- Email: [email protected]
Changes in high affinity sodium independent gamma-aminobutyric acid binding in cerebral cortex and hippocampus of the rat following electroshock
Life Sciences, Vol. 31, pp. 2499-2505 Printed in the U.S.A.
Pergamon Press
CHANGES IN HIGH AFFINITY SODIUM INDEPENDENT GAMMA-AMINOBUTYRIC ACID BINDING IN CEREBRAL CORTEX AND HIPPOCAMPUS OF THE RAT FOLLOWING ELECTROSHOCK Stephen M. Ross and Charles R. Craig Dept. of Pharmacology and Toxicology, West Virginia University Medical Center~ Morgantown, West Virginia 26506 (Received in final form August 17, 1982) Summary Electroshock induced seizures in the rat enhanced high affinity specific Na + independent binding of 3H-gamma aminobutyric acid (GABA) to frozen, Triton X-100 treated cerebral synaptic membranes 30 minutes after exposures to electroshock, although no change in 3H GABA binding was observed in similarly treated preparations from hippocampus. Scatchard analysis of the binding isotherms from cortical membranes indicated that the increase in 3H GABA binding at 30 minutes was due to a rapid increase (39%) in the number of available GABA receptor binding sites (Bma x) rather than an alteration in receptor affinity (KD). The number of binding sites returned to control values within 1 hour and remained so throughout the duration of this study. It is now accepted that the amino acid, gamma-aminobutyric acid (GABA) plays a major role in mammalian brain function as an inhibitory neurotransmitter (9, 14). It is also likely that GABA as well as its synthetic and degradative enzymes are involved in seizure activity, (For a review, see 15). Recently, the existence of at least two classes of high and low affinity, saturable stereospecific Na + independent GABA binding sites, presumably localized post synaptically, have been demonstrated after treatment of crude cortical synaptic membranes with a freeze-thawing and Triton X-100 procedure (i0, 21). The physiological significance and role of these two binding sites, however, remain unclear. There is evidence which indicates that benzodiazepines may facilitate GABA activity (2, 8, 23). Some reports suggest that benzodiazepine and GABA receptors reside on the same cell membrane (i, 13) and Gavish and Snyder have presented evidence (12) that the benzodiazepine and GABA receptor may be part of the same macromolecular complex, yet other data indicate, in certain species that the regional distribution of GABA and benzodiazepine receptors do not coincide (3, 4, 5, 6). Similarly to GABA, high affinity saturable specific binding sites for benzodiazepines also have been demonstrated in cortical preparations from a variety of species including man (4, 6, 18, 19, 20). Regardless of the relationship between benzodiazepine and GABA receptors, both receptor classes probably serve as the central site of action for their respective endogenous ligands and play a pivotal role in neurotransmission. Recently, a rapid enhancement of high affinity benzodiazepine receptors has been shown to occur in rat cerebral cortical preparations after seizures induced by electroshock or pentylenetetrazol (27). These investigators suggest that the increase in receptor density may be related to the mechanism of action of the benzodiazepines in the treatment of sustained recurrent seizure 0024-3205/82/222499-07503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2500
Brain GABA Receptors After Electroshock
Vol. 31, No. 22, 1982
disorders such as status epilepticus. Likewise, after repeated seizures induced either by electroshock or kindling 3H-diazepam binding sites were reported to increase in hippocampal membranes (18). Other studies have reported benzodiazepine receptor alterations after various drug treatments (Ii, 17, 25) and suggest these drug induced changes in benzodiazepine binding may be relevant to the anticonvulsant activity of this class of compounds. In addition, Robertson (24) has shown alterations in brain benzodiazepine binding in mice susceptible to audiogenic induced seizures, and associated the observed increase in benzodiazepine binding in the susceptible strain to a disorder of the GABA/benzodiazepine receptor complex as it relates to seizure susceptibility. Moreover, differences in high affinity GABA receptor sensitivity in inbred strains of mice have been the focus of investigation (27) as have GABA receptor alterations after acute and chronic ethanol treatments in mice (28). In light of the observations in regard to benzodiazepine and GABA receptor alterations after various treatment protocols and the role of GABA as a CNS inhibitory neurotransmitter and its recognized involvement in seizure activity, it was of interest to examine the properties associated with the high affinity, Na + independent GABA receptor from frozen, Triton X-IO0 treated crude synaptic membranes in rat cerebral cortex and hippocampus after electroshock. Materials and Methods Animals Male Sprague-Dawley rats weighing 150-180 g, obtained from Zivic-Miller, were utilized in these studies. The animals were housed in the WVU Medical Center Animal Quarters for approximately 7 days prior to use. During this period, all rats were exposed to alternating light-dark cycles and constant temperature, and had free access to