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Benzodiazepine receptors: Effect of tissue preincubation at 37°C
Neuroseience Letters, 13 (1979) 2 4 3 - - 2 4 7
~) Elsevier/North-HollandScientific Publishers Ltd.
BENZODIAZEPINE R E C U R S : AT 37 °C
243
EFFECT OF TISSUE PREINCUB.ATION
R.C. ~PETH, G.J. WASTEK,T.D. REISINE and H.I. YAMAMURA* De~nt of Pharmacology, University of Arizona School of Medicine, Tucson, AZ 86724 (U.S.A.)
(Receivecl~February 19th, 1979) (Revised version received April 17th, 1979) (Accepted May 4th, 1979)
8UMMARY [3H]Flunitrazepam binding to rat brain homogenates was assayed in two ways: by 37--0°C incubations or O°C only incubations. The affinity of brain benzodiazepine receptors for [ 3H ]flunitrazepam was significantly greater in 37--0°C incubated samples than in 0°C incubated samples, while there was no change in the number of benzodiazepine receptors. The possible mechanisms for this enhanced binding affinity as well as the methodological implications for this observation are discussed.
The identification and characterization of specific, pharmacologically relevant, high affinity benzodiazepine receptors suggests that these receptors mediate the psychotropic effects of benzodiazepines [9]. Early in vitro studies of the binding of [ 3H]diazepam to rat brain homogenates revealed that [JHldiazepam binding at 37°C was drastically reduced in comparison to binding at 0*C ['/,9]. However, brain homogenates incubated at 37°C and s u ~ u e n f l y cooled to 0°C, showed increased [aH]diazepam binding compared to binding observed with only 0°C incubation [2]. The lower levels of. [3Hldiazepam [3] and [3H]flunitrazepam [10] b:nding detected at 370C results from a lower affinity of brain tissue homogcnates for benzo-
diazt
es at 370C than at 0°C.
study, h ~ investigated the effects of 37 ° C incubation prior to 0 *~C incUbation on the affinity of [~H]flunitrazepam binding to rat brain homogenate and has demonstrated significant effects which have functional as well as m e t h o d o l o g k ~ relevance.**
* Author to whom reprint ~equests ahtmld ~ sent. i:, A pmliminm'yreport of thue Irmdings was prew.n~d at the Society for Neur~cienc¢: tn~er;mg, St. Louis, MO, Nov., 1978.
244 AMays of [JH]flunitn~q=m binding to rat brain homoSenat~s were performed e~ntial]y m dear'bed p~evicmly [10]. ~ , crude, unwzau~ h o m ~
of w h o l ~ m t bndns equivalent t o 1 . 6 5 - - ~ 5 nag w e t weisht were
incubated with [~Hlflunitmzepmn (85 Ci/mmok 0.1--5.0 nM final eonce~ tratJoll) in sod/um-potass/um phosphate ~ (40.5 HIM N a = ~ 4 - ' - 9 . 5 mM KH2PO4) (pI~ '7.4) in a total volume of 2 m!: Free and bound [3H]fl~pam were sepiuaZed on 10assfiber (GF/B) filtens and filter bound mdiosctiv~ was memured by fiquid . c i n ~ o n ~ e t ~ . V~
presented are for specific (1 .M clonazepsm displaceable) 1~4] f
l
~
