- Email: [email protected]
The estrous cycle: its influence on single unit activity in the forebrain
SHORT
435
COMMUNICATIONS
The estrous cycle: its influence on single unit activity in the forebrain The role of the forebrain in the secretion of sex hormones and the display of sexual behavior has recently received considerable attention 1. The influence of ovarian hormones on electrical activity of the forebrain has been reportedZ,5, 7-10. Electrophysiological studies on the forebrain have demonstrated cyclic changes in electrical activity, i.e. changes in EEG and multi-unit activity, during the estrous cycle14,~L Such gross recording techniques although useful do not usually permit one to determine whether the behavior of the individual cells tends to be excitatory, inhibitory, or a combination of these. The present experiment was carried out in the female rat to inquire into the possibility of specific changes in single cell activity in the cingulate cortex, lateral septal area, preoptic area and anterior hypothalamic area associated with cyclic variations in the estrous cycle. Extracellular recordings of single cell activity with glass capillary microelectrodes (filled with 3 M KC1, tip diameter 1 /~m, tip impedance 2-5 Mf~) was effected under urethane (1.2 g/kg; injected 2 5 ~ w/v solution) anesthesia. Action potentials were monitored visually and acoustically, stored on magnetic tape and filmed when required. The electrodes were stereotaxically inserted so as to pass through the cingulate cortex (CC), lateral septal area (LS), preoptic area (POA) and the anterior hypothalamic area (AHA). After a spontaneously firing unit had been contacted, an interval of 5-10 min was allowed for its stabilization. Cells that showed signs of deterioration were discarded. All cells were held for a sufficiently long time to establish a stable firing rate. Recording sites were confirmed by reconstructing the electrode tract with the aid of stereotaxically determined depth readings. In the present study we recorded from 778 cells in the forebrain of 80 rats, a maximum of l0 ceils per rat. The animals had a 4-day estrous cycle. Vaginal smears ]
Merest rus.Diestrus
E~ Proestrus ]
Estrus N : 778 units
0,80 o) 0
5 D. 0 . 4 0 o Q.
CC
LS
POA
I
AHA
Fig. 1. Effect of the estrous cycle on single cell activity in the cingulate cortex, lateral septal area, preoptic area and anterior hypothalamic area. Brain Research, 30 (1971) 435-438
436
SHORT C O M M U N I C A T I O N S Ill Metest rus.Diestrus [ ] I~oestrus
Lateral Septum N" 198 units
Cingulate Cortex N- 159 units
Yo
[ ] Estrus
4Q
20
<1
~
1-2
2-3
3-4
4-5
>5
<1
Preoptic Area N- 235 units
Yo
1-2
2-3
3-4
4-5
>5
Anterior Hypothalamus N- 186 units
4Q
20
<1
1-2
2-3
3-4
4-5
>5
<1
1-2
2-3
3-4
4-5
>5
Spikes per Second
Fig. 2. Frequency distribution of the firing rates for the cingulate cortex, lateral septal area, preoptic area and anterior hypothalamic area as recorded during the estrous cycle.
