Development of adenoviral vectors that transduce Purkinje cells and other cerebellar cell-types in the cerebellum of a humanized mouse model.

Viral vector gene therapy has immense promise for treating central nervous system (CNS) disorders. Although adeno-associated virus vectors (AAVs) have had success, their small packaging capacity limits their utility to treat the root cause of many CNS disorders. Adenoviral vectors (Ad) have tremendous potential for CNS gene therapy approaches. Currently, the most common vectors utilize the Group C Ad5 serotype capsid proteins, which rely on the Coxsackievirus-Adenovirus receptor (CAR) to infect cells. However, these Ad5 vectors are unable to transduce many neuronal cell types that are dysfunctional in many CNS disorders. The human CD46 (hCD46) receptor is widely expressed throughout the human CNS and is the primary attachment receptor for many Ad serotypes. Therefore, to overcome the current limitations of Ad vectors to treat CNS disorders, we created chimeric first generation Ad vectors that utilize the hCD46 receptor. Using a "humanized" hCD46 mouse model, we demonstrate these Ad vectors transduce cerebellar cell types, including Purkinje cells, that are refractory to Ad5 transduction. Since Ad vector transduction properties are dependent on their capsid proteins, these chimeric first generation Ad vectors open new avenues for high-capacity helper-dependent adenovirus (HdAd) gene therapy approaches for cerebellar disorders and multiple neurological disorders.

CaV 2.1 α1 subunit motifs that control presynaptic CaV 2.1 subtype abundance are distinct from CaV 2.1 preference.

Presynaptic voltage-gated Ca2+ channel (CaV ) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system (CNS), most presynaptic terminals are CaV 2.1 dominant with a developmental reduction in CaV 2.2 and CaV 2.3 levels, and CaV 2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV 2 subtype levels are largely unsolved. Because the CaV 2 α1  subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV 2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV 2.1 α1  subunits containing swapped motifs with the CaV 2.2 and CaV 2.3 α1  subunit on a CaV 2.1/CaV 2.2 null background at the calyx of Held presynaptic terminals. We found that expression of CaV 2.1 α1  subunit chimeras containing the CaV 2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV 2.1 α1  subunit. However, the Ca2+ current sensitivity to the CaV 2.1 blocker agatoxin-IVA was the same between the chimeras and the CaV 2.1 α1  subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV 2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV 2.1 loop II-III and CT do not individually regulate CaV 2.1 preference, although these motifs control CaV 2.1 levels and the CaV 2.3 CT contains motifs that negatively regulate presynaptic CaV 2.3 levels. We propose that the motifs controlling presynaptic CaV 2.1 preference are distinct from those regulating CaV 2.1 levels and may act synergistically to impact pathways regulating CaV 2.1 preference and abundance. KEY POINTS: Presynaptic CaV 2 subtype abundance regulates neuronal circuit properties, although the mechanisms regulating presynaptic CaV 2 subtype abundance and preference remain enigmatic. The CaV α1  subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. The CaV 2.1 α1  subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic CaV 2.1 abundance but do not regulate preference. The CaV 2.3 α1  subunit cytoplasmic C-terminus negatively regulates presynaptic CaV 2 subtype abundance but not preference, whereas the CaV 2.2 α1  subunit cytoplasmic C-terminus is not a key regulator of presynaptic CaV 2 subtype abundance or preference. The CaV 2 α1  subunit motifs determining the presynaptic CaV 2 preference are distinct from abundance.

Confocal Imaging and 3D Reconstruction to Determine How Genetic Perturbations Impact Presynaptic Morphology at the Mouse Calyx of Held.

