Developmental trajectories of isolation-induced ultrasonic vocalizations in rat pups exposed to early life stress.

Rodent infants emit ultrasonic vocalizations (USVs) when separated from the nest, producing a communicative signal that elicits maternal care. These calls are considered as a sign of isolation distress, and call emission is acutely altered by stressful early life experiences. However, the selective developmental trajectory of these stress-induced USV alterations remains unknown and may have implications for neurodevelopmental processes underlying social dysfunction. Here, we employed a 3 h/day maternal separation (MS) procedure in Wistar rat pups (male and female) and recorded isolation-induced USVs every third day between the postnatal days (PND) 2-14. We observed systematic changes in both the quantity and spectro-temporal acoustic features of USVs, including shifts in frequency, duration and temporal emission patterns. Clustering of acoustic features revealed two USV types emerging after PND 8: short, high-frequency calls and highly frequency-modulated calls, alongside earlier flat, low-frequency calls. MS reduced overall USV emission on PND 8, selectively reduced and shortened flat low-frequency calls on PND 11, and shortened both low- and high frequency calls on PND 14. MS also disrupted the temporal organization of USVs including inter-vocalization intervals and bout structure across development. In contrast, the developmental emergence and diversification of high-frequency and high frequency-modulated call types were largely preserved under MS. These findings suggest a maturation process in rat pup vocalizations between PND 8-11, characterized by a rapid increase in acoustic complexity and changes in temporal structure, while early life stress exerts selective, time-specific effects on particular call categories, notably reducing emission and shortening flat low-frequency calls.