Reduction in ultrasonic vocalizations in pups born to rapid eye movement sleep restricted mothers in rat model

Abstract

The effects of rapid eye movement sleep restriction (REMSR) in rats during late pregnancy were studied on the ultrasonic vocalizations (USVs) made by the pups. USVs are distress calls inaudible to human ears. Rapid eye movement (REM) sleep was restricted in one group of pregnant rats for 22 hours, starting from gestational day 14 to 20, using standard single platform method. The USVs of male pups were recorded after a brief isolation from their mother for two minutes on alternate post-natal days, from day one till weaning. The USVs were recorded using microphones and were analysed qualitatively and quantitatively using SASPro software. Control pups produced maximum vocalization on post-natal days 9 to 11. In comparison, the pups born to REMSR mothers showed not only a reduction in vocalization but also a delay in peak call making days. The experimental group showed variations in the types and characteristics of call types, and alteration in temporal profile. The blunting of distress call making response in these pups indicates that maternal sleep plays a role in regulating the neural development involved in vocalizations and possibly in shaping the emotional behaviour in neonates. It is suggested that the reduced ultrasonic vocalizations can be utilized as a reliable early marker for affective state in rat pups. Such impaired vocalization responses could provide an important lead in understanding mother-child bonding for an optimal cognitive development during post-partum life. This is the first report showing a potential link between maternal REM sleep deprivation and the vocalization in neonates and infants.

Figure 1. Average no of USVs/min in pups after maternal separation (N = 19 each).

Vocalizations are presented as averaged calls/min ± SEM (Standard error of mean) in the Y axis and different developmental days on X axis. ‘D’ refers to days, Control values are represented in blue bars while the REMSR group values are shown in brown bars. The comparison of values between Control v/s REMSR group is depicted in ‘*’. The significance values are given as ‘*’ for p<0.05, ‘***’ for p<0.001.

Figure 2. Sonograph of different call types produced by pups in the isolation paradigm.

The frequency is shown in kHz on the Y axis and time scale is expressed in sec on the X axis. The CF and MF refers to constant and modulated frequency respectively. In single syllable MF category, numeral i, ii, iii and iv denotes Upward, Downward, U-shaped and Inverted U-shaped calls.

Figure 3. Distribution pattern of isolation calls during different developmental days in the control and REMSR group.

The percent distribution of various calls grouped in four categories obtained on isolation at postnatal day 1, 11 and 21 is shown in pie diagram for pups in the control and REMSR group. A denotes category consisting of flat calls; B having upwards (i), downwards (ii), U shaped (iii), and inverted-U (iv); C is sum of 2 and 3 syllable and D contains wave and complex calls.

Figure 4. Distribution pattern of isolation calls grouped in four categories in the control and REMSR group.

Distribution pattern of isolation calls grouped in four categories during different developmental days in the control and REMSR group. Developmental days are shown in horizontal axis and averaged numbers of calls are shown in Y axis.

Figure 5. Distribution pattern of dot calls during different developmental days in the control and REMSR group.

Distribution pattern of dot calls during different developmental days in the control and REMSR group. Developmental days are shown in horizontal axis and averaged numbers of calls are shown in Y axis.

Figure 6. Plot of excess time spent in ultrasonic vocalization by the pups in sleep challenged mothers.

Plot of excess time spent in ultrasonic vocalization by the pups in sleep challenged mothers (difference taken from total time spent in vocalization by control pups) during testing days in the isolation paradigm.

Figure 7. Representative intensities in 2-syllable ultrasonic calls made by pups in control and sleep challenged mothers.

Sonograph of 2-syllable ultrasonic calls showing alteration in intensities (depicted in colour coding of increasing intensity from blue to red) obtained in control and the REMSR group pups on day 11.

Figure 8. Latency of retrieval of first pup and time taken for retrieving all the pups on different postnatal days in control and REMSR group rats.

Latency to retrieval of first pup is shown in A. The post-natal days are shown on horizontal axis and time in sec is shown on the vertical axis. The open bars depict Control and closed bars depict REMSR group data. The hatched closed bar depict retrieval by few mothers (such number of cases are written above the bar).