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Actions of 3α,5α-tetrahydrodeoxycorticosterone on single neurones of the mesencephalic reticular formation in the rat
Neuroscience Letters, 104 (1989) 115 120 Elsevier Scientific Publishers Ireland Ltd.
115
NSL 06322
Actions of 3c ,5e-tetrahydrodeoxycorticosterone on single neurones of the mesencephalic reticular formation in the rat Rosa Ermirio, Donatella Blanchi, Piero Ruggeri, Carla E. Cogo and Claudio Molinari lstituto di Fisiologia Umana, Universitg~ degli Studi, Genova (Italy) (Received 31 January 1989; Revised version received 15 May 1989; Accepted 19 May 1989)
Key word~." Steroid hormone; Tetrahydrodeoxycorticosterone; Brainstem; Reticular formation; Microinjection; Rat The effects of microinjections of 3ct,5ct-tetrahydrodeoxycorticosterone (3ct-THDOC) on single neurones of the mesencephalic reticular formation (M RF) were investigated in rats anaesthetized with urethan. Microinjections of approximately 100 nl of 0.5 2 ~M 3~-THDOC inhibited firing of 105 of 112 neurones (94%). Microinjections of approximately 100 nl of 100-250 nM of 3ct-THDOC did not alter neuronal activity, but in 52 of 68 cases (76%) it potentiated the inhibitory action of microiontophoretically applied 7-aminobutyric acid (GABA). The 3fl-isomer of tetrahydrodeoxycorticosterone did not elicit any changes in neuronal firing. The effects of 3ct-THDOC were reversibly antagonized by microiontophoretically applied bicuculline. This 'in vivo' study supports the hypothesis that 3ct-THDOC may function as endogenous modulator of GABAA-mediated inhibition in various physiopathological conditions.
Steroid hormones act on the central nervous system (CNS) to produce both rapid and delayed neuroendocrine and behavioural effects [6, 9]. Rapid neurotropic effects, such as alterations of neuronal excitability and rapid feedback control of the release of hypothalamic hormones may account for various physiological, pathological and behavioural phenomena [I, 2, 6, 10]. Among steroids affecting neuronal excitability, recent 'in vitro' studies indicate that 3~,5c(-tetrahydrodeoxycorticosterone (3c(-THDOC), a metabolite of deoxycorticosterone, is a potent agonist of the 7-aminobutyric acid (GABA) receptor-chloride ion channel complex [5, 6, 9]. The mammalian adrenal cortex secretes, along with classical hormones, 3~-THDOC and its precursor [14] and the enzymes converting deoxycorticosterone to 3~-THDOC are present in brain areas such as the midbrain tegmentum, medulla, hypothalamus and other limbic structures [12, 13]. These observations suggest a role of 3~-THDOC in the regulation of certain nervous functions, such as emotions, sleep and wakefulness, postural control and other vital activities [6, 9]. The mesencephalic reticular formation (MRF) Correspondence." R. Ermirio, Istituto di Fisiologia Umana, Universitfi degli Studi, Viale Benedetto XV, 3, 1-16132 Genova, Italy. 0304-3940/89/$ 03.50 ~ 1989 Elsevier Scientific Publishers Ireland Ltd.
116
is believed to play an important role in some of the nervous functions in which steroids have been implicated [4, 15]. The aim of this study was to investigate "in vivo' the effects of microinjections of different concentrations of 3:~-THDOC on the firing rate of single M RF neurones. Experiments were done in 25 Wistar male rats weighing 280 320 g. The animals were anaesthetized with urethan (150 mg/100 g b.wt., i.p.: Sigma, St. Louis, MO, U.S.A.). The trachea was cannulated and rectal temperature was controlled and maintained at 37.5 + 0.5'C. Burr holes (3 mm diam.) were made in the skull near the midline in one or both sides and at different locations between bregma and 7 mm posterior. These procedures allowed the exposure of small areas of the cerebral cortex. Alter the dura was removed, multibarrel micropipettes were stereotaxically introduced from 0.5 to 2 mm lateral to the midline in the mesencephalon, i.e., from +0.8 to + 2.2 AP coordinates, according to De Groot as reported in the atlas of Pellegrino et al. [11]. Five-barrel micropipettes were used. The central barrel, filled with 4 M NaC1, was used lbr extracellular recording, while the outer barrels were used for the application of drugs. A
4
6
50
Q. O3
~JM
B
3c~-THDOC
(Inpsl}
4__
rain
4__ 5
5
5O
0 l O 0 n M 3c~-THDOC =
GABA
(inps~)
rain
{m n A )
Fig. 1. Frequency histograms of unitary activity showing effects of microinjections of 3~,5ct-tetrahydrodeoxycorticosterone (3~-THDOC) on firing rate of two mesencephalic reticular formation (MRF) neurones. A: direct inhibitory effect of 1 pM 3c~-THDOC. B: indirect effect of 100 nM 3~-THDOC by potentiation of inhibitory action of microiontophoretically applied 7-aminobutyric acid (GABA).
