after chronic drug administration, whereas no alterations were observed in other brain
in vitro characteristics of GABAA/benzodiazepine receptor complexes after in vivo administration of the
prototypic .5-HTl* receptor agonist 8-OH-DPAT at a low dose (32 pglkg, S.C. -10’) that previously has been demonstrated to produce anxiolytic-like effects in the elevated plus-maze (Sederpalm et al., 1989). Recently, 8-OH-DPAT has been reported to interfere also with 5HT, receptors. Whether the present results are due to interference with 5-HTI* or 5-HT, receptors, or to some other effect of 8-OH-DPAT, cannot be firmly determined on the basis of the present experiments. However, 8-OHDPAT appears to possessa lo- to 50-fold higher affinity for 5-HTlA receptors than for 5-HT7 receptors (Hoyer, 1988; Ruat et al., 1993; Shen et al., 1993; Gobbi et al., 1996), perhaps making an interference with 5-HT1.4 receptors more likely after the very low dose applied here. Systemic administration of 5-HTlA agonists in this dose-range has also been demonstrated to affect 5-HT neuronal firing most likely via interference with 5-HTl* receptors. In addition, other drugs with 5-HTl* agonistic properties may alter benzodiazepine receptor binding. In the first series of experiments, in vitro radioligand binding (3H-flunitrazepam) to cortical membranes prepared from rats treated acutely with 8-OH-DPAT demonstrated a decreased benzodiazepine receptor affinity (increased &), whereas there was no change of the receptor number (B,,). Other workers have reported alterations of benzodiazepine receptor binding after in uivo administration of 5-HTl* receptor agonists. Thus, both Oakley and Jones (1983) (buspirone) and Koe et al. (1987) (8-OH-DPAT, ipsapirone, buspirone), after applying the radioligand in vivo and subjecting the tissue to homogenization and washing ex vivo (to reduce un-
40 r
Vehicle
*,f
35 30 25 20 == .s ‘G
15
R s z ‘Z
5
10
0
-I
z ‘Z
40 r
SOH-DPAT
20 15 10 5 0 3
3 1
3 3
3 10
GABA PM Diazepam pM
Fig. 2. Effect of in vitro addition of diazepamon GABAstimulated (3 PM) 36C1- influx in corticohippocampal synaptoneurosomes from rats treated in vivo with vehicle (top) or 8-
OH-DPAT(bottom;32pg/kgs.c.,10min prior to decapitation). Shown are the means + SEM of six experiments performed in triplicate. Statistics: paired t-test, * = p < 0.05, ** = p < 0.01 and Student’s t-test * =p x0.05, all compared to the effect of GABA 3 PM in synaptoneurosomes from vehicle treated
controls.
8-OH-DPAT and GABA*/benzodiazepinereceptors regions, However neither buspirone nor E-OH-DPAT alters benzodiazepine receptor binding when added to cortical membrane preparations in vitro (Koe et al., 1987; Van Wijngaarden et al., 1990). Thus, it appears as if in vivo administration of these substances triggers events that may alter the subsequentin vitro radioligand binding to benzodiazepine receptors. The differences between the present and previous studies with respect to the type of receptor alteration observed could be related to the different methods used or to the doses and predecapitation injection times applied. In the present study, the increase observed in & for 3H-flunitrazepam binding after in vivo administration of E-OH-DPAT could indicate, (1) that an affinity change of the receptor has occurred in viva due to a direct or indirect effect of E-OH-DPAT at one of the many binding sites of the GABA,&enzodiazepine receptor complex, or (2) that E-OH-DPAT in viva has liberated an endogenous competitive ligand for the benzodiazepine receptor that is also present in the membrane preparation. The latter explanation appearsless likely, however, considering that the tissue is extensively washed and diluted during preparation of the membranes. That the observed decreaseof benzodiazepine receptor affinity may be of functional significance is indicated by the 36C1- flux studies. While in vitro addition of E-OHDPAT did not alter basal or GABA-induced 36C1accumulation, the GABA-induced 36C1- influx was significantly larger in synaptoneurosomes from in vivo E-OH-DPAT-treated animals than from controls. This is a change in the same direction as that observed after acute administration of benzodiazepines, e.g. diazepam (Yu et aE., 1988; Siiderpalm et al., unpublished). The results suggest that in vivo administration of an “anxiolytic” dose of E-OH-DPAT (32 &kg, S.C. -lo’), like, for example, benzodiazepines and barbiturates, enhancesthe function of rat corticohippocampal GABAA/benzodiazepine receptor complexes. One explanation of the enhanced GABA-induced 36Cl- flux observed in vitro after in vivo administration of E-OH-DPAT could also in this case be a tentative presence of significant concentrations of positive modulators of the GABA,Jbenzodiazepine receptor complex in membrane vesicles from drug-treated animals. This explanation is, however, not likely for at least two reasons. Firstly, during the preparation of the synaptoneurosomes the tissue and tissue fluids are considerably diluted, making the presence of residual positive modulators in biologically significant amounts unlikely. Secondly, previous studies of GABA,&enzodiazepine receptor function after in viva administration of benzodiazepines have shown that the enhanced response observed in vitro can not be blocked by in vitro addition of the benzodiazepine receptor antagonist flumazenil (Yu et al., 1988). The enhanced GABA function observed in vitro is, therefore, most likely not due to an action exerted by residual benzodiazepines but is instead probably explained by an altered responsiveness to GABA
1075
