Background <p>The prefrontal cortex (PFC) plays a pronounced role in cognitive and emotional functions, which may be compromised by dismal sleep quality. This study intended to clarify the impact of Slc1a2 ectopic expression in the PFC on sleep deprivation (SD)-induced disturbances in the glutamate (Glu)/GABA-glutamine cycle and the role of astrocyte (AC)-neuron (Neu) communication.</p> Methods <p>Single-cell RNA sequencing was adopted to illuminate cell-specific changes in the brainstem, cortex, and hypothalamus of mice under NS, SD, and post-SD conditions. Cell communication analysis was applied to study interactions between ACs and Neus, which altered after the SD. Slc1a2 was ectopically expressed in the PFC and subjected to SD, followed by electrophysiological, immunofluorescence staining, and [¹H-¹³C]-nuclear magnetic resonance (NMR) assays to examine neural activity and metabolic status. Behavioral tests, including the open field, novel object recognition, and Y-maze, were conducted to examine cognitive functions and emotional states.</p> Results <p>SD caused notable changes in cellular distribution and downregulation of metabolic and synaptic genes in affected brain regions. Cell communication studies highlighted a reduction in AC-Neu interactions, with corresponding metabolic disruptions in the Glu/GABA-glutamine cycle as depicted by [¹H-¹³C]-NMR results. Behavior tests confirmed anxiety and cognitive deficits in SD mice, which were substantially alleviated by Slc1a2 ectopic expression in the PFC.</p> Conclusions <p>Slc1a2 ectopic expression in the PFC negates SD-induced GABA dysfunction through vital AC-Neu communication. This study sheds light on the mechanisms through which SD affects neural function and suggesting potential treatments for sleep-related disorders.</p>