27
Neuroscience Letters, 130 (1991) 27 31 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 0304394091004677 NSL 07987
The induction of corticosteroid actions on membrane properties of hippocampal CA1 neurons requires protein synthesis H. Karst* a n d M. JoWls* Institute of Molecular Biology, University of Utrecht, Utrecht ( The Netherlands) (Received 28 March 1991; Revised version received 28 May 1991; Accepted 28 May 1991)
Key words.
Corticosterone; Mineralocorticoid receptor; Glucocorticoid receptor; Hippocampus; CA1; Cycloheximide; Electrophysiology
Corticosterone can affect electrical properties of CAl pyramidal neurons via binding to two corticoid receptor types, the mineralocorticoid (MR) and glucocorticoid receptor (GR). Previously we have shown that MR-activation leads to attenuation of serotonin (5-HT)-induced membrane hyperpolarization, while GR-activation induces an increase in the amplitude of the afterhyperpolarization (AHP) following a short current pulse. In this study we show that the MR- and GR-mediated changes of the membrane properties are prevented in the presence of the protein synthesis inhibitor cycloheximide, thus suggesting a genomic action of the steroids.
Corticosteroid hormones, ' released from the adrenal gland, can pass the blood-brain barrier and bind to intracellular receptors in the brain [4, 12]. Two intracellular corticosteroid receptor types have been distinguished: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) [7, 15]. Pyramidal cells in the rat hippocampal CA1 area contain both MR and GR. After binding of corticosteroids, the receptor-steroid complex shows increased affinity to the nuclear compartment and affects gene expression (see for review ref. 13). In previous electrophysiological studies it was shown that both MR and GR stimulation cause receptor-specific changes in the membrane properties of CA1 pyramidal neurons. Stimulation of the MR causes, with a delay of at least 15 min, a decrease of the accommodation and afterhyperpolarization (AHP) induced by a short depolarizing current pulse in CAI neurons [10]. The MRmediated action can be gradually overridden and reversed by a GR-mediated increase of the AHP amplitude [9]. In addition to these current-induced phenomena, corticosteroids can also affect transmitter-evoked changes in membrane properties, with a delay of several hours. Activation of GR was found to counteract fl-adrenoreceptor mediated actions in CA1 pyramidal cells [9], while it was recently shown that stimulation of MR attenuates *Present address: Department of Experimental Zoology, University of Amsterdam, Kruislaan 320, I098 SM Amsterdam, The Netherlands. Correspondence: H. Karst, Department of Experimental Zoology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands.
a 5HTiA-receptor mediated membrane hyperpolarization [11]. The fact that (1) these corticosteroid actions were also achieved by selective ligands for the intracellular MR or GR receptor and (2) all corticosteroid actions occurred with a considerable delay in time (15 min for MR [10] to approximately 1 h for GR actions [9]) suggests that corticosteroid hormones affect membrane properties via a genomic interaction. To test this hypothesis, we studied corticosteroid-mediated actions in the presence of a protein synthesis inhibitor, cycloheximide. As an index for MR-mediated actions we examined the effect of 30 nM corticosterone in the presence of the GR-antagonist RU38486, thus selectively activating the MRs, on hyperpolarizing responses to serotonin (5-HT). As an index for GR-mediated effects we studied the effect of 30 nM corticosterone on the amplitude of the AHP. Male Wistar rats (120-170 g) were adrenalectomized (ADX) 5-7 days before the experiments, in order to create a situation in which the receptors are unoccupied at the start of the electrophysiological experiments. Under these circumstances the receptors can be selectively occupied by MR or GR ligands. The animals were housed in an animal room with alternating light/dark (08.00-20.00 h/20.00-08.00 h) cycle and received food and saline (after ADX) ad libitum. On the day of the experiment the rat was placed in a clean cage 30-60 min before decapitation. After decapitation, trunk blood was collected for measurement of plasma corticosterone levels. The hippocampus was removed from the brain and dipped in ice cold oxygenated (95 % 02, 5 % CO2) artifi-
28
