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Effects of ACTH and Corticosteroids on Single Neurons in the Hypothalamus
Effects of ACTH and Corticosteroids on Single Neurons in the Hypothalamus FELIX A. STEINER Department of Experimental Medicine, F. Hoffmnn-La Roche & Co. Ltd., Basle, and Institute for Brain Research, University of Zurich, Zurich (Switzerland)
I N T R O D U C T I O N A N D METHODS
In the past, effects of corticosteroids on the brain have been studied by a variety of experimental approaches. Overall effects-e.g., EEG changes-have been described by Woodbury and Vernadakis (1967), and the effects of intravenously applied steroids on single units have been studied by Slusher, Hyde and Laufer (1966), and by Feldman and Dafny (1966). Experiments in which steroid crystals were implanted into various parts of the brain (e.g., Endroczi, Lissiik and Tekeres (1961); Smelik and Sawyer (1962); Chowers, Feldman and Davidson (1963)) have further defined the site of action of these steroids. However, none of these approaches has made it possible to study the direct effects of these substances on defined single neurons. The use of micro-electrophoresis or iontophoresis (Curtis, 1964) is suited for this latter type of study. In this method, certain ionizable drugs may be applied through multibarrelled micropipettes to the extracellular environment of single neurons. The rate of delivery (or the retention) of these substances is accurately controlled by small electrical currents of opposite polarity. The outside diameter of the common tip of these micropipettes measures between 1-3 p, and one of the barrels may therefore be used for the simultaneous recording of action potentials. Susceptible neurons change their rate of discharge in response to the applied drugs, and their accurate histological identification is possible through the ejection of dyes from one of the barrels according to the same principle. In this study, dexamethasone-21-phosphate has been applied micro-electrophoretically to single neurons in rat brain, and the following problems have been examined : 1) Localization of steroid-sensitive neurons in the hypothalamus and the midbrain, 2) sensitivity of these and other neurons to synthetic ACTH*, 3) responsiveness of steroid-sensitive single neurons to neurohumours (acetylcholine, noradrenaline, dopamine) which are normally present in the hypothalamus. Preference was given to dexamethasone-21-phosphate(rather than corticosterone or cortisol) because of its ready water solubility. Methodological details and most of the results have been previously described (Steiner, Ruf and Akert, 1969; Steiner, Pieri and Kaufmann, 1968; Ruf and Steiner, 1967a; Ruf and Steiner, 1967b). *l,%-beta-tetracosactide, SynacthenB.
EFFECTS OF
1
6
5
4
3
ACTH
2
1
103
AND CORTICOSTEROIDS
u7
6
5
4
3
2
1
Fig. 1. Localization of single neurons inhibited ( 0 )and not influenced (0)by dexamethasone phosphate on parasagittal sections according to De Groot (1959). Neurons in L 0 to L 0.5 are projected on L 0.2; neurons in L 0.6 to L 1.5 on L 1.1.
n
141210
-
8 6 4 2 -
0-
F
10 sec
Fig. 2. Frequency of discharge of a single neuron in the area hypothalamica anterior, plotted against Duratime. Inhibition by micro-electrophoreticallyapplied dexamethasone phosphate (20 nA). tion of micro-electrophoresis. ~
RESULTS
Eflects of dexamethasone In 69 different experiments, the steroid sensitivity of 386 hypothalamic and mesencephalic neurons has been assessed. 66 of these neurons decreased their rate of discharge in response to micro-electrophoretic application of dexamethasone, whereas only 7 were activated. The anatomical distribution of these neurons (Fast Green technique, Thomas and Wilson, 1965) is shown in Fig. 1, and a typical exa.mple of the change in the rate of discharge is given in Fig. 2. No steroid-sensitive neurons were found in representative samplings in the cortex, the dorsal hippocampus or the thalamus. Dexamethasone was applied by micro-electrophoresis for time periods ranging between 30-90 sec, and its effects, if present, were usually seen during this interval. In some cases, the effect was almost instantaneous, and neuronal inhibition persisted after the termination of micro-electrophoresis for 5-100 sec. References p. 106
104
F. A. S T E I N E R
TABLE 1 RESPONSE
~
OF DEXAMETHASONE PHOSPHATE-SENSITIVE
N E U R O N S TO N E U R O H U M O U R S
Acfivation
Inhibition
No effect
16 4 0
2 13
12 10
Total
~
Acetylcholine Noradrenaline Dopamine
7
2
30
27 9
TABLE 2 RESPONSE OF D E X A M E T H A S O N E P H O S P H A T E - S E N S I T I V E N E U R O N S TO BOTH ACETYLCHOLINEAND NORADRENALINE
(t increase, 4. decrease of the discharge rate, 0 no effect.) Acetylcholine 12 t
11
0
Noradrenuline
3t Of
7J.