117
3~-THDOC and 3fl,5~-tetrahydrodeoxycorticosterone (3fl-THDOC) were first dissolved in 48% ethanol at a concentration of 1 mM and then diluted in phosphatebuffered saline (PBS; pH 7.4) to the desired concentration. Pressure microinjection of 37-THDOC, 3fl-THDOC or vehicle was performed by connecting injection barrels through polyethylene tubing to pneumatic pumps (Module PPM-2, Medical Systems Corp., U.S.A.). Injection volumes were measured by direct observation of the fluid meniscus in the micropipette lumen through a microscope fitted with an ocular micrometer. The timing and the amount of each pressure pulse were regulated so that the maximum injection volume did not exceed 100 nl. y-Aminobutyric acid (GABA; 0.1 M, pH 5.6) and bicuculline (5 mM, pH 3) were applied by microiontophoresis (Neurophore Microiontophoresis System, BH2, Medical Systems Corp., U.S.A.). GABA was applied by positive currents between 5 and 15 nA. Bicuculline was applied by positive currents between 20 and 60 nA. Backing currents ranged from 8 to 12 nA. One barrel containing 1 M NaCI was used to pass a balancing current. In 8 experiments Pontamine sky blue (2.5% solution in sodium acetate buffer, pH 5.6), according to Boakes et al. [3], was used in place of the saline in the current balancing barrel as a marker of the position of the electrode tip for histological examination of the recording sites. Standard recording procedures were followed and the neuronal spikes were counted by a D130 Spike Processor (Digitimer Ltd., U.K.). Neuronal activity and times of application of drugs were recorded on magnetic tape (SE Eight-Four, EMI, U.K.) for later analysis. The critical ratio (CR) test was used to statistically evaluate changes in the neuronal firing rate. Values of CR > 1.96 were considered significant and correspond to approximately a 30% change in baseline firing rate [16]. The effects of different concentrations of 3~-THDOC on neuronal firing rate were studied in 14 rats. Microinjections of approximately 100 nl of 0.5 2/tM 3c~-THDOC inhibited firing of 105 of 112 M R F neurones (94%). The effects were maximal within 10-15 s and recovery occurred 20-30 s after the end of the application (see Fig. I A). Microinjections of approximately 100 nl of 100-250 nM 3~-THDOC did not cause statistically significant changes in neuronal firing, but in 52 of 68 cases (76%) it potentiated the inhibitory action of microiontophoreticatly applied GABA (see Fig. 1B). 50 nM 3c~-THDOC failed to potentiate GABA action. 3fl-THDOC microinjected in 100 nl volumes at concentrations ranging from 0.5 to 4/zM on 48 M R F neurones previously or successively tested with 3~-THDOC failed to produce changes in neuronal firing either directly (Fig. 2) or by potentiation of GABA action. Microinjection of vehicle on 16 M R F neurones also failed to elicit any responses. In 11 rats the efficacy of bicuculline as antagonist of neuronal depression produced by 37-THDOC was studied in comparison with its selective GABA antagonism. In 27 of the 34 neurones (79%), in which bicuculline blocked GABA-induced inhibition of firing, it was also capable of reducing the inhibitory effects of 0.5-2/tM 3~-THDOC (see Fig. 3). This antagonism was observed as a reversible slowing of the onset of action and/or a reduction or an abolition of the maximum inhibitory effect of 3~-THDOC. Complete recovery of the effectiveness of 3~-THDOC occurred within
118 3 50-
3
--
6. CO
0 rain 1 pM
3~-THDOC
(in psi.)