binding, When brain homogenates were inculmted for 60 rain at 37"C prior t o incubation for varying time at 0 ~C, there was an mcreme in [3H]flunitrazepam binding with time (Fig. 1). Temperature measurements of the m a y media after placement on ice indicated t h a t tempeml~u~ e q u ~ b m f i o n occurred rapidly: the temperature of the media was IOc after 6 rain on ice. The equilibration of [3H]flunitrazepam binding was increased 33% in 37 ° C preincubated ~ n p l e s in contrast to samptes incubated at 0 °C only (P <~
0.001). To determine the inmate of the increased [~H]flunitrazepmn binding
observed after the 37"C preincubation, t b e . ~
constant (Kd) and
the maximal binding capacity ( B m u ) of [ZH]fltmitmzepam binding were determined. Table I, Expt. 1, shows that there was a dgnificsnt (P < 0.02) decrease in the Kd of 37°C preincubated rat brain h o m ~ g e n a t u for [~14]flunitrazepam compared to 0°C only incubated rat brain homogenatm. The Bmu-values did not differ significantly between the two ~ o u p s (P < 0.20),
.° I
io I o6
2o 4o so so K~o ,2o TIME AT 0°C AFTER 80 rain AT 37°C
Fig. 1. Effects of cooling rat brain homolenatas on icm for varying periods of t/me after 37"C preincubation on [*H]flunitrazelmm bindin¢ Assays of [SH]flunitmzepsm binding were performed essentially as described in the text. Samples were incubated at 37"C for 60 rain and then placed in an ice-water bath for periods of a time range O-120 rain prior to rdtration. Diaeonal dashed lines relmmmt mean • S.D. of samples incubated at 0",C for 90 rain. Initial [SH]flunitrazepsm eom~entration was 0.25 nM, rat brain homogenate equivalent to 2.5 mg tissue was present in each sample.
245 TABLE I C O M P A R I S O N O F 3 7 - - 0 ° C I N C U B A T E D R A T B R A I N H O M O G E N A T E S WITH 0°C ONLY INCUBATED HOMOOENATE8 BY 8CATCHARD ANALYSIS 8 a ~ isotherms o f [~H]fluniia-agepam binding to rat brain h o m o g e n a t e s were analys ~ l b y S c a t c h a r d analysis using 4 different concentrations o f [3H]flunitrazepam; 0.1, 0.4, 1.5 a n d 5.0 nbi. Values presented are m e a n s ± S.E_M. In E x p t . 1 statistical comparboris were m a d e t~i~g a paired t ° t ~ t , n ffi 5. In Expt. 2, statistical comparisons were m a d e using a o n e way anaIys~s o f variance and multiple t-test [ 4 ] n = 2. l.-,cubation c o u d i t i o ~
K d (nM)
Expt. 1 1 5 0 min at 0 ° C 6 0 rain at 37"C then 9 0 rain at 0°C
0.94 ± 0 . 0 9 0.63 ± 0 . 0 2 a
105 +_ 8 114 _~ ,5
Expt. 2 9 0 min at 0 ° C 15 mia at 37"C then 9 0 min at 0 " C 6 0 min at 37"C then 9 0 rain at 0 ° C 180 rain st 37°C then 9 0 rain at 0 ° C
1.17 0.74 0.62 0.44
122 113 114 104
• Significantly b 8ignit'umntly e Significantly different from
± ± ± ±
0.01 0.04 b 0.06 b 0.03 b,c
" Bm~ ( f m o l / m g tissue)
_~ 4 ±7 ± 11 ± ,5
different from 0°C o n l y incubated ~amples P < 0.02. different from 0 ° C o n l y i n c u b a t e d samples P < 0 . 0 1 . different from 15 min at 3 7 ° C , 90 min at 0°C, P < 0 . 0 1 ; significantly 60 min at 37"C, 90 min at 0°C, P < 0.05.