cial cerebrospinal fluid (ACSF: 124 mM NaCl, 3.5 mM KCI, 1.25 mM NaHzPO4, 1.5 mM MgSO4, 2 mM CaCI2, 25 mM NaHCO3, 10 mM glucose). Transverse slices of the dorsal hippocampus were cut (_+ 350 /~m) on a McIwain tissue chopper and submerged in a perfusing chamber. The slices were continuously perfused with oxygenated ACSF (32°C) at a constant rate of 2-3 ml/ min. Corticosterone (Organon International, Oss, the Netherlands) and RU38486 (Roussel-Uclaf, Romainville, France) were, for each experiment, dissolved in ethanol (1 mM) and diluted to the intended concentration in oxygenated ACSF immediately before application. Cycloheximide (100/~M, Sigma) and 5-HT (treat±nine sulfate complex, 100/IM stock-solution, diluted to the intended concentration immediately before the experiment, Sigma) were dissolved in ACSF. Intracellular recordings of pyramidal CAI neurons were performed with 4 M KAc-filled microelectrodes (impedance: 80-150M[2). From each neuron we recorded the resting membrane potential, input resistance, spike accommodation/AHP evoked by 50 ms (0.2-1.0 nA) or 500 ms (0.5 nA) depolarizing current pulses. In one series of experiments we tested the 'control' MR-effect on 5-HT responsiveness by recording responses to 1, 3, 10 and 30/~M 5-HT 1-2 h after a 20 min perfusion of 30 nM corticosterone, in the presence of the GR-antagonist RU38486 (500 nM perfused in total for 60 min, starting 20 min before corticosterone application). In another series of experiments we tested the 'control' GR-mediated responses by establishing the amplitude of the AHP, 1-2 h after a 20 min corticosterone application (30 nM). We subsequently tested the effect of cycloheximide (100 /tM) alone, by comparing membrane properties and responses to 5-HT obtained before cycloheximide application with responses recorded 1-2 h after onset of cycloheximide. Finally, we examined the MR- and GR- mediated actions in the presence of cycloheximide. Administration of the protein synthesis inhibitor was started 20 min before application of corticosterone and continued for at least 2 h. In total we recorded from 45 identified [17, 18] CAI pyramidal neurons. The averaged membrane potentials and input resistances of the experimental groups (Table I) ranged from -65.8 to -71.2 mV and from 35.7 to 44.9 M~, respectively. No significant differences were observed for membrane potential or resistance between neurons recorded in slices treated with corticosterone and cycloheximide (GR effect in the presence of cycloheximide) and neurons in slices treated with either corticosterone (GR effect), with cycloheximide or nontreated ADX cells. Similarly, no differences in these parameters were found between neurons in slices perfused with corticosterone, RU38486 and cycloheximide (MR
TABLE I RESTING MEMBRANE POTENTIAL (RMP; + S.E.M.) AND INPUT RESISTANCE (R~n) OF THE GROUPS OF HIPPOCAMPAL CA1 NEURONS INCORPORATED IN THIS STUDY No significant differences were observed for membrane potential and resistance between groups of neurons compared in this study: Group III (GR effect) with I and II, and group IV (MR effect) with I and II. *Resting membrane potential for cells treated with corticosterone in the presence of cycloheximide was found to be significantly different from the cells treated with corticosterone and RU38486. As these groups of neurons were not compared in the rest of this study, our data were not affected by this difference. Statistics were done with the oneway analysis of variance with the Student-Newman-Keuls test for multiple comparisons between means (P < 0.05). Treatment
n
RMP (mV)
Rin (MI2)
1,
ADX
7
69.6±1.7
38.2±1.95
II.
l 2h 100/aM cycloheximide
6
66.2±1.5
39.2±3.13
III.
30 nM corticosterone
9
68.1±0.7
41.9±2.1
100/aM cycloheximide 30 nM corticosterone
7
71.2±2.1"
43.3±2.2
100/aM cycloheximide 500 nM RU38486/ 30 nM corticosterone
7
68.7±1.0
35.7±2.2
500 nM RU38486/ 30 nM corticosterone
9
65.8±1.1"
~.9±5.3
IV.
effect in the presence of cycloheximide) and neurons in slices treated with either corticosterone/RU38486, with cycloheximide or non-treated slices. Resting membrane potentials for the cells treated with corticosterone and cycloheximide (71.2 _+ 2. I mV) were significantly different from cells treated with corticosterone and RU38486 (65.8 + 1.1 mV). As these groups of neurons were not compared in the rest of the study, our conclusions were not affected by this difference. As reported previously by us and others [1, 3, 11] we observed that 5-HT induces a hyperpolarization of the CA1 pyramidal cell membrane potential and a decrease of the input resistance. The lowest effective dose is 1/~M, while near-maximal responses are obtained with 30/tM 5-HT (see Fig. 1). Activation of the MR significantly attenuated the membrane hyperpolarization and resistance decrease induced by 3, 10 or 30/tM 5-HT (Fig. 1). The MR-mediated decrease of hyperpolarizations induced by 3, 10 or 30/IM 5-HT was no longer observed in the presence of the protein synthesis inhibitor cycloheximide. Similarly, the MR-mediated attenuation of the resistance change evoked by 10 and 30/tM was pre-
29 A1
A2 30 laM 5HT
A3 30/aM 5HT
30 taM 5HT
A 30 nM corticosterone
100 taM cycloheximide
500 nM RU38486
100 laM cycloheximide
30 nM corlicosterone _
500 nM RU38486
_
110my
I min
B
C 10 ¸ ~
> IE
e-
t.9
40. T
8 6,
°
E E 0-
30. 20.
.5
e~
~. e-
o~ .~-
9.
l
"~
10.
