European Journal of Pharmacology, 210 (1992) 247-251
247
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52235
Acute handling stress downregulates benzodiazepine receptors: reversal by diazepam Nick A n d r e w s , A l e x a n d e r Z h a r k o v s k y a n d S a n d r a E. File Psychopharmacology Research Unit, UMDS Division of Pharmacology, Guy's Hospital, London SE1 9RT, U.K.
Received 1 July 1991,revised MS received 8 October 1991, accepted 29 October 1991
In rats naive to handling, the effects of an acute handling stress led to lower [3H]flunitrazepam binding in the frontal cortex, compared with animals previously habituated to handling for 2 or 21 days. This decreased binding was due to reductions in the number of receptors, not to a change in affinity. Pre-incubation with diazepam (0.3, 3.0 or 30/~M) of frontal cortex homogenates taken from naive rats exposed to acute handling stress (followed by extensive washing to remove residual diazepam and endogenous modulators) led to a concentration-dependent increase in the number of benzodiazepine receptors, without any change in KD. Acute in vivo administration of diazepam (4 mg/kg) prior to the handling stress was without significant effect in handling-habituated animals, but increased [3H]flunitrazepam binding in handling naive rats. Thus, handling habituation and diazepam treatment have similar actions on benzodiazepine binding and represent two ways of adapting to the stress of handling by increasing the number of benzodiazepine receptors. Benzodiazepine binding; Handling; Stress
I. Introduction
T h e GABAA-benzodiazepine r e c e p t o r - c h l o r i d e ionophore complex has been shown to change in response to a variety of stressors, such as forced swimming (Medina et al., 1983), immobilisation (Braestrup et al., 1979), white noise (Lai and Carino, 1990) and handling (Biggio et al., 1990). Acute exposure of naive animals to handling stress induced a rapid decrease in G A B A n receptor binding in rat cortical membranes, compared with animals habituated to handling for 15 days. A foot-shock applied to these handling-habituated animals induced a fall in G A B A A receptor numbers, but was without effect in handling-naive animals (Biggio et al., 1981). There was a greater 36C1- flUX in synaptosomes from handling-habituated animals compared with handling-naive animals (Concas et al., 1987, 1988b). An additional footshock stress failed to further decrease the basal 36C1- flUX in handling-naive animals but significantly reduced it in handling-habituated animals (Concas et al., 1988a). The changes in 36C1- flux and G A B A A receptor binding after acute stress appear to be consistent,
Correspondence to: S.E. File, PsychopharmacologyResearch Unit, UMDS Divisionof Pharmacology,Guy's Hospital, London SE1 9RT, U.K.
regardless of the type of stressor, but this is not the case with benzodiazepine receptor binding. It appears that the type of stressor can determine the direction of change of the binding. Thus, benzodiazepine binding was found to decrease after electrical footshock and post-natal isolation of new-born rat pups, increase after immobilisation, and remain unchanged after isolation-induced aggression in mice (Braestrup et al., 1979). There are even different findings for different conditions of swim-stress. Thus, Soubrie et al. (1980), found that cold water swim stress caused an increase in benzodiazepine binding, Braestrup et al. (1979) reported that there was no change after ambient swim stress and Medina et al. (1983) found forced swimming in ambient temperatures (18°C) caused a decrease in binding. There has been a recent report that acute handling stress applied to naive animals, results in lower benzodiazepine binding in cortical membranes when compared with the binding observed in membranes of handling-habituated animals (Mennini et al., 1988). The change was found to be due to a decrease in Bmax with no effect on K D. In experiment 1 we investigated whether these changes could be observed using our handling procedure and strain of rats. The functioning of the GABA-benzodiazepine receptor complex is also changed by anxiolytic and anxiogenic drugs. Diazepam decreases [35S]t-butylbicyclo-
248 phosphorothionate ([35S]TBPS) binding (Concas et al., 1986) and increases GABA A receptor binding (Skerritt and Johnston, 1983). Acute handling stress and administration of an anxiogenic /3-carboline both decrease GABA A receptor binding to cortical membranes; an effect that can be antagonised by in vitro addition of diazepam to the membranes (Biggio et al., 1984). In experiment 2 we investigated whether the changes in benzodiazepine binding induced by handling stress could be reversed by in vitro pre-incubation of the membranes with diazepam, or prevented by in-vivo administration of the drug 30 min prior to killing.