To determine whether [ 3H] flunitrazepa~ underwent rr ~tabolic destruction at 37°C and to see if the presence of [~H]flunitrazepam at 37°C was neceam~ for the induction of changes in binding at 0 ° C, an experiment was performed in which [3H]flunitrazepam was added after the 60 rnin incubation at 37°C. There w u no difference in'the enhancement of {3H Jflur,~raz epam binding by 37 ° C preincubation as a function of whether [ 3H ] flunitrazepam was present during ~his 37 ° C incubation (data not shown). Incubation of brain ~unpl~ for Varying lengths of time at 37 ° C before the addition of [3H]flunitrazepam and subsequent, incubation at 0°C caused a s'~t, (F = 69.2, P < 0.01) systematic decrease in the Kd of rat brain h o m ~ e n a t ~ for [3H]flunitrazepam (Table I, Expt. 2). Once again, there was no sigp.iflcant change in Bmax between groups (F = 1.15, P :> 0.25). The results of this study indicate that the affinity of rat brain benzodiazepine recepto~ for |~H]flvnitrazepam at 0°C is enhanced by in vitro preincubation at 37 ° C. These results extend the preliminary observations of Braestrup and Squires [2]. The o ~ a t i o n s raise the question of which condition most acc-dra~ly recreants the in vivo affinity of benzodiazepine recept~r~ for the benzodiazepines. The changes in the affinity for benzodiazepines could reflect postmortem tissue degradation in which membrane component~ which
24e
rem~ benz~Uz~~ ~ undergo a ~ ~ ~ e incre..ed eou~ abo ~ ~ " ~ d e S n , d a ~ of an endosma~ bensod . f f i ~ ~ a k e s u ~ e e tt~-~oet~ with ~ . for the benzo~ n ~ e ~ . A third ~ whereb~ ~ e bersodiasel~ ~
m ~
~ 7~m~bu~ ~ :(CXSX) [ S ] , m GABA h~ ~ .ao,~n to the bmdmgaffm~y of both [ ' H ] d ~ m ~ a [ U ] m d [3H]flm~m~
epam [12] to rat brain. Thus, at the ~ t time, it is not possible to d e ~ mine which conait/on, or w h ~ both conditions represent in vivo benzodia~pine receptor binding af~nity.
Several invest/~ton, have rout/rely mayed [~H]d/azepa~ ~
.sing
37 ° C incubations and placement of the samples on ice to bring the incubation" medium to 0° C [1,6,8,9]. The results of this study ~ tlmt the 37 °C incubat/on was m m e c e m ~ because equilibration of ['H]diszepam binding would have occurred if the assays had been run for a ~ period of time at 0 ° C. This technique could have introduced a dgnificmt en~r in the me~urement of [JH]diazepmn binding at 0°C ff ~luilibdum binding had not been atta/ned. Foflamately, [3H]diazepam binding at 0"C equilil~ rates rapidly and the amount of error in these ~ was minimal If these techniques had been used to mesmtre [~H]flunitrszepmn binding however, a sizeable error would have been introduced unless samples were incubated at 0°C for 60 min. The binding of other neurotransmittm~ and drugs to ~ may also show changes in Kc with temperature and require long incubations to reach e~uilibr/um at C'C. Unle~ investigators speciflcafly wish to ~ i n ~ the effect~ of 37°C prei~cubation on binding zt 0"C (e.g. to ~ an e n d ~ o u s subetance) or they:have demonstrated that there is no c h s n ~ tn affinity with ~.mperat~re, the use of a 37"C incubation and a chilling of the samples at 0 ° ~" is u n w a r m n t ~ . ACKNOWLEDGEMENT8
The authors acknowledge the excellent technical smistance of Mr. Andrew ~ . e n and the secretawi~ usistance of Ms. Cathy Kmmm. We also thank Dr. Kevin Beaumont and Dr. W ~ Koeske for thelr critical a p ~ of this manuscript. This research was m P ~ by USPH8 ~ants MH-25267, MH. 30626 and the Hereditsry D ~ Foundat/on and the Huntington's Chov~ Foundation. H.I.Y. is a recipient of a USPHS Resurch 8citmtkt Development Award (MH-00095). R.8.C. is supported by a USPHS Clinicsl Phsmmcology Training Grant (GM.07533), REFERENCES ~ Bosmann, H.G., C,a~, K.R. sad DiStefano, P., D i ~ receptor eharaeteri~tioa: Specific binding of a l~mmdJ~zepine to mscromolecules in varim~ areas of rat brain, FEBS Lett., 82 (1977) 368--372,
