Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release.

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.

Molecular Basis for Synaptotagmin-1-Associated Neurodevelopmental Disorder.

At neuronal synapses, synaptotagmin-1 (syt1) acts as a Ca2+ sensor that synchronizes neurotransmitter release with Ca2+ influx during action potential firing. Heterozygous missense mutations in syt1 have recently been associated with a severe but heterogeneous developmental syndrome, termed syt1-associated neurodevelopmental disorder. Well-defined pathogenic mechanisms, and the basis for phenotypic heterogeneity in this disorder, remain unknown. Here, we report the clinical, physiological, and biophysical characterization of three syt1 mutations from human patients. Synaptic transmission was impaired in neurons expressing mutant variants, which demonstrated potent, graded dominant-negative effects. Biophysical interrogation of the mutant variants revealed novel mechanistic features concerning the cooperative action, and functional specialization, of the tandem Ca2+-sensing domains of syt1. These mechanistic studies led to the discovery that a clinically approved K+ channel antagonist is able to rescue the dominant-negative heterozygous phenotype. Our results establish a molecular cause, basis for phenotypic heterogeneity, and potential treatment approach for syt1-associated neurodevelopmental disorder.

Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin.

Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

Excitatory and Inhibitory Neurons Utilize Different Ca2+ Sensors and Sources to Regulate Spontaneous Release.

Spontaneous neurotransmitter release (mini) is an important form of Ca2+-dependent synaptic transmission that occurs in the absence of action potentials. A molecular understanding of this process requires an identification of the underlying Ca2+ sensors. Here, we address the roles of the relatively low- and high-affinity Ca2+ sensors, synapotagmin-1 (syt1) and Doc2α/ß, respectively. We found that both syt1 and Doc2 regulate minis, but, surprisingly, their relative contributions depend on whether release was from excitatory or inhibitory neurons. Doc2α promoted glutamatergic minis, while Doc2ß and syt1 both regulated GABAergic minis. We identified Ca2+ ligand mutations in Doc2 that either disrupted or constitutively activated the regulation of minis. Finally, Ca2+ entry via voltage-gated Ca2+ channels triggered miniature GABA release by activating syt1, but had no effect on Doc2-driven minis. This work reveals an unexpected divergence in the regulation of spontaneous excitatory and inhibitory transmission in terms of both Ca2+ sensors and sources.

Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties.

The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores and enabled the escape of larger cargoes. To determine whether this effect was due to changes in nascent pore size or to changes in stability, we developed an approach that uses nanodiscs and planar lipid bilayer electrophysiology to afford microsecond resolution at the single event level. Both pore size and stability were affected by SNARE copy number. Increasing the number of vesicle (v)-SNAREs per nanodisc from three to five caused a twofold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, pairing of v-SNAREs and target (t)-SNAREs to form trans-SNARE complexes was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, which mimics the action of botulinum neurotoxin A, markedly reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determines fundamental properties of individual pores.

Mechanisms of 5-HT1A receptor-mediated transmission in dorsal raphe serotonin neurons.

KEY POINTS: In the dorsal raphe nucleus, it is known that serotonin release activates metabotropic 5-HT1A autoreceptors located on serotonin neurons that leads to an inhibition of firing through the activation of G-protein-coupled inwardly rectifying potassium channels. We found that in mouse brain slices evoked serotonin release produced a 5-HT1A receptor-mediated inhibitory postsynaptic current (IPSC) that resulted in only a transient pause in firing. While spillover activation of receptors contributed to evoked IPSCs, serotonin reuptake transporters prevented pooling of serotonin in the extrasynaptic space from activating 5-HT1A -IPSCs. As a result, the decay of 5-HT1A -IPSCs was independent of the intensity of stimulation or the probability of transmitter release. These results indicate that evoked serotonin transmission in the dorsal raphe nucleus mediated by metabotropic 5-HT1A autoreceptors may occur via point-to-point synapses rather than by paracrine mechanisms. ABSTRACT: In the dorsal raphe nucleus (DRN), feedback activation by Gαi/o -coupled 5-HT1A autoreceptors reduces the excitability of serotoninergic neurons, which decreases serotonin release both locally within the DRN and in projection regions. Serotonin transmission within the DRN is thought to occur via transmitter spillover and paracrine activation of extrasynaptic receptors. Here, we tested the volume transmission hypothesis in mouse DRN brain slices by recording 5-HT1A receptor-mediated inhibitory postsynaptic currents (5-HT1A -IPSCs) generated by the activation of G-protein-coupled inwardly rectifying potassium channels (GIRKs). We found that in the DRN of ePET1-EYFP mice, which selectively express enhanced yellow fluorescent protein in serontonergic neurons, the local release of serotonin generated 5-HT1A -IPSCs in serotonin neurons that rose and fell within a second. The transient activation of 5-HT1A autoreceptors resulted in brief pauses in neuron firing that did not alter the overall firing rate. The duration of 5-HT1A -IPSCs was primarily shaped by receptor deactivation due to clearance via serotonin reuptake transporters. Slowing diffusion with dextran prolonged the rise and reduced the amplitude the IPSCs and the effects were potentiated when uptake was inhibited. By examining the decay kinetics of IPSCs, we found that while spillover may allow for the activation of extrasynaptic receptors, efficient uptake by serotonin reuptake transporters (SERTs) prevented the pooling of serotonin from prolonging the duration of transmission when multiple inputs were active. Together the results suggest that the activation of 5-HT1A receptors in the DRN results from the local release of serotonin rather than the extended diffusion throughout the extracellular space.

