Chronic antidepressants reduce depolarization-evoked glutamate release and protein interactions favoring formation of SNARE complex in hippocampus

Abstract

Glutamate neurotransmission was recently implicated in the action of stress and in antidepressant mechanisms. We report that chronic (not acute) treatment with three antidepressants with different primary mechanisms (fluoxetine, reboxetine, and desipramine) markedly reduced depolarization-evoked release of glutamate, stimulated by 15 or 25 mm KCl, but not release of GABA. Endogenous glutamate and GABA release was measured in superfused synaptosomes, freshly prepared from hippocampus of drug-treated rats. Interestingly, treatment with the three drugs only barely changed the release of glutamate (and of GABA) induced by ionomycin. In synaptic membranes of chronically treated rats we found a marked reduction in the protein-protein interaction between syntaxin 1 and Thr286-phosphorylated alphaCaM kinase II (alpha-calcium/calmodulin-dependent protein kinase II) (an interaction previously proposed to promote neurotransmitter release) and a marked increase in the interaction between syntaxin 1 and Munc-18 (an interaction proposed to reduce neurotransmitter release). Furthermore, we found a selective reduction in the expression level of the three proteins forming the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. These findings suggest that antidepressants work by stabilizing glutamate neurotransmission in the hippocampus and that they may represent a useful tool for the study of relationship between functional and molecular processes in nerve terminals.

Figure 5.

Effect of Ca²⁺ omission or glutamate transporter block on the release of glutamate from rat hippocampal synaptosomes. Synaptosomes were exposed in superfusion to a 90s pulse of 25 mm KCl or 1 μm ionomycin, and the stimulus-evoked overflow of endogenous glutamate was measured. The stimulus-evoked overflow was calculated by subtracting the neurotransmitter content in the basal outflow from the release evoked by stimulation (see the legend to Fig. 1). dl-TBOA (100 μm) was introduced 9 min before KCl. Ca²⁺ was omitted 19 min before KCl. Data represent the means ± SEM of three to four separate experiments run in triplicate. ^*p < 0.001 versus the overflow induced by KCl or ionomycin in the absence of drugs (two-tailed Student's t test). Open bars, Controls; gray bars, Ca²⁺-free; filled bars, 100 μm dl-TBOA.

Figure 1.

A, Schematic time course of the release of a putative neurotransmitter from synaptosomes stimulated in superfusion by a depolarizing pulse. Synaptosomes were stratified on microporous filter, and neurotransmitter release was monitored during superfusion. After 36 min were allowed to equilibrate the system, two 3 min fractions (t = 36-39 min and t = 45-48 min) were collected before and after one 6 min fraction (t = 39-45 min). Synaptosomes were exposed to the stimulus (90 s) at the end of the first fraction collected (t = 39 min; see line). B, C, Characteristics of the release of glutamate and GABA from hippocampal synaptosomes exposed in superfusion to KCl or ionomycin. Synaptosomes were stratified on microporous filters, superfused, and stimulated by a 90 s pulse of KCl (15 or 25 mm) or ionomycin (0.5 or 1 μm) at t = 39 min of superfusion: the spontaneous or the stimulus-evoked release of endogenous glutamate (B) and GABA (C) was monitored. Basal outflow represents the neurotransmitter content in the two 3 min fractions collected before and after the stimulation fraction; the stimulus-evoked release represents the neurotransmitter content in the 6 min stimulation fraction, collected during and after application of the releasing pulse. ^*p < 0.005, when compared with respective basal outflow value; °p < 0.005, when compared with the release evoked by 15 mm KCl or 0.1 μm ionomycin, as appropriate (two-tailed Student's t test). Open bars, Basal release; filled bars, stimulus-evoked release. Error bars indicate SEM.

Figure 2.

Effect of chronic treatment with reboxetine on the release of glutamate and GABA from rat hippocampal synaptosomes. Reboxetine was administered by means of osmotic minipumps (10 mg/kg daily). After 14 d of treatment, animals were killed for release experiments. Synaptosomes were exposed in superfusion to a 90 s pulse of KCl (15 or 25 mm) or ionomycin (0.5 or 1 μm) at t = 39 min, and the stimulus-evoked overflow of endogenous glutamate (A) and GABA (B) was monitored. The stimulus-evoked overflow was calculated by subtracting the neurotransmitter content in the basal outflow from the release evoked by stimulation (see the legend to Fig. 1). Data represent the means ± SEM of four to six separate experiments run in triplicate. ^*p < 0.05, ^**p < 0.01 versus the respective control value (two-tailed Student's t test). Open bars, Vehicle-treated animals; filled bars, reboxetine-treated animals.

Figure 3.

