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Adrenal steroid-induced changes in ß-adrenergic receptor binding in rat hippocampus
European Journal of Pharmacology, 74 ( 1981) 37- 41
37
Elsevier/North-Holland Biomedical Press
S T E R O I D - I N D U C E D C H A N G E S IN ~ - A D R E N E R G I C R E C E P T O R B I N D I N G IN RAT HIPPOCAMPUS
ADRENAL
DAVID C.S. ROBERTS * and FLOYD E. BLOOM Arthur V. Davis Center for Behavioral Neurobiologv, The Salk Institute. La Jolla, CA 92037, U.S.A.
Received 6 February 1981, revised MS received 19 May 1981, accepted 26 May 1981
D.C.S. ROBERTS and F.E. BLOOM, Adrenal steroid-induced changes in 13-adrenergic receptor binding in rat htppocampus, European J. Pharmacol. 74 (1981) 37-41. Bilateral 6-hydroxydopamine(6-OHDA) lesions of the dorsal noradrenergic bundle have been shown to increase [3H]dihydroalprenolol (DHA) binding in the rat hippocampus. The effect of adrenalectomy on this increase in receptor binding was examined. Groups of rats received stereotaxicallyplaced 6-OHDA injections aimed at the dorsal bundle (DB) or vehicle injections (control). Half of each group subsequently received bilateral adrenalectomies (ADX) and the other half received sham operations, yielding four groups: control, DB, ADX, and DB+ADX. The DB lesions produced a 41% increase in maximum binding in close agreement with a previous report. No statistically significant change in [3H]DHA receptor binding was observed following adrenalectomy treatment alone. By contrast, the DB + ADX group showed a significant increase in maximum [3H]DHA receptor binding compared to the DB group. This adrenalectomy-induced increase was reversed after 1 week of corticosterone treatment (1.0 mg/kg/12 h, s.c.). These results suggest an interaction between noradrenergic mechanisms and corticosterone in the hippocampus. Adrenalectomy
/3-Adrenergicreceptors
Corticosterone Dihydroalprenolol Hippocampus
I. I n t r o d u c t i o n
In most peripheral organs, steroids act by binding intracellularly to cytosol receptors, after which they are transported to the cell nucleus. There they may act on genomic expression or enzyme induction. Target cells for steroids contain specific steroid receptors, and it has been argued that the demonstration of specific steroid binding indicates a normal physiological role for the steroid in that cell. McEwen et al. (1969) have shown corticosterone is specifically bound in brain tissue, and further that the hippocampus displays the highest specific neuronal binding capacity for corticosterone of any major brain region. These data suggest that corticosterone may act physiologically in the hippocampus although to date most attempts to demonstrate an effect of this steroid on enzyme induction have failed (see Meyer et al., 1979). * Present address: Department of Psychology, Carleton University, Ottawa, Ontario, Canada, K1S 5B6.
6-Hydroxydopamine
There is considerable support for the idea that a major role of steroids is to modulate the action of other neurotransmitters or polypeptide hormones, which act on the cell surface. For example, corticosterone plays a permissive role in the action of prolactin on m a m m a r y tissue (Astwood, 1970), of A C T H on adipose tissue (Braun and Hechter, 1970) and glucagon and catecholamines on hepatic tissue (Exton et al., 1972). The idea that corticosterone might perform a similar function in the brain was entertained. Specifically, we sought support for the hypothesis that corticosterone might interact with noradrenergic mechanisms in the hippocampus. Such an interaction occurs in liver, where corticosterone is necessary for gluconeogenesis and glycogenolysis which results from adrenergic receptor stimulation. Following removal of the adrenal gland, noradrenaline-induced glucose metabolism is impaired, and possibly as a compensatory response, the liver shows an increase in the number of/~-adrenergic receptors (Wolfe et al., 1976) and an increase in catecholamine-stimulated c A M P production (Exton et al., 1972). While the
0014-2999/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
38
end biochemical response of NA in the hippocampus is not yet known, we chose to investigate a possible NA-corticosterone interaction at the receptor level. If the postulated interaction in the hippocampus is similar to that shown in liver, then adrenalectomy should produce steroid reversible alterations in B-receptor binding. In addition, 6-hydroxydopamine-induced lesions of the dorsal NA bundle cause a significant increase in B-receptors in the hippocampus (U'Pritchard et al., 1980). We therefore sought to determine whether the steroid deprivation of adrenalectomy might modulate the post-lesion effects on central adrenergic receptors.