E ~
0-
i f
-1
-10 1
1'0
100
[5HT] ([LtM)
100
[5HT] (pM)
Fig. 1. A: the hyperpolarization of hippocampal CAI neurons by serotonin (30 pM 5-HT) is attenuated during MR stimulation by corticosterone in the presence of a GR antagonist (RU38486) (A1). In the presence of a protein synthesis inhibitor, cycloheximide, this MR-specific effect is prevented (A2). Cycloheximide alone does not affect the 5-HT response (A3). The downward deflexions in the registrations are hyperpolarizing current pulses ( - 0 . 3 nA, 100 ms) passed through the recording electrode to measure membrane resistance. The membrane potential was repolarized during the hyperpolarized 5-HT response, by applying a positive current, to measure real resistance changes. Black bars indicate the duration of the 5-HT-perfusion. B,C: stimulation of the 5-HT1A receptors with 5-HT hyperpolarizes hippocampal CA 1 neurons and decreases the input resistance in a dose-dependent manner (ADX, ©). Corticosterone (30 nM) in the presence of a GR antagonist, RU38486, thus causing MR activation, significantly inhibits the actions evoked by 3, 10 and 30 pM 5-HT ( • ) (P < 0.05). The MR-mediated attenuation of the 5-HT responses does not occur in the presence of cycloheximide (A), a protein synthesis inhibitor. Cycloheximide itself does not affect this 5-HT hyperpolarizing response (O). Statistics were done with a one-way analysis of variance followed by the Student-Newman-Keuls test for multiple comparisons (P < 0.05).
vented by cycloheximide. The action of cycloheximide was less clear for 3 pM 5-HT, although statistical analysis showed that, here too, the inhibition of the 5-HTmediated change in resistance by M R activation was no longer observed. As can be seen in Fig. 1, cycloheximide alone affected neither the 5-HT-evoked hyperpolarization nor the change in resistance. In the present study we furthermore observed that neurons recorded 30-90 min after a 20 min perfusion with 30 nM corticosterone show a significantly increased AHP amplitude over the entire range of current intensities tested (Fig. 2). This extends a previous observation that a 20 min application of corticosterone induces after a delay of ca. 1-2 h, a significant increase in the AHP evoked by a 0.5 nA depolarizing current pulse [10]. We found that, in the presence of cycloheximide, corticoste-
rone is no longer able to affect the AHP. As described for the MR-mediated effects on 5-HT responsiveness, cycloheximide by itself did not affect the AHP amplitude. In this study we extended previously reported MRand GR-mediated changes in electrical properties of CA1 pyramidal neurons. Neither M R nor G R occupation appeared to affect passive membrane properties of identified CA1 neurons. These observations are in agreement with previous studies by us [9, 10], but contrast with work by others where rapid steroid-induced changes in passive membrane properties were reported [8]. We show, furthermore, that the previously reported attenuation of responses to 10/zM 5-HT by M R activation [11] also occurs for two other doses of 5-HT, i.e. 3 and 30 pM. Finally, we have extended the previously
30
A
A1
A3
I
B
10 mV
ls
12 11
E
je
I0 9
I
8
4 .3 2 I 0 O.
~e
jO j /jQ j
0.2
0.,3 0.4
0,5
0.6
0.7
0.8
0.9
1.0
1.
current (nA)
Fig. 2. A. The A H P of hippocampal CAI neurons, induced by a 1.0 nA depolarizing current pulse of 50 ms duration, increases in the presence of 30 n M corticosterone (A l). This specific GR-effect is impeded during treatment of corticosterone in the presence of a protein synthesis inhibitor, cycloheximide (A2). Cycloheximide itself does not affect the AHP-amplitude (A3). In A all records on the left represent a typical A H P obtained in slices from A D X rats, while the records on the right show the AHP-amplitude obtained in the same slices after steroid and/ or cycloheximide treatment. B. During depolarization with increasing positive currents, the AHP-amplitude also increases. From 0.2 to 1.0 nA the A H P of ADX-rat CAI neurons increases to ca. 6 mV (@). Stimulation of the GR-receptors by corticosterone in the presence of cycloheximide (A), contrarily to corticosterone (@) application alone, does not affect the AHP-amplitude. Cycloheximide does not affect the A H P ( • ) . Statistics were done with a multivariate analysis of variance test for repeated measurements.
