Periaqueductal Gray Glutamatergic Transmission Governs Chronic Stress-Induced Depression

Abstract

The mechanisms underlying chronic stress-induced dysfunction of glutamatergic transmission that contribute to helplessness-associated depressive disorder are unknown. We investigated the relationship of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and stress, and the neuroplastic changes of stress-induced depression-like behavior in the ventrolateral periaqueductal gray (vlPAG). We conducted whole-cell patch-clamp electrophysiological recordings in the vlPAG neurons. Depression-like behavior was assayed using tail suspension test and sucrose preference test. Surface and cytosolic glutamate receptor 1 (GluR1) AMPA receptor expression was analyzed using western blotting. Phosphorylated GluR1 expression was quantified using western blotting and immunohistochemical analysis. Unpredictable inescapable foot shock stress caused reduction in glutamatergic transmission originating from both presynaptic and postsynaptic loci in the vlPAG that was associated with behavioral despair and anhedonia in chronic stress-induced depression. Pharmacological inhibition of GluR1 function in the vlPAG caused depression-like behavior. Diminished glutamatergic transmission was due to reduced glutamate release presynaptically and enhanced GluR1-endocytosis from the cell surface postsynaptically. Chronic stress-induced neuroplastic changes and maladaptive behavior were reversed and mimicked by administration of glucocorticoid receptor (GR) antagonist and agonist, respectively. However, chronic stress did not affect γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission in the vlPAG. These results demonstrate that depression-like behavior is associated with remarkable reduction in glutamatergic, but not GABAergic, transmission in the vlPAG. These neuroplastic changes and maladaptive behavior are attributed to GR-dependent mechanisms. As reduced GluR1-associated responses in the vlPAG contribute to chronic stress-induced neuroplastic changes, this cellular mechanism may be a critical component in the pathogenesis of stress-associated neuropsychiatric disorders.

Figure 1

Inescapable unpredictable foot shock stress elicits helplessness behavior and depression-like behavior. (a) Schematic experimental procedure of learned helplessness (LH) model. (b) Training and testing protocol of LH model. (c, d) Vertical scatterplots depicting the distribution of helpless behaviors, number of failure (c) and escape latency (d), from control (n=9) and LH groups (n=16). (e) Vertical scatterplot depicting the distribution of dispirited behavior, immobility time of tail suspension test, from control (n=11) and LH (n=10) groups. (f) Vertical scatterplot depicting the distribution of anhedonic behavior, sucrose consumption of sucrose preference test, from control (n=9) and LH (n=8) groups. Data are expressed as mean±SEM; **p<0.01 vs Control.

Figure 2

Chronic stress leads to depression of excitatory synaptic transmission and pGluR1 protein expression in the ventrolateral periaqueductal gray (vlPAG). (a) The current–voltage (I–V) curves of EPSC_AMPAs of vlPAG neurons recorded at holding potentials ranging from −70 to +70 mV in control and LH groups. Left inset: a diagram illustrating the positions of the stimulating (S) and recording (R) electrodes, respectively, in the vlPAG slice. Right inset: represented traces of EPSC_AMPAs obtained from control (n=5) and LH (n=6) groups. Calibration: 50 pA, 50 ms. (b) Vertical scatterplot depicting the distribution of paired-pulse ratio (PPR) evoked by two identical electric stimuli with 50 ms interpulse interval. Inset: represented traces of PPR obtained from control (n=7) and LH (n=8) groups. Calibration: 50 pA, 50 ms. (c) Vertical scatterplot depicting the distribution of AMPA (1 μM)-induced inward currents in control (n=7) and LH (n=6) groups. Horizontal bars denote the periods that AMPA were applied. The dashed line indicates the baseline. Calibration: 50 pA, 1 min. (d–f) Vertical scatterplots depicting the distribution of total GluR1 (d), surface and cytoplasm (e), and pGluR1 (f) expressions in the vlPAG. Inset: represented western blot of protein expression from control (n=5) and LH (n=5) groups. (g, h) Immunohistochemistry images (g) and quantitative analysis (h) of pGluR1 and NeuN in the vlPAG slices dissected from control (n=5) and LH (n=5) groups. Calibration: 50 μm. Data are expressed as mean±SEM; *p<0.01, **p<0.01 vs Control.

