The influence of ovariectomy, estrogen, and progesterone on the catecholamine content of hypothalamic nerve cells in the rat

BRA1N R E S E A R C H

199

T H E I N F L U E N C E OF OVARIECTOMY, ESTROGEN, AND P R O G E S T E R O N E ON THE C A T E C H O L A M I N E C O N T E N T OF HYPOYHALAMIC NERVE CELLS IN THE RAT

WALTER LICHTENSTEIGER, KARI KORPELA, HEINRICH LANGEMANN AND PAUL J. KELLER Department of Pharmacology and (P.J.K.) Hormone Laboratory of the Department of Gynecology and Obstetrics, University of Zurich, Zurich (Switzerland)

(Accepted April 4th, 1969)

INTRODUCTION

When the catecholamine fluorescence of tuberal nerve cells, treated according to the method of Falck and Hillarp, was analyzed microfluorimetrically in the rat, characteristic changes in fluorescence intensity were found during the 4-day estrous cyclelS, 19. Since these nerve cells belong to tubero-infundibular neurons which terminate in the region of the primary plexus of the hypophysial portal system14,15,21, the variations could reflect changes related to the control of anterior pituitary function. This hypothesis is further supported by the finding of a different reactive pattern in the catecholamine-containing nerve cells of the substantia nigra 19. Hence, we have studied the intensity of the catecholamine fluorescence in tuberal nerve cells of female rats with a disturbed pituitary-gonadal axis 22. Simultaneously the secretory activity of the pituitary gland was examined by determinations of the serum levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH). MATERIAL AND METHODS

Animals

The albino rats were descended from the same random-bred CF (Carworth Farm) strain that had been used in one of the previous studies 19. They were provided by the Animal Breeding Institute of the Department of Veterinary Medicine, University of Zurich. After weaning, the females of each litter were housed together at a controlled day-and-night rhythm (light period from 05.00 to 19.00 h) and at constant temperature (22°C). They were fed with a standard diet (Altromin R®) and received water ad libitum. The animals belonged to 10 litters. Their age at death ranged between 12 and 27 weeks (200-280 g body weight). Bran Research, 16 (1969) 199-214

200

W, LI(t-ITI-.,,S'Iti/(;H~, t'l d~.

Steroids

The preparations ofestradiol dipropionate (Ct BA) were diluted with neutralized olive oil. The injected volume was 0.1 ml. Progesterone (C1BA) was also used in an oily solution (0.2 ml). Both hormones were administered subcutaneousl3, always at 14.00 h. Experimental groups

In the investigations on the early phase o f ovariectomy, vaginal smears were taken daily between 07.00 and 09.00 h for at least 3 estrous cycles before ovariectomy was performed. Three groups o f 5 animals with a regular 4-day estrous cycle were ovariectomized in light ether anesthesia on diestrous day 1 (diestrus 1), and studied 4, 7 and 8 days later. One g r o u p of 5 rats remained ovariectomized for a period of 3-3.5 weeks (17, 2 x 20, 2 × 23 days) and 3 rats for 38, 45 and 49 days, respectively. These two groups were used as controls in the experiments with steroid hormones. The steroids were administered according to the schedule given in Table 1. In these groups vaginal smears were taken only during the experiments. The animals were killed by decapitation between 14.00 and 14.15 h, i.e., at the same time as in our previous studies, in order to avoid possible diurnal variations. Ovariectomy was verified at autopsy. TABLE I TREATMENT OF OVARIECTOMIZED ANIMALS

Number of animals

Drug

1 5 5 5

Estradiol dipropionate Estradiol dipropionate Estradiol dipropionate Estradiol dipropionate

5

Progesterone Estradio1100 #g and progesterone 2 mg [- estradio1100 fig and progesterone 2 rng

Dose

1 fig 10 fig 10 #g lOOpg 100#g 2 mg i 2rag

Interval between injection of the drug and death of the animal

Duration of ovariectomy in individual animals"

24 h 24 h 48 h 48 h ~ 24 h 48 h t 24h

23 days 2 x 23, 2 x 27, 28 days 21,23, 2 :~, 27, 41 days 3 x 38, 2 .~ 45 days 3 x 21, 52, 60 days

48 h 2 x 21,2 x 27, 28 days 24 h !

Microfluorimetry

In one experimental series, frontal brain pieces that comprised the tuberal region f r o m 3 or 4 rats were worked up according to a strictly standardized version o f the fluorescence m e t h o d o f Falck and Hillarp 19, Small blocks o f a gelatin standard Brain Research, 16 (1969) 199-214

