- Email: [email protected]
anterior hypothalamic neurone responses to stimulation of the lateral septum
Brain Research, 262 (1983) 136-142 Elsevier Biomedical Press
137
Effect of Testosterone on Medial Preoptic/Anterior Hypothalamic Neurone Responses to Stimulation of the Lateral Septum K. M. KENDRICK Department of Reproduction, Institute of Zoology, Regent's Park, London NW1 4R Y (U.K.) (Accepted August 3rd, 1982) Key words: castration - - testosterone - - medial preoptic/anterior hypothalamus - - lateral septum - medial forebrain bundle - - arcuate/median eminence
Electrophysiological recordings were made from medial preoptic/anterior hypothalamic (MPOAH) neurones which could be orthodromically excited from the lateral septum (LS) in gonadally intact and castrated male rats. Castration significantlyreduced the mean percentage of MPOAH neurones which responded reliably to 0.6 Hz stimulation (from 8t. 1~ to 54.1 ~) and this could be reversed by testosterone propionate (from 52.5 ~ to 90.1 ~). Both in the gonadally intact (58.1 Y/oo)and castrated (53.8~) groups these MPOAH neurones with LS inputs also had inputs from the contralateral fimbria (CFIMB). A further experiment investigated whether these MPOAH neurones responding to LS stimulation had connections with the arcuate/median eminence (ARC/ME) region or the medial forebrain bundle (MFB). Altogether, 23.2 ~o projected directly into the MFB and 9.5 % into the ARC/ME. A further 28.4 ~ and 31.6}/ooof these MPOAH neurones received inputs from these respective brain regions. A small population of MPOAH neurones were also found which could be driven antidromically both from the LS and the MFB. Results show that testosterone alters the responsiveness of MPOAH neurones to stimulation of the LS. Since subpopulations of these neurones project to the MFB and ARC/ME the effectsof castration on them may reflect both changes in sexual behavior and, for example, gonadotropin release. INTRODUCTION Neurones of the medial preoptic/anterior hypothalamus (MPOAH) are less responsive to their input from the fimbria after castration 8. This input goes via the corticomedial amygdala (CMA) s which has been implicated in an excitatory control of sexual behaviour in the male rat2, 4. Corticomedial amygdala neurones which project to the M P O A H show lengthened absolute refractory periods 10 and are less responsive to electrical stimulation of the fimbria after castration 7. The reduced responsiveness o f M P O A H neurones to fimbria stimulation is consequently mainly attributed to a reduced excitatory input from the C M A . Sexual behaviour deficits following castration may therefore be partly accounted for by a reduction in excitatory input to the MPOAH. The lateral septum (LS) also projects to the M P O A H 1 and like the M P O A H and C M A takes up radioactively labelled testosterone 13. The role of the 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
lateral septum in the control of male sexual behaviour is ambiguous however. In the male rat septal lesions do not affect sexual behaviour3 but in the squirrel monkey intracranial stimulation of the septal area evokes penile erectionlL This LS projection to the M P O A H may also be involved in the control of pituitary gonadotrophin release. Progesterone implants into the LS area cause changes in levels of both LH 7 and FSHL Also, L H - R H containing neurones have been shown to be present in the LS 5. Electrophysiological studies to date have shown that castration does not alter the absolute refractory periods of M P O A H neurones projecting to the LSL The present study further investigates the electrophysiological effects of castration and testosterone propionate on M P O A H neurones with inputs from the LS. At the same time the extent of convergence of this LS input with that via the fimbria/CMA was determined. A further experiment investigated whether M P O A H neurones responding to stimulation of the LS had connections with the medial fore-
138 brain bundle (MFB) and the arcuate/median eminence (ARC/ME) region. If the LS input to the M P O A H is involved in the control of male sexual behaviour then one would expect that some of the responding M P O A H neurones should project directly into the MFB. The connections between the M P O A H and the MFB are essential for normal copulatory responses in the male raO 4,16. If, on the other hand, these neurones project to the A R C / M E one would expect them to be involved in pituitary hormone release. MATERIALS AND METHODS Animals and hormone treatments All experiments were performed on adult male sexually naive Porton albino Wistar rats (400-650 g) housed in conditions of controlled lighting (lights on from 24.00 h to 12.00 h). Castrations were carried out under ether anaesthesia and the animals left for a minimum of 8 weeks prior to use. For the first experiment, 16 rats were divided at random into two groups of 8. One group was then castrated and the other group left gonadally intact. For the second experiment 12 castrated rats were divided at random into two groups of 6. One group was then given daily injections of 200 /~g testosterone propionate (TP; Sigma (London) given s.c. in 0.1 ml arachis oil) for 18-22 days and the other group received 0.1 ml injections of oil alone. Electrophysiology In all experiments rats were anaesthetised with urethane (1.3 g/kg intraperitoneally) at 09.00 h on the day of the experiment, and mounted in a conventional stereotaxic frame ( K o p f Instruments, Tujtmga, CA, U.S.A.). Stainless steel monopolar electrodes insulated to within 0.5 mm of the tip were used to stimulate the LS, CFIMB, MFB and ARC/ ME with cathodal monophasic pulses (50 /~A-1 mA; duration 0.5 ms). Glass micropipettes filled with 2 ~ pontamine blue in 0.5 M sodium acetate were used to record single unit activity extracellularly. Co-ordinates for electrode placement were calculated in accordance with a stereotaxic atlas 11. Recordings were started at 10.00 h and generally completed by 16.00 h. At the end of the experiment a small current was
passed through the stimulating and recording electrodes for marking purposes. The rat was then given an overdose of Nembutal and perfused through the heart with 5 ~ formol-saline solution to which a small amount of potassium ferrocyanide had been added. Brains were mounted in paraffin wax, sectioned at 10 # m and stained in Luxol fast blue and neutral red. The criteria for antidromic invasion for silent neutones were constant latency and following of a stimulus pulse pair above 150 Hz. For spontaneously active neurones an additional collision test was used between a spontaneous action potential and a stimulus-evoked potential. Neurones which were excited but did not fulfill these criteria for antidromic invasion were classed as being orthodromically stimulated. For orthodromically stimulated neurones in experiment one, the following measures were taken in each animal: the percentage of neurones capable of following the routine stimulation frequency of 0.6 Hz; the mean activation latency; the mean latency variation; the mean threshold current for stimulation and the median frequency following at 1.2 times the threshold current. This latter frequency following test was only carried out on neurones which could follow 0.6 Hz stimulation of the LS. Statistics Statistical analysis was carried out on data in experiments one and two using the Mann-Whitney U-test.
