Loss of cGMP-dependent protein kinase II alters ultrasonic vocalizations in mice, a model for speech impairment in human microdeletion 4q21 syndrome

Abstract

Chromosome 4q21 microdeletion leads to a human syndrome that exhibits restricted growth, facial dysmorphisms, mental retardation, and absent or delayed speech. One of the key genes in the affected region of the chromosome is PRKG2, which encodes cGMP-dependent protein kinase II (cGKII). Mice lacking cGKII exhibit restricted growth and deficits in learning and memory, as seen in the human syndrome. However, vocalization impairments in these mice have not been determined. The molecular pathway underlying vocalization impairment in humans is not fully understood. Here, we employed cGKII knockout (KO) mice as a model for the human microdeletion syndrome to test whether vocalizations are affected by loss of the PRKG2 gene. Mice emit ultrasonic vocalizations (USVs) to communicate in social situations, stress, and isolation. We thus recorded ultrasonic vocalizations as a model for human speech. We isolated postnatal day 5-7 pups from the nest to record and analyze USVs and found significant differences in vocalizations of KO mice relative to wild-type and heterozygous mutant mice. KO mice produced fewer calls that were shorter duration and higher frequency. Because neuronal activation in the arcuate nucleus in the hypothalamus is important for the production of animal USVs following isolation from the nest, we assessed neuronal activity in the arcuate nucleus of KO pups following isolation. We found significant reduction of neuronal activation in cGKII KO pups after isolation. Taken together, our studies indicate that cGKII is important for neuronal activation in the arcuate nucleus, which significantly contributes to the production of USVs in neonatal mice. We further suggest cGKII KO mice can be a valuable animal model to investigate pathophysiology of human microdeletion 4q21 syndrome.

Keywords: And the arcuate nucleus; Human microdeletion 4q21 syndrome; Speech impairment; Ultrasonic vocalization; cGMP-dependent protein kinase II.

Figure 1.. Spectrogram of mouse pup ultrasonic vocalizations.

Spectrogram generated from P5-P7 pup isolation calls using Avisoft SASLab Pro software. Four individual vocalizations are shown in the representative spectrogram. The horizontal axis shows time (in seconds) from which duration of each USV could be determined as well as a total duration of vocalization during the 5-minute testing period. The vertical axis shows frequency (kHZ) of vocalizations emitted by mouse pups. The amplitude of calls (dB) can also be determined by the color (scale shown in top bar).

Figure 2.. Abnormal ultrasonic vocalizations in isolated cGKII KO pups relative to WT and HET pups.

a) Total number of USVs emitted, b) duration of individual calls, c) peak frequency, d) peak amplitude (or intensity), and e) total duration of calls in each genotype during 5-minute isolation period. (n=14 WT, 12 KO, and 26 HET animals, *p<0.05 and **p<0.01, one-way ANOVA, Tukey test).

Figure 3.. cGKII protein expression levels in each genotype.

Representative immunoblots of cGKII protein expression in brain tissues from 4 animals in each genotype. Actin protein expression is used as a loading control. Normalized cGKII protein levels are shown in graph below. KO pups have no protein expression, while WT and HET pups have the same amount of protein present.

Figure 4.. Reduction of neuronal activation in the arcuate nucleus in isolated cGKII KO pups.

Left panels: Representative c-Fos immunoreactivity (Magenta), DAPI (Blue), merge images, and c-Fos signals colocalized with DAPI images in each condition. The graph on the right shows that neuronal activation is significantly reduced in the KO arcuate nucleus following isolation from the nest (n= WT Nest; 39 sections from 4 animals, WT Isolation; 19 sections from 4 animals, KO Nest; 26 sections from 4 animal, and KO Isolation; 19 sections from 4 animals, *p<0.05 and ****p<0.0001, Kruskal-Walls test, Dunn test). A scale bar indicates 100μm. 3V, the third ventricle.