Taurine Promotes Neurite Outgrowth and Synapse Development of Both Vertebrate and Invertebrate Central Neurons.

Taurine is a sulfur-containing amino acid that is widely expressed throughout the human brain, heart, retina, and muscle tissues. Taurine deficiency is associated with cardiomyopathy, renal dysfunction, abnormalities of the developing nervous system, and epilepsy which suggests a role specific to excitable tissues. Like vertebrates, invertebrates maintain high levels of taurine during embryonic and larval development, which decline during aging, indicating a potential developmental role. Notwithstanding its extensive presence throughout, taurine's precise role/s during early brain development, function, and repair remains largely unknown in both vertebrate and invertebrate. Here, we investigated whether taurine affects neurite outgrowth, synapse formation, and synaptic transmission between postnatal day 0 rat cortical neurons in vitro, whereas its synaptogenic role was tested more directly using the Lymnaea soma-soma synapse model. We provide direct evidence that when applied at physiological concentrations, taurine exerts a significant neurotrophic effect on neuritic outgrowth and thickness of neurites as well as the expression of synaptic puncta as revealed by immunostaining of presynaptic synaptophysin and postsynaptic PSD95 proteins in rat cortical neurons, indicating direct involvement in synapse development. To demonstrate taurine's direct effects on neurons in the absence of glia and other confounding factors, we next exploited individually identified pre- and postsynaptic neurons from the mollusk Lymnaea stagnalis. We found that taurine increased both the incidence of synapse formation (percent of cells that form synapses) and the efficacy of synaptic transmission between the paired neurons. This effect was comparable, but not additive, to Lymnaea trophic factor-induced synaptogenesis. This study thus provides direct morphological and functional evidence that taurine plays an important role in neurite outgrowth, synaptogenesis, and synaptic transmission during the early stages of brain development and that this role is conserved across both vertebrate and invertebrate species.

Neurotrophic factors and target-specific retrograde signaling interactions define the specificity of classical and neuropeptide cotransmitter release at identified Lymnaea synapses.

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.

A novel bio-mimicking, planar nano-edge microelectrode enables enhanced long-term neural recording.

Our inability to accurately monitor individual neurons and their synaptic activity precludes fundamental understanding of brain function under normal and various pathological conditions. However, recent breakthroughs in micro- and nano-scale fabrication processes have advanced the development of neuro-electronic hybrid technology. Among such devices are three-dimensional and planar electrodes, offering the advantages of either high fidelity or longer-term recordings respectively. Here, we present the next generation of planar microelectrode arrays with "nano-edges" that enable long-term (≥1 month) and high fidelity recordings at a resolution 15 times higher than traditional planar electrodes. This novel technology enables better understanding of brain function and offers a tremendous opportunity towards the development of future bionic hybrids and drug discovery devices.

Two proteolytic fragments of menin coordinate the nuclear transcription and postsynaptic clustering of neurotransmitter receptors during synaptogenesis between Lymnaea neurons.

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.

Trophic factor-induced activity 'signature' regulates the functional expression of postsynaptic excitatory acetylcholine receptors required for synaptogenesis.

Highly coordinated and coincidental patterns of activity-dependent mechanisms ("fire together wire together") are thought to serve as inductive signals during synaptogenesis, enabling neuronal pairing between specific sub-sets of excitatory partners. However, neither the nature of activity triggers, nor the "activity signature" of long-term neuronal firing in developing/regenerating neurons have yet been fully defined. Using a highly tractable model system comprising of identified cholinergic neurons from Lymnaea, we have discovered that intrinsic trophic factors present in the Lymnaea brain-conditioned medium (CM) act as a natural trigger for activity patterns in post- but not the presynaptic neuron. Using microelectrode array recordings, we demonstrate that trophic factors trigger stereotypical activity patterns that include changes in frequency, activity and variance. These parameters were reliable indicators of whether a neuron expressed functional excitatory or inhibitory nAChRs and synapse formation. Surprisingly, we found that the post- but not the presynaptic cell exhibits these changes in activity patterns, and that the functional expression of excitatory nAChRs required neuronal somata, de novo protein synthesis and voltage gated calcium channels. In summary, our data provides novel insights into trophic factor mediated actions on neuronal activity and its specific regulation of nAChR expression.

Neuronal somata and extrasomal compartments play distinct roles during synapse formation between Lymnaea neurons.

Proper synapse formation is pivotal for all nervous system functions. However, the precise mechanisms remain elusive. Moreover, compared with the neuromuscular junction, steps regulating the synaptogenic program at central cholinergic synapses remain poorly defined. In this study, we identified different roles of neuronal compartments (somal vs extrasomal) in chemical and electrical synaptogenesis. Specifically, the electrically synapsed Lymnaea pedal dorsal A cluster neurons were used to study electrical synapses, whereas chemical synaptic partners, visceral dorsal 4 (presynaptic, cholinergic), and left pedal dorsal 1 (LPeD1; postsynaptic) were explored for chemical synapse formation. Neurons were cultured in a soma-soma or soma-axon configuration and synapses explored electrophysiologically. We provide the first direct evidence that electrical synapses develop in a soma-soma, but not soma-axon (removal of soma) configuration, indicating the requirement of gene transcription regulation in the somata of both synaptic partners. In addition, the soma-soma electrical coupling was contingent upon trophic factors present in Lymnaea brain-conditioned medium. Further, we demonstrate that chemical (cholinergic) synapses between soma-soma and soma-axon pairs were indistinguishable, with both exhibiting a high degree of contact site and target cell type specificity. We also provide direct evidence that presynaptic cell contact-mediated, clustering of postsynaptic cholinergic receptors at the synaptic site requires transmitter-receptor interaction, receptor internalization, and a protein kinase C-dependent lateral migration toward the contact site. This study provides novel insights into synaptogenesis between central neurons revealing both distinct and synergistic roles of cell-cell signaling and extrinsic trophic factors in executing the synaptogenic program.