RESULTS Effect o f castration on M P O A H neurone responses to stimulation o f the L S Altogether 199 M P O A H neurones were orthodromically excited after LS stimulation in gonadally intact (89 neurones from 8 rats) and castrated (110 neurones from 8 rats) animals. Between 6 and 21 neuronal responses were recorded from each animal. Fig. 1A shows that castration significantly reduced the percentage of neurones which could follow 0.6 Hz stimulation of the LS (mean = from 89.1 to 54.1 ~ ; U = 0, P < 0.001). For both groups a large percentage of these M P O A H neurones responding to LS stimulation were also orthodromi-
139
ioo-
A
,o
c5 0 0") f-
5o-
"0 tO r¢
d'
H n +TP
+ OIL
Fig. 1. A: percentage of MPOAH neurones following 0.6 Hz stimulation of the LS in gonadally intact (~ - - 89 neurones from 8 rats) and castrated (~ - - 110 neurones from 8 rats) animals. B: percentage of MPOAH neurones following 0.6 Hz stimulation of the LS in castrated rats treated with testosterone propionate (~ ÷ TP - - 110 neurones from 6 rats) or oil (g + oil - - 92 neurones from 6 rats). Figure gives the overall mean and the dots represent the individual means.
n u m b e r o f M P O A H n e u r o n e s o r t h o d r o m i c a l l y excited f r o m the LS (mean n u m b e r per electrode p e n e t r a t i o n = 1.55, r a n g e 1.00-2.33 - - f o r the g o n a d a l l y intact g r o u p ; a n d 1.99, r a n g e 1.00-3.00 - - for the c a s t r a t e d g r o u p , U = 17, P > 0.05); their m e a n t h r e s h o l d s t i m u l a t i o n c u r r e n t (650 F A , range 5407 6 0 / ~ A ; v 690 # A , range 5 7 0 - 7 6 0 / ~ A , U = 22.5, P > 0.05); m e a n activation latency (10.4 ms, range 7.9-16.3 ms; v 9.4 ms, range 6.4--12.2 ms, U = 28, P > 0.05; m e a n latency v a r i a t i o n (1.4 ms, range 1.0-1.7 ms; v 1.6 ms, range 1.2-2.1 ms, U ~ 22.5, P > 0.05) o r m e d i a n frequency following at 1.2 times t h r e s h o l d c u r r e n t (35 Hz, range 23.2-75.2 H z ; v 36 Hz, range 22-76.9 Hz, U ~ 28.5, P > 0.05). A 50 "o o
-~
40
E t,r) tn ~_...=
cally excited f r o m the C F I M B ( m e a n = 5 8 . 7 ~ , range 33.3 ~o-85.7 ~o for the g o n a d a l l y intact g r o u p ; a n d 5 3 . 8 ~ , range 28.6~o-71.4~o for the c a s t r a t e d group). This s u b - p o p u l a t i o n o f M P O A H n e u r o n e s w i t h the a d d i t i o n a l i n p u t f r o m C F I M B also showed the significant r e d u c t i o n in their a b i l i t y to follow 0.6 H z s t i m u l a t i o n o f the LS after c a s t r a t i o n (from 9 8 . 0 ~ to 62.7 ~ ; U = 1, P < 0.001) as d i d the neurones r e s p o n d i n g to LS s t i m u l a t i o n alone (from 7 8 . 9 ~ to 41.1~o; U = 12, P = 0.02). T h o s e M P O A H n e u r o n e s s t i m u l a t e d f r o m b o t h the LS a n d the C F I M B s h o w e d the same effects o f c a s t r a t i o n after s t i m u l a t i o n o f the C F I M B as have been previously r e p o r t e d s, including a similar r e d u c e d ability to follow 0.6 H z s t i m u l a t i o n . A small p e r c e n t a g e o f M P O A H n e u r o n e s activ a t e d f r o m the LS r e s p o n d e d to a single pulse stimul a t i o n o f this site b y p r o d u c i n g a p a i r o f action potentials. This m o d e o f n e u r o n a l response was previously r e p o r t e d for M P O A H n e u r o n e s s t i m u l a t e d f r o m the C F I M B s. B o t h this previous study, a n d the p r e s e n t one confirmed t h a t c a s t r a t i o n r e d u c e d the p e r c e n t a g e o f n e u r o n e s r e s p o n d i n g to C F I M B stim u l a t i o n in this way. In the present study, however, the percentage o f M P O A H n e u r o n e s r e s p o n d i n g to LS s t i m u l a t i o n like this was n o t affected b y castration ( m e a n = 2 1 . 2 ~ vs 13.5 ~ , U - - 20, P > 0.05). C a s t r a t i o n was also f o u n d to have no effect on the
30
oE
Z~
•
e
o
20
~
o~o,o
MFB
ARC ME
MFB
anti
anti
ortho
ARC ME ortho
MFB ÷ ARC ME ortho
5O O =~
40
E
u)(n
~
O
,o I 10
0~6 ~mee MFB
A RC/M F
MFB
anti
anti
ottho
ARC/.~ . F . ortho
AR~ME ortho
e~e
.F.
