Presynaptic Calcium Channels Research Articles

Modeling analysis of subthreshold voltage signaling along hippocampal mossy fiber axons.

Axons are classically thought of as electrically well isolated from other parts of the neurons due to the shape of a long cable-like structure. In contrast to this classical view on axonal compartmentalization, recent studies revealed that subthreshold depolarization of soma and dendrite passively propagates to the axons for a substantial distance, as demonstrated in some experimentally accessible axons including hippocampal mossy fibers and cortical pyramidal cell axons. Passive propagation of subthreshold dendritic EPSPs to the axons, defined as EPreSPs (excitatory presynaptic potentials), has been demonstrated to affect transmitter release from the axon terminals. To further characterize and explore the functional significance of passive subthreshold voltage signaling along the axons, the model of EPreSPs along hippocampal mossy fibers, proposed by Alle and Geiger, was reconstructed on the NEURON simulator. To test the effect of EPreSPs on action potentials and transmitter release from the axon terminals, additional conductances were incorporated into the previous passive propagation model. These include the axonal sodium, potassium, and leak channels as well as presynaptic calcium channels composed of P/Q-, N-, and R-types, which are reconstructed from the properties of those recorded from mossy fiber boutons experimentally. In this revised model, the preceding subthreshold EPreSPs slightly reduced the action potential-evoked presynaptic calcium currents by a decrease in the amplitude of action potentials due to the slow depolarization. It should be mentioned that EPreSPs by themselves elicited small calcium currents during subthreshold depolarization through these high-voltage activated calcium channels. Since the previous experimental study by simultaneous pre and postsynaptic recordings demonstrated that EPreSPs enhanced action potential-evoked transmitter release from the mossy fiber terminals, it has been suggested that different mechanisms from the enhancement of action potential-evoked presynaptic calcium entry may involve enhanced transmitter release by EPreSP. Small calcium entry by subthreshold EPreSPs may enhance transmitter release from the mossy fiber terminals by acting as high-affinity calcium sensors for enhancing transmitter release. Another form of axonal subthreshold voltage signaling, GABA-EPreSPs elicited by a spillover of GABA from surrounding interneurons, was also explored. Functional consequences of the two modes of axonal subthreshold voltage signaling were discussed with the simulation results.

Microphysiological Modeling of the Structure and Function of Neuromuscular Transmitter Release Sites.

The general mechanism of calcium-triggered chemical transmitter release from neuronal synapses has been intensely studied, is well-known, and highly conserved between species and synapses across the nervous system. However, the structural and functional details within each transmitter release site (or active zone) are difficult to study in living tissue using current experimental approaches owing to the small spatial compartment within the synapse where exocytosis occurs with a very rapid time course. Therefore, computer simulations offer the opportunity to explore these microphysiological environments of the synapse at nanometer spatial scales and on a sub-microsecond timescale. Because biological reactions and physiological processes at synapses occur under conditions where stochastic behavior is dominant, simulation approaches must be driven by such stochastic processes. MCell provides a powerful simulation approach that employs particle-based stochastic simulation tools to study presynaptic processes in realistic and complex (3D) geometries using optimized Monte Carlo algorithms to track finite numbers of molecules as they diffuse and interact in a complex cellular space with other molecules in solution and on surfaces (representing membranes, channels and binding sites). In this review we discuss MCell-based spatially realistic models of the mammalian and frog neuromuscular active zones that were developed to study presynaptic mechanisms that control transmitter release. In particular, these models focus on the role of presynaptic voltage-gated calcium channels, calcium sensors that control the probability of synaptic vesicle fusion, and the effects of action potential waveform shape on presynaptic calcium entry. With the development of these models, they can now be used in the future to predict disease-induced changes to the active zone, and the effects of candidate therapeutic approaches.

Neuromuscular Active Zone Structure and Function in Healthy and Lambert-Eaton Myasthenic Syndrome States.