CATECHOLAMINE NEURONS AND GONADOTROPINS

201

were processed together with the tissues. They were derived from a 2 % gelatin solution adjusted to pH 7.5, which contained 2.78 • l0 -4 M noradrenaline (NA, base), and 1.39 • l0 -3 M ascorbic acid as a reducing agent. The negative standards were devoid of NA. The brain pieces were ultimately embedded in paraffin in vacuo together with several blocks of standard. They were serially cut at 7 # from the anterior border of the external layer of the median eminence to the point of attachment of the dorsoposterior wall of the infundibular stem. The microfluorimetric technique has recently been described in detail 19. It uses epi-illumination: the exciting light from a stabilized xenon lamp passes through appropriate filters and an illumination field diaphragm, and is conducted through the objective to the preparation. The fluorescent light emitted from the preparation again passes through the objective, then a barrier filter and a second diaphragm which is adjusted to give a measuring field of 2.8 by 2.8/t in the preparation (magnification 700 x ). The light is then detected by a photomultiplier tube, and its intensity is recorded. Measurements were performed on every 8th section (distance = 49 #), i.e., in about 22 sections per animal. The intensity of the light within the measuring field was determined in every nerve cell of the arcuate and periventricular hypothalamic nuclei that exhibited a visible catecholamine fluorescence in the monocular tube. Additional measurements were carried out on neighboring brain tissue and on the standards. The relative intensity Ir of the catecholamine fluorescence of individual nerve cells of the same section was obtained from the equation lr = (c - - t)/(gNA - gf), where c is the absolute fluorescence intensity of the cell, t the mean intensity of the background fluorescence of that section (3 measurements), and gxa and gf are the mean intensities of the NA-containing and of the NA-free standards of the same section (4-6 and 3 measurements, respectively). Statistical analysis. The nerve cells were grouped according to their relative fluorescence intensity into classes, each class representing a 10% change in lr. Frequency distributions of relative fluorescence intensity were established from the total cell counts of each experimental group. They were compared with each other according to the chi-square method. The changes observed in the frequency distributions were further characterized by means of two parameters, the mean relative fluorescence intensity and the skewness. Differences between the mean relative fluorescence intensities of the various groups were tested by the approximation method of Welch aa for comparing means when the population variances are different. The symmetry of the frequency distributions was expressed in terms of the coefficient fll -- #a'~/ /~2~ of the skewness, where/~z and/~3 are the 2nd and 3rd central moments, respectively. This coefficient becomes equal to zero in a completely symmetrical distribution (#3 = 0), and increases with decreasing symmetry. The estimates of ill that were calculated from the actual frequency distributions will be called bl. The coefficients bl of the various groups were compared with each other according to a procedure previously described 19. Differences in the mean number of visibly fluorescent nerve cells per section were tested between the various groups by means of the Wilcoxon rank test.

Brain Research, 16 (1969) 199-214

202

w. LICHTENSTEIGERet aL

Determination of F S H and L H in th~ serum

At decapitation, the blood of each rat was collected in an ice-cold plastic tube and centrifuged immediately. The serum (approximately 4 ml) was stored at --22"C until processed. FSH was determined by means of the ovarian augmentation assay it~ immature female mice treated with 50 I.U. of human chorionic gonadotropin ~. Because this method had a comparatively low sensitivity, the individual serum samples of each group were pooled. Three ml of unextracted serum were injected per animal. The 2nd International Reference Preparation (2.1RP) for human menopausal gonadotropin served as standard material. Results are expressed as l.U./ml serum. All experiments were conducted as 3-point assays, using 10-12 animals per assay. LH was estimated by means of the ovarian ascorbic acid depletion method in immature pseudopregnant female rats 27. The serum samples were tested individually at 0.4 and 1.2 ml. Again the 2. IRP was used as standard material and the results expressed as l.U./ml serum. All experiments were conducted as 4-point assays, using 12-14 animals per assay. Statistical analysis was performed as recommended by Borth et al. 4. RESULTS h~uence o f ovariectomy on the fluorescence intensity of catecholamine-containing nerve cells in the nucleus arcuatus and nucleus periventricularis hypothalami

When the rats were ovariectomized on diestrus 1 and investigated 4, 7 and 8 days later, the resulting frequency distributions did not correspond to the type that would have been expected in an undisturbed continuation of the cycle (Fig. 1, Tables Ii and Ill). The frequency distributions fluctuated between those of diestrus 1 and 2. They differed from each other as well as from the later stages of ovariectomy and from the frequency distributions of the estrous cycle 19 according to the chisquare test (P ~< 0.001 except for day 7 vs. diestrus 1, where P ~< 0.01). With respect to the mean relative fluorescence intensity and the symmetry (bl), the fi'equency distribution found 7 days after ovariectomy (day 7) appeared to be closely related to that of diestrus 1 (no significant difference), whereas the values of the remaining 2 days could not be correlated with a typical phase of the estrous cycle. Distributions with high values of mean relative fluorescence intensity and high degrees of symmetry were not observed on the 3 days that were studied during the early period. At 3-3.5 weeks after ovariectomy (Fig. 1 and Tables II and 1II), the mean relative fluorescence intensity approached the value of diestrus 2, which, however, remained slightly but significantly higher (P ~< 0.01). The symmetry of the frequency distribution became identical with that of diestrus 2. The two groups remained different according to the chi-square test (P ~< 0.001). The stage reached at this time continued until 5.5-7 weeks after ovariectomy (no significant differences at all). Therefore, this period was chosen for studying the effect of the steroid hormones. Brain Research, 16 (1969) 199-214

203

CATECHOLAMINE NEURONS AND GONADOTROPINS

Trio 70.