247 2 ~ p , C. and Squires, R.F., Specific benzodiazepine receptors in rat brain characterized by high-aff'mity [~H]diazepam binding, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3805--3809. 3 Braestrup,,C. and Squires, R.F., Brain specific benzodiazepine receptors, Brit. J. Psydfiat., 133 (1978) 249--260. 4 Kir~ R.E., ,Experimental Desi~- Procedures for the Behavioral Sciences, Wacisworth, Belmont, CA., 1968, 577 pp. 5 Loveil, R.A. and Elliott, K.A.C., The ~-aminobutyric acid and factor I content of brain, J. Neurochem., 10 (1963) 470--488. 6 M~kerer, C.R., Kochman, R.L., Bierschenk, B.A. and Bremner, S.S., The binding of [~H~pam to rat brain homogenates, J. Pharmaeol. exp. Ther., 206 (1978)
40~--413. 7 Mohler, H. and Okada, T., Benzodia,~epine receptor: demonstration in the central ne~mm system, Science, 198 (1977) 849--851. 8 Mmll~r, W.E., 8ehlafer, U. and Wallert, U., Benzodiazepine receptor binding in rat spinal ~ membrane, Neurosei. Lett., 9 (1978) 239--243. 9 ~iuirm, R.F. and Braestrup, C., Benzodiazepi~te receptors in rat brain, Nature (Lond.), 266 (1977) 732"-734. 10 Spet~ R.C., Wastek, G.J. and Yamamura, H.I., Benzodiazepine recept~ ~: Temperatram ~ n e e of [~H]flunitrazelmm binding, Life Sci., 24 (1979) 351--358. 11 ~ J.F., Thomas, J.W. and Gallagher, D.W., GABAergic modulation of benzodimmpimD site aensitivity, Nature (Lond.), 274 (1978) 383--385. 12 WItek, O,J., 8peth, R.C., Reisine, T.D. and Yamamura, H.I., The effect of ~-aminoIm~ acid on tH-flunitr~epam binding in rat brain, Europ. J. Pharmacol., 50 (1~78} 445--447.
~) Elsevier/North-HollandScientific Publishers Ltd.
BENZODIAZEPINE R E C U R S : AT 37 °C
243
EFFECT OF TISSUE PREINCUB.ATION
R.C. ~PETH, G.J. WASTEK,T.D. REISINE and H.I. YAMAMURA* De~nt of Pharmacology, University of Arizona School of Medicine, Tucson, AZ 86724 (U.S.A.)
(Receivecl~February 19th, 1979) (Revised version received April 17th, 1979) (Accepted May 4th, 1979)
8UMMARY [3H]Flunitrazepam binding to rat brain homogenates was assayed in two ways: by 37--0°C incubations or O°C only incubations. The affinity of brain benzodiazepine receptors for [ 3H ]flunitrazepam was significantly greater in 37--0°C incubated samples than in 0°C incubated samples, while there was no change in the number of benzodiazepine receptors. The possible mechanisms for this enhanced binding affinity as well as the methodological implications for this observation are discussed.
The identification and characterization of specific, pharmacologically relevant, high affinity benzodiazepine receptors suggests that these receptors mediate the psychotropic effects of benzodiazepines [9]. Early in vitro studies of the binding of [ 3H]diazepam to rat brain homogenates revealed that [JHldiazepam binding at 37°C was drastically reduced in comparison to binding at 0*C ['/,9]. However, brain homogenates incubated at 37°C and s u ~ u e n f l y cooled to 0°C, showed increased [aH]diazepam binding compared to binding observed with only 0°C incubation [2]. The lower levels of. [3Hldiazepam [3] and [3H]flunitrazepam [10] b:nding detected at 370C results from a lower affinity of brain tissue homogcnates for benzo-
diazt
es at 370C than at 0°C.
study, h ~ investigated the effects of 37 ° C incubation prior to 0 *~C incUbation on the affinity of [~H]flunitrazepam binding to rat brain homogenate and has demonstrated significant effects which have functional as well as m e t h o d o l o g k ~ relevance.**
* Author to whom reprint ~equests ahtmld ~ sent. i:, A pmliminm'yreport of thue Irmdings was prew.n~d at the Society for Neur~cienc¢: tn~er;mg, St. Louis, MO, Nov., 1978.