54.
20 60
Effects of ACTH ACTH was administered to 11 dexamethasone-sensitive neurons. It activated 8 of these and was without influence on one. The remaining 2 neurons showed a biphasic effect. Four further neurons, which had not changed their rate of discharge in response to dexamethasone, also remained uninfluenced by ACTH. 9 experiments were performed in this series. Response of steroid-sensitive neurons to neurohumours Acetylcholine, noradrenaline, and dopamine were Iocally administered to a number of dexamethasone-sensitive single neurons. In some cases, it was possible to test the responsiveness of a neuron to more than one neurohumour in consecutive applications. The following changes in the rate of discharge were observed, see Table 1. It can be seen that acetylcholine had a tendency to activate these neurons, whereas noradrenaline, and especially dopamine, exerted a depressive effect. A similar tendency was observed in those steroid-sensitive neurons which were exposed to the influence of both acetylcholine and noradrenaline. These results are shown in Table 2. DISCUSSION
These results demonstrate the existence of dexamethasone-sensitive neurons in rather extended areas of the hypothalamus and the midbrain. Most of these neurons are inhibited by dexamethasone, a few are activated, ACTH, in turn, activates certain of
EFFECTS OF
ACTH
AND CORTICOSTEROIDS
105
these neurons under the experimental conditions of the study. Neurohumours such as acetylcholine, noradrenaline and dopamine also influence the rate of discharge of steroid-sensitive neurons : acetylcholine predominantly in the sense of activation, noradrenaline and dopamine mainly in the sense of depression. The technique of micro-electrophoresis offers the possibility of a direct assessment of steroid effects on single neurons which would not be obtainable by other techniques. In particular, this approach excludes the possibility of diffusion to neighbouring structures (Bogdanove, 1963), such as the pituitary. Micro-electrophoresis also circumvents diffusion problems caused by the “blood-brain barrier”. However, certain questions inherent in this approach must also be considered. The method operates on a hit-or-miss basis and as such, may be subject to a sampling bias with regard to the cell population examined. Certain neurons may not be detectable due to their slow rate of discharge, whereas others may be picked up preferentially because of aparticular size or abnormally high rate of discharge induced by stressful experimental conditions. Nevertheless, the demonstration of selective steroid effects on certain neurons, which are part of a sizeable hypothalamic and mesencephalic population, may be of some physiological significance. It is conceivable that these neurons function as measuring devices for circulating steroids and thus play a role in the feedback control of corticotropin-releasing factor/ACTH secretion. This mode of action of dexamethasone phosphate could represent a negative feedback mechanism, while the ACTH effect may represent a positive (short) feedback mechanism (Sawyer, Kawakami, Meyerson, Whitmoyer and Lilley, 1968), both in the regulation of ACTH production. Some of these steroid-sensitive neurons may have othei functions, e.g., in the mediation of behavioural correlates of stress. The simultaneous identification of these neurons by other techniques (e.g., peripheral activation of incoming pathways, experimental lowering of circulating steroids) would greatly facilitate the interpretation of these results. It should be emphasized that the demonstration of steroid-sensitive neurons in the brain does not exclude feedback effects of corticosteroids in other organs, particularly the anterior pituitary (Kraicer, Milligan, Gosbee, Conrad and Branson, 1969). SUMMARY