,,cJ.E
anti
anti
"i" ARC/ME M ; B ortho
ortho
Fig. 2. A: percentage of MPOAH neurones stimulated antidromically from the LS (35 neurones from 5 rats) which are orthodromically (oitho) or antidromically (anti) stimulated from the MFB or the ARC/ME or both. B: percentage of MPOAH neurones stimulated orthodromically from the LS (100 neurones from 5 rats) which are orthodromically or antidromically stimulated from the MFB or ARC/ME or both. Figure gives the overall mean and the dots represent the individual means.
140
D ~-
t ~
".
/, ~.~
V/ll,~
x ~
,
/
l
'.o,,~/j ,/
B t,,/ C ...... )
-'(
/,\
E
.----~.-//'/_~ !,iu"..~-" -7~- ,,. o.,.,,! % .... , ~ - -
C
( I \"'¢/l:',~
'q~"¢
I F
i ilt'= ," 1 mm
Fig. 3. Location of M P O A H neurones stimulated antidromically (17) or orthodromically (O) from the LS. Neurones which are also antidromically stimulated from the MFB are shown by ( l l or 0 ) and from the A R C / M E by (©). Neurones driven orthodromically from the M F B are shown by 'J' and from the A R C / M E by ' - - ' . Schematic coronal sections are adapted from the atlas of KSnig and Klippe111. Co-ordinates are: A, A7020 # m ; B, A6860/~m; C, A6790/~m; D, A6670 Fm; E, A6570/~m; F, A6360 F m and A6280/~m. pom = medial preoptic area; ah = anterior hypothalamus.
141
Effect of TP on M P O A H neurone responses to stimulation of the L S A total of 202 M P O A H neurones orthodromically excited from the LS were recorded from castrated animals treated with TP (110 neurones from 6 rats) or oil alone (92 neurones from 6 rats). Fig. 1B shows that TP significantly increased the percentage of neurones which could follow 0.6 Hz stimulation of the LS (from mean 52.5~o to 90.1 ~o, U -~ 0, P < 0.001). The mean percentage of M P O A H neurones which could follow 0.6 Hz stimulation of the LS did not differ significantly for gonadally intact rats (Experiment 1) and TP-treated rats (U = 23.5, P > 0.05). Responses of M P O A H neurones orthodromically excited from L S to stimulation of the MFB and ARC~ME In 5 gonadally intact male rats the responses of 135 M P O A H neurones stimulated from the LS (35 neurones antidromically stimulated and 100 orthodromically stimulated) were monitored after MFB and A R C / M E stimulation. Fig. 2A shows the mean percentage of antidromically stimulated neurones driven from the MFB and A R C / M E and Fig. 2B shows the same for the orthodromically stimulated neurones. For M P O A H neurones stimulated antidromically from the LS, 10.8 ~o were also stimulated antidromically from the MFB and a further 11.5 ~o orthodromically excited. None of these neurones were antidromically stimulated from A R C / M E although 7.9 ~ were orthodromically stimulated. For M P O A H neurones stimulated orthodromically from the LS 23.2 ~o were also antidromically stimulated from the MFB and 9.5 ~ from ARC/ME. A further 2 8 . 4 ~ and 3 1 . 6 ~ of neurones were stimulated orthodromically from respective structures. 12.1~ of neurones were orthodromically excited from both sites; 5 ~ antidromically stimulated from the MFB and orthodromically stimulated from A R C / M E and 3 . 4 ~ vice versa. Fig. 3 shows the approximate locations of all types of responsive neurones within the MPOAH. DISCUSSION
Castration significantly reduces the percentage of M P O A H neurones which can follow 0.6 Hz stimula-
tion of the LS and this can be reversed by treatment with testosterone propionate. The subpopulations of neurones driven only from the LS or from both the LS and CFIMB show the same effect of castration as the overall population. Castration has no effect on the number of stimulated neurones, their stimulation thresholds, activation latencies, latency variations or frequency following at 1.2 times threshold current. Those M P O A H neurones which can be stimulated from the CFIMB (in addition to the LS) show the same characteristics and the same effects of castration as have been previously reported s. A quantitative analysis of the connections of LS driven M P O A H neurones with the MFB and the A R C / M E reveals contributions from both these structures. M P O A H neurones identified as projecting to the LS have a small input from both the MFB and ARC/ME. A small number of these neurones have additional outputs into the MFB, but not the ARC/ME. This latter finding indicates that some M P O A H neurones have bifurcating axons to the LS and the MFB. A quarter of MPOAH neurones with inputs from the LS have outputs into the MFB and a somewhat lesser number have outputs to the ARC/ ME. A large number of this group of neurones also have inputs from the MFB and A R C / M E and some have inputs from both or alternatively inputs from one and an output to the other. The effect of testosterone on the ability of M P O A H neurones to follow 0.6 Hz stimulation of the LS could be due to the hormone acting either on the LS or the MPOAH. That is, either the LS is rendered less excitable by castration or the M P O A H less responsive. The results suggest, however, that the M P O A H is the relevant site of action. Firstly, there is no significant rise in the threshold current needed to stimulate M P O A H neuronal responses from the LS after castration. If castration had altered the excitability of the LS one might have expected a consequent change in this parameter. Secondly, the other findings that activation latencies, latency variations and frequency following parameters are unchanged by castration again supports the argument that the LS neurones are not the critical site. The absence of latency variation changes following castration also suggests that the synapse is not responsible for the effect since any change in synaptic efficiency might be expected to
142 produce more variable synaptic delays. Thirdly, the M P O A H neurones which also receive inputs from C F I M B show the same effect of castration after stimulation of this site. Although these M P O A H neurones receiving inputs from C F I M B show other effects of castration (reduction in percentage of paired action potential responses and decrease in frequency following at 1.2 times threshold current) they have been shown to be probably due to changes in corticomedial amygdala (CMA) neurones projecting to the M P O A H (via which the fimbria input to the M P O A H projects 8) and not to changes in the M P O A H . Overall, however, the results demonstrate that M P O A H neurones receiving projections from the LS are excited less by LS stimulation after castration and this effect can be reversed by testosterone. This provides further evidence for the hypothesis that sexual behaviour deficits following castration may be partly caused by changes in the effectiveness of excitatory inputs to the M P O A H (whether the critical site is the neuronal group proiecting to the M P O A H or the M P O A H neurones themselves).
