Skyler L. Jackman

The calcium sensor synaptotagmin 7 is required for synaptic facilitation

It has been known for more than 70 years that synaptic strength is dynamically regulated in a use-dependent manner. At synapses with a low initial release probability, closely spaced presynaptic action potentials can result in facilitation, a short-term form of enhancement in which each subsequent action potential evokes greater neurotransmitter release. Facilitation can enhance neurotransmitter release considerably and can profoundly influence information transfer across synapses, but the underlying mechanism remains a mystery. One proposed mechanism is that a specialized calcium sensor for facilitation transiently increases the probability of release, and this sensor is distinct from the fast sensors that mediate rapid neurotransmitter release. Yet such a sensor has never been identified, and its very existence has been disputed. Here we show that synaptotagmin 7 (Syt7) is a calcium sensor that is required for facilitation at several central synapses. In Syt7-knockout mice, facilitation is eliminated even though the initial probability of release and the presynaptic residual calcium signals are unaltered. Expression of wild-type Syt7 in presynaptic neurons restored facilitation, whereas expression of a mutated Syt7 with a calcium-insensitive C2A domain did not. By revealing the role of Syt7 in synaptic facilitation, these results resolve a longstanding debate about a widespread form of short-term plasticity, and will enable future studies that may lead to a deeper understanding of the functional importance of facilitation.

The Mechanisms and Functions of Synaptic Facilitation

The ability of the brain to store and process information relies on changing the strength of connections between neurons. Synaptic facilitation is a form of short-term plasticity that enhances synaptic transmission for less than a second. Facilitation is a ubiquitous phenomenon thought to play critical roles in information transfer and neural processing. Yet our understanding of the function of facilitation remains largely theoretical. Here we review proposed roles for facilitation and discuss how recent progress in uncovering the underlying molecular mechanisms could enable experiments that elucidate how facilitation, and short-term plasticity in general, contributes to circuit function and animal behavior.

Release from the cone ribbon synapse under bright light conditions can be controlled by the opening of only a few Ca(2+) channels.

Light hyperpolarizes cone photoreceptors, causing synaptic voltage-gated Ca(2+) channels to open infrequently. To understand neurotransmission under these conditions, we determined the number of L-type Ca(2+) channel openings necessary for vesicle fusion at the cone ribbon synapse. Ca(2+) currents (I(Ca)) were activated in voltage-clamped cones, and excitatory postsynaptic currents (EPSCs) were recorded from horizontal cells in the salamander retina slice preparation. Ca(2+) channel number and single-channel current amplitude were calculated by mean-variance analysis of I(Ca). Two different comparisons-one comparing average numbers of release events to average I(Ca) amplitude and the other involving deconvolution of both EPSCs and simultaneously recorded cone I(Ca)-suggested that fewer than three Ca(2+) channel openings accompanied fusion of each vesicle at the peak of release during the first few milliseconds of stimulation. Opening fewer Ca(2+) channels did not enhance fusion efficiency, suggesting that few unnecessary channel openings occurred during strong depolarization. We simulated release at the cone synapse, using empirically determined synaptic dimensions, vesicle pool size, Ca(2+) dependence of release, Ca(2+) channel number, and Ca(2+) channel properties. The model replicated observations when a barrier was added to slow Ca(2+) diffusion. Consistent with the presence of a diffusion barrier, dialyzing cones with diffusible Ca(2+) buffers did not affect release efficiency. The tight clustering of Ca(2+) channels, along with a high-Ca(2+) affinity release mechanism and diffusion barrier, promotes a linear coupling between Ca(2+) influx and vesicle fusion. This may improve detection of small light decrements when cones are hyperpolarized by bright light.