The effects of micro-electrophoretically applied dexamethasone phosphate on neuronal activity in the brain of rat anaesthetized with chloralose-urethane was studied. Dexamethasone phosphate-sensitive neurons were localized in the hypothalamus, and in the midbrain scattered over wide areas. Of these neurons, the large majority was clearly inhibited and a small number activated. No steroid-sensitive neurons were found in the cortex, the dorsal hippocampus, or in the thalamus. Locally delivered synthetic ACTH activated the steroid-sensitive neurons. Noradrenaline and dopamine inhibited, and acetylcholine activated dexamethasone-sensitive neurons. These results could indicate that specific nerve cells in the hypothalamus and midbi ain are sensitive to both hormonal and neurohumoural factors. Rejkrences p . 106
106
F. A. S T E I N E R
REFERENCES BOGDANOVE, E. M. (1963) Direct gonad-pituitary feedback: an analysis of effects of intracranial estrogenic depots on gonadotrophin secretion. Endocrinology, 73, 696712. J., FELDMAN, S. AND DAVIDSON, J. M. (1963) Effects of intrahypothalamic crystalline CHOWERS, steroids on acute ACTH secretion. Am. J. Physiol., 205, 671-673. CURTIS,D. R. (1964) Micro-electrophoresis. In Physical Techniques in Biological Research, vol. 5, (Ed.), Academic Press, London, pp. 144190. Part A, W. L. NASTUK E N D R ~ ZE., I , LISSAK,K. AND TEKERES, M. (1961) Hormonal feedback regulation of pituitary adrenocortical activity. Acta physiol. Acad. Sci. Hung., 18, 291-299. FELDMAN, S. AND DAFNY,N. (1966) Effect of hydrocortisone on single cell activity in the anterior hypothalamus. Israel J. med. Sci., 2, 621-623. DE GROOT,J. (1959) The rat forebrain in stereotaxic coordinates. Trans. Roy. Netherlands Acad. Sci., 52, 1-40. KRAICER, J., MILLIGAN, J. V., GOSBEE, J. L., CONRAD, R. G. AND BRANSON, C. M. (1969) Potassium, corticosterone, and adrenocorticotropic hormone release in vifro. Science, 161,426. RUF,K. AND STEINER, F. A. (1967a) Steroid-sensitive single neurons in rat hypothalamus and midbrain : identification by micro-electrophoresis. Science, 156, 667-669. RUF,K. AND STEINER, F.A. (1967b) Feedback regulation of ACTH secretion. Suppression of single neurons in rat brain by dexamethasone micro-electrophoresis. Acfa Endocrinol., Suppl. 119, 38. B., WHITMOYER, D. I. AND LILLEY, J. J. (1968)Effectsof SAWYER, C. H., KAWAKAMI, M., MEYERSON, ACTH, dexamethasone and asphyxia on electrical activity of the rat hypothalamus. Brain Res., 10, 213-226. SLUSHER, M. A., HYDE,J. E. AND LAUFER, M. (1966) Effect of intracerebral hydrocortisone on unit activity of diencephalon and midbrain in cats. J. Neurophysiof., 29, 157-169. C. H. (1962) Effects of implantation of cortisol into the brain stem or SMELIK,P. G . AND SAWYER, pituitary gland on the adrenal response to stress in the rabbit. Acta Endocrinol., 41, 561-570. F. A., PIERI, L. AND KAUFMANN, L. (1968) Effects of dopamine and ACTH on steroidSTEINER, sensitive single neurons in the basal hypothalamus. Experientia, 24, 1133-1 134. STEINER, F. A., RUF, K. AND AKERT,K. (1969) Steroid-sensitive neurons in rat brain: anatomic localization and responses to neurohumours and ACTH. Brain Res., 12,74-85. THOMAS, R. C. AND WILSON,V. J. (1965) Precise localization of Renshaw cells with a new marking technique. Nature, 206, 211-213. A. (1967) Influence of hormones on brain activity. In: NeuroWOODBURY, D. M. AND VERNADAKIS, AND MARTINI (Eds.), Academic Press, New York and London, endocrinology, Vol. IT, GANONG pp. 335-375.
DISCUSSION
Is the density of steroid-sensitive neurons higher in any particular part of the hypoFELDMAN: thalamus? STEINER: So far, we have restricted our investigations to neurons situated in or near the midsagittal plane. In these regions no conspicuous differences of density have been observed. HENKIN:What percentage of the number of units measured was steroid sensitive? STEINER: Sixteen per cent of spontaneouslyactive neurons were depressed by microelectrophoretically applied dexamethasone phosphate.