reported corticosterone-induced increase of the AHP, associated with a 0.5 nA depolarizing current pulse [9], for a wide range of current intensities. The key finding of the present report is that neither the attenuation of the 5-HT response caused by MR activation nor the enhancement of the AHP amplitude caused by GR-activation occur in the presence of the protein synthesis inhibitor cycloheximide. This blockade of the steroid effect cannot be attributed to changes in membrane properties caused by cycloheximide itself since it failed to induce any changes in either membrane potential or resistance. This was also observed if neurons were
continuously recorded before, during or after cycloheximide application (unpublished observations). The latter means that the passive membrane properties are apparently largely independent of protein synthesis during slice incubation. The data with steroid treatment in the presence of cycloheximide support the hypothesis that the action of cycloheximide on in vitro m R N A translation, and thus protein synthesis, is responsible for preventing the development of MR- or GR-mediated effects on electrical properties of CA I neurons. Importantly, previous studies by others yielded a 99.5% inhibition of protein synthesis with the same dose of cycloheximide (100/~M) as used by us, under comparable in vitro conditions in rat cerebellar cell cultures [2]. Our results with cycloheximide have been confirmed in a limited series of experiments where we observed that a more potent protein synthesis inhibitor, anisomycin (20 /zM), also inhibits the MR-mediated effect on the hyperpolarizing 5-HT response (unpublished observations). The results are also in line with biochemical studies showing that corticosteroid treatment changes the expression of various mRNA's and proteins in the hippocampus [5, 6, 14, 16]. Since it is not known if the MRand GR-mediated actions on electrical properties actually involve DNA transcription, studies with transcription-inhibitors are in progress. A role of DNA transcription in corticosteroid actions is favored by the work of Etgen et al. [5], who showed that the enhancement of amino acid incorporation ([3H]leucine) by corticosterone can be blocked by ~-amanitin, an inhibitor of m R N A synthesis. In conclusion, we have shown that both MR- and GR-mediated changes of membrane properties fail to occur in the presence of a protein synthesis inhibitor, cycloheximide. The present report is, to our knowledge, the first to demonstrate that corticosteroid-induced effects on electrical membrane properties of CA 1 neurons requires protein synthesis, and thus suggest a genomic action of the steroids. The authors wish to thank Prof. Dr. H.O. Voorma for his biochemical advice and the gift of cycloheximide, Dr. N. Ramsey for his statistical advice and Prof. Dr. E.R. de Kloet for his comments on this manuscript. We furthermore thank Roussel-Uclaf, (Romainville, France) for the gift of RU38486 and Organon (Oss, The Netherlands) for the gift of corticosterone. This study was supported by grants to M.J. (900-533-028; H88-145) from the Dutch organization for scientific research (NWO). I Andrade, R., Malenka, C. and Nicoll, R.A., A G protein couples serotonin and GABAB receptors to the same channels in hippocampus, Science, 234 (1986) 1261 1264.
31 2 Burry, R.W., Protein synthesis requirement for the formation of synaptic elements, Brain Res., 344 (1985) 109-119. 3 Colino, A. and Halliwell, J.V., Differential modulation of three separate K-conductances in hippocampal CA1 neurons by serotonin, Nature, 328 (1987) 73-77. 4 De Kloet, E.R., Wallach, G. and McEwen, B.S., Differences in corticosterone and dexamethasone binding to rat brain and pituitary, Endocrinology, 96 (1975) 598409. 5 Etgen, A.M., Lee, K.S. and Lynch, G., Glucocorticoid modulation of specific protein metabolism in hippocampal slices, maintained in vitro, Brain Res., 165 (1979) 37-45. 6 Etgen, A.M., Martin, M., Gilbert, R. and Lynch, G., Characterization of corticosterone-induced protein synthesis in hippocampal slices, J. Neurochem., 35 (1980) 598-602. 7 Funder, J.W. and Sheppard, K., Adrenocortical steroids and the brain, Ann. Rev. Physiol., 49 (1987) 397-411. 8 Hua, S.H. and Chen, Y.Z., Membrane receptor mediated electrophysiological effects of glucocorticoid on mammalian neurons, Endocrinology, 124 (1989) 687491. 9 Jo61s, M. and de Kloet, E.R., Effects of glucocorticoids and norepinephrine on the excitability in the hippocampus, Science, 245 (1989) 1502-1505. 10 JoWls, M. and de Kloet, E.R., Mineralocorticoid receptor-mediated changes in membrane properties of rat CA1 pyramidal neurons in vitro, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 4495-4498.
11 JoWls, M., Hesen, W. and de Kloet, E.R., Mineralocorticoid hormones suppress serotonin-induced hyperpolarization of rat hippocampal CA 1 neurons, J. Neurosci. in press. 12 McEwen, B.S., Weiss, J.M. and Schwartz, L.S., Selective retention of corticosterone by limbic structures in rat brain, Nature, 220 (1968) 911412. 13 Munck, A., Guyre, P.M. and Holbrook, N.J., Physiological functions of glucocorticoids in stress and their relation to pharmacological actions, Endocr. Rev., 5 (1984) 25-44. 14 Nichols, N.R., Masters, J.N., May, P.C., de Vellis, J. and Finch, C.E., Corticosterone-induced responses in rat brain RNA are also evoked in hippocampus by acute vibratory stress, Neuroendocrinology, 49 (1989) 40-46. 15 Reul, J.M.H.M. and de Kloet, E.R., Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation, Endocrinology, 117 (1985) 2505-2512. 16 Schlatter, L.K., Ting, S.M., Meserve, L.A. and Dokas, L.A., Characterization of a glucocorticoid-sensitive hippocampal protein, Brain Res., 22 (1990) 215~23. 17 Schwarzkroin, P.A., Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation, Brain Res., 85 (1975) 423-436. 18 Schwarzkroin, P.A., Further characteristics of hippocampal CA1 cells in vitro, Brain Res., 128 (1977) 5348.
