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. Author manuscript; available in PMC: 2017 Oct 19.
Published in final edited form as: Neuron. 2016 Oct 6;92(2):493–504. doi: 10.1016/j.neuron.2016.09.029

Figure 7. Stress Weakens Cl− Extrusion and Decreases the Function of KCC2 in the VTA.

Figure 7

A) GABAergic input onto VTA GABA neurons was recorded using gramicidin perforated patches at different holding potentials to measure stress-induced alterations in anion homeostasis. GABAA IPSCs were evoked by electrical stimulation in the presence of DNQX, AP5, and CGP55845.

B) Representative IPSCs recordings from control (black) and stressed (red) animals at the given holding potentials. The IPSCs reverse direction at the EGABA. For display, the traces were filtered and stimulus artifacts were removed.

C) VTA GABA neurons from stressed animals (red, **, p < 0.01 by t-test) demonstrated a significantly more positive EGABA value compared to neurons from unstressed control animals (black square), n = 10 cells/group.

D) Cl− accumulation was estimated by stimulating repetitive GABAA receptor input. Upon stimulation (20 Hz, Vh = 0 mV), a representative GABA neuron from a control (black) animal demonstrated a minor depression of IPSC amplitude compared to the significantly greater depression seen in a GABA neuron from a stressed animal (red).

E) At 0 mV, VTA GABA neurons from stressed animals (red) demonstrated a significantly higher rate of evoked IPSC amplitude depression compared to control animals (black). **Significantly different from the control by F-test, p < 0.01, n = 10–17 cells/group.

F) Western blot analysis was conducted for total KCC2 and phosphorylated-S940 KCC2 with GAPDH as a loading control. A representative western blot indicates no differences in total KCC2 expression after stress. However, stressed animals showed reduced expression of pS940 KCC2 relative to total KCC2 when compared to non-stressed controls.

G) Densiometric analysis revealed a significant reduction in the ratio of pS940 KCC2 to total KCC2 protein in stressed animals compared to non-stress controls (horizontal dashed line). *Significantly different from the control by t-test, p < 0.05, n = 10 animals/group.

H) Densiometric analysis revealed a significant reduction in the ratio of pS940 KCC2 to total KCC2 protein in corticosterone-incubated slices from controls *Significantly different from the control by t-test, p < 0.05, n = 16 animals/group.