were taken daily, and unit activity was correlated with a specific stage in the estrous cycle, either proestrus, estrus or metestrus-diestrus. A comparison of the cyclic variations in firing rates with changes in the estrous cycle is shown in Fig. 1. It is evident that changes in the estrous cycle can alter firing rates in specific forebrain sites. In fact, changes in firing rates as seen in the preoptic area (P < 0.001) and anterior hypothalamic area (P < 0.05) are greater during proestrus than during the other two stages. Thus preoptic and anterior hypothalamic cells fire faster in the proestrus rat than in the estrus and metestrus-diestrus rats. While in the cingulate cortex, unit activity was greatest during metestrus-diestrus (P < 0.05), in comparison with the other two stages. No differences were found in the lateral septal area. The frequency distribution of the firing rates for the various forebrain sites for each stage of the estrous cycle are presented in Fig. 2. From the histograms it is obvious that in the preoptic area, lateral septal area and anterior hypothalamic area there is a larger percentage of cells firing at a slower rate of spontaneous activity than in the cingulate cortex where firing rates are comparatively faster. The effect ofestrous on the shape of the frequency distribution was evaluated by K o l m o g o r o v - S m i r n o v twosample test adjusted for large and unequal N's 12. Comparison of the frequency distributions obtained from the lateral septal area showed no significant difference over the stages of the estrous cycle. On the other hand, in the cingulate cortex, frequency distributions obtained were shown to be significantly different (P < 0.001). Analysis of the preoptic and anterior hypothalamic histograms revealed no significant difference in the frequency distribution of unit activity between estrus and metestrus-diBrain ReSearch, 30 (1971) 435-438
SHORT COMMUNICATIONS
437
estrus while a significant difference was observed between proestrus and the remaining two stages of the cycle (POA: P < 0.001 ; A H A : P < 0.05). It is concluded that changes in single cell activity, i.e., a change in the rate of activity and in the shape of the frequency distribution as seen in the cingulate cortex: preoptic area and anterior hypothalamic area, are associated with the cyclic variations in the estrous cycle. The increase in unit activity in the preoptic and anterior hypothalamic areas occurs on the day of proestrus. Interestingly, it has been proposed that an increase in hypothalamic cell activity might be a prerequisite for various reproductive functions such as ovulation la or the onset of sexual behavior 6. Both of these assumptions are supported by the present findings as well as observations in the intact animal using macro-recording techniques14,15, and in hypothalamic deafferentation experiments, i.e., hypothalamic island preparations utilizing microelectrode recording techniques 4. The increase seen in cingulate cortex cell activity during metestrus-diestrus is extremely interesting and totally unexpected. Although it has been reported that hemidecortication of the female rat resulted in endocrine changes as well as alterations in cyclicity2,1L The cyclic changes in cingulate cortex cell activity tend to emphasize the importance of extrahypothalamic nervous influence on reproductive functions. All the above mentioned cells (778) were tested with various hormonal, peripheral, and sensory stimuli that will be reported in a future publication. We wish to thank Mrs. Christa Jenkins for her very able technical assistance. The research was supported by grants from U.S. Public Health Service (8RO-1HD-00343-03 and 3-RO-HDO-343-03S3) and a grant from the Hancock Foundation. Dr. Robert L. Moss is presently a postdoctoral fellow from the National Institutes of Mental Health at the University of Bristol Medical School, Department of Anatomy, Bristol, Great Britain. Department of Psychology, Claremont Graduate School, Claremont, Calif. ( U.S.A,)
ROBERT L. MOSS O. THOMAS LAW