The mouse neuromuscular junction (NMJ) has long been used as a model synapse for the study of neurotransmission in both healthy and disease states of the NMJ. Neurotransmission from these neuromuscular nerve terminals occurs at highly organized structures called active zones (AZs). Within AZs, the relationships between the voltage-gated calcium channels and docked synaptic vesicles govern the probability of acetylcholine release during single action potentials, and the short-term plasticity characteristics during short, high frequency trains of action potentials. Understanding these relationships is important not only for healthy synapses, but also to better understand the pathophysiology of neuromuscular diseases. In particular, we are interested in Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disorder in which neurotransmitter release from the NMJ decreases, leading to severe muscle weakness. In LEMS, the reduced neurotransmission is traditionally thought to be caused by the antibody-mediated removal of presynaptic voltage-gated calcium channels. However, recent experimental data and AZ computer simulations have predicted that a disruption in the normally highly organized active zone structure, and perhaps autoantibodies to other presynaptic proteins, contribute significantly to pathological effects in the active zone and the characteristics of chemical transmitters.

Dissecting the Component Parts of the Excitation‐Contraction Coupling Machinery in Drosophila melanogaster

The Drosophila neuromuscular system is widely used to characterize synaptic development and function. However, little is known about how specific synaptic and muscular alterations effect neuromuscular transduction and muscle contractility that ultimately dictate behavioral output. Here a system developed to characterize excitation‐contraction coupling at Drosophila larval neuromuscular junctions (NMJs) was exploited to examining how specific neuronal, synaptic, and muscle manipulations disrupt muscle contractility and locomotion. Increases in motor neuron stimulation frequency and duration increased muscle force in a predictable fashion, but show considerable plasticity between 5–40 Hz and saturating above 50 Hz. Fictive contraction recordings from larvae with CNS intact revealed motor neuron burst frequencies of 20‐30 Hz, consistent with the endogenous machinery operating within the range of contractility of greatest plasticity. Experimental parameters such as saline composition (e.g. calcium and magnesium) temperature, larval size and orientation, were explored to examine their impacts on muscle force production. Subsequently, genetic and pharmacological approaches were taken to dissect several molecular pathways which contribute to muscle contraction and modulation of force. First, well‐established calcium regulation pathways were manipulated via application of ryanodine, caffeine, thapsigargin, and felodipine to demonstrate the role of the ryanodine receptors, SERCA pump, and postsynaptic calcium channels in modulating the magnitude and time‐course of muscle contractions. Presynaptic calcium channels and ryanodine receptor expression were genetically altered to further examine their role in muscle force production. Next using both null and knock‐down (Gal4/UAS system) approaches combined with tissue‐specific expression of drivers, screens for motor, synaptic, and muscle proteins contributing to excitation‐contraction coupling (ECC) were conducted. Altering the expression of the motor proteins kinesin, dynein or dynactin all resulted in a significant reduction in muscle force production. Critical synaptic proteins like synaptotagmin, complexin, cacophony, gbb (homolog of BMP), significantly impacted ECC in Drosophila larvae. Several critical muscle‐specific proteins alter the magnitude and time‐course of muscle contractions when altering their expression in a sub‐set of muscle fibers. Lastly a screen for modulators of muscle contractility led to identification and characterization of the molecular and cellular pathway by which the FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to correlate synaptic dysfunction, regulation, and modulation, to alterations in excitation‐contraction coupling.

BaseScope™ Approach to Visualize Alternative Splice Variants in Tissue.

Defining the cell-specific alternative splicing landscape in complex tissues is an important goal to gain functional insights. Deep-sequencing techniques coupled to genetic strategies for cell identification has provided important cues on cell-specific exon usage in complex tissues like the nervous system. BaseScope™ has emerged as a powerful and highly sensitive alternative to in situ hybridization to determine exon composition in tissue with spatial and morphological context. In this protocol, we will review how BaseScope was utilized to detect the e37a-Cacna1b splice variant of the presynaptic calcium channel CaV2.2 or N-type. This splice variant arises from a pair of mutually exclusive exons (e37a and e37b). E37a-Cacna1b is heavily underrepresented relative to e37b-Cacna1b and both exons share 60% of their sequence. By using BaseScope™, we were able to discover that e37a-Cacna1b is expressed in excitatory pyramidal neurons of hippocampus and cortex, as well as motor neurons of the ventral horn of the spinal cord.