Neuroscience Letters, 130 (1991) 27 31 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 0304394091004677 NSL 07987
The induction of corticosteroid actions on membrane properties of hippocampal CA1 neurons requires protein synthesis H. Karst* a n d M. JoWls* Institute of Molecular Biology, University of Utrecht, Utrecht ( The Netherlands) (Received 28 March 1991; Revised version received 28 May 1991; Accepted 28 May 1991)
Key words.
Corticosterone; Mineralocorticoid receptor; Glucocorticoid receptor; Hippocampus; CA1; Cycloheximide; Electrophysiology
Corticosterone can affect electrical properties of CAl pyramidal neurons via binding to two corticoid receptor types, the mineralocorticoid (MR) and glucocorticoid receptor (GR). Previously we have shown that MR-activation leads to attenuation of serotonin (5-HT)-induced membrane hyperpolarization, while GR-activation induces an increase in the amplitude of the afterhyperpolarization (AHP) following a short current pulse. In this study we show that the MR- and GR-mediated changes of the membrane properties are prevented in the presence of the protein synthesis inhibitor cycloheximide, thus suggesting a genomic action of the steroids.
Corticosteroid hormones, ' released from the adrenal gland, can pass the blood-brain barrier and bind to intracellular receptors in the brain [4, 12]. Two intracellular corticosteroid receptor types have been distinguished: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) [7, 15]. Pyramidal cells in the rat hippocampal CA1 area contain both MR and GR. After binding of corticosteroids, the receptor-steroid complex shows increased affinity to the nuclear compartment and affects gene expression (see for review ref. 13). In previous electrophysiological studies it was shown that both MR and GR stimulation cause receptor-specific changes in the membrane properties of CA1 pyramidal neurons. Stimulation of the MR causes, with a delay of at least 15 min, a decrease of the accommodation and afterhyperpolarization (AHP) induced by a short depolarizing current pulse in CAI neurons [10]. The MRmediated action can be gradually overridden and reversed by a GR-mediated increase of the AHP amplitude [9]. In addition to these current-induced phenomena, corticosteroids can also affect transmitter-evoked changes in membrane properties, with a delay of several hours. Activation of GR was found to counteract fl-adrenoreceptor mediated actions in CA1 pyramidal cells [9], while it was recently shown that stimulation of MR attenuates *Present address: Department of Experimental Zoology, University of Amsterdam, Kruislaan 320, I098 SM Amsterdam, The Netherlands. Correspondence: H. Karst, Department of Experimental Zoology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands.
a 5HTiA-receptor mediated membrane hyperpolarization [11]. The fact that (1) these corticosteroid actions were also achieved by selective ligands for the intracellular MR or GR receptor and (2) all corticosteroid actions occurred with a considerable delay in time (15 min for MR [10] to approximately 1 h for GR actions [9]) suggests that corticosteroid hormones affect membrane properties via a genomic interaction. To test this hypothesis, we studied corticosteroid-mediated actions in the presence of a protein synthesis inhibitor, cycloheximide. As an index for MR-mediated actions we examined the effect of 30 nM corticosterone in the presence of the GR-antagonist RU38486, thus selectively activating the MRs, on hyperpolarizing responses to serotonin (5-HT). As an index for GR-mediated effects we studied the effect of 30 nM corticosterone on the amplitude of the AHP. Male Wistar rats (120-170 g) were adrenalectomized (ADX) 5-7 days before the experiments, in order to create a situation in which the receptors are unoccupied at the start of the electrophysiological experiments. Under these circumstances the receptors can be selectively occupied by MR or GR ligands. The animals were housed in an animal room with alternating light/dark (08.00-20.00 h/20.00-08.00 h) cycle and received food and saline (after ADX) ad libitum. On the day of the experiment the rat was placed in a clean cage 30-60 min before decapitation. After decapitation, trunk blood was collected for measurement of plasma corticosterone levels. The hippocampus was removed from the brain and dipped in ice cold oxygenated (95 % 02, 5 % CO2) artifi-
28