KLEIN:Is it fair to state that all cells that did not respond were intrinsically different, i.e. “non steroidresponsive”, or could this be a technical problem? STEINER: We do think that technical reasons cannot account for this difference and therefore these non-responding neurons are probably different from the steroid-sensitive neurons. LARON: Is it possible that the sensitive and the non-sensitive cells represent interchangeablestates of
EFFECTS OF
ACTH
A N D CORTICOSTEROIDS
107
the same kind of cells rather than that they represent two types of cells which permanently act differently. STEINER: We cannot exclude the possibility that we are dealing with interchangeable states of the same kind of neuron, but we think that the two types of cells represent different populations. FELDMAN: There are a number of neurons that are non-responsive to sensory stimulation and are also non-responsive to hormones. Maybe there exists some connection between these properties. MARKS:In your model you propose that dexamethasone acts on the nerve cell membrane. How d o you distinguish between pre- and post-synaptic effects? STEINER: The technique of microelectrophoresis with extracellular recording of action potentials does not allow us to distinguish between pre- and post-synaptic effects.
MCEWEN:Could you comment on the time course of the action of hormones? The cellular action of hormones may be distinguished on the basis of whether they act with a short lag period and for a short time (membrane effects) or with a longer lag period and for a longer time (protein synthesis, metabolic effects). Protein synthesis effects may involve a considerable lag period for production and transport of the protein to the site of action and may last for a long time until the proteins formed are used up. STEINER: We have observed short term effects. Still, it is possible to divide these short term effects in two classes: 1, Effects with a very short latency, 2, Effects with a longer latency (several seconds). We have not been able to observe long term effects. Sawyer and co-workers have described short term (activation) and long term (inhibition) effects after ACTH.
BOHUS:Do you possess any evidence concerning steroid and ACTH sensitivity of thalamic and mesencephalic reticular neurons?
STEINER: We have not found steroid-sensitive cells in the thalamus. ACTH was not applied to thalamic neurons. In the mesencephalic area some steroid-sensitive cells were observed, but we have no data about ACTH. SMELIK: It is tempting to conclude from your data that the steroid-sensitive cells are directly involved in the control of the pituitary-adrenal system. This need not be so, it is equally possible that these effects of dexamethasone are on cells belonging to other systems. The fact that application of much greater amounts of dexamethasone phosphate in this area inhibits the secretion of ACTH after a considerable delay of several hours, should caution us in interpreting your results, since in your experiments the effect on the rate of firing can be observed within a few seconds. STEINER: I agree with you; the interpretation of these results is difficult and should be done with caution. We have discussed possibilities other than a direct action on the control of the pituitaryadrenal system in our papers.
I N T R O D U C T I O N A N D METHODS
In the past, effects of corticosteroids on the brain have been studied by a variety of experimental approaches. Overall effects-e.g., EEG changes-have been described by Woodbury and Vernadakis (1967), and the effects of intravenously applied steroids on single units have been studied by Slusher, Hyde and Laufer (1966), and by Feldman and Dafny (1966). Experiments in which steroid crystals were implanted into various parts of the brain (e.g., Endroczi, Lissiik and Tekeres (1961); Smelik and Sawyer (1962); Chowers, Feldman and Davidson (1963)) have further defined the site of action of these steroids. However, none of these approaches has made it possible to study the direct effects of these substances on defined single neurons. The use of micro-electrophoresis or iontophoresis (Curtis, 1964) is suited for this latter type of study. In this method, certain ionizable drugs may be applied through multibarrelled micropipettes to the extracellular environment of single neurons. The rate of delivery (or the retention) of these substances is accurately controlled by small electrical currents of opposite polarity. The outside diameter of the common tip of these micropipettes measures between 1-3 p, and one of the barrels may therefore be used for the simultaneous recording of action potentials. Susceptible neurons change their rate of discharge in response to the applied drugs, and their accurate histological identification is possible through the ejection of dyes from one of the barrels according to the same principle. In this study, dexamethasone-21-phosphate has been applied micro-electrophoretically to single neurons in rat brain, and the following problems have been examined : 1) Localization of steroid-sensitive neurons in the hypothalamus and the midbrain, 2) sensitivity of these and other neurons to synthetic ACTH*, 3) responsiveness of steroid-sensitive single neurons to neurohumours (acetylcholine, noradrenaline, dopamine) which are normally present in the hypothalamus. Preference was given to dexamethasone-21-phosphate(rather than corticosterone or cortisol) because of its ready water solubility. Methodological details and most of the results have been previously described (Steiner, Ruf and Akert, 1969; Steiner, Pieri and Kaufmann, 1968; Ruf and Steiner, 1967a; Ruf and Steiner, 1967b). *l,%-beta-tetracosactide, SynacthenB.