Convergent, functionally independent signaling by mu and delta opioid receptors in hippocampal parvalbumin interneurons.

Functional interactions between G protein-coupled receptors are poised to enhance neuronal sensitivity to neuromodulators and therapeutic drugs. Mu and delta opioid receptors (MORs and DORs) can interact when overexpressed in the same cells, but whether co-expression of endogenous MORs and DORs in neurons leads to functional interactions is unclear. Here, in mice, we show that both MORs and DORs inhibit parvalbumin-expressing basket cells (PV-BCs) in hippocampal CA1 through partially occlusive signaling pathways that terminate on somato-dendritic potassium channels and presynaptic calcium channels. Using photoactivatable opioid neuropeptides, we find that DORs dominate the response to enkephalin in terms of both ligand sensitivity and kinetics, which may be due to relatively low expression levels of MOR. Opioid-activated potassium channels do not show heterologous desensitization, indicating that MORs and DORs signal independently. In a direct test for heteromeric functional interactions, the DOR antagonist TIPP-Psi does not alter the kinetics or potency of either the potassium channel or synaptic responses to photorelease of the MOR agonist [d-Ala2, NMe-Phe4, Gly-ol5]enkephalin (DAMGO). Thus, aside from largely redundant and convergent signaling, MORs and DORs do not functionally interact in PV-BCs in a way that impacts somato-dendritic potassium currents or synaptic transmission. These findings imply that cross-talk between MORs and DORs, either in the form of physical interactions or synergistic intracellular signaling, is not a preordained outcome of co-expression in neurons.

Editors' Note: What Is the Mechanism of Therapeutic and Adverse Effects of Gabapentinoids?

Dr. Benarroch reviewed the mechanisms underlying the therapeutic and adverse effects of gabapentinoids such as gabapentin and pregabalin. The author noted that the efficacy of these drugs for neuropathic pain, restless legs syndrome, and focal seizures is primarily attributed to their inhibition of the α2δ subunit of presynaptic voltage-gated calcium channels, which decreases neurotransmitter release. He discussed the relevance of this inhibitory action in the potential induction of cerebellar ataxia by gabapentinoids. In response, Dr. Gazulla et al. argue that GABAergic effects of these medications are also important, noting that gabapentin has been shown to increase the expression of δGABAA receptors, inhibitory tone in the cerebellum, and brain GABA concentration in patients, whereas pregabalin has been shown to increase neuronal calcium influx, which facilitates neurotransmission. They argue that gabapentinoids could actually help treat ataxia in diseases involving GABA deficiency. Responding to these comments, Dr. Benarroch notes that he did cite work finding increased expression of GABAA receptors with gabapentin use but did not cite a pilot study of pregabalin for ataxia because the review was focused on mechanisms relating to the current clinical use of gabapentinoids. He agrees that increased GABAergic transmission by these drugs may benefit disorders affecting Purkinje cells and other inhibitory neurons such as periodic alternating nystagmus but cautions that the mechanisms of ataxia are complex and that further studies are needed to confirm potential beneficial effects of gabapentinoids in cerebellar ataxia. This exchange highlights uncertainties regarding the potential therapeutic vs adverse effects of gabapentinoids in ataxia and other cerebellar disorders. Dr. Benarroch reviewed the mechanisms underlying the therapeutic and adverse effects of gabapentinoids such as gabapentin and pregabalin. The author noted that the efficacy of these drugs for neuropathic pain, restless legs syndrome, and focal seizures is primarily attributed to their inhibition of the α2δ subunit of presynaptic voltage-gated calcium channels, which decreases neurotransmitter release. He discussed the relevance of this inhibitory action in the potential induction of cerebellar ataxia by gabapentinoids. In response, Dr. Gazulla et al. argue that GABAergic effects of these medications are also important, noting that gabapentin has been shown to increase the expression of δGABAA receptors, inhibitory tone in the cerebellum, and brain GABA concentration in patients, whereas pregabalin has been shown to increase neuronal calcium influx, which facilitates neurotransmission. They argue that gabapentinoids could actually help treat ataxia in diseases involving GABA deficiency. Responding to these comments, Dr. Benarroch notes that he did cite work finding increased expression of GABAA receptors with gabapentin use but did not cite a pilot study of pregabalin for ataxia because the review was focused on mechanisms relating to the current clinical use of gabapentinoids. He agrees that increased GABAergic transmission by these drugs may benefit disorders affecting Purkinje cells and other inhibitory neurons such as periodic alternating nystagmus but cautions that the mechanisms of ataxia are complex and that further studies are needed to confirm potential beneficial effects of gabapentinoids in cerebellar ataxia. This exchange highlights uncertainties regarding the potential therapeutic vs adverse effects of gabapentinoids in ataxia and other cerebellar disorders.

