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Hypothalamic neurons responsive to increased plasma level of testosterone in the male rat
Brain Research, 212 (1981) 489-493 © Elsevier/North-Holland Biomedical Press
489
Hypothalamic neurons responsive to increased plasma level of testosterone in the male rat
JEAN-CLAUDE ORSINI* Ddpartement de Neurophysiologie Vdg~tative, INP. 1 - - C.N.R.S., B.P. 71 F-13277, Marseille, Cedex 9 (France)
(Accepted December 25th, 1980) Key words: testosterone - - medial forebrain bundle - - steroid action - - sexual motivation - - elec-
trophysiology - - male rat
Unitary activity was recorded from several forebrain structures. Out of 108 cells, 35 reacted to a testosterone i.v. injection by an increase (24 cells) or a decrease (11 cells) of firing rate. This response was early (latency: 5.0 _-k 0.6 min), lasted 25.7 .-k 1.9 rain and was reproducible. Responsive neurons were found to be significantly more numerous in the lateral hypothalamic area than in other structur,~3.
R a d i o i m m u n o a s s a y techniques have recently demonstrated that, in males o f several mammalian species, the level of circulating testosterone is not steady but constantly changes during the day. In an individual male rat, for example, 2- to 5-fold diurnal fluctuations have been detected 1,~° and such changes can occur even within a few minutes 1,16. These data suggest that a reexamination o f androgen-dependent phen o m e n a is necessary to determine if they can follow rapid modifications of testosterone level. More specifically, the question arises as to whether the electrical activity o f some brain cells could be modified with a short latency by a rise in testosterone level, as has been demonstrated in the hypothalamus of female rats following a rise in estrogen concentration 4,1a. Such brain cells could hypothetically receive and convey an information concerning short-term oscillations in the h o r m o n a l state o f the subject. In a preliminary report la, responses to changes in testosterone level were found by us in several forebrain structures, notably the hypothalamus, whose role is essential in neuroendocrine and behavioral aspects o f reproductive functions 9. In the present study, we focus primarily on the lateral hypothalamic area through which passes the medial forebrain bundle I,L H A - M F B ) which has been shown to be involved in the mediation o f male sexual behavior7,8, ~7. Experiments were performed on 46 adult male Wi star rats ( 180-320 g) anesthetized with urethane (1.5 g/kg i.p.). Eleven o f the animals were castrated at least 2 h before recording. Extracellular unit potentials were recorded from stereotaxically placed a glass micropipettes (tip diameter 1-2/~m) filled with a KCI solution (3 M), or with a NaCI (2 M) plus pontamine ( 4 ~ ) solution for precise histological localization. * Assistant, Universit6 de Nice.
490 R e c o r d e d potentials were amplified, displayed on an oscilloscope and filmed for further analysis. Testosterone and solvent injections were m a d e via a j u g u l a r cannula. Three testosterone esters were used: testosterone acetate or testosterone p r o p i o n a t e dissolved in propylene-glycol (10/,g//~l) or testosterone hemisuccinate dissolved in saline (0.1 /~g/t,l). A d m i n i s t e r e d doses (100-500 /~g), although higher than physiological levels c o r r e s p o n d to those usually applied in o r d e r to detect effects o f testosterone and especially neural reactivity 14. After each experiment, the brain was perfused with 80-100 ml o f 10% formalin, and postfixed in the same solution during 24 h. Frozen sections o f the brain, 50/~m thick, were cut in a coronal plane and stained with crcsyl violet, in o r d e r to confirm electrode position. In some cases the tip of the electrode had been m a r k e d before sacrificing the animal, by an electrolitic lesion or by iontophoresing the dye P o n t a m i n e sky blue 6 BX ~. F o r each cell, changes in the tiring rate were
A
A
15
-m,
~-
F
IF
cc (D
Z uJ 5
B
T0
~OTIM~
p < O 0 5 ~
(mien0)
Testosterooe
8
1
10
¢)
CE (9
z Z
:E
i~
[
~
i0
7~
3e
~ TIME
40 (ram)
Fig. 1. Histograms showing averaged firing rate and standard error in response to testosterone injection. Each column represents the mean firing rate counted during 10-30 sec every 5 rain (except before and immediately after the injection, where the intervals between recordings were systematically shortened). A: average of 20 responses with an increase in firing rate. B: average of 8 responses with a decrease in firing rate. Significant differences in firing rate before and after injection is shown for each histogram.