food and water. Electroshock Seizures Maximal Electroshock seizures were induced with an Electroshock Seizure Apparatus (Model 2-C Hans Technical Assoc.) using corneal electrodes and a current of 150 mA for 0.2 seconds. Control rats (sham) were subjected to the same procedure but no current was applied. Only those rats exhibiting full tonic-clonic seizures were used for further studies. GABA Receptor Binding Tissue Preparation and GABA Binding Assay Rats were decapitated and the brains rapidly removed. Depending on the study, cerebral cortices from 2 rats or hippocampi from 4 rats were pooled, placed in 15 volumes of ice cold 0.32 M sucrose and homogenized with a Teflon-glass homogenizer (12-13 hand strokes). The tissue was next centrifuged at i000 xg (3000 rpm) for i0 minutes in a Sorvall (SS-34 head) and was prepared essentially according to the method of Enna and Snyder (i0). The tissue suspension resulting from this technique (crude synaptic membranes) was used for subsequent assays. The binding assay was initiated when 1 ml aliquots of crude synaptic membranes (0.3-1.2 mg prot.) were incubated together in 0.05 M Tris-Citrate buffer (pH 7.1) with known amounts of 3H GABA and excess unlabeled GABA (to determine non-specific binding) at 4°C for 7 minutes. The final reaction volume in all cases was 2 ml and was agitated immediately with a vortex mixer after addition of tissue. Bound and free 3H GABA were separated by high speed centrifugation according to the method of Enna and Snyder (i0).
Vol. 31, No. 22, 1982
Brain GABA Receptors After Electroshock
2501
Bound radioactivity was extracted in 1 ml of Protosol (New England Nuclear) and allowed to dissolve either overnight or at 37°C for 45 minutes. Afterwards, i0 ml of liquid scintillation cocktail was added and the radioactivity counted. Specific 3H GABA binding was obtained by subtracting from the total bound radioactivity the amount not displaced by high concentrations of unlabeled GABA (2~M). When high affinity GABA receptor binding characteristics (KD and Bma x) were examined, non-specific binding was determined for each substrate concentration used and the concentration of 3H GABA was varied between 1 and 64 nM. This value was subtracted from the total binding obtained at the GABA concentration and was used as specific binding. Preliminary time course experiments indicated that specific 3H GABA binding reached equilibrium by 3 min. and remained constant for more than 14 min. when 1 nM 3H GABA was used. Hence, a 7 min. incubation time was used in all studies. Also, specific 3H GABA binding was linear at least up to 1.5 mg protein at the lowest and highest concentration of 3H GABA. All experiments in this study utilized between 0.3 and 1.2 mg protein. Specific high affinity 3H GABA binding was saturable at 64 ~i 3H GABA. Kinetic assessment of GABA binding characteristics, K D and Bmax. were determined according to linear regression Scatchard Plot Analysis (Scatchard, 1949) and levels of significance were calculated by the Student's Independent Sample unpaired t-test. Results 3H GABA Binding In Rat Cerebral Cortex After Electroshock After electroshock induced seizures in rats, a marked increase in specific high affinity 3H GABA binding to crude cortical synaptic membranes was recorded. The binding increased by 37% and 45% at 15 and 30 minutes, respectively, (Figure i). Specific Na + independent binding of 3H GABA to cortical postsynaptic GABA receptors returned to preseizure values by 1 hour and remained there for at least 3 hours. Since the greatest increase in specific high affinity 3H GABA receptor binding occurred 30 min after electroshock, we performed Scatchard analysis on data generated from both electroshock and sham controls after 30 min to determine whether the marked increase in binding resulted from an alteration in receptor affinity (KD) or an enhanced number of binding sites (Bmax), (Figure 2). Thirty minutes after seizures were elicited by electroshock, the total number of binding sites (Bma x) increased by 39% (1.58 to 2.18 pmol/mg prot.) in crude cortical synaptic membranes while the apparent dissociation constant (KD) for 3H GABA binding did not differ from controls (Table i). Furthermore, no change in Bma x or K D parameters were noted , relative to controls, in crude cortical membranes 1 to 3 hours after electrically induced seizures (Table i).
2502
Brain GABA Receptors After Electroshock
Vol. 31, No. 22, 1982
1.0 r
.9
m
c
.8
0 rn >._I ._I
r
0 u_ l
0 LLI a. u~
.5
I
I
l
5
15
30
l~
' I
Minutes
i 2
J
3
Hours
TIME FIG. i GABA receptor binding in rat cerebral cortex at various times after electroshock. The tissue consisted of frozen, Triton X-IO0 treated cortical crude membrane preparations. Specific high affinity 3H GABA binding was obtained by subtracting from the total GABA binding values, those values derived from non-specific binding studies in samples containing 2~M unlabeled GABA. The concentration of 3H-GABA was 16 ruM; further details are provided in the methods section. Maximal specific 3H GABA binding was observed in cortical tissue 15 to 30 min after electroshock. Each value represents the mean of 6-12 experiments, run in duplicate. The hatched area represents control (sham) values ± S.E. *p < .001.