2. Materials and methods
2.1. Animals Male hooded Lister rats (Olac Ltd., Bicester), weighing 200-250 g at the start of the experiment, were housed in groups of 5 with food and water freely available, in a room maintained at 20°C, with lights on from 06:00 to 18:00 h.
2.2. Handling procedure The daily handling consisted of picking up the rat by its body, weighing it and then giving an intraperitoneal (i.p.) injection of water. The animal was then returned to its home cage. Animals were divided into three groups according to the amount of handling they would receive. One group received no handling until the day of killing (handling-naive), one received handling one day prior to killing and also on the day of killing (2 days of handling) and the third group received handling on 20 successive days and also on the day of killing (handling-habituated). The handling procedure on the day of killing was identical to that on previous days and occurred 30 min prior to killing by cervical dislocation. In experiment 2, handling-naive and handling-habituated animals received i.p. injections of water or diazepam (4 mg/kg) 30 min prior to killing.
2.3. Drugs and solutions Diazepam (gift of Roche products Ltd.), was suspended in a Tween 20 and distilled water vehicle and left in an ultrasonic water bath for 30 min before injection. The drug was administered i.p. in a volume of 2 ml/kg. For in vitro studies, diazepam was dissolved in 98% ethanol in a concentration of 10 mM and dilutions made from this stock. Care was taken that the final concentration of ethanol, added to the assay as part of the diazepam solution, was not greater than 0. 1%.
2.4. Biochemical studies
2.4.1. Preparation of the membranes Animals were stunned and killed by cervical dislocation, the brains rapidly removed and frontal cortex dissected out on ice. The tissue was homogenized in ice cold distilled water using a Polytron homogeniser (setting 6.5 for 15 s). The homogenate was then centrifuged at 38 000 x g for 20 min. The resulting pellet was resuspended in ice cold 50 mM Tris HC1 buffer (pH = 7.4 at 20°C) and incubated for 30 min on ice. To observe the effects of in vitro addition of diazepam, the homogenate was resuspended in Tris HCI buffer containing varying concentrations of diazepam. In order to remove all residual diazepam and endogenous modulators of benzodiazepine binding present in the membrane preparation, the following extensive washing procedure was employed. Briefly, the membrane preparation was centrifuged at 20000 X g for 20 min and then subsequently resuspended in Tris HCI buffer and washed an additional 3 times. The resulting pellet was frozen overnight. Eighteen hours later the pellet was thawed, resuspended in Tris HCI buffer and centrifuged at 20000 x g for 20 min. This pellet was then stored on ice for 30 min. The pellet was subsequently resuspended in Tris HC1 buffer and washed" an additional 5 times. The final pellet was resuspended in 300 volumes of Tris HCI buffer and taken to the binding assay.
2.4.2. [3H]Flunitrazepam binding assay Aliquots (0.1-0.2 mg protein/ml final concentration) of membrane suspension were incubated with 0.5 nM (final concentration) [3H]flunitrazepam (Amersham plc., specific activity 85 Ci/mmol) in a final volume of 1 ml for 40 min at room temperature. Non-specific binding was determined in the presence of 3 /zM diazepam and represented 10-15% of total binding. Specific and non-specific binding were both performed in triplicate. Following incubation, the samples were filtered through Whatman G F / B filters using a Millipore manifold and washed rapidly 3 times, with 4 ml of cold Tris HCI buffer. The filters were put into scintillation vials containing 8 ml of Emulsifier-Safe scintillation fluid (Canberra Packard Ltd.). The samples were counted 18 h later, using a Pharmacia/LKB RackBeta 1214 liquid scintillation counter. In order to determine whether any changes in binding were due to a change in Bmax or K o, a saturation curve was constructed using seven concentrations of [3H]flunitrazepam (0.2516 nM), and the data analyzed using the iterative computer program 'Enzfit'. The protein concentration of the homogenates was determined according to the method of Lowry et al. (1951).
