plasma lh levels in the long term ovariectomized rat after anterolateral deafferentation of the...

Brain Research 3ufleti~, Vol. 8, pp. 2.55-259, 1982. Printed in the U.S.A.

Plasma LH Levels in the Long Term Ovariectomized Rat

After Anterolateral Deafferentation of the Medial Basal Hypothalamus

and Lesions in the Medial Preoptic Area’

ALBERT0 J. CARRILLO

Department of Anatomy, The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive, San Antonio, TX 78284

Received 14 December 1981

CARRILLO, A. J. Plasma LH levels in the long term ovnriectomized rat after anterolaterai dea~erentat~on of the media/ basal hypothalamus and lesions in the medial preoptic area. BRAIN RES. BULL. 8(3) 255-259, 1982.-This study was designed to investigate the effect of anterolateral hypothalamic deafferentation (ALHD) and medial preoptic area (MPGA) lesions on plasma LH levels in the long term ovariectomized rat. The deafferentations were carried out with a Halasz-Pupp knife (radius of 1.5 mm and height of 2.0 mm) and the MPGA lesions with a platinum electrode. Sham treated and an intact group served as controls. Blood samples were obtained from the jugular vein under light ether anesthesia before and at 1,2, 4 and 6 weeks after brain surgery. After the sixth week sample all rats were treated with 50 /~g of estradiol benzoate (EB) and two days later blood samples were collected during the morning and afternoon. Hypothalamic deafferentation resulted in a more significant (p<O.Ol) drop in plasma LH levels in one half of the group (ALHD-1) than in the other half (p~O.05) (ALHD-2) when compared to the controls. Treatment of the controls with EB resulted in a significant @<O.Ol) depression of LH levels in the morning and an LH surge during the afternoon. EB also resulted in a suppression @<O.Ol) of LH levels during the morning in all of the ALHD rats; however, only the ALHD-1 group had an LH surge during the afternoon following EB. Plasma LH levels in the ALHD-2 remained suppressed during the afternoon after EB treatment. Lesions in the MPGA had no effect on plasma LH levels at 1 to 6 weeks when compared to controls. Treatment of the MPGA lesion group with EB resulted in a significant fp<O.Ol) drop in plasma LH levels during the morning as well as the afternoon. These data suggest that the fibers that are critical for the control of tonic and phasic LH secretion enter the medial basal hypothalamus laterally and that the deafferentations carried out here were selective in interrupting fibers involved with tonic LH secretion in some rats and those involved with the phasic secretion in others. These data also suggest that the MOPA components involved with tonic LH secretion are separate from those controlling phasic LH secretion.

Hypothalamic deafferentation Medial basal hypothalamus Medial preoptic area Luteinizing hormone

FOR some two decades the technique of hypothalamic deafferentation with a Halasz-Pupp [ 181 knife has been routinely used to study the capacity of the medial basal hy~thal~us (MBH) to control adenohy~physeal function [2, 3, 24, 301. This de~erentation procedure, however, can result in some damage to the M~H-adenohypophyseal unit [21] followed by a period of recovery during which the regeneration of blood vessels [21,24] and nerve fibers [33] across the cut has been reported to take place. Other changes may also take place as a result of the possible loss of trophic influences to the MBH neurons [8,16]. Although there is a vast literature on the effects of hypothalamic deafferentation on gonadotropin secretion, most of these studies have consisted of determining adenohypophyseal function at a single time point after the

‘Supported by USPHS Grant NS 15454.

hypothalamic isolation, thus making it very difficult to detect any changes in hormone secretion across time resulting from the alterations described above.

This study was designed to determine the effects of anterolateral hy~thal~c de~erentation (ALHD) and medial preoptic area (MFOA) lesions on plasma LH levels at various times before and after brain surgery in long term ovariectomized rats. The effects of these hypothalamic lesions on the negative and positive feedback of estrogen on LH secretion were also determined.

