Uncovering the Cellular and Molecular Mechanisms of Synapse

Mar 12, 2018 - Uncovering the Cellular and Molecular Mechanisms of Synapse .... (e.g., chemical or electrical synapses(9−11)), directionality (e.g.,...
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Uncovering the Cellular and Molecular Mechanisms of Synapse Formation and Functional Specificity Using Central Neurons of Lymnaea stagnalis Angela M. Getz,†,‡,∥ Pierre Wijdenes,†,§ Saba Riaz,† and Naweed I. Syed*,† †

Department of Cell Biology & Anatomy, Hotchkiss Brain Institute and Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta T2N 1N4, Canada ‡ Department of Neuroscience, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 1N4, Canada § Biomedical Engineering Graduate Program, University of Calgary, Calgary, Alberta T2N 1N4, Canada

ABSTRACT: 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, semiintact 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. KEYWORDS: Lymnaea stagnalis, model systems, neuronal networks, respiratory behavior, synaptogenesis, neuroelectrode devices

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INTRODUCTION At the most fundamental level, the field of neuroscience aims to fully understand how neuronal networks control behavior. In order to achieve this, we must first uncover the factors that specify the nature of neuron−neuron interactions; more precisely, what are the mechanisms that determine (i) the formation of appropriate synapses during development, (ii) the proper expression, localization, and function of synaptic machinery that underlies efficient transmission, and (iii) how synapses undergo plastic remodeling in the mature nervous system to enable behavioral flexibility? Answering these questions will allow for a better vantage point from which innovative therapies and technology can be developed in order to treat a range of neurodevelopmental, neurological, and neurodegenerative disorders. To approach these questions exper© XXXX American Chemical Society

imentally, neuroscientists employ a variety of animal models where the relationship between neurons, their synaptic connections, and their roles in the control of behavior, are easily discernible and can be directly and rapidly investigated and manipulated. The use of simple model systems and reductionist experimental approaches has contributed to numerous fundamental advancements in our understanding of the nervous system. Here, we will discuss the utility of identified neurons of the central nervous system (CNS) of Lymnaea stagnalis, an Special Issue: Model Systems in Neuroscience Received: November 15, 2017 Accepted: March 1, 2018

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DOI: 10.1021/acschemneuro.7b00448 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Review

ACS Chemical Neuroscience

recapitulate the same patterns of connectivity (e.g., chemical or electrical synapses9−11), directionality (e.g., one-way or reciprocal synapses12), and synaptic network activity (e.g., rhythmogenesis driven by postinhibitory rebound excitation12) that are observed in the intact CNS. For example, we have found that a single identified presynaptic neuron cultured simultaneously with two identical, otherwise appropriate, postsynaptic targets will selectively innervate only one.13 Similarly, two identical presynaptic neurons have been shown to compete for the same postsynaptic target, where innervation by the first cell prevents the other from forming a functional synaptic connection.14 At the molecular level, we have found that voltage-gated calcium channels (VGCCs) selectively cluster to contact sites between appropriate, but not inappropriate synaptic targets, and that this event coincides with the appearance of synaptic transmission during in vitro synaptogenesis.15 These features make invertebrate neuron culture models an ideal preparation for the study of the mechanisms underlying synaptic specificity, and addressing the question of how a neuron establishes the appropriate numbers and types of synapses with distinct targets. Although Lymnaea lags behind the utility of other invertebrate models such as Drosophila melanogaster and Caenorhabditis elegans in terms of genetic investigation and manipulation, individual cloning efforts16−20 and the generation of CNS transcriptome libraries21 have provided sufficient sequence information to make the Lymnaea model system amenable to unraveling the genetic determinants of the synapse. For example, identification of the glia-derived acetylcholine binding protein (AChBP), a soluble transmitter receptor, in Lymnaea provided some of the first direct evidence that glial cells actively participate in the regulation of synaptic transmission efficacy.22 It is worth noting that the small size of the Lymnaea AChBP and its high degree of sequence homology to nicotinic acetylcholine receptors (nAChRs) was also instrumental for later work in resolving the structure of the ligand-binding domain of nAChRs,23 and for opening new opportunities to pursue the structure-aided design of novel therapeutics targets for neuropsychiatric and neurodegenerative disorders.24 In culture, the recapitulation of synapses between identified neurons can be achieved by selectively plating neurons in distinct configurations that offer unique opportunities to address explicit questions about the nature of synapse formation, function, or specificity. For the study of reduced synaptic networks and their role in behavior, the constituent cells are isolated and plated in a growth-permissive media such as conditioned media (CM), which is a defined medium (DM; e.g. Leibovitz L-15) that has been incubated with isolated brains for 3−4 days prior to cell culture to produce a neurotrophic factor (NTF)-rich media that supports neurite outgrowth and synaptogenesis.4 Cells are maintained in culture for several days, where they establish extensive neurite processes and reform synaptic connections that exhibit the same functional properties and morphological specializations as the synapses observed in the intact CNS.10,15,25 In invertebrate models, neuron culture is also possible directly within the nervous system in situ or in vivo, as donor neurons can be transplanted into the CNS of host animals to study the physiological integration of transplanted cells into existing neuronal networks.14,26 These experimental preparations have, for instance, allowed researchers to directly answer the question of whether it is the intrinsic electrical properties of individual neurons or the synaptic properties of specific neuronal networks that form the basis of rhythmical patterned activity