cial cerebrospinal fluid (ACSF: 124 mM NaCl, 3.5 mM KCI, 1.25 mM NaHzPO4, 1.5 mM MgSO4, 2 mM CaCI2, 25 mM NaHCO3, 10 mM glucose). Transverse slices of the dorsal hippocampus were cut (_+ 350 /~m) on a McIwain tissue chopper and submerged in a perfusing chamber. The slices were continuously perfused with oxygenated ACSF (32°C) at a constant rate of 2-3 ml/ min. Corticosterone (Organon International, Oss, the Netherlands) and RU38486 (Roussel-Uclaf, Romainville, France) were, for each experiment, dissolved in ethanol (1 mM) and diluted to the intended concentration in oxygenated ACSF immediately before application. Cycloheximide (100/~M, Sigma) and 5-HT (treat±nine sulfate complex, 100/IM stock-solution, diluted to the intended concentration immediately before the experiment, Sigma) were dissolved in ACSF. Intracellular recordings of pyramidal CAI neurons were performed with 4 M KAc-filled microelectrodes (impedance: 80-150M[2). From each neuron we recorded the resting membrane potential, input resistance, spike accommodation/AHP evoked by 50 ms (0.2-1.0 nA) or 500 ms (0.5 nA) depolarizing current pulses. In one series of experiments we tested the 'control' MR-effect on 5-HT responsiveness by recording responses to 1, 3, 10 and 30/~M 5-HT 1-2 h after a 20 min perfusion of 30 nM corticosterone, in the presence of the GR-antagonist RU38486 (500 nM perfused in total for 60 min, starting 20 min before corticosterone application). In another series of experiments we tested the 'control' GR-mediated responses by establishing the amplitude of the AHP, 1-2 h after a 20 min corticosterone application (30 nM). We subsequently tested the effect of cycloheximide (100 /tM) alone, by comparing membrane properties and responses to 5-HT obtained before cycloheximide application with responses recorded 1-2 h after onset of cycloheximide. Finally, we examined the MR- and GR- mediated actions in the presence of cycloheximide. Administration of the protein synthesis inhibitor was started 20 min before application of corticosterone and continued for at least 2 h. In total we recorded from 45 identified [17, 18] CAI pyramidal neurons. The averaged membrane potentials and input resistances of the experimental groups (Table I) ranged from -65.8 to -71.2 mV and from 35.7 to 44.9 M~, respectively. No significant differences were observed for membrane potential or resistance between neurons recorded in slices treated with corticosterone and cycloheximide (GR effect in the presence of cycloheximide) and neurons in slices treated with either corticosterone (GR effect), with cycloheximide or nontreated ADX cells. Similarly, no differences in these parameters were found between neurons in slices perfused with corticosterone, RU38486 and cycloheximide (MR
TABLE I RESTING MEMBRANE POTENTIAL (RMP; + S.E.M.) AND INPUT RESISTANCE (R~n) OF THE GROUPS OF HIPPOCAMPAL CA1 NEURONS INCORPORATED IN THIS STUDY No significant differences were observed for membrane potential and resistance between groups of neurons compared in this study: Group III (GR effect) with I and II, and group IV (MR effect) with I and II. *Resting membrane potential for cells treated with corticosterone in the presence of cycloheximide was found to be significantly different from the cells treated with corticosterone and RU38486. As these groups of neurons were not compared in the rest of this study, our data were not affected by this difference. Statistics were done with the oneway analysis of variance with the Student-Newman-Keuls test for multiple comparisons between means (P < 0.05). Treatment
n
RMP (mV)
Rin (MI2)
1,
ADX
7
69.6±1.7
38.2±1.95
II.
l 2h 100/aM cycloheximide
6
66.2±1.5
39.2±3.13
III.
30 nM corticosterone
9
68.1±0.7
41.9±2.1
100/aM cycloheximide 30 nM corticosterone
7
71.2±2.1"
43.3±2.2
100/aM cycloheximide 500 nM RU38486/ 30 nM corticosterone
7
68.7±1.0
35.7±2.2
500 nM RU38486/ 30 nM corticosterone
9
65.8±1.1"
~.9±5.3
IV.
effect in the presence of cycloheximide) and neurons in slices treated with either corticosterone/RU38486, with cycloheximide or non-treated slices. Resting membrane potentials for the cells treated with corticosterone and cycloheximide (71.2 _+ 2. I mV) were significantly different from cells treated with corticosterone and RU38486 (65.8 + 1.1 mV). As these groups of neurons were not compared in the rest of the study, our conclusions were not affected by this difference. As reported previously by us and others [1, 3, 11] we observed that 5-HT induces a hyperpolarization of the CA1 pyramidal cell membrane potential and a decrease of the input resistance. The lowest effective dose is 1/~M, while near-maximal responses are obtained with 30/tM 5-HT (see Fig. 1). Activation of the MR significantly attenuated the membrane hyperpolarization and resistance decrease induced by 3, 10 or 30/tM 5-HT (Fig. 1). The MR-mediated decrease of hyperpolarizations induced by 3, 10 or 30/IM 5-HT was no longer observed in the presence of the protein synthesis inhibitor cycloheximide. Similarly, the MR-mediated attenuation of the resistance change evoked by 10 and 30/tM was pre-
29 A1
A2 30 laM 5HT
A3 30/aM 5HT
30 taM 5HT
A 30 nM corticosterone
100 taM cycloheximide
500 nM RU38486
100 laM cycloheximide
30 nM corlicosterone _
500 nM RU38486
_
110my
I min
B
C 10 ¸ ~
> IE
e-
t.9
40. T
8 6,
°
E E 0-
30. 20.
.5
e~
~. e-
o~ .~-
9.
l
"~
10.