Lambert Eaton Myasthenic Syndrome masquerading as myopathy in asymptomatic lung malignancy

Introduction: Lambert-Eaton Myasthenic Syndrome (LEMS), is a rare disorder characterized by antibodies against presynaptic voltage-gated calcium channels in the neuromuscular junction. It is regarded as a paraneoplastic syndrome, as most patients have an underlying malignancy, commonly a small-cell lung cancer (SCLC).

Presynaptic Paraneoplastic Disorders of the Neuromuscular Junction: An Update.

The neuromuscular junction (NMJ) is the target of a variety of immune-mediated disorders, usually classified as presynaptic and postsynaptic, according to the site of the antigenic target and consequently of the neuromuscular transmission alteration. Although less common than the classical autoimmune postsynaptic myasthenia gravis, presynaptic disorders are important to recognize due to the frequent association with cancer. Lambert Eaton myasthenic syndrome is due to a presynaptic failure to release acetylcholine, caused by antibodies to the presynaptic voltage-gated calcium channels. Acquired neuromyotonia is a condition characterized by nerve hyperexcitability often due to the presence of antibodies against proteins associated with voltage-gated potassium channels. This review will focus on the recent developments in the autoimmune presynaptic disorders of the NMJ.

UNC-2 CaV2 Channel Localization at Presynaptic Active Zones Depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans.

Presynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel localization by active zone proteins is not completely understood. In a Caenorhabditis elegans (C. elegans) forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic localization of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel puncta at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel localization, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that although SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 localization by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel localization. In addition, we find that core active zone proteins are unequal in their abundance. Although the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel localization in redundant yet distinct manners.SIGNIFICANCE STATEMENT Precise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM, our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, a core active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

Locus revealed: Painlessness via loss of NaV1.7 at central terminals of sensory neurons

How genetic loss of the sodium channel NaV1.7 results in painlessness is puzzling. MacDonald etal. (2021) demonstrate that instead of impairing peripheral excitability, NaV1.7 channels at central terminals of pain-sensing afferents play a pivotal role in the balance between pain and analgesia.

Cnr2 Is Important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors.

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.

Male pheromones modulate synaptic transmission at the C. elegans neuromuscular junction in a sexually dimorphic manner.