deriving from a benzodiazepine-induced conformational change of the receptor complex in vivo. By analogy, E-OH-DPAT may in vivo by itself or via liberation of an endogenous positive modulator cause a conformational change of the receptor complex that is maintained in vitro and reflected in an enhanced functional response to GABA. As expected (e.g. Facklam et aE., 1992), in vitro addition of diazepam enhanced the GABA-induced 36C1flux into synaptoneurosomes from vehicle-treated animals. Interestingly, such an enhancement was not observed in synaptoneurosomes from E-OH-DPAT treated rats. Moreover, the increased GABA-induced 36C1- flux observed in synaptoneurosomes from E-OHDPAT-treated rats reached a similar level as that obtained after addition of diazepam to membrane vesicles from controls. It thus appears that the GABA-induced 36C1flux is already enhanced in tissue from E-OH-DPATtreated animals and can not be further increased after addition of diazepam. Taken together with the radioligand binding data a plausible explanation to these findings would be that E-OH-DPAT in viva releases an endogenous positive modulator of the GABAA/benzodiazepine receptor complex that interacts with the benzodiazepine site or some other site on the receptor complex, and that this interaction secondarily decreases the affinity of the benzodiazepine binding site. This decreasedaffinity may explain why diazepam is unable to further enhance 36Cl- flux in synaptoneurosomesfrom EOH-DPAT-treated rats. The present effects on the GABAA/benzodiazepine receptor complex were observed after systemic administration of a low dose of E-OH-DPAT (32 pglkg, S.C. -10’) that previously has been demonstrated to produce an anxiolytic-like effect in the elevated plus-maze (Sbderpalm et al., 1989). The behavioral effect of this dose of E-OH-DPAT is similar to that observed after lesioning of 5-HT neurons by means of 5,7-DHT or decreasing 5-HT synthesis with PCPA in this model (Sijderpalm and Engel, 1990) and was also interpreted to derive from an acute decreasing action on 5-HT neuronal function (Saderpalm et al., 1989). Indeed, low challenge doses of 5-HTl* agonists have in electrophysiological and in viva microdialysis studies been shown to decrease5-HT neuronal firing and release, most likely by interfering with somatodendritic ~-HTIA receptors (De Montigny et aZ., 1984; Sharp et al., 1989a, 1989b; Sprouse and Aghajanian, 1987; VanderMaelen et aZ., 1986). Based on behavioral and neurochemical data, the anxiolytic-like effects of 5,7-DHT or PCPA have been suggested to result from an indirect activation of GABA,Jbenzodiazepine receptor complexes, possibly through disinhibition of neurons containing an endogenous positive modulator of this receptor complex (Siiderpalm and Engel, 1989, 1991; SGderpalm et al., 1992). The present results may similarly suggest that the decreased 5-HT release resulting from acute administration of E-OH-DPAT secondarily releases an endogenous
1076
B. SGderpalm et al.
positive modulator of the GABA*/benzodiazepine receptor complex. In this context it is interesting that immunohistochemical and electrophysiological data suggest that 5-HT neurons form synaptic contacts with GABA neurons in a majority of cases and that the influence probably is inhibitory (Halasy et al., 1992). It should be noted that in a previous study GABAinduced 36C1- flux in synaptoneurosomesfrom 5,7-DHTlesioned animals was decreased rather than increased, compared to that in sham-operated controls (Nderpalm et al., 1992). These results thus appear to be in contrast to the present findings and at variance with the behavioral similarities observed in the elevated plus-maze after treatment with 5,7-DHT or a low dose of 8-OH-DPAT (S6derpalm and Engel, 1990). However, the present experiments were performed on tissue from animals acutely treated with 8-OH-DPAT, whereas in the former experiments tissue from rats sacrificed 2-4 weeks after the 5,7-DHT lesion was used. Thus, these animals, in contrast to the present 8-OH-DPAT treated rats, had been low in 5-HT neurotransmission for an extended period of time. In fact, it was hypothesized that the subsensitivity to GABA observed in vitro in tissue from 5,7-DHT treated animals reflected long-term adaptations of GABA*/ benzodiazepine receptors, that, however, were insufficient to overcome an enhanced liberation of a positive modulator in vivo (SGderpalm et al., 1992). Indeed, the GABAA/benzodiazepine receptor complex is prone to functional and molecular adaptation following prolonged agonistic influence (e.g. Primus and Gallager, 1992; Zhao et al., 1994), and a similar relationship as observed here with regard to in vitro GABA*/benzodiazepine receptor fuction after acute and chronic treatment is observed also after benzodiazepine administration. Thus, after acute in vivo adminstration of a benzodiazepine the in vitro sensitivity to GABA is enhanced, whereas after chronic in vivo treatment the in vitro sensitivity is instead reduced (e.g. Yu et aE., 1988). That the above suggested neuronal arrangement and possible indirect GABAergic effect of 8-OH-DPAT may be of relevance to the behavioral actions of the drug is illustrated by the findings of L6pez-Rubalcava et al. (1992), that anxiolytic-like effects of a higher dose of 8OH-DPAT (125 pg/kg) in mice may be counteracted by flumazenil. Using a rat Vogel’s conflict model we have observed similar results (Siiderpalm et al., 1994), although also in these studies a higher dose of 8-OHDPAT (125 pg/kg) was applied. Taken together, the present neurochemical in vitro data may suggest that in vivo administration of 8-OH-DPAT in the low dose applied here (32 pg/kg, S.C.- 10’) acutely enhances the function of corticohippocampal GABAAI benzodiazepine receptor complexes, possibly by causing release of an endogenous positive modulator of this receptor complex. This action may account for the anxiolytic-like action previously observed after this dose of 8-OH-DPAT. It is possible that a similar mechanism of action accounts for the anxiolytic-like effects also of