invertebrate pulmonate gastropod mollusc, for studying the development, plasticity, and functional specificity of synapses, and how they instruct the expression of simple behaviors. While a complete review of this field is beyond the scope of this Review, we will focus mainly on how the experimental advantages afforded by Lymnaea neurons in culture have enabled researchers to answer specific neurobiological questions and identify novel bioengineering solutions that would have been difficult, if not impossible, to approach in other model systems. In particular, we will provide our perspective on the three primary axes of our research group: (i) the role of simple synaptic networks in the establishment of central pattern generator (CPG) circuits that control rhythmic behaviors, (ii) the precise roles of neurotrophic factors in synaptogenesis, and (iii) the development of novel tools such as neuroelectrode devices that can be used to interrogate the intrinsic and network properties of neurons.

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AMENABLE ATTRIBUTES OF THE Lymnaea stagnalis MODEL SYSTEM The CNS of L. stagnalis is composed of a limited number of cells (only ∼20 000), where individually identified neurons of known neurotransmitter phenotype and defined patterns of synaptic connectivity can be readily identified by their size, color, and position within distinct ganglia.1 In this Review, we will emphasize in particular the utility of studying isolated neurons and reconstructed synapses in culture. While the ultimate goal of studies in neuroscience may be to define the human condition, the sheer complexity of the mammalian CNS, with billions of neurons and trillions of synapses, makes it rather difficult to investigate the formation and function of individual synapses. This increased complexity introduces variability that confounds precise experimental control over variables such as environmental factors, target cell interactions, and spatiotemporal regulation of the synaptogenic program. In order to combat this issue, many laboratories have utilized the simple nervous systems of molluscs to minimize experimental variability and achieve the precision necessary for investigating the environmental and target cell interactions that define synapse formation and function at the resolution of single cells.2 Relative to vertebrate neurons, the larger and more physiologically resilient neurons of invertebrates allow for the extraction and culturing of single cells from the intact CNS, and also permit intracellular microelectrode or patch-clamp recordings to be made repeatedly and/or for longer periods of time. Importantly, these systems easily allow for replicable studies to be made on precisely the same neuron or identified synapse, an experimental feat which is unparalleled in other models. The techniques for culturing invertebrate neurons were first developed in the 1970s to 1980s by several groups working on the freshwater snails Lymnaea stagnalis3 and Helisoma trivolvis,4,5 the marine snail Aplysia californica,6,7 and the leech Hirudo medicinalis.8 The culture procedure typically involves the isolation of visually identified neurons from desheathed ganglia using suction applied through a glass pipet. When isolated cell bodies or cells with attached axon “stumps” are plated on culture dishes coated with a permissive adhesive substrate such as poly-Llysine, neurons maintain their intrinsic membrane properties, regenerate their axonal processes, and re-establish appropriate synaptic connections.1,8 The inherent fidelity of in vitro recapitulated synapses is perhaps the most impressive feature of the invertebrate neuron culture technique, and is strikingly exemplified by the specificity with which synapses form between identified neurons in culture, where appropriate target cells B

DOI: 10.1021/acschemneuro.7b00448 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Figure 1. Respiratory CPG circuit underlying aerial breathing behavior in Lymnaea. (A) Adult L. stagnalis animal exhibiting aerial respiration (open pneumostome, expiration phase) at the water surface. (B) Schematic representation of the Lymnaea central nervous system (dorsal view of the central ring ganglion), depicting the location of the interneurons that comprise the respiratory CPG circuit (VD4, RPeD1, IP3I). (C) Summary diagram of the synaptic connections underlying the respiratory CPG and the pneumostome motor neurons. (D) In vitro reconstruction of the respiratory CPG circuit established by neurite outgrowth from IP3I, VD4, and RPeD1 neurons maintained in culture on a silicon chip. (E) Rhythmic activity of the respiratory CPG network recorded from an isolated Lymnaea CNS. Note that due to the inaccessible position of IP3I in the intact CNS, activity of the follower VJ motor neuron is recorded as an indication of the occurrence of activity in IP3I. (F) Rhythmic activity of the respiratory CPG network recorded from in vitro reconstructed synapses between RPeD1, IP3I, and VD4. Note that the rhythmic pattern observed in vitro mimics the respiratory cycle observed in vivo. Figure adapted and modified with permission from Syed et al.,12 and Janes and Syed.49