Animals were sacrificed at approximately 9:30 a.m., and the brains quickly removed and dissected on ice. The hippocampi from 3 animals from a group were pooled and homogenized in 50 mM Tris-HC1 buffer as described by Bylund and Snyder (1976). All assays were performed on fresh tissue. Binding studies were carried out with various concentrations of [3H]dihydroalprenolol (47.4 Ci/mmol; N.E.N.) at 23°C for 20 min according to the method of Bylund and Snyder (1976). Nonspecific binding was determined in the presence of 1/~M (-)-alprenolol. Each assay was replicated 5-8 times for each group of treated rats. The K D and Bmax values were compared between groups using Student's t-tests.
2. Materials and methods
Male Wistar rats weighing 290-340 g at the start of the experiment were used. Under pentobarbital anesthesia (50 mg/kg) one group of animals received stereotaxically placed bilateral injections of 6-hydroxydopamine (4/.tg/1 #1 expressed as the base, in 0.9% saline containing 0.2 m g / m l ascorbate) at an injection rate of 1 #1/2 min through a 30 gauge needle. The injection coordinates from stereotaxis zero were AP + 2.6 mm; ML__+I.1 mm; DV+37 mm; with the animal's head held in the plane of KOnig and Klippel (1962). Control animals were treated identically except that the 6-OHDA was omitted from the injection vehicle. All animals were maintained on ad libitum food and water and group-housed 2 --3/cage. A 12 h dark-light cycle was maintained throughout the experiment. In addition, some groups of rats received adrenalectomies of sham operations 3-5 weeks following stereotaxic surgery. These animals were housed individually and in the case of the adrenalectomized rats, received 0.9% saline to drink. Animals were sacrificed 4-6 weeks following stereotaxic surgery or exactly 7 days following adrenalectomy (ADX). One group of rats which recei"ced the combined 6-OHDA+ADX treatment was injected twice daily with corticosterone (1.0 mg/kg, s.c.; Sigma; suspended in 20% propylene glycol and 25% EtOH). An identically treated group received the injection vehicle only.
3. Results
The binding characteristics of [3H]DHA are presented in fig. 1. Binding of [3H]DHA in the presence (non-specific) and absence (total) of ( - ) alprenolol is shown with the resultant saturable 'specific' binding curve. The Scatchard curve is inset. Fig. 2 shows representative Scatchard curves of single experiments on control, DB and DB+ ADX animals. The average Bma~ and K o values calculated
'°I.~ o
',
~
sl]
......... ~ ....• / "/TOTAL
o.aE 4
NON-SPE~
oe
aN3 o
~'~
E
2(](]0
i
20
•
~ --
t600
,2oo" SPECIFIC BINOING
1
800
400
25
I0
2.0 4.0 [ 3 H - ] (-} D I H Y D R O A L P R E N O L O L
a 80 (nM)
Fig. I. Characteristics of [3H]dihydroalprenolol binding in the presence (non-specific) and absence (total) of I/~M ( - ) alprenolol. Inset shows Scatchard plot of 'specific' plot of 'specific' binding.
39 TABLE I Effect of 6-hydroxydopamine-inducedlesion of the dorsal tegmental NA bundle (DB) and/or adrenalectomy (ADX) on Bmax and KD values of [3 H]dihydroalprenolol binding in the rat hippocampus. Data represent mean ( ± S.E.M.) of 5-8 replications. Tissues from 3 rats were pooled for each replication.
KD (nM)
Control DB ADX DB + A D X DB + ADX + corticosterone
1.67 ~+0,29 1,72_+0.28 1.38± 1.2 1,51 ±0.33 1.22 -4- 1.3
Bmax
Protein (fmol/mg)
% of control
56.0 ~ 4.3 79.4±3.8 ~ 61.7 ± 7.1 94.4±4.8 ~,b 73.8 -4-8.2 c
100 141 110 168 131
a P<0.05 significantly different from control. b P<0.05 significantly different from DB. c P<0.05 significantly different from DB+ADX.
from Scatchard analysis of the [3H]DHA binding data from 5 - 8 replications are presented in table 1. No differences were observed between unoperated and vehicle injected-sham operated animals. The data from these animals were pooled and are presented as one control group. Control values agree closely with previously reported binding values for rat hippocampus (U'Pritchard et al., 1980). Four weeks following the 6-OHDA treat-
50
40
m
W E
3O
a
20
[]
•
BD
•
•
u.
