Dopamine D2 receptor modulating mPFC-BLA circuit contributes to chronic sleep deprivation-induced memory impairment in mice

Abstract

Background: Chronic sleep deprivation (CSD) affects the orchestration of neural networks, leading to cognitive impairment, but the underlying molecular and neural circuitry mechanisms remain unknown. Methods: Mice underwent a two-week CSD regimen, followed by spatial memory assessment using the Y-maze test and EEG gamma oscillation analysis. Dopamine D2 receptor (Drd2) expression in the medial prefrontal cortex (mPFC) was evaluated using transcriptomic and immunofluorescent analysis. The role of Drd2 in CSD-induced memory deficits was examined through local infusion of Drd2 agonists or antagonists into the mPFC. Neural circuit tracing, fiber photometry, and opto-chemogenetic approaches were used to assess Drd2 in the gating of the mPFC-basolateral amygdala (BLA) circuit-mediated memory impairment induced by CSD. Results: CSD disinhibited dopaminergic input to the mPFC and impaired spatial memory in mice. A significant increase in Drd2 expression was found in the layers II/III of the mPFC after CSD. Infusion of Drd2 agonist into the mPFC induced memory deficits in naïve mice, while administration of the Drd2 antagonist reversed memory impairment caused by CSD. Drd2 was found to co-localize with Ca²⁺/calmodulin-dependent protein kinase IIα (CaMKIIα⁺) neurons in the mPFC that project to the basolateral amygdala (BLA). Activation of CaMKIIα⁺ neurons restored memory impairment induced by CSD through enhancing mPFC-to-BLA output and reversed memory defects induced by the Drd2 agonist. Conclusion: Our findings demonstrated that excessive Drd2 signaling leads to cognitive impairment following CSD by suppressing mPFC-BLA neurotransmission, suggesting a possible therapeutic value of dopamine D2 receptor antagonists in relieving CSD-induced cognitive decline.

Keywords: Basolateral amygdala; Chronic sleep deprivation; Dopamine D2 receptor; Medial prefrontal cortex; Memory impairment.

Figure 1

Chronic sleep deprivation induced learning and memory impairment. (A) Left, schematic drawings of the REM sleep deprivation. Middle, experimental design. Right, Schematic drawings of the Y-maze test. Con, the control group; CSD, the chronic sleep deprivation group; SD, sleep deprivation; S, sleep; EEG, electroencephalogram. (B) Representative heatmap tracing during the Y-maze test. (C) Quantification of average speed and novel arm preference index. (D) Representative signals of EEG before and after the mice entered the novel arm of the Control and CSD groups. (E) Representative spectrogram of the gamma oscillations before and after the mice entered the novel arm between the Control and CSD groups. (F) Representative traces of power spectral density of the EEG before and after the mice entered the novel arm between the Control (left) and CSD (right) groups. (G) Quantification of change in average gamma oscillation pre- and post-novel arm entry. For the Y-maze test, n = 14 mice for each group; For EEG recording, n = 19 trials from 7 mice for the Control group, n = 14 trials from 9 mice for the CSD group.

Figure 2

Chronic sleep deprivation increased dopamine D2 receptor gene expression in the mPFC. (A) Experimental design. SD, sleep deprivation; S, sleep. (B) Volcano plot of mPFC gene expression after CSD. padj, adjusted p-value; ns, no significance. (C) Heatmaps showing differentially expressed genes in the Con and CSD groups. Con, the control group; CSD, the chronic sleep deprivation group. (D) Quantification of Drd2, Npase4, and Baiap3 gene expression between the two groups. (E) Gene ontology functional analysis diagram for the CSD group. (F) Heatmaps showing the gene expression of GABA receptor subunits (Gabrs) in the Con and CSD groups. (G) Heatmaps showing the correlation of Drd2, Npase4, and Gabrs. *p < 0.05. n = 4 mice for each group.