METHOD

Adult female rats (150-200 g, Simonsen) were housed 5

Copyright @ 1982 ANKHO International Ins.-0361-9230/82/080255-03$03.00/O

256 CARRILLO

TABLE 1

EFFECT OF ANTEROLATERAL HYPOTHALAMIC DEAFFERENTATION (ALHD) AND PREOFTIC AREA (POA) LESIONS ON PLASMA LH LEVELS

Group

Plasma LH-RPl (&ml)

AM PM N -1 wk +I wk +2 wk +4 wk +6 wk after EB after EB

Intact 8 69.5 rt 93 867 ir 83 909 * 117 886 5 91 780 5 103 361 t 48 1660 c 320 pco.01 p‘co.01

Sham 9 817 t 109 917 r 143 999 t 179 1037 2 123 1048 * 140 473 ” 57 1234 + 261 p<O.Ol p<O.Ol

ALHD-1 6 998 t 298 321 it 78* 35.5 r 72* 328 ? 49* 345 t 7s* 128 -+ 25* 747 r 235 pCO.01 p-Co.01

ALHD-2 6 703 i: 177 587 z!z 39t 494 -c 53t 5.55 f 49”i 706 r 8% 194 -r- 15* 121 & 21* p<O.OOl p<O.O5

POA 7 6.52 ?z 92 704 it 109 936 -t 137 806 _’ 116 816 t 112 381 ~fr 54 214 t 37* lesion p<O.Ol p co.01

Values given represent the mean * S.E. p values represent levels of significance for that sampling time when compared to the previous sampling time. *p<O.Ol vs controls. tpcO.05 vs controls. $p<O.O5 vs sham.

per cage under controlled lighting (lighting on from 0500 to 1900 hr) with free access to Purina lab chow and water. Two days after arrival the animals were ovariectomized.

Four weeks after ovariectomy the rats were anesthetized with ether and placed on a stereotaxic apparatus. An anterolateral deafferentation (ALHD) of the medial basal hypothalamus was carried out with a Halasz-Pupp knife 1181 with a radius of 1.5 mm and height of 2.0 mm. For the deafferentation procedure the nose bar was set at -7 mm and the knife placed 2 mm posterior to bregma and lowered to the base of the brain. The knife was then rotated 90” to the right and left. With the knife rotated to both sides, the carrier (and knife) was moved posteriorly 2 mm to produce the lateral cut. Each cutting procedure was repeated several times in order to ensure the completeness of the deafferentation. In another group of rats, bilateral lesions were produced in the medial preoptic area (MPOA) by lowering a platinum electrode into the region and passing an anodal direct current of 1 mA for 20 set with the cathode clamped to the tail. For the MPOA lesions, the nose bar was set at +5 mm and the coordinates of DeGroot [lo] were followed. A group of sham control rats had the deafferentation knife lowered to the base of the brain and then removed without rotation. An intact group also served as controls.

Blood samples were collected from all of the rats at 1 week before surgery and at 1,2,4 and 6 weeks after surgery from the external jugular vein under light ether anesthesia. After the 6 week blood sample, all the animals were injected (SC, 0630-0800 hr) with 50 pg of estradiol benzoate (EB). Two days later a blood sample was collected during the morning (08~1000 hr) in the same manner as the previous ones. On that afternoon (1730-1900 hr) all the rats were de- capitated and trunk blood collected in heparinized centrifuge tubes. All of the blood samples were centrifuged and the plasma was separated and frozen for LH determination. The brains were removed and placed in 10% Formalin. After the brains were fixed they were embedded in paraffin, sectioned at 10 pm and every 10th section stained with carbol fuchsin

to determine the location and extent of the deafferentation and lesions.

Plasma LW was measured with the reagents provided by the NIAMDD-RIA kit (Dr. A. F. Parlow). All the samples were determined in a single assay, Because of the large heterogeneity of variance in the means for the LH levels, square root transformations of the data was carried out to achieve homogeneity of variance. Statistical analysis of the transformed data was then carried out be two-way analysis of variance for repeated measures and the Student Newman-Keuls test [32].

RESULTS

Intact and sham operated controls had elevated plasma LH levels throughout the length of the experiment, charac- teristic of long term ovariectomized rats. The injection of EB resulted in the significant (p<O.Ol) suppression of plasma LH levels during the morning and in a significant (p<O.Ol) elevation of plasma LH levels during the afternoon of the same day (Table 1).

