Brain Research, 589 (1992) 97-101
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$1)5.1)1)
97
BRES 18018
Regulation of flunitrazepam binding in the dorsal horn of the spinal cord by adrenalectomy and corticosteroids S u s a n a L. G o n z f i l e z , M o n i c a F e r r i n i , H e c t o r C o i r i n i , M . C l a u d i a G o n z ~ i l e z D e n i s e l l e and A l e j a n d r o F. D e Nicola Laboratorio de Bioqubnica Neuroendocrina, hlstituto de Biologfa y M,,dicina Experimental, Buenos Aires (Argemina)
(Accepted 31 March 1992)
Key words: Benzodiazepine receptor; Spinal cord; Glucocorticoid; Mineralocorticoid; Adrenalectomy
Adrenal corticosteroids and adrenalectomy (ADX) have opposing effects on benzodiazepine binding sites in brain regions. These treatments were employed to study [3H]flunitrazepam (FLU) binding in regions punched out from the rat spinal cord. We found that binding was higher in dorsal horn than in ventral horn, and minimal in white matter. CIonazepam and RO 15-1788 largely displaced [3H]FLU binding, whereas RO 5-4864 was weakly active. Four days post-ADX, binding increased exclusively in the dorsal horn, and this effect was reversed by administration of corticosterone (CORT), but not dexamethasone (DEX) or aldosterone (ALDO) given over 4 days. When endogenous CORT was increased by administration of cold stress to adrenal-intact rats, reduced benzodiazepine (BDZ) binding was also observed in Ihe dorsal horn. When added in vitro, only ALDO and not CORT or DEX, inhibited [3H]FLU binding. It is suggested that steroids with affinity for the type i corticosteroid receptor (CORT, ALDO) decrease ['~H]FLUbinding to a neural-type BDZ receptor in the dorsal horn. Reduction of the inhibitory BDZ system may be physiologicallyimportant, and can partly explain the enhancement of excitatory synaptic transmission produced by corticosteroids at the level of the spinal cord.
INTRODUCTION There is compelling evidence that neurotransmission and membrane excitability are regulated by steroid hormones acting on the central nervous system a°'17'28. These effects have been considered partly genomic and partly direct membrane actions involving a putative neuronal membrane receptor ~Lt7'z3. For effects mediated by intracellular steroid receptors, two receptor types have been described for adrenal steroids, the high affinity type I site and the lower affinity type !I site, which show differential steroid specificity and a distinct regional distribution '~'ms'tT'z7. It has been shown that regulation of neurotransmitter function can occur at the level of the receptor, as exemplified by the changes reported for the inhibitory y-aminobutyric acid (GABAa)-benzodiazepine (BDZ) receptor following adrenalectomy (ADX), and by replacement with active hormones. This regulation occurred in the hippocampus, cerebral cortex, hypothala-
mus, striatum and cerebellum ~'t''7'ta't'~'32'a~. in the case of the spinal cord, one of the most prominent effects of BDZ is the enhancement of GABA-mediated presynaptic inhibition 2~ and inhibition of dorsal horn interneurons ~2. To our knowledge, regulation of BDZ binding sites in the spinal cord by adrenal gland hormones have not yet been reported, although data exist regarding glucocorticoid antagonism of GABA-mediated inhibition of transmission in spinal cord and ganglia 2'1°. Therefore, our objective was to study the effects of glucocorticoids and mineralocorticoids on BDZ binding sites in regions of the spinal cord, after hormonal treatment in vivo and during hormone application in vitro. The possibility of corticoid regulation of this parameter is supported by previous studies on the detection of type I and type II corticoid receptors in the spinal cord ~'zl, enzyme induction 24 and neurophysioiogical evidence that glucocorticoids enhance excitatory synaptic transmission at the spinal cord level ~c~
Correspondence: A.F. De Nicola, Instituto de Biologia y Medicina Experimental, Obligado 2490, (1428) Buenos Aires, Argentina. Fax: (54)
1-786-2564.