Figure 3

Drd2 was involved in learning and memory impairment induced by chronic sleep deprivation. (A) Schematic of the micro-infusion and the Y-maze test. The black line rectangle represents a barrier to close the novel arm during stage 1 of the Y-maze test. mPFC, medial prefrontal cortex; TF, Trifluoperazine 2HCl; NS, normal saline. (B) Experimental design. SD, sleep deprivation; S, sleep. (C) Representative image showing the position of a cannula in the mPFC. (D) Representative tracing heatmaps during the Y-maze test. (E) Quantification of average speed (left) and novel arm preference index (right). Con, the control group; CSD, the chronic sleep deprivation group. n = 12 mice for each group. (F) Schematic of the micro-infusion (left) and representative image showing the position of a cannula in the mPFC (right). (G) Experimental design. (H) Representative tracing heatmaps during the Y-maze test. (I) Quantification of average speed (left) and novel arm preference index (right). QH, quinpirole hydrochloride. n = 8 mice for each group.

Figure 4

Activation of CaMKIIα⁺ neurons rescued learning and memory impairment induced by chronic sleep deprivation. (A) Schematic of virus injection. mPFC, medial prefrontal cortex. (B) Experimental design. SD, sleep deprivation; S, sleep; PBS, phosphate buffer saline; CNO, Clozapine N-oxide. (C) Representative coronal brain section stained with c-Fos antibody (green), hM3Dq (red), and DAPI (blue), and high-magnification image of the white-boxed area. Left, the basal c-Fos level; Right, the c-Fos level after hM3Dq activation. (D) Quantification of average speed (top) and the novel arm preference index (bottom). Con, the control group. n = 8 mice for each group. One mouse was excluded due to a missed viral. (E) Representative tracing heatmaps during the Y-maze tests. (F) Left, Schematic drawing of virus injection. Middle, Representative coronal brain section stained with hM3Dq (green) and DAPI (blue). Right, Representative high-magnification image of the white-boxed area of the middle image. (G) Experimental design. (H) Representative tracing heatmaps during the Y-maze tests. (I) Quantification of average speed (left) and the novel arm preference index (right). n = 8 mice for each group.

Figure 5

The mPFC-BLA circuit contributed to learning and memory impairment induced by chronic sleep deprivation. (A) Schematic of virus injection and optical fiber implantation (left). Representative coronal brain sections of the mPFC (middle) and BLA (right), stained with eNpHR (red) and DAPI (blue). mPFC, medial prefrontal cortex; BLA, basolateral amygdala. (B) Experimental design. (C) Representative tracing heatmaps during the Y-maze tests. (D) Quantification of average speed (left) and the novel arm preference index (right). eNpHR_on or eNpHR_off, the group with or without eNpHR stimulation. n = 8 mice for the group with or without eNpHR stimulation. (E) Left, Schematic drawing of virus injection and optical fiber implantation. Representative coronal brain sections of the mPFC stained with c-Fos antibody (red), ChR2 (green), and DAPI (blue) (right). (F) Representative coronal brain sections of the BLA stained with c-Fos antibody (red), ChR2 (green), and DAPI (blue). (G) Experimental design. (H) A single 10-ms light pulse (top) and a train of 1-ms pulses at 20 Hz (bottom) for the light-evoked action potentials of mPFC CaMKII⁺ neurons. (I) Representative tracing heatmaps during the Y-maze tests. Con, the control group; ChR2_on or ChR2_off, the group with or without ChR2 stimulation. (J, K) Quantification of average speed and the novel arm preference index. n = 7 mice for each group. One mouse was excluded due to an outlier.

Figure 6

Drd2-mediated mPFC-BLA circuit contributed to learning and memory impairment induced by chronic sleep deprivation. (A) Experimental design and schematic of virus injection, optical fiber implantation, micro-infusion, and Y-maze test. (B) Representative coronal brain sections of the mPFC (left) and BLA (right) stained with c-Fos antibody (red), ChR2 (green), and DAPI (blue). (C) Representative tracing heatmaps during the Y-maze tests. (D) Quantification of average speed and the novel arm preference index. n = 7 mice for each group. mPFC, medial prefrontal cortex; BLA, basolateral amygdala. One mouse was excluded due to low viral expression.

Figure 7

Dopamine D2 receptor modulating mPFC-BLA circuit contributes to chronic sleep deprivation-induced memory impairment in mice. Chronic REM sleep deprivation increased the expression of dopamine receptor 2 (Drd2) in CaMKIIα⁺ neurons of the medial prefrontal cortex (mPFC), leading to decreased neuronal activity, dysregulation of the mPFC-basolateral amygdala (BLA) circuit, and learning and memory impairment in mice.