Anterolateral hy~th~~ic deafferentation resulted in a significant (pcO.05) reduction in plasma LH levels when compared with the controls (Table 1). When these rats were treated with EB all had a significant @<O.Ol) reduction in plasma LH levels on the following morning, however only half of the ALHD rats had an LH surge during the afternoon (ALHD-I). The other rats did not have an increase in plasma LH levels (ALHD-2); instead the circulating LH levels dropped further during the afternoon when compared to the morning. Dividing the ALHD rats into these two groups not only revealed a different response to EB, but also a differ- ence in the changes of plasma LH levels after hypothalamic deafferentation. After deafferentation, the ALHD-1 group had significantly lower (p~O.01) plasma LH levels than the ALHD-2. At 6 weeks the ALHD-2 had plasma LH levels that were significantly (p~0.05) different from the sham group, but not the intact group.

HYPOTHALAMIC DEAFFERENTATION AND LH RELEASE 257

FIG. 1. Representative coronal sections through the rat medial basal hypothalamus illustrating the extent of the anterolateral hypothalamic deafferentation in the ALHD-1 and ALHD-2 groups. The thick straight lines represent the cut. Redrawn from DeGroot [IO].

Histological examiniation of the hy~thal~us from these ALHD rats did not reveal any significant differences in the extent of the detierentation (Fig. 1). The cuts extended from the retrochiasmatic area back 2 mm, approximately 1.2 mm from the midline. The hypothalamic area deaf&rented consisted of the arcuate-median eminence region and part of the ventromedial nucleus. In all cases the anterior tip of the hypothalamic island was infarcted. Although there were differences in the hormonal response as a result of the ALHD, careful examiniation of the hypothalamus did not show any obvious diB?erences in the cuts between the two groups of brains to account for these hormonal differences.

L , MFB

POP.

MFB

FIG. 2. Representative coronal sections through the rat preoptic area illustrating the extent of the medial preoptic area lesions. The black areas represent the destroyed brain tissue common to all of the brains (n=7). Redrawn from DeGroot [la].

Placing bilateral lesions in the MPOA had no significant effect on plasma LH levels at 1, 2, 4 and 6 weeks when compared to controls (Table 1). Estradiol benzoate injec- tions resulted in a significant b~O.01) suppression of plasma LH levels during the morning that was not different from the controls. During the afternoon of that day plasma LH levels were further reduced when compared to the morning levels and with the controls @<O.Ol). The histological findings from the MPOA groups are shown in Fig. 2 which illustrates the destroyed area common to all the animals in this group. This area consists of part of the MPOA and part of the posterior end of the diagonal band of Broca on one side.

2.58 CARRILLO

DISCUSSION

Anterolateral deafferentation of the MBH resulted in a significant reduction in plasma LH levels; more in one group of animals (ALHD-I) than in the other (ALHD-2) although at the 6 week period the ALHD-2 had plasma LH levels that were comparable to the intact group suggesting that some recovery had taken place in the LH secreting mechanism. Following the injection of EB, both deafferented groups had a significant reduction in plasma LH levels suggesting that the neuroendoc~ne mechanism responsible for EB negative feedback was not altered by the deafferentation. The ironic findings came during the afternoon when those animals where the ALHD had resulted in the greatest reduction in plasma LH levels (ALHD-1) were able to respond to the positive feedback aspect of EB with an elevation in plasma LH level similar to that observed in the controls. In the ALHD-2 group on the other hand, where the circulating LH levels were less affected by the deafferentation, there was no elevation in plasma LH levels during the afternoon after EB.

That hypothalamic deafferentation resulted in such differences in basal levels of circulating LH has been reported before, following complete deafferentation of the MBH [ 1,4, 53 but not after anterolateral deafferentation. As was reported by these authors, histologic~ examination of the brains did not reveal any significant differences in the extent of the cuts between these two groups to account for such differences in the hormonal levels. It should be taken into account however, that the rats in the present study as well as in the other studies were killed at least 6 weeks after the deafferentation and thus, the extent of the cuts were not as readily identified as they are when the brains are fixed shortly after the deatferentation. This is particularly appli- cable when it comes to determining the posterior extent of the cuts in the brains that have undergone ALHD. Blake and Sawyer [S] found that anterolateral dealferentation of the MBH had no significant effect on the pulsatile discharges and on the basal levels of plasma LH in long term ovariectomized rats. It is possible that the extent of their deafferentation was similar to those in the ALHD-2 group in the present study where partial isolation of the MBH resulted in a small suppression of plasma LH levels.