1 ANANO,B. K., Nervous regulation of reproductive behaviour, Bull. nat. Inst. Sci. (India), 27 (1964) 20-27. 2 COVtAN,M. R., MIGLIORINI,R. a., AND TRAMEZZANI,J. H., Endocrine changes in hemidecorticate rats, Acta physiol, lat.-amer., 9 (1959) 24-34. 3 CROSS,B. A., ANDSmVER,I. A., Effect of luteal hormone on the behavior of hypothalamicneurones in pseudopregnant rats, J. Endocr., 31 (1965) 251-263. 4 CROSS,B. A., AND DYER, R. G., Characterization of unit activity in hypothalamic islands with special reference to hormone effects. In L. MARTINi,M. MOTrA AND F. FRASCHINI(Eds.), The Hypothalamus, Academic Press, New York, 1970, pp. 115-122. 5 KAWAKAMI,M., AND SAWYER,C. H., Neuroendocrine correlates of changes in brain activity threshold by sex steroids and pituitary hormones, Endocrinology, 65 (1959) 652-668. 6 LAW,O. T., ANDMOSS,R. L., Sex and single neuron. Paper presented at the meeting of the Western Psychological Association, Vancouver, B.C., June, 1969. 7 LAW,O. T., ANDSACKET,G. P., Hypothalamic potentials in female rats evoked by hormones and vaginal stimulation, Neuroendocrinology, 1 (1965-66) 31-34. 8 LINCOLN,D. W., Unit activity in the hypothalamus, septum and preoptic area of the rat: Characteristics of spontaneous activity and the effect of oestrogen, J. Endocr., 37 (1967) 177-189. Brain Research, 30 (1971) 435--438
438
SHORT COMMUNICATIONS
9 LINCOLN,D. W., AND CROSS,B. A., Effect of oestrogen on the responsiveness of neurones in the hypothalamus, septum and preoptic area of rats with light induced persistent oestrus, J. Endocr., 37 (1967) 191-203. 10 RAMIREZ,V. D., KOMISARUK,B., WHITMOYER,D. E., AND SAWYER,C. H., Effects of hormones and vaginal stimulation on EEG and hypothalamic units in rats, Amer. J. Physiol., 212 (1967) 13671384. 11 RODRIGUEZ,J. A., Influencia de la corteza cerebral sobre el ciclo sexuel de la rats blanca, Rev. Soc. argent. Biol., 35 (1959) 5-15. 12 SIEGEL, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956, pp. 127-136. 13 TERASAWA,E., AND SAWYER,C. H., Changes in electrical activity in the rat hypothalamus related to electrochemical stimulation of adenohypophyseal function, Endocrinology, 85 (1969) 143-149. 14 TERASAWA,E., AND TIM1RAS, P. S., Electrical activity during the estrous cycle of the rat; cyclic changes in limbic structures, Endocrinology, 83 (1968) 207-216. 15 TERASAWA,E., AND TLMIRAS,P. S., Cyclic changes in electrical activity of the rat midbrain re ticular formation during the estrous cycle, Brain Research, 14 (1969) 189-198. Accepted April 30th, 1971)
Brain Research, 30 (1971) 435-438
435
COMMUNICATIONS
The estrous cycle: its influence on single unit activity in the forebrain The role of the forebrain in the secretion of sex hormones and the display of sexual behavior has recently received considerable attention 1. The influence of ovarian hormones on electrical activity of the forebrain has been reportedZ,5, 7-10. Electrophysiological studies on the forebrain have demonstrated cyclic changes in electrical activity, i.e. changes in EEG and multi-unit activity, during the estrous cycle14,~L Such gross recording techniques although useful do not usually permit one to determine whether the behavior of the individual cells tends to be excitatory, inhibitory, or a combination of these. The present experiment was carried out in the female rat to inquire into the possibility of specific changes in single cell activity in the cingulate cortex, lateral septal area, preoptic area and anterior hypothalamic area associated with cyclic variations in the estrous cycle. Extracellular recordings of single cell activity with glass capillary microelectrodes (filled with 3 M KC1, tip diameter 1 /~m, tip impedance 2-5 Mf~) was effected under urethane (1.2 g/kg; injected 2 5 ~ w/v solution) anesthesia. Action potentials were monitored visually and acoustically, stored on magnetic tape and filmed when required. The electrodes were stereotaxically inserted so as to pass through the cingulate cortex (CC), lateral septal area (LS), preoptic area (POA) and the anterior hypothalamic area (AHA). After a spontaneously firing unit had been contacted, an interval of 5-10 min was allowed for its stabilization. Cells that showed signs of deterioration were discarded. All cells were held for a sufficiently long time to establish a stable firing rate. Recording sites were confirmed by reconstructing the electrode tract with the aid of stereotaxically determined depth readings. In the present study we recorded from 778 cells in the forebrain of 80 rats, a maximum of l0 ceils per rat. The animals had a 4-day estrous cycle. Vaginal smears ]
Merest rus.Diestrus
E~ Proestrus ]
