Brain Research, 514 (1990) 151-154 Elsevier
151
BRES 24043
Regionally specific alterations in the low-affinity GABAA receptor following perinatal exposure to diazepam R a n d J. G r u e n 1, J o h n D. Elsworth 2 and R o b e r t H. R o t h 2 1Departments of Psychology and Psychiatry, New York University, New York, NY 10003 (U. S.A.) and 2Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New Haven, CT 06510 (U.S.A.) (Accepted 26 December 1989) Key words: y-Aminobutyric acid; Bicuculline; Benzodiazepine; Diazepam; Cingulate cortex; Hypothalamus; Development
Alterations in a low affinity form of the GABAA receptor were examined with [3H]bicuculline methylchloride in the adult rat following perinatal exposure to diazepam. Perinatal exposure resulted in a significant reduction in [3H]bicuculline binding in the cingulate cortex. A significant decrease in the ability of GABA to displace bound [3H]bicuculline was observed only in the hypothalamus. The results suggest that the effects of perinatal exposure to diazepam are regionally specific and that benzodiazepine receptors and low affinity GABAA receptors a r e functionally linked during the perinatal period. Perinatal exposure to benzodiazepines has been shown to lead to a variety of behavioral and biochemical changes in animals, including disturbances in locomotor and exploratory behavior 11-14, alterations in dopamine (DA) turnover 12, and alterations in the stress-induced activation of norepinephrine and D A neurons 2'27. Recent studies have also indicated that perinatal exposure to benzodiazepines may result in alterations in the development of benzodiazepine (BZ) receptors 1A°'16-18'24. For example, prenatal exposure to the BZ agonist diazepam (DZ) appears to lead to a down regulation of [3H]DZ binding in the hypothalamus and pre- and postcentral cortex of adult cats 17 and the thalamus of adult rats 16. In contrast, prenatal exposure to Ro 15-1788, the BZ antagonist, leads to an increase in the density of BZ hippocampal receptors labeled with [3H]flunitrazepam 18. The clinical effects of BZs have been associated with a presumed activation of GABAergic neurons 3'2°'2s. The majority of BZ receptors are associated with G A B A A receptors defined by their sensitivity to muscimol and bicuculline 4'6'2°'26. BZ and GABAA receptors are thought to be allosterically linked around a central chloride ionophore, forming a GABA/BZ/barbiturate macromolecular complex 3'5'26'28. BZ agonists enhance G A B A transmission and increase G A B A receptor binding under certain conditions 4"5"23 and G A B A and G A B A mimetic compounds have been shown to enhance [3H]BZ binding in extensively washed preparations 26. Lower affinity G A B A A sites, labeled by bicuculline, are thought
to interact with BZ receptors to mediate the physiological response to G A B A 3'2°-22'29. Given the functional relationship between BZ and G A B A A receptors, we examined the effect of perinatal exposure to D Z on the development of a low affinity form of the G A B A A receptor using [3H]bicuculline. Pregnant Sprague-Dawley dams (Charles River Laboratory) were implanted with 3 silastic pellets containing 90 mg of recrystallized D Z (Hoffmann-LaRoche, Inc.) on embryonic day 8 (E8) following a procedure developed by Gallagher et al. 9. This method has been shown to result in the continuous release of D Z from implanted pellets for 3 weeks (average release: 5.0 mg • kg-1 • 24 h-l). Exposure to D Z occurred from E8 to postnatal day 7 (P7). Postnatal exposure occurred via the mother's milk. The release of D Z from implanted pellets declines to minimal levels following the 3 week exposure period 9. Control dams received no treatment at all. Male offspring were sacrificed by decapitation at P90 and their brains rapidly removed and transferred to a chilled dissecting stage. Dissections of the following brain regions were done as previously describedT: hypothalamus (HT), caudate-putamen (CP), and prefrontal cortex (PFC). The cingulate cortex (CIN) was removed from the slice containing the CP by cutting along the border of the corpus callosum. The cerebellum (CB) was also removed. There were 14 and 13 subjects in the control and D Z exposed groups, respectively. Brain tissue from 3-4 subjects was pooled for assay to form a single case in all