Neurons communicate via synapses-specialized structures that consist of a presynaptic terminal of one neuron and a postsynaptic terminal of another. As knowledge is emerging that mutations in molecules that regulate synaptic function underpin many neurological disorders, it is crucial to elucidate the molecular mechanisms regulating synaptic function to understand synaptic strength, plasticity, modulation, and pathology, which ultimately impact neuronal circuit output and behavior. The presynaptic calyx of Held is a large glutamatergic presynaptic terminal in the auditory brainstem, which due to its accessibility and the possibility to selectively perform molecular perturbations on it, is an ideal model to study the role of presynaptic proteins in regulating synaptic function. In this protocol, we describe the use of confocal imaging and three-dimensional reconstruction of the calyx of Held to assess alterations in gross morphology following molecular perturbation. Using viral-vector delivery to perform molecular perturbations at distinct developmental time points, we provide a fast and cost-effective method to investigate how presynaptic proteins regulate gross morphology such as surface area and synapse volume throughout the lifetime of a neuronal circuit. Key features Confocal imaging and 3D reconstruction of presynaptic terminals. Used with a virus-mediated expression of mEGFP to achieve efficient, cell-type specific labeling of the presynaptic compartment. Protocol was developed with the calyx of Held but is suitable for pre- and postsynaptic compartments of various neurons across multiple mammalian and invertebrate species.

Stereotactic Delivery of Helper-dependent Adenoviral Viral Vectors at Distinct Developmental Time Points to Perform Age-dependent Molecular Manipulations of the Mouse Calyx of Held.

Synapses are specialized structures that enable neuronal communication, which is essential for brain function and development. Alterations in synaptic proteins have been linked to various neurological and neuropsychiatric disorders. Therefore, manipulating synaptic proteins in vivo can provide insight into the molecular mechanisms underlying these disorders and aid in developing new therapeutic strategies. Previous methods such as constitutive knock-out animals are limited by developmental compensation and off-target effects. The current approach outlines procedures for age-dependent molecular manipulations in mice using helper-dependent adenovirus viral vectors (HdAd) at distinct developmental time points. Using stereotactic injection of HdAds in both newborn and juvenile mice, we demonstrate the versatility of this method to express Cre recombinase in globular bushy cells of juvenile Rac1fl/fl mice to ablate presynaptic Rac1 and study its role in synaptic transmission. Separately, we overexpress CaV2 α1 subunits at two distinct developmental time points to elucidate the mechanisms that determine presynaptic CaV2 channel abundance and preference. This method presents a reliable, cost-effective, and minimally invasive approach for controlling gene expression in specific regions of the mouse brain and will be a powerful tool to decipher brain function in health and disease. Key features Virus-mediated genetic perturbation in neonatal and young adult mice. Stereotaxic injection allows targeting of brain structures at different developmental stages to study the impact of genetic perturbation throughout the development.

Development and evaluation of helper dependent adenoviral vectors for inner ear gene delivery.

Viral vector gene therapy is an attractive strategy to treat hearing loss. Since hearing loss is due to a variety of pathogenic signaling cascades in distinct cells, viral vectors that can express large or multiple genes in a cell-type specific manner are needed. Helper-dependent adenoviral vectors (HdAd) are safe viral vectors with a large packaging capacity (-36 kb). Despite the potential of HdAd, its use in the inner ear is largely unexplored. Therefore, to evaluate the utility of HdAd for inner ear gene therapy, we created two HdAd vectors that use distinct cellular receptors for transduction: HdAd Serotype Type 5 (HdAd5), the Coxsackie-Adenovirus Receptor (CAR) and a chimeric HdAd 5/35, the human CD46+ receptor (hCD46). We delivered these vectors through the round window (RW) or scala media in CBA/J, C57Bl6/J and hCD46 transgenic mice. Immunostaining in conjunction with confocal microscopy of cochlear sections revealed that multiple cell types were transduced using HdAd5 and HdAd 5/35 in all mouse models. Delivery of HdAd5 via RW in the C57Bl/6 J or CBA/J cochlea resulted in transduced mesenchymal cells of the peri­lymphatic lining and modiolar region while scala media delivery resulted in transduction of supporting cells and inner hair cells. Hd5/35 transduction was CD46 dependent and RW delivery of HdAd5/35 in the hCD46 mouse model resulted in a similar transduction pattern as HdAd5 in the peri­lymphatic lining and modiolar region in the cochlea. Our data indicate that HdAd vectors are promising vectors for use in inner ear gene therapy to treat some causes of hearing loss.

