Postnatal environment affects auditory development and sensorimotor gating in a rat model for autism spectrum disorder

Abstract

The homozygous Cntnap2 knockout (KO) rat is a well-established genetic model for neurodevelopmental disorders, exhibiting core features of autism spectrum disorder (ASD), including impaired sensory processing and sensorimotor gating. Recent findings indicate that the severity of ASD-like phenotypes in Cntnap2 KO offspring is influenced by the parental genotype, with more pronounced impairments observed in KO rats bred from homozygous pairs compared to heterozygous pairs (Cntnap2 HET). However, it is unclear to what extent this is due to in utero versus postnatal effects. We, therefore, investigated how early postnatal environmental factors, shaped by differences in parental and littermate genotypes, influence auditory processing and sensorimotor gating in Cntnap2 KO rats. To examine this, we cross-fostered Cntnap2 KO pups bred from Cntnap2 KO rats to be reared with litters of Cntnap2 HET dams. Cross-fostering Cntnap2 KO rats reversed or partially reversed delayed hearing sensitivity maturation, heightened acoustic startle responses, and deficits in prepulse inhibition of the acoustic startle response. However, cross-fostering also exacerbated deficits in the neural responsiveness and conductivity in the auditory brainstem, as well as in gap-induced prepulse inhibition of the acoustic startle response. These results emphasize the importance of considering the postnatal environment and breeding strategies in preclinical genetic models of neuropsychiatric disorders. More importantly, they also demonstrate that ASD-like traits, including alterations in brainstem sensory processing, are not strictly determined by genetic factors, but remain malleable by environmental factors during early postnatal development.

Keywords: CNTNAP2; auditory brainstem response; auditory processing; gap detection; neurodevelopmental disorders; prepulse inhibition; rat; startle.

Figure 1

Cross-fostering illustration and summary of experimental timeline. (A) A breeding round consisted of breeding Cntnap2 KO (Cntnap2- KO × Cntnap2 KO) and Cntnap2 HET (Cntnap2 HET × Cntnap2 HET) rats. Experimental groups included: Cntnap2 WT animals bred by Cntnap2 HET rats and reared by a Cntnap2 HET dam (Cntnap2 WT; blue), Cntnap2 KO animals bred by Cntnap2 HET rats and reared by a Cntnap2 HET dam (Cntnap2 KO^het; red), Cntnap2 KO animals bred by Cntnap2 KO rats and reared by a Cntnap2 KO dam (Cntnap2 KO^hom; purple), and Cntnap2 KO animals bred by Cntnap2 KO rats and cross-fostered to be reared by a Cntnap2 HET dam (Cntnap2 KO^CF; green). (B) Experimental timeline outlining the postnatal days (PND) for cross-fostering and behavioral assessments. The auditory brainstem response (ABR) was assessed during the juvenile period and adulthood, while the acoustic startle response and prepulse inhibition (PPI) were evaluated in adulthood.

Figure 2

Cross-fostered Cntnap2 knockout rats have improved development of hearing sensitivity but reduced neural responsiveness and conduction within the auditory brainstem pathway. (A) An example of the acoustically evoked ABRs from a rat in response to click stimuli of increasing sound level with a red arrow to indicate the ABR threshold. Below is a magnification of an ABR trace from the same animal in response to a 70 dB SPL click stimulus. Waves I, II, and IV reflect the synchronized neural activity in the auditory nerve, cochlear nucleus, and inferior colliculus/lateral lemniscus, respectively. Measurements show how amplitude (from negative to positive peak; blue) and peak latency (negative peak; orange) were determined. (B) Change in ABR threshold to click stimuli with age. The hearing threshold of Cntnap2 KO^hom rats does not improve between juvenile age and adulthood as observed in WT and KO^het rats. Cntnap2 KO^CF rats exhibit typical maturation of hearing threshold with age, suggesting they have restored development of hearing sensitivity. (C) Juvenile ABR peak amplitudes. Group WIV/I ratios are indicated under the x-axis. Juvenile Cntnap2 KO^hom and KO^het rats exhibit reduced peak amplitudes for WII and WIV, respectively, compared to WT rats. The WIV amplitude of Cntnap2 KO^CF rats is trending toward being lower than WT rats. (D) Adult ABR peak amplitudes. Group WIV/I ratios are indicated under the x-axis. The reduced peak amplitudes observed in juvenile Cntnap2 KO^hom and KO^het rats do not persist in adulthood. In contrast, Cntnap2 KO^CF rats display reduced WI amplitude compared to WT rats. (E) ABR peak latencies across age. Only juvenile Cntnap2 KO^het rats show slower WII and WIV latencies. This is normalized by adulthood. (F) ABR interpeak latencies. In adulthood, Cntnap2 KO^hom and KO^CF exhibit slower WII-IV interpeak latencies. Adult Cntnap2 KO^CF rats also show a slower WI-IV interpeak latency, indicative of overall slower conduction of the ABR. *p < 0.05, **p < 0.01, ***p < 0.0001.

