Brain Research, 103 (1976) 597-602 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
597
Immunohistological study of the origin of LH-RH-containing nerve fibers of the rat hypothalamus
GYORGY SI~T/~L6, SANDOR VIGH, ANDREW V. SCHALLY, AK1RA ARIMURA AND BELA FLERK6 (B.F., G.S. and S.V.) Department of Anatomy, University Medical School, 7643 Pecs (Hungary) and Department of Medicine, Tulane University School of Medicine, New Orleans, La. 70112, and Endocrine and Polypeptide Laboratories, Veterans Administration Hospital, New Orleans, La. 70146 (U.S.A.)
(Accepted November 7th, 1975)
Recent studies on the LH-RH-synthesizing nerve cells have led to contradiction concerning their localization. The aim of the present wo,'k was to obtain more accurate data on the origin of the LH-RH-containing nerve fibers of the rat. Using rabbit a n t i - L H - R H serum, a system of delicate immunoreactive L H - R H nerve fibers and terminals was demonstrated in the median eminence (ME) of the rat 8. The delicate fiber system could be traced to the retrochiasmatic area and to the arcuate nucleus but the nerve cells in these areas did not show any immunopositive reaction for L H - R H under the experimental conditions of the study. Even intraventricular infusion of colchicine, which resulted in the appearance of L H - R H positive nerve cell bodies in the experiment of Barry et al. 2 in guinea pigs, failed to yield any cell reaction in the rat. However, when we reproduced the conditions of Barry et al. by injecting colchicine into the lateral ventricle of guinea pigs, we also found LH-RH-positive perikarya in the suprachiasmatic area of the guinea pig hypothalamus (Fig. lc). Differences between species appear to account for this discrepancy. Our pcevious observations on the absence of LH-RH-containing perikarya in the hypothalamus of intact adult female rats 8 are in accordance with those of Leonardelli et al. 4 and Baker et al. l, who also were unable to find LH-RH-positive nerve cell bodies in the brain of intact guinea pigs and rats. In the meantime, however, we occasionally found LH-RH-positive perikarya in the suprachiasmatic and medial prechiasmatic areas of intact adult female rats which were killed at the end of diestrus or in proestrus 9. Considering the scarcity of such cells, we speculated, similarly to Barry et al. 2, that this might be due to the low L H - R H content of the nerve cells, possibly below the threshold of the immunohistological technique. In the present experiments, therefore, we tried to increase the neurohormone concentration in the cell bodies of the LH-RH-containing nerve fibers with the aid of various experimental procedures interfering with (1) the release of the neurohormone or (2) the axoplasmic
598 flow. For these purposes, (1) intact adult female rats were kept under Nembutal (Abbott) anesthesia from 9:00 a.m. until midnight on the day of proestrus, and they were killed at 9:00 a.m. the next morning; and (2) in another group o f intact adult female rats a 'half-dome' shaped frontal cut was made at the rear of the optic chiasm, using the technique developed by Half, sz and Pupp a. The length of the cutting edge of the Halfisz knife used was: horizontal position 1.3 mm, vertical 1.8 mm. Delails of the operative technique have been described elsewhere 7. The operations were performed under hexobarbital anesthesia, and the animals were killed two weeks after the operation. The LH-RH-containing neural elements were detected immunohistologically as described previously :~. Rabbit antiserum to synthetic LH-RH, No. 422, was used for most of the study, except in one experiment represented by Fig. l a, in which antiserum No. 710 was used. Neither of these sera cross-reacted with other hypothalamic and pituitary hormones, but antiserum No. 422 was more specific than No. 710, as tested for crossreaction with 18 different synthetic peptides which were related to LH-RH and its fragments. No. 422 showed a slight cross-reaction (1.2 °/o) only with [Glu 1] -LH-R H, whereas No. 710 showed cross-reaction with [desamido]-LH-RH (100°,~;), [des,pGlul]-LH-RH (15/o) and [Glul]-LH-RH (23.8'}so). In the majority of the Nembutal-injected and frontally deafferented rats, we found LH-RH-containing nerve cell bodies which were scattered in the suprachiasmatic area between the anterior commissure and the optic chiasm and in the medial prechiasmatic area near the organum vasculosum of the lamina terminalis
Fig. 1. LH-RH-positive nerve cell bodies in the suprachiasmatic area of rats. a: treated with Nembutal, b: bearing a frontal cut at the rear of the optic chiasm, c: LH-RH-positive nerve cell in the suprachiasmatic area of a guinea pig treated with colchicine.