exhibited by the CPGs that instruct simple behaviors such as respiration12 and feeding.27 One of the usual constraints on studying synapses in vitro is the requirement for neurite outgrowth and its dependency on NTFs.28 To circumvent this experimental limitation, the soma and/or axons of isolated invertebrate neurons can be precisely positioned by manipulating the positive or negative pressure applied through the glass electrode, and thus the cells can be selectively juxtaposed in culture, allowing for synapse formation to occur in the absence of neurite outgrowth.29−31 These “soma− soma”, “soma−axon”, or “axon−axon” configurations provide advantageous preparations where the cellular, molecular, and genetic foundations of the synaptogenic program can be selectively dissected in the absence or presence of NTFs, by culturing cells in NTF-deficient DM, in NTF-rich CM, or in DM supplemented with purified NTFs. Furthermore, because these configurations bypass the requirement for neurite outgrowth, we gain the ability to precisely time synaptogenesis in order to dissect the temporal mechanisms that direct the functional maturation of synapses.32 Importantly, this experimental resolution cannot be equivalently attained with mammalian dissociated cell culture approaches, as neurons do not extend processes, form synapses, or survive without trophic support. The ability to isolate individual invertebrate neurons with or without their axons, and then precisely control the position of these cells in culture also affords the opportunity to define the roles of different cellular compartments and specific target cell signaling interactions during synaptogenesis. Once plated, the selective axotomization, or severing of the cultured axon and soma using an electrode, allows direct access to the question of

whether transcriptional events are required for distinct synaptogenic events. For example, by culturing identified neurons in soma−soma, or axotomized soma−axon configurations, we have found that somal and extrasomal compartments recruit distinct transcription-dependent or -independent mechanisms, and distinct cell-intrinsic or -extrinsic signaling events, during chemical and electrical synapse formation.11 Similarly, we have found that the axotomized growth cones isolated from appropriate pre- and postsynaptic partners, or “growth balls”, have the innate capacity to form synapses when juxtaposed in culture, but that synaptogenic factors must be received from the soma for synaptic consolidation and the longterm maintenance of these nascent synapses.32 Thus, this invertebrate neuron culture model provides an ideal preparation with which to investigate the influence of various intrinsic and extrinsic factors on instructing the development, molecular composition, and function of individual pre- and postsynaptic terminals. We will now highlight some of the contributions to the field of neuroscience that have been made using the Lymnaea model.

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IDENTIFICATION AND RECONSTRUCTION OF SIMPLE NEURONAL CIRCUITS UNDERLYING BEHAVIOR The fact that invertebrate nervous systems are composed of a limited number of large, easily identifiable, and accessible neurons, whose functional roles within specific networks could be defined and directly attributed to observable behaviors, is the most significant factor that propelled the development of invertebrate preparations for studies in synaptic physiology. C

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and sufficiency of a half-center oscillator network in the expression of rhythmic behaviors proved difficult to obtain. A fundamental insight into the mechanism of rhythm generation in CPG networks was made possible by the isolation and in vitro reconstruction of the component neurons of Lymnaea’s respiratory CPG network12 (Figure 1D). When individual IP3I and VD4 neurons were isolated in culture, neither was observed to exhibit spontaneous activity or endogenous repetitive bursting, and the in vitro reconstructed IP3I-VD4 synapse was quiescent, indicating that the respiratory rhythm could not be generated by these two neurons alone. The giant dopaminergic interneuron right pedal dorsal 1 (RPeD1) was identified as a third necessary cellular component of the Lymnaea respiratory CPG, integrated through a reciprocal inhibitory synapse with VD4 and a reciprocal mixed excitatory-inhibitory synapse with IP3I. While none of the respiratory interneurons exhibited the feature of endogenous patterned bursting activity that is required for rhythm initiation (e.g., plateau potentials as in the N1M interneuron that initiates the Lymnaea feeding CPG rhythm38), isolated RPeD1s did exhibit spontaneous activity due to a depolarized resting membrane potential established by a pacemaker sodium leak current.12,46 By reconstructing the threeneuron network in vitro, rhythmogenesis in the respiratory CPG was shown to be driven by postinhibitory rebound (PIR) excitation of IP3I and VD4 in response to synaptic network interactions initiated by RPeD1, where PIR excitation of IP3I elicited bursting activity in RPeD1, and synaptic inhibition of VD4 by IP3I and RPeD1 sequentially elicited PIR bursting in VD4. Importantly, the patterns of bursting activity of the in vitro reconstructed network were found to be indistinguishable from the respiratory rhythm observed in semi-intact brain preparations (Figure 1E,F). The pacemaker sodium leak current of the RPeD1 interneuron is therefore not an endogenous electrophysiological property required for rhythmogenesis, but rather serves to modulate network excitability and CPG frequency. Taken together, these observations demonstrate that the emergent properties of the synaptic network are the sole contributor to the expression of bursting activity underlying rhythm generation in the Lymnaea respiratory CPG. It is important to emphasize that this conclusion could not have been validated without the ability afforded by invertebrate models to maintain identified neurons and reconstruct specific synapses in culture, given that synaptic inputs present in the intact nervous system confound the study of the intrinsic properties of individual neurons and their contributions to the emergence of synaptic network properties.1,12,38 Complementary to in vitro reconstruction approaches, the use of intracellular recordings and in situ transplantation approaches in Lymnaea semi-intact and in vivo preparations enabled further insights into the necessity and sufficiency of these neurons and their synapses in the expression of respiratory behavior. For example, selective ablation of the VD4 interneuron in vivo was found to occlude aerial respiration in Lymnaea, demonstrating its necessity for respiratory CPG rhythm generation and breathing behavior.14 Transplantation of VD4 from a donor animal into a VD4-ablated host resulted in the restoration of respiratory CPG network activity and the functional recovery of breathing behavior, demonstrating the ability of transplanted VD4 interneurons to regenerate their axonal processes and reestablish specific synapses with appropriate target cells in vivo. Together with in vitro cell culture approaches, these techniques offer a unique opportunity to define the precise roles of individual neurons or synaptic networks in the expression of well-defined

