Regenerative responses of long duration recorded intracellularly from dispersed cell cultures of fetal mouse hypothalamus.

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Abstract
To learn the extent to which endogenous mechanisms or synaptic driving determines the bursting impulse activity associated with peptide, especially vasopressin (VP), secretion by magnocellular neurons, electrical activity was recorded intracellularly from neurons differentiated in cultures of dispersed, 14-day fetal mouse hypothalamus. After the 5th wk in culture, neurons of several morphological types are recognizable. One type (15% of all neurons) is distinguished by having large (15-25 .mu.m diameter) somata; with intracellular injection of markers these neurons characteristically have 2 or 3 stout dendritic processes and a thin, long, occasionally branching axonal process. Neurons of all types exhibit resting potentials of -40 to -60 mV, input resistances of 50-200 M80, spontaneous postsynaptic potentials (PSP) and overshooting impulses. PSP, but not impulses, are inhibited by Co (10 mM) or Cd (10 mM) ejected from a 10-.mu.m delivery pipette. Impulses are blocked by tetrodotoxin (TTX) (1 .mu.M). Slow potentials were observed in 15% of the neurons of the large morphological type from which recordings were made. They are regenerative responses to depolarization (PSP or applied current) reaching an absolute potential of -30 mV in .apprx. 50 ms, then repolarizing over 0.2-2 s. They can be evoked from a wide range of membrane holding potentials (Vh). They exhibit graded behavior if evoked at rates exceeding 0.2/s. Plateau potentials in response to depolarization (PSP or applied current) were observed in 80% of the neurons showing slow potentials. No cells exhibited plateaus also without slow potentials. Depolarization to an absolute potential of -20 mV requires 50-100 ms and is then sustained for 0.5-1.5 min. Repolarization occurs after a slight hyperpolarizing drift with a rapid (2 mV/s for 1-2 s) and a slow phase requiring .gtoreq. 0.5 min for the final 10 mV. Plateau potentials can only be evoked from Vh more polarized than -50 mV. Both slow and pleateau potentials involve conductance increases to Ca: both are reversibly blocked by Co or Cd; they are not blocked, but their rise time and amplitude are reduced by TTX. Slow potentials are converted to a form resembling plateau potentials, without responses of intermediate duration, by localized application of 4-aminopyridine (4-AP) (1 mM) or tetraethylammonium (TEA) (1 mM). Neurons that showed only slow potentials also exhibit this conversion. Brief (5 s) TEA application prolongs plateaus by several minutes. During the slow repolarizing phase following a plateau potential, slow potentials of gradually increasing amplitude and duration are evokable until another plateau response occurs. With strong spontaneous synaptic bombardment or repeated depolarizing current pulses, the recurrence of plateau responses is periodic. This periodicity, as well as the duration of plateau responses, including their variability, parallels the periodicity and the duration of in vivo extracellularly recorded impulse bursts of VP neurons observed by others. Preliminary evidence associates the neurons of the morphological type having slow and plateau potentials with those showing immunoreactivity to VP. Positive VP immunoreactivity was observed in specific cells from which slow and plateau potentials were recorded. The unusual, long-duration, presumably endogenous responses, perhaps observable only transiently during maturation, may reflect unique membrane properties developed by VP neurons.