<i>Cacna1b</i> alternative splicing is linked to associative learning

Voltage-gated Ca_V2.2 channels are essential for neurotransmitter release throughout the nervous system including areas related to learning and memory like the hippocampus. Previous results have shown that Ca_V2.2 channels are involved in cognitive processes. However, a link between alternative splicing of the <i>Cacna1b</i> (gene that encodes for Ca_V2.2) pre-mRNA and cognitive processes has not been described. The <i>Cacna1b</i> pre-mRNA undergoes extensive cell-specific alternative splicing. In this body of work, we focus on the cassette exon 18a. Alternative splicing of exon 18a generates two splice variants, +18a-<i>Cacna1b</i> and Δ18a-<i>Cacna1b</i>. Exon 18a encodes a 21-amino acid sequence within the SYNaptic PRotein INTeraction (<i>synprint</i>) site. Splice variants containing exon 18a (+18a-Ca_V2.2) show reduced cumulative inactivation and increased Ca²⁺ current density compared to splice variants lacking exon 18a (Δ18a-Ca_V2.2), suggesting functional specialization. We previously showed that +18a-<i>Cacna1b</i> splice variants are enriched in cholecystokinin-expressing interneurons (CCK⁺INs). This neuronal type is strongly implicated in associative learning. Therefore, we tested whether alternative splicing of exon 18a contributes to associative learning. To test this hypothesis, we used genetically engineered mice that constitutively express either +18a-<i>Cacna1b</i> (+18a) or Δ18a-<i>Cacna1b</i> (Δ18a). We first validated that restricted splicing of exon 18a did not alter downstream alternative or constitutive spliced exons in the <i>Cacna1b</i> pre-mRNA, nor total Ca_V2.2 protein levels. We then performed a comprehensive behavioral analysis that included assessment of associate learning. We found that in the trace fear conditioning task, +18a mice exhibited less freezing during the trace interval in both the acquisition and memory phases compared to WT mice. Whereas Δ18a mice showed enhanced freezing during the same intervals relative to WT mice. These bidirectional phenotypes reveal that exon 18a shapes aversive associative learning. Furthermore, exon 18a splicing did not influence spatial working memory, spatial navigation under stress, nociceptive responses in basal and inflammatory conditions, overall locomotion or exploratory behavior. These results suggest that the behavioral impact of exon 18a splicing is highly selective. Together, our findings identify alternative splicing of exon 18a as a molecular contributor to associative learning.