Trophic factor-induced excitatory synaptogenesis involves postsynaptic modulation of nicotinic acetylcholine receptors.

Neurotrophic factors have well established roles in neuronal development, although their precise involvement in synapse formation and plasticity is yet to be fully determined. Using soma-soma synapses between identified Lymnaea neurons, we have shown recently that trophic factors are required for excitatory but not inhibitory synapse formation. However, neither the precise site (presynaptic versus postsynaptic cell) nor the underlying mechanisms have yet been defined. In the present study, synapse formation between the presynaptic cell visceral dorsal 4 (VD4) and its postsynaptic partner right pedal dorsal 1 (RPeD1) was examined to define the cellular mechanisms mediating trophic factor-induced excitatory synaptogenesis in cell culture. When paired in a soma-soma configuration in the presence of defined media (DM, nonproteinacious), mutually inhibitory synapses were appropriately reconstructed between VD4 and RPeD1. However, when cells were paired in the presence of increasing concentrations of Lymnaea brain-conditioned medium (CM), a biphasic synapse (initial excitatory synaptic component followed by inhibition) developed. The CM-induced excitatory synapse formation required trophic factor-mediated activation of receptor tyrosine kinases in the postsynaptic cell, RPeD1, and a concomitant modulation of existing postsynaptic nicotinic acetylcholine receptors (nAChRs). Specifically, when RPeD1 was isolated in DM, exogenously applied ACh induced a hyperpolarizing response that was sensitive to the AChR antagonist methyllycaconitine (MLA). In contrast, a single RPeD1 isolated in CM exhibited a biphasic response to exogenously applied ACh. The initial depolarizing phase of the biphasic response was sensitive to both mecamylamine and hexamethonium chloride, whereas the hyperpolarizing phase was blocked by MLA. In soma-soma-paired neurons, the VD4-induced synaptic responses in RPeD1 were sensitive to the cholinergic antagonists in a concentration range similar to that used to block cholinergic responses in single RPeD1 cells. Therefore, the modulation of postsynaptic nAChRs was sufficient to account for the trophic factor-induced excitatory synaptogenesis. This study thus provides the first direct evidence that trophic factors act postsynaptically to promote excitatory synapse formation.

Synapse number and synaptic efficacy are regulated by presynaptic cAMP and protein kinase A.

The mechanisms by which neurons regulate the number and strength of synapses during development and synaptic plasticity have not yet been defined fully. This lack of fundamental knowledge in the fields of neurodevelopment and synaptic plasticity can be attributed, in part, to compensatory mechanisms by which neurons accommodate for the loss of function in their synaptic partners. This is generally achieved either by scaling up neuronal transmitter release capabilities or by enhancing the postsynaptic responsiveness. Here, we demonstrate that regulation of synaptic strength and number between identified Lymnaea neurons visceral dorsal 4 (VD4, the presynaptic cell) and left pedal dorsal 1 (LPeD1, the postsynaptic cell) requires presynaptic activation of a cAMP-PKA-dependent signal. Experimental activation of the cAMP-PKA pathway resulted in reduced synaptic efficacy, whereas inhibition of the cAMP-PKA cascade permitted hyperinnervation and an overall enhancement of synaptic strength. Because synaptic transmission between VD4 and LPeD1 does not require a cAMP-PKA pathway, our data show that these messengers may play a novel role in regulating the synaptic efficacy during early synaptogenesis and plasticity.

Identification and functional expression of a family of nicotinic acetylcholine receptor subunits in the central nervous system of the mollusc Lymnaea stagnalis.

We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 subunits that display a high sequence similarity to nAChR subunits, of which 10 are of the alpha-type, 1 is of the beta-type, and 1 was not classified because of insufficient sequence information. Heterologous expression of identified subunits confirms their capacity to form functional receptors responding to acetylcholine. The alpha-type subunits can be divided into groups that appear to underlie cation-conducting (excitatory) and anion-conducting (inhibitory) channels involved in synaptic cholinergic transmission. The expression of the Lymnaea nAChR subunits, assessed by real time quantitative PCR and in situ hybridization, indicates that it is localized to neurons and widespread in the CNS, with the number and localization of expressing neurons differing considerably between subunit types. At least 10% of the CNS neurons showed detectable nAChR subunit expression. In addition, cholinergic neurons, as indicated by the expression of the vesicular ACh transporter, comprise approximately 10% of the neurons in all ganglia. Together, our data suggested a prominent role for fast cholinergic transmission in the Lymnaea CNS by using a number of neuronal nAChR subtypes comparable with vertebrate species but with a functional complexity that may be much higher.