3H GABA Binding In Rat Hippocampus After Electroshock In contrast to the results obtained from the cerebral cortex, in the hippocampus, only minimal and non-significant increases in 3H GABA high affinity specific binding were present between experimental animals one-half and 1 hour after electroshock treatment and controls (figure not shown). Moreover, as shown in Table 2, Scatchard analysis on data derived from controls and animals subjected to electroshock seizures demonstrated no change in receptor affinity (KD) or maximum number of binding sites (Bmax). These data should be interpreted with some caution since GABA receptor density may have changed but was not detected due to smaller sample size and low amounts of GABA binding (compared with the cortex), and a relatively large experimental error.
Vol.
31, No.
22,
1982
Brain
GABA R e c e p t o r s
A
After
Electroshock
2503
1.8
2.5 m
°~.,2 ).~ "
•
2.0 if)
c~ z8 ~ 0
1.5
._1 o
1.0
/
Y
/
g~
L-
~
/
,,,,
p" ~
2 4
8
16(nmol/ i)
32
13.. I
I
.05
.I
l I~ "%.
.15
I
I~
.20
.2,5
BOUND / FREE FIG.
2
Scatchard plot and saturation curve (inset) of GABA receptor binding in tissue from rat cerebral cortex 30 min after electroshock. The tissue consisted of frozen Triton X-100 treated crude synaptic membranes. Specific 3H G A B A binding was observed in rats subject to electroshock. Scatchard analysis of the saturation curves demonstrated that an increase in Bma x (with no change in affinity) accounted for the enhanced 3H GABA binding. Each value represents the mean of 10-12 separate experiments run in duplicate in which 2 rat cerebral cortices were pooled to generate an n of i. Circles represent controls and squares represent treatment groups. TABLE GABA R E C E P T O R CEREBRAL
BINDING
1
CHARACTERISTICS
C O R T E X AFTER
ELECTROSHOCK Hours
Parameter Analyzed
Control (Sham)
KD
10.8+1.41
(nM)
Bma x (pmol/mg
1.6 ±
.5
.I
IN RAT
After
1
Electroshock
2
3
9.5 ± .9
8.0 ± .6
8.2 ± 1.0
8.3 ± i.i
2.2 +_ .2*
1.8 ± .2
1.6 ±
1.8 +
.i
protein)
M e m b r a n e s were prepared and assayed as described in the text. Cortices from 2 rats were pooled to generate an n of 1 and each value represents the mean of 10-12 separate experiments run in duplicate. i = MEAN ± SEM,
* p < 0.02
.4
2504
Brain GABA Receptors After Electroshock
Vol. 31, No. 22, 1982
TABLE 2 GABA RECEPTOR BINDING CHARACTERISTICS IN HIPPOCAMPUS AFTER ELECTROSHOCK
Hours After Electroshock Parameter Analyzed
Control (Sham)
KD
B
(nM)
ma x
.5
i
2
6.4 ± 1.81
6.5 ± 1.7
7.3 ± 1.8
5.9 ± 9
.44 ± .14
.72 ± .17
.81 ± .13
.67 ± .12
(pmol/mg protein)
Membranes were prepared and assayed as described in the text. Hippocampi from 4 rats were pooled to generate an n of 1 and each value represents the mean of 5-6 experiments run in duplicate. 1 = MEAN ± SEM
Discussion Our results extend the original observations of Paul and Skolnick (22) who demonstrated an increase in the number of benzodiazepine receptor binding sites (Bma x) in cerebral cortical preparations 30 min after electroshock induced seizures. Our principal findings in the present study indicate: an increase in specific high affinity 3H GABA binding in rat cerebral cortical preparations 15 and 30 min after electroshock, reaching a maximal level at 30 min. Binding returned to control values by 60 min. Scatchard analysis of the data, at 30 min, indicated the increase in specific binding was associated with an increase in available high affinity receptor binding sites (Bmax) rather than a change in receptor affinity. Moreover, the increase in high affinity, synaptic GABA receptor density observed in this study parallels enhancement of benzodiazepine receptors reported earlier (22) and is consistent with the coupling of those receptor sites. As stated by Paul and Skolnick (22), several explanations are possible to account for the rapid increase in GABA binding sites without a concomitant change in receptor affinity after seizures induced by electroshock. These include: i) alteration of GABA receptor turnover; 2) a conformational change in the binding sites; or 3) a dissociation of an endogenous ligand from the binding site. Since the functional significance of the high and low affinity Na + independent GABA binding sites is still unclear, the results from the present study in regard to high affinity GABA receptor alterations after electroshock in rat cerebral cortex are difficult to evaluate. It is tempting, however, to
Vol. 31, No. 22, 1982
Brain GABA Receptors After Electroshock
2505
speculate that the increase in the number of binding sites ( B _ ~ ) may inIII dicate an attempt by the cortex to enhance the physiological e ~ e c t of GABA at the level of the GABA/benzodiazepine receptor complex. This proposed mechanism could have important implications for the control of neuronal excitability as it relates to seizure suppression. Acknowledgement This research was supported in part by the West Virginia Medical Corporation and NIH Biomedical Research Grant No. 5 SO7-RR05433-18. Dr. Ross was supported by NIGMS predoctoral training grant 2T32 GM07039-05. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21.