249 20O
3. Results
3.1. Effect of handling stress on [3H]flunitrazepam binding Handling significantly increased [3H]flunitrazepam binding (F(2,21)---5.97; P < 0.01), see fig. 1. Comparing the handling-naive and 2 days handling groups the difference just failed to reach significance, but after 21 days the increase was significant (P < 0.05). Scatchard analysis revealed that the change in [3H]flunitrazepam binding was entirely due to the increase in the number of benzodiazepine receptors (naive: Bmax = 1202 + 109 fmol/mg protein; handling-habituated: Bmax -- 1605 + 141 fmol/mg protein (P < 0.05)), without any significant changes in affinity (naive: KD = 8.4 + 1.2 nM; handling-habituated: KD = 9.9 + 1.8 nM).
-7-
150
-7
100
50
CON
0.3
3.0
30.0
OIAZEPAM (uM]
Fig. 2. Effect of in vitro preincubation with diazepam (0.3-30.0/*M) on [3H]flunitrazepam binding to membranes prepared from cerebral cortex of handling naive rats. The data are means+ S.E.M. of 6-10 separate experiments. * P < 0.05; ** P < 0.01 compared with control group.
3.2. In vitro pre-incubation with diazepam of cortical membranes from naive rats Pre-incubation of homogenates, prepared from the cortex of naive rats, for 30 min with diazepam resulted in a significant concentration-dependent increase in specific [3H]flunitrazepam binding (F(3,18)= 3.6; P < 0.03), see fig. 2. Scatchard analysis of the saturation isotherm in the presence of 30 ~M diazepam revealed that these changes in [3H]flunitrazepam binding were due to an increase in the density of binding sites rather than to changes in the affinity, see table 1.
3.3. Effect of in viuo diazepam administration on [SH]flunitrazepam binding in naive and handlinghabituated rats Acute administration of diazepam (4 mg/kg i.p.) 30 rain prior to killing did not induce any changes in
TABLE 1 Effect of in vitro preincubation of homogenate with diazepam (30 /xM) on [3H]flunitrazepam binding to cortical membranes of handling naive animals, n, number of separate experiments Group
n
Bnaax (fmol/mg protein)
KD (nM)
Control (naive) Diazepam (30 p.M)
7 5
1104+ 95 1716+248 a
6.8+0.5 8.1 +0.8
a p < 0.05 (Student's t-test).
handling-habituated animals, see table 2. In contrast, when diazepam was administered to naive animals it significantly reversed the reduction of [3H]flunitrazepam binding (F(1,18)= 7.45; P = 0.01), restoring it to the level seen in handling-habituated animals, see table 2.
250
20O
TABLE 2 t50
-F-
Effect of in vivo administration of diazepam (4.0 mg/kg i.p.) to naive (N) and handling-habituated (HH) rats on [3H]flunitrazepam binding (0.5 nM) to cortical membranes. Diazepam was administered 30 min prior to killing, n, number of separate experiments Group
n
[3H]Flunitrazepam binding (fmol/mg protein)
% of control (HH)
HH HH + diazepam N N + diazepam
6 4 8 5
174.3 + 19.1 193.0 +_23.5 103.6+ 6.7 a 164.1 + 13.5 b
100 110.7 59.5 94.1
50
Handled 2t days
Handled 2 days
Naive
Fig. 1. Effect of handling on [3H]flunitrazepam binding to rat cortical membranes. The data are means+S.E.M, of 6-10 separate experiments. * P < 0.05 compared with naive group.
a p < 0.05 as compared to handling-habituated controls; b p < 0.05 as compared to handling naive controls (Student's t-test).