other ~-HT~A receptor agonists, such as buspirone (Buspa?). Acknowledgements-During
parts of these studies B.S. was a
recipientof the Berth von KantzowScholarship. This study was financially supported by the Swedish Medical Research Council (project no 11583 and no 4247), Ahltn-Stiftelsen, Stiftelsen Wilhelm och Martina Lundgrens VeJenskapsfond, Leons Fond, Magnus Bergvalls Stiftelse, Ake Wibergs Stiftelse, the Gothenburg Medical Society and Svenska Ltiareslllskapets Forskningsfonder. REFERENCES Carli M. and Samanin R. (1988) Potential anxiolytic properties of 8-hydroxy-2-(di-n-propylamino)tetralin, a selective serotonin,* receptor agonist. Psychopharmacology 94: 84-91. De Montigny C., Blier P. and Chaput Y. (1984) Electrophysiologically identified serotonin receptors in the rat CNS. Effect of antidepressant treatment. Neurophannacology 23: 151I-1520. Eison A. S., Eison M. S., Stanley M. and Riblet L. A. (1986) Serotonergic mechanisms in the behavioural effects of buspirone and gepirone. Pharmacology, Biochemistry and Behavior 24: 701-707. Engel L. A., Hjorth S., Svensson K., Carlsson A. and Liljequist S. (1984) Anticonflict effect of the putative serotonin receptor agonist 8-hydroxy-2-(Dl-n-propylamino)tetralin (8-OH-DPAT). European Journal of Pharmacology 105: 365-368. Facklam M., Schoch P. and Haefely W. E. (1992) Relationship between benzodiazepine receptor occupancy and potentiation of y-aminobutyric acid-stimulated chloride flux in vitro, of four ligands of differing intrinsic efficacies. Journal of Pharmacology and Experimental Therapeutics 261: 11061112. Gobbi M., Cavanus S., Miari A. and Mennini T. (1991) Effect of acute and chronic administration of buspirone on serotonin and benzodiazepine receptor subtypes in the rat brain: an autoradiographic study. Neurophamzacology 30: 313-321. Gobbi M., Parotti L. and Mennini T. (1996) Are 5hydroxytryptamine, receptors involved in [3H]5-hydroxytryptamine binding to 5-hydroxytryptamineInod-n,,na receptors in rat hypothalamus. Molecular Pharmacology 49: 556 559. Halasy K., Miettinen R., Szabat E. and Freund T. F. (1992) GABAergic intemeurons are the major postsynaptic targets of median raphe afferents in the rat dentate gyms. European Journal of Neuroscience 4: 153-155. Higgins G. A., Bradbury A. J., Jones B. J. and Oakley N. R. (1988) Behavioural and biochemical consequences following activation of 5-HTl-like and GABA receptors in the dorsal raphC nucleus of the rat. Neurophannacology 27: 993-1001. Hjorth S., Carlsson A., Lindberg P., Sanchez D., Wikstrijm H., Arvidsson L. -E., Hacksell U. and Nilsson J. L. G. (1982) 8-Hydroxy-2-(di-n-propylamino)tetralin, 8-OH-DPAT, a potent and selective simplified ergot congener with central 5-HT-receptor stimulating activity. Journal of Neural Transmission 55: 169-188. Hollingsworth E. B., McNeal E. T., Burton J. L., Williams R. J., Dalv J. W. and Crevelingc C. R. (1985) Biochemical
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Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. Journal of Biological
Chemistry 193: 265-275.
Luu M. D., Morrow A. L., Paul S. D. and Schwartz R. D. (1987) Characterization of GABA* receptor-mediated 36chloride uptake in rat brain synaptoneurosomes. Life Sciences 41: 1277-1287. Oakley N. R. and Jones B. J. (1983) Buspirone enhances (3H)flunitrazepam binding in vivo. European Journal of Pharmacology 87: 499-500.
Pharmacology
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28. Ruat M., Traiffort E., Leurs R., Tardivel-Lacombe J., Diaz J., Arrang J.-M. and Schwartz J. C. (1993) Molecular cloning, characterization, and localization of a high-affinity serotonin receptor (5-HT7) activating CAMP formation. Proceedings of the National Academy of Sciences U.S.A. 90: 8547-855 1. Schreiber R. and DeVry J. (1993) Studies on the neuronal circuits involved in the discriminative stimulus effects of 5hydroxytryptamineiA receptor agonists in the rat. Journal of Pharmacology
and Experimental
Therapeutics
265: 572-
579. Schwartz R. D., Skolnick P., Hollingsworth E. B. and Paul S. M. (1984) Barbiturate and picrotoxin-sensitive chloride efflux in rat cerebral cortical synaptoneurosomes. FEBS Letters 175: 193-196.
Sharp T., Bramwell S. K., Hjorth S. and Grahame-Smith D. G. 1989b Pharmacological characterization of 8-OH-DPATinduced inhibition of rat hippocampal5-I-IT release in vivo as measuredby microdialysis. British Journal of Pharmacology 98: 989-997. Shen Y., Monsma F. J. Jr, Metcalf M. A., Jose P. A., Hamblin M. W. and Sibley D. R. (1993) Molecular cloning and expression of a 5-hydroxytryptamine~ serotonin receptor subtype. Journal of Biological Chemistry 268: 1820018204.
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Soderpalm, B. (1990) On the neuropharmacology of conflict behaviour. Studies on noradrenergic, serotonergic and GABAergic mechanisms in experimental anxiety in the rat. Thesis, GSteborg University, ISBN 91-628-0175-9. Soderpalm B., Andersson G., JohannessenK. and Engel J. A. (1992) Intracerebroventricular 5,7-DHT alters the in vitro function of rat cortical GABAAfbenzodiazepine chloride ionophore receptor complexes. Ltfe Sciences 51: 327-335. Siiderpalm B. and Engel J. A. (1989) Does the PCPA induced anticonflict effect involve activation of the GABA*/benzodiazepine chloride ionophore receptor complex? Journal of Neural Transmission 76: 145-153. Soderpalm B. and Engel J. A. (1990) Serotonergic involvement in conflict behavior. European Neuropsychopharmacology 1: 7-13.