491 considered as responses only if they differed significantly from the spontaneous fluctuations before injections. For different neurons showing a similar pattern of response, the frequencies for corresponding intervals were averaged. The chi square test was used to compare the ratio of responsive vs unresponsive neurons in different brain areas. A total of 108 forebrain cells were tested with testosterone injections. Seventythree appeared unresponsive, whereas 35 responded by an increase (24 cells) or by a decrease (11 cells) of their discharge frequency. The responses had a latency of 5.0 + 0.6 rain and a duration of 25.7 --'_ 1.9 min (mean + S.E.). Fig. l shows the firing rate during tile period immediately preceding and following testosterone administration. As compared to their spontaneous activity, 20 responses averaged in Fig. IA increased their firing rate and 8 responses averaged in Fig. l B showed a decrease in discharge frequency. In the latter diagram, the mean firing rate does not return to initial level because 4 responses showed sustained decrease until the end of recording. Cells which either were not recorded for sufficient time after injection, or displayed too low or too high firing rates were not included in the average graphs. However, the response of these cells followed the general pattern of the above responses. Individual cells appeared to react specifically and consistently to the hormonal stimulus: (l) in 8 responsive neurons, the effect of 2 or 3 successive injections, 45-60 min apart, were compared (this interval is justified by preliminary results 13 which showed that a second administration was always effective if applied after a period of at least 40 min). In all 8 cells, successive responses displayed very similar patterns; (2) cells in close proximity often showed different patterns of response. In 14 cases, individual units recorded simultaneously reacted in entirely different ways to the hormone, in 12 cases, one cell responded whereas the other was unaffected, in 2 other cases, the cells reacted in opposite direction; (3) the possible indirect effects of the high doses of testosterone were not reflected by changes in heart rate, monitored during 99 administrations. In some cases (15 injections) slight modification immediately followed injection, but only 3 of these cardiac responses lasted for more than a few seconds. Moreover, the occurrence of such modifications was not correlated to that of a neuronal response. Response patterns were identical regardless of the type of ester or the solvent (propylene-glycol or saline) administered. Propylene-glycol injected alone modified the firing rate of 14 out of 77 cells. However, the latency (,0.6 _-k0.1 min) and the duration (1.4 --' 0.4 min) of these changes were clearly distinct from that of responses to hormone. Castration appeared to have no effect on the ratio of responsive versus unresponsive cells (7 neurons/22 for castrated rats, 28 neurons/86 for normal rats). However, this does not imply a lack of influence of endogenous testosterone, but rather that the level is low in all animals as a result of the operative procedure 15. Ninety-three neurons have been unequivocally localized through histological control. Among them, 30 responsive cells were distributed in quite different forebrain structures: in L H A - M F B and medial hypothalamus, as well as in extrahypothalamic sites such as the lateral septum (one cell), ventrobasal thalamus (two cells) and even the medial geniculate nucleus (one cell). However, the proportion of responsive neurons was significantly more important (P ,< 0.05) in L H A - M F B than in the other