40.

I0- 0 ,

,

,

|

,

,

,

,

,

,

,

,-

bl

2.01.5-

',

e,

/\'

,

,

~

P

"',

o

,

,

,

,

,

,

~

h

P

o

°1 h

!

]

P

o

3+'~

weeks

sa-?

weeks

Fig. 1. Influence o f ovariectomy. T h e m e a n relative fluorescence intensity ],- (solid line) a n d the coefficient bl of the skewness (broken line) are indicated for a regular 4-day estrous cycle 19. T h e values observed at different time intervals after ovariectomy are s h o w n by bars• D1 = diestrus l; D2 = diestrus 2; P = proestrus" O = estrus. O v a r i e c t o m y was performed on D1. It strongly disturbed the cyclic pattern which would have been expected in rats with a regular 4-day cycle.

Effects of estrogen and progesterone on the fluorescence intensity of catecholaminecontaining tuberal nerve cells in ovariectomized rats The effect of the steroid hormones was compared with both groups of ovariectomized controls (3-3.5 weeks and 5.5-7 weeks). gstradiol dipropionate always provoked a shift of the frequency distribution towards classes of lower relative fluorescence intensity which was accompanied by a decrease in symmetry. The change led to frequency distributions that may be compared with the type of dicstrus 1 (Figs. 2 and 4, Tables II and 11I). They were significantly different from those of the ovariectomized controls (chi-square, mean relative fluorescence intensity and bl, P ~< 0.001). Strongest effects were noted 24 and 48 h after administration of 10 /zg of estradiol dipropionate. The mean relative fluorescence intensity was then even lower (P ~< 0.001), and the asymmetry (bl) higher (P ~< 0.01 for the group studied after 24 h vs. diestrus 1, P ~< 0.001 for the group studied after 48 h vs. diestrus 1), than on diestrus 1. Between 24 and 48 h after the injection there was a certain increase of the coefficient bl, but the two groups were not significantly different from each other. The administration of 1 #g of estradiol dipropionate caused a similar shift within 24 h. Fourty-eight hours after high doses of estradiol dipropionate the frequency distribution occupied a position that was intermediate between those of diestrus I and 2. It differed from them according to chi-square and mean relative fluorescence intensity (P ~< 0.001 ), whereas its symmetry (bl) was similar to that of diestrus 2. The distribution was also different from that observed 48 h after the 10/~g dose (P ~< 0.001). When progesterone was added to the high doses of estrogen, the frequency Brain Research, 16 (1969) 199-214

w. LICHTENSTI~IGEReta/.

204

u~25-

I

ovar iectom ized controls

E

I

estradiol dipropionate I lx lO~tg, 24hr

[7

~r

20-

15

10

10 20 ~0 ~0 s0

s0 70 ~

90 ~ i ~

~'o . . . . .

r

Fig. 2. Effect of estrogen. Frequency distributions of catecholamine-containing tuberal nerve cells classified according to their relative fluorescence intensities at 14.00 h. Abscissa: relative intensities of catecholamine fluorescence as percentage of the fluorescence intensity of the NAstandard. Ordinate: cell frequencies as percentage of the total cell numbers of each group (cf Table !I). As compared with the ovariectomized control group (3-3.5 weeks, white bars), 10 #g of estradiol dipropionate caused a marked shift of the frequency distribution towards classes of lower relative fluorescence intensity within 24 h (black bars). distribution was again displaced toward classes of lower relative fluorescence intensity (Fig. 4, Tables 11 and I11). The effect of the addition of progesterone was manifest in the chi-square test and in the difference of the mean relative fluorescence intensities (P ~< 0.001). On the other hand, the symmetry did not change significantly. In the group treated with progesterone in the absence of estrogen, the mean relative fluorescence intensity remained in the range of that of the ovariectomized controls (Figs. 3 and 4, Tables II and III). The symmetry of the frequency distribution decreased (P ~< 0.01). The resulting distribution differed from the controls also in the chi-square test (P ~< 0.001). It degree of symmetry became similar to that found 48 h after 2 x 100 #g of estradiol dipropionate. However, the lower degree of symmetry observed after progesterone was due to a decrease in the frequencies on both sides of the distribution, and thus differed from the kind of shift induced by estrogen (Fig. 3). The chi-square test revealed a significant difference between the groups treated with progesterone and estradiol dipropionate (P ~< 0.001), and the mean relative fluorescence intensities were also different (P ~< 0.001). (See note added in proof.) Brain Research, 16 (1969) 199-214

CATECHOLAMINE NEURONS AND GONADOTROPINS

25 L)

ovariectomlzed controls

[7

progesterone 2x2mg~ 48hr

B

205

(#

20

15

10

1" 11-" 21" 31- 41- 51- st- 7i- ei- 9i- I0~-1fi- ~121%Ir

10 20 30 40 50 60 70 80 90 100 110 120

Fig. 3. Effect of progesterone. Frequency distributions of catecholamine-containing tuberal nerve cells classified according to their relative fluorescence intensities at 14.00 h. Abscissa and ordinate as in Fig. 2. In the absence of estrogen, 2 × 2 mg of progesterone administered on 2 days had only a weak effect (hatched bars) which consisted in a slight reduction of the frequency of nerve cells on both sides of the distribution, i.e., in classes of low as well as of high fluorescence intensity. The control group ovarieetomized for 3-3.5 weeks is represented by white bars.

