Physiological and pharmacological evidence has suggested that both endogenous opiates and gonadotropin-releasing hormone (GnRH) itself can act centrally to exert a tonic inhibition on gonadotropin secretion via an inhibition of the neurosecretion of GnRH. To determine if the effects of these two peptides might be mediated via a direct synaptic input to the GnRH neuron, we undertook a double label ultrastructural study. We were able to localize in the same tissue section beta-endorphin and GnRH. Analysis of serial sections through GnRH perikarya and dendrites in the male rat diagonal band/preoptic area revealed that almost 10% of the synapses impinging on the GnRH neuron contained beta-endorphin; an additional 10% of the terminals contained GnRH. These data provide anatomical evidence in support of both a direct modulation of GnRH release by opiates and of the presence of an ultrashort feedback loop.
Gonadotropin releasing hormone (GnRH) released into the hypophysial portal vasculature is the major stimulus for gonadotropin secretion. These hormones in turn regulate gonadal function. The GnRH neurosecretory cells receive a sparse (Witkin, 1987) but chemically diverse innervation. Double-label immunocytochemical experiments at the ultrastructural level have demonstrated GABA-(Leranth et al., 1985), serotonin- (Kiss and Halasz, 1985), and proopiomelanocortin- (Chen et al., in press; Leranth et al., 1988) containing inputs. Previous light microscopic studies suggested that GNRH cells were also contacted by numerous tyrosine hyroxylase (TH)- (Hoffman, 1985) and/or dopamine beta hydroxylase (DBHbpositive terminals (Jennes et al., 1982). As all three catecholamines have been implicated in regulation of GnRH secretion (Fink, 1988;Gallo, 1980;Kalra and Kalra, 1983), we have applied immunocytochemical techniques at the ultrastructural level to determine if TH-containing terminals synapse onto GnRH neurons.Adult rats (two of each sex) were anesthetized with pentobarbital (60 m@g) and rapidly perfused with 500 ml of fixative (McLean and Nakane, 1974). Brains were postfixed overnight at 4°C. TH and GnRH were detected sequentially in the same vibratome sections (Chen et al., in press). In brief, sections were incubated in antiserum to TH (EugeneTech) for 48 h at the dilution recommended by the supplier. The diluent for this and all other reagents (unless noted) is 0.1 M phosphate buffer, pH 7.3, containing 0.02% saponin. The antigen-antibody complex was demonstrated using a biotinylated second antibody and an avidin-biotin-HRP complex (Vector Laboratories). The HRP was detected with 3,3' diaminobenzidine (DAB) as the chromogen. The DAB precipitate is known to cover the immunological bridge, making its components unavailable in the next reaction series, (Sternberger and Joseph, 1979). Sections were then pIaced in an antibody directed against GnRH (Silverman, 1984). This antigen was visualized with the same reagents except that tetramethylbenzidine (TMB) was used as the chrornogen (Olucha et al., 1985). The development of the blue crystals was monitored under the microscope for 8-10 min. Sections were next treated with a DAB-cobalt chloride solution (4 C; 0.05% DAB in 0.05 M Tris, pH 7.4; 0.02% CoCl,; 0.01% H,O,) for 5-10 min (Tsai et al., 1988; Chen et al., in press). This step stabilized the TMB crystal, transformed its color to black, and intensified the DAB from the first reaction. Without this step, the blue color faded within 24 h. 0 1989 ALAN R. LISS, INC.Small regions containing GnRH neurons were dissected with a scalpel blade and osmicated for 1 h (2% OsO, plus 1.5% KFeCN). For this study we selected GnRH cells from the medial preoptic area (MPOA) and diagonal band of Broca (DBB) at the level of the organum vasculosum of the lamina terminalis (OVLT). Tissue was embedded in EPON 812, and 1Fm sections were used to identify GnRH neurons or their processes. A series of 20-30 serial ultrathin sections were then cut, m...
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