The third experiment investigating the connections of LS stimulated M P O A H neurones with the MFB and the A R C / M E produced ambiguous results. Around one quarter of these neurones had direct outputs into the MFB and just under 10 ~ to the ARC/ME. Since the output from the M P O A H into the MFB is crucial for normal male copulatory responses14,16 this finding confirms that the LS input to the M P O A H may be involved in the control of male sexual behaviour. The smaller output to the A R C / M E also implies a possible role for LS modulation of pituitary hormone release via the MPOAH. The effect of castration on these M P O A H neurones driven from the LS may therefore reflect both changes in sexual behaviour and, for instance, gonadotrophin release.
REFERENCES
tic/anterior hypothalamic neurone responses to stimulation of the fimbria in the rat, J. Physiol. (Lond.), 315 (1982) 449-461. 9 Kendrick, K. M., Electrophysiological effects of testosterone on the medial preoptic/anterior hypothalamus, J. Endocr., in press. 10 Kendrick, K. M. and Drewett, R. F., Testosterone reduces refractory period of stria terminalis neurones in rat brain, Science, 204 (1979) 877-879. 11 K6nig, J. F. R. and Klippel, R. A., The Rat Brain, Williams and Wilkins, Baltimore, 1963. 12 MacLean, P. D. and Ploog, D. W., Cerebral representation of penile erection, J. Neurophysiol., 25 (1962) 29-55. 13 Sar, M. and Stumpf, W. E., Autoradiographic localisation of radioactivity in the rat brain after injection of [1,2-aH]testosterone, Endocrinology, 92 (1973) 251-256. 14 Scouten, C. W., Burrell, L., Palmer, T. and Cegavske, C. F., Lateral projections of the medial preoptic area are necessary for androgenic influence on urine marking and copulation in rats, Physiol. Behav., 25 (1980) 237-243. 15 Silverman, A. J. and Krey, L. C., The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea pig brain. I. Intra- and extra-hypothalamic projections, Brain Research, 157 (1978) 233-246. 16 Szechtman, H., Caggiula, A. R. and Wulkan, D., Preoptic knife cuts and sexual behaviour in male rats, Brain Research, 150 (1978) 569-591.
1 Conrad, L. C. A. and Pfaff, D. W., Axonal projections of medial preoptic and anterior hypothalamic neurons, Science, 190 (1975) 1112-1114. 2 Giantonio, G. W., Lund, N. L. and Gerall, A. A., Effect of diencephalic and rhinencephalic lesions on the male rat's sexual behaviour, J. comp. physiol. Psychol., 73 (1970) 38-46. 3 Goodman, E. D., Bunnell, B. N., Dewsbury, D. A. and Boland, B., Septal lesions and male rat copulatory behavior, Psychon. Sci., 16 (1969) 123-132. 4 Harris, V. H. and Sachs, B. D., Copulatory behaviour in male rats following amygdaloid lesions, Brain Research, 86 (1975) 514-518. 5 Kawakami, M., Arita, J., Yoshioka, E., Visessuvan, S. and Akema, T., Data on the sites of the stimulatory feedback action of gonadal steroids indispensable for follicle-stimulating hormone release in the rat, Endocrinology, 103 (1978) 752-759. 6 Kawakami, M., Yoshioka, E., Konda, N., Arita, J. and Visessuvan, S., Data on the sites of the stimulatory feedback action of gonadal steroids indispensable for luteinizing hormone release in the rat, Endocrinology, 102 (1978) 791-797. 7 Kendrick, K. M., Inputs to testosterone-sensitive stria terminalis neurones in the rat brain and the effects of castration, J. Physiol. (Lond.), 315 (1982) 437-447. 8 Kendrick, K. M., Effects of castration on medial preop-
ACKNOWLEDGEMENTS This work was supported by the M R C and carried out in the Psychology Department at Durham University. I thank Shirley Whiteley for her help with the histology and Dr. A. F. Dixson for helpful discussion and critical reading of the manuscript.