The mean n u m b e r of visibly fluorescent nerve cells per section was f o u n d to be 11.1 in the control group 3-3.5 weeks after ovariectomy, 8.2 at 5.5-7 weeks after o v a r i e c t o m y ; 13.8 after 1 yg, 13.0 at 24 h after 10 yg, 6.7 at 48 h after 10 y g and 6.1 after 2 × 100 /zg of estradiol d i p r o p i o n a t e ; 8.9 after progesterone a n d 9.3 after estradiol d i p r o p i o n a t e a n d progesterone. The differences between the various experimental groups are n o t significant. F u r t h e r m o r e , the mean n u m b e r s of cells per section did not differ significantly from those previously found d u r i n g the estrous cycle (except for a difference (P ~< 0.01) between the values observed on diestrus I and 2 on one side and 48 h after estradiol on the other side).

Gonadotropic activities The g o n a d o t r o p i c activities were determined in some of the n o r m a l cycling rats that were previously investigated 19 and in 6 experimental groups of the presznt study (Table IV). Three to seven weeks after g o n a d e c t o m y the F S H activity of pooled

Brain Research, 16 (1969) 199-214

206

W. L I ( H I 1 { N S IIZIGI-[:, ~:l t2/.

bl

O

4,0

3.5

ID 3.0

2.5

~X

2.0

1.5

[]

.,0;

1.0

0.5

20

30

40 50 6 0 7 0 % I r

Fig. 4. Comparison of the frequency distributions of relative fluorescence intensity. Abscissa, logarithms of the mean relative fluorescence intensity; ordinate, coefficient bl of the skewness (bl ~-0 if the distribution is completely symmetrical). Four-day estrous cycle according to ref. 19: Di, diestrus 1 ; D2, diestrus 2; P, proestrus; 0, estrus. Experimental groups: ~7, control group ovariectomized for 3-3.5 weeks; A , control group ovariectomized for 5,5-7 weeks: ×, ovariectomized rat 24 h after injection of 1 #g of estradiol dipropionate; tD, 24 h or, O, 48 h after 10 t~g of estradiol dipropionate; (3, 48 h after the first injection of 2 x 100/tg of estradiol dipropionate; [], 48 h after the first injection of 2 x 2 mg of progesterone; ~ , 48 h after the first combined injection of 2 :~ estradiol dipropionate 100 #g and progesterone 2 rag. The values of the estrous cycle show a relationship which in this diagram is represented by the straight line (see Discussion). A shift of the point given by Ir and bt towards the upper left results in a predominance of nerve cells with lower fluorescence intensity and a loss of symmetry o_f the frequency distribution. It was accompanied by a decrease in serum gonadotropin levels. The position of estrus may be due to a certain persistence of the reaction of the nerve cell body.

Brain Research, 16 (t969) 199-214

CATECHOLAMINE NEURONS AND GONADOTROPINS

207

serum was apparently higher than in the intact cycling rats, whereas the LH activity resembled the mean concentration found on diestrus 2. However, the latter values exhibited considerable variations. After administration o f 10/~g o f estradiol dipropionate during 1 or 2 days both levels decreased. The LH level was very low or not detectable and thus similar to that found on diestrus 1. As far as statistical analysis was possible, these differences were estimated to be at the border line of significance (P ~< 0.05). The administration o f 100 # g o f estradiol dipropionate on 2 days showed a rather surprising result. The FSH level increased, whereas the values of the LH level again more resembled those observed on diestrus 2. Neither activity was significantly different from those of ovariectomized control animals. When the high estrogen dose was combined with progesterone, the LH level tended

TABLE I1 DISTRIBUTION

OF

NERVE

CELLS ACCORDING

TO THE RELATIVE

INTENSITY

OF T H E I R

CATECHOLAMINE

FLUORESCENCE

Absolute values representing the total cell counts of 5 rats in each group (except for '5½ 7': 3 rats; 'e 1': 1 rat). L' ':'

1 10 11- 20 21 30 31--4.0 41 50 51- 60 61 70 71 80 81- 90 91 100 10[ 110 111-120 121 130 131 14.0 141-150 151 160 161 170 171-180 181 190 191 200 201 Total