Correspondence: R. Roth, Department of Pharmacology, Yale University School of Medicine, Sterling Hall of Medicine, 333 Cedar Street, New Haven, CT 06510 8066, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V, (Biomedical Division)
152 regions examined, except the CB. In the case of the CB, 4 samples were randomly chosen for assay from each treatment condition. P2 membranes were prepared by centrifugation and resuspension in 0.05 M Tris-HC1 buffer, pH 7.0. The suspension was frozen, thawed, centrifuged (30,900 g for 15 min), and resuspended in Tris buffer 3 times. The pellet was finally resuspended in a 1:25 (w/v) dilution of 25 mM potassium phosphate/0.2 M potassium thiocyanate buffer, pH 7.4. 300/~1 phosphate buffer, 100/A of various concentrations of GABA (0, 10-6 and 10-3 M), 500 /~1 of the membrane suspension, and 100 ~1 [3H]bicuculline methylchloride (5 nM; 78.8 Ci/mM, NEN) were added to each microcentrifuge tube for assay. Following a 30 min incubation at 4 °C, the tubes were centrifuged at 30,900 g for 10 min. Pellets were rinsed with 1 ml ice-cold buffer, solubilized in 500 ~1 Protosol, and dispersed in liquid scintillant. At each concentration of GABA used, samples were run in duplicate, or, if adequate amounts of tissue were available, in triplicate. Specific binding was defined as total binding minus non-specific binding (binding in the presence of 10-3 M GABA). Preliminary experiments with tissue from the PFC and CP of untreated rats indicated that the IC5o value for G A B A (the concentration of GABA which displaced 50% of the specifically bound ligand) was approximately 10- 6 M GABA. The percent specific binding displaced by 10- 6 M GABA was calculated as follows: (total binding - binding in the presence of 10 -6 M GABA)/specific binding. These indices of receptor binding were utilized given the large amount of tissue needed for assay (15-20 rag/tube). Perinatal exposure to D Z resulted in a significant reduction in specific binding in the CIN and a marginally significant 54% decrease in specific binding in the CP (F = 4.14; P = 0.06) (Fig. 1). DZ exposure also resulted in a significant decrease in the ability of GABA to displace specifically bound [3H]bicuculline in the H T (control = 53.3 + 16.3%; D Z exposed = 7.4 + 7.4%) (Fig. 2). Alterations in percent specific binding displaced by 10-6 M G A B A were not observed in any of the other regions examined. The results suggest that perinatal exposure to DZ, a GABA-positive ligand for BZ binding sites, alters development of a low affinity component of the GABAA receptor. The alteration in specific binding observed in the CIN may be due to an increase in the Kd, a decrease in the Bmax, o r both. Decreases in the ability of GABA to displace bound [3H]bicuculline in the H T are probably associated with a change in receptor affinity involving a decrease in the K d for bicuculline or an increase in the K d for GABA.
[] 150
CONTROL DZ-EXPOSED
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25
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PFC
Fig. 1. Effects of perinatal exposure to diazepam on specific [3H]bicuculline binding. Data were analyzed using an analysis of variance technique and are expressed as percent control (+ S.E.M.). Numbers in each bar refer to the number of cases in each treatment condition. Values for control subjects, expressed in fmol [3H]bicuculline bound/mg protein, are as follows: cerebellum (CB), 57.9 + 14.4; hypothalamus (HT), 50.2 + 8.9; cingulate cortex (CIN), 289.7 + 36.2; caudate-putamen (CP), 74.8 + 15.1 and prefrontal cortex (PFC), 144.0 + 13.4. *P < 0.01.
