A suitable preparation to study the effect of dopamine (DA) on in vitro LHRH secretion is presented. Enzymatic degradation of LHRH in the incubation medium is completely inhibited by bacitracin (2 X 10(-5) M). Ahigh potassium concentration (56mM) induces an increase in LHRH release from entire pieces of mediobasal hypothalamus (MBH), but not from a synaptosomal pellet; this release is inhibited when calcium is omitted. The depolarization-induced LHRH in the MBH. In contrast, DA (10(-6)M) is not effective in vitro on MBH from ovariectomized rats but induces a fast release of LHRH from MBH of normal male and estradiol-pretreated ovariectomized rats. The preparation presented here appears to be of great interest in investigating amine-steroid-LHRH interactions at the cellular level.

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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...