D
[]
z 0 m
ment, a 41% increase in maximum [3H]DHA binding was observed. No statistical difference was observed between control and adrenalectomized animals. Adrenalectomy, when combined with the 6O H D A treatment, was found to increase significantly the Bm~x values above that which was produced by the 6-OHDA treatment alone, the DB + ADX group which received daily injections of corticosterone was found to have significantly lower Bm~ values compared to the untreated or vehicle injected D B + A D X groups. (These latter two groups did not differ and their data were pooled.) No statistically significant differences in K D values were observed between groups. Hill plot coefficients from each group were not significantly different and were close to 1.0.
4. Discussion
10
CONTROL 10
20
fmoles
DB
30
BOUND
DB + ADX
40
J
10 mg
50
60
tissue
Fig. 2. Representative Scatchard plots of [3H]DHA binding in hippocampal tissue from various groups of treated animals. Each assay was performed on pooled tissue from 3 rats. One group received 6-OHDA lesions of dorsal NA bundle (DB), while another group a combined adrenalectomy and DB treatment ( D B + A D X ) . Control animals received vehicle injections and sham adrenalectomies. See table 1 for statistical comparisons.
Injections of 6-OHDA into the dorsal tegmental NA bundle have been shown to produce near total depletions of forebrain NA (Roberts et al., 1976). The present data show that this treatment also produces a 41% increase in maximum [3H]DHA binding in the hippocampus, which indicates a denervation supersensitivity of adrenergic receptors in the hippocampus. These data are in close agreement with those of U'Pritchard et al. (1980) who previously have shown a similar in-
40
crease in [3H]DHA binding after 6-OHDA lesions of the dorsal NA bundle. Adrenalectomy alone did not produce a significant alteration in either the Bm~, or K D of [3H]DHA binding. By contrast, adrenalectomy when combined with the 6-OHDA treatment, produced an increase in maximum [3H]DHA binding. To determine whether adrenal steroids would reverse this effect of adrenalectomy a group of rats with combined 6-OHDA and adrenalectomy treatments received daily corticosterone injections. Corticosterone was found to reverse the effect of the adrenalectomy. These data indicate that corticosterone interacts with NA mechanisms in the hippocampus, although how this interaction occurs is not yet clear. It is possible that corticosterone acts directly on receptor synthesis or transport into the membrane. These results parallel data reported in liver. Wolfe et al. (1976) have shown a substantial increase in fl-adrenergic receptor binding in liver following adrenalectomy, which was reversed by glucocorticoid replacement. It is interesting to point out that the adrenalectomy would deprive the liver of both medullary (catecholamine) and steroid influence and would therefore be analogous to the DB + ADX treatment reported here for the hippocampus. The liver also shows an increase in N A stimulated adenylate cyclase activity following adrenalectomy even though the normal biochemi-, cal response to NA (glucose metabolism) is impaired (Exton et al., 1972). These data support the view that corticosterone has a 'permissive' action on this NA-stimulated mechanism. When the steroid is removed the end result is blocked and a compensatory increase in receptor number and NA-stimulated adenylate cyclase activity occurs. We postulate that a similar interaction between NA mechanisms and corticosterone exists in brain. While the end biochemical reaction which is initiated by adrenergic receptor stimulation is not yet known, some data suggest that corticosterone may interact with these events. The present results show changes in B-receptors following steroid manipulations, and Mobley and Sulser (1980) have demonstrated similar alterations in NA-stimulated adenylate cyclase in frontal cortex.