The alterations in binding parameters observed in the present study may be associated with alterations in GABAergic transmission induced by perinatal D Z treatment. The findings provide support for the idea that BZ binding sites and some low affinity forms of the GABA A receptor are functionally linked during the perinatal period. The type of alteration induced by perinatal DZ exposure varied in different brain regions; an alteration in specific binding was observed in the CIN, while an alteration in the ability of G A B A to displace bound [3H]bicuculline was observed in the HT. BZ binding sites and G A B A receptors appear to follow a similar developmental course; receptor development in cortical areas occurs relatively late in ontogeny 15. Variations in the type
,,m
125
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151
BRES 24043
Regionally specific alterations in the low-affinity GABAA receptor following perinatal exposure to diazepam R a n d J. G r u e n 1, J o h n D. Elsworth 2 and R o b e r t H. R o t h 2 1Departments of Psychology and Psychiatry, New York University, New York, NY 10003 (U. S.A.) and 2Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New Haven, CT 06510 (U.S.A.) (Accepted 26 December 1989) Key words: y-Aminobutyric acid; Bicuculline; Benzodiazepine; Diazepam; Cingulate cortex; Hypothalamus; Development
Alterations in a low affinity form of the GABAA receptor were examined with [3H]bicuculline methylchloride in the adult rat following perinatal exposure to diazepam. Perinatal exposure resulted in a significant reduction in [3H]bicuculline binding in the cingulate cortex. A significant decrease in the ability of GABA to displace bound [3H]bicuculline was observed only in the hypothalamus. The results suggest that the effects of perinatal exposure to diazepam are regionally specific and that benzodiazepine receptors and low affinity GABAA receptors a r e functionally linked during the perinatal period. Perinatal exposure to benzodiazepines has been shown to lead to a variety of behavioral and biochemical changes in animals, including disturbances in locomotor and exploratory behavior 11-14, alterations in dopamine (DA) turnover 12, and alterations in the stress-induced activation of norepinephrine and D A neurons 2'27. Recent studies have also indicated that perinatal exposure to benzodiazepines may result in alterations in the development of benzodiazepine (BZ) receptors 1A°'16-18'24. For example, prenatal exposure to the BZ agonist diazepam (DZ) appears to lead to a down regulation of [3H]DZ binding in the hypothalamus and pre- and postcentral cortex of adult cats 17 and the thalamus of adult rats 16. In contrast, prenatal exposure to Ro 15-1788, the BZ antagonist, leads to an increase in the density of BZ hippocampal receptors labeled with [3H]flunitrazepam 18. The clinical effects of BZs have been associated with a presumed activation of GABAergic neurons 3'2°'2s. The majority of BZ receptors are associated with G A B A A receptors defined by their sensitivity to muscimol and bicuculline 4'6'2°'26. BZ and GABAA receptors are thought to be allosterically linked around a central chloride ionophore, forming a GABA/BZ/barbiturate macromolecular complex 3'5'26'28. BZ agonists enhance G A B A transmission and increase G A B A receptor binding under certain conditions 4"5"23 and G A B A and G A B A mimetic compounds have been shown to enhance [3H]BZ binding in extensively washed preparations 26. Lower affinity G A B A A sites, labeled by bicuculline, are thought
to interact with BZ receptors to mediate the physiological response to G A B A 3'2°-22'29. Given the functional relationship between BZ and G A B A A receptors, we examined the effect of perinatal exposure to D Z on the development of a low affinity form of the G A B A A receptor using [3H]bicuculline. Pregnant Sprague-Dawley dams (Charles River Laboratory) were implanted with 3 silastic pellets containing 90 mg of recrystallized D Z (Hoffmann-LaRoche, Inc.) on embryonic day 8 (E8) following a procedure developed by Gallagher et al. 9. This method has been shown to result in the continuous release of D Z from implanted pellets for 3 weeks (average release: 5.0 mg • kg-1 • 24 h-l). Exposure to D Z occurred from E8 to postnatal day 7 (P7). Postnatal exposure occurred via the mother's milk. The release of D Z from implanted pellets declines to minimal levels following the 3 week exposure period 9. Control dams received no treatment at all. Male offspring were sacrificed by decapitation at P90 and their brains rapidly removed and transferred to a chilled dissecting stage. Dissections of the following brain regions were done as previously describedT: hypothalamus (HT), caudate-putamen (CP), and prefrontal cortex (PFC). The cingulate cortex (CIN) was removed from the slice containing the CP by cutting along the border of the corpus callosum. The cerebellum (CB) was also removed. There were 14 and 13 subjects in the control and D Z exposed groups, respectively. Brain tissue from 3-4 subjects was pooled for assay to form a single case in all