The development of functional synapses in the nervous system is important for animal physiology and behaviors, and its disturbance has been linked with many neurodevelopmental disorders. The synaptic transmission efficacy can be modulated by the environment to accommodate external changes, which is crucial for animal reproduction and survival. However, the underlying plasticity of synaptic transmission remains poorly understood. Here we show that in Caenorhabditis elegans, the male environment increases the hermaphrodite cholinergic transmission at the neuromuscular junction (NMJ), which alters hermaphrodites' locomotion velocity and mating efficiency. We identify that the male-specific pheromones mediate this synaptic transmission modulation effect in a developmental stage-dependent manner. Dissection of the sensory circuits reveals that the AWB chemosensory neurons sense those male pheromones and further transduce the information to NMJ using cGMP signaling. Exposure of hermaphrodites to the male pheromones specifically increases the accumulation of presynaptic CaV2 calcium channels and clustering of postsynaptic acetylcholine receptors at cholinergic synapses of NMJ, which potentiates cholinergic synaptic transmission. Thus, our study demonstrates a circuit mechanism for synaptic modulation and behavioral flexibility by sexual dimorphic pheromones.

Presynaptic α2δ subunits are key organizers of glutamatergic synapses

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

Functions of Presynaptic Voltage-gated Calcium Channels.

Voltage-gated calcium channels are the principal conduits for depolarization-mediated Ca2+ entry into excitable cells. In this review, the biophysical properties of the relevant members of this family of channels, those that are present in presynaptic terminals, will be discussed in relation to their function in mediating neurotransmitter release. Voltage-gated calcium channels have properties that ensure they are specialized for particular roles, for example, differences in their activation voltage threshold, their various kinetic properties, and their voltage-dependence of inactivation. All these attributes play into the ability of the various voltage-gated calcium channels to participate in different patterns of presynaptic vesicular release. These include synaptic transmission resulting from single action potentials, and longer-term changes mediated by bursts or trains of action potentials, as well as release resulting from graded changes in membrane potential in specialized sensory synapses.

Retinal teratogenicity of pregabalin in chick embryo model

Background Pregabalin is a gamma-aminobutyric acid analog that binds to the α2-δ subunits of the pre-synaptic voltage-dependent calcium channels of nerves with a high affinity and selectivity. In this study, the retinal teratogenic potential of pregabalin was investigated in a chick embryo model. Materials and methods Fertilised chicken eggs were divided into groups for administration with different doses of pregabalin. All eggs were opened on the 10th day of incubation. The embryos were dissected and the effects of pregabalin on the retina were investigated histopathologically, morphometrically, and immunohistochemically (Caspase-3). Results There was no statistically significant difference between the low dose pregabalin, control, or vehicle control groups in terms of the number of retina layers and retinal thickness. Medium and high dose pregabalin caused a statistically significant decrease in the number of retina layers, as well as sensory retinal and pigment epithelium layer thicknesses. The outer nuclear and outer plexiform layer did not form in the group administered a medium dose. Similarly, the outer nuclear, outer plexiform, inner nuclear, and inner plexiform layer did not form in the high-dose group. No statistically significant difference was observed between the groups in terms of cellular damage and Caspase-3 expression. Conclusion The use of pregabalin during pregnancy compromises retinal development in a dose-dependent manner. The use of pregabalin in pregnancy causes the aforementioned defects in this system and it may have developmental effects that needs to be further evaluated.

Physiological involvement of presynaptic L-type voltage-dependent calcium channels in GABA release of cerebellar molecular layer interneurons.

While high threshold voltage-dependent Ca2+ channels (VDCCs) of the N and P/Q families are crucial for evoked neurotransmitter release in the mammalian CNS, it remains unclear to what extent L-type Ca2+ channels (LTCCs), which have been mainly considered as acting at postsynaptic sites, participate in the control of transmitter release. Here, we investigate the possible role of LTCCs in regulating GABA release by cerebellar molecular layer interneurons (MLIs) from rats. We found that BayK8644 (BayK) markedly increases mIPSC frequency in MLIs and Purkinje cells (PCs), suggesting that LTCCs are expressed presynaptically. Furthermore, we observed (1) a potentiation of evoked IPSCs in the presence of BayK, (2) an inhibition of evoked IPSCs in the presence of the LTCC-specific inhibitor Compound 8 (Cp8), and (3) a strong reduction of mIPSC frequency by Cp8. BayK effects are reduced by dantrolene, suggesting that ryanodine receptors act in synergy with LTCCs. Finally, BayK enhances presynaptic AP-evoked Ca2+ transients and increases the frequency of spontaneous axonal Ca2+ transients observed in TTX. Taken together, our data demonstrate that LTCCs are of primary importance in regulating GABA release by MLIs.