A number of behavioural experiments have also shown an apparent interaction between the telencephalic projection of the locus coeruleus and corticosterone. While neither 6-OHDA lesions of the dorsal NA bundle nor adrenalectomy alone significantly affected the acquisition of a number of shock avoidance behaviours in rats, animals which received both treatments were found to be markedly impaired in their ability to learn the appropriate response (Ogren and Fuxe, 1974, 1977: Roberts and Fibiger, 1979; Mason et al., 1979). This deficit could be reversed by daily corticosterone injections (Ogren and Fuxe, 1977). It is interesting to point out that the 6-OHDA treated group showed no behavioural deficit and the similarly treated animals in the present study showed only a sub-maximal increase in receptor binding. We speculate that this increase, in addition to other mechanisms such as up-regulation of adenylate cyclase, may have been sufficient to compensate for the NA depletions. Such compensation following the lesion may explain the lack of behavioural deficits reported in these animals (e.g., Mason and Iversen, 1975; Roberts et al., 1976: Crow et al., 1978). On the other hand, the group which showed the behavioural impairment also showed the greatest increase in receptor binding in the present experiment (6-OHDA+ADX). The compensatory increases may have been maximal but insufficient to allow for recovery of function and thus the animals show a deficit. Following identical corticosterone treatments, the behavioural deficit is abolished and the increase in receptor binding is reduced. No significant change in binding was observed after adrenalectomy alone. In the strain of rat used here, some steroid producing cells remain following adrenalectomy, and circulating steroid levels are reduced only to 25% (Mason et al., 1979). It is possible that the system can function with the remaining circulating corticosterone, which may explain why no significant change in binding was observed following adrenalectomy alone. Mobley and Sulser (1980) have also reported only nonsignificant increases in [3H]DHA binding following adrenalectomy in rat frontal cortex. Molinoff et al. (1979) have examined fl-receptor subtypes in rat forebrain following adrenalectomy
41
and found increases in/~2 but not the fll subtypes. They report that these increases were reversed with epinephrine but not cortisone and speculate that these fl2-receptors are on the peripheral side of the blood-brain barrier. Molinoff et al. (1979) also report an increase in the fl~ subtype following 6-OHDA treatment, and these receptors constitute 85% of fl-receptors in the forebrain. These data suggest that the present results are probably due to alterations in the/3~ subtype; however, additional experiments are required to address this point directly. The possibility must be recognized that the 6-OHDA lesions and adrenalectomy produce independent and unrelated effects which are additive when measured biochemically or behaviourally. It is our working hypothesis, however, that a direct physiological interaction may account for these data, and we propose that corticosterone interacts with NA-stimulated mechanisms in the hippocampus to perform a critical role in some learning situations.
Acknowledgements The assistance of S. Back and Dr. W. Shoemaker is gratefully acknowledged. Supported by the Medical Research Council (of Canada).
References Astwood, E.B., 1970, in: The Pharmacological Basis of Therapeutics, eds. L.S. Goodman and A. Gilman (Macmillan, Toronto) p. 1538. Braun, T. and O. Hechter, 1970, Glucocorticoid regulation of ACTH sensitivity of adeny/ cyclase in rat fat cell membranes, Proc. Nat. Acad. Sci. U.S.A. 66, 995. Bylund, D.B. and S.H. Snyder, 1976, Beta-adrenergic receptor binding in membrane preparations from mammalian brain, Mol. Pharmacol. 12, 568. Crow, T.J., J.F.W. Deakin, S.E. File, A. Longden and S.