250
4. Discussion The results of the present study confirm those of Mennini et al. (1988) that handling changes the number of benzodiazepine receptors. Our data also show that this change develops gradually and depends on the number of times the rat has been previously handled. Downregulation of benzodiazepine receptors after acute handling stress correlates well with the findings of Biggio's group who have reported a decreased density of GABA A receptors and decreased 36C1- efflux and uptake into the brain of acutely handled animals compared with handling-habituated rats (Biggio et al., 1981, 1984, 1990; Concas et al., 1987). We think that it is possible to exclude GABA as the mediator of these changes in benzodiazepine binding. The modulatory effect of GABA on benzodiazepine binding is to alter the affinity constant, without affecting the number of binding sites (Guidotti et al., 1978). Secondly, we have previously found higher K+-evoked release of [14C]GABA from cortical and hippocampal slices of handling-naive rats compared with handlinghabituated animals (File et al., 1990). If changes in release of GABA were the cause of the changes in benzodiazepine binding seen in our study and that of Mennini et al. (1988), then naive rats should have displayed higher binding than handling-habituated rats. Mennini et al. (1988) suggested that the rapid decrease in binding sites found after acute handling stress may be due to internalisation and consequent alteration in the receptor turnover. It may also be envisaged that a conformational change, or partial occlusion by an endogenous ligand of a proportion of the free binding sites, may lead to a decreased accessibility to the radioligand. Addition of diazepam (0-30 /~M) in vitro during preparation of the membranes or in vivo administration of diazepam (4 mg/kg i.p. 30 min prior to killing), followed by an extensive washing procedure, resulted in an increase in the number of benzodiazepine receptors. There were no changes in the affinity constant indicating a complete removal of the added diazepam during the extensive washing procedure. It is possible that this extensive washing accounts for the difference between our results and those of Korneyev and Factor (1981) who found that in vitro pre-incubation with diazepam increased both Bmax and K D. Our results suggest that handling habituation and diazepam treatment have similar actions on benzodiazepine binding and represent two ways of adapting to the stress of handling by increasing the number of benzodiazepine receptors. The two effects were not additive, suggesting that benzodiazepines will have less effect in handling-habituated animals. This has been demonstrated behaviourally in an animal test of anxiety, where diazepam has less anxiolytic effect in ban-
dling-habituated compared with handling-naive animals (Andrews et al., 1991; Brett and Pratt, 1990). In conclusion, this study provides evidence that specific effects of benzodiazepines may be due not only to the occupation of benzodiazepine receptors and subsequent augmentation of GABAergic neurotransmission, but also due to the increase of benzodiazepine receptor binding sites on neuronal membranes. It also demonstrates that the number of benzodiazepine binding sites can be increased by non-pharmacological means.
Acknowledgements These experiments were supported by grants from The Wellcome Trust and the Special Trustees of Guy's Hospital.
References Andrews, N., A. Zharkovsky and S.E. File, 1991, Handling stress: benzodiazepine binding and behaviour in the elevated plus-maze test of anxiety in the rat, Br. J. Pharmacol. 102, 305P. Biggio, G., M.G. Corda, A. Concas, G. Demontis, Z. Rosetti and G.L. Gessa, 1981, Rapid changes in GABA binding induced by stress in different areas of rat brain, Brain Res. 229, 363. Biggio, G., A. Concas, M. Serra, M.G. Corda, V. Nurchi, C. Crisponi and G.L. Gessa, 1984, Stress and /3-carbolines decrease the density of low affinity GABA binding sites: an effect reversed by diazepam, Brain Res. 305, 13. Biggio, G., A. Concas, M.G. Corda, O. Giorgi, E. Sanna and M. Serra, 1990, GABAergic and dopaminergic transmission in the rat cerebral cortex: effect of stress, anxiolytic and anxiogenic drugs, Pharmacol. Ther. 48, 121. Braestrup, C., M. Nielsen, E.B. Nielsen and M. Lyon, 1979, Benzodiazepine receptors in the brain are affected by different experimental stresses: The changes are small and not unidirectional, Psychopharmacology 65, 273. Brett, R.R. and J.A. Pratt, 1990, Chronic handling modifies the anxiolytic effect of diazepam in the elevated plus-maze, Enr. J. Pharmacol. 178, 135. Coneas, A., D.R. Gehlert, J.K. Walmsley and H.I. Yamamura, 1986, Effect of benzodiazepine site ligands on 35S t-butylbicyclopbosphorothionate (3sS-TBPS) binding to rat brain: an autoradiographic study, in: GABAergic Transmission and Anxiety, eds. G. Biggio and E. Costa (Raven Press, New York) p. 227. Concas, A., S. Mele and G. Biggio, 1987, Footshock stress decreases chloride efflux from rat brain synaptosomes, Eur. J. Pharmacol. 135, 423. Concas, A., M. Serra, T. Astoggiu and G. Biggio, 1988a, Footshock stress and anxiogenic /3-carbolines increase t-[35Sl-butylbicyclo phophorothionate binding in the rat cerebral cortex; an effect opposite to anxiolytics and y-amino butyric acid mimetics, J. Neurochem. 5, 1868. Concas, A., M. Serra, M.G. Corda and G. Biggio, 1988b, Changes in 36C1- flux and 35S-TBPS binding induced by stress and GABAergic drugs, in: Chloride Channels and Their Modulation by Neurostransmitters and Drugs, eds. G. Biggio and E. Costa (Raven Press, New York) p. 227. File, S.E., N. Andrews and A. Zbarkovsky, 1990, Handling habituation and chlordiazepoxide have different effects on GABA and 5-HT function in the frontal cortex and hippocampus, Eur. J. Pharmacol. 190, 229.
