Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants

Abstract

Background: Behavioral stress is recognized as a main risk factor for neuropsychiatric diseases. Converging evidence suggested that acute stress is associated with increase of excitatory transmission in certain forebrain areas. Aim of this work was to investigate the mechanism whereby acute stress increases glutamate release, and if therapeutic drugs prevent the effect of stress on glutamate release.

Methodology/findings: Rats were chronically treated with vehicle or drugs employed for therapy of mood/anxiety disorders (fluoxetine, desipramine, venlafaxine, agomelatine) and then subjected to unpredictable footshock stress. Acute stress induced marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex in superfusion, and the chronic drug treatments prevented the increase of glutamate release. Stress induced rapid increase in the circulating levels of corticosterone in all rats (both vehicle- and drug-treated), and glutamate release increase was blocked by previous administration of selective antagonist of glucocorticoid receptor (RU 486). On the molecular level, stress induced accumulation of presynaptic SNARE complexes in synaptic membranes (both in vehicle- and drug-treated rats). Patch-clamp recordings of pyramidal neurons in the prefrontal cortex revealed that stress increased glutamatergic transmission through both pre- and postsynaptic mechanisms, and that antidepressants may normalize it by reducing release probability.

Conclusions/significance: Acute footshock stress up-regulated depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex. Stress-induced increase of glutamate release was dependent on stimulation of glucocorticoid receptor by corticosterone. Because all drugs employed did not block either elevation of corticosterone or accumulation of SNARE complexes, the dampening action of the drugs on glutamate release must be downstream of these processes. This novel effect of antidepressants on the response to stress, shown here for the first time, could be related to the therapeutic action of these drugs.

Figure 1. Basal and depolarization-evoked release of amino acid neurotransmitters from synaptosomes after acute footshock stress.

A) Basal and 15 mM KCl evoked glutamate release from hippocampal (HPC) and prefrontal/frontal cortex (P/FC) synaptosomes of vehicle-treated (control, CNT) and subjected to acute footshock (FS)-stress (−/+ Stress) rats. Data are expressed as means±SEM. *p<0.05, two-tailed Student's t test). Open bars, basal release; filled bars, 15 mM KCl evoked release (n = 4–6 rats/group). B) Basal and 15 mM KCl evoked GABA release from HPC and P/FC synaptosomes of vehicle-treated (CNT) and subjected to FS-stress (−/+ Stress) rats. Data are expressed as means±SEM. Open bars, basal release; filled bars, 15 mM KCl evoked release.

Figure 2. Effect of chronic antidepressant treatments on depolarization-evoked release of amino acid neurotransmitters from prefrontal/frontal cortex synaptosomes.

A) 15 mM KCl evoked glutamate and GABA release from P/FC synaptosomes of vehicle-treated (CNT), chronically treated with fluoxetine (FLX), subjected to FS-stress (STRESS) or chronically treated with FLX and then subjected to FS-stress (FLX+STRESS) rats. Data are expressed as means±SEM. *p<0.05, Newman-Keuls post-hoc tests following one-way ANOVA (n = 6–9 rats/group). B) 15 mM KCl evoked glutamate and GABA release from P/FC synaptosomes of vehicle-treated (CNT), chronically treated with desipramine (DMI), subjected to FS-stress (STRESS) or chronically treated with DMI and then subjected to FS-stress (DMI+STRESS) rats. Data are as in A. *p<0.05, statistics as above. C) 15 mM KCl evoked glutamate and GABA release from P/FC synaptosomes of vehicle-treated (CNT), chronically treated with venlafaxine (VFX), subjected to FS-stress (STRESS) or chronically treated with VFX and then subjected to FS-stress (VFX+STRESS) rats. Data are as in A. *p<0.05; **p<0.01, statistics as above. D) 15 mM KCl evoked glutamate and GABA release from P/FC synaptosomes of vehicle-treated (CNT), chronically treated with agomelatine (AGO), subjected to FS-stress (STRESS) or chronically treated with AGO and then subjected to FS-stress (AGO+STRESS) rats. Data are as in A. **p<0.01, statistics as above.

Figure 3. The increase of glutamate release induced in P/FC by footshock stress is dependent on release of corticosterone and activation of glucocorticoid receptor.

A) Corticosterone serum levels in vehicle-treated (CNT), subjected to FS-stress (STRESS) and chronically treated with FLX or DMI and then subjected to FS-stress (FLX+STRESS; DMI+STRESS) rats. Data are expressed as means±SEM. *** p<0.001 vs CNT, Newman-Keuls post-hoc tests following one-way ANOVA (n = 5 rats/group). B) Corticosterone serum levels in vehicle-treated (CNT), subjected to FS-stress (STRESS) and chronically treated with VFX or AGO and then subjected to FS-stress (VFX+STRESS; AGO+STRESS) rats. Data are expressed as means±SEM. *** p<0.001 vs CNT, Newman-Keuls post-hoc tests following one-way ANOVA (n = 10 rats/group). C–D) Depolarization evoked glutamate release from P/FC synaptosomes of vehicle-treated (CNT) and subjected to FS-stress (STRESS) rats pretreated or not with RU 486 (C), a selective antagonist of glucocorticoid receptor or RU 28318 (D), a selective antagonist of mineralocorticoid receptor. Data are expressed as means±SEM. **p<0.01 and ***p<0.001, Bonferroni post-hoc tests following two-way ANOVA (n = 4 rats/group).

