| In neurons cultured from neonatal rat hypothalamus and brainstem, angiotensin II (Ang II) caused an increase in Ca2+ current. Molecular and pharmacological analyses revealed the presence of all high voltage activated Ca2+ current subtypes in the neurons: L, N, P/Q, and R. The Ca2+ current could be facilitated with a depolarizing voltage pulse, which is indicative of voltage-dependent Ca2+ current inhibition by Gβγ subunits. Recovery of inhibition following a depolarizing voltage pulse occurs with a time constant consistent with rates of Gβγ reassociation. Ang II partially prevented facilitation, consistent with relief of Gβγ inhibition. These data suggest that Ang II, via the Ang II type 1 receptor (AT1R), may increase Ca2+ current by relieving tonic G protein inhibition in a PKC dependent manner.; Embryonic or neonatal rat neurons retain plasticity and are readily cultured, but neurons of the adult brain are thought to be non-replenishable, and therefore difficult to culture. Adult neural cell cultures were prepared from brainstem or hypothalamus. Very few adherent cells were apparent in cultures for up to one week, at which time the cell population expanded dramatically. The predominant cell type was immunopositive for α-internexin, a neurofilament expressed in developing neurons of the CNS. α-internexin positive cells co-immunostained for neuronal markers including MAP2, β-tubulin III, and tetanus toxin, but were negative for glial markers and for the neurofilaments characteristic of mature neurons. α-internexin positive cells incorporated BrdU, suggesting that the neuron like cells retain the ability to proliferate. Patch clamp analysis revealed voltage gated Na+ currents and small Ca2+ currents, but the cells were unable to fire action potentials, consistent with an immature phenotype. These results show that the cultures are immunologically and electrophysiologically more similar to neurons than glia. The immature neuronal phenotype makes these cells an attractive model system for several neurobiology applications. |