CaV2.1 α1 subunit motifs that control presynaptic CaV2.1 subtype abundance are distinct from CaV2.1 preference.

Presynaptic voltage-gated Ca2+ channels (CaV) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system, most presynaptic terminals are CaV2.1 dominant with a developmental reduction in CaV2.2 and CaV2.3 levels, and CaV2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV2 subtype levels are largely unsolved. Since the CaV2 α1 subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV2.1 α1subunits containing swapped motifs with the CaV2.2 and CaV2.3 α1 subunit on a CaV2.1/CaV2.2 null background at the calyx of Held presynaptic terminal. We found that expression of CaV2.1 α1 subunit chimeras containing the CaV2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV2.1 α1 subunit. However, the Ca2+ current sensitivity to the CaV2.1 blocker Agatoxin-IVA, was the same between the chimeras and the CaV2.1 α1 subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV2.1 loop II-III and CT do not individually regulate CaV2.1 preference, but these motifs control CaV2.1 levels and the CaV2.3 CT contains motifs that negatively regulate presynaptic CaV2.3 levels. We propose that the motifs controlling presynaptic CaV2.1 preference are distinct from those regulating CaV2.1 levels and may act synergistically to impact pathways regulating CaV2.1 preference and abundance.

Presynaptic Rac1 controls synaptic strength through the regulation of synaptic vesicle priming.

Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1's roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability.

VikAD, a Vika site-specific recombinase-based system for efficient and scalable helper-dependent adenovirus production.

Recombinant viral vectors have become integral tools for basic in vivo research applications. Helper-dependent adenoviral (HdAd) vectors have a large packaging capacity of ∼36 kb of DNA that mediate long-term transgene expression in vitro and in vivo. The large carrying capacity of HdAd enables basic research or clinical applications requiring the delivery of large genes or multiple transgenes, which cannot be packaged into other widely used viral vectors. Currently, common HdAd production systems use an Ad helper virus (HV) with a packaging signal (Ψ) that is flanked by either loxP or FRT sites, which is excised in producer cells expressing Cre or Flp recombinases to prevent HV packaging. However, these production systems prevent the use of HdAd vectors for genetic strategies that rely on Cre or Flp recombination for cell-type-specific expression. To overcome these limitations, we developed the VikAD production system, which is based on producer cells expressing the Vika recombinase and an HV that contains a Ψ flanked by vox sites. The availability of this production system will greatly expand the utility and flexibility of HdAd vectors for use in research applications to monitor and manipulate cellular activity with increased specificity.

Presynaptic voltage-gated calcium channels in the auditory brainstem.

Sound information encoding within the initial synapses in the auditory brainstem requires reliable and precise synaptic transmission in response to rapid and large fluctuations in action potential (AP) firing rates. The magnitude and location of Ca2+ entry through voltage-gated Ca2+ channels (CaV) in the presynaptic terminal are key determinants in triggering AP-mediated release. In the mammalian central nervous system (CNS), the CaV2.1 subtype is the critical subtype for CNS function, since it is the most efficient CaV2 subtype in triggering AP-mediated synaptic vesicle (SV) release. Auditory brainstem synapses utilize CaV2.1 to sustain fast and repetitive SV release to encode sound information. Therefore, understanding the presynaptic mechanisms that control CaV2.1 localization, organization and biophysical properties are integral to understanding auditory processing. Here, we review our current knowledge about the control of presynaptic CaV2 abundance and organization in the auditory brainstem and impact on the regulation of auditory processing.

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

KEY POINTS: 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. ABSTRACT: 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.

Using Sox2 to alleviate the hallmarks of age-related hearing loss.