492 structures considered together: 24 neurons/60 and 6 neurons/33 respectively. A monop o l a r projection o f recorded units on a parasagittal plane failed to display any spatial pattern o f responsive neuron distribution. Modifications o f brain activity, n o t a b l y in h y p o t h a l a m u s , by testosterone have been already describedl~, ~t.z0. M a n g a t et al. ~ mentionncd an " i m m e d i a t e effect" of i.v. injections on evoked potentials, but this effect was not tested before 15 min. Pfaffand Pfaffman ~~ observed unit responses to i.p. or local a d m i n i s t r a t i o n s with a latency o f 5-.15 min which could be explained at the cellular level by a slow and classical mechanism o f steroid action. Conversely, the responses to a p l a s m a rise o f testosterone which we obtained, because o f their short latency, can be classified as a "'non-genomic effect ''xz, and perhaps involve a direct coupling o f steroid molecules with m e m b r a n e reccptors e. Such a direct action has also been observed by Y a m a d a "o who found that neurons in the preoptic a r e a and lateral septum o f the male rat respond in a few seconds to iontophoretically applied testosterone. The longer latency o f the present responses could involve internal events between the injection site and the sensitive cells. A short-term effect o f testosterone on brain cells is c o m p a t i b l e not only with the rapid onset o f the responses we observed, but also with their short d u r a t i o n : the effect generally ends a b o u t 30 min after the injection, i.e. the a p p r o x i m a t e delay for disappearance o f radioactivity from h y p o t h a l a m i c tissue after an intravenous bolus o f labelled testosterone ~. It could be hypothesized that the " t e s t o s t e r o n e early responsive n e u r o n s ' , notably in L H A - M F B , regulate male sexual motivationT, 8 according to diurnal fluctuations o f circulating androgens. As an alternative, these neurons could play a role in the control o f g o n a d o t r o p h i n secretion, as suggested for estrogen responsive neurons in the female hypothalamus4,18,19. T h a n k s are given to Dr. N. Mei for continuous help and advice, to Dr. P. MacLeod for usefull suggestions and critical comments, and to Dr. H. M. C o o p e r for assistance with the English language.
I Bartke, A., Steele, R. E., Musto, N. and Caldwell, B. V., Fluctuations in plasma testosterone levels in adult male rats and mice, Ettdocrinology, 92 (1973) 1223-1228. 2 Beyer, C., Larsson, K. and Cruz, M. L., Neuronal mechanisms probably related to the effect of sex steroids on sexual behavior. In C. Beyer (Ed.), Endocrine ControlofSexual Behavior, Raven Press, New York, 1979, pp. 365--387. 3 De Groot, J., The Rat Forebrain in Stereotaxk" Coordinates, North-Holland Publishing-Company, Amsterdam, 1972, 40 pp. 4 Dufy, B., Partouche, C., Poulain, D., l)ufy-Barbe, L. and Vincent, J. D., Effects of estrogen on the electrical activity of identified and unidentified hypothalamic units, Neuroendocrinoh~gy, 22 (1976) 38- 47. 5 Godfraind, J. M., Localisation dc l'extrcmit6 dc microelectrodes de verre dans le syst6me nerveux central par 61ectrophor6se de pontamine, J. Physiol. IParis), 61 Suppl. I (1969) 436 437. 6 Henderson, S. B., Ciaccio, L. A. and Kincl, F. A., Dynamics of estradiol and testosterone uptake in the brain of adult male rats, J. steroid Biochem., II (1979) 1601-1607. 7 Hitt, J. C., Hendricks, S. E., Ginsberg, S. I. and Le~is, J. H., Disruption of male, but not female, sexual behavior in rats by medial forebrain bundle lesions, J. comp. physiol. Psychol., 73 (1970) 377-384.