Neurotensin Induces Presynaptic Depression of D2 Dopamine Autoreceptor-Mediated Neurotransmission in Midbrain Dopaminergic Neurons.

Increased dopaminergic signaling is a hallmark of severe mesencephalic pathologies such as schizophrenia and psychostimulant abuse. Activity of midbrain dopaminergic neurons is under strict control of inhibitory D2 autoreceptors. Application of the modulatory peptide neurotensin (NT) to midbrain dopaminergic neurons transiently increases activity by decreasing D2 dopamine autoreceptor function, yet little is known about the mechanisms that underlie long-lasting effects. Here, we performed patch-clamp electrophysiology and fast-scan cyclic voltammetry in mouse brain slices to determine the effects of NT on dopamine autoreceptor-mediated neurotransmission. Application of the active peptide fragment NT8-13 produced synaptic depression that exhibited short- and long-term components. Sustained depression of D2 autoreceptor signaling required activation of the type 2 NT receptor and the protein phosphatase calcineurin. NT application increased paired-pulse ratios and decreased extracellular levels of somatodendritic dopamine, consistent with a decrease in presynaptic dopamine release. Surprisingly, we observed that electrically induced long-term depression of dopaminergic neurotransmission that we reported previously was also dependent on type 2 NT receptors and calcineurin. Because electrically induced depression, but not NT-induced depression, was blocked by postsynaptic calcium chelation, our findings suggest that endogenous NT may act through a local circuit to decrease presynaptic dopamine release. The current research provides a mechanism through which augmented NT release can produce a long-lasting increase in membrane excitability of midbrain dopamine neurons. SIGNIFICANCE STATEMENT: Whereas plasticity of glutamate synapses in the brain has been studied extensively, demonstrations of plasticity at dopaminergic synapses have been more elusive. By quantifying inhibitory neurotransmission between midbrain dopaminergic neurons in brain slices from mice we have discovered that the modulatory peptide neurotensin can induce a persistent synaptic depression by decreasing dopamine release. This depression of inhibitory synaptic input would be expected to increase excitability of dopaminergic neurons. Induction of the plasticity can be pharmacologically blocked by antagonists of either the protein phosphatase calcineurin or neurotensin receptors, and persists surprisingly long after a brief exposure to the peptide. Since neurotensin-dopamine interactions have been implicated in hyperdopaminergic pathologies, these findings describe a synaptic mechanism that could contribute to addiction and/or schizophrenia.

The timing of dopamine- and noradrenaline-mediated transmission reflects underlying differences in the extent of spillover and pooling.

Metabotropic transmission typically occurs through the spillover activation of extrasynaptic receptors. This study examined the mechanisms underlying somatodendritic dopamine and noradrenaline transmission and found that the extent of spillover and pooling varied dramatically between these two transmitters. In the mouse ventral tegmental area, the time course of D2-receptor-mediated IPSCs (D2-IPSCs) was consistent between cells and was unaffected by altering stimulation intensity, probability of release, or the extent of diffusion. Blocking dopamine reuptake with cocaine extended the time course of D2-IPSCs and suggested that transporters strongly limited spillover. As a result, individual release sites contributed independently to the duration of D2-IPSCs. In contrast, increasing the release of noradrenaline in the rat locus ceruleus prolonged the duration of α2-receptor-mediated IPSCs even when reuptake was intact. Spillover and subsequent pooling of noradrenaline activated distal α2-receptors, which prolonged the duration of α2-IPSCs when multiple release sites were activated synchronously. By using the rapid application of agonists onto large macropatches, we determined the concentration profile of agonists underlying the two IPSCs. Incorporating the results into a model simulating extracellular diffusion predicted that the functional range of noradrenaline diffusion was nearly fivefold greater in the locus ceruleus than dopamine in the midbrain. This study demonstrates that catecholamine synapses differentially regulate the extent of spillover and pooling to control the timing of local inhibition and suggests diversity in the roles of uptake and diffusion in governing metabotropic transmission.

Species differences in somatodendritic dopamine transmission determine D2-autoreceptor-mediated inhibition of ventral tegmental area neuron firing.

The somatodendritic release of dopamine within the ventral tegmental area (VTA) and substantia nigra pars compacta activates inhibitory postsynaptic D2-receptors on dopaminergic neurons. The proposed mechanisms that regulate this form of transmission differ between electrochemical studies using rats and guinea pigs and electrophysiological studies using mice. This study examines the release and resulting dopamine D2-autoreceptor-mediated IPSCs (D2-IPSCs) in the VTA of mouse, rat, and guinea pig. Robust D2-IPSCs were observed in all recordings from neurons in slices taken from mouse, whereas D2-IPSCs in rat and guinea pig were observed less frequently and were significantly smaller in amplitude. In slices taken from guinea pig, dopamine release was more persistent under conditions of reduced extracellular calcium. The decline in the concentration of dopamine was also prolonged and not as sensitive to inhibition of reuptake by cocaine. This resulted in an increased duration of D2-IPSCs in the guinea pig. Therefore, unlike the mouse or the rat, the time course of dopamine in the extracellular space of the guinea pig determined the duration the D2-IPSC. Functionally, differences in D2-IPSCs resulted in inhibition of dopamine neuron firing only in slices from mouse. The results suggest that the mechanisms and functional consequences of somatodendritic dopamine transmission in the VTA vary among species. This highlights the complexity that underlies dopamine-dependent transmission in one brain area. Differences in somatodendritic transmission would be expected in vivo to affect the downstream activity of the mesocorticolimbic dopamine system and subsequent terminal release.