Saderpalm B. and Engel J. A. (1991) Involvement of the GABA&enzodiazepine chloride ionophore receptor complex in the 5,7-DHT induced anticonflict effect. Life Sciences 49: 139-153. Sijderpalm B., Hjorth S. and Engel J. A. (1989) Effects of 5HTm receptor agonists and L-5-HTP in Montgomery’s conflict test. Pharmacology, Biochemistry and Behavior 32: 259-265. Soderpalm B., Lundin B. and Hjorth S. (1993) Sustained 5hydroxytryptamine release-inhibitory and anxiolytic-like action of the partial 5-HTiA receptor agonist, buspirone, after prolonged chronic administration. European Journal of Pharmacology
Primus R. J. and Gallager D. W. (1992) GABAA receptor subunit mRNA levels are differentially influenced by chronic FG 7142 and diazepam exposure. European Journal of
receptors
239: 69-73.
Sijderpalm B., SWerpalm A., Lundin B. and Engel J. A. (1994) Endozepine involvement in non-benzodiazepine anxiolyticlike action? Neuropsychopharmacology 10 (suppl. 2): 2598. Soubrie P. (1986) Reconciling the role of central serotonin neurons in human and animal behaviour. The Behavioural and Brain Sciences 9: 319-364. SprouseJ. S. and Aghajanian G. K. (1987) Electrophysiological responses of serotonergic dorsal raphe neurons to ~-HT~,A, and 5-HTia agonists. Synapse 1: 3-9. Traber J. and Glaser T. (1987) 5-HTm receptor-related anxiolytics. TIPS 8: 432-437. VanderMaelen C. P., Matheson G. K., Wilderman R. C. and Patterson L. A. (1986) Inhibition of serotonergic dorsal raphe neurons by systemic and iontophoretic administration of buspirone, a non-benzodiazepine anxiolytic drug. European Journal of Pharmacology 129: 123-130. Van Wijngaarden I., Tulp M. Th. M. and Soudijn W. (1990) The concept of selectivity in 5-I-IT receptor research. European Journal of Pharmacology ogy Section) 188: 301-312.
(Molecular
Pharmacol-
Yu O., Chiu T. H. and Rosenberg H. C. (1988) Modulation of GABA-gated chloride ion flux in rat brain by acute and chronic benzodiazepine administration. Journal of Pharmacology and Experimental Therapeutics 246: 107-l 13. Zhao T.-J., Chiu T. H. and Rosenberg H. C. (1994) Reduced expression of y-aminobutyric acid type Afbenzodiazepine receptor y2 and x5 subunit mRNAs in brain regions of Rurazepam-treated rats. Molecular Pharmacology 45: 657663.
Pergamon PII: SOO2S-3908(97)00105-6
In Viva Administration of the 5-HTl* Receptor Agonist
8-OH-DPAT Interferes with Brain GABA*/ Benzodiazepine Receptor Complexes B. SGDERPALM, G. ANDERSSON, C. ENERBACK and J. A. ENGEL Institute of Physiology and Pharmacology, Department of Pharmacology, Medicinaregatan 7 S-413 90, Giiteborg, Sweden
Gateborg University,
(Accepted 2 May 1997)
Summary-In the present study the influence of in vivo administration, or in vitro addition, of the prototypic 5-HTi* receptor agonist 8-OH-DPAT on in vitro characteristics of GABAA/benzodiazepine receptor complexes was examined. In viva administration of 8OH-DPAT at a dose (32 &kg, S.C.- 10’) that has been reported to produce anxiolytic-like effects in the elevated plus-maze doubled the Kd for in vitro binding of 3Hflunitrazepam to rat cortical membranes (B,,, was unchanged) and enhanced GABA-stimulated (3, lo,30 and 100 PM) 36C1l influx in corticohippocampal synaptoneurosomes. In synaptoneurosomesfrom vehicle treated rats, diazepam (1, 3 and 10 /LM) potentiated GABA-stimulated (3 PM) 36C11 influx. No such effect was observed in tissue from 8-OH-DPAT treated rats, in which the GABA-stimulated (3 PM) 36C1l influx was similar to that caused by GABA + diazepam in tissue from vehicle treated rats. When added in vitro, S-OHDPAT failed to alter basal or GABA-stimulated 36C1-uptake. In vivo administration of a low “anxiolytic” dose of 8-OH-DPAT thus appears to interfere with GABAA/benzodiazepine receptor complexes, whereas in vitro application does not. The underlying mechanism remains to be elucidated but could involve in viilo release of positive modulators of GABAA/benzodiazepine receptor complexes, e.g. GABA, endozepines or neurosteroids. 0 1997 Elsevier Science Ltd.
Keywords-Anxiety, %OH-DPAT,5-HTi* receptor,GABAAlbenzodiazepinereceptorcomplex.