Estrus N : 778 units
0,80 o) 0
5 D. 0 . 4 0 o Q.
CC
LS
POA
I
AHA
Fig. 1. Effect of the estrous cycle on single cell activity in the cingulate cortex, lateral septal area, preoptic area and anterior hypothalamic area. Brain Research, 30 (1971) 435-438
436
SHORT C O M M U N I C A T I O N S Ill Metest rus.Diestrus [ ] I~oestrus
Lateral Septum N" 198 units
Cingulate Cortex N- 159 units
Yo
[ ] Estrus
4Q
20
<1
~
1-2
2-3
3-4
4-5
>5
<1
Preoptic Area N- 235 units
Yo
1-2
2-3
3-4
4-5
>5
Anterior Hypothalamus N- 186 units
4Q
20
<1
1-2
2-3
3-4
4-5
>5
<1
1-2
2-3
3-4
4-5
>5
Spikes per Second
Fig. 2. Frequency distribution of the firing rates for the cingulate cortex, lateral septal area, preoptic area and anterior hypothalamic area as recorded during the estrous cycle.
were taken daily, and unit activity was correlated with a specific stage in the estrous cycle, either proestrus, estrus or metestrus-diestrus. A comparison of the cyclic variations in firing rates with changes in the estrous cycle is shown in Fig. 1. It is evident that changes in the estrous cycle can alter firing rates in specific forebrain sites. In fact, changes in firing rates as seen in the preoptic area (P < 0.001) and anterior hypothalamic area (P < 0.05) are greater during proestrus than during the other two stages. Thus preoptic and anterior hypothalamic cells fire faster in the proestrus rat than in the estrus and metestrus-diestrus rats. While in the cingulate cortex, unit activity was greatest during metestrus-diestrus (P < 0.05), in comparison with the other two stages. No differences were found in the lateral septal area. The frequency distribution of the firing rates for the various forebrain sites for each stage of the estrous cycle are presented in Fig. 2. From the histograms it is obvious that in the preoptic area, lateral septal area and anterior hypothalamic area there is a larger percentage of cells firing at a slower rate of spontaneous activity than in the cingulate cortex where firing rates are comparatively faster. The effect ofestrous on the shape of the frequency distribution was evaluated by K o l m o g o r o v - S m i r n o v twosample test adjusted for large and unequal N's 12. Comparison of the frequency distributions obtained from the lateral septal area showed no significant difference over the stages of the estrous cycle. On the other hand, in the cingulate cortex, frequency distributions obtained were shown to be significantly different (P < 0.001). Analysis of the preoptic and anterior hypothalamic histograms revealed no significant difference in the frequency distribution of unit activity between estrus and metestrus-diBrain ReSearch, 30 (1971) 435-438
SHORT COMMUNICATIONS
437
estrus while a significant difference was observed between proestrus and the remaining two stages of the cycle (POA: P < 0.001 ; A H A : P < 0.05). It is concluded that changes in single cell activity, i.e., a change in the rate of activity and in the shape of the frequency distribution as seen in the cingulate cortex: preoptic area and anterior hypothalamic area, are associated with the cyclic variations in the estrous cycle. The increase in unit activity in the preoptic and anterior hypothalamic areas occurs on the day of proestrus. Interestingly, it has been proposed that an increase in hypothalamic cell activity might be a prerequisite for various reproductive functions such as ovulation la or the onset of sexual behavior 6. Both of these assumptions are supported by the present findings as well as observations in the intact animal using macro-recording techniques14,15, and in hypothalamic deafferentation experiments, i.e., hypothalamic island preparations utilizing microelectrode