98 MATERIALS AND METHODS
Experimental anbnals Male Sprague-Dawley rats (200-250 g) were used. A group of animals was adrenalectomized under ether anesthesia and used 4 days after surgery. A second group of ADX rats received over 4 days one of the following compounds (Sigma Chemical, St.Louis, MO): a: corticosterone (CORT), 3 mg/day s.c. dissolved in vegetable oil; b: dexamethasone (DEX), 1 me% dissolved in drinking saline or c: aldosterone (ALDO), released at a rate of 1 /zg//~l/h from implanted ALZET minipumps (model 2001, ALZA Corp., CA) containing ! mg hormone/ml propylene glycol. Specific biological effects were reported previously for these treatments ~7"34. For studies on the in vitro effects of steroids, 10 p.M CORT, DEX, ALDO or 3a,21-dihydroxy-5a-pregnan.20.one (tetrahydrodeoxy corticosterone, THDOC) were added to incubation medium in 2
~.[ ethanol: controls received the vehicle only. The effects of stress were studied in adrenal-intact rats kept in the cold room (4°C) for 2 days. To ascertain the effectiveness of the stressor, serum CORT was measured by a competitive protein binding assay~-', with serum samples obtained at the time of sacrifice in control and cold stressed rats. For extraction of tissues, ether anesthetized animals were perfused intracardially with 60 ml cold 0.9% NaCI and the spinal cords were removed following dorsal laminectomy. Cervical regions (C~C7) were embedded in Tissue-Tek (OCT Compound, Miles Labs., Buenos Aires) and cut every 300 /~m in a cryostat precooled at -10°C. Punches were obtained from the ventral horn, dorsal horn and lateral funiculus, following the technique described in the microdissection atlas of Palkovits and Brownstein 2s.
l ~Hl Fhmimlzepam hireling Punches obtained from a pool of 7 spinal cords per experimental group were homogenized in 300/.tl of 50 mM Tris-HCI (pH 7.4) and centrifuged at I(I,(X)0x g for ! rain, The membrane pellets were washed twice in the same media, suspended in 300 /zl buffer and frozen-thawed to remove as much endogenous GABA as possibie'. One hundred /zl of tissue suspension, containing 0,25-0.35 mg protein, was incubated with a saturating amount (10 nM) of [~H]Flunitrazepam (FLU, 74.8 Ci/mmol, NEN-Dupont, Boston, MA). This concentration is at least 5 times higher than the K d (I.6-2.0 nM) determined by saturation analysis. Incubations were carried out during I h at 4°C, with or without addition of 500-fold excess of Diazepam (Roemmers Lab., Buenos Aires) to determine non-specific binding. Receptor characterization was performed by addition to incubation media of 3/zM RO 15-1788, RO 5-4864 (a kind gift of Dr, J. Apud, Fidia Res. Foundation, Washington, D.C.) or clonazepam (a gift from Dr. M. Ritta of this Institute). Incubations were stopped by addition of I ml cold 50 mM Tris-HCI, and rapidly filtered under vacuum through G F / B filters (2.4 cm, Whatman, Maidstone, UK) contained in a filter manifold holder (Millipore Corp., Bedford, MA), Each tube was subsequently washed 5 times with buffer, and washes were passed through the filters. The filters were dried under an air jet, placed into glass vials and 10 ml of a Triton X-100 toluene-based liquid scintillation solution was added. Results were expressed as fmol specifically bound ['~H]FLU per mg protein. Proteins were determined according to Lowry et al. I'~.
~D Z
~
• R0 IS- 1788
~-
1
• R0 s- 406~.
Z
• CLONAZEPAM
N~
a
1004
e
I-El
50
~:¢i'e
~unsideredsignificantif P < 0.05.