The reconstruction of rudimentary synaptic networks in culture enabled transformative insights into how the functional characteristics of individual neurons and their synapses controlled the expression and plasticity of simple behaviors. Perhaps the most widely known example of this is the learning model pioneered by Eric Kandel using the gill-withdrawal reflex of Aplysia,33 the first behaviorally relevant network to be reconstructed in culture.1 In vitro, these synapses exhibited the same aspects of plasticity that are associated with habituation, sensitization, and long-term facilitation as observed in the intact nervous system; observations that were key to identifying the molecular mechanisms underlying synaptic plasticity, and how changes in synaptic strength and structural connectivity were associated with learning and memory behavior.34−37 These foundational studies in Aplysia demonstrated the utility of neuron culture approaches for investigating the cellular and molecular determinants of synapse formation and function, and were readily adapted for use in other model systems for their opportunities to target distinct neurobiological questions. In the Lymnaea model, functional studies identifying the component neurons of CPG networks and behavioral studies on the modulation of simple observable behaviors by environmental stimuli have similarly contributed significantly to our understanding of the synaptic mechanisms that produce and modulate simple behaviors. Reductionist approaches using cell culture and semi-intact preparations have allowed for the identification of the synaptic circuitry involved in the respiratory behavior of Lymnaea, and have provided the first direct evidence for the fundamental network properties that underlie rhythm generation in CPG circuits.12,14 In addition, the roles of other identified Lymnaea neurons have been well-defined in the control of simple behaviors,38,39 most notably the feeding CPG (composed of the N1M, N2, and N3t interneurons) and the associated modulatory input (serotonergic cerebral giant cells; CGCs) and motoneuron output (B1, B3, B4) networks, which have been reviewed in detail elsewhere.40,41 In the interest of space, we will focus here on the neurophysiological insights that have been gained by studying the neuronal circuitry that elicits and modulates breathing behavior in Lymnaea. Under normoxic conditions, Lymnaea are bimodal breathers, achieving their oxygen intake requirements via aerial respiration and cutaneous diffusional exchange.42 Aerial respiration is conducted through the pneumostome, an external respiratory orifice connected to the lung cavity, the opening and closing of which is associated with the expiration and inspiration phases of breathing, respectively14 (Figure 1A). The animals can be made to rely entirely on aerial respiration by exposure to hypoxic conditions such as nitrogenated water, which makes the analysis of this behavior easy to achieve under strictly regulated and reproducible experimental conditions. Aerial respiration in Lymnaea is controlled by a reciprocal inhibitory synapse between the visceral dorsal 4 (VD4) and input 3 interneurons (IP3I), which activate distinct visceral K (VK) and visceral J (VJ) motoneurons that drive inspiration and expiration, respectively43,44 (Figure 1B,C). This arrangement of a mutually inhibitory synapse driving antagonistic motor functions via alternating bursts of activity paralleled the “half-center” oscillator model that had been proposed to underlie the expression of many rhythmic behaviors under study in both vertebrate and invertebrate models, where rhythmic network activity could result from reciprocal antagonistic synaptic connections between two neurons or two populations of neurons.1,45 However, direct and unequivocal experimental evidence supporting the necessity D