2 pM
313-THDOC (in p s i )
Fig. 2. Frequency histogram of a single unit in the mesencephalic reticular formation showing that microinjection of 2/lM 3fl,5~-tetrahydrodeoxycorticosterone (3fl-THDOC) did not elicit any response, compared with the inhibitory effect induced by microinjection of 1 /tM 3c~,5~-tetrahydrodeoxycorticosterone (3~-THDOC). A
B1cuculhne
10
50hA 10
10
50
I3. (/3
rain
GABA Qn nA)
B
Bicuculllne 4
50hA 4
4
50
Q.
ca0
0 I
~ pM
3o(-THDOC
{in p s i )
min
Fig. 3. Frequency histograms of a single unit in the mesencephalic reticular formation showing comparison of the effectiveness of bicuculline as antagonist, in A, of x-aminobutyric acid (GABA) and, in B, of 3c~,5~tetrahydrodeoxycorticosterone (3~-THDOC). In both cases, complete recoveries of the actions both of GABA and of 3~-THDOC are not illustrated in the figure.
119 3-4 min. In each case the effect of bicuculline alone was monitored for at least 1 min, before evaluating its antagonistic properties (see Fig. 3). The occasional changes in the neuronal firing rate induced by bicuculline were not correlated with GABA and 3ct-THDOC antagonism. The results of this study demonstrate that 3ct-THDOC exerts a potent inhibitory action on neuronal firing rate when applied in very small amounts to M R F neurones. The short onset latency of the effects and their brief duration after the end of the application suggest that they are non-genomic effects presumably due to interactions with membrane components [10]. The potentiation of GABA inhibitory action by 3at-THDOC and the observation that the depressant effects by this steroid could be blocked by bicuculline demonstrate interaction of 3~-THDOC with the GABAA receptor complex. The effects of 3~-THDOC appear to be specific. This is supported by the observation that microinjection of comparable volumes of vehicle did not elicit any response. 3fl-THDOC also failed to elicit changes in neuronal firing either directly or by potentiation of GABA action. This shows that there is a high degree of structural discrimination at the steroid binding sites and precise steric requirem,ents for 3~-THDOC action at the GABAA receptor complex are needed, as already observed by other laboratories for other steroids [5, 6]. These results are in agreement with 'in vitro' studies concerning the effects of 3ct-THDOC on membrane conductance, action potential generation and selective binding of GABA agonists or antagonists [5, 9]. 'In vitro' evidence has shown that glucocorticoid hormones can also affect neuronal excitability through interactions with the GABA receptor system [6-8]. However, our previous reports [1, 2] have shown that local or systemic administration of corticosterone to neurones of the brainstem reticular formation can induce both excitatory and inhibitory effects on neuronal firing rate. This supports the hypothesis [6, 7] that additional factors, varying from cell to cell, should be important in determining the agonistic- or antagonistic-type of interaction of glucocorticoids with the G A B A receptor system. On the contrary, the uniform inhibitory effect obtained in this study by local applications of 3~-THDOC on M R F neurones suggests that 3~-THDOC interaction with GABA receptors is independent of local neurochemical factors. In conclusion, this 'in vivo' study supports the hypothesis that 3~-THDOC may function as endogenous modulator of GABA-mediated inhibition in various physiological, pathological and behavioural states. The authors thank Prof. F.R. Calaresu for critical comments and suggestions on the manuscript. 1 Avanzino, G.L., Celasco, G., Cogo, C.E., Ermirio, R. and Ruggeri, P., Actions of microelectrophoretically applied glucocorticoidhormones on reticular formation neurones in the rat, Neurosci. Lett., 38 (1983)4549. 2 Avanzino,G.L., Ermirio, R., Ruggeri, P. and Cogo, C.E., Effectsof corticosteroneon neurons of reticular formation in rats, Am. J. Physiol.,253 (1987) R25 R30.