Effect of chronic treatment with fluoxetine on the release of glutamate and GABA from rat hippocampal synaptosomes. Fluoxetine was administered by means of osmotic minipumps (10 mg/kg daily). After 14 d of treatment, animals were killed for release experiments. Synaptosomes were exposed in superfusion to KCl (15 or 25 mm) or ionomycin (0.5 or 1 μm), and the stimulus-evoked overflow of endogenous glutamate (A) and GABA (B) was monitored. The stimulus-evoked overflow was calculated by subtracting the neurotransmitter content in the basal outflow from the release evoked by stimulation (see the legend to Fig. 1). Data represent the means ± SEM of four to five separate experiments run in triplicate. ^*p < 0.05 versus the respective control value (two-tailed Student's t test). Open bars, Vehicle-treated animals; filled bars, fluoxetine-treated animals.

Figure 4.

The effect of acute treatment (A) with fluoxetine, reboxetine, and desipramine on the release of glutamate from rat hippocampal synaptosomes. The three drugs were administered intraperitoneally (10 mg/kg), and the rats were killed after 3 h. Synaptosomes were superfused and stimulated as in previous figures; the stimulus-evoked overflow is reported. Data represent the means ± SEM of three to four separate experiments run in triplicate. No significant differences were found (two-tailed Student's t test). Expression levels of total αCaM kinase II (B) and αCaM kinase II phosphorylated on Thr²⁸⁶ (C) in synaptosomes from rats acutely treated with antidepressants are shown. Expression levels were normalized for level of β-actin in the same blotted membrane. Representative immunoreactive bands are shown. Data represent the means ± SEM (percentage treated vs control) of three separate experiments run in triplicate; n.a., not assessed; iono, ionomycin. No significant differences were found (two-tailed Student's t test).

Figure 6.

Expression levels of presynaptic proteins, measured by Western analysis, in total homogenate, whole synaptosomes, synaptic membranes, synaptic vesicles from hippocampus of vehicle- and drug-treated rats (chronic treatment). CNT, Control; FLX, fluoxetine; RBX, reboxetine. Expression levels were normalized for level of β-actin in the same blotted membrane. A, Representative immunoreactive bands. SYN., Synaptic; syt, synaptotagmin I; syph, synaptophysin; stx, syntaxin 1; syb, VAMP (vesicle-associated membrane protein)-synaptobrevin 2. B, Quantitation of protein expression level for each single protein in synaptic membrane fraction. Data represent the means ± SEM (percentage treated vs control) of six separate experiments run in triplicate. ^*p < 0.05, ^**p < 0.01 versus the respective control value (two-tailed Student's t test).

Figure 7.

Expression levels of total αCaM kinase II (A, B) and αCaM kinase II phosphorylated on Thr²⁸⁶ (C, D) in synaptosomes and synaptic membranes (chronic treatment). Expression levels were normalized for level of β-actin in the same blotted membrane. Representative immunoreactive bands are shown. Data represent the means ± SEM (percentage treated vs control) of six separate experiments run in triplicate. ^*p < 0.05, ^**p < 0.01 versus the respective control value (two-tailed Student's t test).

Figure 8.

Activation of αCaM kinase II in vitro increases phosphorylation of Thr²⁸⁶ and binding of syntaxin 1 to αCaM kinase II. αCaM kinase II was activated by adding to lysed synaptosomes 1 mm CaCl₂, 20 μg/ml CaM, or 1 mm CaCl₂, 20 μg/ml CaM, and 500 μm ATP. Control samples contained 20 mm EGTA. The reaction was performed at 37°C for 5 min and stopped by adding immunoprecipitation buffer. After immunoprecipitation with antibody for αCaM kinase II, immunoprecipitated protein was separated by SDS-electrophoresis and electroblotted on membrane. The membrane was probed with antibodies for αCaM kinase II, phospho-αCaM kinase II, and syntaxin 1. Representative of four separate experiments run in duplicate.

Figure 9.

Chronic treatment with fluoxetine or reboxetine increases the binding of syntaxin 1 to αCaM kinase II and decreases the binding of syntaxin 1 to Munc-18 in synaptic membranes. A, αCaM kinase II was immunoprecipitated; αCaM kinase II and syntaxin 1 in the immunoprecipitate were analyzed by Western analysis. Representative immunoreactive bands are shown. Data represent the means ± SEM (percentage ratio syntaxin 1/αCaM kinase II) of four separate experiments in duplicate. ^*p < 0.05, ^**p < 0.01 versus the respective control value (two-tailed Student's t test). B, Munc-18 was immunoprecipitated; Munc-18 and syntaxin 1 in the immunoprecipitate were analyzed by Western analysis. CNT, Control; FLX, fluoxetine; RBX, reboxetine. Data represent the means ± SEM (percentage ratio syntaxin 1/Munc-18) of four separate experiments in duplicate. ^*p < 0.05, ^**p < 0.01 versus the respective control value (two-tailed Student's t test). Inset, Protein expression level of Munc-18 in synaptosomes and synaptic membranes.