244 AMays of [JH]flunitn~q=m binding to rat brain homoSenat~s were performed e~ntial]y m dear'bed p~evicmly [10]. ~ , crude, unwzau~ h o m ~
of w h o l ~ m t bndns equivalent t o 1 . 6 5 - - ~ 5 nag w e t weisht were
incubated with [~Hlflunitmzepmn (85 Ci/mmok 0.1--5.0 nM final eonce~ tratJoll) in sod/um-potass/um phosphate ~ (40.5 HIM N a = ~ 4 - ' - 9 . 5 mM KH2PO4) (pI~ '7.4) in a total volume of 2 m!: Free and bound [3H]fl~pam were sepiuaZed on 10assfiber (GF/B) filtens and filter bound mdiosctiv~ was memured by fiquid . c i n ~ o n ~ e t ~ . V~
presented are for specific (1 .M clonazepsm displaceable) 1~4] f
l
~
binding, When brain homogenates were inculmted for 60 rain at 37"C prior t o incubation for varying time at 0 ~C, there was an mcreme in [3H]flunitrazepam binding with time (Fig. 1). Temperature measurements of the m a y media after placement on ice indicated t h a t tempeml~u~ e q u ~ b m f i o n occurred rapidly: the temperature of the media was IOc after 6 rain on ice. The equilibration of [3H]flunitrazepam binding was increased 33% in 37 ° C preincubated ~ n p l e s in contrast to samptes incubated at 0 °C only (P <~
0.001). To determine the inmate of the increased [~H]flunitrazepmn binding
observed after the 37"C preincubation, t b e . ~
constant (Kd) and
the maximal binding capacity ( B m u ) of [ZH]fltmitmzepam binding were determined. Table I, Expt. 1, shows that there was a dgnificsnt (P < 0.02) decrease in the Kd of 37°C preincubated rat brain h o m ~ g e n a t u for [~14]flunitrazepam compared to 0°C only incubated rat brain homogenatm. The Bmu-values did not differ significantly between the two ~ o u p s (P < 0.20),
.° I
io I o6
2o 4o so so K~o ,2o TIME AT 0°C AFTER 80 rain AT 37°C
Fig. 1. Effects of cooling rat brain homolenatas on icm for varying periods of t/me after 37"C preincubation on [*H]flunitrazelmm bindin¢ Assays of [SH]flunitmzepsm binding were performed essentially as described in the text. Samples were incubated at 37"C for 60 rain and then placed in an ice-water bath for periods of a time range O-120 rain prior to rdtration. Diaeonal dashed lines relmmmt mean • S.D. of samples incubated at 0",C for 90 rain. Initial [SH]flunitrazepsm eom~entration was 0.25 nM, rat brain homogenate equivalent to 2.5 mg tissue was present in each sample.