251 Guidotti, A., G. Toffano and E. Costa, 1978, An endogenous protein modulates the affinity of GABA and benzodiazepine receptors in rat brain, Nature (London) 257, 553. Korneyev, A.Ya and M.I. Factor, 1981, Increase of benzodiazepine binding to the membranes isolated in the presence of diazepam, Eur. J. Pharmacol. 71, 127. Lai, H. and M.A. Carino, 1990, Acute white noise exposure affects the concentration of benzodiazepine receptors in the brain of the rat, Pharm. Biochem. Behav. 36, 985. Lowry, O.H., N.J. Rosebrough, A.L Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Medina, J,H., M.L. Novas, C.N.V. Wolfman, M. Levi De Stein and E. De Robertis, 1983, Benzodiazepine receptors in rat cerebral
cortex and hippocampus undergo rapid and reversible changes after acute stress, Neuroscience 9, 331. Mennini, T., M. Gobbi, L. Perin and M. Salmona, 1988, Rapid internalization of benzodiazepine receptors in the rat cortex induced by handling, in: Chloride Channels and Their Modulation by Neurotransmitters and Drugs, eds. G. Biggio and E. Costa (Raven Press, New York) p. 263. Skerritt, J.H. and G.A.R. Johnston, 1983, Enhancement of GABA binding by benzodiazepines and related anxiolytics, Eur. J. Pharmacol. 89, 193. Soubrie, P., M.H. Thiebot, A. Jobert, J.L. Montastruc, F. Hery and M. Hamon, 1980, Decreased convulsant potency of picrotoxin and pentetrazol and enhanced [3H]flunitrazepam cortical binding following stressful manipulations in rats, Brain Res. 189, 505.
247
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52235
Acute handling stress downregulates benzodiazepine receptors: reversal by diazepam Nick A n d r e w s , A l e x a n d e r Z h a r k o v s k y a n d S a n d r a E. File Psychopharmacology Research Unit, UMDS Division of Pharmacology, Guy's Hospital, London SE1 9RT, U.K.
Received 1 July 1991,revised MS received 8 October 1991, accepted 29 October 1991
In rats naive to handling, the effects of an acute handling stress led to lower [3H]flunitrazepam binding in the frontal cortex, compared with animals previously habituated to handling for 2 or 21 days. This decreased binding was due to reductions in the number of receptors, not to a change in affinity. Pre-incubation with diazepam (0.3, 3.0 or 30/~M) of frontal cortex homogenates taken from naive rats exposed to acute handling stress (followed by extensive washing to remove residual diazepam and endogenous modulators) led to a concentration-dependent increase in the number of benzodiazepine receptors, without any change in KD. Acute in vivo administration of diazepam (4 mg/kg) prior to the handling stress was without significant effect in handling-habituated animals, but increased [3H]flunitrazepam binding in handling naive rats. Thus, handling habituation and diazepam treatment have similar actions on benzodiazepine binding and represent two ways of adapting to the stress of handling by increasing the number of benzodiazepine receptors. Benzodiazepine binding; Handling; Stress
I. Introduction
T h e GABAA-benzodiazepine r e c e p t o r - c h l o r i d e ionophore complex has been shown to change in response to a variety of stressors, such as forced swimming (Medina et al., 1983), immobilisation (Braestrup et al., 1979), white noise (Lai and Carino, 1990) and handling (Biggio et al., 1990). Acute exposure of naive animals to handling stress induced a rapid decrease in G A B A n receptor binding in rat cortical membranes, compared with animals habituated to handling for 15 days. A foot-shock applied to these handling-habituated animals induced a fall in G A B A A receptor numbers, but was without effect in handling-naive animals (Biggio et al., 1981). There was a greater 36C1- flUX in synaptosomes from handling-habituated animals compared with handling-naive animals (Concas et al., 1987, 1988b). An additional footshock stress failed to further decrease the basal 36C1- flUX in handling-naive animals but significantly reduced it in handling-habituated animals (Concas et al., 1988a). The changes in 36C1- flux and G A B A A receptor binding after acute stress appear to be consistent,
Correspondence to: S.E. File, PsychopharmacologyResearch Unit, UMDS Divisionof Pharmacology,Guy's Hospital, London SE1 9RT, U.K.