22. 23. 24. 25. 26. 27. 28. 29.
M. BARALDI, A. GUIDOTTI, J.P. SCHWARTZ, and E. COSTA, Science 205: 821823, (1979). G. BIGGIO, B.B. BRODIE, E. COSTA, and A. GUIDOTTI, Proc. Nat. Acad. Sci. (Wash) 74: 3592-3596, (1977). G. BIGGIO, M.G. CORDA, G. DeMONTIS, E. STEFFANINI, and G.L. GESSA, Brain Res. 193: 589-593, (1980). C. BRAESTRUP, R. ALBRECHTSEN, and R.F. SQUIRES, Nature (London) 269: 702-704, (1977). C. BRAESTRUP and M. NIELSEN, Brain Res. 147: 170-173, (1978). C. BRAESTRUP and R.F. SQUIRES, Proc. Nat. Acad. Sci. (Wash) 74: 38053809, (1977). C. BRAESTRUP and R.F. SQUIRES, Brit. J. Psychiat. 133: 249-260, (1978). E. COSTA, A. GUIDOTTI, C.C. MAO, and A. SURIA, Life Sci. 17: 167-186, (1975). D.R. CURTIS and G.A.R. JOHNSTON, Ergeb. Physiol. Biol. Chem. Exp. Pharmakoc. 69: 98-188, (1974). S.S. ENNA and S.H. SNYDER, Molecular Pharmacol. 13: 442-453, (1977). D.W. GALLAGHER, P. MALLORGA, and J.F. TALLMAN, Brain Res. 189: 209-220, (1980). M. GAVlSH and S.H. SNYDER, P r o c . N a t l . Acad. S c i . 78: 1939-1942, (1981). A. GUIDOTTI, G. TOFFANO, and E. COSTA, Nature (London) 275: 553-555, (1978). K. KRNJEVIC, Physiol. Rev. 54: 418-450, (1974). K.G. LLOYD, C. MUNARI, P. WORMS, L. BOSSI, J. BANCAUD, J. TALARIACH, and P.L. MORSELLI, In: GABA-Neurotransmitters ed. by E. Costa, G. Dichiara and C.L. Gessa; pp. 199-216, Raven Press, NY (1981). J.O. McNAMARA, A.M. PEPER and V. PATRONE, Proc. Nat. Acad. Sci. 77: 30293032, (1980). T. MIMAKI, P.P. DESHMIfKH and H.I. YAMAMURA, J. Neurochem. 35: 14731475, (1980). H. MOHLER and T. OKADA, Life Sci. 22: 985-996, (1978). H. MOHLER and T. OKADA, Science 198~: 849-851, (1977). H. MOHLER and T. OKADA, Brain Res. 156: 391-395, (1978). R.W. OLSEN, M.K. TICKU, P. GREENLEE, and P. VAN NESS, in GABA Neurotransmitters, (Ed. P. Krogsgaard-Larsen, J. Scheel-Kruger, and H. Kofod), pp. 165-178, Munksgaard, Copenhagen, (1979). S.M. PAUL and P. SKOLNICK, Science 202: 892-893, (1978). P. POLC, H. MOHLER and W. HAEFELY, Naunyn-Schmiedeberg's Arch. Pharmacol. 284: 319-337, (1974). H.A. ROBERTSON, Eur. J. Pharmacol. 66: 249-252, (1980). H.C. ROSENBERG and T.H. CHIU, Life Sci. 24: 803-808, (1979). G. SCATCHARD, Ann. N.Y. Acad. Sci. 51: 6--60-672, (1949). M.K. TICKU, J. Neurochem. 33: 1135-1138, (1979). M.K. TICKU and T. BURCH, J. Neurochem. 34: 417-423, (1980). G. TOFFANO, A. GUIDOTTI, A. SURIA and E. COSTA, Fed. Proc. 37: 17251729, (1978).
