A primary culture technique of neurones from adult mammals and an electrophysiological study of their sodium and calcium spike components

$2 A PRIMARY CULTI/rtE TECHNIQUE OF NEURONES FROM ADULT MAMMALS AND AN ELECTROPHYSIOLOGICAL STUDY OF THEIR SODIUM AND CALCIUM SPIKE COMPONENTS. JUN FUKUDA .'~mdMASAKI KAMEYAMA*, D~pt. P h y s i o l . , Fac. Med., Univ. Tokyo~ Bunkyo-ku, Tokyo 113. We have recently established a technique to obtain p r i m a r y culture of dissociated neuroues from adult m a m m a l s . Dorsal root ganglia w e r e di~ssected from adult guinea pigs, and their neurones were di:~sociated by collagenase. These neurones were incubated on collagen-coated dishes with growth medium containing Eagle's Medium, horse s e r u m , chick embryo extr~tct and ant,ibiotics. In a few days o£ incubation, tl~e cells began to extend 1heir neurit:es ~n s e v e r a l d i r e c t i o n s . Neurones thus obtained w e r e penetrated by glass m i c r o e l e c t r o d e s while being viewed directly with an inverted phase contrast microscope. ~ntracellular injection of depolarizing pulses elicited Na spikes. C mtinuous hyperpolarization of the m e m b r a n e potential removed inactivation of Na chan~els~ membrane capability of generating Na spikes may be compared among neurones by using this hyperpolarized cells. Ca spikes were elicited in neurones bathed in a Na-free (tris+), TEA-containing medium. Steady hyperpo!arization remeved inactivation of Ca channels as well, and the membrane exhibited the maximum capability of generating the Ca spikes. Na and Ca spikes were compared among neurones of various ages in tissue culture~ Na spike components did not s e e m to change during 3 weeks of the cell culture. However, changes in Ca spike components w e r e suggested in an early period of axonal regeneration. CALCIUM SPIKES IN FROG MUSCLE SPINDLE. FUMIO ITO, TADASHI AKAIKE* and YUK!O KOMATSU*. P?~t. PhTsiol., Nagoya Univ. Sch. Med., Sho~va-ku, Nagoya 466~ Calcium spikes were observed at the afferent nerve t e r m i n a l of isolated f~-og muscle spindles during stretching at high velocity in Binger solution with TTX. The responses at the nerve t e r m i n a l were r e c o r d e d acro~ s a r a i r - g a p made upon the myelinated segment of the sensory axon jL~st outside the capsule. The amplitude of the calci~um spikes deper~ds upou the concentration of external calcium from 1 to 10 mM. Removal of Ca 2+ from the Ringer or administration of EGTA decayed the amplitude and ritually removed the spikes. The calcium spikes were r e v e r s i b l y blocked by addition of 1 mM Co 2+ or Mn2+~ or of 5 mM Mg z+. Str3ntium and barium w e r e able to substitute, for calcium as a c u r r e n t c a r r i e r in calcium spikes for muscle spindle° The calcium spikes could alBo be elicited by depolarizing current stimulations over 2.5 - 3.5 n~k in choline Ringer or in TTX Ringer. This suggests that ~he CaLcium spikes may be initiated in the vicinity of the site ~,: generating receptor potential and that the spike may play an important role in prod~:~cing afferent impulse~ {sodium spikes).