regardless of the type of stressor, but this is not the case with benzodiazepine receptor binding. It appears that the type of stressor can determine the direction of change of the binding. Thus, benzodiazepine binding was found to decrease after electrical footshock and post-natal isolation of new-born rat pups, increase after immobilisation, and remain unchanged after isolation-induced aggression in mice (Braestrup et al., 1979). There are even different findings for different conditions of swim-stress. Thus, Soubrie et al. (1980), found that cold water swim stress caused an increase in benzodiazepine binding, Braestrup et al. (1979) reported that there was no change after ambient swim stress and Medina et al. (1983) found forced swimming in ambient temperatures (18°C) caused a decrease in binding. There has been a recent report that acute handling stress applied to naive animals, results in lower benzodiazepine binding in cortical membranes when compared with the binding observed in membranes of handling-habituated animals (Mennini et al., 1988). The change was found to be due to a decrease in Bmax with no effect on K D. In experiment 1 we investigated whether these changes could be observed using our handling procedure and strain of rats. The functioning of the GABA-benzodiazepine receptor complex is also changed by anxiolytic and anxiogenic drugs. Diazepam decreases [35S]t-butylbicyclo-
248 phosphorothionate ([35S]TBPS) binding (Concas et al., 1986) and increases GABA A receptor binding (Skerritt and Johnston, 1983). Acute handling stress and administration of an anxiogenic /3-carboline both decrease GABA A receptor binding to cortical membranes; an effect that can be antagonised by in vitro addition of diazepam to the membranes (Biggio et al., 1984). In experiment 2 we investigated whether the changes in benzodiazepine binding induced by handling stress could be reversed by in vitro pre-incubation of the membranes with diazepam, or prevented by in-vivo administration of the drug 30 min prior to killing.
2. Materials and methods
2.1. Animals Male hooded Lister rats (Olac Ltd., Bicester), weighing 200-250 g at the start of the experiment, were housed in groups of 5 with food and water freely available, in a room maintained at 20°C, with lights on from 06:00 to 18:00 h.
2.2. Handling procedure The daily handling consisted of picking up the rat by its body, weighing it and then giving an intraperitoneal (i.p.) injection of water. The animal was then returned to its home cage. Animals were divided into three groups according to the amount of handling they would receive. One group received no handling until the day of killing (handling-naive), one received handling one day prior to killing and also on the day of killing (2 days of handling) and the third group received handling on 20 successive days and also on the day of killing (handling-habituated). The handling procedure on the day of killing was identical to that on previous days and occurred 30 min prior to killing by cervical dislocation. In experiment 2, handling-naive and handling-habituated animals received i.p. injections of water or diazepam (4 mg/kg) 30 min prior to killing.
2.3. Drugs and solutions Diazepam (gift of Roche products Ltd.), was suspended in a Tween 20 and distilled water vehicle and left in an ultrasonic water bath for 30 min before injection. The drug was administered i.p. in a volume of 2 ml/kg. For in vitro studies, diazepam was dissolved in 98% ethanol in a concentration of 10 mM and dilutions made from this stock. Care was taken that the final concentration of ethanol, added to the assay as part of the diazepam solution, was not greater than 0. 1%.