Age-related hearing loss (ARHL) is the most prevalent sensory deficit. ARHL reduces the quality of life of the growing population, setting seniors up for the enhanced mental decline. The size of the needy population, the structural deficit, and a likely research strategy for effective treatment of chronic neurosensory hearing in the elderly are needed. Although there has been profound advancement in auditory regenerative research, there remain multiple challenges to restore hearing loss. Thus, additional investigations are required, using novel tools. We propose how the (1) flat epithelium, remaining after the organ of Corti has deteriorated, can be converted to the repaired-sensory epithelium, using Sox2. This will include (2) developing an artificial gene regulatory network transmitted by (3) large viral vectors to the flat epithelium to stimulate remnants of the organ of Corti to restore hair cells. We hope to unite with our proposal toward the common goal, eventually restoring a functional human hearing organ by transforming the flat epithelial cells left after the organ of Corti loss.

Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures.

Synapse loss and dendritic damage correlate with cognitive decline in many neurodegenerative diseases, underlie neurodevelopmental disorders, and are associated with environmental and drug-induced CNS toxicities. However, screening assays designed to measure loss of synaptic connections between live cells are lacking. Here, we describe the design and validation of automated synaptic imaging assay (ASIA), an efficient approach to label, image, and analyze synapses between live neurons. Using viral transduction to express fluorescent proteins that label synapses and an automated computer-controlled microscope, we developed a method to identify agents that regulate synapse number. ASIA is compatible with both confocal and wide-field microscopy; wide-field image acquisition is faster but requires a deconvolution step in the analysis. Both types of images feed into batch processing analysis software that can be run on ImageJ, CellProfiler, and MetaMorph platforms. Primary analysis endpoints are the number of structural synapses and cell viability. Thus, overt cell death is differentiated from subtle changes in synapse density, an important distinction when studying neurodegenerative processes. In rat hippocampal cultures treated for 24 h with 100 µM 2-bromopalmitic acid (2-BP), a compound that prevents clustering of postsynaptic density 95 (PSD95), ASIA reliably detected loss of postsynaptic density 95-enhanced green fluorescent protein (PSD95-eGFP)-labeled synapses in the absence of cell death. In contrast, treatment with 100 µM glutamate produced synapse loss and significant cell death, determined from morphological changes in a binary image created from co-expressed mCherry. Treatment with 3 mM lithium for 24 h significantly increased the number of fluorescent puncta, showing that ASIA also detects synaptogenesis. Proof of concept studies show that cell-specific promoters enable the selective study of inhibitory or principal neurons and that alternative reporter constructs enable quantification of GABAergic or glutamatergic synapses. ASIA can also be used to study synapse loss between human induced pluripotent stem cell (iPSC)-derived cortical neurons. Significant synapse loss in the absence of cell death was detected in the iPSC-derived neuronal cultures treated with either 100 µM 2-BP or 100 µM glutamate for 24 h, while 300 µM glutamate produced synapse loss and cell death. ASIA shows promise for identifying agents that evoke synaptic toxicities and screening for compounds that prevent or reverse synapse loss.

Presynaptic Mitochondria Volume and Abundance Increase during Development of a High-Fidelity Synapse.

The calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem undergoes developmental changes to support the high action-potential firing rates required for auditory information encoding. In addition, calyx terminals are morphologically diverse, which impacts vesicle release properties and synaptic plasticity. Mitochondria influence synaptic plasticity through calcium buffering and are crucial for providing the energy required for synaptic transmission. Therefore, it has been postulated that mitochondrial levels increase during development and contribute to the morphological-functional diversity in the mature calyx. However, the developmental profile of mitochondrial volumes and subsynaptic distribution at the calyx of Held remains unclear. To provide insight on this, we developed a helper-dependent adenoviral vector that expresses the genetically encoded peroxidase marker for mitochondria, mito-APEX2, at the mouse calyx of Held. We developed protocols to detect labeled mitochondria for use with serial block face scanning electron microscopy to carry out semiautomated segmentation of mitochondria, high-throughput whole-terminal reconstruction, and presynaptic ultrastructure in mice of either sex. Subsequently, we measured mitochondrial volumes and subsynaptic distributions at the immature postnatal day (P)7 and the mature (P21) calyx. We found an increase of mitochondria volumes in terminals and axons from P7 to P21 but did not observe differences between stalk and swelling subcompartments in the mature calyx. Based on these findings, we propose that mitochondrial volumes and synaptic localization developmentally increase to support high firing rates required in the initial stages of auditory information processing.SIGNIFICANCE STATEMENT Elucidating the developmental processes of auditory brainstem presynaptic terminals is critical to understanding auditory information encoding. Additionally, morphological-functional diversity at these terminals is proposed to enhance coding capacity. Mitochondria provide energy for synaptic transmission and can buffer calcium, impacting synaptic plasticity; however, their developmental profile to ultimately support the energetic demands of synapses following the onset of hearing remains unknown. Therefore, we created a helper-dependent adenoviral vector with the mitochondria-targeting peroxidase mito-APEX2 and expressed it at the mouse calyx of Held. Volumetric reconstructions of serial block face electron microscopy data of immature and mature labeled calyces reveal that mitochondrial volumes are increased to support high firing rates upon maturity.

CaV2.1 α1 Subunit Expression Regulates Presynaptic CaV2.1 Abundance and Synaptic Strength at a Central Synapse.

The abundance of presynaptic CaV2 voltage-gated Ca2+ channels (CaV2) at mammalian active zones (AZs) regulates the efficacy of synaptic transmission. It is proposed that presynaptic CaV2 levels are saturated in AZs due to a finite number of slots that set CaV2 subtype abundance and that CaV2.1 cannot compete for CaV2.2 slots. However, at most AZs, CaV2.1 levels are highest and CaV2.2 levels are developmentally reduced. To investigate CaV2.1 saturation states and preference in AZs, we overexpressed the CaV2.1 and CaV2.2 α1 subunits at the calyx of Held at immature and mature developmental stages. We found that AZs prefer CaV2.1 to CaV2.2. Remarkably, CaV2.1 α1 subunit overexpression drove increased CaV2.1 currents and channel numbers and increased synaptic strength at both developmental stages examined. Therefore, we propose that CaV2.1 levels in the AZ are not saturated and that synaptic strength can be modulated by increasing CaV2.1 levels to regulate neuronal circuit output. VIDEO ABSTRACT.

Inhibitory Signaling to Ion Channels in Hippocampal Neurons Is Differentially Regulated by Alternative Macromolecular Complexes of RGS7.

The neuromodulatory effects of GABA on pyramidal neurons are mediated by GABAB receptors (GABABRs) that signal via a conserved G-protein-coupled pathway. Two prominent effectors regulated by GABABRs include G-protein inwardly rectifying K+ (GIRK) and P/Q/N type voltage-gated Ca2+ (CaV2) ion channels that control excitability and synaptic output of these neurons, respectively. Regulator of G-protein signaling 7 (RGS7) has been shown to control GABAB effects, yet the specificity of its impacts on effector channels and underlying molecular mechanisms is poorly understood. In this study, we show that hippocampal RGS7 forms two distinct complexes with alternative subunit configuration bound to either membrane protein R7BP (RGS7 binding protein) or orphan receptor GPR158. Quantitative biochemical experiments show that both complexes account for targeting nearly the entire pool of RGS7 to the plasma membrane. We analyzed the effect of genetic elimination in mice of both sexes and overexpression of various components of RGS7 complex by patch-clamp electrophysiology in cultured neurons and brain slices. We report that RGS7 prominently regulates GABABR signaling to CaV2, in addition to its known involvement in modulating GIRK. Strikingly, only complexes containing R7BP, but not GPR158, accelerated the kinetics of both GIRK and CaV2 modulation by GABABRs. In contrast, GPR158 overexpression exerted the opposite effect and inhibited RGS7-assisted temporal modulation of GIRK and CaV2 by GABA. Collectively, our data reveal mechanisms by which distinctly composed macromolecular complexes modulate the activity of key ion channels that mediate the inhibitory effects of GABA on hippocampal CA1 pyramidal neurons.SIGNIFICANCE STATEMENT This study identifies the contributions of distinct macromolecular complexes containing a major G-protein regulator to controlling key ion channel function in hippocampal neurons with implications for understanding molecular mechanisms underlying synaptic plasticity, learning, and memory.