Regulation of presynaptic strength by controlling Ca2+ channel mobility: effects of cholesterol depletion on release at the cone ribbon synapse

Synaptic communication requires proper coupling between voltage-gated Ca(2+) (Ca(V)) channels and synaptic vesicles. In photoreceptors, L-type Ca(V) channels are clustered close to synaptic ribbon release sites. Although clustered, Ca(V) channels move continuously within a confined domain slightly larger than the base of the ribbon. We hypothesized that expanding Ca(V) channel confinement domains should increase the number of channel openings needed to trigger vesicle release. Using single-particle tracking techniques, we measured the expansion of Ca(V) channel confinement domains caused by depletion of membrane cholesterol with cholesterol oxidase or methyl-β-cyclodextrin. With paired whole cell recordings from cones and horizontal cells, we then determined the number of Ca(V) channel openings contributing to cone Ca(V) currents (I(Ca)) and the number of vesicle fusion events contributing to horizontal cell excitatory postsynaptic currents (EPSCs) following cholesterol depletion. Expansion of Ca(V) channel confinement domains reduced the peak efficiency of release, decreasing the number of vesicle fusion events accompanying opening of each Ca(V) channel. Cholesterol depletion also inhibited exocytotic capacitance increases evoked by brief depolarizing steps. Changes in efficiency were not due to changes in I(Ca) amplitude or glutamate receptor properties. Replenishing cholesterol restored Ca(V) channel domain size and release efficiency to control levels. These results indicate that cholesterol is important for organizing the cone active zone. Furthermore, the finding that cholesterol depletion impairs coupling between channel opening and vesicle release by allowing Ca(V) channels to move further from release sites shows that changes in presynaptic Ca(V) channel mobility can be a mechanism for adjusting synaptic strength.

Synaptotagmin 7 confers frequency invariance onto specialized depressing synapses

At most synapses in the brain, short-term plasticity dynamically modulates synaptic strength. Rapid frequency-dependent changes in synaptic strength have key roles in sensory adaptation, gain control and many other neural computations. However, some auditory, vestibular and cerebellar synapses maintain constant strength over a wide range of firing frequencies, and as a result efficiently encode firing rates. Despite its apparent simplicity, frequency-invariant transmission is difficult to achieve because of inherent synaptic nonlinearities. Here we study frequency-invariant transmission at synapses from Purkinje cells to deep cerebellar nuclei and at vestibular synapses in mice. Prolonged activation of these synapses leads to initial depression, which is followed by steady-state responses that are frequency invariant for their physiological activity range. We find that synaptotagmin 7 (Syt7), a calcium sensor for short-term facilitation, is present at both synapses. It was unclear why a sensor for facilitation would be present at these and other depressing synapses. We find that at Purkinje cell and vestibular synapses, Syt7 supports facilitation that is normally masked by depression, which can be revealed in wild-type mice but is absent in Syt7 knockout mice. In wild-type mice, facilitation increases with firing frequency and counteracts depression to produce frequency-invariant transmission. In Syt7-knockout mice, Purkinje cell and vestibular synapses exhibit conventional use-dependent depression, weakening to a greater extent as the firing frequency is increased. Presynaptic rescue of Syt7 expression restores both facilitation and frequency-invariant transmission. Our results identify a function for Syt7 at synapses that exhibit overall depression, and demonstrate that facilitation has an unexpected and important function in producing frequency-invariant transmission.

Synaptic Specializations Support Frequency-Independent Purkinje Cell Output from the Cerebellar Cortex

The output of the cerebellar cortex is conveyed to the deep cerebellar nuclei (DCN) by Purkinje cells (PCs). Here, we characterize the properties of the PC-DCN synapse in juvenile and adult mice and find that prolonged high-frequency stimulation leads to steady-state responses that become increasingly frequency independent within the physiological firing range of PCs in older animals, resulting in a linear relationship between charge transfer and activation frequency. We used a low-affinity antagonist to show that GABAA-receptor saturation occurs at this synapse but does not underlie frequency-invariant transmission. We propose that PC-DCN synapses have two components of release: one prominent early in trains and another specialized to maintain transmission during prolonged activation. Short-term facilitation offsets partial vesicle depletion to produce frequency-independent transmission.