It is well established that decreased5-HT neurotransmission results in anxiolytic-like effects in various animal anxiety models. Thus extensive lesioning of brain 5-HT neurons by means of selective neurotoxins (5,6- and 5,7dihydroxytryptamine (DHT)) or inhibition of 5-HT synthesis with parachlorophenylalanine (PCPA) have been shown to produce anxiolytic-like effects both in classical punished conflict models and in models devoid of punishment, e.g. the elevated plus-maze and the social interaction test (Soubrie, 1986; Kahn et al., 1988; Siiderpalm, 1990). The non-benzodiazepine anxiolytics, e.g. buspirone (Buspar@), ipsapirone and gepirone, are believed to produce their anxiolytic effects by stimulating brain 5HTiA receptors (Traber and Glaser, 1987). These compounds, as well as the prototypic ~-HT~A receptor agonist 8-OH-DPAT (Hjorth et aZ., 1982), also produce anxiolytic-like effects in animal anxiety models, although
the effects are usually less pronounced than those obtained after the benzodiazepines (e.g. Engel et al., 1984; Eison et al., 1986). Some authors have suggested that the anxiolytic-like effects of these substancesare due to stimulation of 5-HT iA autoreceptors located on 5-HT cell-bodies in the raphe nuclei, leading to a decreaseof 5HT neuronal firing and release (e.g. Engel et al., 1984; Traber and Glaser, 1987; Carli and Samanin, 1988; Higgins et al., 1988; Sijderpalm et al., 1989; Schreiber and DeVry, 1993) whereas others have claimed that interaction with postsynaptic receptors accounts for this effect (cf. Schreiber and DeVry, 1993). According to the former view the net action of these compounds on 5-HT neurotransmission would be similar to that of extensive lesioning of 5-HT neurons, i.e. a decrease of 5-HT neurotransmission in neuronal circuits involved in the regulation of anxiety-related behaviors. Evidence was recently presented indicating that the anxiolytic-like effects observed after depletion of brain 5-HT by means of PCPA or 5,7-DHT pretreatment *To whom correspondence should be addressed. Tel: 46 31 involve an indirect activation of y-amino-butyric-acidA 773 34 20; Fax: 46 31 82 17 95; E-mail: bo.soderpalm@ (GABAA)/benzodiazepine receptor complexes located distally to 5-HT neurons, and that this activation may pharm.gu.se 1071
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B. Sijderpalmet al.
involve release of an endogenous agonist for this receptor complex (Stiderpalm and Engel, 1989, 1991; Siiderpalm et aZ., 1992). Interestingly, a recent report indicates that the anxiolytic-like effects of some 5-HTrA receptor agonists may be counteracted by the benzodiazepine receptor antagonist flumazenil (Lopez-Rubalcava et al., 1992), further supporting the hypothesis that the anxiolytic-like effects of .5-HTI* receptor agonists may in fact involve an indirect activation of GABA*benzodiazepine receptor complexes (Siiderpalm et aI.. 1993). In the present study we have examined the effect of in viva administration of 8-OH-DPAT in a low dose (32 pg/kg, s.c.) that produces anxiolytic-like effects in the elevated plus-maze (Soderpalm et al., 1989) on in vitro binding of 3H-flunitrazepam to rat cortical membranes and on the in vitro function (36C1- flux) of GABA*benzodiazepine receptor complexes in corticohippocampal synaptoneurosomes, according to the method of Schwartz and co-workers (Schwartz et cd., 1984; Luu et al., 1987; Sijderpalm et aZ., 1992). The effect of in vitro addition of 8-OH-DPAT in the 36C1-flux model was also investigated. MATERIALS
AND METHODS
Animals
Male Sprague-Dawley rats (Alab, Sollentuna, Sweden) weighing 250-350 g were used. The animals were kept under regular light-dark conditions (light on at 5:00 a.m. and off at 7:00 p.m.) and at constant temperature (25°C) and humidity (65%) with free accessto food and water. An adaptation period of at least seven days to the animal maintenance facilities of the department was allowed prior to the start of the experiments. This study was approved by the Ethics Committee for Animal Experiments, Gbteborg, Sweden. In vitro 3H-junitrazepam binding. Brain tissue from rat cortex cerebri was homogenized in 20 volumes of 0.32 M sucrose and 5 mM HEPES (N-2hydroxyethylpiperazinyl-N-2 ethene sulfonic acid; pH 7.4) using a glass teflon homogenizer (8-10 strokes). The homogenate was centrifuged at 1OOOgfor 10 min and the pellet was discarded. The recovered supernatant was centrifuged at 48000g for 20 min and the recovered pellet was washed five times with cold buffer (50 mM NaKP04, 100 mM NaCl; pH 7.4). The final pellet was resuspended in the phosphate buffer to give a protein concentration of about 0.25 mg protein/ml. 3H-Flunitrazepam (74.8 Ci/mmol; New England Nuclear, MA, U.S.A.) binding was measured by incubating 0.4 ml aliquots of the membrane suspension at 0°C with various concentrations of 3H-flunitrazepam in a total volume of 0.5 ml. Non-specific binding was determined in the presence of 10 PM diazepam (generously supplied by Hoffmann-La Roche, Switzerland). After 75 min, aliquots of the incubation mixture were filtered over
Whatman GF/B filters and the filters were washed three times with 3 ml ice-cold buffer. The filters were placed into scintillation vials and dried overnight. Radioactivity of the samples was determined by conventional scintillation counting procedures and Scatchard plots were established from which B,, and Kd were determined. Preparation
of synaptoneurosomes
Synaptoneurosomes were prepared essentially according to the method of Hollingsworth et aZ. (1985). Rats were decapitated and their brains rapidly taken out and placed in ice-cold preparation buffer (PB = NaCl 118.5 mM, KC1 4.7 mM, MgS04 1.18 mM, CaCll 2.5 mM, HEPES (2-(4-(2-Hydroxyethyl)-l-piperazinyl)ethansulfonic acid) 20 mM and Tris-Base 9mM). The brains were then placed on a chilled Petri dish on a filter paper soaked with PB. The cerebral cortices and hippocampi were dissected free and the white matter and the dura and pia mater were removed. Thereafter the cortices and hippocampi from each single animal were pooled and placed in ice-cold PB. The tissue was then decanted over a filter paper, weighed and homogenized in 7 ml of PB in a glass-to-glass homogenizer (five strokes). The homogenate was gravity filtered through three layers of nylon-filter (160 PM) in a Sweenex filter holder, and the glass-to-glass homogenizer was rinsed with 30 ml of PB. The resultant material was filtered once more over a 10 pm Millipore filter. The filtrate was centrifuged at 1OOOgfor 15 min and the supematant was decanted. The pellet was gently resuspended in 2 ml of PB in a glassteflon-homogenizer, and was thereafter diluted with 30 ml of PB and centrifuged once more at 1OOOgfor 15 min. The supematant was again decanted and the remaining pellet was diluted with PB, to yield a final protein concentration of approximately 10 mg/ml. The resulting material after a very similar cortical preparation has been described in detail and is considered to mainly be made up of sealed neuronal membranes, often arranged in a “snowman”-like fashion (“synaptoneurosomes”), with a very limited number of other “cells” (a “cell-free” preparation) (Schwartz et al., 1984; Luu et al., 1987). In the present study we added hippocampus to the preparation, since (1) besides being implicated in anxiety, this brain region shows a high density of GABAJbenzodiazepine receptors, and (2) this procedure increases the tissue yield from each animal. 36Cl- uptake in synaptoneurosomes
Assay tubes containing 300 ~1 assay-buffer (PB with pH 7.4 at 30°C were prewarmed in a water-bath (30”(Z), before addition of 100 ~1 of the synaptoneurosomal suspension previously described. The suspension was left to incubate for 20 min before the addition of 0.5 &i of 36C1- The mixture was rapidly vortexed and the flux of 36C1-‘terminated 15 set later by the addition of 5 ml icecold PB containing 100 PM picrotoxin. The mixture was then immediately vacuum-filtered (Schleicher and Scheull filters, GF31, 24 mm) and the tube and the filter
8-OH-DPAT and GABAAibenzodiazepinereceptors Table 1. Effect of in vivo administration of 8-OH-DPAT (32 /*g/kg s.c., 10 min prior to decapitation) on the in vitro binding characteristics for 3H-flunitrazepam to cortical membranes. Shown arc the means f SEM of five separate experiments. Statistics: Student’s t-test.