Wendlandt, 1978, The locus coeruleus noradrenaline system --evidence against a role in attention, habituation, anxiety and motor activity, Brain Res. 155, 249. Exton, J.H., N. Freidman, E., H.-A. Wong, LP. Brineaux, J.D. Corbin and C.R. Park, 1972, Interaction of glucocorticoids with glucagon and epinephrine in the control of gluconeogenesis and glycogenolysis in liver and of lipolysis in adipose tissue, J. Biol. Chem. 247, 3579. K6nig, J.F.R. and R.A. Klippel, 1962, The Rat Brain (Williams and Wilkins, Baltimore). Mason, S.T. and S.D. Iversen, 1975, Learning in the absence of forebrain noradrenaline, Nature 258, 422. Mason, S.T., D.C.S. Roberts and H.C. Fibiger, 1979, Interaction of brain noradrenaline and the pituitary-adrenal axis in learning and extinction, Pharmacol. Biochem. Behav. 10, 11. McEwen, B.S., J.M. Weiss and L.S. Schwartz, 1969, Uptake of corticosterone by rat brain and its concentration by certain limbic structures, Brain Res. 16, 227. Meyer, J.S., V.N. Luine, R.I. Khylchevskaya and B.S. McEwen, 1979, Glucocorticoids and hippocampal enzyme activity, Brain Res. 166, 172. Mobley, P.L. and F. Sulser, 1980, Adrenal corticoids regulate sensitivity of noradrenaline receptor-coupled adenylate cyclase in brain, Nature 286, 608. Molinoff, P.B., K.P. Minneman, B.B. Wolfe, R.N. Pittman and M.D. Dibner, 1979, r-1 and fl-2 adrenergic receptors in rat cerebral cortex are independently regulated, Annual Meeting of Soc. Psychoneuroendocrin. (Abstr.). Ogren, S.-O and K. Fuxe, 1974, Learning, brain noradrenaline and the pituitary-adrenal axis, Med. Biol. 52, 399. Ogren, S.-O. and K. Fuxe, 1977, On the role of brain noradrenaline and the pituitary-adrenal axis in avoidance learning. I. Studies with corticosterone, Neurosci. Lett. 5, 291. Roberts, D.C.S. and H.C. Fibiger, 1979, Evidence for interactions between central noradrenaline neurons and adrenal hormones in learning and memory, Pharmacol. Biochem. Behav. 7, 191. Roberts, D.C.S., M.T.C. Price and H.C. Fibiger, 1976, The dorsal tegmental noradrenergic projection: an analysis of its role in maze learning, J. Comp. Physiol. Psych. 90, 363. U'Pritchard, D.C., T.D. Reisine, S.T. Mason, H.C. Fibiger and H.I. Yamamura, 1980, Modulation of rat brain a- and fl-adrenergic receptor populations by lesion of the dorsal noradrenergic bundle, Brain Res. 187, 143. Wolfe, B.B., T.K. Harden and P.B. MolinofL 1976, flAdrenergic receptors in rat liver. Effects of adrenalectomy, Proc. Nat. Acad. Sci. 73, 1343.
37
Elsevier/North-Holland Biomedical Press
S T E R O I D - I N D U C E D C H A N G E S IN ~ - A D R E N E R G I C R E C E P T O R B I N D I N G IN RAT HIPPOCAMPUS
ADRENAL
DAVID C.S. ROBERTS * and FLOYD E. BLOOM Arthur V. Davis Center for Behavioral Neurobiologv, The Salk Institute. La Jolla, CA 92037, U.S.A.
Received 6 February 1981, revised MS received 19 May 1981, accepted 26 May 1981
D.C.S. ROBERTS and F.E. BLOOM, Adrenal steroid-induced changes in 13-adrenergic receptor binding in rat htppocampus, European J. Pharmacol. 74 (1981) 37-41. Bilateral 6-hydroxydopamine(6-OHDA) lesions of the dorsal noradrenergic bundle have been shown to increase [3H]dihydroalprenolol (DHA) binding in the rat hippocampus. The effect of adrenalectomy on this increase in receptor binding was examined. Groups of rats received stereotaxicallyplaced 6-OHDA injections aimed at the dorsal bundle (DB) or vehicle injections (control). Half of each group subsequently received bilateral adrenalectomies (ADX) and the other half received sham operations, yielding four groups: control, DB, ADX, and DB+ADX. The DB lesions produced a 41% increase in maximum binding in close agreement with a previous report. No statistically significant change in [3H]DHA receptor binding was observed following adrenalectomy treatment alone. By contrast, the DB + ADX group showed a significant increase in maximum [3H]DHA receptor binding compared to the DB group. This adrenalectomy-induced increase was reversed after 1 week of corticosterone treatment (1.0 mg/kg/12 h, s.c.). These results suggest an interaction between noradrenergic mechanisms and corticosterone in the hippocampus. Adrenalectomy
/3-Adrenergicreceptors
Corticosterone Dihydroalprenolol Hippocampus
I. I n t r o d u c t i o n
In most peripheral organs, steroids act by binding intracellularly to cytosol receptors, after which they are transported to the cell nucleus. There they may act on genomic expression or enzyme induction. Target cells for steroids contain specific steroid receptors, and it has been argued that the demonstration of specific steroid binding indicates a normal physiological role for the steroid in that cell. McEwen et al. (1969) have shown corticosterone is specifically bound in brain tissue, and further that the hippocampus displays the highest specific neuronal binding capacity for corticosterone of any major brain region. These data suggest that corticosterone may act physiologically in the hippocampus although to date most attempts to demonstrate an effect of this steroid on enzyme induction have failed (see Meyer et al., 1979). * Present address: Department of Psychology, Carleton University, Ottawa, Ontario, Canada, K1S 5B6.