A planar microelectrode array for simultaneous detection of electrically evoked dopamine release from distinct locations of a single isolated neuron.

Neurotransmission is a key process of communication between neurons. Although much is known about this process and the influence it has on the function of the body, little is understood about the dynamics of signalling from structural regions of a single neuron. In this study we have fabricated and characterised a microelectrode array (MEA) which was utilised for simultaneous multi-site recordings of dopamine release from an isolated single neuron. The MEA consisted of gold electrodes that were created in plane with the insulation layer using a chemical mechanical planarization process. The detection limit for dopamine measurements was 11 ± 3 nM and all the gold electrodes performed in a consistent fashion during amperometric recordings of 100 nM dopamine. Fouling of the gold electrode was investigated, where no significant change in the current was observed over 4 hours when monitoring 100 nM dopamine. The MEA was accessed using freshly isolated dopaminergic somas from the pond snail, Lymnaea stagnalis, where electrically evoked dopamine release was clearly observed. Measurements were conducted at four structural locations of a single isolated neuron, where electrically evoked dopamine release was observed from the cell body, axonal regions and the terminal. Over time, the release of dopamine varied over the structural regions of the neuron. Such information can provide an insight into the signalling mechanism of neurons and how they potentially form synaptic connections.

The antidepressant fluoxetine but not citalopram suppresses synapse formation and synaptic transmission between Lymnaea neurons by perturbing presynaptic and postsynaptic machinery.

Depression is a debilitating mental disorder, and selective serotonin reuptake inhibitors (SSRIs) constitute the first-line antidepressant treatment choice for the clinical management of this illness; however, the mechanisms underlying their therapeutic actions and side effects remain poorly understood. Here, we compared the effects of two SSRIs, fluoxetine and citalopram, on synaptic connectivity and the efficacy of cholinergic synaptic transmission between identified presynaptic and postsynaptic neurons from the mollusc Lymnaea. The in vitro paired cells were exposed to clinically relevant concentrations of the two SSRIs under chronic and acute experimental conditions, and the incidence of synapse formation and the efficacy of synaptic transmission were tested electrophysiologically and with fluorescent Ca(2+) imaging. We demonstrate that chronic exposure to fluoxetine, but not to citalopram, inhibits synapse formation and reduces synaptic strength, and that these effects are reversible following prolonged drug washout. At the structural level, we demonstrate that fluoxetine, but not citalopram, prevents the expression and localization of the presynaptic protein synaptophysin. Acute exposure to fluoxetine substantially reduced synaptic transmission and synaptic plasticity (post-tetanic potentiation) in established synapses, whereas citalopram reduced synaptic transmission, but not short-term synaptic plasticity. We further demonstrate that fluoxetine, but not citalopram, directly inhibits voltage-gated Ca(2+) currents in the presynaptic neuron, as well as postsynaptic responsiveness to exogenously applied neurotransmitter. This study provides the first direct evidence that fluoxetine and citalopram exert characteristic, non-specific side effects that are unrelated to their function as SSRIs, and that fluoxetine is more detrimental to synaptic physiology and structure than citalopram.

Antidepressant fluoxetine suppresses neuronal growth from both vertebrate and invertebrate neurons and perturbs synapse formation between Lymnaea neurons.

Current treatment regimes for a variety of mental disorders involve various selective serotonin reuptake inhibitors such as Fluoxetine (Prozac). Although these drugs may 'manage' the patient better, there has not been a significant change in the treatment paradigm over the years and neither have the outcomes improved. There is also considerable debate as to the effectiveness of various selective serotonin reuptake inhibitors and their potential side-effects on neuronal architecture and function. In this study, using mammalian cortical neurons, a dorsal root ganglia cell line (F11 cells) and identified Lymnaea stagnalis neurons, we provide the first direct and unequivocal evidence that clinically relevant concentrations of Fluoxetine induce growth cone collapse and neurite retraction of both serotonergic and non-serotonergic neurons alike in a dose-dependent manner. Using intracellular recordings and calcium imaging techniques, we further demonstrate that the mechanism underlying Fluoxetine-induced effects on neurite retraction from Lymnaea neurons may involve lowering of intracellular calcium and a subsequent retardation of growth cone cytoskeleton. Using soma-soma synapses between identified presynaptic and postsynaptic Lymnaea neurons, we provide further direct evidence that clinically used concentrations of Fluoxetine also block synaptic transmission and synapse formation between cholinergic neurons. Our study raises alarms over potentially devastating side-effects of this antidepressant drug on neurite outgrowth and synapse formation in a developing/regenerating brain. Our data also demonstrate that drugs such as Fluoxetine may not just affect communication between serotonergic neurons but that the detrimental effects are widespread and involve neurons of various phenotypes from both vertebrate and invertebrate species.