Vehicle 8-OH-DPAT
&, (pmokd
Kd
2.05 * 0.2 n.s. 2.51 * 0.3
138.4 f 22 p = 0.026 233.9 T 21
(nM)
was rinsed twice more with 5 ml of the picrotoxincontaining buffer. The filters were placed into scintillation vials and 4 ml of sqintillation fluid (Beckmann Ready-Safe) were added. Radioactivity was counted over night (DPM), using conventional liquid scintillation techniques. Except when indicated data are expressed as percent stimulation of baseline 36C1- accumulation (15 set in the absence of GABA) of the respective animal. In all experiments GABA was added simultaneously with 36C1-. Protein was determined by the method of Lowry et aE.(1951).
RESULTS
In uivo administration of S-OH-DPAT (32 pg/kg, s.c., 10 min prior to decapitation) significantly increased (Student’s t-test, p = 0.026) the dissociation constant (I&) for 3H-flunitrazepam. binding to cortical membranes
250
r
200
-
(Table 1). There was no significant change of B,, (Student’s t-test, ~~0.05). The same dose of 8-OH-DPAT injected in viuo increased GABA-stimulated 36C1-flux into corticohippocampal synaptoneurosomes at all concentrations of GABA tested, whereas in vitro addition of the drug (1 PM) did not influence the GABA-response (Fig. 1). A two factor repeated measures ANOVA on seven pooled in vivo experiments revealed significant treatment (8OH-DPAT versus vehicle; F( 1,52) = 4.99, p = 0.0298) and concentration (GABA 3, 10, 30, 100pM; F(3,52) = 183.74, p c 0.0001) effects, but no significant interaction (F(3,52) = 0.46, p = 0.713). For three pooled in vitro experiments the ANOVA showed a significant concentration effect (F(1,12) = 678.7, p < 0.0001) but a non-significant treatment effect (F(2,12) = 0.40, p = 0.5623) and no interaction (F(2,12) = 0.384, p = 0.6932). There was no statistically significant difference between the basal accumulation of 36C1- in synaptoneurosomes obtained from vehicle- or 8-OH-DPAT-treated animals (Student’s t-test, p >0.05), and in vitro addition of various concentrations of 8-OH-DPAT did not influence the basal accumulation of 36C1- in synaptoneurosomes (paired t-tests versus [0] nM, p > 0.05; Table 2). As illustrated in Fig. 2, addition of various concentrations of diazepam to the test-tubes enhanced (paired ttest, p c 0.01 or p < 0.05) GABA-induced (3 PM) 36C1flux into synaptoneurosomes obtained from vehicletreated animals, whereas no such enhancement was
250 -
-eveh - 0 - 8-OH-DPAT (in vitro)
200
-eveh - 0 - 8-OH-DPAT (in vivo)
A
-
E: .g $ 150 u e:
E zz 150 ‘F m 25 2 F: .F 100 i.i I8 50
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5 f
100
-
Y
-
50 -
o-
o-5.5
-4.5 -5 log[GABA]M
-4
I
-5.5
I
I
-4.5 -5 log[GABA]M
I
-4
after in vitro Fig. 1. Effect of 8-OH-DPAT on basal36C1-influx in rat corticohippocampalsynaptoneurosomes addition (left; 1 PM) or in viva administration(right; 32 pg/kg s.c., 10 min proir to decapitation).Shownare the results of representative experiments performed in triplicate and repeated at least three times with similar results.