6-Hydroxydopamine
There is considerable support for the idea that a major role of steroids is to modulate the action of other neurotransmitters or polypeptide hormones, which act on the cell surface. For example, corticosterone plays a permissive role in the action of prolactin on m a m m a r y tissue (Astwood, 1970), of A C T H on adipose tissue (Braun and Hechter, 1970) and glucagon and catecholamines on hepatic tissue (Exton et al., 1972). The idea that corticosterone might perform a similar function in the brain was entertained. Specifically, we sought support for the hypothesis that corticosterone might interact with noradrenergic mechanisms in the hippocampus. Such an interaction occurs in liver, where corticosterone is necessary for gluconeogenesis and glycogenolysis which results from adrenergic receptor stimulation. Following removal of the adrenal gland, noradrenaline-induced glucose metabolism is impaired, and possibly as a compensatory response, the liver shows an increase in the number of/~-adrenergic receptors (Wolfe et al., 1976) and an increase in catecholamine-stimulated c A M P production (Exton et al., 1972). While the
0014-2999/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
38
end biochemical response of NA in the hippocampus is not yet known, we chose to investigate a possible NA-corticosterone interaction at the receptor level. If the postulated interaction in the hippocampus is similar to that shown in liver, then adrenalectomy should produce steroid reversible alterations in B-receptor binding. In addition, 6-hydroxydopamine-induced lesions of the dorsal NA bundle cause a significant increase in B-receptors in the hippocampus (U'Pritchard et al., 1980). We therefore sought to determine whether the steroid deprivation of adrenalectomy might modulate the post-lesion effects on central adrenergic receptors.
Animals were sacrificed at approximately 9:30 a.m., and the brains quickly removed and dissected on ice. The hippocampi from 3 animals from a group were pooled and homogenized in 50 mM Tris-HC1 buffer as described by Bylund and Snyder (1976). All assays were performed on fresh tissue. Binding studies were carried out with various concentrations of [3H]dihydroalprenolol (47.4 Ci/mmol; N.E.N.) at 23°C for 20 min according to the method of Bylund and Snyder (1976). Nonspecific binding was determined in the presence of 1/~M (-)-alprenolol. Each assay was replicated 5-8 times for each group of treated rats. The K D and Bmax values were compared between groups using Student's t-tests.
2. Materials and methods
Male Wistar rats weighing 290-340 g at the start of the experiment were used. Under pentobarbital anesthesia (50 mg/kg) one group of animals received stereotaxically placed bilateral injections of 6-hydroxydopamine (4/.tg/1 #1 expressed as the base, in 0.9% saline containing 0.2 m g / m l ascorbate) at an injection rate of 1 #1/2 min through a 30 gauge needle. The injection coordinates from stereotaxis zero were AP + 2.6 mm; ML__+I.1 mm; DV+37 mm; with the animal's head held in the plane of KOnig and Klippel (1962). Control animals were treated identically except that the 6-OHDA was omitted from the injection vehicle. All animals were maintained on ad libitum food and water and group-housed 2 --3/cage. A 12 h dark-light cycle was maintained throughout the experiment. In addition, some groups of rats received adrenalectomies of sham operations 3-5 weeks following stereotaxic surgery. These animals were housed individually and in the case of the adrenalectomized rats, received 0.9% saline to drink. Animals were sacrificed 4-6 weeks following stereotaxic surgery or exactly 7 days following adrenalectomy (ADX). One group of rats which recei"ced the combined 6-OHDA+ADX treatment was injected twice daily with corticosterone (1.0 mg/kg, s.c.; Sigma; suspended in 20% propylene glycol and 25% EtOH). An identically treated group received the injection vehicle only.