RESULTS
In order to characterize the receptor type expressed in our spinal cord preparation, we studied the effect of
i
0 Fig. I. Characterization of benzodiazepine (BDZ) binding sites in the dorsal horn and the ventral horn. Incubations were carried out in 50 mM Tris-HCI buffer pH 7.4 alone (control, open columns), with the antagonists of central-type BDZ receptor RO 15-1788 and clonazepam, or the antagonist of the peripheral-type BDZ receptor RO 5-4864. Results are expressed as % of control [3H]flunitrazepam (FLU) binding. Statistical analysis demonstrated that the following differences were significant at P < 0.01: a vs. b; a vs. d; e vs. f and e vs. h.
specific antagonists and GABA on [3H]FLU binding. As shown in Fig. 1, binding of radioligand in the dorsal and ventral horn was significantly inhibited (P < 0.05 or less) by drugs acting at central-type BDZ receptors such as clonazepam and RO 15-1788, in contrast to tho antagonist of the peripheral BDZ receptor RO 5-4864 which was barely active 4.2~,2'~,32. Furthermore, addition of 50-150 /zM GABA to the incubation medium slightly increased ['~H]FLU binding (controls: 209 ± 27.5 fmol/mg prot, n = 3; GABA: 233 :t: 27.8, P:NS). Fig. 2 shows the effects of ADX and treatment with steroids on [3H]FLU binding. In intact rats, binding
e
a
300-
~
o
I E~3INTACT 1 1ADX v~ICORT ~DEX nmIALDO
//11
= 200" ~oo-
an " L ~ l....... li~l~nces 0.95 and 0t ....
g
h
Statistical analysis Results are presented as the mean ± S.E.M. For evaluation of the effect of different treatments on ['~H]FLU binding, we used ANOVA. Those comparisons yielding a significant F value were further analyzed using the Newman-Keuls post-hoe test, set for l-or values of
~
DORSAL HORN
VENTRAL HORN
LATERAL FUNICULUS
Fig. 2. Effect of in vivo steroid administration on BDZ binding sites in the dorsal horn, ventral horn and lateral funiculus. Five groups of animals were studied (n -- 25 animals per group): adrenal intact rats (open columns); adrenalectomized rats (black columns); rats treated over 4 days with corticosterone (CORT, 3 mg/day s.c., diagonal lines), dexamethasone (DEX, 1 rag% of drinking saline, dotted columns) or aldosterone (ALDO, 24 ~ g / d a y released from miniosmotic pumps, vertical lines), Statistical analysis demonstrated that a vs. b and b vs. c were significantly different at P < 0.01.
99
o. L~
I [~ CONTROL ISTRESS
o
E Z
m--Z 200-
,
:E
1
LIJ N
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$1)5.1)1)
97
BRES 18018
Regulation of flunitrazepam binding in the dorsal horn of the spinal cord by adrenalectomy and corticosteroids S u s a n a L. G o n z f i l e z , M o n i c a F e r r i n i , H e c t o r C o i r i n i , M . C l a u d i a G o n z ~ i l e z D e n i s e l l e and A l e j a n d r o F. D e Nicola Laboratorio de Bioqubnica Neuroendocrina, hlstituto de Biologfa y M,,dicina Experimental, Buenos Aires (Argemina)
(Accepted 31 March 1992)
Key words: Benzodiazepine receptor; Spinal cord; Glucocorticoid; Mineralocorticoid; Adrenalectomy
Adrenal corticosteroids and adrenalectomy (ADX) have opposing effects on benzodiazepine binding sites in brain regions. These treatments were employed to study [3H]flunitrazepam (FLU) binding in regions punched out from the rat spinal cord. We found that binding was higher in dorsal horn than in ventral horn, and minimal in white matter. CIonazepam and RO 15-1788 largely displaced [3H]FLU binding, whereas RO 5-4864 was weakly active. Four days post-ADX, binding increased exclusively in the dorsal horn, and this effect was reversed by administration of corticosterone (CORT), but not dexamethasone (DEX) or aldosterone (ALDO) given over 4 days. When endogenous CORT was increased by administration of cold stress to adrenal-intact rats, reduced benzodiazepine (BDZ) binding was also observed in Ihe dorsal horn. When added in vitro, only ALDO and not CORT or DEX, inhibited [3H]FLU binding. It is suggested that steroids with affinity for the type i corticosteroid receptor (CORT, ALDO) decrease ['~H]FLUbinding to a neural-type BDZ receptor in the dorsal horn. Reduction of the inhibitory BDZ system may be physiologicallyimportant, and can partly explain the enhancement of excitatory synaptic transmission produced by corticosteroids at the level of the spinal cord.