Figure 4. The increase of glutamate release induced in P/FC by acute footshock stress is accounted for by accumulation of SNARE complexes in presynaptic membranes of prefrontal/frontal cortex.

A) Representative SNARE complexes of P/FC synaptosomes of vehicle-treated (CNT), subjected to FS-stress (STRESS) and chronically treated with FLX or DMI and then subjected to FS-stress (FLX+STRESS and DMI+STRESS) rats. B) Quantitation of syntaxin-1 in the rat groups as in (B). Data are expressed as means±SEM. C) Quantitation of normalized 100 kDa SNARE complex. Data expressed as above. Each single SNARE complex was normalized on monomeric syntaxin-1 in the same lane. **p<0.01 vs CNT, Newman-Keuls post-hoc tests following one-way ANOVA (n = 6–8 rats/group). D) Quantitation of normalized 80 kDa SNARE complex. E) Representative SNARE complexes of P/FC synaptosomes of vehicle-treated (CNT), subjected to FS-stress (STRESS) and chronically treated with venlafaxine (VFX) or agomelatine (AGO) and then subjected to FS-stress (VFX+STRESS and AGO+STRESS) rats. F) Quantitation of syntaxin-1 in the rat groups as in (F). Data expressed as above. G) Quantitation of normalized 100 kDa SNARE complex. *p<0.05 vs CNT, ***p<0.001 vs CNT; Statistics as above (n = 8–12 rats/group). H) Quantitation of normalized 80 kDa SNARE complex. ***p<0.001 vs CNT; Statistics as above.

Figure 5. Acute footshock stress enhances the amplitude of spontaneous EPSCs, and desipramine pretreatment completely counteracts this effect.

A) Representative traces of EPSCs evoked by spontaneous action potentials (sEPSCs) recorded in pyramidal neurons of layer III of prefrontal cortex obtained from vehicle-treated (CNT) (black trace) and subjected to FS-stress (Stress) (grey trace) rats. B) Mean cumulative analysis of sEPSCs amplitude evaluated in vehicle-treated (CNT) (n = 11), subjected to FS-stress (Stress) (n = 7), chronically treated with DMI (DMI) (n = 9) or chronically treated with DMI and then subjected to FS-stress (DMI+Stress) (n = 9) rats. Cumulative curves were analyzed by using the Kolgomorov-Smirnov test; *p<0.0001 FS-stressed rats vs all the other groups). C,D) Amplitude (C), and frequency (D) of sEPSCs (means±SEM) recorded in neurons obtained from vehicle-treated (CNT) (n = 10), subjected to FS-stress (Stress) (n = 7), chronically treated with DMI (DMI) (n = 8) and chronically treated with DMI and then subjected to FS-stress (DMI+Stress) (n = 9) rats. *p<0.05 Bonferroni post-hoc tests following two-way ANOVA.

Figure 6. The synaptic facilitation and its Ca²⁺-dependence are significantly reduced in footshock stressed rats and the effect is efficiently prevented by desipramine pre-treatment.

A) Specimen paired-pulse eEPSCs at increasing Ca²⁺ concentrations were obtained in vehicle-treated (CNT) and subjected to FS-stress (Stress) rats by stimulating layer V at a time interval of 50 ms and recording from pyramidal neurons. B) The mean paired-pulse ratio (PPR) of the second to the first eEPSC in neurons from vehicle-treated (CNT) (closed squares; n = 11), subjected to FS-stress (Stress) (open circles; n = 11), chronically treated with DMI (DMI) (closed triangles; n = 9) and chronically treated with DMI and then subjected to FS-stress (DMI+Stress) (open triangles; n = 10) rats were plotted as a function of the inter-stimulus interval (from 10 to 250 ms). PPR, at all tested intervals, were significantly lower in cells from FS-stressed rats than in neurons from all the other groups (*p<0.05, Bonferroni post-hoc tests following two-way ANOVA). C) PPR calculated at 50 ms inter-stimulus interval clearly decreased with increasing Ca²⁺ concentrations in neurons of vehicle-treated (CNT) (n = 6), chronically treated with DMI (DMI) (n = 8) and chronically treated with DMI and then subjected to FS-stress (DMI+Stress) (n = 9) rats. On the contrary, neurons from FS-stress rats (n = 6) showed a lower sensitivity to the changes in external Ca²⁺ concentration (*p<0.05, FS-stressed vs all the other groups, Bonferroni post-hoc tests following two-way ANOVA). D) Representative eEPSCs traces recorded in vehicle-treated (CNT) (black trace) and subjected to FS-stress (Stress) (grey trace) rats, showing the rise and decay kinetics fitted by mono-exponential curves. E,F) Rise (E), and decay (F) times (means±SEM) of eEPSCs estimated in neurons obtained from vehicle-treated (CNT) (n = 11), subjected to FS-stress (Stress) (n = 11), chronically treated with DMI (DMI) (n = 8) and chronically treated with DMI and then subjected to FS-stress (DMI+Stress) (n = 8) rats. *p<0.05; **p<0.01, Bonferroni post-hoc tests following two-way ANOVA.