493 8 Hitt, J. C., Bryon, D. M. and Modianos, D. T., Effects of rostral medial forebrain bundle and olfactory tubercle lesions upon sexual behavior of male rats, J. comp. physiol. Psychol., 82 (1973) 30-36. 9 Karli, P., Neurophysiologie du comportement. In C. Kayser (Ed.), Physiologie, VoL 2, Editions MSdicales Flammarion, Paris, 1976, pp. 1331-1454. 10 Keating, R. J. and Tcholakian, R. K., In vivo patterns of circulating steroids in adult male rats. I. Variations in testosterone during 24- and 48-hour standard and reverse light-dark cycles, Endocrinology, 104 (1979) 184-188. I 1 Mangat, H. K., Chhina, G. S., Singh, B. and Anand, B. K., Neuroendocrine correlates of testosterone-induced changes in brain evoked responses to afferent input, Acta Neurobiol., 39 (1979) 353-360. 12 Mc Ewen, B. S., Krey, L. C. and Luine, V. N., Steroid hormone action in the neuroendocrine system: when is the genome involved? In S. Reichlin, R. J. Baldessarini, J. B. Martin (Eds.), The Hypothalarnus, Raven Press, New York, 1978, pp. 255-268. 13 Orsini, J. C. and Mei, N., Reponse pr6coce des neurones hypothalamiques ~ une injection de testost6rone chez le rat m~le, C.R. Soc. Biol. (Paris), 173 (1979) 96-102. 14 Pfaff, D. W. and Pfaffmann, C., Olfactory and hormonal influences on the basal forebrain of the male rat, Brain Research, 15 (1969) 137-156. 15 Resko, J. A. and Phoenix, C. H., Sexual behavior and testosterone concentrations in the plasma of the rhesus monkey before and after castration, Endocrinology, 91 (1972) 499-503. 16 Tcholakian, R. K. and Keating, R. J., In vivo patterns of circulating steroids in adult male rats. IV. Evidence for rapid oscillations in testosterone in normal and totally parenterally nourished animals, Steroids, 32 (1978) 269-278. 17 Vaughan, E. and Fisher, A. E., Male sexual behavior induced by intracranial electrical stimulation, Science, 137 (1962) 758-760. 18 Whitehead, S. A. and Ruf, K. B., Responses of antidromically identified preoptic neurons in the rat to neurotransmitters and to estrogen, Brain Research, 79 (1974) 185-198. 19 Yagi, K., Changes in firing rates of single preoptic and hypothalamic units following an intravenous administration of estrogen in the castrated female rat, Brain Research, 53 (1973) 343-352. 20 Yamada, Y., Effects of testosterone on unit activity in rat hypothalamus and septum, Brain Research, 172 (1979) 165-168.
489
Hypothalamic neurons responsive to increased plasma level of testosterone in the male rat
JEAN-CLAUDE ORSINI* Ddpartement de Neurophysiologie Vdg~tative, INP. 1 - - C.N.R.S., B.P. 71 F-13277, Marseille, Cedex 9 (France)
(Accepted December 25th, 1980) Key words: testosterone - - medial forebrain bundle - - steroid action - - sexual motivation - - elec-
trophysiology - - male rat
Unitary activity was recorded from several forebrain structures. Out of 108 cells, 35 reacted to a testosterone i.v. injection by an increase (24 cells) or a decrease (11 cells) of firing rate. This response was early (latency: 5.0 _-k 0.6 min), lasted 25.7 .-k 1.9 rain and was reproducible. Responsive neurons were found to be significantly more numerous in the lateral hypothalamic area than in other structur,~3.
R a d i o i m m u n o a s s a y techniques have recently demonstrated that, in males o f several mammalian species, the level of circulating testosterone is not steady but constantly changes during the day. In an individual male rat, for example, 2- to 5-fold diurnal fluctuations have been detected 1,~° and such changes can occur even within a few minutes 1,16. These data suggest that a reexamination o f androgen-dependent phen o m e n a is necessary to determine if they can follow rapid modifications of testosterone level. More specifically, the question arises as to whether the electrical activity o f some brain cells could be modified with a short latency by a rise in testosterone level, as has been demonstrated in the hypothalamus of female rats following a rise in estrogen concentration 4,1a. Such brain cells could hypothetically receive and convey an information concerning short-term oscillations in the h o r m o n a l state o f the subject. In a preliminary report la, responses to changes in testosterone level were found by us in several forebrain structures, notably the hypothalamus, whose role is essential in neuroendocrine and behavioral aspects o f reproductive functions 9. In the present study, we focus primarily on the lateral hypothalamic area through which passes the medial forebrain bundle I,L H A - M F B ) which has been shown to be involved in the mediation o f male sexual behavior7,8, ~7. Experiments were performed on 46 adult male Wi star rats ( 180-320 g) anesthetized with urethane (1.5 g/kg i.p.). Eleven o f the animals were castrated at least 2 h before recording. Extracellular unit potentials were recorded from stereotaxically placed a glass micropipettes (tip diameter 1-2/~m) filled with a KCI solution (3 M), or with a NaCI (2 M) plus pontamine ( 4 ~ ) solution for precise histological localization. * Assistant, Universit6 de Nice.