Figure 3

Cross-fostered Cntnap2 knockout males show less exaggerated startle responses. (A) Baseline startle response curves. Black arrows point to the maximum startle response value (Top) for WT animals. Goodness of fit Sy.x: male WT = 449.7, male KO^het = 2,485, male KO^hom = 2,413, male KO^CF = 2,736, female WT = 181.1, female KO^het = 841.6, female KO^hom = 932.0, female KO^CF = 2,223. (B) Maximum startle response (Top). The gray arrow indicates that there are values outside the limits of the y-axis, but the graph was zoomed in to visualize the data more clearly. Unlike male Cntnap2 KO^hom rats, KO^CF males do not have a statistically higher Top than WT males. This is not apparent in females where all Cntnap2 KO rats have a higher Top than WT rats. (C) Scaled startle response curves. Black arrows point to the threshold, ES50, and saturation point for WT animals. Goodness of fit Sy.x: male WT = 0.2048, male KO^het = 0.1263, male KO^hom = 0.1311, male KO^CF = 0.1673, female WT = 0.1956, female KO^het = 0.1961, female KO^hom = 0.1823, female KO^CF = 0.2003. (D) Slope. In males and females, Cntnap2 KO^hom rats but not KO^CF rats have a lower slope than WT rats. (E) Threshold. Male Cntnap2 KO^CF rats have a higher threshold than KO^hom rats, but still a lower threshold than WT rats. (F) ES50. Male Cntnap2 KO^CF rats have a higher ES50 than KO^hom rats, but still a lower ES50 than WT rats. (G) Saturation. There are no group differences in saturation point. *p < 0.05, **p < 0.01, ***p < 0.0001, ns indicates non-significance of the comparison.

Figure 4

Cross-fostered Cntnap2 knockout males and females show improved PPI. (A) PPI (% inhibition) across startle stimulus intensity levels with the 75 dB prepulse. # denotes startle stimulus intensities where there were group differences in PPI. Stimulus levels highlighted by gray rectangles indicate significant improvements by cross-fostering and are presented in the bottom panel. (B) PPI analysis across startle stimulus intensity with the 85 dB prepulse. (C) PPI of males with 100 dB startle stimulus with a 75 dB prepulse. PPI in male Cntnap2 KO^CF is not different from WT. (D) PPI in males at the 110 dB startle stimulus with a 75 dB prepulse. Again, PPI of male Cntnap2 KO^CF is not different from WT. (E) PPI of females at the 110 dB startle stimulus with a 75 dB prepulse. PPI of Cntnap2 KO^CF females is not different from WT females. (F) PPI of males at the 100 dB startle stimulus with an 85 dB prepulse. PPI of Cntnap2 KO^CF males is not different from WT males. *p < 0.05, **p < 0.01, ***p < 0.0001.

Figure 5

Gap-PPI impairment found in Cntnap2 knockout rats is not improved by cross-fostering. (A) PPI (% inhibition) with short gap lengths (2 ms, 5 ms, and 10 ms). Male Cntnap2 KO^hom showed a lower PPI with a 5 ms gap than WT rats, whereas KO^CF rats had a lower %PPI than WT rats at all short gap lengths. PPI did not differ between female Cntnap2 WT and KO rats for short gap lengths. (B) PPI with long gap lengths (20 ms, 40 ms, 50 ms, 75 ms, and 100 ms). All Cntnap2 KO males had a lower PPI than WT males for gap lengths between 40 ms and 100 ms. At 20 ms, Cntnap2 KO^CF males had a lower PPI than KO^hom males. Cntnap2 KO^hom and KO^CF females had lower PPI than WT females with a 40 ms gap length. Female Cntnap2 KO^CF but not KO^hom showed a lower PPI than WT with a 50 ms gap length, and female Cntnap2 KO^hom but not KO^CF had a lower PPI than WT with a 75 ms gap length. # denotes gap lengths where there were group differences in PPI.