120 3 Boakes, R.J., Bramwell, G.J., Briggs, I., Candy, J.M. and Tempesta, E., Localization with Pontamine sky blue of neurones in the brainstem responding to microiontophoretically applied compounds, Neuropharmacology, 13 (1974) 475 479. 4 Brodal, A., Neurological Anatomy (3rd edn.), Oxford Univ. Press, New York, 1981, pp. 394 447. 5 Harrison, N.L., Majewska, M.D., Harrington, J.W. and Barker, J.L., Structure~ctivity relationships for steroid interaction with the y-aminobutyric acida receptor complex, J. Pharmacol. Exp. Ther., 241 (1987) 34(~353. 6 Majewska, M.D., Steroids and brain activity. Essential dialogue between body and mind, Biochem. Pharmacol., 36 (t987) 3781 3788. 7 Majewska, M.D., Antagonist-type interaction of glucocorticoids with the GABA receptor-coupled chloride channel, Brain Res., 418 (1987) 377 382. 8 Majewska, M.D., Bisserbe, J.C. and Eskay, R.L., Glucocorticoids are modulators of GABAA receptors in brain, Brain Res., 339 (1985) 178 182. 9 Majewska, M.D., Harrison, N.L., Schwartz, R.D., Barker, J.L. and Paul, S.M., Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor, Science, 232 (1986) 1004 1007. l0 McEwen, B.S., Davis, P.G., Parsons, B. and Pfaff, D.W., The brain as a target for steroid hormone action, Annu. Rev. Neurosci., 2 (1979) 65 112. I1 Pellegrino, L.J., Pellegrino, A.S. and Cushman, A.J.. A Stereotaxic Atlas of the Rat Brain (2nd edn.), Plenum Press, New York, 1981, 35 pp. 12 Rommerts, F.F.G. and Van Der Molen, H.J., Occurrence and localization of 5c~-steroidreductase, 3:~and 17fl-hydroxysteroid dehydrogenases in hypothalamus and other brain tissues of the male rat, Biochim. Biophys. Acta, 248 ( 1971 ) 489 502. 13 Roselli, ('.E. and Snipes, ('.A., Progesterone-5:~-reductase in mouse brain, Brain Res., 305 (1984) 197 202. 14 Schambelan, M. and Biglieri, E.G., Deoxycorticosterone production and regulation in man, J. Clin. Endocrinol., 34 ~1972) 695 703. 15 Scheibel, A.B., The brain stem reticular core and sensory function. In Handbook of Physiology, The Nervous System. Sensory Processes, Sect. I, Vol. Ill, Bethesda, MD, 1984, pp. 213 256. 16 Stone, T.W., Microiontophoresis and pressure ejection. In A.D. Smith (Ed.), IBRO Handbook Series: Methods in the Neurosciences. Vol. 8, Wiley, Chichester, U.K., 1985, pp. 96, 98.
115
NSL 06322
Actions of 3c ,5e-tetrahydrodeoxycorticosterone on single neurones of the mesencephalic reticular formation in the rat Rosa Ermirio, Donatella Blanchi, Piero Ruggeri, Carla E. Cogo and Claudio Molinari lstituto di Fisiologia Umana, Universitg~ degli Studi, Genova (Italy) (Received 31 January 1989; Revised version received 15 May 1989; Accepted 19 May 1989)
Key word~." Steroid hormone; Tetrahydrodeoxycorticosterone; Brainstem; Reticular formation; Microinjection; Rat The effects of microinjections of 3ct,5ct-tetrahydrodeoxycorticosterone (3ct-THDOC) on single neurones of the mesencephalic reticular formation (M RF) were investigated in rats anaesthetized with urethan. Microinjections of approximately 100 nl of 0.5 2 ~M 3~-THDOC inhibited firing of 105 of 112 neurones (94%). Microinjections of approximately 100 nl of 100-250 nM of 3ct-THDOC did not alter neuronal activity, but in 52 of 68 cases (76%) it potentiated the inhibitory action of microiontophoretically applied 7-aminobutyric acid (GABA). The 3fl-isomer of tetrahydrodeoxycorticosterone did not elicit any changes in neuronal firing. The effects of 3ct-THDOC were reversibly antagonized by microiontophoretically applied bicuculline. This 'in vivo' study supports the hypothesis that 3ct-THDOC may function as endogenous modulator of GABAA-mediated inhibition in various physiopathological conditions.
