Stress Exposure in Dopamine D4 Receptor Knockout Mice Induces Schizophrenia-Like Behaviors via Disruption of GABAergic Transmission

Abstract

A combination of genetic and environmental risk factors has been considered as the pathogenic cause for mental disorders including schizophrenia. Here, we sought to find out whether the abnormality of the dopamine system, coupled with the exposure to modest stress, is sufficient to trigger the manifestation of schizophrenia-like behaviors. We found that exposing dopamine D4 receptor knockout (D4KO) mice with 1-week restraint stress (2 h/d) induced significant deficits in sensorimotor gating, cognitive processes, social engagement, as well as the elevated exploratory behaviors, which are reminiscent to schizophrenia phenotypes. Electrophysiological studies found that GABAergic transmission was significantly reduced in prefrontal cortical neurons from stressed D4KO mice. Additionally, administration of diazepam, a GABA enhancer, restored GABAergic synaptic responses and ameliorated some behavioral abnormalities in stressed D4KO mice. These results have revealed that the combination of 2 key genetic and environmental susceptibility factors, dopamine dysfunction and stress, is a crucial trigger for schizophrenia-like phenotypes, and GABA system in the prefrontal cortex is a downstream convergent target that mediates some behavioral outcomes.

Keywords: GABA; dopamine D4 receptor; schizophrenia; stress.

Fig. 1.

Stressed D4KO mice exhibit deficits in sensorimotor gating, cognition, and sociability. (A) Plot of the startle response to white noise background (66 dB), and different acoustic stimulus intensities (90, 100, 110, and 120 dB) in non-stressed wild-type (WT) mice (WT, n = 6), WT mice with 7-d restraint stress (Stress, n= 9), non-stressed D4KO mice (D4KO, n = 7), and D4KO mice with 7-d restraint stress (D4KO+Stress, n = 12). (B) Plot of PPI with the pre-pulse intensity of 70, 76 and 85 dB in the 4 mouse groups. (C) Plot of discrimination ratio in TOR tests of WT (n = 11), Stress (n = 6), D4KO (n = 10), and D4KO+Stress (n = 9) mice. (D) Plot of interaction time with novel and old objects in TOR tests of the 4 mouse groups. (E, F) Representative heat maps (E) and plot of social contact time and social approach numbers (F) in the social engagement test of the 4 mouse groups (n = 6 per group). *P < .05, **P < .01, 2-way ANOVA.

Fig. 2.

Stressed D4KO mice display elevated exploratory behaviors, but no anxiety or depression-like behaviors. (A, B) Representative heat maps (A) and plot of time in the open arm (B) of the elevated plus maze test of wild-type (WT; n = 6), Stress (n = 11), D4KO (n = 8), and D4KO+Stress (n = 11) mice. (C) Plot of the percentage of sucrose preference on day 4 of sucrose preference test of WT (n = 7), Stress (n = 9), D4KO (n = 7), and D4KO+Stress (n = 12) mice. (D) Plot of total moving distance in the locomotion test of WT (n = 11), Stress (n = 10), D4KO (n = 12), and D4KO+Stress (n = 11) mice. (E) Plot of total immobility time in the forced-swimming test of WT (n = 7), Stress (n = 5), D4KO (n = 10), and D4KO+Stress (n = 10) mice. *P < .05, **P < .01, 2-way ANOVA.

Fig. 3.

Stressed D4KO mice show little changes in excitatory synaptic signaling in prefrontal cortex. (A, B, C) Input-output curves of AMPAR-EPSC (A), representative traces (B), and plot of paired-pulse ratio of AMPA-EPSC (C) in prefrontal cortex (PFC) pyramidal neurons from wild-type (WT; n = 20 cells), Stress (n = 15 cells), D4KO (n = 21 cells), and D4KO+Stress (n = 12 cells) mice. (D, E, F) Miniature EPSC amplitude (D), frequency (E) and representative traces (F) in PFC pyramidal neurons from WT (n = 30 cells), Stress (n = 11 cells), D4KO (n = 20 cells), and D4KO+Stress (n = 18 cells) mice. (G, H) Input-output curves (G) and representative traces (H) of evoked NMDAR-EPSC in PFC pyramidal neurons from WT (n = 9 cells), Stress (n = 15 cells), D4KO (n = 14 cells), and D4KO+Stress (n = 8 cells) mice.

Fig. 4.

Stressed D4KO mice have the significantly diminished GABAergic synaptic inhibition in prefrontal cortex. (A, B, C) Input-output curves of GABA_AR-IPSC (A), representative traces (B), and plot of paired-pulse ratio of GABA_AR-IPSC (C) in prefrontal cortex (PFC) pyramidal neurons from wild-type (WT; n = 23 cells), Stress (n = 15 cells), D4KO (D4KO, n = 11 cells), and D4KO+Stress (n = 17 cells) mice. (D, E, F) Miniature IPSC amplitude (D), frequency (E) and representative traces (F) in PFC pyramidal neurons from WT (n = 21 cells), Stress (n = 24 cells), D4KO (n = 12 cells), and D4KO+Stress (n = 22 cells) mice. *P < .05, **P < .01, ***P < .001; 2-way ANOVA.

Fig. 5.

Stressed D4KO mice have the significantly reduced number of parvalbumin (PV)-positive interneurons in the prefrontal cortex. (A) Representative confocal images of PV staining in prefrontal cortex (PFC) slices from wild-type (WT) and D4KO mice without or with 7-d restraint stress. (B) Bar graphs of the number of PV+ interneurons in PFC slices from WT (n = 23 slices), Stress (n = 16 slices), D4KO (D4KO, n = 46 slices), and D4KO+Stress (n = 37 slices) mice (3–4 each group). *P < .05, **P < .01, ***P < .001, 2-way ANOVA.

Fig. 6.

The GABA enhancer diazepam elevates GABAergic responses and reverses some behavioral abnormalities in stressed D4KO mice. (A) Input-output curves of GABA_AR-IPSC in prefrontal cortex (PFC) pyramidal neurons from wild-type (WT) or stressed D4KO mice injected with saline (KO+S+sal, n = 15 cells) or diazepam (KO+S+DZ, n = 11 cells). Inset: representative GABA_AR-IPSC traces. (B) Bar graphs of spontaneous IPSC amplitude and frequency in PFC pyramidal neurons from stressed D4KO mice injected with saline (KO+S+sal, n = 11 cells) or DZ (KO+S+DZ, n = 16 cells). Inset: representative sIPSC traces. (C) Plot of basal startle response to different acoustic stimulus intensities in WT mice injected with saline (WT+sal, n = 5) or diazepam (WT+DZ, n = 4), or stressed D4KO mice injected with saline (KO+S+sal, n = 9) or diazepam (KO+S+DZ, n = 12). (D) Plot of PPI with the pre-pulse intensity of 70, 76 and 85 dB in the 4 groups. (E) Plot of discrimination ratio in TOR tests of WT+sal (n = 5), KO+S+sal (n = 9), and KO+S+DZ (n = 12) mice. (F) Plot of time in the open arm of elevated plus maze test from WT+sal (n = 5), KO+S+sal (n = 9) and KO+S+DZ (n = 12) mice. (G) Plot of social contact time and social approach numbers in the social engagement test of WT+sal (n = 5), KO+S+sal (n = 6) and KO+S+DZ (n = 6) mice. (H, I) Plot of the latency to fall in the rotarod test (H) and total moving distance within 10 and 30 min of the locomotion test (I) from the 3 groups. (A, D, E, F, G): *P < .05, **P < .01; ANOVA. (B): ***P < .001, unpaired t test.