Correspondence: R. Roth, Department of Pharmacology, Yale University School of Medicine, Sterling Hall of Medicine, 333 Cedar Street, New Haven, CT 06510 8066, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V, (Biomedical Division)
152 regions examined, except the CB. In the case of the CB, 4 samples were randomly chosen for assay from each treatment condition. P2 membranes were prepared by centrifugation and resuspension in 0.05 M Tris-HC1 buffer, pH 7.0. The suspension was frozen, thawed, centrifuged (30,900 g for 15 min), and resuspended in Tris buffer 3 times. The pellet was finally resuspended in a 1:25 (w/v) dilution of 25 mM potassium phosphate/0.2 M potassium thiocyanate buffer, pH 7.4. 300/~1 phosphate buffer, 100/A of various concentrations of GABA (0, 10-6 and 10-3 M), 500 /~1 of the membrane suspension, and 100 ~1 [3H]bicuculline methylchloride (5 nM; 78.8 Ci/mM, NEN) were added to each microcentrifuge tube for assay. Following a 30 min incubation at 4 °C, the tubes were centrifuged at 30,900 g for 10 min. Pellets were rinsed with 1 ml ice-cold buffer, solubilized in 500 ~1 Protosol, and dispersed in liquid scintillant. At each concentration of GABA used, samples were run in duplicate, or, if adequate amounts of tissue were available, in triplicate. Specific binding was defined as total binding minus non-specific binding (binding in the presence of 10-3 M GABA). Preliminary experiments with tissue from the PFC and CP of untreated rats indicated that the IC5o value for G A B A (the concentration of GABA which displaced 50% of the specifically bound ligand) was approximately 10- 6 M GABA. The percent specific binding displaced by 10- 6 M GABA was calculated as follows: (total binding - binding in the presence of 10 -6 M GABA)/specific binding. These indices of receptor binding were utilized given the large amount of tissue needed for assay (15-20 rag/tube). Perinatal exposure to D Z resulted in a significant reduction in specific binding in the CIN and a marginally significant 54% decrease in specific binding in the CP (F = 4.14; P = 0.06) (Fig. 1). DZ exposure also resulted in a significant decrease in the ability of GABA to displace specifically bound [3H]bicuculline in the H T (control = 53.3 + 16.3%; D Z exposed = 7.4 + 7.4%) (Fig. 2). Alterations in percent specific binding displaced by 10-6 M G A B A were not observed in any of the other regions examined. The results suggest that perinatal exposure to DZ, a GABA-positive ligand for BZ binding sites, alters development of a low affinity component of the GABAA receptor. The alteration in specific binding observed in the CIN may be due to an increase in the Kd, a decrease in the Bmax, o r both. Decreases in the ability of GABA to displace bound [3H]bicuculline in the H T are probably associated with a change in receptor affinity involving a decrease in the K d for bicuculline or an increase in the K d for GABA.
[] 150
CONTROL DZ-EXPOSED
-5 125
L
o
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~
T
(9 Z Z
~
75
--u_
5o
u.I
25
a.
0
i
14
5 CB
HT
CIN
CP
PFC
Fig. 1. Effects of perinatal exposure to diazepam on specific [3H]bicuculline binding. Data were analyzed using an analysis of variance technique and are expressed as percent control (+ S.E.M.). Numbers in each bar refer to the number of cases in each treatment condition. Values for control subjects, expressed in fmol [3H]bicuculline bound/mg protein, are as follows: cerebellum (CB), 57.9 + 14.4; hypothalamus (HT), 50.2 + 8.9; cingulate cortex (CIN), 289.7 + 36.2; caudate-putamen (CP), 74.8 + 15.1 and prefrontal cortex (PFC), 144.0 + 13.4. *P < 0.01.
The alterations in binding parameters observed in the present study may be associated with alterations in GABAergic transmission induced by perinatal D Z treatment. The findings provide support for the idea that BZ binding sites and some low affinity forms of the GABA A receptor are functionally linked during the perinatal period. The type of alteration induced by perinatal DZ exposure varied in different brain regions; an alteration in specific binding was observed in the CIN, while an alteration in the ability of G A B A to displace bound [3H]bicuculline was observed in the HT. BZ binding sites and G A B A receptors appear to follow a similar developmental course; receptor development in cortical areas occurs relatively late in ontogeny 15. Variations in the type
,,m
125
~3NTROL )Z-EXPOSED
w 0