Presynaptic development is controlled by the core active zone proteins CAST/ELKS.

CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held. CAST/ELKS regulate all three CaV 2 subtype channel levels in the presynaptic terminal and not just CaV 2.1. The half-life of ELKS is on the timescale of days and not weeks. Synaptic transmission was not impacted by the loss of CAST/ELKS. CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z-stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV 2 channel subtype currents that correlated with a loss in presynaptic CaV 2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV 2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV 2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation.

Early Metazoan Origin and Multiple Losses of a Novel Clade of RIM Presynaptic Calcium Channel Scaffolding Protein Homologs.

The precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which, Rab3-interacting molecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channels in vitro via a PDZ domain that binds to the extreme C-termini of the channels at acidic ligand motifs of D/E-D/E/H-WC-COOH, and knockout of RIM in vertebrates and invertebrates disrupts CaV2 channel synaptic localization and synapse function. Here, we describe a previously uncharacterized clade of RIM proteins bearing domain architectures homologous to those of known RIM homologs, but with some notable differences including key amino acids associated with PDZ domain ligand specificity. This novel RIM emerged near the stem lineage of metazoans and underwent extensive losses, but is retained in select animals including the early-diverging placozoan Trichoplax adhaerens, and molluscs. RNA expression and localization studies in Trichoplax and the mollusc snail Lymnaea stagnalis indicate differential regional/tissue type expression, but overlapping expression in single isolated neurons from Lymnaea. Ctenophores, the most early-diverging animals with synapses, are unique among animals with nervous systems in that they lack the canonical RIM, bearing only the newly identified homolog. Through phylogenetic analysis, we find that CaV2 channel D/E-D/E/H-WC-COOH like PDZ ligand motifs were present in the common ancestor of cnidarians and bilaterians, and delineate some deeply conserved C-terminal structures that distinguish CaV1 from CaV2 channels, and CaV1/CaV2 from CaV3 channels.

Presynaptic Calcium Channel Open Probability and Changes in Calcium Influx Throughout the Action Potential Determined Using AP-Waveforms.

Action potentials arriving at a nerve terminal activate voltage-gated calcium channels and set the electrical driving force for calcium entry which affects the amount and duration of neurotransmitter release. During propagation, the duration, amplitude, and shape of action potentials often changes. This affects calcium entry, and can cause large changes in neurotransmitter release. Here, we have used a series of amplitude and area matched stimuli to examine how the shape and amplitude of a stimulus affect calcium influx at a presynaptic nerve terminal in the mammalian brain. We identify fundamental differences in calcium entry following calcium channel activation by a standard voltage jump vs. an action potential-like stimulation. We also tested a series of action potential-like stimuli with the same amplitude, duration, and stimulus area, but differing in their rise and decay times. We find that a stimulus that matches the rise and decay times of a physiological action potential produces a calcium channel response that is optimized over a range of peak amplitudes. Next, we determined the relative number of calcium channels that are active at different times during an action potential, which is important in the context of how local calcium domains trigger neurotransmitter release. We find the depolarizing phase of an AP-like stimulus only opens ~20% of the maximum number of calcium channels that can be activated. Channels continue to activate during the falling phase of the action potential, with peak calcium channel activation occurring near 0 mV. Although less than 25% of calcium channels are active at the end of the action potential, these calcium channels will generate a larger local calcium concentration that will increase the release probability for nearby vesicles. Determining the change in open probability of presynaptic calcium channels, and taking into account how local calcium concentration also changes throughout the action potential are both necessary to fully understand how the action potential triggers neurotransmitter release.