EFFECTS OF
1
6
5
4
3
ACTH
2
1
103
AND CORTICOSTEROIDS
u7
6
5
4
3
2
1
Fig. 1. Localization of single neurons inhibited ( 0 )and not influenced (0)by dexamethasone phosphate on parasagittal sections according to De Groot (1959). Neurons in L 0 to L 0.5 are projected on L 0.2; neurons in L 0.6 to L 1.5 on L 1.1.
n
141210
-
8 6 4 2 -
0-
F
10 sec
Fig. 2. Frequency of discharge of a single neuron in the area hypothalamica anterior, plotted against Duratime. Inhibition by micro-electrophoreticallyapplied dexamethasone phosphate (20 nA). tion of micro-electrophoresis. ~
RESULTS
Eflects of dexamethasone In 69 different experiments, the steroid sensitivity of 386 hypothalamic and mesencephalic neurons has been assessed. 66 of these neurons decreased their rate of discharge in response to micro-electrophoretic application of dexamethasone, whereas only 7 were activated. The anatomical distribution of these neurons (Fast Green technique, Thomas and Wilson, 1965) is shown in Fig. 1, and a typical exa.mple of the change in the rate of discharge is given in Fig. 2. No steroid-sensitive neurons were found in representative samplings in the cortex, the dorsal hippocampus or the thalamus. Dexamethasone was applied by micro-electrophoresis for time periods ranging between 30-90 sec, and its effects, if present, were usually seen during this interval. In some cases, the effect was almost instantaneous, and neuronal inhibition persisted after the termination of micro-electrophoresis for 5-100 sec. References p. 106
104
F. A. S T E I N E R
TABLE 1 RESPONSE
~
OF DEXAMETHASONE PHOSPHATE-SENSITIVE
N E U R O N S TO N E U R O H U M O U R S
Acfivation
Inhibition
No effect
16 4 0
2 13
12 10
Total
~
Acetylcholine Noradrenaline Dopamine
7
2
30
27 9
TABLE 2 RESPONSE OF D E X A M E T H A S O N E P H O S P H A T E - S E N S I T I V E N E U R O N S TO BOTH ACETYLCHOLINEAND NORADRENALINE
(t increase, 4. decrease of the discharge rate, 0 no effect.) Acetylcholine 12 t
11
0
Noradrenuline
3t Of
7J.
54.
20 60
Effects of ACTH ACTH was administered to 11 dexamethasone-sensitive neurons. It activated 8 of these and was without influence on one. The remaining 2 neurons showed a biphasic effect. Four further neurons, which had not changed their rate of discharge in response to dexamethasone, also remained uninfluenced by ACTH. 9 experiments were performed in this series. Response of steroid-sensitive neurons to neurohumours Acetylcholine, noradrenaline, and dopamine were Iocally administered to a number of dexamethasone-sensitive single neurons. In some cases, it was possible to test the responsiveness of a neuron to more than one neurohumour in consecutive applications. The following changes in the rate of discharge were observed, see Table 1. It can be seen that acetylcholine had a tendency to activate these neurons, whereas noradrenaline, and especially dopamine, exerted a depressive effect. A similar tendency was observed in those steroid-sensitive neurons which were exposed to the influence of both acetylcholine and noradrenaline. These results are shown in Table 2. DISCUSSION
These results demonstrate the existence of dexamethasone-sensitive neurons in rather extended areas of the hypothalamus and the midbrain. Most of these neurons are inhibited by dexamethasone, a few are activated, ACTH, in turn, activates certain of
EFFECTS OF
ACTH
AND CORTICOSTEROIDS
105
these neurons under the experimental conditions of the study. Neurohumours such as acetylcholine, noradrenaline and dopamine also influence the rate of discharge of steroid-sensitive neurons : acetylcholine predominantly in the sense of activation, noradrenaline and dopamine mainly in the sense of depression. The technique of micro-electrophoresis offers the possibility of a direct assessment of steroid effects on single neurons which would not be obtainable by other techniques. In particular, this approach excludes the possibility of diffusion to neighbouring structures (Bogdanove, 1963), such as the pituitary. Micro-electrophoresis also circumvents diffusion problems caused by the “blood-brain barrier”. However, certain questions inherent in this approach must also be considered. The method operates on a hit-or-miss basis and as such, may be subject to a sampling bias with regard to the cell population examined. Certain neurons may not be detectable due to their slow rate of discharge, whereas others may be picked up preferentially because of aparticular size or abnormally high rate of discharge induced by stressful experimental conditions. Nevertheless, the demonstration of selective steroid effects on certain neurons, which are part of a sizeable hypothalamic