490 R e c o r d e d potentials were amplified, displayed on an oscilloscope and filmed for further analysis. Testosterone and solvent injections were m a d e via a j u g u l a r cannula. Three testosterone esters were used: testosterone acetate or testosterone p r o p i o n a t e dissolved in propylene-glycol (10/,g//~l) or testosterone hemisuccinate dissolved in saline (0.1 /~g/t,l). A d m i n i s t e r e d doses (100-500 /~g), although higher than physiological levels c o r r e s p o n d to those usually applied in o r d e r to detect effects o f testosterone and especially neural reactivity 14. After each experiment, the brain was perfused with 80-100 ml o f 10% formalin, and postfixed in the same solution during 24 h. Frozen sections o f the brain, 50/~m thick, were cut in a coronal plane and stained with crcsyl violet, in o r d e r to confirm electrode position. In some cases the tip of the electrode had been m a r k e d before sacrificing the animal, by an electrolitic lesion or by iontophoresing the dye P o n t a m i n e sky blue 6 BX ~. F o r each cell, changes in the tiring rate were
A
A
15
-m,
~-
F
IF
cc (D
Z uJ 5
B
T0
~OTIM~
p < O 0 5 ~
(mien0)
Testosterooe
8
1
10
¢)
CE (9
z Z
:E
i~
[
~
i0
7~
3e
~ TIME
40 (ram)
Fig. 1. Histograms showing averaged firing rate and standard error in response to testosterone injection. Each column represents the mean firing rate counted during 10-30 sec every 5 rain (except before and immediately after the injection, where the intervals between recordings were systematically shortened). A: average of 20 responses with an increase in firing rate. B: average of 8 responses with a decrease in firing rate. Significant differences in firing rate before and after injection is shown for each histogram.
491 considered as responses only if they differed significantly from the spontaneous fluctuations before injections. For different neurons showing a similar pattern of response, the frequencies for corresponding intervals were averaged. The chi square test was used to compare the ratio of responsive vs unresponsive neurons in different brain areas. A total of 108 forebrain cells were tested with testosterone injections. Seventythree appeared unresponsive, whereas 35 responded by an increase (24 cells) or by a decrease (11 cells) of their discharge frequency. The responses had a latency of 5.0 + 0.6 rain and a duration of 25.7 --'_ 1.9 min (mean + S.E.). Fig. l shows the firing rate during tile period immediately preceding and following testosterone administration. As compared to their spontaneous activity, 20 responses averaged in Fig. IA increased their firing rate and 8 responses averaged in Fig. l B showed a decrease in discharge frequency. In the latter diagram, the mean firing rate does not return to initial level because 4 responses showed sustained decrease until the end of recording. Cells which either were not recorded for sufficient time after injection, or displayed too low or too high firing rates were not included in the average graphs. However, the response of these cells followed the general pattern of the above responses. Individual cells appeared to react specifically and consistently to the hormonal stimulus: (l) in 8 responsive neurons, the effect of 2 or 3 successive injections, 45-60 min apart, were compared (this interval is justified by preliminary results 13 which showed that a second administration was always effective if applied after a period of at least 40 min). In all 8 cells, successive responses displayed very similar patterns; (2) cells in close proximity often showed different patterns of response. In 14 cases, individual units recorded simultaneously reacted in entirely different ways to the hormone, in 12 cases, one cell responded whereas the other was unaffected, in 2 other cases, the cells reacted in opposite direction; (3) the possible indirect effects of