INTRODUCTION There is compelling evidence that neurotransmission and membrane excitability are regulated by steroid hormones acting on the central nervous system a°'17'28. These effects have been considered partly genomic and partly direct membrane actions involving a putative neuronal membrane receptor ~Lt7'z3. For effects mediated by intracellular steroid receptors, two receptor types have been described for adrenal steroids, the high affinity type I site and the lower affinity type !I site, which show differential steroid specificity and a distinct regional distribution '~'ms'tT'z7. It has been shown that regulation of neurotransmitter function can occur at the level of the receptor, as exemplified by the changes reported for the inhibitory y-aminobutyric acid (GABAa)-benzodiazepine (BDZ) receptor following adrenalectomy (ADX), and by replacement with active hormones. This regulation occurred in the hippocampus, cerebral cortex, hypothala-
mus, striatum and cerebellum ~'t''7'ta't'~'32'a~. in the case of the spinal cord, one of the most prominent effects of BDZ is the enhancement of GABA-mediated presynaptic inhibition 2~ and inhibition of dorsal horn interneurons ~2. To our knowledge, regulation of BDZ binding sites in the spinal cord by adrenal gland hormones have not yet been reported, although data exist regarding glucocorticoid antagonism of GABA-mediated inhibition of transmission in spinal cord and ganglia 2'1°. Therefore, our objective was to study the effects of glucocorticoids and mineralocorticoids on BDZ binding sites in regions of the spinal cord, after hormonal treatment in vivo and during hormone application in vitro. The possibility of corticoid regulation of this parameter is supported by previous studies on the detection of type I and type II corticoid receptors in the spinal cord ~'zl, enzyme induction 24 and neurophysioiogical evidence that glucocorticoids enhance excitatory synaptic transmission at the spinal cord level ~c~
Correspondence: A.F. De Nicola, Instituto de Biologia y Medicina Experimental, Obligado 2490, (1428) Buenos Aires, Argentina. Fax: (54)
1-786-2564.
98 MATERIALS AND METHODS
Experimental anbnals Male Sprague-Dawley rats (200-250 g) were used. A group of animals was adrenalectomized under ether anesthesia and used 4 days after surgery. A second group of ADX rats received over 4 days one of the following compounds (Sigma Chemical, St.Louis, MO): a: corticosterone (CORT), 3 mg/day s.c. dissolved in vegetable oil; b: dexamethasone (DEX), 1 me% dissolved in drinking saline or c: aldosterone (ALDO), released at a rate of 1 /zg//~l/h from implanted ALZET minipumps (model 2001, ALZA Corp., CA) containing ! mg hormone/ml propylene glycol. Specific biological effects were reported previously for these treatments ~7"34. For studies on the in vitro effects of steroids, 10 p.M CORT, DEX, ALDO or 3a,21-dihydroxy-5a-pregnan.20.one (tetrahydrodeoxy corticosterone, THDOC) were added to incubation medium in 2
~.[ ethanol: controls received the vehicle only. The effects of stress were studied in adrenal-intact rats kept in the cold room (4°C) for 2 days. To ascertain the effectiveness of the stressor, serum CORT was measured by a competitive protein binding assay~-', with serum samples obtained at the time of sacrifice in control and cold stressed rats. For extraction of tissues, ether anesthetized animals were perfused intracardially with 60 ml cold 0.9% NaCI and the spinal cords were removed following dorsal laminectomy. Cervical regions (C~C7) were embedded in Tissue-Tek (OCT Compound, Miles Labs., Buenos Aires) and cut every 300 /~m in a cryostat precooled at -10°C. Punches were obtained from the ventral horn, dorsal horn and lateral funiculus, following the technique described in the microdissection atlas of Palkovits and Brownstein 2s.