and mesencephalic population, may be of some physiological significance. It is conceivable that these neurons function as measuring devices for circulating steroids and thus play a role in the feedback control of corticotropin-releasing factor/ACTH secretion. This mode of action of dexamethasone phosphate could represent a negative feedback mechanism, while the ACTH effect may represent a positive (short) feedback mechanism (Sawyer, Kawakami, Meyerson, Whitmoyer and Lilley, 1968), both in the regulation of ACTH production. Some of these steroid-sensitive neurons may have othei functions, e.g., in the mediation of behavioural correlates of stress. The simultaneous identification of these neurons by other techniques (e.g., peripheral activation of incoming pathways, experimental lowering of circulating steroids) would greatly facilitate the interpretation of these results. It should be emphasized that the demonstration of steroid-sensitive neurons in the brain does not exclude feedback effects of corticosteroids in other organs, particularly the anterior pituitary (Kraicer, Milligan, Gosbee, Conrad and Branson, 1969). SUMMARY
The effects of micro-electrophoretically applied dexamethasone phosphate on neuronal activity in the brain of rat anaesthetized with chloralose-urethane was studied. Dexamethasone phosphate-sensitive neurons were localized in the hypothalamus, and in the midbrain scattered over wide areas. Of these neurons, the large majority was clearly inhibited and a small number activated. No steroid-sensitive neurons were found in the cortex, the dorsal hippocampus, or in the thalamus. Locally delivered synthetic ACTH activated the steroid-sensitive neurons. Noradrenaline and dopamine inhibited, and acetylcholine activated dexamethasone-sensitive neurons. These results could indicate that specific nerve cells in the hypothalamus and midbi ain are sensitive to both hormonal and neurohumoural factors. Rejkrences p . 106
106
F. A. S T E I N E R
REFERENCES BOGDANOVE, E. M. (1963) Direct gonad-pituitary feedback: an analysis of effects of intracranial estrogenic depots on gonadotrophin secretion. Endocrinology, 73, 696712. J., FELDMAN, S. AND DAVIDSON, J. M. (1963) Effects of intrahypothalamic crystalline CHOWERS, steroids on acute ACTH secretion. Am. J. Physiol., 205, 671-673. CURTIS,D. R. (1964) Micro-electrophoresis. In Physical Techniques in Biological Research, vol. 5, (Ed.), Academic Press, London, pp. 144190. Part A, W. L. NASTUK E N D R ~ ZE., I , LISSAK,K. AND TEKERES, M. (1961) Hormonal feedback regulation of pituitary adrenocortical activity. Acta physiol. Acad. Sci. Hung., 18, 291-299. FELDMAN, S. AND DAFNY,N. (1966) Effect of hydrocortisone on single cell activity in the anterior hypothalamus. Israel J. med. Sci., 2, 621-623. DE GROOT,J. (1959) The rat forebrain in stereotaxic coordinates. Trans. Roy. Netherlands Acad. Sci., 52, 1-40. KRAICER, J., MILLIGAN, J. V., GOSBEE, J. L., CONRAD, R. G. AND BRANSON, C. M. (1969) Potassium, corticosterone, and adrenocorticotropic hormone release in vifro. Science, 161,426. RUF,K. AND STEINER, F. A. (1967a) Steroid-sensitive single neurons in rat hypothalamus and midbrain : identification by micro-electrophoresis. Science, 156, 667-669. RUF,K. AND STEINER, F.A. (1967b) Feedback regulation of ACTH secretion. Suppression of single neurons in rat brain by dexamethasone micro-electrophoresis. Acfa Endocrinol., Suppl. 119, 38. B., WHITMOYER, D. I. AND LILLEY, J. J. (1968)Effectsof SAWYER, C. H., KAWAKAMI, M., MEYERSON, ACTH, dexamethasone and asphyxia on electrical activity of the rat hypothalamus. Brain Res., 10, 213-226. SLUSHER, M. A., HYDE,J. E. AND LAUFER, M. (1966) Effect of intracerebral hydrocortisone on unit activity of diencephalon and midbrain in cats. J. Neurophysiof., 29, 157-169. C. H. (1962) Effects of implantation of cortisol into the brain stem or SMELIK,P. G . AND SAWYER, pituitary gland on the adrenal response to stress in the rabbit. Acta Endocrinol., 41, 561-570. F. A., PIERI, L. AND KAUFMANN, L. (1968) Effects of dopamine and ACTH on steroidSTEINER, sensitive single neurons in the basal hypothalamus. Experientia, 24, 1133-1 134. STEINER, F. A., RUF, K. AND AKERT,K. (1969) Steroid-sensitive neurons in rat brain: anatomic localization and responses to neurohumours and ACTH. Brain Res., 12,74-85. THOMAS, R. C. AND WILSON,V. J. (1965) Precise localization of Renshaw cells with a new marking technique. Nature, 206, 211-213. A. (1967) Influence of hormones on brain activity. In: NeuroWOODBURY, D. M. AND VERNADAKIS, AND MARTINI (Eds.), Academic Press, New York and London, endocrinology, Vol. IT, GANONG pp. 335-375.
