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Many neurons concurrently and/or differentially release multiple neurotransmitter substances to selectively modulate the activity of distinct postsynaptic targets within a network. However, the molecular mechanisms that produce synaptic heterogeneity by regulating the cotransmitter release characteristics of individual presynaptic terminals remain poorly defined. In particular, we know little about the regulation of neuropeptide corelease, despite the fact that they mediate synaptic transmission, plasticity and neuromodulation. Here, we report that an identified Lymnaea neuron selectively releases its classical small molecule and peptide neurotransmitters, acetylcholine and FMRFamide-derived neuropeptides, to differentially influence the activity of distinct postsynaptic targets that coordinate cardiorespiratory behaviour. Using a combination of electrophysiological, molecular, and pharmacological approaches, we found that neuropeptide cotransmitter release was regulated by cross-talk between extrinsic neurotrophic factor signaling and target-specific retrograde arachidonic acid signaling, which converged on modulation of glycogen synthase kinase 3. In this context, we identified a novel role for the Lymnaea synaptophysin homologue as a specific and synapse-delimited inhibitory regulator of peptide neurotransmitter release. This study is among the first to define the cellular and molecular mechanisms underlying the differential release of cotransmitter substances from individual presynaptic terminals, which allow for context-dependent tuning and plasticity of the synaptic networks underlying patterned motor behaviour.

In the central nervous system (CNS), cholinergic transmission induces synaptic plasticity that is required for learning and memory. However, our understanding of the development and maintenance of cholinergic circuits is limited, as the factors regulating the expression and clustering of neuronal nicotinic acetylcholine receptors (nAChRs) remain poorly defined. Recent studies from our group have implicated calpain-dependent proteolytic fragments of menin, the product of the MEN1 tumor suppressor gene, in coordinating the transcription and synaptic clustering of nAChRs in invertebrate central neurons. Here, we sought to determine whether an analogous cholinergic mechanism underlies menin’s synaptogenic function in the vertebrate CNS. Our data from mouse primary hippocampal cultures demonstrate that menin and its calpain-dependent C-terminal fragment (C-menin) regulate the subunit-specific transcription and synaptic clustering of neuronal nAChRs, respectively. MEN1 knockdown decreased nAChR α5 subunit expression, the clustering of α7 subunit-containing nAChRs at glutamatergic presynaptic terminals, and nicotine-induced presynaptic facilitation. Moreover, the number and function of glutamatergic synapses was unaffected by MEN1 knockdown, indicating that the synaptogenic actions of menin are specific to cholinergic regulation. Taken together, our results suggest that the influence of menin on synapse formation and synaptic plasticity occur via modulation of nAChR channel subunit composition and functional clustering.

All functions of the nervous system are contingent upon the precise organization of neuronal connections that are initially patterned during development, and then continually modified throughout life. Determining the mechanisms that specify the formation and functional modulation of synaptic circuitry are critical to advancing both our fundamental understanding of the nervous system as well as the various neurodevelopmental, neurological, neuropsychiatric, and neurodegenerative disorders that are met in clinical practice when these processes go awry. Defining the cellular and molecular mechanisms underlying nervous system development, function, and pathology has proven challenging, due mainly to the complexity of the vertebrate brain. Simple model system approaches with invertebrate preparations, on the other hand, have played pivotal roles in elucidating the fundamental mechanisms underlying the formation and plasticity of individual synapses, and the contributions of individual neurons and their synaptic connections that underlie a variety of behaviors, and learning and memory. In this Review, we discuss the experimental utility of the invertebrate mollusc Lymnaea stagnalis, with a particular emphasis on in vitro cell culture, semi-intact and in vivo preparations, which enable molecular and electrophysiological identification of the cellular and molecular mechanisms governing the formation, plasticity, and specificity of individual synapses at a single-neuron or single-synapse resolution.

Synapse formation and plasticity depend on nuclear transcription and site-specific protein targeting, but the molecular mechanisms that coordinate these steps have not been well defined. The MEN1 tumor suppressor gene, which encodes the protein menin, is known to induce synapse formation and plasticity in the CNS. This synaptogenic function has been conserved across evolution, however the underlying molecular mechanisms remain unidentified. Here, using central neurons from the invertebrate Lymnaea stagnalis, we demonstrate that menin coordinates subunit-specific transcriptional regulation and synaptic clustering of nicotinic acetylcholine receptors (nAChR) during neurotrophic factor (NTF)-dependent excitatory synaptogenesis, via two proteolytic fragments generated by calpain cleavage. Whereas menin is largely regarded as a nuclear protein, our data demonstrate a novel cytoplasmic function at central synapses. Furthermore, this study identifies a novel synaptogenic mechanism in which a single gene product coordinates the nuclear transcription and postsynaptic targeting of neurotransmitter receptors through distinct molecular functions of differentially localized proteolytic fragments.