137
Effect of Testosterone on Medial Preoptic/Anterior Hypothalamic Neurone Responses to Stimulation of the Lateral Septum K. M. KENDRICK Department of Reproduction, Institute of Zoology, Regent's Park, London NW1 4R Y (U.K.) (Accepted August 3rd, 1982) Key words: castration - - testosterone - - medial preoptic/anterior hypothalamus - - lateral septum - medial forebrain bundle - - arcuate/median eminence
Electrophysiological recordings were made from medial preoptic/anterior hypothalamic (MPOAH) neurones which could be orthodromically excited from the lateral septum (LS) in gonadally intact and castrated male rats. Castration significantlyreduced the mean percentage of MPOAH neurones which responded reliably to 0.6 Hz stimulation (from 8t. 1~ to 54.1 ~) and this could be reversed by testosterone propionate (from 52.5 ~ to 90.1 ~). Both in the gonadally intact (58.1 Y/oo)and castrated (53.8~) groups these MPOAH neurones with LS inputs also had inputs from the contralateral fimbria (CFIMB). A further experiment investigated whether these MPOAH neurones responding to LS stimulation had connections with the arcuate/median eminence (ARC/ME) region or the medial forebrain bundle (MFB). Altogether, 23.2 ~o projected directly into the MFB and 9.5 % into the ARC/ME. A further 28.4 ~ and 31.6}/ooof these MPOAH neurones received inputs from these respective brain regions. A small population of MPOAH neurones were also found which could be driven antidromically both from the LS and the MFB. Results show that testosterone alters the responsiveness of MPOAH neurones to stimulation of the LS. Since subpopulations of these neurones project to the MFB and ARC/ME the effectsof castration on them may reflect both changes in sexual behavior and, for example, gonadotropin release. INTRODUCTION Neurones of the medial preoptic/anterior hypothalamus (MPOAH) are less responsive to their input from the fimbria after castration 8. This input goes via the corticomedial amygdala (CMA) s which has been implicated in an excitatory control of sexual behaviour in the male rat2, 4. Corticomedial amygdala neurones which project to the M P O A H show lengthened absolute refractory periods 10 and are less responsive to electrical stimulation of the fimbria after castration 7. The reduced responsiveness o f M P O A H neurones to fimbria stimulation is consequently mainly attributed to a reduced excitatory input from the C M A . Sexual behaviour deficits following castration may therefore be partly accounted for by a reduction in excitatory input to the MPOAH. The lateral septum (LS) also projects to the M P O A H 1 and like the M P O A H and C M A takes up radioactively labelled testosterone 13. The role of the 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
lateral septum in the control of male sexual behaviour is ambiguous however. In the male rat septal lesions do not affect sexual behaviour3 but in the squirrel monkey intracranial stimulation of the septal area evokes penile erectionlL This LS projection to the M P O A H may also be involved in the control of pituitary gonadotrophin release. Progesterone implants into the LS area cause changes in levels of both LH 7 and FSHL Also, L H - R H containing neurones have been shown to be present in the LS 5. Electrophysiological studies to date have shown that castration does not alter the absolute refractory periods of M P O A H neurones projecting to the LSL The present study further investigates the electrophysiological effects of castration and testosterone propionate on M P O A H neurones with inputs from the LS. At the same time the extent of convergence of this LS input with that via the fimbria/CMA was determined. A further experiment investigated whether M P O A H neurones responding to stimulation of the LS had connections with the medial fore-
138 brain bundle (MFB) and the arcuate/median eminence (ARC/ME) region. If the LS input to the M P O A H is involved in the control of male sexual behaviour then one would expect that some of the responding M P O A H neurones should project directly into the MFB. The connections between the M P O A H and the MFB are essential for normal copulatory responses in the male raO 4,16. If, on the other hand, these neurones project to the A R C / M E one would expect them to be involved in pituitary hormone release. MATERIALS AND METHODS Animals and hormone treatments All experiments were performed on adult male sexually naive Porton albino Wistar rats (400-650 g) housed in conditions of controlled lighting (lights on from 24.00 h to 12.00 h). Castrations were carried out under ether anaesthesia and the animals left for a minimum of 8 weeks prior to use. For the first experiment, 16 rats were divided at random into two groups of 8. One group was then castrated and the other group left gonadally intact. For the second experiment 12 castrated rats were divided at random into two groups of 6. One group was then given daily injections of 200 /~g testosterone propionate (TP; Sigma (London) given s.c. in 0.1 ml arachis oil) for 18-22 days and the other group received 0.1 ml injections of oil alone. Electrophysiology In all experiments rats were anaesthetised with urethane (1.3 g/kg intraperitoneally) at 09.00 h on the day of the experiment, and mounted in a conventional stereotaxic frame ( K o p f Instruments, Tujtmga, CA, U.S.A.). Stainless steel monopolar electrodes insulated to within 0.5 mm of the tip were used to stimulate the LS, CFIMB, MFB and ARC/ ME with cathodal monophasic pulses (50 /~A-1 mA; duration 0.5 ms). Glass micropipettes filled with 2 ~ pontamine blue in 0.5 M sodium acetate were used to record single unit activity extracellularly. Co-ordinates for electrode placement were calculated in accordance with a stereotaxic atlas 11. Recordings were started at 10.00 h and generally completed by 16.00 h. At the end of the experiment a small current was
passed through the stimulating and recording electrodes for marking purposes. The rat was then given an overdose of Nembutal and perfused through the heart with 5 ~ formol-saline solution to which a small amount of potassium ferrocyanide had been added. Brains were mounted in paraffin wax, sectioned at 10 # m and stained in Luxol fast blue and neutral red. The criteria for antidromic invasion for silent neutones were constant latency and following of a stimulus pulse pair above 150 Hz. For spontaneously active neurones an additional collision test was used between a spontaneous action potential and a stimulus-evoked potential. Neurones which were excited but did not fulfill these criteria for antidromic invasion were classed as being orthodromically stimulated. For orthodromically stimulated neurones in experiment one, the following measures were taken in each animal: the percentage of neurones capable of following the routine stimulation frequency of 0.6 Hz; the mean activation latency; the mean latency variation; the mean threshold current for stimulation and the median frequency following at 1.2 times the threshold current. This latter frequency following test was only carried out on neurones which could follow 0.6 Hz stimulation of the LS. Statistics Statistical analysis was carried out on data in experiments one and two using the Mann-Whitney U-test.