599
Fig. 2. Microphotographs showing paramedian-sagittal sections through the median eminence (ME) of rats having (a) frontally or (b) completely isolated medial basal hypothalamus. The frontally isolated ME (a) contains a fairly large amount of LH-RH-positive nerve fibers and terminals. In the completely isolated ME (b), only very few LH-RH-containing fibers are visible in the most superficial layer of the ME. These fibers have been knife-cut.
(OVLT) (Fig. la and b). Since the pathway of the LH-RH-containing nerve fibers in the ME of the rat coincides with the course of the nerve fibers belonging to the tuberoinfundibular tract s, and this tract receives numerous fibers from the arcuate nuclei10,11, these nuclei were assumed to contain LH-RH-producing nerve cells. This speculation, however, was not supported by the observation made on rats with completely isolated medial basal hypothalamus (MBH). Using the same Halgtsz knife as mentioned above, MBH was isolated between Pl and P4.7 according to the stereotaxic atlas of Szentfigothai et al. 11. Two weeks after surgery, the hypothalamic island contained only a very few LH-RH-positive nerve fibers, which entered the island in the most superficial layer of the ME and, hence, may well have escaped the knife cut. The region of the arcuate nuclei of the intact adult female rat is trespassed by numerous LH-RH-positive nerve fibers, although their cells are always negative s. Conversely, the completely isolated MBH was devoid of LH-RH-containing neural elements (Fig. 2b). When, however, only a frontal cut was made behind the optic chiasm, the frontally deafferented M E, including the region oI the arcuate nuclei, did contain a fair number of LH-RH-positive nerve fibers and terminals but no L H - R H positive cell bodies (Fig. 2a). This finding suggests that a considerable number ot the LH-RH-producing nerve cells, situated in the suprachiasmatic area in front of the
600
I
~'~
r ¸
d'
i!~+!!i~!i~!i¸!il
ilJ)!i!~i ~
5~
! lilliput'
597
Immunohistological study of the origin of LH-RH-containing nerve fibers of the rat hypothalamus
GYORGY SI~T/~L6, SANDOR VIGH, ANDREW V. SCHALLY, AK1RA ARIMURA AND BELA FLERK6 (B.F., G.S. and S.V.) Department of Anatomy, University Medical School, 7643 Pecs (Hungary) and Department of Medicine, Tulane University School of Medicine, New Orleans, La. 70112, and Endocrine and Polypeptide Laboratories, Veterans Administration Hospital, New Orleans, La. 70146 (U.S.A.)
(Accepted November 7th, 1975)
Recent studies on the LH-RH-synthesizing nerve cells have led to contradiction concerning their localization. The aim of the present wo,'k was to obtain more accurate data on the origin of the LH-RH-containing nerve fibers of the rat. Using rabbit a n t i - L H - R H serum, a system of delicate immunoreactive L H - R H nerve fibers and terminals was demonstrated in the median eminence (ME) of the rat 8. The delicate fiber system could be traced to the retrochiasmatic area and to the arcuate nucleus but the nerve cells in these areas did not show any immunopositive reaction for L H - R H under the experimental conditions of the study. Even intraventricular infusion of colchicine, which resulted in the appearance of L H - R H positive nerve cell bodies in the experiment of Barry et al. 2 in guinea pigs, failed to yield any cell reaction in the rat. However, when we reproduced the conditions of Barry et al. by injecting colchicine into the lateral ventricle of guinea pigs, we also found LH-RH-positive perikarya in the suprachiasmatic area of the guinea pig hypothalamus (Fig. lc). Differences between species appear to account for this discrepancy. Our pcevious observations on the absence of LH-RH-containing perikarya in the hypothalamus of intact adult female rats 8 are in accordance with those of Leonardelli et al. 4 and Baker et al. l, who also were unable to find LH-RH-positive nerve cell bodies in the brain of intact guinea pigs and rats. In the meantime, however, we occasionally found LH-RH-positive perikarya in the suprachiasmatic and medial prechiasmatic areas of intact adult female rats which were killed at the end of diestrus or in proestrus 9. Considering the scarcity of such cells, we speculated, similarly to Barry et al. 2, that this might be due to the low L H - R H content of the nerve cells, possibly below the threshold of the immunohistological technique. In the present experiments, therefore, we tried to increase the neurohormone concentration in the cell bodies of the LH-RH-containing nerve fibers with the aid of various experimental procedures interfering with (1) the release of the neurohormone or (2) the axoplasmic