Synaptogenesis in the CNS: an odyssey from wiring together to firing together.

To acquire a better comprehension of nervous system function, it is imperative to understand how synapses are assembled during development and subsequently altered throughout life. Despite recent advances in the fields of neurodevelopment and synaptic plasticity, relatively little is known about the mechanisms that guide synapse formation in the central nervous system (CNS). Although many structural components of the synaptic machinery are pre-assembled prior to the arrival of growth cones at the site of their potential targets, innumerable changes, central to the proper wiring of the brain, must subsequently take place through contact-mediated cell-cell communications. Identification of such signalling molecules and a characterization of various events underlying synaptogenesis are pivotal to our understanding of how a brain cell completes its odyssey from "wiring together to firing together". Here we attempt to provide a comprehensive overview that pertains directly to the cellular and molecular mechanisms of selection, formation and refinement of synapses during the development of the CNS in both vertebrates and invertebrates.

Neurotrophic activities of trk receptors conserved over 600 million years of evolution.

The trk family of receptor tyrosine kinases is crucial for neuronal survival in the vertebrate nervous system, however both C. elegans and Drosophila lack genes encoding trks or their ligands. The only invertebrate representative of this gene family identified to date is Ltrk from the mollusk Lymnaea. Did trophic functions of trk receptors originate early in evolution, or were they an innovation of the vertebrates? Here we show that the Ltrk gene conserves a similar exon/intron order as mammalian trk genes in the region encoding defined extracellular motifs, including one exon encoding a putative variant immunoglobulin-like domain. Chimeric receptors containing the intracellular and transmembrane domains of Ltrk undergo ligand-induced autophosphorylation followed by MAP kinase activation in transfected cells. The chimeras are internalized similarly to TrkA in PC12 cells, and their stimulation leads to differentiation and neurite extension. Knock-down of endogenous Ltrk expression compromises outgrowth and survival of Lymnaea neurons cultured in CNS-conditioned medium. Thus, Ltrk is required for neuronal survival, suggesting that trophic activities of the trk receptor family originated before the divergence of molluscan and vertebrate lineages approximately 600 million years ago.

Differential proteomics reveals multiple components in retrogradely transported axoplasm after nerve injury.

Information on axonal damage is conveyed to neuronal cell bodies by a number of signaling modalities, including the post-translational modification of axoplasmic proteins. Retrograde transport of a subset of such proteins is thought to induce or enhance a regenerative response in the cell body. Here we report the use of a differential 2D-PAGE approach to identify injury-correlated retrogradely transported proteins in nerves of the mollusk Lymnaea. A comprehensive series of gels at different pI ranges allowed resolution of approximately 4000 spots by silver staining, and 172 of these were found to differ between lesioned versus control nerves. Mass spectrometric sequencing of 134 differential spots allowed their assignment to over 40 different proteins, some belonging to a vesicular ensemble blocked by the lesion and others comprising an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament termed RGP51 (retrograde protein of 51 kDa). Inhibition of RGP51 expression by RNA interference inhibits regenerative outgrowth of adult Lymnaea neurons in culture. These results implicate regulated proteolysis in the formation of retrograde injury signaling complexes after nerve lesion and suggest that this signaling modality utilizes a wide range of protein components.

Calcium channel structural determinants of synaptic transmission between identified invertebrate neurons.

We report here that unlike what was suggested for many vertebrate neurons, synaptic transmission in Lymnaea stagnalis occurs independent of a physical interaction between presynaptic calcium channels and a functional complement of SNARE proteins. Instead, synaptic transmission in Lymnaea requires the expression of a C-terminal splice variant of the Lymnaea homolog to mammalian N- and P/Q-type calcium channels. We show that the alternately spliced region physically interacts with the scaffolding proteins Mint1 and CASK, and that synaptic transmission is abolished following RNA interference knockdown of CASK or after the injection of peptide sequences designed to disrupt the calcium channel-Mint1 interactions. Our data suggest that Mint1 and CASK may serve to localize the non-L-type channels at the active zone and that synaptic transmission in invertebrate neurons utilizes a mechanism for optimizing calcium entry, which occurs independently of a physical association between calcium channels and SNARE proteins.