the high doses of testosterone were not reflected by changes in heart rate, monitored during 99 administrations. In some cases (15 injections) slight modification immediately followed injection, but only 3 of these cardiac responses lasted for more than a few seconds. Moreover, the occurrence of such modifications was not correlated to that of a neuronal response. Response patterns were identical regardless of the type of ester or the solvent (propylene-glycol or saline) administered. Propylene-glycol injected alone modified the firing rate of 14 out of 77 cells. However, the latency (,0.6 _-k0.1 min) and the duration (1.4 --' 0.4 min) of these changes were clearly distinct from that of responses to hormone. Castration appeared to have no effect on the ratio of responsive versus unresponsive cells (7 neurons/22 for castrated rats, 28 neurons/86 for normal rats). However, this does not imply a lack of influence of endogenous testosterone, but rather that the level is low in all animals as a result of the operative procedure 15. Ninety-three neurons have been unequivocally localized through histological control. Among them, 30 responsive cells were distributed in quite different forebrain structures: in L H A - M F B and medial hypothalamus, as well as in extrahypothalamic sites such as the lateral septum (one cell), ventrobasal thalamus (two cells) and even the medial geniculate nucleus (one cell). However, the proportion of responsive neurons was significantly more important (P ,< 0.05) in L H A - M F B than in the other
492 structures considered together: 24 neurons/60 and 6 neurons/33 respectively. A monop o l a r projection o f recorded units on a parasagittal plane failed to display any spatial pattern o f responsive neuron distribution. Modifications o f brain activity, n o t a b l y in h y p o t h a l a m u s , by testosterone have been already describedl~, ~t.z0. M a n g a t et al. ~ mentionncd an " i m m e d i a t e effect" of i.v. injections on evoked potentials, but this effect was not tested before 15 min. Pfaffand Pfaffman ~~ observed unit responses to i.p. or local a d m i n i s t r a t i o n s with a latency o f 5-.15 min which could be explained at the cellular level by a slow and classical mechanism o f steroid action. Conversely, the responses to a p l a s m a rise o f testosterone which we obtained, because o f their short latency, can be classified as a "'non-genomic effect ''xz, and perhaps involve a direct coupling o f steroid molecules with m e m b r a n e reccptors e. Such a direct action has also been observed by Y a m a d a "o who found that neurons in the preoptic a r e a and lateral septum o f the male rat respond in a few seconds to iontophoretically applied testosterone. The longer latency o f the present responses could involve internal events between the injection site and the sensitive cells. A short-term effect o f testosterone on brain cells is c o m p a t i b l e not only with the rapid onset o f the responses we observed, but also with their short d u r a t i o n : the effect generally ends a b o u t 30 min after the injection, i.e. the a p p r o x i m a t e delay for disappearance o f radioactivity from h y p o t h a l a m i c tissue after an intravenous bolus o f labelled testosterone ~. It could be hypothesized that the " t e s t o s t e r o n e early responsive n e u r o n s ' , notably in L H A - M F B , regulate male sexual motivationT, 8 according to diurnal fluctuations o f circulating androgens. As an alternative, these neurons could play a role in the control o f g o n a d o t r o p h i n secretion, as suggested for estrogen responsive neurons in the female hypothalamus4,18,19. T h a n k s are given to Dr. N. Mei for continuous help and advice, to Dr. P. MacLeod for usefull suggestions and critical comments, and to Dr. H. M. C o o p e r for assistance with the English language.
