Cyclic nucleotide-gated channels (CNGCs) are important transducers of external signals in sensory processes. These channels are ubiquitously expressed in a variety of neurons, and are necessary to transduce signals for growth cone guidance and plasticity. Here, we demonstrate that the CNGC subunits (CNGA1 and CNGB1, presumably the 1b isoform) are expressed in rat cerebellar granule cells and that they combine to form functional channels. The expression of the mRNAs that encode these proteins is maximal after 7 days in cell culture, when the channels are expressed at synapses and co-localize with the synaptic marker synapsin I. These ligand-gated channels are functional and can be blocked by Mg(2+) or L-cis-diltiazem. Moreover, channel opening in response to increases in intracellular cGMP results in Ca(2+) entry into the cell. Chronic blockade (96 h) of these channels with L-cis-diltiazem significantly decreases the number of functional boutons, as determined by their capacity to load and unload the styryl dye FM1-43 when stimulated. Moreover, the unloading kinetics is modified from a biphasic to a monophasic profile in a subset of synaptic boutons. These channels are also expressed in early developmental stages, both in the soma and in emerging processes, and CNGA1 can be detected in growth cones. Pharmacological blockade of these channels with L-cis-diltiazem causes an overall change in growth cone morphology, impairing the formation of lamellipodia between filopodia and increasing the number of filopodia. J

The increased expression of different soluble guanylyl cyclase (sGC) subunits during development is consistent with these proteins participating in the formation and establishment of interneuronal contacts. Functional sGC is generated by the dimerization of an a-subunit (sGCa1/2) with the b1-subunit (sGCb1), and both depletion of the sGCb1 subunit and inhibiting sGC activity impair neurite outgrowth. Similarly, impairing sGC activity diminishes the amount of growth-associated protein (GAP-43) and synapsin I, two proteins that participate in axon elongation and synaptogenesis, suggesting a role for sGC in these processes. Indeed, fewer synapses form when sGC is inhibited, as witnessed by FM1-43 imaging and synapsin I immunostaining, and the majority of synapses that do form remain functionally immature. These findings highlight the importance of sGC in the regulation of neurite outgrowth and synapse formation, and in the functional maturation of cerebellar granule cells in vitro. During neuronal development, cGMP signaling is important to modulate growth cone activity in a variety of cell types. [1][2][3] Indeed, intracellular cGMP synthesis is linked to and mediates the neurogenic effects of different factors, including that of nerve growth factor (NGF). 4 The expression of the different guanylyl cyclase subunits (sGC), the main physiological receptor for nitric oxide (NO), augments throughout development in different areas of the rat brain and in in vitro models of neuronal maturation. 5-7 Accordingly, these proteins may be important for the formation and growth of neuronal processes, as well as for the establishment of intercellular contacts and the refinement of functional neuronal connections within the peripheral and central nervous system. 1,8,9 There are two known isoforms of sGC, the ubiquitous a 1 b 1 and a 2 b 1 , which has a more limited distribution. 10 This latter isoform interacts with synaptic scaffolding proteins through its PDZ domains, suggesting that this enzyme is accumulated in synapses 11,12 and that it is responsible for cGMP synthesis in this cellular compartment. 13 GAP-43 is an integral membrane protein associated with the cytoplasmic surface of axonal growth cones in developing neurons, and it is generally considered an intrinsic determinant of neurite outgrowth and plasticity. 14,15 In addition, GAP-43 also plays important roles in synaptogenesis as well as in regulating the cytoskeletal organization of the nerve ending. 16 Indeed, neurite outgrowth and axonal pathfinding are affected when GAP-43 expression is altered, both during development and in cultured cells. Likewise, synapsins are also thought to participate in the processes of axon elongation and synapse formation during development, and the expression of synapsin I correlates well with synapse maturation. 17 Synapsins are exclusively associated with small synaptic vesicles and they are virtually excluded from other tissues or neuroendocrine cells. 18 Indeed, synapsin I is probably the most specific marker of synapses in the ...