Most neurons use more than one type of neurotransmitter to mediate synaptic transmission. The use of multiple neurotransmitters enhances a neuron’s capacity to encode information within a circuit, allowing for more complex patterns of synaptic interactions to emerge, and the opportunity to differentially influence the activity of synaptic partners over multiple timescales. Many neurons form hundreds of presynapses, innervate a variety of postsynaptic targets, and modulate their transmitter output in response to a wide range of intracellular and extracellular signals. There is accumulating evidence that a neuron’s individual presynaptic terminals can exhibit distinct cotransmitter composition and release characteristics, and that this can be dynamically regulated in order to meet the spatiotemporal functional requirements of each synapse. However, our understanding of the cellular and molecular mechanisms that allow a neuron to regulate the use of its cotransmitters at distinct synaptic sites is considerably lacking.
The concurrent use of classical small molecule and peptide neurotransmitters is common throughout the nervous system and across evolution. Neuropeptides are important modulatory signals that regulate numerous aspects of synaptic development, plasticity and network function, as well as animal physiology and behaviour. Although peptide neurotransmitters are traditionally viewed as slow-acting neuromodulators released from perisynaptic sites in response to Ca 2+ diffusion during high-frequency bursting, their release has been demonstrated to occur over a wide range of firing frequencies. Peptidergic large dense-core vesicles (LDCVs) have also been shown to cluster directly at presynaptic active zones and be released by single action potentials. Furthermore, several studies have indicated that postsynaptic target identity influences the selective use of classical and peptide neurotransmitters at the different presynaptic terminals of a given neuron. Taken together, these observations suggest that the peptidergic characteristics of cotransmitting terminals can be highly variable, although the molecular mechanisms that give rise to such differences are yet to be defined.
In this study we sought to identify the cellular and molecular mechanisms underlying presynaptic specificity, with a focus on two unresolved questions: (i) how does a neuron establish functionally distinct presynapses with specific postsynaptic targets, and (ii) how does a presynaptic neuron generate the appropriate patterns of cotransmitter release that are required for the coordinated actions of neurons in behaviourally-relevant networks? To address these outstanding questions, we used cardiorespiratory neurons from the central nervous system (CNS) of the invertebrate mollusc Lymnaea stagnalis to study how the selective use of cotransmitters at synapses with distinct postsynaptic targets influences the assembly and function of the neuronal circuits involved in cardiorespiratory regulation. Reductionist approaches using the simple nervous systems of invertebrate models have enabled fundamental insights into cotransmission because their large identified neurons with known transmitter phenotypes participate in behaviourally-defined synaptic networks that can be studied directly in situ or reconstructed and manipulated in vitro. Moreover, invertebrate models are especially valuable for functional studies on peptidergic transmission because they are one of the few systems in which electrophysiologically measurable synaptic responses can be directly attributed to the actions of identified peptide neurotransmitters at individual synapses.
Here, we found that presynaptic neuropeptide release competency was regulated in a target- and context-dependent manner. This involved an interplay between extrinsic neurotrophic factors (NTF) and synapse-specific retrograde arachidonic acid (AA) signaling interactions, which converged on glycogen synthase kinase 3 (GSK-3) activity. In this context, we identified a surprising role for the Lymnaea synaptophysin (Syp) homologue in the inhibitory regulation of peptide neurotransmitter release. These findings on the regulation of cotransmitter use at individual synapses uncover a previously undefined mechanism for presynaptic cotransmitter specificity in synaptic networks, with implications for the appropriate expression and plasticity of patterned motor behaviours.
To investigate the mechanisms that differentiate cotransmitter use at individual synapses, and the consequences of this for specifying network function and behaviour, we first sought to monitor synaptic transmission by classical and peptide transmitters at functionally-defined synapses. The Lymnaea cardiorespiratory interneuron visceral dorsal 4 (VD4) was of interest for this study as it is well known to use the classical small molecule neurotransmitter acetylcholine (ACh) alongside a mixture of neuropeptides derived from the heptapeptide transcript of the FMRFamide gene (primarily G/SDPFLRFamide; discussed in subsequent text as FMRF neuropeptides). VD4 is integrated into a three-neuron network via reciprocal inhibitory synapses with the FMRFamidergic input 3 interneuron (IP3I) and the giant dopaminergic neuron right pedal dorsal 1 (RPeD1). These neurons and their reciprocal synapses establish Lymnaea’s respiratory central pattern generator (rCPG) network in vivo, are indispensable for the expression of respiratory behaviour, and recapitulate appropriate synaptic networks and rhythmic activity when reconstructed in vitro. VD4 is also known to form one-way excitatory synapses with a number of other identified neurons that act to coordinate cardiorespiratory behaviour, including the FMRFamidergic visceral F group cells (VF)