Blocking slow exocytosis with slow Ca2+ buffers slows recovery from depression

Synaptic transmission is a hungry process. During periods of elevated neuronal activity, sustained exocytosis can consume the pool of available synaptic vesicles and lead to profound synaptic depression (Betz, 1970). At some synapses depression may act as a useful computational feature, employed by neural circuits for the purposes of gain control and novelty detection (Abbott & Regehr, 2004). But many synapses must maintain strength in order to reliably transmit information during high-frequency firing. This requires efficient resupply of vesicles to the readily releasable pool (RRP). For years it has been assumed that synapses employ an active mechanism to increase the rate of vesicle replenishment during periods of intense activity, a phenomenon known as “Ca2+-dependent recovery from depression”. In this issue of The Journal of Physiology Ritzau-Jost et al. (2018) provide an elegant analysis and a new perspective on Ca2+-dependent recovery from depression, and suggest that – for at least one synapse – the phenomenon that has been observed may have little to do with vesicle replenishment. Twenty years ago a seeming paradox was discovered: recovery from depression can be accelerated by strong stimulation, despite the fact that strong activation also drives greater depletion of the RRP. Following 100 Hz stimulation, recovery at the calyx of Held proceeds with a single exponential time constant of 4–5 s (at room temperature), a rate typical for many central synapses (von Gersdorff et al. 1997). Stimulating the calyx of Held at higher frequencies (300 Hz) reveals a component of recovery with a much faster time constant ( 80 ms) (Wang & Kaczmarek, 1998). This accelerated vesicle replenishment appears to be driven by the elevated cytoplasmic Ca2+ that persists after action potentials, known as “residual calcium”. Both high-frequency stimulation and manipulations that increase Ca2+ influx during action potentials accelerate recovery from depression. Recovery from depression is slowed when residual calcium is clamped at lower levels by introducing the slow Ca2+ buffer EGTA into nerve terminals (Dittman & Regehr, 1998; Stevens & Wesseling, 1998). This led to the hypothesis that an active, Ca2+-sensitive mechanism speeds up replenishment and helps synapses avoid premature vesicle depletion. Subsequent studies suggested a mechanism for Ca2+-dependent recovery from depression. Using the calyx of Held, Sakaba & Neher (2001) showed a role for calmodulin in accelerating recovery. Calmodulin binds to Ca2+ with relatively high affinity, making it a promising candidate for sensing the sub-micromolar residual Ca2+ signals. Calmodulin inhibitors slow the rate of recovery from depression (Sakaba & Neher, 2001). Munc13, which plays an important role in regulating the RRP, binds to Ca2+-activated calmodulin to form a complex that recruits

The high-affinity calcium sensor synaptotagmin-7 serves multiple roles in regulated exocytosis

Synaptotagmin (Syt) proteins comprise a 17-member family, many of which trigger exocytosis in response to calcium. Historically, most studies have focused on the isoform Syt-1, which serves as the primary calcium sensor in synchronous neurotransmitter release. Recently, Syt-7 has become a topic of broad interest because of its extreme calcium sensitivity and diversity of roles in a wide range of cell types. Here, we review the known and emerging roles of Syt-7 in various contexts and stress the importance of its actions. Unique functions of Syt-7 are discussed in light of recent imaging, electrophysiological, and computational studies. Particular emphasis is placed on Syt-7–dependent regulation of synaptic transmission and neuroendocrine cell secretion. Finally, based on biochemical and structural data, we propose a mechanism to link Syt-7’s role in membrane fusion with its role in subsequent fusion pore expansion via strong calcium-dependent phospholipid binding.

Silk Fibroin Films Facilitate Single-Step Targeted Expression of Optogenetic Proteins

SUMMARY Optical methods of interrogating neural circuits have emerged as powerful tools for understanding how the brain drives behaviors. Optogenetic proteins are widely used to control neuronal activity, while genetically encoded fluorescent reporters are used to monitor activity. These proteins are often expressed by injecting viruses, which frequently leads to inconsistent experiments due to misalignment of expression and optical components. Here, we describe how silk fibroin films simplify optogenetic experiments by providing targeted delivery of viruses. Films composed of silk fibroin and virus are applied to the surface of implantable optical components. After surgery, silk releases the virus to transduce nearby cells and provide localized expression around optical fibers and endoscopes. Silk films can also be used to express genetically encoded sensors in large cortical regions by using cranial windows coated with a silk/virus mixture. The ease of use and improved performance provided by silk make this a promising approach for optogenetic studies.