3. Results
The binding characteristics of [3H]DHA are presented in fig. 1. Binding of [3H]DHA in the presence (non-specific) and absence (total) of ( - ) alprenolol is shown with the resultant saturable 'specific' binding curve. The Scatchard curve is inset. Fig. 2 shows representative Scatchard curves of single experiments on control, DB and DB+ ADX animals. The average Bma~ and K o values calculated
'°I.~ o
',
~
sl]
......... ~ ....• / "/TOTAL
o.aE 4
NON-SPE~
oe
aN3 o
~'~
E
2(](]0
i
20
•
~ --
t600
,2oo" SPECIFIC BINOING
1
800
400
25
I0
2.0 4.0 [ 3 H - ] (-} D I H Y D R O A L P R E N O L O L
a 80 (nM)
Fig. I. Characteristics of [3H]dihydroalprenolol binding in the presence (non-specific) and absence (total) of I/~M ( - ) alprenolol. Inset shows Scatchard plot of 'specific' plot of 'specific' binding.
39 TABLE I Effect of 6-hydroxydopamine-inducedlesion of the dorsal tegmental NA bundle (DB) and/or adrenalectomy (ADX) on Bmax and KD values of [3 H]dihydroalprenolol binding in the rat hippocampus. Data represent mean ( ± S.E.M.) of 5-8 replications. Tissues from 3 rats were pooled for each replication.
KD (nM)
Control DB ADX DB + A D X DB + ADX + corticosterone
1.67 ~+0,29 1,72_+0.28 1.38± 1.2 1,51 ±0.33 1.22 -4- 1.3
Bmax
Protein (fmol/mg)
% of control
56.0 ~ 4.3 79.4±3.8 ~ 61.7 ± 7.1 94.4±4.8 ~,b 73.8 -4-8.2 c
100 141 110 168 131
a P<0.05 significantly different from control. b P<0.05 significantly different from DB. c P<0.05 significantly different from DB+ADX.
from Scatchard analysis of the [3H]DHA binding data from 5 - 8 replications are presented in table 1. No differences were observed between unoperated and vehicle injected-sham operated animals. The data from these animals were pooled and are presented as one control group. Control values agree closely with previously reported binding values for rat hippocampus (U'Pritchard et al., 1980). Four weeks following the 6-OHDA treat-
50
40
m
W E
3O
a
20
[]
•
BD
•
•
u.
D
[]
z 0 m
ment, a 41% increase in maximum [3H]DHA binding was observed. No statistical difference was observed between control and adrenalectomized animals. Adrenalectomy, when combined with the 6O H D A treatment, was found to increase significantly the Bm~x values above that which was produced by the 6-OHDA treatment alone, the DB + ADX group which received daily injections of corticosterone was found to have significantly lower Bm~ values compared to the untreated or vehicle injected D B + A D X groups. (These latter two groups did not differ and their data were pooled.) No statistically significant differences in K D values were observed between groups. Hill plot coefficients from each group were not significantly different and were close to 1.0.
4. Discussion
10
CONTROL 10
20
fmoles
DB
30
BOUND
DB + ADX
40
J
10 mg
50
60
tissue
Fig. 2. Representative Scatchard plots of [3H]DHA binding in hippocampal tissue from various groups of treated animals. Each assay was performed on pooled tissue from 3 rats. One group received 6-OHDA lesions of dorsal NA bundle (DB), while another group a combined adrenalectomy and DB treatment ( D B + A D X ) . Control animals received vehicle injections and sham adrenalectomies. See table 1 for statistical comparisons.