Pergamon Press
CHANGES IN HIGH AFFINITY SODIUM INDEPENDENT GAMMA-AMINOBUTYRIC ACID BINDING IN CEREBRAL CORTEX AND HIPPOCAMPUS OF THE RAT FOLLOWING ELECTROSHOCK Stephen M. Ross and Charles R. Craig Dept. of Pharmacology and Toxicology, West Virginia University Medical Center~ Morgantown, West Virginia 26506 (Received in final form August 17, 1982) Summary Electroshock induced seizures in the rat enhanced high affinity specific Na + independent binding of 3H-gamma aminobutyric acid (GABA) to frozen, Triton X-100 treated cerebral synaptic membranes 30 minutes after exposures to electroshock, although no change in 3H GABA binding was observed in similarly treated preparations from hippocampus. Scatchard analysis of the binding isotherms from cortical membranes indicated that the increase in 3H GABA binding at 30 minutes was due to a rapid increase (39%) in the number of available GABA receptor binding sites (Bma x) rather than an alteration in receptor affinity (KD). The number of binding sites returned to control values within 1 hour and remained so throughout the duration of this study. It is now accepted that the amino acid, gamma-aminobutyric acid (GABA) plays a major role in mammalian brain function as an inhibitory neurotransmitter (9, 14). It is also likely that GABA as well as its synthetic and degradative enzymes are involved in seizure activity, (For a review, see 15). Recently, the existence of at least two classes of high and low affinity, saturable stereospecific Na + independent GABA binding sites, presumably localized post synaptically, have been demonstrated after treatment of crude cortical synaptic membranes with a freeze-thawing and Triton X-100 procedure (i0, 21). The physiological significance and role of these two binding sites, however, remain unclear. There is evidence which indicates that benzodiazepines may facilitate GABA activity (2, 8, 23). Some reports suggest that benzodiazepine and GABA receptors reside on the same cell membrane (i, 13) and Gavish and Snyder have presented evidence (12) that the benzodiazepine and GABA receptor may be part of the same macromolecular complex, yet other data indicate, in certain species that the regional distribution of GABA and benzodiazepine receptors do not coincide (3, 4, 5, 6). Similarly to GABA, high affinity saturable specific binding sites for benzodiazepines also have been demonstrated in cortical preparations from a variety of species including man (4, 6, 18, 19, 20). Regardless of the relationship between benzodiazepine and GABA receptors, both receptor classes probably serve as the central site of action for their respective endogenous ligands and play a pivotal role in neurotransmission. Recently, a rapid enhancement of high affinity benzodiazepine receptors has been shown to occur in rat cerebral cortical preparations after seizures induced by electroshock or pentylenetetrazol (27). These investigators suggest that the increase in receptor density may be related to the mechanism of action of the benzodiazepines in the treatment of sustained recurrent seizure 0024-3205/82/222499-07503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2500
Brain GABA Receptors After Electroshock
Vol. 31, No. 22, 1982
disorders such as status epilepticus. Likewise, after repeated seizures induced either by electroshock or kindling 3H-diazepam binding sites were reported to increase in hippocampal membranes (18). Other studies have reported benzodiazepine receptor alterations after various drug treatments (Ii, 17, 25) and suggest these drug induced changes in benzodiazepine binding may be relevant to the anticonvulsant activity of this class of compounds. In addition, Robertson (24) has shown alterations in brain benzodiazepine binding in mice susceptible to audiogenic induced seizures, and associated the observed increase in benzodiazepine binding in the susceptible strain to a disorder of the GABA/benzodiazepine receptor complex as it relates to seizure susceptibility. Moreover, differences in high affinity GABA receptor sensitivity in inbred strains of mice have been the focus of investigation (27) as have GABA receptor alterations after acute and chronic ethanol treatments in mice (28). In light of the observations in regard to benzodiazepine and GABA receptor alterations after various treatment protocols and the role of GABA as a CNS inhibitory neurotransmitter and its recognized involvement in seizure activity, it was of interest to examine the properties associated with the high affinity, Na + independent GABA receptor from frozen, Triton X-IO0 treated crude synaptic membranes in rat cerebral cortex and hippocampus after electroshock. Materials and Methods Animals Male Sprague-Dawley rats weighing 150-180 g, obtained from Zivic-Miller, were utilized in these studies. The animals were housed in the WVU Medical Center Animal Quarters for approximately 7 days prior to use. During this period, all rats were exposed to alternating light-dark cycles and constant temperature, and had free access to food and water. Electroshock Seizures Maximal Electroshock seizures were induced with an Electroshock Seizure Apparatus (Model 2-C Hans Technical Assoc.) using corneal electrodes and a current of 150 mA for 0.2 seconds. Control rats (sham) were subjected to the same procedure but no current was applied. Only those rats exhibiting full tonic-clonic seizures were used for further studies. GABA Receptor Binding Tissue Preparation and GABA Binding Assay Rats were decapitated and the brains rapidly removed. Depending on the study, cerebral cortices from 2 rats or hippocampi from 4 rats were pooled, placed in 15 volumes of ice cold 0.32 M sucrose and homogenized with a Teflon-glass homogenizer (12-13 hand strokes). The tissue was next centrifuged at i000 xg (3000 rpm) for i0 minutes in a Sorvall (SS-34 head) and was prepared essentially according to the method of Enna and Snyder (i0). The tissue suspension resulting from this technique (crude synaptic membranes) was used for subsequent assays. The binding assay was initiated when 1 ml aliquots of crude synaptic membranes (0.3-1.2 mg prot.) were incubated together in 0.05 M Tris-Citrate buffer (pH 7.1) with known amounts of 3H GABA and excess unlabeled GABA (to determine non-specific binding) at 4°C for 7 minutes. The final reaction volume in all cases was 2 ml and was agitated immediately with a vortex mixer after addition of tissue. Bound and free 3H GABA were separated by high speed centrifugation according to the method of Enna and Snyder (i0).
