Npas4 regulates synaptic development in the patch/striosome compartment and early affective vocalization in neonatal mice

Early vocal communication, such as isolation-induced ultrasonic vocalization (USV) in neonatal rodents, is critical for infant survival and represents a primary readout of early affective states. The patch/striosome compartment in the striatum has been proposed to serve as a limbic-motor interface integrating emotion and motivation. Developmentally, this compartment undergoes early maturation, during which activity-dependent transcription factors might enable the functional assembly of limbic-striosomal circuits to regulate infant vocal-motor behavior. Npas4, an activity-dependent early-response transcription factor, controls the developmental balance of excitatory and inhibitory synaptic activity. Here, we investigated how patch/ striosome compartment-expressing Npas4 shapes early synaptic development and neonatal vocalization. We found that Npas4 transcripts were transiently enriched in patch/striosomes at postnatal days (P) 4 and P8, before adopting a homogenous striatal distribution by P14. To investigate its biological function, we generated a patch/striosome-specific Npas4 conditional knockout. Anatomically, Npas4 deletion disrupted patch/striosomal synaptic balance, evidenced by a significant reduction in excitatory (Vglut1) and an increase in inhibitory (Vgat) presynaptic terminals. Behaviorally, acoustic and Markov-chain syntactical analyses of isolation-induced USVs revealed a hyper-vocal phenotype at P8, characterized by a significant increase in total USV call numbers and augmented temporal clustering of vocal bouts and sequences. Furthermore, acoustic analysis demonstrated a shift toward more complex syllable types. While the structure of global transition networks was preserved, Npas4 knockout mice exhibited an elevated sequence entropy rate, indicating greater moment-to-moment structural variability. Our study suggests that Npas4 shapes the development of limbic-striosomal circuits to provide top-down gating of early affective vocalization in neonates.