The photoreceptors of Drosophila express a nitric oxide-sensitive guanylate cyclase during the first half of metamorphosis, when postsynaptic elements in the optic lobe are being selected. Throughout this period, the optic lobes show NADPH-diaphorase activity and stain with an antibody to nitric oxide synthase (NOS). The NOS inhibitor L-NAME, the NO scavenger PTIO, the sGC inhibitor ODQ, and methylene blue, which inhibits NOS and guanylate cyclase, each caused the disorganization of retinal projections and extension of photoreceptor axons beyond their normal synaptic layers in vitro. The disruptive effects of L-NAME were prevented with the addition of 8-bromo-cGMP. These results suggest NO and cGMP act to stabilize retinal growth cones at the start of synaptic assembly.

Genetic screens for synaptogenesis mutants have been performed in many organisms, but few if any have simultaneously screened for defects in pre- and postsynaptic specializations. Here, we report the results of a small-scale genetic screen, the first in vertebrates, for defects in synaptogenesis. Using zebrafish as a model system, we identified seven mutants that affect different aspects of neuromuscular synapse formation. Many of these mutant phenotypes have not been previously reported in zebrafish and are distinct from those described in other organisms. Characterization of mutant and wild-type zebrafish, from the time that motor axons first arrive at target muscles through adulthood, has provided the new information about the cellular events that occur during neuromuscular synaptogenesis. These include insights into the formation and dispersal of prepatterned AChR clusters, the relationship between motor axon elongation and synapse size, and the development of precise appositions between presynaptic clusters of synaptic vesicles in nerve terminals and postsynaptic receptor clusters. In addition, we show that the mechanisms underlying synapse formation within the myotomal muscle itself are largely independent of those that underlie synapse formation at myotendinous junctions and that the outgrowth of secondary motor axons requires at least one cue not necessary for the outgrowth of primary motor axons, while other cues are required for both. One-third of the mutants identified in this screen did not have impaired motility, suggesting that many genes involved in neuromuscular synaptogenesis were missed in large scale motility-based screens. Identification of the underlying genetic defects in these mutants will extend our understanding of the cellular and molecular mechanisms that underlie the formation and function of neuromuscular and other synapses.

Many studies have revealed the free radical nitric oxide (NO) to be an important modulator of vascular and neuronal physiology. It also plays a developmental role in regulating synapse formation and patterning. Recent studies suggest that NO may also mediate the switch from proliferation to differentiation during neurogenesis. Many mechanisms of this response are conserved between neuronal precursor cells and the cells of the vascular system, where NO can inhibit the proliferative response of endothelial and smooth-muscle cells to injury. In cultured neuroblastoma cells, NO synthase (NOS) expression is increased in the presence of various growth factors and mitogens. Subsequent production of NO leads to cessation of cell division and the acquisition of a differentiated phenotype. The inhibitory action of NO on neuroblast proliferation has also been demonstrated in vivo for vertebrate and invertebrate nervous systems, as well as in the adult brain. Potential downstream effectors of NO include the second messenger cyclic GMP, activation of the tumor-suppressor genes p53 and Rb, and the cyclin-dependent kinase inhibitor p21. These studies highlight a new role for NO in the nervous system, as a coordinator of proliferation and patterning during neural development and adult neurogenesis.

Granule cells in the guinea pig dentate gyrus release kappa opioid neuropeptides, dynorphins, from dendrites as well as from axon terminals. We have found that both L- and N-type calcium channel antagonists inhibited dendritic dynorphin release. In contrast, N-type but not L-type calcium channel antagonists inhibited axonal dynorphin release. Neither L- nor N-type channel antagonists directly altered the effects of kappa opioid receptor activation. By inhibiting dynorphin release, L-type channel antagonists also facilitated the induction of long-term potentiation of the perforant path-granule cell synapse. These studies establish that a single cell type can release a transmitter from two different cellular domains and provide new distinction between axonal and dendritic transmitter release mechanisms.

A requirement for nitric oxide (NO) in visual system development has been demonstrated in many model systems, but the role of potential downstream effector molecules has not been established. Developing Drosophila photoreceptors express an NO-sensitive soluble guanylate cyclase (sGC), whereas the optic lobe targets express NO synthase. Both of these molecules are expressed after photoreceptor outgrowth to the optic lobe, when retinal growth cones are actively selecting their postsynaptic partners. We have previously shown that inhibition of the NO-cGMP pathway in vitro leads to overgrowth of retinal axons. Here we examined flies mutant for the alpha subunit gene of the Drosophila sGC (Gcalpha1). This mutation severely reduced but did not abolish GCalpha1 protein levels and NO-stimulated sGC activity in the developing photoreceptors. Although few mutant individuals possessed a disorganized retinal projection pattern, pharmacological NOS inhibition during metamorphosis increased this disorganization in mutants to a greater degree than in the wild type. Adult mutants lacked phototactic behavior, and the off-transient component of electroretinograms was frequently absent or greatly reduced in amplitude. Normal phototaxis and off-transient amplitude were restored by heat shock-mediated Gcalpha1 expression applied during metamorphosis but not in the adult. We propose that diminished sGC activity in the visual system during development causes inappropriate or inadequate formation of first-order retinal synapses, leading to defects in visual system function and visually mediated behavior.