recording techniques 4. The increase seen in cingulate cortex cell activity during metestrus-diestrus is extremely interesting and totally unexpected. Although it has been reported that hemidecortication of the female rat resulted in endocrine changes as well as alterations in cyclicity2,1L The cyclic changes in cingulate cortex cell activity tend to emphasize the importance of extrahypothalamic nervous influence on reproductive functions. All the above mentioned cells (778) were tested with various hormonal, peripheral, and sensory stimuli that will be reported in a future publication. We wish to thank Mrs. Christa Jenkins for her very able technical assistance. The research was supported by grants from U.S. Public Health Service (8RO-1HD-00343-03 and 3-RO-HDO-343-03S3) and a grant from the Hancock Foundation. Dr. Robert L. Moss is presently a postdoctoral fellow from the National Institutes of Mental Health at the University of Bristol Medical School, Department of Anatomy, Bristol, Great Britain. Department of Psychology, Claremont Graduate School, Claremont, Calif. ( U.S.A,)
ROBERT L. MOSS O. THOMAS LAW
1 ANANO,B. K., Nervous regulation of reproductive behaviour, Bull. nat. Inst. Sci. (India), 27 (1964) 20-27. 2 COVtAN,M. R., MIGLIORINI,R. a., AND TRAMEZZANI,J. H., Endocrine changes in hemidecorticate rats, Acta physiol, lat.-amer., 9 (1959) 24-34. 3 CROSS,B. A., ANDSmVER,I. A., Effect of luteal hormone on the behavior of hypothalamicneurones in pseudopregnant rats, J. Endocr., 31 (1965) 251-263. 4 CROSS,B. A., AND DYER, R. G., Characterization of unit activity in hypothalamic islands with special reference to hormone effects. In L. MARTINi,M. MOTrA AND F. FRASCHINI(Eds.), The Hypothalamus, Academic Press, New York, 1970, pp. 115-122. 5 KAWAKAMI,M., AND SAWYER,C. H., Neuroendocrine correlates of changes in brain activity threshold by sex steroids and pituitary hormones, Endocrinology, 65 (1959) 652-668. 6 LAW,O. T., ANDMOSS,R. L., Sex and single neuron. Paper presented at the meeting of the Western Psychological Association, Vancouver, B.C., June, 1969. 7 LAW,O. T., ANDSACKET,G. P., Hypothalamic potentials in female rats evoked by hormones and vaginal stimulation, Neuroendocrinology, 1 (1965-66) 31-34. 8 LINCOLN,D. W., Unit activity in the hypothalamus, septum and preoptic area of the rat: Characteristics of spontaneous activity and the effect of oestrogen, J. Endocr., 37 (1967) 177-189. Brain Research, 30 (1971) 435--438
438
SHORT COMMUNICATIONS
9 LINCOLN,D. W., AND CROSS,B. A., Effect of oestrogen on the responsiveness of neurones in the hypothalamus, septum and preoptic area of rats with light induced persistent oestrus, J. Endocr., 37 (1967) 191-203. 10 RAMIREZ,V. D., KOMISARUK,B., WHITMOYER,D. E., AND SAWYER,C. H., Effects of hormones and vaginal stimulation on EEG and hypothalamic units in rats, Amer. J. Physiol., 212 (1967) 13671384. 11 RODRIGUEZ,J. A., Influencia de la corteza cerebral sobre el ciclo sexuel de la rats blanca, Rev. Soc. argent. Biol., 35 (1959) 5-15. 12 SIEGEL, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956, pp. 127-136. 13 TERASAWA,E., AND SAWYER,C. H., Changes in electrical activity in the rat hypothalamus related to electrochemical stimulation of adenohypophyseal function, Endocrinology, 85 (1969) 143-149. 14 TERASAWA,E., AND TIM1RAS, P. S., Electrical activity during the estrous cycle of the rat; cyclic changes in limbic structures, Endocrinology, 83 (1968) 207-216. 15 TERASAWA,E., AND TLMIRAS,P. S., Cyclic changes in electrical activity of the rat midbrain re ticular formation during the estrous cycle, Brain Research, 14 (1969) 189-198. Accepted April 30th, 1971)
Brain Research, 30 (1971) 435-438