An evaluation of the nitric oxide/cGMP/cGMP-dependent protein kinase cascade in the induction of cerebellar long-term depression in culture

Cerebellar long-term depression (LTD) is a model system of information storage in which a persistent attenuation of the parallel fiber-Purkinje neuron (PN) synapse is induced by conjunctive stimulation of parallel fiber and climbing fiber inputs at low frequency. As some studies have suggested that release of the gaseous second messenger, nitric oxide (NO), in the molecular layer and the consequent activation of soluble guanylate cyclase and cGMP-dependent protein kinase (PKG) in the PN, is necessary for LTD induction, we have further examined this hypothesis using a cell culture protocol. In cerebellar cultures made from transgenic mice in which the gene for neuronal nitric oxide synthase (nNOS) has been rendered null, LTD induced by glutamate/depolarization conjunctive stimulation was indistinguishable from that in cultures from wild-type mice in terms of amplitude, rate of onset, and duration. Bath application of cGMP analogs produced a large (80%), transient attenuation of glutamate-gated inward currents. However, application of an activator of soluble guanylate cyclase or an inhibitor of type V cGMP-phosphodiesterase did not mimic the effect of cGMP analogs, and inclusion of cGMP analogs in the patch pipette did not give rise to a slowly developing attenuation, suggesting that these compounds exert their effects at the cell surface. Free Ca was measured in the distal dendritic arbor of single PNs by fura-2 microfluorimetry. Cyclic ADP-ribose (cADPr), which has been suggested to be formed by a cGMP/PKG-dependent process and to be an endogenous ligand of the type 2 ryanodine receptor, increased the response to a 3 sec depolarizing step from -80 to +10 mV when included in the patch pipette. This effect was completely antagonized by the co-inclusion of a competitive inhibitor (8-NH₂-cADPr). Induction of LTD was not blocked by inclusion in the patch pipette of three different PKG inhibitors or 8-NH2-cADPr. These results suggest that while cGMP/cADPr signaling may be important in neuronal Ca regulation, a NO/ cGMP cascade is not required for cerebellar LTD induction in culture.