Fig 2: The amount of neurotransmitter and hypoactive glutamatergic signaling did not change with insulin treatment. (a) Glu, Gln, and GABA levels in the PFC were analyzed using LC-MS/MS. CTL, n = 5; STZ, n = 5; SI, n = 9. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 between indicated groups according to one-way ANOVA with Newman–Keuls post-hoc tests. (b) Experimental design for electrophysiologic analysis and microscope images of sEPSC recording. sEPSC in the mPFC was measured after 4-week hyperglycemia induction and subsequent 4-week insulin treatment in VGLUT2-CRE::EGFP mice. (c) sEPSC frequency and cumulative amplitude in glutamatergic neurons in the mPFC. CTL, n = 11 cells from 2 mice; STZ, n = 8 cells from 2 mice; SI, n = 16 cells from 3 mice. Data are presented as mean ± SEM. *** p < 0.001 between indicated groups according to one-way ANOVA with Newman–Keuls post-hoc tests for sEPSC frequency and two-way ANOVA with Tukey’s post-hoc tests for cumulative amplitude.

Fig 3: Hyperglycemia reduced insulin receptor β (IRβ) expression and altered its cellular localization in the mPFC. Immunohistochemistry images of (a) IRβ, (b) insulin receptor substrate-1 (IRS-1), (c) vesicular glutamate transporter 1 (VGLUT1), and (d) VGLUT2 with the nuclear marker DAPI in the mPFC. Scale bar = 50 μm. (a) Inset scale bar = 5 μm. The right panel shows each signal quantification result. n = 4/group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 between indicated groups according to one-way ANOVA with Newman–Keuls post-hoc tests.

Fig 4: Hyperglycemia decreased glutamatergic neuronal activity. (a) Immunohistochemical analysis of ΔFOSB in the prelimbic and infralimbic mPFC. The left panel shows representative images, and the right panel shows quantifications. Scale bar = 100 μm. (b,c) Glutamate (Glu), glutamine (Gln), and γ-aminobutyric acid (GABA) levels in the PFC (b) and plasma (c) analyzed by LC-MS/MS. (d) Experimental design for electrophysiologic analysis and microscope images of spontaneous excitatory postsynaptic current (sEPSC) recording. sEPSC in the mPFC was measured 4 weeks after STZ injection in VGLUT2-CRE::tdTomato mice. (e) sEPSC and cumulative amplitude in glutamatergic neurons in the MPFC. n = 10/group (10 cells from 3 mice). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 between indicated groups according to Student’s t-test or two-way ANOVA with Tukey’s post-hoc tests.

Fig 5: Photomicrographs of IC projecting neurons (yellow) and vGLUT2 neurons in the LSO. Principal neurons were labeled via a unilateral injection of the retrograde tracer, FG (yellow), into the right IC and counterstained with vGLUT2 immunohistochemistry (magenta fluorescence). (A) Low magnification montages of the SOC (10x objective) show the contralateral and ipsilateral LSO with retrogradely labeled principal neurons (top panels, yellow) and vGLUT2 staining (bottom panels, magenta). (B) Higher magnification images (25x objective) of the same tissue in A, revealed double-labeled neurons for FG (upper panels) and vGLUT2 (bottom panels) in the contralateral and ipsilateral LSO (red arrowheads) in approximately equal numbers. Some vGLUT2 neurons project to the ipsilateral IC, some to the contralateral IC, and some to neither. vGLUT2, vesicular glutamate transporter 2; others as in Figure 1.