2.4. Biochemical studies
2.4.1. Preparation of the membranes Animals were stunned and killed by cervical dislocation, the brains rapidly removed and frontal cortex dissected out on ice. The tissue was homogenized in ice cold distilled water using a Polytron homogeniser (setting 6.5 for 15 s). The homogenate was then centrifuged at 38 000 x g for 20 min. The resulting pellet was resuspended in ice cold 50 mM Tris HC1 buffer (pH = 7.4 at 20°C) and incubated for 30 min on ice. To observe the effects of in vitro addition of diazepam, the homogenate was resuspended in Tris HCI buffer containing varying concentrations of diazepam. In order to remove all residual diazepam and endogenous modulators of benzodiazepine binding present in the membrane preparation, the following extensive washing procedure was employed. Briefly, the membrane preparation was centrifuged at 20000 X g for 20 min and then subsequently resuspended in Tris HCI buffer and washed an additional 3 times. The resulting pellet was frozen overnight. Eighteen hours later the pellet was thawed, resuspended in Tris HCI buffer and centrifuged at 20000 x g for 20 min. This pellet was then stored on ice for 30 min. The pellet was subsequently resuspended in Tris HC1 buffer and washed" an additional 5 times. The final pellet was resuspended in 300 volumes of Tris HCI buffer and taken to the binding assay.
2.4.2. [3H]Flunitrazepam binding assay Aliquots (0.1-0.2 mg protein/ml final concentration) of membrane suspension were incubated with 0.5 nM (final concentration) [3H]flunitrazepam (Amersham plc., specific activity 85 Ci/mmol) in a final volume of 1 ml for 40 min at room temperature. Non-specific binding was determined in the presence of 3 /zM diazepam and represented 10-15% of total binding. Specific and non-specific binding were both performed in triplicate. Following incubation, the samples were filtered through Whatman G F / B filters using a Millipore manifold and washed rapidly 3 times, with 4 ml of cold Tris HCI buffer. The filters were put into scintillation vials containing 8 ml of Emulsifier-Safe scintillation fluid (Canberra Packard Ltd.). The samples were counted 18 h later, using a Pharmacia/LKB RackBeta 1214 liquid scintillation counter. In order to determine whether any changes in binding were due to a change in Bmax or K o, a saturation curve was constructed using seven concentrations of [3H]flunitrazepam (0.2516 nM), and the data analyzed using the iterative computer program 'Enzfit'. The protein concentration of the homogenates was determined according to the method of Lowry et al. (1951).
249 20O
3. Results
3.1. Effect of handling stress on [3H]flunitrazepam binding Handling significantly increased [3H]flunitrazepam binding (F(2,21)---5.97; P < 0.01), see fig. 1. Comparing the handling-naive and 2 days handling groups the difference just failed to reach significance, but after 21 days the increase was significant (P < 0.05). Scatchard analysis revealed that the change in [3H]flunitrazepam binding was entirely due to the increase in the number of benzodiazepine receptors (naive: Bmax = 1202 + 109 fmol/mg protein; handling-habituated: Bmax -- 1605 + 141 fmol/mg protein (P < 0.05)), without any significant changes in affinity (naive: KD = 8.4 + 1.2 nM; handling-habituated: KD = 9.9 + 1.8 nM).
-7-
150
-7
100
50
CON
0.3
3.0
30.0
OIAZEPAM (uM]
Fig. 2. Effect of in vitro preincubation with diazepam (0.3-30.0/*M) on [3H]flunitrazepam binding to membranes prepared from cerebral cortex of handling naive rats. The data are means+ S.E.M. of 6-10 separate experiments. * P < 0.05; ** P < 0.01 compared with control group.
3.2. In vitro pre-incubation with diazepam of cortical membranes from naive rats Pre-incubation of homogenates, prepared from the cortex of naive rats, for 30 min with diazepam resulted in a significant concentration-dependent increase in specific [3H]flunitrazepam binding (F(3,18)= 3.6; P < 0.03), see fig. 2. Scatchard analysis of the saturation isotherm in the presence of 30 ~M diazepam revealed that these changes in [3H]flunitrazepam binding were due to an increase in the density of binding sites rather than to changes in the affinity, see table 1.
3.3. Effect of in viuo diazepam administration on [SH]flunitrazepam binding in naive and handlinghabituated rats Acute administration of diazepam (4 mg/kg i.p.) 30 rain prior to killing did not induce any changes in
TABLE 1 Effect of in vitro preincubation of homogenate with diazepam (30 /xM) on [3H]flunitrazepam binding to cortical membranes of handling naive animals, n, number of separate experiments Group
n
Bnaax (fmol/mg protein)
KD (nM)
Control (naive) Diazepam (30 p.M)
7 5
1104+ 95 1716+248 a
6.8+0.5 8.1 +0.8
a p < 0.05 (Student's t-test).
handling-habituated animals, see table 2. In contrast, when diazepam was administered to naive animals it significantly reversed the reduction of [3H]flunitrazepam binding (F(1,18)= 7.45; P = 0.01), restoring it to the level seen in handling-habituated animals, see table 2.