245 TABLE I C O M P A R I S O N O F 3 7 - - 0 ° C I N C U B A T E D R A T B R A I N H O M O G E N A T E S WITH 0°C ONLY INCUBATED HOMOOENATE8 BY 8CATCHARD ANALYSIS 8 a ~ isotherms o f [~H]fluniia-agepam binding to rat brain h o m o g e n a t e s were analys ~ l b y S c a t c h a r d analysis using 4 different concentrations o f [3H]flunitrazepam; 0.1, 0.4, 1.5 a n d 5.0 nbi. Values presented are m e a n s ± S.E_M. In E x p t . 1 statistical comparboris were m a d e t~i~g a paired t ° t ~ t , n ffi 5. In Expt. 2, statistical comparisons were m a d e using a o n e way anaIys~s o f variance and multiple t-test [ 4 ] n = 2. l.-,cubation c o u d i t i o ~
K d (nM)
Expt. 1 1 5 0 min at 0 ° C 6 0 rain at 37"C then 9 0 rain at 0°C
0.94 ± 0 . 0 9 0.63 ± 0 . 0 2 a
105 +_ 8 114 _~ ,5
Expt. 2 9 0 min at 0 ° C 15 mia at 37"C then 9 0 min at 0 " C 6 0 min at 37"C then 9 0 rain at 0 ° C 180 rain st 37°C then 9 0 rain at 0 ° C
1.17 0.74 0.62 0.44
122 113 114 104
• Significantly b 8ignit'umntly e Significantly different from
± ± ± ±
0.01 0.04 b 0.06 b 0.03 b,c
" Bm~ ( f m o l / m g tissue)
_~ 4 ±7 ± 11 ± ,5
different from 0°C o n l y incubated ~amples P < 0.02. different from 0 ° C o n l y i n c u b a t e d samples P < 0 . 0 1 . different from 15 min at 3 7 ° C , 90 min at 0°C, P < 0 . 0 1 ; significantly 60 min at 37"C, 90 min at 0°C, P < 0.05.
To determine whether [ 3H] flunitrazepa~ underwent rr ~tabolic destruction at 37°C and to see if the presence of [~H]flunitrazepam at 37°C was neceam~ for the induction of changes in binding at 0 ° C, an experiment was performed in which [3H]flunitrazepam was added after the 60 rnin incubation at 37°C. There w u no difference in'the enhancement of {3H Jflur,~raz epam binding by 37 ° C preincubation as a function of whether [ 3H ] flunitrazepam was present during ~his 37 ° C incubation (data not shown). Incubation of brain ~unpl~ for Varying lengths of time at 37 ° C before the addition of [3H]flunitrazepam and subsequent, incubation at 0°C caused a s'~t, (F = 69.2, P < 0.01) systematic decrease in the Kd of rat brain h o m ~ e n a t ~ for [3H]flunitrazepam (Table I, Expt. 2). Once again, there was no sigp.iflcant change in Bmax between groups (F = 1.15, P :> 0.25). The results of this study indicate that the affinity of rat brain benzodiazepine recepto~ for |~H]flvnitrazepam at 0°C is enhanced by in vitro preincubation at 37 ° C. These results extend the preliminary observations of Braestrup and Squires [2]. The o ~ a t i o n s raise the question of which condition most acc-dra~ly recreants the in vivo affinity of benzodiazepine recept~r~ for the benzodiazepines. The changes in the affinity for benzodiazepines could reflect postmortem tissue degradation in which membrane component~ which
24e
rem~ benz~Uz~~ ~ undergo a ~ ~ ~ e incre..ed eou~ abo ~ ~ " ~ d e S n , d a ~ of an endosma~ bensod . f f i ~ ~ a k e s u ~ e e tt~-~oet~ with ~ . for the benzo~ n ~ e ~ . A third ~ whereb~ ~ e bersodiasel~ ~
m ~
~ 7~m~bu~ ~ :(CXSX) [ S ] , m GABA h~ ~ .ao,~n to the bmdmgaffm~y of both [ ' H ] d ~ m ~ a [ U ] m d [3H]flm~m~
epam [12] to rat brain. Thus, at the ~ t time, it is not possible to d e ~ mine which conait/on, or w h ~ both conditions represent in vivo benzodia~pine receptor binding af~nity.