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Figure 2. NTF- and L-MEN1-dependent excitatory cholinergic synaptogenesis in Lymnaea. (A) Soma-soma paired VD4-LPeD1 neurons. (B) In the absence of NTFs, when cultured in DM, an inhibitory cholinergic synapse forms between VD4-LPeD1 paired neurons. In the presence of NTFs, when cultured in CM, an excitatory cholinergic synapse forms. Microinjection of synthetic L-MEN1 mRNA into LPeD1 neurons is sufficient to induce the formation of excitatory synapses in the absence of NTFs. (C) Proposed model for the “default” formation of inappropriate inhibitory cholinergic synapses between VD4-LPeD1 in the absence of NTFs. (D) Proposed model for the role of NTF signaling, via L-EGF and L-EGFR, in inducing excitatory cholinergic synaptogenesis via (i) transcriptional upregulation of L-MEN1, (ii) proteolytic cleavage of L-menin via NTF-induced calcium oscillations that activate the calcium-dependent protease calpain, (iii) transcriptional upregulation of excitatory L-nAChR subunits via the N-terminal Lmenin proteolytic fragment, and (iv) postsynaptic clustering of excitatory L-nAChRs via the C-terminal L-menin proteolytic fragment. Figure adapted and modified with permission from Getz et al.72

aerial breathing behavior. Using a combination of behavioral and electrophysiological approaches, we have recently reported that Lymnaea possesses a spatially distributed network of peripheral oxygen chemoreceptor sites that respond selectively to graded hypoxic challenges.44,49 These findings suggest that the existence of multiple oxygen chemoreceptor sites is an evolutionarily conserved strategy that underlies the adaptability of respiratory behavior to meet changing environmental demands, and opens to new opportunities to investigate the fundamental synaptic mechanisms that regulate respiratory systems.

and readily observable behaviors. Such invertebrate preparations may thus be considered an ideal system for studies on neuronal transplantation, as both the functional and morphological specificity of regenerated synapses can be approached at a resolution that is still not fully feasible in vertebrate models. In addition to the detailed knowledge of the synaptic mechanisms underlying Lymnaea’s respiratory CPG gained via cell culture approaches, studies on the behavioral plasticity of aerial respiration have also contributed to our understanding of how animals integrate external environmental stimuli in order to appropriately modulate their behavioral responses. For example, in response to an operant conditioning paradigm where respiration-contingent tactile stimulation was applied to the pneumostome in order to activate the escape-withdrawal reflex, it was found that breathing behavior became significantly attenuated in a hypoxic environment that normally stimulates aerial respiration.47 Furthermore, spaced training sessions of this operant conditioning paradigm were subsequently found to produce behavioral changes that persisted for at least 4 weeks, thus identifying an associative form of long-term memory in Lymnaea.48 More recent research efforts of our lab have also focused on dissecting the roles of other environmental signals in the modulation of respiratory CPG activity and the plasticity of

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ROLE OF NEUROTROPHIC FACTORS AND MEN1 TUMOR SUPPRESSOR GENE IN SYNAPTOGENESIS Defining the specific requirements for NTF signaling via receptor tyrosine kinases (RTK) during synaptogenesis, and dissecting the underlying molecular mechanisms at presynaptic versus postsynaptic sites, have been some of our group’s primary areas of research focus. As discussed above, the ability to selectively characterize the synaptogenic effects of NTFs, independent of their influence on neurite outgrowth, is a unique experimental feat made possible by the use of invertebrate cell culture preparations, particularly the soma−soma model, where identified pre- and postsynaptic partners reliably form targetE

