Humidified Gas Mixture Research Articles

Phasic discharge in supraoptic neurones recorded from hypothalamic slices.

An intriguing feature of recordings from the supraoptic (SO) nucleus in vivo is that many of the neurones exhibit a phasic firing pattern when excited by appropriate osmotic (Walters and Hatton, 1974; Arnauld et al., 1975; Brimble and Dyball, 1977; Wakerley et al., 1978) or hypovolaemic (Poulain et al., 1977) stimulation. Phasic firing, which seems to be associated with vasopressin release (Dreifuss et al., 1976; Poulain et al., 1977) is characterized by high frequency (6-12 Hz) trains of spikes, lasting 5-120 sec and alternating with periods of electrical silence of 4-80 sec duration. At present it is not known whether the pacemaker mechanism for timing the intermittent bursts during phasic firing lies in the region of the SO nucleus, or distant from it. To resolve this question, we have investigated the occurrence of phasic firing in SO neurones recorded from thin (300 ~) hypothalamic slices in vitro. In this preparation, it is possible to activate the neurones in the SO nucleus by direct chemical stimulation, and to study their firing characteristics in the absence of connections from other brain areas. Brains were removed from decapitated male rats and a tissue block containing the hypothalamus was prepared. This tissue block was then sectioned at 300 ~t in a coronal plane, using an 'Oxford' vibratome. Throughout sectioning, the tissue remained immersed in standard incubation medium, kept at 37 ~ C. Hypothalamic slices containing parts of the SO nucleus were immediately transferred to an incubation chamber. Their upper surfaces were exposed to a humidified gas mixture (95% 0 2 5 % CO2) while their undersurfaces were supported on a nylon grid and perifused with Yamamoto's solution (pH 7.3-7.5) (Yamamoto, 1972) at a flow rate of 1.4-1.6 ml/min. The osmotic pressure of the medium was in the range 296-300 mOsm/kg, determined using

Transfer of preimplantation pig embryos following in vitro culture for 24 or 48 hours.

Two experiments were conducted to determine the viability of 258 pig ova cultured in vitro for 24 or 48 hr by transfer to 19 recipient gilts. One- to four-cell ova were recovered from donor gilts and cultured in droplets of Brinster's medium under oil. A humidified gas mixture of 5% CO2 and 95% air flowed through the incubator which was maintained at 37 C. In Experiment I, a total of 69 ova was transferred to six unmated gilts after a 24 hr in vitro culture period. At recovery, 63 ova were at the two-cell stage, and at transfer 59 were at the four-cell stage. Five of six (83%) recipient gilts had an average of 7.6 (63%) normal embryos at slaughter on days 26 to 33 post-estrus. In Experiment II, a total of 189 ova were transferred to 13 unmated gilts following a 48 hr in vitro culture period. Fifty-three percent of the ova were recovered at the two-cell stage and the remainder were recovered either as one-(21%) or four-cell (26%) ova. After 48 hr in culture 77% of the ova were transferred at the four- or six-cell stage of development. Two gilts (15%) were pregnant and slaughtered 28 and 34 days post-estrus. Fifteen of 29 (52%) ova transferred to the two pregnant recipients were present as normal embryos. The reduced pregnancy rate in Experiment II may have been due to factors other than the increased length of the culture period. During the course of Experiment I, 12 of 28 ova maintained in culture from the same donors as those which were transferred developed to the blastocyst stage after 96 to 120 hr in culture. None of the 97 ova cultured for 72 additional hr after Experiment II ova were transferred reached the blastocyst stage.