Steroid hormones act on the central nervous system (CNS) to produce both rapid and delayed neuroendocrine and behavioural effects [6, 9]. Rapid neurotropic effects, such as alterations of neuronal excitability and rapid feedback control of the release of hypothalamic hormones may account for various physiological, pathological and behavioural phenomena [I, 2, 6, 10]. Among steroids affecting neuronal excitability, recent 'in vitro' studies indicate that 3~,5c(-tetrahydrodeoxycorticosterone (3c(-THDOC), a metabolite of deoxycorticosterone, is a potent agonist of the 7-aminobutyric acid (GABA) receptor-chloride ion channel complex [5, 6, 9]. The mammalian adrenal cortex secretes, along with classical hormones, 3~-THDOC and its precursor [14] and the enzymes converting deoxycorticosterone to 3~-THDOC are present in brain areas such as the midbrain tegmentum, medulla, hypothalamus and other limbic structures [12, 13]. These observations suggest a role of 3~-THDOC in the regulation of certain nervous functions, such as emotions, sleep and wakefulness, postural control and other vital activities [6, 9]. The mesencephalic reticular formation (MRF) Correspondence." R. Ermirio, Istituto di Fisiologia Umana, Universitfi degli Studi, Viale Benedetto XV, 3, 1-16132 Genova, Italy. 0304-3940/89/$ 03.50 ~ 1989 Elsevier Scientific Publishers Ireland Ltd.
116
is believed to play an important role in some of the nervous functions in which steroids have been implicated [4, 15]. The aim of this study was to investigate "in vivo' the effects of microinjections of different concentrations of 3:~-THDOC on the firing rate of single M RF neurones. Experiments were done in 25 Wistar male rats weighing 280 320 g. The animals were anaesthetized with urethan (150 mg/100 g b.wt., i.p.: Sigma, St. Louis, MO, U.S.A.). The trachea was cannulated and rectal temperature was controlled and maintained at 37.5 + 0.5'C. Burr holes (3 mm diam.) were made in the skull near the midline in one or both sides and at different locations between bregma and 7 mm posterior. These procedures allowed the exposure of small areas of the cerebral cortex. Alter the dura was removed, multibarrel micropipettes were stereotaxically introduced from 0.5 to 2 mm lateral to the midline in the mesencephalon, i.e., from +0.8 to + 2.2 AP coordinates, according to De Groot as reported in the atlas of Pellegrino et al. [11]. Five-barrel micropipettes were used. The central barrel, filled with 4 M NaC1, was used lbr extracellular recording, while the outer barrels were used for the application of drugs. A
4
6
50
Q. O3
~JM
B
3c~-THDOC
(Inpsl}
4__
rain
4__ 5
5
5O
0 l O 0 n M 3c~-THDOC =
GABA
(inps~)
rain
{m n A )
Fig. 1. Frequency histograms of unitary activity showing effects of microinjections of 3~,5ct-tetrahydrodeoxycorticosterone (3~-THDOC) on firing rate of two mesencephalic reticular formation (MRF) neurones. A: direct inhibitory effect of 1 pM 3c~-THDOC. B: indirect effect of 100 nM 3~-THDOC by potentiation of inhibitory action of microiontophoretically applied 7-aminobutyric acid (GABA).
117
3~-THDOC and 3fl,5~-tetrahydrodeoxycorticosterone (3fl-THDOC) were first dissolved in 48% ethanol at a concentration of 1 mM and then diluted in phosphatebuffered saline (PBS; pH 7.4) to the desired concentration. Pressure microinjection of 37-THDOC, 3fl-THDOC or vehicle was performed by connecting injection barrels through polyethylene tubing to pneumatic pumps (Module PPM-2, Medical Systems Corp., U.S.A.). Injection volumes were measured by direct observation of the fluid meniscus in the micropipette lumen through a microscope fitted with an ocular micrometer. The timing and the amount of each pressure pulse were regulated so that the maximum injection volume did not exceed 100 nl. y-Aminobutyric acid (GABA; 0.1 M, pH 5.6) and bicuculline (5 mM, pH 3) were applied by microiontophoresis (Neurophore Microiontophoresis System, BH2, Medical Systems Corp., U.S.A.). GABA was applied by positive currents between 5 and 15 nA. Bicuculline was applied by positive currents between 20 and 60 nA. Backing currents ranged from 8 to 12 nA. One barrel containing 1 M NaCI was used to pass a balancing current. In 8 experiments Pontamine sky blue (2.5% solution in sodium acetate