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mechanisms of synaptogenesis, we found that a Lymnaea homologue of the multiple endocrine neoplasia type 1 tumor suppressor gene (L-MEN1),60 which encodes the transcription factor menin (L-menin), was upregulated during synaptogenesis between VD4-LPeD1.61 With single-cell RNAi-mediated knockdown, synapse formation was shown to require L-MEN1 expression specifically in the postsynaptic neuron. These observations were the first to identify a novel synaptogenic function for the MEN1 gene. Subsequent studies by other groups working on rat and mouse models of peripheral nerve injury found that the upregulation of MEN1 mediates synaptic plasticity and the development of neuropathic pain by inducing synaptic plasticity in the spinal cord dorsal horn.62−64 These observations indicated that menin’s synaptogenic function had likely been conserved across evolution; however, the underlying molecular mechanisms remained to be determined. As a tumor suppressor, the function of menin in cancer biology, particularly with regard to the transcriptional mechanisms underlying cell cycle regulation, had been well characterized.65−70 Conversely, the molecular actions of menin in neurons had never before been investigated. This prompted us to investigate whether L-MEN1-dependent transcriptional activation of excitatory L-nAChR subunits might be recruited by L-EGF/R signaling during excitatory synaptogenesis. Using a combination of single-cell qPCR, cell-specific mRNA microinjection, immunocytochemical, and electrophysiological approaches, we found that L-MEN1 expression in LPeD1 was induced by NTF/R signaling via the MAPK/ERK cascade, and that its activation was sufficient to induce the functional expression of excitatory nAChRs and promote excitatory synaptogenesis in the absence of NTFs71 (Figure 2B). Given that menin is described in the cancer literature as a nuclear protein,65 our initial hypothesis was that the synaptogenic effects of L-menin were mediated by the upregulation of excitatory LnAChR expression. We were, however, surprised to find that protein phosphorylation events induced by NTFs triggered the synaptic localization of L-menin immunoreactivity, suggesting that there was a previously unidentified cytoplasmic mechanism of action underlying MEN1’s synaptogenic effects.72 Further investigation revealed that L-menin was proteolytically cleaved at an evolutionarily conserved calpain consensus site, producing an N-terminal L-menin fragment (N-menin) that targeted to the nucleus and induced the transcriptional upregulation of two excitatory L-nAChR subunits, and a C-terminal L-menin fragment (C-menin) that mediated the functional clustering of excitatory L-nAChRs at the synapse.72 Importantly, these observations identified a novel mechanism underlying excitatory synaptogenesis in which distinct molecular actions of two differentially localized proteolytic fragments of a single gene product coordinated the nuclear transcription and postsynaptic targeting of neurotransmitter receptors. Central to this model for NTF- and MEN1-dependent excitatory synaptogenesis was the activity of the calcium-dependent protease calpain,73,74 which is further supported by earlier observations from our group that CM induces calcium oscillations via L-type VGCCs in postsynaptic LPeD1, but not presynaptic VD4 neurons, which are required for excitatory L-nAChR expression and excitatory synapse formation75 (Figure 2C,D). It is important to emphasize that the single-cell resolution of invertebrate synaptic cell culture, most notably the ability to selectively inject molecular constructs into the large Lymnaea neurons, enabled us to pursue the functional studies necessary to uncover the postsynaptic mechanisms underlying MEN1’s

specific synapses when juxtaposed in culture. Using this model, we initially identified a unique requirement for NTF-RTK (NTF/R) signaling during the formation of appropriate excitatory, but not inhibitory synapses between identified Lymnaea neurons in culture.30,50,51 Much of the work from our lab has focused on defining the mechanisms underlying the formation and plastic remodeling of a cholinergic synapse between the presynaptic VD4 neuron and its postsynaptic target left pedal dorsal 1 (LPeD1), where appropriate excitatory synapses are observed when VD4-LPeD1 pairs are cultured in NTF-rich CM, whereas inappropriate inhibitory synapses are observed in NTF-deficient DM (Figure 2A,B). These inhibitory synapses observed in DM are termed “inappropriate” because they are not observed in the intact CNS.51 Together with previous indications of the phenomenon of “synaptic capture”, where synaptic innervation of appropriate targets prevents supernumerary innervation and thus the inappropriate wiring of neuronal networks (see above),13,14 these observations led us to postulate that the formation of these inappropriate inhibitory synapses between appropriate synaptic partners may be indicative of a mechanism to both functionally modulate the gain of excitatory synapses and ensure proper synaptic circuitry is maintained during changes in NTF/R signaling. Subsequent work revealed that excitatory synapse formation between VD4-LPeD1 was contingent upon molecular signaling events elicited by a Lymnaea epidermal growth factor homologue (L-EGF), and the activation of L-EGF RTK (L-EGFR), expressed in the postsynaptic LPeD1 neuron.52,53 It is also of interest to note that the supplementation of DM with human EGF was found to mimic the excitatory synaptogenic effects of LEGF, highlighting the evolutionary conservation of NTF/R signaling systems, and the utility of Lymnaea for studying the role of NTFs in synaptogenesis. In contrast to the exclusively excitatory (cation-conducting) nAChRs of vertebrates, molecular cloning, recombinant expression and functional electrophysiological studies have demonstrated that both excitatory and inhibitory (anion-conducting) nAChRs are expressed in the Lymnaea CNS (L-nAChRs).18,19 Although this biphasic cholinergic system is unique to invertebrates, it offers an ideal electrophysiological assay with which to investigate the apparently L-EGF/R-mediated effects on the expression of excitatory L-nAChRs in LPeD1 neurons, and further supports a postsynaptic locus for the inhibitory-excitatory phenotypic switch induced by CM. In this context, it is also of interest to note that the VD4-RPeD1 synapse has been reported by different groups to exhibit both purely inhibitory12 and biphasic (excitation followed by inhibition)54 synaptic transmission; as in VD4-LPeD1 synapses, we suspect that this discrepancy may be explained by variability in the expression of excitatory and inhibitory nAChRs in RPeD1 neurons. Complementary to early evidence from our group indicating that NTF-dependent excitatory synapse formation required transcription and translation events,50 numerous reports emerged identifying that the transcriptional upregulation of specific neurotransmitter receptor subunits by NTF/R signaling was a ubiquitous mechanism to tune receptor function during synapse formation and maturation.55−58 In many instances, however, the precise identity of the transcription factors linking NTF/R signaling to the expression of mRNA for specific neurotransmitter receptor subunits, and the mechanisms targeting nascent neurotransmitter receptors to appropriate postsynaptic sites, remained largely undefined.59 Using a singlecell differential display PCR approach to screen for novel genetic F

