A Continuum of Response Properties across the Population of Unipolar Brush Cells in the Dorsal Cochlear Nucleus.

Unipolar brush cells (UBCs) in the cerebellum and dorsal cochlear nucleus (DCN) perform temporal transformations by converting brief mossy fiber bursts into long-lasting responses. In the cerebellar UBC population, mixing inhibition with graded mGluR1-dependent excitation leads to a continuum of temporal responses. In the DCN, it has been thought that mGluR1 contributes little to mossy fiber responses and that there are distinct excitatory and inhibitory UBC subtypes. Here, we investigate UBC response properties using noninvasive cell-attached recordings in the DCN of mice of either sex. We find a continuum of responses to mossy fiber bursts ranging from 100 ms excitation to initial inhibition followed by several seconds of excitation to inhibition lasting for hundreds of milliseconds. Pharmacological interrogation reveals excitatory responses are primarily mediated by mGluR1 Thus, UBCs in both the DCN and cerebellum rely on mGluR1 and have a continuum of response durations. The continuum of responses in the DCN may allow more flexible and efficient temporal processing than can be achieved with distinct excitatory and inhibitory populations.SIGNIFICANCE STATEMENT UBCs are specialized excitatory interneurons in cerebellar-like structures that greatly prolong the temporal responses of mossy fiber inputs. They are thought to help cancel out self-generated signals. In the DCN, the prevailing view was that there are two distinct ON and OFF subtypes of UBCs. Here, we show that instead the UBC population has a continuum of response properties. Many cells show suppression and excitation consecutively, and the response durations vary considerably. mGluR1s are crucial in generating a continuum of responses. To understand how UBCs contribute to temporal processing, it is essential to consider the continuous variations of UBC responses, which have advantages over just having opposing ON/OFF subtypes of UBCs.

Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1.

Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal-regulated kinase (ERK) that mediates CB1R- and mGluR2/3-induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock-induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18-1. Mimicking constitutive phosphorylation of Munc18-1 results in a drastic decrease in synaptic transmission. ERK-mediated phosphorylation of Munc18-1 ultimately leads to degradation by the ubiquitin-proteasome system. Conversely, preventing ERK-dependent Munc18-1 phosphorylation increases synaptic strength. CB1R- and mGluR2/3-induced synaptic inhibition and depolarization-induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK-dependent Munc18-1 phosphorylation is blocked. Thus, ERK-dependent Munc18-1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.

Graded heterogeneity of metabotropic signaling underlies a continuum of cell-intrinsic temporal responses in unipolar brush cells.

Many neuron types consist of populations with continuously varying molecular properties. Here, we show a continuum of postsynaptic molecular properties in three types of neurons and assess the functional correlates in cerebellar unipolar brush cells (UBCs). While UBCs are generally thought to form discrete functional subtypes, with mossy fiber (MF) activation increasing firing in ON-UBCs and suppressing firing in OFF-UBCs, recent work also points to a heterogeneity of response profiles. Indeed, we find a continuum of response profiles that reflect the graded and inversely correlated expression of excitatory mGluR1 and inhibitory mGluR2/3 pathways. MFs coactivate mGluR2/3 and mGluR1 in many UBCs, leading to sequential inhibition-excitation because mGluR2/3-currents are faster. Additionally, we show that DAG-kinase controls mGluR1 response duration, and that graded DAG kinase levels correlate with systematic variation of response duration over two orders of magnitude. These results demonstrate that continuous variations in metabotropic signaling can generate a stable cell-autonomous basis for temporal integration and learning over multiple time scales.

Diverse roles of Synaptotagmin-7 in regulating vesicle fusion.

Synaptotagmin 7 (Syt7) is a multifunctional calcium sensor expressed throughout the body. Its high calcium affinity makes it well suited to act in processes triggered by modest calcium increases within cells. In synaptic transmission, Syt7 has been shown to mediate asynchronous neurotransmitter release, facilitation, and vesicle replenishment. In this review we provide an update on recent developments, and the newly emerging roles of Syt7 in frequency invariant synaptic transmission and in suppressing spontaneous release. Additionally, we discuss Syt7's regulation of membrane fusion in non-neuronal cells, and its involvement in disease. How such diversity of functions is regulated remains an open question. We discuss several potential factors including temperature, presynaptic calcium signals, the localization of Syt7, and its interaction with other Syt isoforms.

Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1.

Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca2+-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca2+-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1D232N) or increasing its hydrophobicity (Syt14W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt19Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1-independent mechanisms that lower the energy barrier and act additively with Syt1-dependent mechanisms to produce PTP by exerting multiplicative effects on release rates.

Publisher Correction: Synaptotagmin-1 enables frequency coding by suppressing asynchronous release in a temperature dependent manner.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Synaptotagmin-1 enables frequency coding by suppressing asynchronous release in a temperature dependent manner.

To support frequency-coded information transfer, mammalian synapses tightly synchronize neurotransmitter release to action potentials (APs). However, release desynchronizes during AP trains, especially at room temperature. Here we show that suppression of asynchronous release by Synaptotagmin-1 (Syt1), but not release triggering, is highly temperature sensitive, and enhances synchronous release during high-frequency stimulation. In Syt1-deficient synapses, asynchronous release increased with temperature, opposite to wildtype synapses. Mutations in Syt1 C2B-domain polybasic stretch (Syt1 K326Q,K327Q,K331Q) did not affect synchronization during sustained activity, while the previously observed reduced synchronous response to a single AP was confirmed. However, an inflexible linker between the C2-domains (Syt1 9Pro) reduced suppression, without affecting synchronous release upon a single AP. Syt1 9Pro expressing synapses showed impaired synchronization during AP trains, which was rescued by buffering global Ca2+ to prevent asynchronous release. Hence, frequency coding relies on Syt1's temperature sensitive suppression of asynchronous release, an aspect distinct from its known vesicle recruitment and triggering functions.

Vti1a/b regulate synaptic vesicle and dense core vesicle secretion via protein sorting at the Golgi.

The SNAREs Vti1a/1b are implicated in regulated secretion, but their role relative to canonical exocytic SNAREs remains elusive. Here, we show that synaptic vesicle and dense-core vesicle (DCV) secretion is indeed severely impaired in Vti1a/b-deficient neurons. The synaptic levels of proteins that mediate secretion were reduced, down to 50% for the exocytic SNARE SNAP25. The delivery of SNAP25 and DCV-cargo into axons was decreased and these molecules accumulated in the Golgi. These defects were rescued by either Vti1a or Vti1b expression. Distended Golgi cisternae and clear vacuoles were observed in Vti1a/b-deficient neurons. The normal non-homogeneous distribution of DCV-cargo inside the Golgi was lost. Cargo trafficking out of, but not into the Golgi, was impaired. Finally, retrograde Cholera Toxin trafficking, but not Sortilin/Sorcs1 distribution, was compromised. We conclude that Vti1a/b support regulated secretion by sorting secretory cargo and synaptic secretion machinery components at the Golgi.

Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate.

The energy required to fuse synaptic vesicles with the plasma membrane ('activation energy') is considered a major determinant in synaptic efficacy. From reaction rate theory, we predict that a class of modulations exists, which utilize linear modulation of the energy barrier for fusion to achieve supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by hypertonic solutions. We show that complexinI/II deficiency or phorbol ester stimulation indeed affects responses to hypertonic solution in a supralinear manner. An additive vs multiplicative relationship between activation energy and fusion rate provides a novel explanation for previously observed non-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca(2+)-dependent release.