buffer, pH 5.6), according to Boakes et al. [3], was used in place of the saline in the current balancing barrel as a marker of the position of the electrode tip for histological examination of the recording sites. Standard recording procedures were followed and the neuronal spikes were counted by a D130 Spike Processor (Digitimer Ltd., U.K.). Neuronal activity and times of application of drugs were recorded on magnetic tape (SE Eight-Four, EMI, U.K.) for later analysis. The critical ratio (CR) test was used to statistically evaluate changes in the neuronal firing rate. Values of CR > 1.96 were considered significant and correspond to approximately a 30% change in baseline firing rate [16]. The effects of different concentrations of 3~-THDOC on neuronal firing rate were studied in 14 rats. Microinjections of approximately 100 nl of 0.5 2/tM 3c~-THDOC inhibited firing of 105 of 112 M R F neurones (94%). The effects were maximal within 10-15 s and recovery occurred 20-30 s after the end of the application (see Fig. I A). Microinjections of approximately 100 nl of 100-250 nM 3~-THDOC did not cause statistically significant changes in neuronal firing, but in 52 of 68 cases (76%) it potentiated the inhibitory action of microiontophoreticatly applied GABA (see Fig. 1B). 50 nM 3c~-THDOC failed to potentiate GABA action. 3fl-THDOC microinjected in 100 nl volumes at concentrations ranging from 0.5 to 4/zM on 48 M R F neurones previously or successively tested with 3~-THDOC failed to produce changes in neuronal firing either directly (Fig. 2) or by potentiation of GABA action. Microinjection of vehicle on 16 M R F neurones also failed to elicit any responses. In 11 rats the efficacy of bicuculline as antagonist of neuronal depression produced by 37-THDOC was studied in comparison with its selective GABA antagonism. In 27 of the 34 neurones (79%), in which bicuculline blocked GABA-induced inhibition of firing, it was also capable of reducing the inhibitory effects of 0.5-2/tM 3~-THDOC (see Fig. 3). This antagonism was observed as a reversible slowing of the onset of action and/or a reduction or an abolition of the maximum inhibitory effect of 3~-THDOC. Complete recovery of the effectiveness of 3~-THDOC occurred within
118 3 50-
3
--
6. CO
0 rain 1 pM
3~-THDOC
(in psi.)
2 pM
313-THDOC (in p s i )
Fig. 2. Frequency histogram of a single unit in the mesencephalic reticular formation showing that microinjection of 2/lM 3fl,5~-tetrahydrodeoxycorticosterone (3fl-THDOC) did not elicit any response, compared with the inhibitory effect induced by microinjection of 1 /tM 3c~,5~-tetrahydrodeoxycorticosterone (3~-THDOC). A
B1cuculhne
10
50hA 10
10
50
I3. (/3
rain
GABA Qn nA)
B
Bicuculllne 4
50hA 4
4
50
Q.
ca0
0 I
~ pM
3o(-THDOC
{in p s i )
min
Fig. 3. Frequency histograms of a single unit in the mesencephalic reticular formation showing comparison of the effectiveness of bicuculline as antagonist, in A, of x-aminobutyric acid (GABA) and, in B, of 3c~,5~tetrahydrodeoxycorticosterone (3~-THDOC). In both cases, complete recoveries of the actions both of GABA and of 3~-THDOC are not illustrated in the figure.
119 3-4 min. In each case the effect of bicuculline alone was monitored for at least 1 min, before evaluating its antagonistic properties (see Fig. 3). The occasional changes in the neuronal firing rate induced by bicuculline were not correlated with GABA and 3ct-THDOC antagonism. The results of this study demonstrate that 3ct-THDOC exerts a potent inhibitory action on neuronal firing rate when applied in very small amounts to M R F neurones. The short onset latency of the effects and their brief duration after the end of the application suggest that they are non-genomic effects presumably due to interactions with membrane components [10]. The potentiation of GABA inhibitory action by 3at-THDOC and the observation that the depressant effects by this steroid could be blocked by bicuculline demonstrate interaction of 3~-THDOC with the GABAA receptor complex. The effects of 3~-THDOC appear to be specific. This is supported by the observation that microinjection of comparable volumes of vehicle did not elicit any response. 