The Pituitary Stalk and Ovulation 69 



pituitary activity. If, for the reasons given above, the median eminence 

 contains the greatest concentration of any agent, then this would be greatly 

 diluted by taking a relatively large mass of surrounding hypothalamic 

 tissue. 



In the present work eight extracts of median eminence tissue have been 

 studied. The first six of these were obtained from rabbits and the last two from 

 cattle (steers). For each extract made from rabbit tissue four normal adult 

 female rabbits, that had been isolated for several weeks, were anesthetized 

 with ether, the skin of the head reflected and the head sawn through in the 

 transverse plane so that the cut severed the midbrain. The forebrain in the 

 front end of the skull was quickly removed from the bone, care being taken 

 to cut the pituitary stalk with fine scissors so that no fragment of pars distalis 

 tissue was removed with the stalk. The median eminence, the hypothalamus 

 and samples of cerebral cortex were dissected and immediately frozen. The 

 cattle material was obtained from a slaughter house, but in this case the 

 animals (castrated) had been killed (by bleeding) |-2 hr previously. The same 

 tissues were taken from each brain ; material from about twelve brains being 

 pooled for each cattle extract. Again the tissues were immediately frozen after 

 dissection. The extracts were prepared by homogenizing the pooled samples 

 in 0.5 % acetic acid and centrifuging. The solutions were then neutralized and 

 made isotonic by addition of appropriate amounts of sohd sodium bi- 

 carbonate and sodium chloride, centrifuged for 15 min at 10,000 r.p.m. at 

 0°C, and the supernatant distributed into sealed ampoules and kept in the 

 frozen state. The volume of the final extracts of the different brain samples 

 was equivalent on the basis of wet tissue weight. 



The technique finally chosen for infusing the extracts directly into the 

 anterior pituitary in the conscious animal was a modification of that used by 

 von Euler and Holmgren (26). In a preUminary operation cannulae consisting 

 of fine platinum tubing (SWG 25) are inserted through a small trephine hole 

 in the vault of the skull so that the tip of the cannula is located in the pars 

 distalis of the pituitary. This is easily and simply performed with the use of a 

 stereotaxic machine and X-ray control. A flange attached to the upper end 

 of the tubing is then fixed to stainless steel screws inserted in the skull with 

 dental cement, a fine wire stilette inserted in the cannula, the skin sutured 

 around the cannula mounting and a protective cap screwed to the mounting 

 (see Figs. 8, 9, 10). 



In early experiments attention was paid to the spread of infused dye, or 

 radioactive P^\ when different rates of administration were used. An infusion 

 rate of 0.06-0.07 ml/hr, for a 2-hr period, was finally chosen. 



Since the majority of the animals in this work were being used to see 

 the eff"ect of the various infusions on both TSH and LH release, the experi- 

 mental procedure was usually as follows. After recovery from the operation 

 of implanting the cannula, the animals received 100 /xc P^^ subcutaneously. 