RESULTS Effect o f castration on M P O A H neurone responses to stimulation o f the L S Altogether 199 M P O A H neurones were orthodromically excited after LS stimulation in gonadally intact (89 neurones from 8 rats) and castrated (110 neurones from 8 rats) animals. Between 6 and 21 neuronal responses were recorded from each animal. Fig. 1A shows that castration significantly reduced the percentage of neurones which could follow 0.6 Hz stimulation of the LS (mean = from 89.1 to 54.1 ~ ; U = 0, P < 0.001). For both groups a large percentage of these M P O A H neurones responding to LS stimulation were also orthodromi-
139
ioo-
A
,o
c5 0 0") f-
5o-
"0 tO r¢
d'
H n +TP
+ OIL
Fig. 1. A: percentage of MPOAH neurones following 0.6 Hz stimulation of the LS in gonadally intact (~ - - 89 neurones from 8 rats) and castrated (~ - - 110 neurones from 8 rats) animals. B: percentage of MPOAH neurones following 0.6 Hz stimulation of the LS in castrated rats treated with testosterone propionate (~ ÷ TP - - 110 neurones from 6 rats) or oil (g + oil - - 92 neurones from 6 rats). Figure gives the overall mean and the dots represent the individual means.
n u m b e r o f M P O A H n e u r o n e s o r t h o d r o m i c a l l y excited f r o m the LS (mean n u m b e r per electrode p e n e t r a t i o n = 1.55, r a n g e 1.00-2.33 - - f o r the g o n a d a l l y intact g r o u p ; a n d 1.99, r a n g e 1.00-3.00 - - for the c a s t r a t e d g r o u p , U = 17, P > 0.05); their m e a n t h r e s h o l d s t i m u l a t i o n c u r r e n t (650 F A , range 5407 6 0 / ~ A ; v 690 # A , range 5 7 0 - 7 6 0 / ~ A , U = 22.5, P > 0.05); m e a n activation latency (10.4 ms, range 7.9-16.3 ms; v 9.4 ms, range 6.4--12.2 ms, U = 28, P > 0.05; m e a n latency v a r i a t i o n (1.4 ms, range 1.0-1.7 ms; v 1.6 ms, range 1.2-2.1 ms, U ~ 22.5, P > 0.05) o r m e d i a n frequency following at 1.2 times t h r e s h o l d c u r r e n t (35 Hz, range 23.2-75.2 H z ; v 36 Hz, range 22-76.9 Hz, U ~ 28.5, P > 0.05). A 50 "o o
-~
40
E t,r) tn ~_...=
cally excited f r o m the C F I M B ( m e a n = 5 8 . 7 ~ , range 33.3 ~o-85.7 ~o for the g o n a d a l l y intact g r o u p ; a n d 5 3 . 8 ~ , range 28.6~o-71.4~o for the c a s t r a t e d group). This s u b - p o p u l a t i o n o f M P O A H n e u r o n e s w i t h the a d d i t i o n a l i n p u t f r o m C F I M B also showed the significant r e d u c t i o n in their a b i l i t y to follow 0.6 H z s t i m u l a t i o n o f the LS after c a s t r a t i o n (from 9 8 . 0 ~ to 62.7 ~ ; U = 1, P < 0.001) as d i d the neurones r e s p o n d i n g to LS s t i m u l a t i o n alone (from 7 8 . 9 ~ to 41.1~o; U = 12, P = 0.02). T h o s e M P O A H n e u r o n e s s t i m u l a t e d f r o m b o t h the LS a n d the C F I M B s h o w e d the same effects o f c a s t r a t i o n after s t i m u l a t i o n o f the C F I M B as have been previously r e p o r t e d s, including a similar r e d u c e d ability to follow 0.6 H z s t i m u l a t i o n . A small p e r c e n t a g e o f M P O A H n e u r o n e s activ a t e d f r o m the LS r e s p o n d e d to a single pulse stimul a t i o n o f this site b y p r o d u c i n g a p a i r o f action potentials. This m o d e o f n e u r o n a l response was previously r e p o r t e d for M P O A H n e u r o n e s s t i m u l a t e d f r o m the C F I M B s. B o t h this previous study, a n d the p r e s e n t one confirmed t h a t c a s t r a t i o n r e d u c e d the p e r c e n t a g e o f n e u r o n e s r e s p o n d i n g to C F I M B stim u l a t i o n in this way. In the present study, however, the percentage o f M P O A H n e u r o n e s r e s p o n d i n g to LS s t i m u l a t i o n like this was n o t affected b y castration ( m e a n = 2 1 . 2 ~ vs 13.5 ~ , U - - 20, P > 0.05). C a s t r a t i o n was also f o u n d to have no effect on the
30
oE
Z~
•
e
o
20
~
o~o,o
MFB
ARC ME
MFB
anti
anti
ortho
ARC ME ortho
MFB ÷ ARC ME ortho
5O O =~
40
E
u)(n
~
O
,o I 10
0~6 ~mee MFB
A RC/M F
MFB
anti
anti
ottho
ARC/.~ . F . ortho
AR~ME ortho
e~e
.F.
