Immunocytochemical Localization of LHRH in Axons and Nerve Terminals of the Rat Median Eminence

Abstract

Immunocytochemical identification of nerve terminals reacting specifically with anti-LHRH antisera has recently been reported in various species, like the guinea pig (1) and the duck (2), employing a double antibody labeling technique, with the second antibody bound to fluorescein isothiocyanate (3). However, visualization of such terminals has not yet been described in the rat; on the other hand, a double labeling with a second, peroxidase bound, antibody does not seem to have been successfully applied to that material as yet. In the present paper, we report identification of fibers containing an antigen reacting with anti-LHRH in the rat median eminence, using both fluorescein and peroxidase labels.Methods. The antibodies were obtained from rabbits injected with synthetic LHRH (Hoffman-LaRoche, Basle) coupled to guinea pig gamma globulin (4), according to the technique described by Avramkas ( 5 ) . In a radioimmunoassay system (6), the titer of the antiserum is approximately 1/100,000; it can detect as little as 1-5 pg of LHRH. Its specificity was tested against oxytocin, lysin-vasopressin and TRH, as well as against LH, FSH and Prolactin; it does not cross react with these hormones at doses up to 500 pg. With this antibody, endogenous levels of LHRH were determined in the rat hypothalamus; values ranged between 1 and 3 ng/hypothalamus of normal male rats. Injected to cyclic female rats (200 pl ip at 1200 noon of the day of proestrus), this antiserum inhibited the preovulatory surge of LH and FSH and blocked ovulation (7).Male rats were perfused by aortal cannulation with various fixatives (200 ml total volume) under light chloral hydrate anesthesia. The fixa-tives used were either Bouin-Hollande without acetic acid (4% formalin, 4% picric acid, 2.5% copper acetate and 10% of a saturated solution of Hg sublimate), 4% paraformaldehyde in saline solution, or picroformol. After dissection, the hypothalamus was postfixed for 4 hr at 4". The tissues were dehydrated in graded butanol series followed by a mixture of paraffin and butanol and embedded in paraffin. Five micrometers serial sections were rehydrated, washed in water overnight, immersed in 0.01 A4 saline phosphate buffer solution (PBS) for 30 min and incubated with the antiserum against LHRH at various dilutions for 90 min. Adjacent sections were incubated for the same time with (a) normal rabbit serum, (b) serum of a rabbit immunized against guinea pig gamma globulins, (c) anti-LHRH serum absorbed previously with an excess of LHRH by overnight incubation at 4" of 100 p1 of pure serum with 20 pl of a 3. lob4 M solution of synthetic LHRH, (d) anti-LH serum (8), (e) anti-LHRH serum incubated overnight with a cytoplasmic fraction from cerebral cortex and subsequently centrifuged ( 17,OOOg for 30 min)in an attempt to eliminate background staining. Some sections were treated for 5 min with 0.5 M HCl, or with 1% trypsin in PBS, prior to incubation with the antisera. After thorough washing in PBS, a second incubation with either peroxidase-or fluorescein-labeled sh...

Immunocytology of Luteinizing Hormone-Releasing Hormone

“…These results would be consistent with Barry's observations in the guinea pig. However, Kordon et al [1974] were unable to demonstrate perikaryal localization of LH-RH in the rat following intraventricular injection of colchicine. Kordon et al [1974] further considered the possibility that fixation may play some role in the ability to localize LH-RH within its neuronal compart ments.…”

“…These results suggested that some interference with microtubular transport or other neuronal function must take place before significant amounts of the neurohormone accumulate in the cell soma. Yet the necessity for pharmacologic treatment of the animal raised questions as to the degree to which neural proteins were altered which might interfere with the selectivity and specificity of the immunocytochemical procedure [Kordon et al, 1974]. Barry's laboratory countered this argument with the demonstration of similarly localized LH-RH perikarya in a number of untreated animal models: pregnant guinea pigs late in gestation [Barry and D ubois, 1973b], fetal guinea pigs [Barry and Dubois, 1974b], and prepuberal guinea pigs, cats and dogs D ubois, 1974a, 1975].…”

“…The median eminence (ME) provides the region of most intense immunocytological visualization of luteinizing hormone-releasing hormone (LH-RH) fibers in a variety of vertebrates, including frog [Alpert et al, 1976;Goos et al, 1976], toad [Doerr-Schott and D ubois, 1975], duck [Calas et al, 1974;McNeill et al, 1976], cockerel [de Reviers and D ubois, 1974], guinea pig [Leonardelli et al, 1973;Barry and D ubois, 1974a; Baker et al, 1974;Silverman, 1976], rat [Baker et al, 1974[Baker et al, , 1975; King et al, 1974;Kordon et al, 1974;Pelletier et al, 1974;G oldsmith and G anong, 1975;Setalo et al, 1975Setalo et al, , 1976Hoffman, 1976], mouse [Zimmerman et al, 1974;Baker et al, 1974;Leonardelli et al, 1973;G ross, 1976;Hoffman, 1976], hamster [Pickard and Silverman, 1976], cat [Barry and D ubois, 1974a[Barry and D ubois, , 1975[Barry and D ubois, , 1976Bugnon et al, 1976b], dog [Bugnon et al, 1976b;Dubois, 1974a, 1975], squirrel monkey [Barry and Carette, 1975], rhesus monkey , and human fetus [Bugnon et al, 1976a]. Electron microscopic examination of the ME identi fied LH-RH within granules [Pelletier et al, 1974;G oldsmith and G anong, 1975;Silverman and D esnoyers, 1975], These could be categorized into 2 groups: granules 90 to 120 nanometers in diameter found mainly in axons, and granules 40 to 70 nanometers in diameter found in nerve terminals [Silverman and D esnoyers, 1975].…”

Immunohistochemical demonstration of LHRH-neurons in young rat hypothalamus: Light and electron microscopy.

“…However, the demonstration of LHRH perikarya in rats was particularly controverted and fragmental, as various research groups failed to find them (KORDON et al, 1974;BAKER et al, 1974BAKER et al, , 1975KING et al, 1974KING et al, , 1978PELLETIER et al, 1974;GOLDSMITH and GANONG, 1975) and NAIK 'S work (1975) was criticized by them. Using an antiserum having a highly specific titer, KRISCH (1980) pronounced the location of the immunoreactive cells to be restricted to the preoptic area.…”

Effect of hypothalamic deafferentation on the distribution of luteinizing hormone-releasing hormone (LHRH) in the rat brain.

“…Recent development of immunohistochemical techniques for the staining of LHRH has demonstrated that a large amount of immunoreactive LHRH is localized in the external layer of the median eminence (ME) of the rat in close contact with the primary capillary network of the hypophyseal portal vessels and around the vascular structure in the organum vasculosum of the lamina terminalis (OVLT) (King et al, 1974;Kordon et al, 1974;Baker et al, 1975;SetalO et al, 1975;Goldsmith & Ganong, 1975;Naik et al, 1976;Ibata, et al, 1978;Nozaki et al, 1979).…”