Figure 3

Inhibition of calcium-permeable GluR1 AMPA receptor in the vlPAG causes depression-like behavior. (a) Effect of bath application of NASPM (100 μM) on the amplitude of EPSCs in vlPAG neurons dissected from control and LH groups. Horizontal bar denotes the periods that NASPM was applied. Inset: representative traces recorded at the time points denoted by the numbers on the time-course graph. Calibration: 50 pA, 20 ms. (b) Vertical scatterplot depicting the distribution of NASPM-induced EPSC inhibition recorded from vlPAG neurons obtained from control (n=6) and LH (n=6) groups. (c) Vertical scatterplot depicting the distribution of immobility time of tail suspension test, from control (n=6) and LH (n=6) groups before (circle) and after (triangle) intra-vlPAG microinjection of NASPM. (d) Vertical scatterplot depicting the distribution of sucrose consumption of sucrose preference test, from control (n=6) and LH (n=6) groups before (circle) and after (triangle) intra-vlPAG microinjection of NASPM. Inset: cannula tip placements for rats infused with NASPM in control (open circle) and LH (filled circle) groups. Data are expressed as mean±SEM; **p<0.01 vs Control.

Figure 4

Chronic stress diminishes excitatory synaptic transmission through both pre- and postsynaptic mechanisms in a glucocorticoid receptor (GR)-dependent manner. (a) Schematic experimental procedure of the effect of glucocorticoid receptor antagonist, RU486, in LH. (b) Rectification index (EPSC_−70 mV/EPSC_+70 mV) of AMPA receptor obtained from control (n=10), LH (n=11), and LH+RU486 (n=7) groups. Inset: represented traces of rectification index obtained from control and LH groups. Calibration: 50 pA, 20 ms. (c) Representative traces of mEPSCs recorded from vlPAG neurons dissected from control, LH, and LH+RU486 groups. Calibration: 25 pA, 600 ms. (d, e) Vertical scatterplots depicting the distribution of frequency (d) and (e) amplitude of mEPSCs recorded from vlPAG neurons obtained from control (n=9), LH (n=9), and LH+RU486 (n=10) groups. (f, g) Vertical scatterplot depicting the distribution of surface and cytoplasm (f) and pGluR1 (g) expressions in the vlPAG. Inset: represented western blot of protein expression from LH (n=5) and LH+RU486 (n=5) groups. (h, i) Immunohistochemistry images (h) and quantitative analysis (i) of pGluR1 and NeuN in the vlPAG slices dissected from control (n=5), LH (n=5), and LH+RU486 (n=5) groups. Calibration: 50 μm. (j, k) Vertical scatterplot depicting the distribution of immobility time of tail suspension test (j) and sucrose consumption of sucrose preference test (k) from control (n=8), LH (n=8), and LH+RU486 (n=6) groups. Data are expressed as mean±SEM; **p<0.01 vs Control, ^##p<0.01 vs LH.

Figure 5

Glucocorticoid receptor activation mimics chronic stress-induced depression of excitatory synaptic transmission via both pre- and postsynaptic mechanisms. (a) Schematic experimental procedure of the effect of glucocorticoid receptor agonist, dexamethasone (DEX), in naive rats. (b) Rectification index of AMPA receptor obtained from control and DEX groups. Inset: represented traces of rectification index obtained from control (n=8) and DEX (n=10) groups. Calibration: 50 pA, 20 ms. (c) Representative traces of mEPSCs recorded from vlPAG neurons dissected from control and DEX groups. Calibration: 25 pA, 600 ms. (d, e) Vertical scatterplots depicting the distribution of frequency (d) and amplitude (e) of mEPSCs recorded from vlPAG neurons obtained from control (n=7) and DEX (n=12) groups. (f, g) Vertical scatterplot depicting the distribution of surface and cytoplasm (f) and pGluR1 (g) expressions in the vlPAG. Inset: represented western blot of protein expression from control (n=5) and DEX (n=5) groups. (h, i) Immunohistochemistry images (h) and quantitative analysis (i) of pGluR1 and NeuN in the vlPAG slices dissected from control (n=6) and DEX (n=6) groups. Calibration: 50 μm. (j, k) Vertical scatterplot depicting the distribution of immobility time of tail suspension test (j) and sucrose consumption of sucrose preference test (k) from control (n=7) and DEX (n=7) groups. Data are expressed as mean±SEM; **p<0.01 vs Control.