Neurovascular Coupling under Chronic Stress Is Modified by Altered GABAergic Interneuron Activity

Abstract

Neurovascular coupling (NVC), the interaction between neural activity and vascular response, ensures normal brain function by maintaining brain homeostasis. We previously reported altered cerebrovascular responses during functional hyperemia in chronically stressed animals. However, the underlying neuronal-level changes associated with those hemodynamic changes remained unclear. Here, using in vivo and ex vivo experiments, we investigate the neuronal origins of altered NVC dynamics under chronic stress conditions in adult male mice. Stimulus-evoked hemodynamic and neural responses, especially beta and gamma-band local field potential activity, were significantly lower in chronically stressed animals, and the NVC relationship, itself, had changed. Further, using acute brain slices, we discovered that the underlying cause of this change was dysfunction of neuronal nitric oxide synthase (nNOS)-mediated vascular responses. Using FISH to check the mRNA expression of several GABAergic subtypes, we confirmed that only nNOS mRNA was significantly decreased in chronically stressed mice. Ultimately, chronic stress impairs NVC by diminishing nNOS-mediated vasodilation responses to local neural activity. Overall, these findings provide useful information in understanding NVC dynamics in the healthy brain. More importantly, this study reveals that impaired nNOS-mediated NVC function may be a contributory factor in the progression of stress-related diseases.SIGNIFICANCE STATEMENT The correlation between neuronal activity and cerebral vascular dynamics is defined as neurovascular coupling (NVC), which plays an important role for meeting the metabolic demands of the brain. However, the impact of chronic stress, which is a contributory factor of many cerebrovascular diseases, on NVC is poorly understood. We therefore investigated the effects of chronic stress on impaired neurovascular response to sensory stimulation and their underlying mechanisms. Multimodal approaches, from in vivo hemodynamic imaging and electrophysiology to ex vivo vascular imaging with pharmacological treatment, patch-clamp recording, FISH, and immunohistochemistry revealed that chronic stress-induced dysfunction of nNOS-expressing interneurons contributes to NVC impairment. These findings will provide useful information to understand the role of nNOS interneurons in NVC in normal and pathological conditions.

Keywords: GABAergic interneuron; cerebral blood flow; chronic stress; nNOS; neurovascular coupling.

Figure 1.

Behavioral and physiological validation of the chronic stress mouse model. A, Representative heatmaps showing the exploration of mice during a 5 min EPM test. Each image was obtained from a single mouse. Red represents more time spent in an area. Blue represents less time spent in an area. B, Average duration of time spent in each area of the EPM (control, n = 117; stressed, n = 123). C, Comparison of baseline plasma corticosterone concentrations (control, n = 25; stressed, n = 25). Data are mean ± SEM. ^††p < 0.01; ^†††p < 0.001; n.s., not significant; Mann–Whitney U test.

Figure 2.

Reduced hemodynamic responses to forelimb stimulus in chronically stressed animals. A, Experimental settings for in vivo OIS imaging. Light reflected from the exposed S1FL cortex (i.e., the forelimb region of the primary somatosensory cortex) was filtered at the 546 nm wavelength, which is a measure of the Hbt. To assess the NVC relationship during functional hyperemia, LFPs were measured simultaneously with the OIS imaging. B, Representative Hbt-weighted OIS images during forelimb stimulation in control (left) and stressed mice (right). Each image represents the average of 18 trials from a single individual. Scale bars, 1 mm. C, Time course traces of relative changes in the evoked Hbt signal following forelimb stimulation, relative to baseline levels (control, black, n = 10; stressed, red, n = 10). D, Average of the maximum evoked Hbt change. E, Comparison of the area of activated region (spatial extent) for the time point at which the maximum Hbt change was observed. F, Time of onset. G, Time to reach half-maximum of the first peak of the Hbt response. H, Time course traces of heart rate monitored during OIS imaging sessions, including the sensory stimulation period (indicated with the yellow box). I, Average of the heart rate measured throughout the experiments (control, n = 10; stressed, n = 10). J, Blood pressure measured in urethane-anesthetized mice (control, n = 3; stressed, n = 3) using a tail-cuff blood pressure measurement system. Data are mean ± SEM. ^†p < 0.05 (Mann–Whitney U test). *p < 0.05 (independent t test); n.s., not significant.

