Socio-affective communication in Tph2-deficient rat pups: communal nesting aggravates growth retardation despite ameliorating maternal affiliation deficits

Abstract

Background: A lack of serotonin (also known as 5-hydroxytryptamine, 5-HT) in the brain due to deficiency of the rate-limiting enzyme in 5-HT synthesis, tryptophan hydroxylase 2 (TPH2), was recently reported to result in impaired maternal affiliation across species, including mice, rats, and monkeys. In rodents, this was reflected in a lack of preference for maternal odors and reduced levels of isolation-induced ultrasonic vocalizations (USV), possibly contributing to a severe growth retardation phenotype.

Methods: Here, we tested whether growth retardation, maternal affiliation deficits, and/or impairments in socio-affective communication caused by Tph2 deficiency can be rescued through early social enrichment in rats. To this aim, we compared male and female Tph2^-/- knockout and Tph2^+/- heterozygous rat pups to Tph2^+/+ wildtype littermate controls, with litters being randomly assigned to standard nesting (SN; one mother with her litter) or communal nesting (CN; two mothers with their two litters).

Results: Our results show that Tph2 deficiency causes severe growth retardation, together with moderate impairments in somatosensory reflexes and thermoregulatory capabilities, partially aggravated by CN. Tph2 deficiency further led to deficits in socio-affective communication, as evidenced by reduced emission of isolation-induced USV, associated with changes in acoustic features, clustering of subtypes, and temporal organization. Although CN did not rescue the impairments in socio-affective communication, CN ameliorated the maternal affiliation deficit caused by Tph2 deficiency in the homing test. To close the communicative loop between mother and pup, we assessed maternal preference and showed that mothers display a preference for Tph2^+/+ controls over Tph2^-/- pups, particularly under CN conditions. This is consistent with the aggravated growth phenotype in Tph2^-/- pups exposed to the more competitive CN environment.

Conclusion: Together, this indicates that CN aggravates growth retardation despite ameliorating maternal affiliation deficits in Tph2-deficient rat pups, possibly due to reduced and acoustically altered isolation-induced USV, hindering efficient socio-affective communication between mother and pup.

Keywords: Autism; Neurodevelopmental disorders; Serotonin; Social behavior; Tryptophan hydroxylase 2; Ultrasonic vocalizations.

Fig. 1

Overview of experimental design, genotype distribution, and survival rates. (A) A heterozygous breeding strategy was applied to obtain male and female Tph2^−/− knockout and Tph2^+/− heterozygous rat pups, together with Tph2^+/+ wildtype littermate controls. Litters were randomly assigned to standard nesting (SN; one mother with her litter) or communal nesting (CN; two mothers with their two litters). An intensive health monitoring and care system was applied to mitigate for the low survival rates previously reported to be associated with Tph2 deficiency in mice and rats. (B) Genotype distribution of rat pups obtained through applying a heterozygous breeding strategy, including all N = 33 litters obtained. (C) Survival rate of Tph2^−/− (KO) and Tph2^+/− (HET) rat pups, as compared to Tph2^+/+ (WT) littermate controls; including all N = 33 litters obtained, with rat pups removed through culling from the litter to mitigate for the low survival rates not included in calculating survival rates as percentages. (D) Litter sizes of the N = 15 experimental litters from SN and CN included in the analyses on postnatal day 0 (P0) and postnatal day 3 (P3). (E) Genotype distribution of experimental rat pups included in the analyses, split according to sex