CAST/ELKS Proteins Control Voltage-Gated Ca2+ Channel Density and Synaptic Release Probability at a Mammalian Central Synapse.

In the presynaptic terminal, the magnitude and location of Ca2+ entry through voltage-gated Ca2+ channels (VGCCs) regulate the efficacy of neurotransmitter release. However, how presynaptic active zone proteins control mammalian VGCC levels and organization is unclear. To address this, we deleted the CAST/ELKS protein family at the calyx of Held, a CaV2.1 channel-exclusive presynaptic terminal. We found that loss of CAST/ELKS reduces the CaV2.1 current density with concomitant reductions in CaV2.1 channel numbers and clusters. Surprisingly, deletion of CAST/ELKS increases release probability while decreasing the readily releasable pool, with no change in active zone ultrastructure. In addition, Ca2+ channel coupling is unchanged, but spontaneous release rates are elevated. Thus, our data identify distinct roles for CAST/ELKS as positive regulators of CaV2.1 channel density and suggest that they regulate release probability through a post-priming step that controls synaptic vesicle fusogenicity.

Orphan receptor GPR158 controls stress-induced depression.

Stress can be a motivational force for decisive action and adapting to novel environment; whereas, exposure to chronic stress contributes to the development of depression and anxiety. However, the molecular mechanisms underlying stress-responsive behaviors are not fully understood. Here, we identified the orphan receptor GPR158 as a novel regulator operating in the prefrontal cortex (PFC) that links chronic stress to depression. GPR158 is highly upregulated in the PFC of human subjects with major depressive disorder. Exposure of mice to chronic stress also increased GPR158 protein levels in the PFC in a glucocorticoid-dependent manner. Viral overexpression of GPR158 in the PFC induced depressive-like behaviors. In contrast GPR158 ablation, led to a prominent antidepressant-like phenotype and stress resiliency. We found that GPR158 exerts its effects via modulating synaptic strength altering AMPA receptor activity. Taken together, our findings identify a new player in mood regulation and introduce a pharmacological target for managing depression.

Triple Function of Synaptotagmin 7 Ensures Efficiency of High-Frequency Transmission at Central GABAergic Synapses.

Synaptotagmin 7 (Syt7) is thought to be a Ca2+ sensor that mediates asynchronous transmitter release and facilitation at synapses. However, Syt7 is strongly expressed in fast-spiking, parvalbumin-expressing GABAergic interneurons, and the output synapses of these neurons produce only minimal asynchronous release and show depression rather than facilitation. To resolve this apparent contradiction, we examined the effects of genetic elimination of Syt7 on synaptic transmission at the GABAergic basket cell (BC)-Purkinje cell (PC) synapse in cerebellum. Our results indicate that at the BC-PC synapse, Syt7 contributes to asynchronous release, pool replenishment, and facilitation. In combination, these three effects ensure efficient transmitter release during high-frequency activity and guarantee frequency independence of inhibition. Our results identify a distinct function of Syt7: ensuring the efficiency of high-frequency inhibitory synaptic transmission.

A novel region in the CaV2.1 α1 subunit C-terminus regulates fast synaptic vesicle fusion and vesicle docking at the mammalian presynaptic active zone.

In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by CaV2.1 calcium channels (CaV2.1) and is highly dependent on the physical distance between CaV2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of CaV2.1 through direct interactions with the CaV2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant CaV2.1 α1 subunits on a CaV2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of CaV2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for CaV2.1 abundance and coupling. Therefore, our work advances our molecular understanding of CaV2.1 regulation of neurotransmitter release in mammalian CNS synapses.