Serial Long-Term Assessment of in vivo LHRH Release from a Discrete Area of the Ewe Median Eminence Using Multiple Guide Cannula Assembly and Removable Push-Pull Cannulae

Abstract

Analysis of neuronal interactions at the median eminence (ME) that control anterior pituitary function requires sampling of in vivo release of specific hypophysiotropic components and of putative inputs that might regulate such a release. We developed a multiple guide cannula assembly (MGCA) to sample repetitively discrete areas of the ewe ME, using removable push-pull cannula (PPC) probes. The MGCA is attached to the skull over the ME using stereotaxic surgery. A specific guide cannula of the assembly (1 of 48 guides) located on the midline and directly on top of the central portion of the ME is selected based on roentgenograms obtained after infusion of radiopaque contrast material into the third ventricle. The MGCA and the large size of the ewe brain allows the simultaneous positioning of a PPC probe, an infusion cannula or another removable probe in additional discrete hypothalamic and extrahypothalamic areas. To determine the capabilities of this sampling technique, we assessed in vivo release of LHRH from the extracellular space at the posterior-lateral ME, under various reproductive conditions that have well-defined LH-secretory patterns. The pulsatile profile of both LHRH and LH was significantly lower in anestrus than during the follicular phase of cycling ewes. In vivo pulsatile release of LHRH was higher in the follicular than in the midluteal phase of ewes sampled repetitively from the same ME site during three consecutive estrous cycles. All ewes showed increased midluteal progesterone levels. In vivo LHRH release, as determined by PPC sampling of the extracellular fluid at the posterior-lateral ME, probably reflects a mix of hypophysiotropic and nonhypophysiotropic LHRH components. Histological analysis revealed a well-organized, pallisade-like array of astroglial processes along the track of chronically implanted cannulae. This glial scar is disrupted along the tracks of acutely reimplanted cannulae but without apparent difference in the yield of the method. Our method increases the efficiency and versatility of ME PPC sampling providing an additional tool to assess the role of the ME as the final neuroendocrine control site of anterior pituitary function.

Feed Restriction in Prepubertal Lambs: Effect on Puberty Onset and on in vivo Release of Luteinizing-Hormone-Releasing Hormone, Neuropeptide Y and Beta-Endorphin from the Posterior-Lateral Median Eminence

“…In vivo release of neuropeptides from the posterior-lateral ME was assessed using a PPC technique and a multiple guide cannula assembly designed in our laboratory [15]. The latter is a plastic plat form with a circular opening into which a plug containing an array of vertical guide cannulae fits.…”

Regulation of Gonadotropin Releasing Hormone Release by Neuropeptide Y at the Median Eminence during the Preovulatory Period in Ewes

“…Stereotaxic surgery was performed as previously reported by us [22]. Briefly, fasted ewes (12–16 h before surgery) having the dorsal aspect of their heads shaved were anesthetized (Brevital, i.v., 50 mg/10 kg BW, followed by halothane, 1.5% through endotracheal tube) and operated employing aseptic techniques.…”

“…For this, the removable plug was replaced by a multiple guide cannula (MGC) plug to direct a PPC probe into the ME through one of its parallel guide holes positioned 1.5 mm apart (48 holes in a 6 × 8 array). The use of this MGC assembly, removable PPC probes, and specific radioimmunoassays (see below) allowed for the serial long-term assessment of GnRH and NPY release from a discrete area of the ewe ME without disturbing the in vivo neuropeptides secretion, the synchronized LH surge, and the spontaneous estrous cyclicity thereafter [22]. …”

“…CRR 11B73, provided by Dr. V.D. Ramirez, University of Illinois, Urbana, Ill., USA) as previously reported [16, 22, 27]. 50 µl of PPC sample perfusate was used for NPY and GnRH RIAs.…”

“…However, physiological evidence in the ewe for the regulation of in vivo preovulatory GnRH release by NPY at the ME is lacking. To directly assess the physiological relevance of an action of NPY on GnRH neuronal terminals at the ME we used a removable push-pull cannula (PPC) approach [22]and a modified synchronized follicular model [23]in intact cycling ewes. Using these approaches, we determined the simultaneous release of NPY and GnRH in the ME, as well as the plasma LH levels, during the luteal, follicular and surge stages of a synchronized estrous cycle.…”