DISCUSSION
Is the density of steroid-sensitive neurons higher in any particular part of the hypoFELDMAN: thalamus? STEINER: So far, we have restricted our investigations to neurons situated in or near the midsagittal plane. In these regions no conspicuous differences of density have been observed. HENKIN:What percentage of the number of units measured was steroid sensitive? STEINER: Sixteen per cent of spontaneouslyactive neurons were depressed by microelectrophoretically applied dexamethasone phosphate.
KLEIN:Is it fair to state that all cells that did not respond were intrinsically different, i.e. “non steroidresponsive”, or could this be a technical problem? STEINER: We do think that technical reasons cannot account for this difference and therefore these non-responding neurons are probably different from the steroid-sensitive neurons. LARON: Is it possible that the sensitive and the non-sensitive cells represent interchangeablestates of
EFFECTS OF
ACTH
A N D CORTICOSTEROIDS
107
the same kind of cells rather than that they represent two types of cells which permanently act differently. STEINER: We cannot exclude the possibility that we are dealing with interchangeable states of the same kind of neuron, but we think that the two types of cells represent different populations. FELDMAN: There are a number of neurons that are non-responsive to sensory stimulation and are also non-responsive to hormones. Maybe there exists some connection between these properties. MARKS:In your model you propose that dexamethasone acts on the nerve cell membrane. How d o you distinguish between pre- and post-synaptic effects? STEINER: The technique of microelectrophoresis with extracellular recording of action potentials does not allow us to distinguish between pre- and post-synaptic effects.
MCEWEN:Could you comment on the time course of the action of hormones? The cellular action of hormones may be distinguished on the basis of whether they act with a short lag period and for a short time (membrane effects) or with a longer lag period and for a longer time (protein synthesis, metabolic effects). Protein synthesis effects may involve a considerable lag period for production and transport of the protein to the site of action and may last for a long time until the proteins formed are used up. STEINER: We have observed short term effects. Still, it is possible to divide these short term effects in two classes: 1, Effects with a very short latency, 2, Effects with a longer latency (several seconds). We have not been able to observe long term effects. Sawyer and co-workers have described short term (activation) and long term (inhibition) effects after ACTH.
BOHUS:Do you possess any evidence concerning steroid and ACTH sensitivity of thalamic and mesencephalic reticular neurons?
STEINER: We have not found steroid-sensitive cells in the thalamus. ACTH was not applied to thalamic neurons. In the mesencephalic area some steroid-sensitive cells were observed, but we have no data about ACTH. SMELIK: It is tempting to conclude from your data that the steroid-sensitive cells are directly involved in the control of the pituitary-adrenal system. This need not be so, it is equally possible that these effects of dexamethasone are on cells belonging to other systems. The fact that application of much greater amounts of dexamethasone phosphate in this area inhibits the secretion of ACTH after a considerable delay of several hours, should caution us in interpreting your results, since in your experiments the effect on the rate of firing can be observed within a few seconds. STEINER: I agree with you; the interpretation of these results is difficult and should be done with caution. We have discussed possibilities other than a direct action on the control of the pituitaryadrenal system in our papers.