Vol. 31, No. 22, 1982
Brain GABA Receptors After Electroshock
2501
Bound radioactivity was extracted in 1 ml of Protosol (New England Nuclear) and allowed to dissolve either overnight or at 37°C for 45 minutes. Afterwards, i0 ml of liquid scintillation cocktail was added and the radioactivity counted. Specific 3H GABA binding was obtained by subtracting from the total bound radioactivity the amount not displaced by high concentrations of unlabeled GABA (2~M). When high affinity GABA receptor binding characteristics (KD and Bma x) were examined, non-specific binding was determined for each substrate concentration used and the concentration of 3H GABA was varied between 1 and 64 nM. This value was subtracted from the total binding obtained at the GABA concentration and was used as specific binding. Preliminary time course experiments indicated that specific 3H GABA binding reached equilibrium by 3 min. and remained constant for more than 14 min. when 1 nM 3H GABA was used. Hence, a 7 min. incubation time was used in all studies. Also, specific 3H GABA binding was linear at least up to 1.5 mg protein at the lowest and highest concentration of 3H GABA. All experiments in this study utilized between 0.3 and 1.2 mg protein. Specific high affinity 3H GABA binding was saturable at 64 ~i 3H GABA. Kinetic assessment of GABA binding characteristics, K D and Bmax. were determined according to linear regression Scatchard Plot Analysis (Scatchard, 1949) and levels of significance were calculated by the Student's Independent Sample unpaired t-test. Results 3H GABA Binding In Rat Cerebral Cortex After Electroshock After electroshock induced seizures in rats, a marked increase in specific high affinity 3H GABA binding to crude cortical synaptic membranes was recorded. The binding increased by 37% and 45% at 15 and 30 minutes, respectively, (Figure i). Specific Na + independent binding of 3H GABA to cortical postsynaptic GABA receptors returned to preseizure values by 1 hour and remained there for at least 3 hours. Since the greatest increase in specific high affinity 3H GABA receptor binding occurred 30 min after electroshock, we performed Scatchard analysis on data generated from both electroshock and sham controls after 30 min to determine whether the marked increase in binding resulted from an alteration in receptor affinity (KD) or an enhanced number of binding sites (Bmax), (Figure 2). Thirty minutes after seizures were elicited by electroshock, the total number of binding sites (Bma x) increased by 39% (1.58 to 2.18 pmol/mg prot.) in crude cortical synaptic membranes while the apparent dissociation constant (KD) for 3H GABA binding did not differ from controls (Table i). Furthermore, no change in Bma x or K D parameters were noted , relative to controls, in crude cortical membranes 1 to 3 hours after electrically induced seizures (Table i).
2502
Brain GABA Receptors After Electroshock
Vol. 31, No. 22, 1982
1.0 r
.9
m
c
.8
0 rn >._I ._I
r
0 u_ l
0 LLI a. u~
.5
I
I
l
5
15
30
l~
' I
Minutes
i 2
J
3
Hours
TIME FIG. i GABA receptor binding in rat cerebral cortex at various times after electroshock. The tissue consisted of frozen, Triton X-IO0 treated cortical crude membrane preparations. Specific high affinity 3H GABA binding was obtained by subtracting from the total GABA binding values, those values derived from non-specific binding studies in samples containing 2~M unlabeled GABA. The concentration of 3H-GABA was 16 ruM; further details are provided in the methods section. Maximal specific 3H GABA binding was observed in cortical tissue 15 to 30 min after electroshock. Each value represents the mean of 6-12 experiments, run in duplicate. The hatched area represents control (sham) values ± S.E. *p < .001.