Several invest/~ton, have rout/rely mayed [~H]d/azepa~ ~
.sing
37 ° C incubations and placement of the samples on ice to bring the incubation" medium to 0° C [1,6,8,9]. The results of this study ~ tlmt the 37 °C incubat/on was m m e c e m ~ because equilibration of ['H]diszepam binding would have occurred if the assays had been run for a ~ period of time at 0 ° C. This technique could have introduced a dgnificmt en~r in the me~urement of [JH]diazepmn binding at 0°C ff ~luilibdum binding had not been atta/ned. Foflamately, [3H]diazepam binding at 0"C equilil~ rates rapidly and the amount of error in these ~ was minimal If these techniques had been used to mesmtre [~H]flunitrszepmn binding however, a sizeable error would have been introduced unless samples were incubated at 0°C for 60 min. The binding of other neurotransmittm~ and drugs to ~ may also show changes in Kc with temperature and require long incubations to reach e~uilibr/um at C'C. Unle~ investigators speciflcafly wish to ~ i n ~ the effect~ of 37°C prei~cubation on binding zt 0"C (e.g. to ~ an e n d ~ o u s subetance) or they:have demonstrated that there is no c h s n ~ tn affinity with ~.mperat~re, the use of a 37"C incubation and a chilling of the samples at 0 ° ~" is u n w a r m n t ~ . ACKNOWLEDGEMENT8
The authors acknowledge the excellent technical smistance of Mr. Andrew ~ . e n and the secretawi~ usistance of Ms. Cathy Kmmm. We also thank Dr. Kevin Beaumont and Dr. W ~ Koeske for thelr critical a p ~ of this manuscript. This research was m P ~ by USPH8 ~ants MH-25267, MH. 30626 and the Hereditsry D ~ Foundat/on and the Huntington's Chov~ Foundation. H.I.Y. is a recipient of a USPHS Resurch 8citmtkt Development Award (MH-00095). R.8.C. is supported by a USPHS Clinicsl Phsmmcology Training Grant (GM.07533), REFERENCES ~ Bosmann, H.G., C,a~, K.R. sad DiStefano, P., D i ~ receptor eharaeteri~tioa: Specific binding of a l~mmdJ~zepine to mscromolecules in varim~ areas of rat brain, FEBS Lett., 82 (1977) 368--372,
247 2 ~ p , C. and Squires, R.F., Specific benzodiazepine receptors in rat brain characterized by high-aff'mity [~H]diazepam binding, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3805--3809. 3 Braestrup,,C. and Squires, R.F., Brain specific benzodiazepine receptors, Brit. J. Psydfiat., 133 (1978) 249--260. 4 Kir~ R.E., ,Experimental Desi~- Procedures for the Behavioral Sciences, Wacisworth, Belmont, CA., 1968, 577 pp. 5 Loveil, R.A. and Elliott, K.A.C., The ~-aminobutyric acid and factor I content of brain, J. Neurochem., 10 (1963) 470--488. 6 M~kerer, C.R., Kochman, R.L., Bierschenk, B.A. and Bremner, S.S., The binding of [~H~pam to rat brain homogenates, J. Pharmaeol. exp. Ther., 206 (1978)
40~--413. 7 Mohler, H. and Okada, T., Benzodia,~epine receptor: demonstration in the central ne~mm system, Science, 198 (1977) 849--851. 8 Mmll~r, W.E., 8ehlafer, U. and Wallert, U., Benzodiazepine receptor binding in rat spinal ~ membrane, Neurosei. Lett., 9 (1978) 239--243. 9 ~iuirm, R.F. and Braestrup, C., Benzodiazepi~te receptors in rat brain, Nature (Lond.), 266 (1977) 732"-734. 10 Spet~ R.C., Wastek, G.J. and Yamamura, H.I., Benzodiazepine recept~ ~: Temperatram ~ n e e of [~H]flunitrazelmm binding, Life Sci., 24 (1979) 351--358. 11 ~ J.F., Thomas, J.W. and Gallagher, D.W., GABAergic modulation of benzodimmpimD site aensitivity, Nature (Lond.), 274 (1978) 383--385. 12 WItek, O,J., 8peth, R.C., Reisine, T.D. and Yamamura, H.I., The effect of ~-aminoIm~ acid on tH-flunitr~epam binding in rat brain, Europ. J. Pharmacol., 50 (1~78} 445--447.