Combined Autoradiographic and Immunohistochemical Evidence for an Association of Somatostatin Binding Sites with Growth Hormone‐Releasing Factor‐Containing Nerve Cell Bodies in the Rat Arcuate Nucleus

Abstract

The regulation of growth hormone secretion depends upon the complex interplay between two hypothalamic hypophysiotropic factors: growth hormone-releasing factor and somatotropin release inhibiting factor or somatostatin. Interactions between these two neurohormones appear to be exerted both distally, at the level of pituitary somatotropes, and proximally, within the hypothalamus. In an attempt to detect a possible anatomical substrate for central interactions between the two neurohormones, we compared the autoradiographic distribution of specifically labeled somatostatin binding sites with the immunohistochemical distribution of growth hormone-releasing factor-containing neurons in the hypothalamus of adult rats. Somatostatin binding sites were labeled in vitro by incubating serial brain sections with [1251]TyrO-DTrp8-somatostatin. Growth hormone-releasing factor-immunoreactive neurons were visualized in a second set of animals, using an antiserum raised against synthetic rat growth hormone-releasing factor (1-29) NH,. In light microscopic autoradiograms of sections incubated with [ '251]somatostatin the label was found to be concentrated over small, round or oval neuronal perikarya clustered within the ventrolateral aspect of the arcuate nucleus. The topographic distribution of these [ '251]somatostatin-labeled cells was similar to that of growth hormone-releasing factor-immunoreactive neurons detected within the same region. Moreover, the number of [1251]somatostatin-labeled cells was found to vary in parallel with that of growth hormone-releasing factor-immunoreactive neurons throughout the rostro-caudal extent of the arcuate nucleus (coefficient of correlation r = 0.80). These results suggest that somatostatin binding sites may be directly associated with the perikarya of arcuate growth hormone-releasing factor neurons. Such an association would provide an anatomical substrate for a direct regulation of growth hormone-releasing factor secretion by somatostatin at the hypothalamic level.

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

“…This binding was specific, as demonstrated by the fact that sections incubated in the presence of a hundred-fold excess of nonlabeled ligand showed only background fluorescence. At the regional level, the labeled pattern was comparable to that previously observed by autoradiography using 125 I-SRIF (30). However, confocal microscopy allowed for a more clear-cut localization of the ligand to small neuronal perikarya distributed throughout the structure.…”

Chronic growth hormone (GH) hypersecretion induces reciprocal and reversible changes in mRNA levels from hypothalamic GH-releasing hormone and somatostatin neurons in the rat.

“…'251-SRIH binding experiments were performed on series of adjacent coronal sections as previously described (20). Sections were preincubated for 15 min at room temperature in 0.05 M Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose and 0.2% BSA.…”

“…Hypothalamic SRIH and GHRH mRNA levels were determined at 6 and 18 wk by in situ hybridization in female rats bearing ectopic GHproducing tumors. As SRIH-specific receptors have recently been located on GHRH neurons (20)(21)(22), we also used quantitative light microscopic autoradiography (23) In situ hybridization was carried out as described elsewhere (25). Briefly, 45-base oligoprobes (bases 31-75 of rat GHRH cDNA [33] and bases 96-111 of rat SRIH cDNA [34] GHRH, respectively.…”

Joint pituitary-hypothalamic and intrahypothalamic autofeedback construct of pulsatile growth hormone secretion

“…Individual peptide elimination rates are assumed to be distinct and stable. The primary network-like connections (and their experimental derivation) are 1) GHRH's acute stimulation of GH release from somatotrope cells (8,18,26,30,37,42,43,45,46), 2) SRIF's antagonism of GHRHinduced GH secretion (18,20,34,42), 3) GH's delayed feedforward on SRIF release (6,9,29,31,34,35,37), 4) SRIF's inhibition of GHRH secretion (8,12,16,24,30), 5) GH's repression of GHRH outflow (7,9,16,31,32), 6) GHRH's time-lagged induction of pituitary SRIF secretion (1, 2, 11, 24, 27, 29, 32-34, 37, 46), and 7) GHRH's induction of the de novo synthesis and accumulation of (releasable) GH in the pituitary gland (20,34,42,45,46). This ensemble formulation extends an earlier basic construct by including intrahypothalamic GHRH-evoked SRIF outflow and GHRH-stimulated synthesis and accumulation of GH stores (see DISCUS-SION).…”