3H GABA Binding In Rat Hippocampus After Electroshock In contrast to the results obtained from the cerebral cortex, in the hippocampus, only minimal and non-significant increases in 3H GABA high affinity specific binding were present between experimental animals one-half and 1 hour after electroshock treatment and controls (figure not shown). Moreover, as shown in Table 2, Scatchard analysis on data derived from controls and animals subjected to electroshock seizures demonstrated no change in receptor affinity (KD) or maximum number of binding sites (Bmax). These data should be interpreted with some caution since GABA receptor density may have changed but was not detected due to smaller sample size and low amounts of GABA binding (compared with the cortex), and a relatively large experimental error.
Vol.
31, No.
22,
1982
Brain
GABA R e c e p t o r s
A
After
Electroshock
2503
1.8
2.5 m
°~.,2 ).~ "
•
2.0 if)
c~ z8 ~ 0
1.5
._1 o
1.0
/
Y
/
g~
L-
~
/
,,,,
p" ~
2 4
8
16(nmol/ i)
32
13.. I
I
.05
.I
l I~ "%.
.15
I
I~
.20
.2,5
BOUND / FREE FIG.
2
Scatchard plot and saturation curve (inset) of GABA receptor binding in tissue from rat cerebral cortex 30 min after electroshock. The tissue consisted of frozen Triton X-100 treated crude synaptic membranes. Specific 3H G A B A binding was observed in rats subject to electroshock. Scatchard analysis of the saturation curves demonstrated that an increase in Bma x (with no change in affinity) accounted for the enhanced 3H GABA binding. Each value represents the mean of 10-12 separate experiments run in duplicate in which 2 rat cerebral cortices were pooled to generate an n of i. Circles represent controls and squares represent treatment groups. TABLE GABA R E C E P T O R CEREBRAL
BINDING
1
CHARACTERISTICS
C O R T E X AFTER
ELECTROSHOCK Hours
Parameter Analyzed
Control (Sham)
KD
10.8+1.41
(nM)
Bma x (pmol/mg
1.6 ±
.5
.I
IN RAT
After
1
Electroshock
2
3
9.5 ± .9
8.0 ± .6
8.2 ± 1.0
8.3 ± i.i
2.2 +_ .2*
1.8 ± .2
1.6 ±
1.8 +
.i
protein)
M e m b r a n e s were prepared and assayed as described in the text. Cortices from 2 rats were pooled to generate an n of 1 and each value represents the mean of 10-12 separate experiments run in duplicate. i = MEAN ± SEM,
* p < 0.02
.4
2504
Brain GABA Receptors After Electroshock
Vol. 31, No. 22, 1982
TABLE 2 GABA RECEPTOR BINDING CHARACTERISTICS IN HIPPOCAMPUS AFTER ELECTROSHOCK
Hours After Electroshock Parameter Analyzed
Control (Sham)
KD
B
(nM)
ma x
.5
i
2
6.4 ± 1.81
6.5 ± 1.7
7.3 ± 1.8
5.9 ± 9
.44 ± .14
.72 ± .17
.81 ± .13
.67 ± .12
(pmol/mg protein)
Membranes were prepared and assayed as described in the text. Hippocampi from 4 rats were pooled to generate an n of 1 and each value represents the mean of 5-6 experiments run in duplicate. 1 = MEAN ± SEM
Discussion Our results extend the original observations of Paul and Skolnick (22) who demonstrated an increase in the number of benzodiazepine receptor binding sites (Bma x) in cerebral cortical preparations 30 min after electroshock induced seizures. Our principal findings in the present study indicate: an increase in specific high affinity 3H GABA binding in rat cerebral cortical preparations 15 and 30 min after electroshock, reaching a maximal level at 30 min. Binding returned to control values by 60 min. Scatchard analysis of the data, at 30 min, indicated the increase in specific binding was associated with an increase in available high affinity receptor binding sites (Bmax) rather than a change in receptor affinity. Moreover, the increase in high affinity, synaptic GABA receptor density observed in this study parallels enhancement of benzodiazepine receptors reported earlier (22) and is consistent with the coupling of those receptor sites. As stated by Paul and Skolnick (22), several explanations are possible to account for the rapid increase in GABA binding sites without a concomitant change in receptor affinity after seizures induced by electroshock. These include: i) alteration of GABA receptor turnover; 2) a conformational change in the binding sites; or 3) a dissociation of an endogenous ligand from the binding site. Since the functional significance of the high and low affinity Na + independent GABA binding sites is still unclear, the results from the present study in regard to high affinity GABA receptor alterations after electroshock in rat cerebral cortex are difficult to evaluate. It is tempting, however, to
Vol. 31, No. 22, 1982
Brain GABA Receptors After Electroshock
2505
speculate that the increase in the number of binding sites ( B _ ~ ) may inIII dicate an attempt by the cortex to enhance the physiological e ~ e c t of GABA at the level of the GABA/benzodiazepine receptor complex. This proposed mechanism could have important implications for the control of neuronal excitability as it relates to seizure suppression. Acknowledgement This research was supported in part by the West Virginia Medical Corporation and NIH Biomedical Research Grant No. 5 SO7-RR05433-18. Dr. Ross was supported by NIGMS predoctoral training grant 2T32 GM07039-05. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21.