B. SGderpalmet al.
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Table 2. Lack of effect of S-OH-DPAT on basal 36C1Finflux in rat corticohippocampal synaptoneurosomes after in viva administration (32 pg/kg s.c., 10 min prior to decapitation) or in virro addition (various concentrations). In viva treatment (n = 6)
36C1- influx (DPIWmg protein)
Vehicle S-OH-DPAT (32 &kg
350.6 k 13.1 341.4 f 14.0
s.c., - 10’)
In vitro addition &OH-DPAT]
nM (n = 3)
0 1 10 100 1000 10000
observed in synaptoneurosomes from 8-OH-DPATtreated rats. As in Fig. 1, 36C1- influx after GABA 3 PM was, however, greater in synaptoneurosomes from 8-OH-DPAT-treated rats (Student’s t-test, p c 0.05; Fig. 2). DISCUSSION
The present study has demonstrated alterations of the
36Cl- influx (percentage stimulation) 0f 7.3 * 10.0 ) 13.7 k 14.7 & 7.0 +
8.6 4.7 6.2 5.8 12.3 4.5
specific binding), reported enhancement of 3H-flunitrazepam and 3H-Ro-15-1788 binding, respectively. Whether this enhancement was due to alterations of & or Bmaxis, however, not clear. Using an autoradiographic approach Gobbi et al. (1991) reported a decrease of benzodiazepine receptor binding in the substantia nigra after acute drug treatment and an enhancement in the same brain region after chronic drug administration, whereas no alterations were observed in other brain
in vitro characteristics of GABAA/benzodiazepine receptor complexes after in vivo administration of the
prototypic .5-HTl* receptor agonist 8-OH-DPAT at a low dose (32 pglkg, S.C. -10’) that previously has been demonstrated to produce anxiolytic-like effects in the elevated plus-maze (Sederpalm et al., 1989). Recently, 8-OH-DPAT has been reported to interfere also with 5HT, receptors. Whether the present results are due to interference with 5-HTI* or 5-HT, receptors, or to some other effect of 8-OH-DPAT, cannot be firmly determined on the basis of the present experiments. However, 8-OHDPAT appears to possessa lo- to 50-fold higher affinity for 5-HTlA receptors than for 5-HT7 receptors (Hoyer, 1988; Ruat et al., 1993; Shen et al., 1993; Gobbi et al., 1996), perhaps making an interference with 5-HT1.4 receptors more likely after the very low dose applied here. Systemic administration of 5-HTlA agonists in this dose-range has also been demonstrated to affect 5-HT neuronal firing most likely via interference with 5-HTl* receptors. In addition, other drugs with 5-HTl* agonistic properties may alter benzodiazepine receptor binding. In the first series of experiments, in vitro radioligand binding (3H-flunitrazepam) to cortical membranes prepared from rats treated acutely with 8-OH-DPAT demonstrated a decreased benzodiazepine receptor affinity (increased &), whereas there was no change of the receptor number (B,,). Other workers have reported alterations of benzodiazepine receptor binding after in uivo administration of 5-HTl* receptor agonists. Thus, both Oakley and Jones (1983) (buspirone) and Koe et al. (1987) (8-OH-DPAT, ipsapirone, buspirone), after applying the radioligand in vivo and subjecting the tissue to homogenization and washing ex vivo (to reduce un-
40 r
Vehicle
*,f
35 30 25 20 == .s ‘G
15
R s z ‘Z
5
10
0
-I
z ‘Z
40 r
SOH-DPAT
20 15 10 5 0 3
3 1
3 3
3 10
GABA PM Diazepam pM
Fig. 2. Effect of in vitro addition of diazepamon GABAstimulated (3 PM) 36C1- influx in corticohippocampal synaptoneurosomes from rats treated in vivo with vehicle (top) or 8-
OH-DPAT(bottom;32pg/kgs.c.,10min prior to decapitation). Shown are the means + SEM of six experiments performed in triplicate. Statistics: paired t-test, * = p < 0.05, ** = p < 0.01 and Student’s t-test * =p x0.05, all compared to the effect of GABA 3 PM in synaptoneurosomes from vehicle treated
controls.
8-OH-DPAT and GABA*/benzodiazepinereceptors regions, However neither buspirone nor E-OH-DPAT alters benzodiazepine receptor binding when added to cortical membrane preparations in vitro (Koe et al., 1987; Van Wijngaarden et al., 1990). Thus, it appears as if in vivo administration of these substances triggers events that may alter the subsequentin vitro radioligand binding to benzodiazepine receptors. The differences between the present and previous studies with respect to the type of receptor alteration observed could be related to the different methods used or to the doses and predecapitation injection times applied. In the present study, the increase observed in & for 3H-flunitrazepam binding after in vivo administration of E-OH-DPAT could indicate, (1) that an affinity change of the receptor has occurred in viva due to a direct or indirect effect of E-OH-DPAT at one of the many binding sites of the GABA,&enzodiazepine receptor complex, or (2) that E-OH-DPAT in viva has liberated an endogenous competitive ligand for the benzodiazepine receptor that is also present in the membrane preparation. The latter explanation appearsless likely, however, considering that the tissue is extensively washed and diluted during preparation of the membranes. That the observed decreaseof benzodiazepine receptor affinity may be of functional significance is indicated by the 36C1- flux studies. While in vitro addition of E-OHDPAT did not alter basal or GABA-induced 36C1accumulation, the GABA-induced 36C1- influx was significantly larger in synaptoneurosomes from in vivo E-OH-DPAT-treated animals than from controls. This is a change in the same direction as that observed after acute administration of benzodiazepines, e.g. diazepam (Yu et aE., 1988; Siiderpalm et al., unpublished). The results suggest that in vivo administration of an “anxiolytic” dose of E-OH-DPAT (32 &kg, S.C. -lo’), like, for example, benzodiazepines and barbiturates, enhancesthe function of rat corticohippocampal GABAA/benzodiazepine receptor complexes. One explanation of the enhanced GABA-induced 36Cl- flux observed in vitro after in vivo administration of E-OH-DPAT could also in this case be a tentative presence of significant concentrations of positive modulators of the GABA,Jbenzodiazepine receptor complex in membrane vesicles from drug-treated animals. This explanation is, however, not likely for at least two reasons. Firstly, during the preparation of the synaptoneurosomes the tissue and tissue fluids are considerably diluted, making the presence of residual positive modulators in biologically significant amounts unlikely. Secondly, previous studies of GABA,&enzodiazepine receptor function after in viva administration of benzodiazepines have shown that the enhanced response observed in vitro can not be blocked by in vitro addition of the benzodiazepine receptor antagonist flumazenil (Yu et al., 1988). The enhanced GABA function observed in vitro is, therefore, most likely not due to an action exerted by residual benzodiazepines but is instead probably explained by an altered responsiveness to GABA