Figure 3.

Reduced neural response to forelimb stimulus and alteration of the NVC relationship in chronically stressed animals. A, Representative raw LFP traces acquired from control and stressed mice. B, Time course traces of relative changes in the evoked neural activity following forelimb stimulation, relative to prestimulus baseline levels (control, black, n = 10; stressed, red, n = 10). C, Power spectra of LFP recorded during stimulation (solid lines; control group in black and stressed group in red) and prestimulus baseline (dashed lines) periods. Gray box represents the frequency range (55–65 Hz) excluded from all analyses due to use of a 60 Hz notch filter. D, Comparison of baseline neural activity in different frequency ranges. E, Comparison of the evoked neural activity in different frequency ranges. B–E, Data are mean ± SEM. F, Box plot (box-and-whisker diagram) of the ratio of the evoked Hbt response to the evoked neural response. ^†p < 0.05; ^††p < 0.01; Mann–Whitney U test. **p < 0.01; ***p < 0.001; independent-samples t test; n.s., not significant.

Figure 4.

Altered vascular dynamics in chronically stressed mice, as measured in acute brain slices. A, The schematic setup showing the experiment. B, Representative images of baseline and postelectrical 20 Hz stimulation arterioles in the somatosensory cortex of control and stressed mice. Red vertical lines were traced along vascular walls for visualization of penetrating arterioles. C, D, Time course traces and maximal relative amplitudes of arteriolar diameter changes following electrical stimulation for control (black, n = 10) and stressed brain (red, n = 11). Clearly, stimulation induces dilation for the control, but constriction for the stressed brain slice. E, F, Time course traces and maximal relative amplitudes of vascular diameter changes after stimulation in the presence (green, n = 6) or absence of TTX (gray, n = 5) in the control mouse brain slice. In the control brain slice, stimulation induces vascular dilation (1.08 ± 0.01), which is abolished by TTX application (0.98 ± 0.01, U_(6,5) = 0_, p = 0.004, U test). G–J, Time course traces and maximal relative amplitudes of vessel diameter change induced by 2 min application (horizontal black line) of 30 μm NMDA (G, H; control, n = 9; stressed, n = 7) and 10 μm AMPA (I, J; control, n = 5; stressed, n = 5) for control (black) and stressed brain slice (red). Both NMDA and AMPA mimic the action of glutamate. Neither compounds produce any significant difference in vascular diameter for control versus stressed slice. K–M, Time course traces (K, M) and maximal relative amplitudes (L, N) of vessel diameter change induced by 2 min treatment of GABA receptor agonists (100 μm muscimol + 50 μm baclofen) in the absence (K, M) and presence of TTX (L, N) for control (black, n = 13 without and 8 with TTX) and stressed brain slices (red, n = 11 without and 11 with TTX). Data are mean ± SEM. **p < 0.01, ***p < 0.001, n.s., not significant.

Figure 5.

Reduction of the frequency of sIPSCs from pyramidal cells in chronically stressed mice. A, The schematics showing patch-clamp recording from pyramidal cells in somatosensory cortex. B, C, Representative traces of EPSCs (B) and IPSCs (C) in control (top, black) and stressed mice (bottom, red). D, The amplitude of sEPSCs (control: n = 10; stressed: n = 11) and sIPSCs (control: n = 10; stressed: n = 13). E, The frequency of sEPSCs (control: n = 10; stressed: n = 11) and sIPSCs (control: n = 10; stressed: n = 13). F, G, Representative traces of tonic GABA_AR currents in control (left, black) and stressed mice (right, red). H, The amplitude of tonic GABA_AR currents measured in two groups (control: n = 10; stressed: n = 11) and the amplitude of tonic GABA_AR currents measured with treatment of 5 μm GABA in two groups (control: 13.28 ± 2.26 pA, n = 9; stressed: 12.3 ± 2.13, n = 10; U_(9,10) = 38, p = 0.567, U test). Data are mean ± SEM. *p < 0.05, n.s., not significant.