22. 23. 24. 25. 26. 27. 28. 29.
M. BARALDI, A. GUIDOTTI, J.P. SCHWARTZ, and E. COSTA, Science 205: 821823, (1979). G. BIGGIO, B.B. BRODIE, E. COSTA, and A. GUIDOTTI, Proc. Nat. Acad. Sci. (Wash) 74: 3592-3596, (1977). G. BIGGIO, M.G. CORDA, G. DeMONTIS, E. STEFFANINI, and G.L. GESSA, Brain Res. 193: 589-593, (1980). C. BRAESTRUP, R. ALBRECHTSEN, and R.F. SQUIRES, Nature (London) 269: 702-704, (1977). C. BRAESTRUP and M. NIELSEN, Brain Res. 147: 170-173, (1978). C. BRAESTRUP and R.F. SQUIRES, Proc. Nat. Acad. Sci. (Wash) 74: 38053809, (1977). C. BRAESTRUP and R.F. SQUIRES, Brit. J. Psychiat. 133: 249-260, (1978). E. COSTA, A. GUIDOTTI, C.C. MAO, and A. SURIA, Life Sci. 17: 167-186, (1975). D.R. CURTIS and G.A.R. JOHNSTON, Ergeb. Physiol. Biol. Chem. Exp. Pharmakoc. 69: 98-188, (1974). S.S. ENNA and S.H. SNYDER, Molecular Pharmacol. 13: 442-453, (1977). D.W. GALLAGHER, P. MALLORGA, and J.F. TALLMAN, Brain Res. 189: 209-220, (1980). M. GAVlSH and S.H. SNYDER, P r o c . N a t l . Acad. S c i . 78: 1939-1942, (1981). A. GUIDOTTI, G. TOFFANO, and E. COSTA, Nature (London) 275: 553-555, (1978). K. KRNJEVIC, Physiol. Rev. 54: 418-450, (1974). K.G. LLOYD, C. MUNARI, P. WORMS, L. BOSSI, J. BANCAUD, J. TALARIACH, and P.L. MORSELLI, In: GABA-Neurotransmitters ed. by E. Costa, G. Dichiara and C.L. Gessa; pp. 199-216, Raven Press, NY (1981). J.O. McNAMARA, A.M. PEPER and V. PATRONE, Proc. Nat. Acad. Sci. 77: 30293032, (1980). T. MIMAKI, P.P. DESHMIfKH and H.I. YAMAMURA, J. Neurochem. 35: 14731475, (1980). H. MOHLER and T. OKADA, Life Sci. 22: 985-996, (1978). H. MOHLER and T. OKADA, Science 198~: 849-851, (1977). H. MOHLER and T. OKADA, Brain Res. 156: 391-395, (1978). R.W. OLSEN, M.K. TICKU, P. GREENLEE, and P. VAN NESS, in GABA Neurotransmitters, (Ed. P. Krogsgaard-Larsen, J. Scheel-Kruger, and H. Kofod), pp. 165-178, Munksgaard, Copenhagen, (1979). S.M. PAUL and P. SKOLNICK, Science 202: 892-893, (1978). P. POLC, H. MOHLER and W. HAEFELY, Naunyn-Schmiedeberg's Arch. Pharmacol. 284: 319-337, (1974). H.A. ROBERTSON, Eur. J. Pharmacol. 66: 249-252, (1980). H.C. ROSENBERG and T.H. CHIU, Life Sci. 24: 803-808, (1979). G. SCATCHARD, Ann. N.Y. Acad. Sci. 51: 6--60-672, (1949). M.K. TICKU, J. Neurochem. 33: 1135-1138, (1979). M.K. TICKU and T. BURCH, J. Neurochem. 34: 417-423, (1980). G. TOFFANO, A. GUIDOTTI, A. SURIA and E. COSTA, Fed. Proc. 37: 17251729, (1978).