Characterization of Mitotic Neurons Derived From Adult Rat Hypothalamus and Brain Stem

1 Department of Physiology and the McKnight Brain Institute and 2 Department of Neuroscience, University of Florida College of Medicine, Gainesville, Florida 32610 Evans, Jenafer, Colin Sumners, Jennifer Moore, Matthew J. Huentelman, Jie Deng, Craig H. Gelband, and Gerry Shaw. Characterization of Mitotic Neurons Derived From Adult Rat Hypothalamus and Brain Stem. J. Neurophysiol. 87: 1076-1085, 2002. Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker -internexin, while the other stained with the astrocyte marker GFAP. The -internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These -internexin positive cells coimmunostained for the neuronal markers MAP2, type III -tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocyte marker GalC and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these -internexin positive cells revealed small Ca 2+ currents with a peak current of 0.5 ± 0.1 pA/pF at a membrane potential of 20 mV ( n = 5) and half-maximal activation at 30 mV ( n = 5). Na + currents with a peak current density of 154.5 ± 49.8 pA/pF at a membrane potential of 15 mV ( n = 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.