E ~
0-
i f
-1
-10 1
1'0
100
[5HT] ([LtM)
100
[5HT] (pM)
Fig. 1. A: the hyperpolarization of hippocampal CAI neurons by serotonin (30 pM 5-HT) is attenuated during MR stimulation by corticosterone in the presence of a GR antagonist (RU38486) (A1). In the presence of a protein synthesis inhibitor, cycloheximide, this MR-specific effect is prevented (A2). Cycloheximide alone does not affect the 5-HT response (A3). The downward deflexions in the registrations are hyperpolarizing current pulses ( - 0 . 3 nA, 100 ms) passed through the recording electrode to measure membrane resistance. The membrane potential was repolarized during the hyperpolarized 5-HT response, by applying a positive current, to measure real resistance changes. Black bars indicate the duration of the 5-HT-perfusion. B,C: stimulation of the 5-HT1A receptors with 5-HT hyperpolarizes hippocampal CA 1 neurons and decreases the input resistance in a dose-dependent manner (ADX, ©). Corticosterone (30 nM) in the presence of a GR antagonist, RU38486, thus causing MR activation, significantly inhibits the actions evoked by 3, 10 and 30 pM 5-HT ( • ) (P < 0.05). The MR-mediated attenuation of the 5-HT responses does not occur in the presence of cycloheximide (A), a protein synthesis inhibitor. Cycloheximide itself does not affect this 5-HT hyperpolarizing response (O). Statistics were done with a one-way analysis of variance followed by the Student-Newman-Keuls test for multiple comparisons (P < 0.05).
vented by cycloheximide. The action of cycloheximide was less clear for 3 pM 5-HT, although statistical analysis showed that, here too, the inhibition of the 5-HTmediated change in resistance by M R activation was no longer observed. As can be seen in Fig. 1, cycloheximide alone affected neither the 5-HT-evoked hyperpolarization nor the change in resistance. In the present study we furthermore observed that neurons recorded 30-90 min after a 20 min perfusion with 30 nM corticosterone show a significantly increased AHP amplitude over the entire range of current intensities tested (Fig. 2). This extends a previous observation that a 20 min application of corticosterone induces after a delay of ca. 1-2 h, a significant increase in the AHP evoked by a 0.5 nA depolarizing current pulse [10]. We found that, in the presence of cycloheximide, corticoste-
rone is no longer able to affect the AHP. As described for the MR-mediated effects on 5-HT responsiveness, cycloheximide by itself did not affect the AHP amplitude. In this study we extended previously reported MRand GR-mediated changes in electrical properties of CA1 pyramidal neurons. Neither M R nor G R occupation appeared to affect passive membrane properties of identified CA1 neurons. These observations are in agreement with previous studies by us [9, 10], but contrast with work by others where rapid steroid-induced changes in passive membrane properties were reported [8]. We show, furthermore, that the previously reported attenuation of responses to 10/zM 5-HT by M R activation [11] also occurs for two other doses of 5-HT, i.e. 3 and 30 pM. Finally, we have extended the previously
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Fig. 2. A. The A H P of hippocampal CAI neurons, induced by a 1.0 nA depolarizing current pulse of 50 ms duration, increases in the presence of 30 n M corticosterone (A l). This specific GR-effect is impeded during treatment of corticosterone in the presence of a protein synthesis inhibitor, cycloheximide (A2). Cycloheximide itself does not affect the AHP-amplitude (A3). In A all records on the left represent a typical A H P obtained in slices from A D X rats, while the records on the right show the AHP-amplitude obtained in the same slices after steroid and/ or cycloheximide treatment. B. During depolarization with increasing positive currents, the AHP-amplitude also increases. From 0.2 to 1.0 nA the A H P of ADX-rat CAI neurons increases to ca. 6 mV (@). Stimulation of the GR-receptors by corticosterone in the presence of cycloheximide (A), contrarily to corticosterone (@) application alone, does not affect the AHP-amplitude. Cycloheximide does not affect the A H P ( • ) . Statistics were done with a multivariate analysis of variance test for repeated measurements.
