In Vivo Release of Luteinizing Hormone-Releasing Hormone Estimated with Push-Pull Cannulae from the Mediobasal Hypothalami of Ovariectomized, Steroid- Primed Rats*

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Abstract
The in vivo release of LHRH from the mediobasal hypothalami (MBH) of conscious, freely moving rats was estimated by means of a push-pull perfusion system. The effects of local 15-mM K+ infusions as well as a regimen of estradiol benzoate and progesterone (P) were assessed. Ovariectomized (Ovx) rats received 22-gauge cannula implants fitted with 28- gauge stylettes. Ten to 22 days post Ovx and 6 –-18 days post implantation, a rat was placed in a testing chamber, the stylette was removed, and an inner cannula assembly (28 gauge) was inserted, its tip protruding 0.75 mm past the outer permanent cannula into the MBH at the following coordinates: 5.8 mm anterior, 0.6 mm lateral, and 9.8 mm ventral. Artificial cerebrospinal fluid was pushed through the inner cannula and pulled up between the cannulae at 18 μl/min by two identically calibrated peristaltic pumps, effectively perfusing a sphere approximately 1 mm in diameter, as determined by dye experiments. Tenminute fractions were collected continuously on ice, acidified to 0.1 N HC1, and stored at -20 C along with acidified medium blanks. All tubes were subsequently neutralized, centrifuged, and assayed for LHRH by RIA. Placement of cannulae was verified histologically. The basal output of LHRH was markedly pulsatile (0.5–2 pulses/h), with pulse magnitude ranging between less than 0.2 to 1.0 pg/10 min. A 2- to 6-fold increase in LHRH perfusate levels was elicited by the infusion of 15 HIM K+ through the cannula. Another group of Ovx rats received 5 jug estradiol benzoate sc at 1000 h on day 0, followed 48 h later by either 1.5 mg P sc (n = 5) or oil (O) vehicle sc (n = 5). Animals were then perfused between 1200-1800 h on day 2. Rats in which P was administered showed a significant (P < 0.01) 40 min elevation of LHRH secretion at approximately 1600 h compared to prior basal output or oil control animals during the same time period. The presence of LHRH in perfusates was directly correlated with e placement of the cannula tip approximately 0.5 mm from the rostral median eminence. These results demonstrate that P can stimulate the release of LHRH from terminals in the MBH. Of greater general significance is the first demonstration that neuropeptide release can be estimated intracerebrally in the unanesthetized rat with push-pull cannulae.