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Figure 3. Role of Lymnaea neurons in the development of novel neuroelectronic devices. (A) Schematic representation of the connection between preand postsynaptic neurons used to develop the analogue passive electrical circuit of an electrode−neuron interface. (B) Three-dimensional rendering of a 30 μm diameter microelectrode with a nanoedge (arrows), tilted 40°, using atomic force microscopy. (C) Traditional 8 × 8 MEAs with cultured individual or soma−soma paired LPeD1 and VD4 neurons allow long-term recordings at a single-cell level. (D) Patterned action potentials recorded from a single LPeD1 neuron cultured on a nanoedge MEA exhibit 15-fold increased SNR resolution compared to traditional planar microelectrodes MEAs. (E) Long-term recording capability of MEAs enables the identification of distinctive patterns of spontaneous activity in LPeD1 and RPeD1 neurons. Figure adapted and modified with permission from Luk et al.89 and Wijdenes et al.95

development, function, and maintenance of central cholinergic synapses. This may hold potential for the regulation of learning and memory behavior,79,80 as well as neurodegenerative diseases such as Alzheimer’s disease, which has been linked to reduced NTF/R signaling,81 tumor suppressor gene dysfunctions,82,83 and cholinergic deficits.84−87

synaptogenic effect. It is also of interest to note that the presence of a faster-migrating C-terminal menin immunoreactive fragment had been previously observed in Western blots from various mammalian preparations, however, the significance of this fastermigrating band had been dismissed as it was thought to be due to a nonspecific cross-reaction of the antibody.65,76,77 However, the presence of a calpain cleavage site that was conserved in menin orthologues from Drosophila to human suggested to us that menin proteolytic fragments had been selected for during evolution, and prompted our initial investigation into the functional roles of menin proteolytic fragments.72 Using mouse primary hippocampal cultures and shRNA-mediated MEN1 knockdown, we have recently reported that menin and the calpain-dependent C-menin fragment similarly regulate the subunit-specific transcriptional regulation (α5) and synaptic clustering of neuronal nAChRs in vertebrate central neurons, indicating that molecular mechanisms underlying menin’s synaptogenic effects are conserved.78 As the formation and function of glutamatergic synapses was not influenced by MEN1 knockdown, our results furthermore indicate that MEN1 is a specific genetic mechanism employed in the regulation of central cholinergic synaptogenesis. Current research efforts of the lab are now aimed at employing a neuron-specific conditional MEN1 knockout mouse to understand the role of MEN1 in the

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CONTRIBUTIONS TO THE ADVANCEMENT OF NEUROELECTRONIC DEVICES In addition to the utility of the Lymnaea model for addressing neurobiological questions, the large size, physiological robustness, and ability to precisely position these Lymnaea neurons in culture has been instrumental for numerous innovations in the bioengineering of neuroelectronic technologies. While traditional intracellular recording electrodes and/or patch clamp electrodes remain the gold standard for recording and analyzing neuronal activity at the single-cell level, these approaches are limited, as only a few neurons can be recorded concurrently, and the invasiveness of these techniques only permit short-term recordings (minutes to hours). To overcome these limitations, researchers have turned to the use of neuroelectronic devices that permit the noninvasive electrical stimulation and recording of activity, and the ability to collect data from multiple electrodes G

