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.

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.

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.

Loss of inhibitory synapses causes locomotor network dysfunction of the rat spinal cord during prolonged maintenance in vitro.

The isolated spinal cord of the neonatal rat is widely employed to clarify the basic mechanisms of network development or the early phase of degeneration after injury. Nevertheless, this preparation survives in Krebs solution up to 24 h only, making it desirable to explore approaches to extend its survival for longitudinal studies. The present report shows that culturing the spinal cord in oxygenated enriched Basal Medium Eagle (BME) provided excellent preservation of neurons (including motoneurons), glia and primary afferents (including dorsal root ganglia) for up to 72 h. Using DMEM medium was unsuccessful. Novel characteristics of spinal networks emerged with strong spontaneous activity, and deficit in fictive locomotion patterns with stereotypically slow cycles. Staining with markers for synaptic proteins synapsin 1 and synaptophysin showed thoroughly weaker signal after 3 days in vitro. Immunohistochemical staining of markers for glutamatergic and glycinergic neurons indicated significant reduction of the latter. Likewise, there was lower expression of the GABA-synthesizing enzyme GAD65. Thus, malfunction of locomotor networks appeared related to loss of inhibitory synapses. This phenomenon did not occur in analogous opossum preparations of the spinal cord kept in vitro. In conclusion, despite histological data suggesting that cultured spinal cords were undamaged (except for inhibitory biomarkers), electrophysiological data revealed important functional impairment. Thus, the downregulation of inhibitory synapses may account for the progressive hyperexcitability of rat spinal networks despite apparently normal histological appearance. Our observations may help to understand the basis of certain delayed effects of spinal injury like chronic pain and spasticity.

ASIC channel inhibition enhances excitotoxic neuronal death in an in vitro model of spinal cord injury.

In the spinal cord high extracellular glutamate evokes excitotoxic damage with neuronal loss and severe locomotor impairment. During the cell dysfunction process, extracellular pH becomes acid and may activate acid-sensing ion channels (ASICs) which could be important contributors to neurodegenerative pathologies. Our previous studies have shown that transient application of the glutamate analog kainate (KA) evokes delayed excitotoxic death of spinal neurons, while white matter is mainly spared. The present goal was to enquire if ASIC channels modulated KA damage in relation to locomotor network function and cell death. Mouse spinal cord slices were treated with KA (0.01 or 0.1mM) for 1h, and then washed out for 24h prior to analysis. RT-PCR results showed that KA (at 0.01mM concentration that is near-threshold for damage) increased mRNA expression of ASIC1a, ASIC1b, ASIC2 and ASIC3, an effect reversed by the ASIC inhibitor 4',6-diamidino-2-phenylindole (DAPI). A KA neurotoxic dose (0.1mM) reduced ASIC1a and ASIC2 expression. Cell viability assays demonstrated KA-induced large damage in spinal slices from mice with ASIC1a gene ablation. Likewise, immunohistochemistry indicated significant neuronal loss when KA was followed by the ASIC inhibitors DAPI or amiloride. Electrophysiological recording from ventral roots of isolated spinal cords showed that alternating oscillatory cycles were slowed down by 0.01mMKA, and intensely inhibited by subsequently applied DAPI or amiloride. Our data suggest that early rise in ASIC expression and function counteracted deleterious effects on spinal networks by raising the excitotoxicity threshold, a result with potential implications for improving neuroprotection.

A study of cannabinoid-1 receptors during the early phase of excitotoxic damage to rat spinal locomotor networks in vitro.

Endocannabinoids acting on cannabinoid-1 receptors (CB1Rs) are proposed to protect brain and spinal neurons from excitotoxic damage. The ability to recover from spinal cord injury (SCI), in which excitotoxicity is a major player, is usually investigated at late times after modulation of CB1Rs whose role in the early phases of SCI remains unclear. Using the rat spinal cord in vitro as a model for studying SCI initial pathophysiology, we investigated if agonists or antagonists of CB1Rs might affect SCI induced by the excitotoxic agent kainate (KA) within 24h from a transient (1h) application of this glutamate agonist. The CB1 agonist anandamide (AEA or pharmacological block of its degradation) did not limit excitotoxic depolarization of spinal networks: cyclic adenosine monophosphate (cAMP) assay demonstrated that CB1Rs remained functional 24h later and similarly expressed among dead or survived cells. Locomotor-like network activity recorded from ventral roots could not recover with such treatments and was associated with persistent depression of synaptic transmission. Motoneurons, that are particularly vulnerable to KA, were not protected by AEA. Application of 2-arachidonoylglycerol also did not attenuate the electrophysiological and histological damage. The intensification of damage by the CB1 antagonist AM251 suggested that endocannabinoids were operative after excitotoxic stimulation, yet insufficient to contrast it efficiently. The present data indicate that the early phases of excitotoxic SCI could not be arrested by pharmacologically exploiting the endocannabinoid system, consistent with the notion that AEA and its derivatives are more useful to treat late SCI phases.

Altered development in GABA co-release shapes glycinergic synaptic currents in cultured spinal slices of the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

KEY POINTS: Increased environmental risk factors in conjunction with genetic susceptibility have been proposed with respect to the remarkable variations in mortality in amyotrophic lateral sclerosis (ALS). In vitro models allow the investigation of the genetically modified counter-regulator of motoneuron toxicity and may help in addressing ALS therapy. Spinal organotypic slice cultures from a mutant form of human superoxide dismutase 1 (SOD1G93A) mouse model of ALS allow the detection of altered glycinergic inhibition in spinal microcircuits. This altered inhibition improved spinal cord excitability, affecting motor outputs in early SOD1(G93A) pathogenesis. ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurological disease characterized by a progressive degeneration of motoneurons (MNs). In a previous study, we developed organotypic spinal cultures from an ALS mouse model expressing a mutant form of human superoxide dismutase 1 (SOD1(G93A) ). We reported the presence of a significant synaptic rearrangement expressed by these embryonic cultured networks, which may lead to the altered development of spinal synaptic signalling, which is potentially linked to the adult disease phenotype. Recent studies on the same ALS mouse model reported a selective loss of glycinergic innervation in cultured MNs, suggestive of a contribution of synaptic inhibition to MN dysfunction and degeneration. In the present study, we further exploit organotypic cultures from wild-type and SOD1(G93A) mice to investigate the development of glycine-receptor-mediated synaptic currents recorded from the interneurons of the premotor ventral circuits. We performed single cell electrophysiology, immunocytochemistry and confocal microscopy and suggest that GABA co-release may speed the decay of glycine responses altering both temporal precision and signal integration in SOD1(G93A) developing networks at the postsynaptic site. Our hypothesis is supported by the finding of an increased MN bursting activity in immature SOD1(G93A) spinal cords and by immunofluorescence microscopy detection of a longer persistence of GABA in SOD1(G93A) glycinergic terminals in cultured and ex vivo spinal slices.