Group* * day4

day7

day8

week week e l 3-3.5 5.5 7 24h

e 10 24h

elO 48h

e200 p 2 x 2 48h 48h

e : p 48h

10 71 198 266 222 180 14,9 97 53 39 17 14 5 3 2 3 0 0 0 0 0

20 92 182 232 171 81 4.9 22 12 15 4 4 5 7 0 0 0 0 I 0 3

6 48 147 262 194 149 87 52 22 22 16 9 4 4 4 1 2 2 0 2 2

21 58 157 229 210 182 117 81 60 53 29 13 18 II 9 5 3 2 0 3 5

2 25 64 73 49 35 17 5 7 4 5 2 1 0 1 0 0 0 0 0 0

22 195 400 377 200 112 65 37 17 6 4 8 5 I 4 1 0 0 1 0 1

4 ll2 233 175 112 60 30 19 II 5 2 5 3 5 2 0 1 1 0 1 0

20 64 118 169 117 63 42 37 24 18 9 8 7 5 2 2 4 1 1 0 3

0 38 125 267 211 138 107 84 37 27 10 13 16 6 4 3 2 1 1 1 2

34 171 303 261 183 87 44 39 9 12 6 3 1 0 0 0 1 0 1 2 1

1329

900

1035

1266

290

1456

781

714

1033

1158

8 41 89 106 99 69 63 49 27 27 I1 7 8 1 5 2 2 1 2 1 5 623

* Relative fluorescence intensities as percentage of the noradrenaline standard. ** 4 (day 4), 7 (day 7) and 8 (day 8) days and 3-3.5 and 5.5-7 weeks after ovariectomy; estradiol dipropionate 1 t~g (e I), 10 ,ug (e 10), 2 x 100 :~g (e 200); progesterone 2 x 2 mg (p 2 x 2); estradiol dipropionate ~ progesterone (e ~ p). Brain Research, 16 (1969) 199 214

208

W. IA('HTENSI'EIGER el cI/.

TABLE 111 MEAN RELATIVE FLUORESCENCE INTENSITY i r AND COEFFICIENT b l OF THE SKEWNESS

Group*

lr* *

99 % confidence limits

bl

99 % confidence limits

day4 day 7 day 8 week 3-3.5 week 5.5-7 e1 24 h e 10 24h e 10 48 h e200 48h p2~<2 48h e p 48h

49.4 40.9 48.3 54.5 54. I 41.9 36.8 37.7 47.0 52.9 36.4

:17.7 51.1 38.9 43.0 46.3-56.4 52.3--56.2 5C.7-57.5 38.6-45.2 35.4-38.2 35.6-39.8 44.1-49.9 50.7~55. I 34.8-38.1

0.75 2.69 1.80 0.82 0.70 2.05 3.13 4.05 1.53 1.34 2.09

0.42- 1.08 1.80-3.58 1.31 2.29 0.58-1.06 0.31-1,09 2.10-4.17 2.72-5.38 0.95-2.10 0.90 1.78 1.14-3.04

DI*** D2 P O

41.2 57.5 63.4 67.2

40.0-42.3 55.9-59.0 61.6-65.1 65.4-69.0

1.73 0.74 0.42 0.27

1.31-2.15 0.52-0.96 0.26-0.58 0.15-0.39

* 4 (day 4), 7 (day 7) and 8 (day 8) days and 3-3.5 and 5.5-7 weeks after ovariectomy; estradiol dipropionate 1 pg (e 1), 10/hg (e 10), 2 ~.: 100/~g (e 200); progesterone 2 × 2 mg (p 2 ;<2); estradiol dipropionate + progesterone (e i- p); diestrous days 1 (D1) and 2 (D2), proestrous day (P), estrous day (O). ** Ir in per cent of the noradrenaline standard fluorescence. *** Taken from LichtensteigerlL

to decrease further a n d the F S H level b e c a m e very low. T h e effect o f 2 mg o f p r o gesterone on 2 d a y s was a p p a r e n t l y less h o m o g e n e o u s . The F S H level decreased whereas the L H levels varied c o n s i d e r a b l y . T h e m e a n L H c o n c e n t r a t i o n w o u l d be s o m e w h a t lower than in the o v a r i e c t o m i z e d controls. All assays were r e g a r d e d valid with respect to the statistical analysis p e r f o r m e d . N o significant d e v i a t i o n f r o m parallelism was f o u n d in any o f the 4-point assays. The precision was satisfactory, the 2 values ranging between 0.15 and 0 A I (mean 0.28) in the F S H assays, a n d between 0.09 a n d 0.23 ( m e a n 0.15) in the L H assays. The vaginal smears showed the changes t h a t w o u l d be expected after the a d m i n i s t r a t i o n o f the steroid h o r m o n e s 24. DISCUSSION It a p p e a r s from our results that b o t h o v a r i e c t o m y itself and the subsequent a d m i n i s t r a t i o n o f sexual steroids influenced the frequency d i s t r i b u t i o n o f relative fluorescence intensity o f the c a t e c h o l a m i n e - c o n t a i n i n g t u b e r a l nerve cells. T h e changes can be described in terms o f the m e a n relative fluorescence intensity Ir a n d the coefficient b 1 o f the skewness, which is a m e a s u r e o f s y m m e t r y . The values o f Ir and o f bx t h a t were previously f o u n d on the 4 d a y s o f the estrous cycle la a p p e a r to be related to each other. O n an empirical basis this r e l a t i o n s h i p is expressed by the Brain Research, 16 (1969) 199-214