598 flow. For these purposes, (1) intact adult female rats were kept under Nembutal (Abbott) anesthesia from 9:00 a.m. until midnight on the day of proestrus, and they were killed at 9:00 a.m. the next morning; and (2) in another group o f intact adult female rats a 'half-dome' shaped frontal cut was made at the rear of the optic chiasm, using the technique developed by Half, sz and Pupp a. The length of the cutting edge of the Halfisz knife used was: horizontal position 1.3 mm, vertical 1.8 mm. Delails of the operative technique have been described elsewhere 7. The operations were performed under hexobarbital anesthesia, and the animals were killed two weeks after the operation. The LH-RH-containing neural elements were detected immunohistologically as described previously :~. Rabbit antiserum to synthetic LH-RH, No. 422, was used for most of the study, except in one experiment represented by Fig. l a, in which antiserum No. 710 was used. Neither of these sera cross-reacted with other hypothalamic and pituitary hormones, but antiserum No. 422 was more specific than No. 710, as tested for crossreaction with 18 different synthetic peptides which were related to LH-RH and its fragments. No. 422 showed a slight cross-reaction (1.2 °/o) only with [Glu 1] -LH-R H, whereas No. 710 showed cross-reaction with [desamido]-LH-RH (100°,~;), [des,pGlul]-LH-RH (15/o) and [Glul]-LH-RH (23.8'}so). In the majority of the Nembutal-injected and frontally deafferented rats, we found LH-RH-containing nerve cell bodies which were scattered in the suprachiasmatic area between the anterior commissure and the optic chiasm and in the medial prechiasmatic area near the organum vasculosum of the lamina terminalis
Fig. 1. LH-RH-positive nerve cell bodies in the suprachiasmatic area of rats. a: treated with Nembutal, b: bearing a frontal cut at the rear of the optic chiasm, c: LH-RH-positive nerve cell in the suprachiasmatic area of a guinea pig treated with colchicine.
599
Fig. 2. Microphotographs showing paramedian-sagittal sections through the median eminence (ME) of rats having (a) frontally or (b) completely isolated medial basal hypothalamus. The frontally isolated ME (a) contains a fairly large amount of LH-RH-positive nerve fibers and terminals. In the completely isolated ME (b), only very few LH-RH-containing fibers are visible in the most superficial layer of the ME. These fibers have been knife-cut.
(OVLT) (Fig. la and b). Since the pathway of the LH-RH-containing nerve fibers in the ME of the rat coincides with the course of the nerve fibers belonging to the tuberoinfundibular tract s, and this tract receives numerous fibers from the arcuate nuclei10,11, these nuclei were assumed to contain LH-RH-producing nerve cells. This speculation, however, was not supported by the observation made on rats with completely isolated medial basal hypothalamus (MBH). Using the same Halgtsz knife as mentioned above, MBH was isolated between Pl and P4.7 according to the stereotaxic atlas of Szentfigothai et al. 11. Two weeks after surgery, the hypothalamic island contained only a very few LH-RH-positive nerve fibers, which entered the island in the most superficial layer of the ME and, hence, may well have escaped the knife cut. The region of the arcuate nuclei of the intact adult female rat is trespassed by numerous LH-RH-positive nerve fibers, although their cells are always negative s. Conversely, the completely isolated MBH was devoid of LH-RH-containing neural elements (Fig. 2b). When, however, only a frontal cut was made behind the optic chiasm, the frontally deafferented M E, including the region oI the arcuate nuclei, did contain a fair number of LH-RH-positive nerve fibers and terminals but no L H - R H positive cell bodies (Fig. 2a). This finding suggests that a considerable number ot the LH-RH-producing nerve cells, situated in the suprachiasmatic area in front of the
600
I
~'~
r ¸
d'
i!~+!!i~!i~!i¸!il
ilJ)!i!~i ~
5~
! lilliput'