,,cJ.E
anti
anti
"i" ARC/ME M ; B ortho
ortho
Fig. 2. A: percentage of MPOAH neurones stimulated antidromically from the LS (35 neurones from 5 rats) which are orthodromically (oitho) or antidromically (anti) stimulated from the MFB or the ARC/ME or both. B: percentage of MPOAH neurones stimulated orthodromically from the LS (100 neurones from 5 rats) which are orthodromically or antidromically stimulated from the MFB or ARC/ME or both. Figure gives the overall mean and the dots represent the individual means.
140
D ~-
t ~
".
/, ~.~
V/ll,~
x ~
,
/
l
'.o,,~/j ,/
B t,,/ C ...... )
-'(
/,\
E
.----~.-//'/_~ !,iu"..~-" -7~- ,,. o.,.,,! % .... , ~ - -
C
( I \"'¢/l:',~
'q~"¢
I F
i ilt'= ," 1 mm
Fig. 3. Location of M P O A H neurones stimulated antidromically (17) or orthodromically (O) from the LS. Neurones which are also antidromically stimulated from the MFB are shown by ( l l or 0 ) and from the A R C / M E by (©). Neurones driven orthodromically from the M F B are shown by 'J' and from the A R C / M E by ' - - ' . Schematic coronal sections are adapted from the atlas of KSnig and Klippe111. Co-ordinates are: A, A7020 # m ; B, A6860/~m; C, A6790/~m; D, A6670 Fm; E, A6570/~m; F, A6360 F m and A6280/~m. pom = medial preoptic area; ah = anterior hypothalamus.
141
Effect of TP on M P O A H neurone responses to stimulation of the L S A total of 202 M P O A H neurones orthodromically excited from the LS were recorded from castrated animals treated with TP (110 neurones from 6 rats) or oil alone (92 neurones from 6 rats). Fig. 1B shows that TP significantly increased the percentage of neurones which could follow 0.6 Hz stimulation of the LS (from mean 52.5~o to 90.1 ~o, U -~ 0, P < 0.001). The mean percentage of M P O A H neurones which could follow 0.6 Hz stimulation of the LS did not differ significantly for gonadally intact rats (Experiment 1) and TP-treated rats (U = 23.5, P > 0.05). Responses of M P O A H neurones orthodromically excited from L S to stimulation of the MFB and ARC~ME In 5 gonadally intact male rats the responses of 135 M P O A H neurones stimulated from the LS (35 neurones antidromically stimulated and 100 orthodromically stimulated) were monitored after MFB and A R C / M E stimulation. Fig. 2A shows the mean percentage of antidromically stimulated neurones driven from the MFB and A R C / M E and Fig. 2B shows the same for the orthodromically stimulated neurones. For M P O A H neurones stimulated antidromically from the LS, 10.8 ~o were also stimulated antidromically from the MFB and a further 11.5 ~o orthodromically excited. None of these neurones were antidromically stimulated from A R C / M E although 7.9 ~ were orthodromically stimulated. For M P O A H neurones stimulated orthodromically from the LS 23.2 ~o were also antidromically stimulated from the MFB and 9.5 ~ from ARC/ME. A further 2 8 . 4 ~ and 3 1 . 6 ~ of neurones were stimulated orthodromically from respective structures. 12.1~ of neurones were orthodromically excited from both sites; 5 ~ antidromically stimulated from the MFB and orthodromically stimulated from A R C / M E and 3 . 4 ~ vice versa. Fig. 3 shows the approximate locations of all types of responsive neurones within the MPOAH. DISCUSSION
Castration significantly reduces the percentage of M P O A H neurones which can follow 0.6 Hz stimula-
tion of the LS and this can be reversed by treatment with testosterone propionate. The subpopulations of neurones driven only from the LS or from both the LS and CFIMB show the same effect of castration as the overall population. Castration has no effect on the number of stimulated neurones, their stimulation thresholds, activation latencies, latency variations or frequency following at 1.2 times threshold current. Those M P O A H neurones which can be stimulated from the CFIMB (in addition to the LS) show the same characteristics and the same effects of castration as have been previously reported s. A quantitative analysis of the connections of LS driven M P O A H neurones with the MFB and the A R C / M E reveals contributions from both these structures. M P O A H neurones identified as projecting to the LS have a small input from both the MFB and ARC/ME. A small number of these neurones have additional outputs into the MFB, but not the ARC/ME. This latter finding indicates that some M P O A H neurones have bifurcating axons to the LS and the MFB. A quarter of MPOAH neurones with inputs from the LS have outputs into the MFB and a somewhat lesser number have outputs to the ARC/ ME. A large number of this group of neurones also have inputs from the MFB and A R C / M E and some have inputs from both or alternatively inputs from one and an output to the other. The effect of testosterone on the ability of M P O A H neurones to follow 0.6 Hz stimulation of the LS could be due to the hormone acting either on the LS or the MPOAH. That is, either the LS is rendered less excitable by castration or the M P O A H less responsive. The results suggest, however, that the M P O A H is the relevant site of action. Firstly, there is no significant rise in the threshold current needed to stimulate M P O A H neuronal responses from the LS after castration. If castration had altered the excitability of the LS one might have expected a consequent change in this parameter. Secondly, the other findings that activation latencies, latency variations and frequency following parameters are unchanged by castration again supports the argument that the LS neurones are not the critical site. The absence of latency variation changes following castration also suggests that the synapse is not responsible for the effect since any change in synaptic efficiency might be expected to
142 produce more variable synaptic delays. Thirdly, the M P O A H neurones which also receive inputs from C F I M B show the same effect of castration after stimulation of this site. Although these M P O A H neurones receiving inputs from C F I M B show other effects of castration (reduction in percentage of paired action potential responses and decrease in frequency following at 1.2 times threshold current) they have been shown to be probably due to changes in corticomedial amygdala (CMA) neurones projecting to the M P O A H (via which the fimbria input to the M P O A H projects 8) and not to changes in the M P O A H . Overall, however, the results demonstrate that M P O A H neurones receiving projections from the LS are excited less by LS stimulation after castration and this effect can be reversed by testosterone. This provides further evidence for the hypothesis that sexual behaviour deficits following castration may be partly caused by changes in the effectiveness of excitatory inputs to the M P O A H (whether the critical site is the neuronal group proiecting to the M P O A H or the M P O A H neurones themselves).