209

CATECHOLAMINE NEURONS AND GONADOTROPINS TABLE IV SERUM F S H

AND

Groupt

3-7 weeks

LH

CONCENTRATIONS

1. U. FSH/ml serum (SD)

I.U. LH/ml serum Mean

( SD)

Individual values

0.44

(0.10)

0.31

(0.09)

0.21/0.41/0.31 0.39/0.25 nd*/nd* nd*/nd*/nd* nd*/0.19 0.19/0.21/0.21 0.29/0.39

e 10 e 10

24 h 48 h

0.26 0.24

(0.06) (0.07)

nd* nd*

e200

48h

0.69

(0.29)

0.26

p2~2

48 h

0.15

(0.08)

e ~ p

48h

nd**

0.19

(0.08)

nd*** nd*** nd*** nd***

0.14 0.30 0.76 0.18

(0.08) (0.23) (0.31) (0.10)

Dirt Dz P O

(0.09)

0.25/0.27/0.44

nd*/nd* 0.13/0.12/0.12 0.20/0.30 0.23/0.10/0.09 0.09/0.16/0.31/0.33/0.62 0.58/0.62/1.10 0.12/0.13/0.30

t Control rats ovariectomized for 3-7 weeks; ovariectomized rats treated with estradiol dipropionate 10 /~g (e 10) and 2 × 100/zg (e200), progesterone 2 × 2 mg (p 2×2) or estradiol dipropionate 2 × 100 #g and progesterone 2 × 2 mg (e H p); diestrous days 1 (D1) and 2 (Dz), proestrous day (P), estrous day (O). t t Animals from a previous investigation1'°. * Not detectable at 1.2 ml/animal. ** Not detectable at 1.5 ml/animal. *** Not detectable at 3.0 ml/animal.

equation Ia e hl/3 ~ It, where Ia is the mean relative fluorescence intensity o f a c o m pletely symmetrical d i s t r i b u t i o n (b 1 = 0). The theoretical implications are presently being studied (Lichtensteiger, in p r e p a r a t i o n , see also note a d d e d in proof). On Fig. 4 the r e l a t i o n s h i p is shown in a s e m i l o g a r i t h m i c d i a g r a m , with the values o f the 4 days o f the estrous cycle lying on a straight line which follows the e q u a t i o n bl --~ 3 In (Ia - - l r ) . We refer to this relationship, since f u n c t i o n a l changes o f a similar type might express themselves in a m o v e m e n t o f the same direction in the d i a g r a m , if n e u r o n a l activity and fluorescence intensity o f the cell were in some way related to each other. As far as p r i m a r y c a t e c h o l a m i n e s are concerned, changes in fluorescence intensity reflect variations in the c a t e c h o l a m i n e c o n c e n t r a t i o n , whereas in the presence o f s e c o n d a r y catec h o l a m i n e s their lower fluorescence yield w o u l d have to be considered as well ~9. Since the a v a i l a b l e evidence indicates that these n e u r o n s contain a p r i m a r y c a t e c h o l a m i n e ( p r o b a b l y m a i n l y d o p a m i n e ) , the discussion will be limited to the effect o f changes in a m i n e c o n c e n t r a t i o n 19. A n increase o f the c a t e c h o l a m i n e c o n c e n t r a t i o n in central nerve cell bodies1, s, as well as e n h a n c e d t r a n s m i t t e r synthesis in p e r i p h e r a l adrenergic neurons 3,3°,3~, is r e p o r t e d to a c c o m p a n y increased neuronal activity. If the t u b e r o i n f u n d i b u l a r c a t e c h o l a m i n e - c o n t a i n i n g neurons are involved in the c o n t r o l o f g o n a d o Brain Research, 16 (1969) 199-214

210

W. LICHIENSIEIGER C.t ~t/.

tropin secretion, it should thus be possible to disclose certain connexions between the fluorescence intensity of the nerve cell bodies and the serum levels of LH and FSH. It is, however, evident that the action of these neurons need no1 be limited to the gonadotropins.