Fig. 2

Tph2 deficiency in rats causes severe growth retardation, together with moderate impairments in somatosensory reflexes and thermoregulatory capabilities, partially aggravated by communal nesting (CN), as compared to standard nesting (SN). (A) Overview of the assessment of developmental milestones, somatosensory reflexes, and thermoregulatory capabilities in Tph2^−/− (KO) and Tph2^+/− (HET) rat pups, as compared to Tph2^+/+ (WT) littermate controls. (B) Exemplary image of WT and KO rat pups on P10. (C) Exemplary thermal image of WT, HET, and KO across P2 to P14 obtained during body temperature measurements. (D) Absolute body weight of WT, HET, and KO rat pups across P2 to P14, depending on nesting condition. (D′) Normalized body weight of HET and KO rat pups across P2 to P14 relative to WT littermate controls, depending on nesting condition. (E) Body temperature before the 10-min isolation period for WT, HET, and KO rat pups across P2 to P14, depending on nesting condition. (F) Body temperature after the 10-min isolation period for WT, HET, and KO rat pups across P2 to P14, depending on nesting condition. (G) Body temperature change during the 10-min isolation period for WT, HET, and KO across P2 to P14, depending on nesting condition. (H) Upright latency in the negative geotaxis assay for WT, HET, and KO rat pups across P2 to P14, depending on nesting condition. SN = standard nesting (blue), CN = communal nesting (orange). P = postnatal day. Data are expressed as mean ± SEM. Effect of genotype: ***^(black) WT vs. KO p < .001; **^(black) WT vs. KO p < .01. ***^(gray) HET vs. KO p < .001; *^(gray) HET vs. KO p < .01; *^(gray) HET vs. KO p < .05. Effect of nesting: +  +  + p < .001; +  + p < .01; + p < .05. Interaction nesting x genotype: @@ p < .01; @ p < .05. N(WT-SN) = 16, N(HET-SN) = 18, N(KO-SN) = 19, N(WT-CN) = 12, N(HET-CN) = 12, N(KO-CN) = 17

Fig. 3

Tph2 deficiency in rats leads to severe deficits in socio-affective communication during the first two weeks of life, as evidenced by reduced call emission rates. (A) Overview of the assessment of isolation-induced ultrasonic vocalizations in Tph2^−/− (KO) and Tph2^+/− (HET) rat pups, as compared to Tph2^+/+ (WT) littermate controls. (B) Exemplary spectrograms of isolation-induced ultrasonic vocalizations emitted by WT, HET, and KO on P12, depending on nesting condition. (C) Average number of isolation-induced ultrasonic vocalizations emitted across P2 to P14. (D) Average total calling time across P2 to P14. Developmental trajectories of (C′) the number of isolation-induced ultrasonic vocalizations and (D′) total calling time across P2 to P14. SN = standard nesting (blue), CN = communal nesting (orange). P = postnatal day. Data are expressed as mean ± SEM. Effect of genotype: ***^(black) WT vs. KO p < .001; **^(black) WT vs. KO p < .01. ***^(gray) HET vs. KO p < .001; Effect of nesting: + p < .05. N(WT-SN) = 16, N(HET-SN) = 18, N(KO-SN) = 19, N(WT-CN) = 12, N(HET-CN) = 12, N(KO-CN) = 17

Fig. 4

Tph2 deficiency in rats is results in changes in the acoustic features of isolation-induced ultrasonic vocalizations. (A) Overview of the acoustic features of isolation-induced ultrasonic vocalizations assessed in Tph2^−/− (KO) and Tph2^+/− (HET) rat pups, as compared to Tph2^+/+ (WT) littermate controls: call duration, peak frequency, peak amplitude, and frequency modulation. (B) Average call duration across P2 to P14. (C) Average peak frequency across P2 to P14. (D) Average peak amplitude across P2 to P14. (E) Average frequency modulation across P2 to P14. Developmental trajectories of (B′) call duration, (C′) peak frequency, (D′) peak amplitude, and (E′) frequency modulation of isolation-induced ultrasonic vocalizations emitted across P2 to P14. SN = standard nesting (blue), CN = communal nesting (orange). P = postnatal day. Data are expressed as means ± SEM. Effect of genotype: ***^(black) WT vs. KO p < .001; **^(black) WT vs. KO p < .01; *^(black) WT vs. KO p < .05. ***^(gray) HET vs. KO p < .001; **^(gray) HET vs. KO p < .01; *^(gray) HET vs. KO p < .05. Effect of nesting: +  +  + p < .001; + p < .05. N(WT-SN) = 16, N(HET-SN) = 18, N(KO-SN) = 19, N(WT-CN) = 12, N(HET-CN) = 12, N(KO-CN) = 17

