Neuronal deletion of CaV1.2 is associated with sex-specific behavioral phenotypes in mice.

The gene CACNA1C, which encodes the pore forming subunit of the L-type calcium channel CaV1.2, is associated with increased risk for neuropsychiatric disorders including schizophrenia, autism spectrum disorder, major depression, and bipolar disorder. Previous rodent work identified that loss or reduction of CaV1.2 results in cognitive, affective, and motor deficits. Most previous work has either included non-neuronal cell populations (haploinsufficient and Nestin-Cre) or investigated a discrete neuronal cell population (e.g. CaMKII-Cre, Drd1-Cre), but few studies have examined the effects of more broad neuron-specific deletion of CaV1.2. Additionally, most of these studies did not evaluate for sex-specific effects or used only male animals. Here, we sought to clarify whether there are sex-specific behavioral consequences of neuron-specific deletion of CaV1.2 (neuronal CaV1.2 cKO) using Syn1-Cre-mediated conditional deletion. We found that neuronal CaV1.2 cKO mice have normal baseline locomotor function but female cKO mice display impaired motor performance learning. Male neuronal CaV1.2 cKO display impaired startle response with intact pre-pulse inhibition. Male neuronal CaV1.2 cKO mice did not display normal social preference, whereas female neuronal CaV1.2 cKO mice did. Neuronal CaV1.2 cKO mice displayed impaired associative learning in both sexes, as well as normal anxiety-like behavior and hedonic capacity. We conclude that deletion of neuronal CaV1.2 alters motor performance, acoustic startle reflex, and social behaviors in a sex-specific manner, while associative learning deficits generalize across sexes. Our data provide evidence for both sex-specific and sex-independent phenotypes related to neuronal expression of CaV1.2.

The voltage-gated Ca2+ channel subunit α2δ-4 regulates locomotor behavior and sensorimotor gating in mice.

Voltage-gated Ca2+ channels are critical for the development and mature function of the nervous system. Variants in the CACNA2D4 gene encoding the α2δ-4 auxiliary subunit of these channels are associated with neuropsychiatric and neurodevelopmental disorders. α2δ-4 is prominently expressed in the retina and is crucial for vision, but extra-retinal functions of α2δ-4 have not been investigated. Here, we sought to fill this gap by analyzing the behavioral phenotypes of α2δ-4 knockout (KO) mice. α2δ-4 KO mice (both males and females) exhibited significant impairments in prepulse inhibition that were unlikely to result from the modestly elevated auditory brainstem response thresholds. Whereas α2δ-4 KO mice of both sexes were hyperactive in various assays, only females showed impaired motor coordination in the rotarod assay. α2δ-4 KO mice exhibited anxiolytic and anti-depressive behaviors in the elevated plus maze and tail suspension tests, respectively. Our results reveal an unexpected role for α2δ-4 in sensorimotor gating and motor function and identify α2δ-4 KO mice as a novel model for studying the pathophysiology associated with CACNA2D4 variants.

A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins.

The self-labeling protein HaloTag has been used extensively to determine the localization and turnover of proteins of interest at the single-cell level. To this end, halogen-substituted alkanes attached to diverse fluorophores are commercially available that allow specific, irreversible labeling of HaloTag fusion proteins; however, measurement of protein of interest half-life by pulse-chase of HaloTag ligands is not widely employed because residual unbound ligand continues to label newly synthesized HaloTag fusions even after extensive washing. Excess unlabeled HaloTag ligand can be used as a blocker of undesired labeling, but this is not economical. In this study, we screened several inexpensive, low-molecular-weight haloalkanes as blocking agents in pulse-chase labeling experiments with the cell-permeable tetramethylrhodamine HaloTag ligand. We identified 7-bromoheptanol as a high-affinity, low-toxicity HaloTag-blocking agent that permits protein turnover measurements at both the cell population (by blotting) and single-cell (by imaging) levels. We show that the HaloTag pulse-chase approach is a nontoxic alternative to inhibition of protein synthesis with cycloheximide and extend protein turnover assays to long-lived proteins.