The influence of ovariectomy In rats with a regular 4-day estrous cycle, ovariectomy strongly disturbs the cyclic activity that would be expected theoretically (Fig. 1 and Tables I1 and lI1). There are certain oscillations of the catecholamine content of the tuberal nerve cells in this early phase. The question whether the changes are completely irregular or whether they correspond to residual parts of the previous cyclic activity, cannot be answered, since only 3 days out of 8 have been studied. The stage reached between 3 and 7 weeks after ovariectomy strongly resembles diestrus 2 (Fig. 4). As compared with the estrous cycle, this stage occupies an intermediary level of fluorescence int~,nsity. With respect to the degree of activity of the neuron system, such as it may be deduced from the catecholamine content of the nerve cell bodies, it is therefore interesting to note that the LH level found between 3 and 7 weeks after ovariectomy roughly corresponds to the mean observed on diestrus 2 in rats (Table 1V). However, the considerable variations at the latter stage leave some doubts about the reliability of this mean. On the other hand, there is a considerable difference between the FSH levels of the two phases. However, it should be emphasized that our FSH levels are much less reliable because they are merely based on single 3-point assays of pooled serum from each of the groups. Furthermore, processes not involving the tubero-infundibular neurons might be at work in ovariectomized animals. So far frequency distributions similar to those of proestrus or estrus, with high values of mean relative fluorescence intensity and high degrees of symmetry, have not been found in the ovariectomized animals. Since very high levels of LH, for example, are only seen after considerably longer periods of ovariectomy 17, a slow shift of the frequency distributions towards classes of higher relative fluorescence intensity might be expected for later phases of ovariectomy. The comparatively moderate change observed 3-7 weeks after ovariectomy is probably responsible for the negative results that were obtained in the same nerve cells in histochemicat studies without microfluorimetry ~6. Biochemical studies have also disclosed changes of brain catecholamine levels after ovariectomy. However, the increase of noradrenaline that was observed 10 and 20 days after ovariectomy and orchidectomy l°,az was limited to the anterior hypothalamus, no change occurring in the middle hypothalamus which contains the tubero-infundibular neurons. As mentioned above, the latter do not appear to contain noradrenaline, but rather dopamine. Therefore, these findings might reflect changes in the numerous noradrenergic nerve terminals of this region and thus be due to a reaction of other adrenergic systems. Similarly, the increase of brain noradrenaline turnover seen 6 days after ovariectomy z is at present difficult to assign to a distinct catecholamine-containing system.

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The eff'ect of sexual steroids in ovariectomized an&zals Since the frequency distributions did not significantly change between 3 and 7 weeks after ovariectomy, the steroid hormones were administered during this period. The strong reduction of the LH level and the decrease in the FSH level that resulted from the administration of 10 /~g of estradiol dipropionate (Table IV), indicate a marked negative feedback action. At this stage, the frequency distribution of relative fluorescence intensity was shifted towards classes of lower intensity and became strongly unsymmetrical (Figs. 2 and 4, Tables 1l and Ill). If we consider the evidence for a relationship between stimulation and increase in catecholamine content of the nerve cell body, the decrease in fluorescence intensity rather points to a reduction of neuronal activity. This reduction could be brought about by a negative feedback action of the estrogen. This hypothesis implies that the catecholamine-containing tubero-infundibular neurons exert a stimulating action on the release of the gonadotropins, whether this be direct or indirect. It agrees with the findings of the estrous cycle. The artificially induced frequency distribution resembles the type of diestrus 1, but the dose of estrogen used provoked a shift that exceeded the one that leads to diestrus I (Fig. 4). The marked effect which, in a single experiment, 1 /zg of estradiol dipropionate exerted on the frequency distribution indicates that lower doses are also effective. At present we do not know which neural mechanism is responsible for the feedback effects of the steroid on the frequency distribution. Although we would rather think of one of the possible feedback actions of the steroid itself, indirect effects mediated by a 'short' feedback of the gonadotropinsV,9, la cannot be entirely ruled out. It is also conceivable that the change in the nerve cells might reflect a reaction to the lowered gonadotropin concentrations. This would then lead to a different interpretation of their functional role. A similar association between a predominance of nerve cells with lower fluorescence intensity and reduced serum levels of LH and FSH occurred in the group treated with high doses of estrogen in combination with progesterone (Fig. 4, Tables II, Ill and IV). This finding appears to be in keeping with experiments in rats pretreated with estrogen which indicate a negative feedback action of progesterone after a similar time interval 6. In the absence of progesterone the same dose of estrogen left the LH level almost unchanged and induced only a minor shift of the frequency distribution. Whether the increase of FSH is significant remains doubtful, because the value is derived from a single 3-point assay of pooled serum. With respect to gonadotropin level and fluorescence intensity, the results of the two groups seem compatible with the scheme presented above. An explanation for the small changes observed after 200/zg of estradiol could be sought in an interference of positive and negative feedback influences. Indications of a positive feedback action of estrogen have not only been observed after low dosesll, 28, but also after considerably higher ones 1'-', and after implantation ofestradiol into the median eminence e~. It is difficult to consider the relevance of these results for the present study, because they were obtained in rats with intact ovaries. As in the group treated with the 10#g dose for 48 h, the mean number of visibly fluorescent nerve cells per section is here remarkably low. It is

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LI('HTENS['IiI(iER

el a[.