250
20O
TABLE 2 t50
-F-
Effect of in vivo administration of diazepam (4.0 mg/kg i.p.) to naive (N) and handling-habituated (HH) rats on [3H]flunitrazepam binding (0.5 nM) to cortical membranes. Diazepam was administered 30 min prior to killing, n, number of separate experiments Group
n
[3H]Flunitrazepam binding (fmol/mg protein)
% of control (HH)
HH HH + diazepam N N + diazepam
6 4 8 5
174.3 + 19.1 193.0 +_23.5 103.6+ 6.7 a 164.1 + 13.5 b
100 110.7 59.5 94.1
50
Handled 2t days
Handled 2 days
Naive
Fig. 1. Effect of handling on [3H]flunitrazepam binding to rat cortical membranes. The data are means+S.E.M, of 6-10 separate experiments. * P < 0.05 compared with naive group.
a p < 0.05 as compared to handling-habituated controls; b p < 0.05 as compared to handling naive controls (Student's t-test).
250
4. Discussion The results of the present study confirm those of Mennini et al. (1988) that handling changes the number of benzodiazepine receptors. Our data also show that this change develops gradually and depends on the number of times the rat has been previously handled. Downregulation of benzodiazepine receptors after acute handling stress correlates well with the findings of Biggio's group who have reported a decreased density of GABA A receptors and decreased 36C1- efflux and uptake into the brain of acutely handled animals compared with handling-habituated rats (Biggio et al., 1981, 1984, 1990; Concas et al., 1987). We think that it is possible to exclude GABA as the mediator of these changes in benzodiazepine binding. The modulatory effect of GABA on benzodiazepine binding is to alter the affinity constant, without affecting the number of binding sites (Guidotti et al., 1978). Secondly, we have previously found higher K+-evoked release of [14C]GABA from cortical and hippocampal slices of handling-naive rats compared with handlinghabituated animals (File et al., 1990). If changes in release of GABA were the cause of the changes in benzodiazepine binding seen in our study and that of Mennini et al. (1988), then naive rats should have displayed higher binding than handling-habituated rats. Mennini et al. (1988) suggested that the rapid decrease in binding sites found after acute handling stress may be due to internalisation and consequent alteration in the receptor turnover. It may also be envisaged that a conformational change, or partial occlusion by an endogenous ligand of a proportion of the free binding sites, may lead to a decreased accessibility to the radioligand. Addition of diazepam (0-30 /~M) in vitro during preparation of the membranes or in vivo administration of diazepam (4 mg/kg i.p. 30 min prior to killing), followed by an extensive washing procedure, resulted in an increase in the number of benzodiazepine receptors. There were no changes in the affinity constant indicating a complete removal of the added diazepam during the extensive washing procedure. It is possible that this extensive washing accounts for the difference between our results and those of Korneyev and Factor (1981) who found that in vitro pre-incubation with diazepam increased both Bmax and K D. Our results suggest that handling habituation and diazepam treatment have similar actions on benzodiazepine binding and represent two ways of adapting to the stress of handling by increasing the number of benzodiazepine receptors. The two effects were not additive, suggesting that benzodiazepines will have less effect in handling-habituated animals. This has been demonstrated behaviourally in an animal test of anxiety, where diazepam has less anxiolytic effect in ban-
dling-habituated compared with handling-naive animals (Andrews et al., 1991; Brett and Pratt, 1990). In conclusion, this study provides evidence that specific effects of benzodiazepines may be due not only to the occupation of benzodiazepine receptors and subsequent augmentation of GABAergic neurotransmission, but also due to the increase of benzodiazepine receptor binding sites on neuronal membranes. It also demonstrates that the number of benzodiazepine binding sites can be increased by non-pharmacological means.
Acknowledgements These experiments were supported by grants from The Wellcome Trust and the Special Trustees of Guy's Hospital.
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