3fl-THDOC also failed to elicit changes in neuronal firing either directly or by potentiation of GABA action. This shows that there is a high degree of structural discrimination at the steroid binding sites and precise steric requirem,ents for 3~-THDOC action at the GABAA receptor complex are needed, as already observed by other laboratories for other steroids [5, 6]. These results are in agreement with 'in vitro' studies concerning the effects of 3ct-THDOC on membrane conductance, action potential generation and selective binding of GABA agonists or antagonists [5, 9]. 'In vitro' evidence has shown that glucocorticoid hormones can also affect neuronal excitability through interactions with the GABA receptor system [6-8]. However, our previous reports [1, 2] have shown that local or systemic administration of corticosterone to neurones of the brainstem reticular formation can induce both excitatory and inhibitory effects on neuronal firing rate. This supports the hypothesis [6, 7] that additional factors, varying from cell to cell, should be important in determining the agonistic- or antagonistic-type of interaction of glucocorticoids with the G A B A receptor system. On the contrary, the uniform inhibitory effect obtained in this study by local applications of 3~-THDOC on M R F neurones suggests that 3~-THDOC interaction with GABA receptors is independent of local neurochemical factors. In conclusion, this 'in vivo' study supports the hypothesis that 3~-THDOC may function as endogenous modulator of GABA-mediated inhibition in various physiological, pathological and behavioural states. The authors thank Prof. F.R. Calaresu for critical comments and suggestions on the manuscript. 1 Avanzino, G.L., Celasco, G., Cogo, C.E., Ermirio, R. and Ruggeri, P., Actions of microelectrophoretically applied glucocorticoidhormones on reticular formation neurones in the rat, Neurosci. Lett., 38 (1983)4549. 2 Avanzino,G.L., Ermirio, R., Ruggeri, P. and Cogo, C.E., Effectsof corticosteroneon neurons of reticular formation in rats, Am. J. Physiol.,253 (1987) R25 R30.
120 3 Boakes, R.J., Bramwell, G.J., Briggs, I., Candy, J.M. and Tempesta, E., Localization with Pontamine sky blue of neurones in the brainstem responding to microiontophoretically applied compounds, Neuropharmacology, 13 (1974) 475 479. 4 Brodal, A., Neurological Anatomy (3rd edn.), Oxford Univ. Press, New York, 1981, pp. 394 447. 5 Harrison, N.L., Majewska, M.D., Harrington, J.W. and Barker, J.L., Structure~ctivity relationships for steroid interaction with the y-aminobutyric acida receptor complex, J. Pharmacol. Exp. Ther., 241 (1987) 34(~353. 6 Majewska, M.D., Steroids and brain activity. Essential dialogue between body and mind, Biochem. Pharmacol., 36 (t987) 3781 3788. 7 Majewska, M.D., Antagonist-type interaction of glucocorticoids with the GABA receptor-coupled chloride channel, Brain Res., 418 (1987) 377 382. 8 Majewska, M.D., Bisserbe, J.C. and Eskay, R.L., Glucocorticoids are modulators of GABAA receptors in brain, Brain Res., 339 (1985) 178 182. 9 Majewska, M.D., Harrison, N.L., Schwartz, R.D., Barker, J.L. and Paul, S.M., Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor, Science, 232 (1986) 1004 1007. l0 McEwen, B.S., Davis, P.G., Parsons, B. and Pfaff, D.W., The brain as a target for steroid hormone action, Annu. Rev. Neurosci., 2 (1979) 65 112. I1 Pellegrino, L.J., Pellegrino, A.S. and Cushman, A.J.. A Stereotaxic Atlas of the Rat Brain (2nd edn.), Plenum Press, New York, 1981, 35 pp. 12 Rommerts, F.F.G. and Van Der Molen, H.J., Occurrence and localization of 5c~-steroidreductase, 3:~and 17fl-hydroxysteroid dehydrogenases in hypothalamus and other brain tissues of the male rat, Biochim. Biophys. Acta, 248 ( 1971 ) 489 502. 13 Roselli, ('.E. and Snipes, ('.A., Progesterone-5:~-reductase in mouse brain, Brain Res., 305 (1984) 197 202. 14 Schambelan, M. and Biglieri, E.G., Deoxycorticosterone production and regulation in man, J. Clin. Endocrinol., 34 ~1972) 695 703. 15 Scheibel, A.B., The brain stem reticular core and sensory function. In Handbook of Physiology, The Nervous System. Sensory Processes, Sect. I, Vol. Ill, Bethesda, MD, 1984, pp. 213 256. 16 Stone, T.W., Microiontophoresis and pressure ejection. In A.D. Smith (Ed.), IBRO Handbook Series: Methods in the Neurosciences. Vol. 8, Wiley, Chichester, U.K., 1985, pp. 96, 98.