I Bartke, A., Steele, R. E., Musto, N. and Caldwell, B. V., Fluctuations in plasma testosterone levels in adult male rats and mice, Ettdocrinology, 92 (1973) 1223-1228. 2 Beyer, C., Larsson, K. and Cruz, M. L., Neuronal mechanisms probably related to the effect of sex steroids on sexual behavior. In C. Beyer (Ed.), Endocrine ControlofSexual Behavior, Raven Press, New York, 1979, pp. 365--387. 3 De Groot, J., The Rat Forebrain in Stereotaxk" Coordinates, North-Holland Publishing-Company, Amsterdam, 1972, 40 pp. 4 Dufy, B., Partouche, C., Poulain, D., l)ufy-Barbe, L. and Vincent, J. D., Effects of estrogen on the electrical activity of identified and unidentified hypothalamic units, Neuroendocrinoh~gy, 22 (1976) 38- 47. 5 Godfraind, J. M., Localisation dc l'extrcmit6 dc microelectrodes de verre dans le syst6me nerveux central par 61ectrophor6se de pontamine, J. Physiol. IParis), 61 Suppl. I (1969) 436 437. 6 Henderson, S. B., Ciaccio, L. A. and Kincl, F. A., Dynamics of estradiol and testosterone uptake in the brain of adult male rats, J. steroid Biochem., II (1979) 1601-1607. 7 Hitt, J. C., Hendricks, S. E., Ginsberg, S. I. and Le~is, J. H., Disruption of male, but not female, sexual behavior in rats by medial forebrain bundle lesions, J. comp. physiol. Psychol., 73 (1970) 377-384.
493 8 Hitt, J. C., Bryon, D. M. and Modianos, D. T., Effects of rostral medial forebrain bundle and olfactory tubercle lesions upon sexual behavior of male rats, J. comp. physiol. Psychol., 82 (1973) 30-36. 9 Karli, P., Neurophysiologie du comportement. In C. Kayser (Ed.), Physiologie, VoL 2, Editions MSdicales Flammarion, Paris, 1976, pp. 1331-1454. 10 Keating, R. J. and Tcholakian, R. K., In vivo patterns of circulating steroids in adult male rats. I. Variations in testosterone during 24- and 48-hour standard and reverse light-dark cycles, Endocrinology, 104 (1979) 184-188. I 1 Mangat, H. K., Chhina, G. S., Singh, B. and Anand, B. K., Neuroendocrine correlates of testosterone-induced changes in brain evoked responses to afferent input, Acta Neurobiol., 39 (1979) 353-360. 12 Mc Ewen, B. S., Krey, L. C. and Luine, V. N., Steroid hormone action in the neuroendocrine system: when is the genome involved? In S. Reichlin, R. J. Baldessarini, J. B. Martin (Eds.), The Hypothalarnus, Raven Press, New York, 1978, pp. 255-268. 13 Orsini, J. C. and Mei, N., Reponse pr6coce des neurones hypothalamiques ~ une injection de testost6rone chez le rat m~le, C.R. Soc. Biol. (Paris), 173 (1979) 96-102. 14 Pfaff, D. W. and Pfaffmann, C., Olfactory and hormonal influences on the basal forebrain of the male rat, Brain Research, 15 (1969) 137-156. 15 Resko, J. A. and Phoenix, C. H., Sexual behavior and testosterone concentrations in the plasma of the rhesus monkey before and after castration, Endocrinology, 91 (1972) 499-503. 16 Tcholakian, R. K. and Keating, R. J., In vivo patterns of circulating steroids in adult male rats. IV. Evidence for rapid oscillations in testosterone in normal and totally parenterally nourished animals, Steroids, 32 (1978) 269-278. 17 Vaughan, E. and Fisher, A. E., Male sexual behavior induced by intracranial electrical stimulation, Science, 137 (1962) 758-760. 18 Whitehead, S. A. and Ruf, K. B., Responses of antidromically identified preoptic neurons in the rat to neurotransmitters and to estrogen, Brain Research, 79 (1974) 185-198. 19 Yagi, K., Changes in firing rates of single preoptic and hypothalamic units following an intravenous administration of estrogen in the castrated female rat, Brain Research, 53 (1973) 343-352. 20 Yamada, Y., Effects of testosterone on unit activity in rat hypothalamus and septum, Brain Research, 172 (1979) 165-168.