The third experiment investigating the connections of LS stimulated M P O A H neurones with the MFB and the A R C / M E produced ambiguous results. Around one quarter of these neurones had direct outputs into the MFB and just under 10 ~ to the ARC/ME. Since the output from the M P O A H into the MFB is crucial for normal male copulatory responses14,16 this finding confirms that the LS input to the M P O A H may be involved in the control of male sexual behaviour. The smaller output to the A R C / M E also implies a possible role for LS modulation of pituitary hormone release via the MPOAH. The effect of castration on these M P O A H neurones driven from the LS may therefore reflect both changes in sexual behaviour and, for instance, gonadotrophin release.
REFERENCES
tic/anterior hypothalamic neurone responses to stimulation of the fimbria in the rat, J. Physiol. (Lond.), 315 (1982) 449-461. 9 Kendrick, K. M., Electrophysiological effects of testosterone on the medial preoptic/anterior hypothalamus, J. Endocr., in press. 10 Kendrick, K. M. and Drewett, R. F., Testosterone reduces refractory period of stria terminalis neurones in rat brain, Science, 204 (1979) 877-879. 11 K6nig, J. F. R. and Klippel, R. A., The Rat Brain, Williams and Wilkins, Baltimore, 1963. 12 MacLean, P. D. and Ploog, D. W., Cerebral representation of penile erection, J. Neurophysiol., 25 (1962) 29-55. 13 Sar, M. and Stumpf, W. E., Autoradiographic localisation of radioactivity in the rat brain after injection of [1,2-aH]testosterone, Endocrinology, 92 (1973) 251-256. 14 Scouten, C. W., Burrell, L., Palmer, T. and Cegavske, C. F., Lateral projections of the medial preoptic area are necessary for androgenic influence on urine marking and copulation in rats, Physiol. Behav., 25 (1980) 237-243. 15 Silverman, A. J. and Krey, L. C., The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea pig brain. I. Intra- and extra-hypothalamic projections, Brain Research, 157 (1978) 233-246. 16 Szechtman, H., Caggiula, A. R. and Wulkan, D., Preoptic knife cuts and sexual behaviour in male rats, Brain Research, 150 (1978) 569-591.
1 Conrad, L. C. A. and Pfaff, D. W., Axonal projections of medial preoptic and anterior hypothalamic neurons, Science, 190 (1975) 1112-1114. 2 Giantonio, G. W., Lund, N. L. and Gerall, A. A., Effect of diencephalic and rhinencephalic lesions on the male rat's sexual behaviour, J. comp. physiol. Psychol., 73 (1970) 38-46. 3 Goodman, E. D., Bunnell, B. N., Dewsbury, D. A. and Boland, B., Septal lesions and male rat copulatory behavior, Psychon. Sci., 16 (1969) 123-132. 4 Harris, V. H. and Sachs, B. D., Copulatory behaviour in male rats following amygdaloid lesions, Brain Research, 86 (1975) 514-518. 5 Kawakami, M., Arita, J., Yoshioka, E., Visessuvan, S. and Akema, T., Data on the sites of the stimulatory feedback action of gonadal steroids indispensable for follicle-stimulating hormone release in the rat, Endocrinology, 103 (1978) 752-759. 6 Kawakami, M., Yoshioka, E., Konda, N., Arita, J. and Visessuvan, S., Data on the sites of the stimulatory feedback action of gonadal steroids indispensable for luteinizing hormone release in the rat, Endocrinology, 102 (1978) 791-797. 7 Kendrick, K. M., Inputs to testosterone-sensitive stria terminalis neurones in the rat brain and the effects of castration, J. Physiol. (Lond.), 315 (1982) 437-447. 8 Kendrick, K. M., Effects of castration on medial preop-
ACKNOWLEDGEMENTS This work was supported by the M R C and carried out in the Psychology Department at Durham University. I thank Shirley Whiteley for her help with the histology and Dr. A. F. Dixson for helpful discussion and critical reading of the manuscript.