Although histological examination of the deafferented MBH did not reveal any major differences, the hormonal responses indicate that there were significant differences in the cuts between the two ALHD groups in the present study. These variations had to be present in the lateral aspect of the cut because in all the cases the anterior part of the hypothalamic peninsula was infarcted, thus assuring the completeness of the deafferentation there.

The stimulation of an LH surge in the ALHD-1 group could be evidence for the deatferentation being incomplete in this group; that is, in some places along the path of the knife, the blade did not go all the way down to the ventral surface of the hypothalamus. This however, would not seem possible in light of the marked reduction in plasma LH levels observed in the ALHD-1 group after deafferentation.

Thus, it appears that in the present study ALHD selectively disrupted the elements responsible for tonic LH release but not those involved in the phasic LH release in the ALHD-1 group and that the opposite was the case in the ALHD-2 group. Whether these elements consisted partly of LHRH neurons in the MBH 16, 11, 201 or LHRH fibers entering the lateral aspect of the MBH [19,23] can not be fully determined, however, since EB acts on the MPOA to

induce the LH surge [ 1 I] and some of the connections between the MPOA and the MBH must remain intact in order for EB to induce an LH surge [28] the ALHD-2 group must have had these connections interrupted. The ALHD-1 group on the other hand appears to have had most of these MPOA-MBH connections spared. As indicated previously these variations in the extent of the cuts had to take place in the lateral aspect of the MBH which would be in agreement with the data of other [7, 15, 17, 21, 271 which suggest that the lateral input to the MBH is involved with the release of LH. The present data also corroborates that of Phelps and coworkers [25,26] which showed that this lateral input to the MBH was involved in the MPOA mediated release of LH. Just how the deafferentation selectively disrupted the components involved with tonic LH secretion in one group and those involved with phasic LH secretion in the other cannot be determined in the present study. These data serve to point out the difficulty associated with the technique of hypothalamic deafferentation.

Bilateral lesions in the MPOA had no effect on basal levels of plasma LH or on the inhibition of LH secretion by EB. These lesions did result in the complete suppression of the afternoon LH surge observed in the controls after EB injection. This is in partial agreement with the work of others [14, 27, 28, 341 who reported that lesions in the MPOA at- tenuated the LH surge induced by progesterone injection in the EB primed ovariectomized rats. It is not appropriate however, to compare these other studies with the present one because these other investigators elicited the LH surge with EB followed by progesterone whereas in the present study it was done with EB alone.

Whereas the MPOA lesions described here eliminated the positive feedback of EB, these lesions had no effect on the tonic levels of plasma LH nor on the negative feeback mechanism. This may be because as suggested by Samson and McCann [27] either the number of LHRH neurons destroyed in the preoptic area was relatively small and therefore of little consequence to LH levels, or the neurons involved with positive feedback are separate from the LHRH neurons. Based on the similarity in their response to deafferentation and EB it appears that the same neural components were disrupted in the MPOA lesion and ALHD-2 group.

Regardless of the type of lesion or deafferentation and the effect these had on the tonic or phasic LH release, EB resulted in the suppression of plasma LH levels during the morning in all the groups. Thus, the negative feedback effects of EB were not compromised by any of these proce- dures. Since all the experimental groups had the MBH deafferented to varying degrees this data could suggest that the MBH is the site where EB exerts its negative feedback effects, which would be in agreement with the findings of others [4, 13, 211. On the other hand, since ALHD did not interrupt all the connections with areas anterior and lateral to the MBH, EB could therefore be having its negative feedback effect outside the MBH. This had recently been suggested from studies which have shown that the hyperse- cretion of LH is still present in ovariectomized rats after the destruction of the arcuate nucleus 17,151.

ACKNOWLEDGEMENTS

The author would like to thank Ms. Sarah Kohn, Ms. Kathy Evans and Ms. Judith Omillian for their excellent technical and sec- retarial assistance.

HYPOTHALAMIC DEAFFERENTATION AND LH RELEASE 259

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plasma lh levels in the long term ovariectomized rat after anterolateral deafferentation of the...

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