Injections of 6-OHDA into the dorsal tegmental NA bundle have been shown to produce near total depletions of forebrain NA (Roberts et al., 1976). The present data show that this treatment also produces a 41% increase in maximum [3H]DHA binding in the hippocampus, which indicates a denervation supersensitivity of adrenergic receptors in the hippocampus. These data are in close agreement with those of U'Pritchard et al. (1980) who previously have shown a similar in-
40
crease in [3H]DHA binding after 6-OHDA lesions of the dorsal NA bundle. Adrenalectomy alone did not produce a significant alteration in either the Bm~, or K D of [3H]DHA binding. By contrast, adrenalectomy when combined with the 6-OHDA treatment, produced an increase in maximum [3H]DHA binding. To determine whether adrenal steroids would reverse this effect of adrenalectomy a group of rats with combined 6-OHDA and adrenalectomy treatments received daily corticosterone injections. Corticosterone was found to reverse the effect of the adrenalectomy. These data indicate that corticosterone interacts with NA mechanisms in the hippocampus, although how this interaction occurs is not yet clear. It is possible that corticosterone acts directly on receptor synthesis or transport into the membrane. These results parallel data reported in liver. Wolfe et al. (1976) have shown a substantial increase in fl-adrenergic receptor binding in liver following adrenalectomy, which was reversed by glucocorticoid replacement. It is interesting to point out that the adrenalectomy would deprive the liver of both medullary (catecholamine) and steroid influence and would therefore be analogous to the DB + ADX treatment reported here for the hippocampus. The liver also shows an increase in N A stimulated adenylate cyclase activity following adrenalectomy even though the normal biochemi-, cal response to NA (glucose metabolism) is impaired (Exton et al., 1972). These data support the view that corticosterone has a 'permissive' action on this NA-stimulated mechanism. When the steroid is removed the end result is blocked and a compensatory increase in receptor number and NA-stimulated adenylate cyclase activity occurs. We postulate that a similar interaction between NA mechanisms and corticosterone exists in brain. While the end biochemical reaction which is initiated by adrenergic receptor stimulation is not yet known, some data suggest that corticosterone may interact with these events. The present results show changes in B-receptors following steroid manipulations, and Mobley and Sulser (1980) have demonstrated similar alterations in NA-stimulated adenylate cyclase in frontal cortex.
A number of behavioural experiments have also shown an apparent interaction between the telencephalic projection of the locus coeruleus and corticosterone. While neither 6-OHDA lesions of the dorsal NA bundle nor adrenalectomy alone significantly affected the acquisition of a number of shock avoidance behaviours in rats, animals which received both treatments were found to be markedly impaired in their ability to learn the appropriate response (Ogren and Fuxe, 1974, 1977: Roberts and Fibiger, 1979; Mason et al., 1979). This deficit could be reversed by daily corticosterone injections (Ogren and Fuxe, 1977). It is interesting to point out that the 6-OHDA treated group showed no behavioural deficit and the similarly treated animals in the present study showed only a sub-maximal increase in receptor binding. We speculate that this increase, in addition to other mechanisms such as up-regulation of adenylate cyclase, may have been sufficient to compensate for the NA depletions. Such compensation following the lesion may explain the lack of behavioural deficits reported in these animals (e.g., Mason and Iversen, 1975; Roberts et al., 1976: Crow et al., 1978). On the other hand, the group which showed the behavioural impairment also showed the greatest increase in receptor binding in the present experiment (6-OHDA+ADX). The compensatory increases may have been maximal but insufficient to allow for recovery of function and thus the animals show a deficit. Following identical corticosterone treatments, the behavioural deficit is abolished and the increase in receptor binding is reduced. No significant change in binding was observed after adrenalectomy alone. In the strain of rat used here, some steroid producing cells remain following adrenalectomy, and circulating steroid levels are reduced only to 25% (Mason et al., 1979). It is possible that the system can function with the remaining circulating corticosterone, which may explain why no significant change in binding was observed following adrenalectomy alone. Mobley and Sulser (1980) have also reported only nonsignificant increases in [3H]DHA binding following adrenalectomy in rat frontal cortex. Molinoff et al. (1979) have examined fl-receptor subtypes in rat forebrain following adrenalectomy
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and found increases in/~2 but not the fll subtypes. They report that these increases were reversed with epinephrine but not cortisone and speculate that these fl2-receptors are on the peripheral side of the blood-brain barrier. Molinoff et al. (1979) also report an increase in the fl~ subtype following 6-OHDA treatment, and these receptors constitute 85% of fl-receptors in the forebrain. These data suggest that the present results are probably due to alterations in the/3~ subtype; however, additional experiments are required to address this point directly. The possibility must be recognized that the 6-OHDA lesions and adrenalectomy produce independent and unrelated effects which are additive when measured biochemically or behaviourally. It is our working hypothesis, however, that a direct physiological interaction may account for these data, and we propose that corticosterone interacts with NA-stimulated mechanisms in the hippocampus to perform a critical role in some learning situations.
Acknowledgements The assistance of S. Back and Dr. W. Shoemaker is gratefully acknowledged. Supported by the Medical Research Council (of Canada).
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