l ~Hl Fhmimlzepam hireling Punches obtained from a pool of 7 spinal cords per experimental group were homogenized in 300/.tl of 50 mM Tris-HCI (pH 7.4) and centrifuged at I(I,(X)0x g for ! rain, The membrane pellets were washed twice in the same media, suspended in 300 /zl buffer and frozen-thawed to remove as much endogenous GABA as possibie'. One hundred /zl of tissue suspension, containing 0,25-0.35 mg protein, was incubated with a saturating amount (10 nM) of [~H]Flunitrazepam (FLU, 74.8 Ci/mmol, NEN-Dupont, Boston, MA). This concentration is at least 5 times higher than the K d (I.6-2.0 nM) determined by saturation analysis. Incubations were carried out during I h at 4°C, with or without addition of 500-fold excess of Diazepam (Roemmers Lab., Buenos Aires) to determine non-specific binding. Receptor characterization was performed by addition to incubation media of 3/zM RO 15-1788, RO 5-4864 (a kind gift of Dr, J. Apud, Fidia Res. Foundation, Washington, D.C.) or clonazepam (a gift from Dr. M. Ritta of this Institute). Incubations were stopped by addition of I ml cold 50 mM Tris-HCI, and rapidly filtered under vacuum through G F / B filters (2.4 cm, Whatman, Maidstone, UK) contained in a filter manifold holder (Millipore Corp., Bedford, MA), Each tube was subsequently washed 5 times with buffer, and washes were passed through the filters. The filters were dried under an air jet, placed into glass vials and 10 ml of a Triton X-100 toluene-based liquid scintillation solution was added. Results were expressed as fmol specifically bound ['~H]FLU per mg protein. Proteins were determined according to Lowry et al. I'~.
~D Z
~
• R0 IS- 1788
~-
1
• R0 s- 406~.
Z
• CLONAZEPAM
N~
a
1004
e
I-El
50
~:¢i'e
~unsideredsignificantif P < 0.05.
RESULTS
In order to characterize the receptor type expressed in our spinal cord preparation, we studied the effect of
i
0 Fig. I. Characterization of benzodiazepine (BDZ) binding sites in the dorsal horn and the ventral horn. Incubations were carried out in 50 mM Tris-HCI buffer pH 7.4 alone (control, open columns), with the antagonists of central-type BDZ receptor RO 15-1788 and clonazepam, or the antagonist of the peripheral-type BDZ receptor RO 5-4864. Results are expressed as % of control [3H]flunitrazepam (FLU) binding. Statistical analysis demonstrated that the following differences were significant at P < 0.01: a vs. b; a vs. d; e vs. f and e vs. h.
specific antagonists and GABA on [3H]FLU binding. As shown in Fig. 1, binding of radioligand in the dorsal and ventral horn was significantly inhibited (P < 0.05 or less) by drugs acting at central-type BDZ receptors such as clonazepam and RO 15-1788, in contrast to tho antagonist of the peripheral BDZ receptor RO 5-4864 which was barely active 4.2~,2'~,32. Furthermore, addition of 50-150 /zM GABA to the incubation medium slightly increased ['~H]FLU binding (controls: 209 ± 27.5 fmol/mg prot, n = 3; GABA: 233 :t: 27.8, P:NS). Fig. 2 shows the effects of ADX and treatment with steroids on [3H]FLU binding. In intact rats, binding
e
a
300-
~
o
I E~3INTACT 1 1ADX v~ICORT ~DEX nmIALDO
//11
= 200" ~oo-
an " L ~ l....... li~l~nces 0.95 and 0t ....
g
h
Statistical analysis Results are presented as the mean ± S.E.M. For evaluation of the effect of different treatments on ['~H]FLU binding, we used ANOVA. Those comparisons yielding a significant F value were further analyzed using the Newman-Keuls post-hoe test, set for l-or values of
~
DORSAL HORN
VENTRAL HORN
LATERAL FUNICULUS
Fig. 2. Effect of in vivo steroid administration on BDZ binding sites in the dorsal horn, ventral horn and lateral funiculus. Five groups of animals were studied (n -- 25 animals per group): adrenal intact rats (open columns); adrenalectomized rats (black columns); rats treated over 4 days with corticosterone (CORT, 3 mg/day s.c., diagonal lines), dexamethasone (DEX, 1 rag% of drinking saline, dotted columns) or aldosterone (ALDO, 24 ~ g / d a y released from miniosmotic pumps, vertical lines), Statistical analysis demonstrated that a vs. b and b vs. c were significantly different at P < 0.01.
99
o. L~
I [~ CONTROL ISTRESS
o
E Z
m--Z 200-
,
:E
1
LIJ N