Figure 6.

Alteration of GABA_A receptor-mediated vascular responses induced by 20 Hz focal electrical stimulation in stressed mice. A–C, E–G, Time course traces of arteriolar diameter change in control (A–C) and stressed brain tissues (E–G) exposed to two conditions: under ACSF circulation (black or red) or with treatment of drug (blue); 100 μm bicuculline, a GABA_A antagonist (A, E), 100 μm bicuculline with glutamergic antagonists (10 μm AP5 + 5 μm NBQX) (B, F), and 10 μm CGP 55845, a GABAB antagonist (C, G) were used as drug circulation. D, Maximum diameter change amplitude in the control group following each drug treatment (ACSF: n = 10; bicuculline: n = 8; bicuculline with AP5 and NBQX: n = 5; CGP 55845: n = 7). H, Maximum diameter change amplitude in the chronically stressed group following each drug treatment (ACSF: n = 11; bicuculline: n = 8; bicuculline with AP5 and NBQX: n = 5; CGP 55845: n = 7). Data are mean ± SEM. ***p < 0.001, n.s., not significant. Statistical significance was tested with the one-way ANOVA followed by Bonferroni post hoc tests according to the results of a normal distribution test (Shapiro–Wilk test).

Figure 7.

Alteration of nNOS-mediated vascular responses induced by 20 Hz focal electrical stimulation in stressed mice. A–C, E–G, Time course traces of arterial diameter change in control (A–C) and stressed brain tissues (E–G) under ACSF (black or red) or with drug treatment (green). Drug was 1 μm SOM (A, E), a neuropeptide that agonist for SOM, 1 μm BIBP 3226 (B, F), an antagonist for the neuropeptide Y receptor, and 10 μm l-NPA (C, G), a nNOS inhibitor. D, Maximum diameter change amplitude following each drug treatment in the control group (ACSF: n = 10; SOM: n = 13, BIBP 3226: n = 6, l-NPA: n = 10). H, Maximum diameter change amplitude following each drug treatment in the chronically stressed group (ACSF: n = 11; SOM: n = 12, BIBP 3226: n = 5; l-NPA: n = 12). Data are mean ± SEM. **p < 0.01, n.s., not significant. A one-way ANOVA followed by Bonferroni post hoc tests was used for analysis of a drug treatment effect in the control group, and the Kruskal–Wallis test was used for statistical analysis of the chronically stressed group according to the results of a normal distribution test (Shapiro–Wilk test).

Figure 8.

Decreased mRNA expression of nNOS in the somatosensory cortex of chronically stressed mice. A, E, SOM mRNA expression in the somatosensory cortex of both control and chronically stressed mice (control: n = 7; stressed: n = 5). B, F, VIP mRNA expression (control: n = 7; stressed: n = 5). C, G, NPY mRNA expression (control: n = 7; stressed: n = 5). D, H, nNOS mRNA expression (control: n = 5; stressed: n = 5). Data are mean ± SEM. *p < 0.05 (Mann–Whitney U test); n.s., not significant.

Figure 9.

Decreased protein expression of nNOS-expressing neurons in the somatosensory cortex of chronically stressed mice. A, C, Cortical layer-dependent (left) and expanded IHC images (right) of nNOS-expressing neurons from control and chronically stressed mice. Type 1 nNOS-expressing neurons have large soma with strong IHC intensity, whereas Type 2 nNOS neurons have small soma with weak intensity. Scale bars: subplots, 20 μm. B, D, The number of Type 1 (control: n = 12; stressed: n = 11) and Type 2 nNOS neurons (control: n = 6; stressed: n = 6). Data are mean ± SEM. *p < 0.05, Mann–Whitney U test.