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ACS Chemical Neuroscience simultaneously. The Lymnaea model has had a considerable impact on the development of bionic hybrid devices, and their use in enabling fundamental discoveries in the field of neuroscience. For example, the experimental advantages afforded by Lymnaea neurons were key for the success of the first proof-ofprinciple experiment coupling a silicon chip with the excitatory chemical synapse between VD4-LPeD1 neurons, achieving a “neuro-chip” interface. Here, using a conventional capacitortransistor array approach to stimulate neurons and record activity, respectively, stimulation of VD4 elicited by the capacitor evoked postsynaptic excitation in LPeD1, recorded as field potentials by the transistor.88 Moreover, the ability to detect activity-dependent synaptic plasticity indicated that the semiconductor chip reliably induced and appropriately recorded the bioelectronic features of neuronal works. The interfacing of Lymnaea neurons with multielectrode array (MEA) devices has also enabled the detailed study of the role of NTFs in eliciting specific changes in the frequency and variance of spontaneous activity patterns.89 The emergence of stereotypical activity patterns that could be classified into unique “signatures” was found to have the power to predict whether or not individual LPeD1 neurons would exhibit functional expression of excitatory or inhibitory nAChRs, and whether excitatory synapses would form between VD4−LPeD1 pairs. This discovery, afforded by the ability to perform noninvasive long-term recordings using bionic hybrids, demonstrated the importance of long-term patterned spontaneous activity for the appropriate development and function of synapses. Together with the development of machine learning algorithms, this insight may ultimately lead to new opportunities for the development of models to understand how specific activity-dependent mechanisms serve as inductive signals during synaptogenesis. Technological breakthroughs in micro- and nanoscale fabrication processes have contributed to the development of neuroelectronic hybrids, including patch-clamp chips,90−92 planar and three-dimensional MEAs,93 that can more accurately record activity at the single-cell resolution. In particular, the advancement of MEA technology with the ability to locally switch between stimulating and recording neuronal activity now allows many major types of electrophysiological experiments to be conducted noninvasively, although the resolution (signal-tonoise ratio; SNR) is significantly lower than traditional intracellular techniques. Using Lymnaea neurons as a model, we have recently demonstrated that designing neuroelectronic devices that biomimic cellular architecture could enhance the recording capability of MEAs in terms of both signal resolution and recording stability. The use of Lymnaea neurons supported the development of computational models required to understand electrophysical interfaces between firing neurons and microelectrodes,94 which were applied to the development of a “nanoedge” planar MEA that enabled high-fidelity recordings with a 15-fold improvement of the SNR95 (Figure 3). These realizations have subsequently been translated to the development of related devices that enable the study of more complex neuronal networks from mammalian neuron culture and tissue slice preparations (Wijdenes et al., manuscript in preparation), thus offering new opportunities to examine advanced neuronal phenomena such as network formation and dysfunction.

understand the incredible structural and functional complexity of the brain and its capacity for flexibility, it is imperative to elucidate the cellular and molecular mechanisms responsible for assembling synaptic networks and establishing the molecular specificity of individual pre- and postsynaptic structures. However, the complexity of the brain and behavior of vertebrate animals makes it difficult to approach the underlying synaptic mechanisms, especially at a single-cell resolution. Reductionist approaches using the simple nervous systems of invertebrate model organisms as an experimental tool to unravel the basic tenants of neural and synaptic physiology have enabled significant progress in identifying these evasive mechanisms. From exploring fundamental concepts in neurobiology to developing novel electrophysiology biosensors and neurochips, the experimental advantages of the Lymnaea model system have enabled researchers to bridge numerous gaps in our understanding of how single genes, individual neurons, and their synapses interact to produce simple behaviors, and are modulated to ensure synaptic network function is matched to the behavioral requirements of an animal.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Naweed I. Syed: 0000-0002-6124-9745 Present Address ∥

A.M.G.: Interdisciplinary Institute for Neuroscience, University of Bordeaux, CNRS, UMR 5297, F-33000, Bordeaux, France. Author Contributions

A.M.G., P.W., and S.R. wrote and edited the manuscript. A.G. and P.W. generated the figures. N.I.S. contributed to the organization and suggestions on content and edited the manuscript. Funding

This work was supported by Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada (NSERC) grants to N.I.S. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. Tara A. Janes for contributions to figures and thoughtful discussions on the manuscript.

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ABBREVIATIONS ACh, acetylcholine; AChBP, acetylcholine binding protein; CM, conditioned media, NTF-rich; CNS, central nervous system; CPG, central pattern generator; DM, defined medial, NTFdeficient; ERK, extracellular signal-regulated kinase; IP3I, input 3 interneuron; L-EGF, Lymnaea epidermal growth factor homologue; L-EGFR, Lymnaea epidermal growth factor receptor tyrosine kinase homologue; L-nAChR, Lymnaea nAChR homologue; L-MEN1, Lymnaea MEN1 homologue; LPeD1, left pedal dorsal 1 neuron; MAPK, mitogen activated protein kinase; MEA, multielectrode array; MEN1, multiple endocrine neoplasia type 1 tumor suppressor gene; nAChR, nicotinic acetylcholine receptor; NTF, neurotrophic factor; PIR, postinhibitory rebound; PCR, polymerase chain reaction; qPCR, quantitative PCR; RNAi, RNA interference; RPeD1, right pedal

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CONCLUSIONS Synapses form the foundation of behavior and, through plastic remodeling, enable animals to appropriately adapt their behavior to constantly changing environmental conditions. In order to H

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ACS Chemical Neuroscience

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dorsal 1 neuron; RTK, receptor tyrosine kinase; shRNA, smallhairpin RNA; SNR, signal-to-noise ratio; VD4, visceral dorsal 4 interneuron; VGCC, voltage-gated calcium channel; VJ, visceral J group motor neuron; VK, visceral K group motor neuron

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