Fig. 5

Tph2 deficiency in rats is associated with changes in the clustering of subtypes of isolation-induced ultrasonic vocalizations. (A-L) Density plots depicting the distribution of individual isolation-induced ultrasonic vocalizations in Tph2^−/− (KO) and Tph2^+/− (HET) rat pups, as compared to Tph2^+/+ (WT) littermate controls, depending on nesting condition, i.e. WT-SN (A, G; ~ 35,000 calls), WT-CN (D, J; ~ 24,000 calls), HET-SN (B, H; ~ 34,000 calls), HET-CN (E, K; ~ 25,000 calls), KO-SN (C, I; ~ 20,000 calls), and KO-CN (F, L; ~ 14,000 calls). SN = standard nesting (blue), CN = communal nesting (orange). Color coding reflects frequencies as percentages. Individual isolation-induced ultrasonic vocalizations depicted here were recorded on postnatal day 12

Fig. 6

Tph2 deficiency in rats affects the temporal organization of the emission of isolation-induced ultrasonic vocalizations. (A-C) Correlation coefficients (r) between call durations reflecting the sequential organization of isolation-induced ultrasonic vocalizations in Tph2^−/− (KO) and Tph2^+/− (HET) rat pups, compared to Tph2^+/+ (WT) littermate controls. Correlation coefficients (r) indicate the level of correlations between the durations of a given isolation-induced ultrasonic vocalizations with the call durations of the previous ones (A, N-1), the ones two before (B, N-2), and the ones three before (C, N-3) on postnatal days (P) 4, 8, and 12. SN = standard nesting (blue), CN = communal nesting (orange). Data are presented as mean ± SEM. Effect of age: *** p < .001; ** p < .01: * p < .05

Fig. 7

Tph2 deficiency in rats leads to maternal affiliation deficits, which can be rescued through communal nesting (CN), as compared to standard nesting (SN). (A) Overview of the homing test as a proxy for maternal affiliation in in Tph2^−/− (KO) and Tph2^+/− (HET) rat pups, compared to Tph2^+/+ (WT) littermate controls. (B) Maternal affiliation as measured through the time spent in the clean bedding zone versus the soiled bedding zone containing maternal odors, depending on nesting condition. (C) Total number of line crossings, depending on nesting condition. (D) Total number of isolation-induced ultrasonic vocalizations, depending on nesting condition. (E) Total calling time, depending on nesting condition. SN = standard nesting (blue), CN = communal nesting (orange). Data are expressed as mean ± SEM. Effect of genotype: *** p < .001; Effect of zone: ### p < .001, ## p < .01. N(WT-SN) = 16, N(HET-SN) = 18, N(KO-SN) = 19, N(WT-CN) = 12, N(HET-CN) = 12, N(KO-CN) = 17

Fig. 8

Tph2 deficiency in rats causes a reduction in maternal preference, which is emphasized through communal nesting (CN), as compared to standard nesting (SN). (A) Overview of the maternal preference test to close the communicative loop between mother and pup and to compare maternal preferences between Tph2^−/− (KO) rat pups and Tph2^+/+ (WT) littermate controls. (B) Time spent by the mother in proximity to WT versus KO rat pups. Of note, the individual data points of an individual WT rat pup and an individual KO rat pup are connected by a line in order to indicate that the two connected rat pups were simultaneously exposed to their mother. Mothers were always exposed to their own pups. SN = standard nesting (blue), CN = communal nesting (orange). Data are expressed as mean ± SEM. Effect of pup genotype: *p < .05. N(SN) = 12 WT-KO pairs, N(CN) = 12 WT-KO pairs