conceivable that some nerve cells might have been lost because o f an extremely low a m i n e c o n c e n t r a t i o n , with a consequent alteration o f the frequency distribution. However, such an influence does not seem to be o f critical importance, since the differences in the m e a n n u m b e r o f nerve cells per section are not statistically significant. The type o f change o f the frequency d i s t r i b u t i o n t h a t was p r o v o k e d by 2 , 2 mg o f p r o g e s t e r o n e alone has never been observed so far (Fig. 3, Tables II and II1). The decrease in the frequency o f nerve cells on b o t h sides o f the frequency d i s t r i b u t i o n a p p e a r s to indicate that the c a t e c h o l a m i n e content increased in some nerve cells while it decreased in others, and in p a r t o f them it m a y have remained unchanged (see note a d d e d in proof). N o clear-cut tendency is shown by the LH levels either, whereas the ( p o o l e d ) F S H level is lowered (Table 1V). F r o m earlier investigations it a p p e a r s that c o n s i d e r a b l y higher single doses o f progesterone are required for a clear-cut decrease o f p l a s m a LH levels, if the steroid is a d m i n i s t e r e d in the absence o f estrogen 2a. D o s e s s i m i l a r to that used in the present study lowered LH levels within 4 htT, but o t h e r investigators have failed to find a n y influence on plasm~ LH in o v a r i e c t o m i z e d rats 2a,~)~. It may be o f some significance that the differences in the f e e d b a c k a c t i o n s o f progesterone, whether given a l o n e o r in c o m b i n a t i o n with estrogen. are reflected in b o t h the m i c r o f l u o r i m e t r i c a l findings a n d the g o n a d o t r o p i n e levels.

Conc&sions W h e n c o n s i d e r e d t o g e t h e r with the findings o f the estrous c y c l e l S : 9, the present experiments indicate a close i n t e r d e p e n d e n c e between the t u b e r o - i n f u n d i b u l a r catec h o l a m i n e - c o n t a i n i n g neurons and the ftmctional state o f the p i t u i t a r y - g o n a d a l axis. W h i l e these neurons have thus been shown to r e s p o n d to such influences, one should n o t disregard a possible involvement o f at least p a r t o f t h e m in the control o f other a n t e r i o r pituitary h o r m o n e s . Such an a d d i t i o n a l role is even strongly suggested by experiments which d e m o n s t r a t e a reaction o f the same neuronal system to cold exposure z0. The present results do not answer the q u e s t i o n o f h o w the relative specificity o f functional c o n t r o l is effected. It m a y be l i n k e d with a n o t h e r p r o b l e m , which is the m e c h a n i s m o f action o f the c a t e c h o l a m i n e t h a t is released in the m e d i a n eminence. NOTE ADDED IN PROOF

1. During the estrous cycle there are differences between the anterior and posterior tuberal region ~°. The reaction of the ovariectomized animals to the steroids was similar in both halves. 2. The course of the means (and of their logarithms) during the estrous cycle can be approximated by a quadratic function of time (Lichtensteiger, in preparation). The coefficients of skewness show a similar change in the opposite direction. The reason for this finding is unknown, but it explains the relation between the two sets of values discussed above: At a given time, the quotient of the slopes of the two curves is --0.210/0.069 =~ --3.04 -: dfll/d In It. This corresponds to the value (-- 3) mentioned above. 3. The various frequency distributions can be accurately described as lognormal ones (Lichtensteiger, in preparation). In this way the various states are characterized more precisely. The differences between ovariectomized controls and steroid-treated rats have been confirmed by a statistical analysis of the logarithmically transformed distributions. The mean and the variance (in natural logarithms) of 3 groups are given in the order: whole tuberal region-anterior part-posterior part: (a) Rats ovariectomized for 3-3J, weeks: 3.855 (0.3338)-3.835 (0.3478)-3.884 (0.3122). (b) Estradiol dipro-

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pionate 1 × 10 ltg/24 h: 3.497 (0.2555) 3.443 (0.2668) 3.574 (0.2296). (c) Progesterone 2 x 2 rag/ 48 h: 3.860 (0.2256)-3.853 (0.2437)-3.871 (0.1976). With respect to the action of progesterone, it is evident from these values that it did not influence the mean but induced a decrease in the variance similar to that observed after estrogen. This indicates that in both situations the populations have become more homogeneous. SUMMARY

In female rats the intensity of the catecholamine fluorescence was measured in nerve cell bodies of t u b e r o - i n f u n d i b u l a r n e u r o n s by microfluorimetry, and the serum LH and FSH levels were determined. Ovariectomy interrupted the typical sequence in the frequency distributions of relative fluorescence intensity of the n o r m a l estrous cycle. A frequency distribution c o m p a r a b l e to the type of diestrous day 2 was observed between 3 and 7 weeks after ovariectomy together with an LH level similar to diestrous day 2 but an FSH level higher than during the cycle. Ten #g of estradiol d i p r o p i o n a t e caused a shift of the frequency distribution which then resembled the type of diestrous day 1, and a fall of LH and FSH levels. Very high doses were less effective in both respects. Effects of progesterone depended on whether it was administered with or without estrogen. The findings further support the hypothesis of a functional relationship between different states of activity of these t u b e r o - i n f u n d i b u l a r n e u r o n s and the control of g o n a d o t r o p i n secretion. ACKNOWLEDGEMENTS

The study was supported by the Swiss F o u n d a t i o n for Scientific Research ( G r a n t s Nos. 4309 a n d 4563). The skillful technical assistance of Mrs. S. Winter is gratefully acknowledged.

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