reported corticosterone-induced increase of the AHP, associated with a 0.5 nA depolarizing current pulse [9], for a wide range of current intensities. The key finding of the present report is that neither the attenuation of the 5-HT response caused by MR activation nor the enhancement of the AHP amplitude caused by GR-activation occur in the presence of the protein synthesis inhibitor cycloheximide. This blockade of the steroid effect cannot be attributed to changes in membrane properties caused by cycloheximide itself since it failed to induce any changes in either membrane potential or resistance. This was also observed if neurons were
continuously recorded before, during or after cycloheximide application (unpublished observations). The latter means that the passive membrane properties are apparently largely independent of protein synthesis during slice incubation. The data with steroid treatment in the presence of cycloheximide support the hypothesis that the action of cycloheximide on in vitro m R N A translation, and thus protein synthesis, is responsible for preventing the development of MR- or GR-mediated effects on electrical properties of CA I neurons. Importantly, previous studies by others yielded a 99.5% inhibition of protein synthesis with the same dose of cycloheximide (100/~M) as used by us, under comparable in vitro conditions in rat cerebellar cell cultures [2]. Our results with cycloheximide have been confirmed in a limited series of experiments where we observed that a more potent protein synthesis inhibitor, anisomycin (20 /zM), also inhibits the MR-mediated effect on the hyperpolarizing 5-HT response (unpublished observations). The results are also in line with biochemical studies showing that corticosteroid treatment changes the expression of various mRNA's and proteins in the hippocampus [5, 6, 14, 16]. Since it is not known if the MRand GR-mediated actions on electrical properties actually involve DNA transcription, studies with transcription-inhibitors are in progress. A role of DNA transcription in corticosteroid actions is favored by the work of Etgen et al. [5], who showed that the enhancement of amino acid incorporation ([3H]leucine) by corticosterone can be blocked by ~-amanitin, an inhibitor of m R N A synthesis. In conclusion, we have shown that both MR- and GR-mediated changes of membrane properties fail to occur in the presence of a protein synthesis inhibitor, cycloheximide. The present report is, to our knowledge, the first to demonstrate that corticosteroid-induced effects on electrical membrane properties of CA 1 neurons requires protein synthesis, and thus suggest a genomic action of the steroids. The authors wish to thank Prof. Dr. H.O. Voorma for his biochemical advice and the gift of cycloheximide, Dr. N. Ramsey for his statistical advice and Prof. Dr. E.R. de Kloet for his comments on this manuscript. We furthermore thank Roussel-Uclaf, (Romainville, France) for the gift of RU38486 and Organon (Oss, The Netherlands) for the gift of corticosterone. This study was supported by grants to M.J. (900-533-028; H88-145) from the Dutch organization for scientific research (NWO). I Andrade, R., Malenka, C. and Nicoll, R.A., A G protein couples serotonin and GABAB receptors to the same channels in hippocampus, Science, 234 (1986) 1261 1264.
31 2 Burry, R.W., Protein synthesis requirement for the formation of synaptic elements, Brain Res., 344 (1985) 109-119. 3 Colino, A. and Halliwell, J.V., Differential modulation of three separate K-conductances in hippocampal CA1 neurons by serotonin, Nature, 328 (1987) 73-77. 4 De Kloet, E.R., Wallach, G. and McEwen, B.S., Differences in corticosterone and dexamethasone binding to rat brain and pituitary, Endocrinology, 96 (1975) 598409. 5 Etgen, A.M., Lee, K.S. and Lynch, G., Glucocorticoid modulation of specific protein metabolism in hippocampal slices, maintained in vitro, Brain Res., 165 (1979) 37-45. 6 Etgen, A.M., Martin, M., Gilbert, R. and Lynch, G., Characterization of corticosterone-induced protein synthesis in hippocampal slices, J. Neurochem., 35 (1980) 598-602. 7 Funder, J.W. and Sheppard, K., Adrenocortical steroids and the brain, Ann. Rev. Physiol., 49 (1987) 397-411. 8 Hua, S.H. and Chen, Y.Z., Membrane receptor mediated electrophysiological effects of glucocorticoid on mammalian neurons, Endocrinology, 124 (1989) 687491. 9 Jo61s, M. and de Kloet, E.R., Effects of glucocorticoids and norepinephrine on the excitability in the hippocampus, Science, 245 (1989) 1502-1505. 10 JoWls, M. and de Kloet, E.R., Mineralocorticoid receptor-mediated changes in membrane properties of rat CA1 pyramidal neurons in vitro, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 4495-4498.
11 JoWls, M., Hesen, W. and de Kloet, E.R., Mineralocorticoid hormones suppress serotonin-induced hyperpolarization of rat hippocampal CA 1 neurons, J. Neurosci. in press. 12 McEwen, B.S., Weiss, J.M. and Schwartz, L.S., Selective retention of corticosterone by limbic structures in rat brain, Nature, 220 (1968) 911412. 13 Munck, A., Guyre, P.M. and Holbrook, N.J., Physiological functions of glucocorticoids in stress and their relation to pharmacological actions, Endocr. Rev., 5 (1984) 25-44. 14 Nichols, N.R., Masters, J.N., May, P.C., de Vellis, J. and Finch, C.E., Corticosterone-induced responses in rat brain RNA are also evoked in hippocampus by acute vibratory stress, Neuroendocrinology, 49 (1989) 40-46. 15 Reul, J.M.H.M. and de Kloet, E.R., Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation, Endocrinology, 117 (1985) 2505-2512. 16 Schlatter, L.K., Ting, S.M., Meserve, L.A. and Dokas, L.A., Characterization of a glucocorticoid-sensitive hippocampal protein, Brain Res., 22 (1990) 215~23. 17 Schwarzkroin, P.A., Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation, Brain Res., 85 (1975) 423-436. 18 Schwarzkroin, P.A., Further characteristics of hippocampal CA1 cells in vitro, Brain Res., 128 (1977) 5348.
