Dendritic spines are small protrusions on dendrites that endow neurons with the ability to receive and transform synaptic input. Dendritic spine number and morphology are altered as a consequence of synaptic plasticity and circuit refinement during adolescence. Dendritic spine density (DSD) is significantly different based on sex in subcortical brain regions associated with the generation of sexspecific behaviors. It is largely unknown if sex differences in DSD exist in auditory and visual brain regions and if there are sex-specific changes in DSD in these regions that occur during adolescent development. We analyzed dendritic spines in 4-week-old (P28) and 12-week-old (P84) male and female mice and found that DSD is lower in female mice due in part to fewer short stubby, long stubby and short mushroom spines. We found striking layer-specific patterns including a significant age by layer interaction and significantly decreased DSD in layer 4 from P28 to P84. Together these data support the possibility of developmental sex differences in DSD in visual and auditory regions and provide evidence of layer-specific refinement of DSD over adolescent brain development.

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The canonical voltage-gated calcium channel (VGCC) subunit complex is comprised of the α1 subunit, the ion permeable channel, plus three auxiliary subunits: β, α2δ and γ. β is the most extensively studied auxiliary subunit and is necessary for proper forward trafficking of the α1 subunit to the plasma membrane. α1 subunits mediate voltagedependent movement of calcium ions into the cytoplasm of neurons, including at dendritic sites, where increased intracellular calcium initiates signaling cascades that shape structural and functional plasticity of dendritic spines. Genetic studies strongly implicate calcium signaling dysfunction in the etiology of neurodevelopmental disorders including schizophrenia. Dendritic spine density (DSD) is significantly decreased in schizophrenia in primary auditory cortex where DSD is driven by loss of small spines, and small spine loss is associated with increased peptide levels of ALFDFLK found in the VGCC β subunit β4. Overexpessing CACNB4 to increase β4 levels selectively reduced small spine density in cortical neuron cultures. The studies described herein set out to validate this in vitro observation in an intact mammalian system within a neurodevelopmental context. We overexpressed CACNB4 in neurodevelopment and assessed DSD and morphology in cerebral cortex of male and female mice at an adult timpoint. We then characterized β protein levels and β4 protein-protein interactions in male and female mouse cortex. Overexpression selectively reduced small dendritic spine density but this effect was present only in female mice and did not appear to result from estrous stage. Instead, the sex-dependent effect on DSD corresponded to sex differences in the β4 interactome of male versus female mice: the VGCC β subunit β1b was significantly enriched in the β4 interactome of brain tissue of male mice, and thus may have served to mitigate VGCC overexpression-mediated spine loss in male mice.

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The voltage-gated calcium channel (VGCC) subunit complex is comprised of the α1 subunit, the ion permeable channel, and three auxiliary subunits: β, α2δ and γ. β is the most extensively studied auxiliary subunit and is necessary for forward trafficking of the α1 subunit to the plasma membrane. VGCCs mediate voltage-dependent movement of calcium ions into neuronal cytoplasm, including at dendrites, where intracellular calcium spikes initiate signaling cascades that shape the structural plasticity of dendritic spines. Genetic studies strongly implicate calcium signaling dysfunction in the etiology of neurodevelopmental disorders including schizophrenia. Dendritic spine density is significantly decreased in schizophrenia in primary auditory cortex where it is driven by loss of small spines, and small spine loss associated with increased peptide levels of ALFDFLK found in the VGCC β subunit β4. Overexpessing CACNB4 selectively reduced small spine density in cortical neuron cultures. We set out to validate this observation in an intact mammalian system within the relevant neurodevelopmental context. We overexpressed CACNB4 in early development and assessed spine density and morphology in adult male and female mouse cortex. We characterized β1–4 protein levels and β4 protein-protein interactions. Overexpression selectively reduced small spine density in female mice. This effect was not dependent on estrous stage. Instead it corresponded to sex differences in the murine β4 interactome. The VGCC subunit β1b was significantly enriched in the β4 interactome of male relative to female mice, and thus may have served to mitigate VGCC overexpression-mediated spine loss in male mice.