1075
deriving from a benzodiazepine-induced conformational change of the receptor complex in vivo. By analogy, E-OH-DPAT may in vivo by itself or via liberation of an endogenous positive modulator cause a conformational change of the receptor complex that is maintained in vitro and reflected in an enhanced functional response to GABA. As expected (e.g. Facklam et aE., 1992), in vitro addition of diazepam enhanced the GABA-induced 36C1flux into synaptoneurosomes from vehicle-treated animals. Interestingly, such an enhancement was not observed in synaptoneurosomes from E-OH-DPAT treated rats. Moreover, the increased GABA-induced 36C1- flux observed in synaptoneurosomes from E-OHDPAT-treated rats reached a similar level as that obtained after addition of diazepam to membrane vesicles from controls. It thus appears that the GABA-induced 36C1flux is already enhanced in tissue from E-OH-DPATtreated animals and can not be further increased after addition of diazepam. Taken together with the radioligand binding data a plausible explanation to these findings would be that E-OH-DPAT in viva releases an endogenous positive modulator of the GABAA/benzodiazepine receptor complex that interacts with the benzodiazepine site or some other site on the receptor complex, and that this interaction secondarily decreases the affinity of the benzodiazepine binding site. This decreasedaffinity may explain why diazepam is unable to further enhance 36Cl- flux in synaptoneurosomesfrom EOH-DPAT-treated rats. The present effects on the GABAA/benzodiazepine receptor complex were observed after systemic administration of a low dose of E-OH-DPAT (32 pglkg, S.C. -10’) that previously has been demonstrated to produce an anxiolytic-like effect in the elevated plus-maze (Sbderpalm et al., 1989). The behavioral effect of this dose of E-OH-DPAT is similar to that observed after lesioning of 5-HT neurons by means of 5,7-DHT or decreasing 5-HT synthesis with PCPA in this model (Sijderpalm and Engel, 1990) and was also interpreted to derive from an acute decreasing action on 5-HT neuronal function (Saderpalm et al., 1989). Indeed, low challenge doses of 5-HTl* agonists have in electrophysiological and in viva microdialysis studies been shown to decrease5-HT neuronal firing and release, most likely by interfering with somatodendritic ~-HTIA receptors (De Montigny et aZ., 1984; Sharp et al., 1989a, 1989b; Sprouse and Aghajanian, 1987; VanderMaelen et aZ., 1986). Based on behavioral and neurochemical data, the anxiolytic-like effects of 5,7-DHT or PCPA have been suggested to result from an indirect activation of GABA,Jbenzodiazepine receptor complexes, possibly through disinhibition of neurons containing an endogenous positive modulator of this receptor complex (Siiderpalm and Engel, 1989, 1991; SGderpalm et al., 1992). The present results may similarly suggest that the decreased 5-HT release resulting from acute administration of E-OH-DPAT secondarily releases an endogenous
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positive modulator of the GABA*/benzodiazepine receptor complex. In this context it is interesting that immunohistochemical and electrophysiological data suggest that 5-HT neurons form synaptic contacts with GABA neurons in a majority of cases and that the influence probably is inhibitory (Halasy et al., 1992). It should be noted that in a previous study GABAinduced 36C1- flux in synaptoneurosomesfrom 5,7-DHTlesioned animals was decreased rather than increased, compared to that in sham-operated controls (Nderpalm et al., 1992). These results thus appear to be in contrast to the present findings and at variance with the behavioral similarities observed in the elevated plus-maze after treatment with 5,7-DHT or a low dose of 8-OH-DPAT (S6derpalm and Engel, 1990). However, the present experiments were performed on tissue from animals acutely treated with 8-OH-DPAT, whereas in the former experiments tissue from rats sacrificed 2-4 weeks after the 5,7-DHT lesion was used. Thus, these animals, in contrast to the present 8-OH-DPAT treated rats, had been low in 5-HT neurotransmission for an extended period of time. In fact, it was hypothesized that the subsensitivity to GABA observed in vitro in tissue from 5,7-DHT treated animals reflected long-term adaptations of GABA*/ benzodiazepine receptors, that, however, were insufficient to overcome an enhanced liberation of a positive modulator in vivo (SGderpalm et al., 1992). Indeed, the GABAA/benzodiazepine receptor complex is prone to functional and molecular adaptation following prolonged agonistic influence (e.g. Primus and Gallager, 1992; Zhao et al., 1994), and a similar relationship as observed here with regard to in vitro GABA*/benzodiazepine receptor fuction after acute and chronic treatment is observed also after benzodiazepine administration. Thus, after acute in vivo adminstration of a benzodiazepine the in vitro sensitivity to GABA is enhanced, whereas after chronic in vivo treatment the in vitro sensitivity is instead reduced (e.g. Yu et aE., 1988). That the above suggested neuronal arrangement and possible indirect GABAergic effect of 8-OH-DPAT may be of relevance to the behavioral actions of the drug is illustrated by the findings of L6pez-Rubalcava et al. (1992), that anxiolytic-like effects of a higher dose of 8OH-DPAT (125 pg/kg) in mice may be counteracted by flumazenil. Using a rat Vogel’s conflict model we have observed similar results (Siiderpalm et al., 1994), although also in these studies a higher dose of 8-OHDPAT (125 pg/kg) was applied. Taken together, the present neurochemical in vitro data may suggest that in vivo administration of 8-OH-DPAT in the low dose applied here (32 pg/kg, S.C.- 10’) acutely enhances the function of corticohippocampal GABAAI benzodiazepine receptor complexes, possibly by causing release of an endogenous positive modulator of this receptor complex. This action may account for the anxiolytic-like action previously observed after this dose of 8-OH-DPAT. It is possible that a similar mechanism of action accounts for the anxiolytic-like effects also of
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