ABSTRACT
Title
The impact of behavioral stress and antidepressant/anxiolytic treatments on glutamate transmission: relevance for neuropsychiatric disorders
Authors
G. Treccani1,2, L. Musazzi1, M. Milanese3, C. Perego4, A. Villa5, A. Corbelli5, G. Racagni1, G. Bonanno3, M. Popoli1
1Center of Neuropharmacology - Department of Pharmacological Sciences and Center of Excellence for Neurodegenerative Diseases, University of Milano
2Department of Experimental and Clinical Pharmacology, University of Catania
3Department of Experimental Medicine, Section of Pharmacology and Toxicology and Center of Excellence for Biomedical Research, University of Genova
4Department of Molecular Sciences Applied to Biosystems, University of Milano
5Microscopy and Image Analysis Consortium, University Milano-Bicocca, Monza, Italy.
1Center of Neuropharmacology - Department of Pharmacological Sciences and Center of Excellence for Neurodegenerative Diseases, University of Milano
2Department of Experimental and Clinical Pharmacology, University of Catania
3Department of Experimental Medicine, Section of Pharmacology and Toxicology and Center of Excellence for Biomedical Research, University of Genova
4Department of Molecular Sciences Applied to Biosystems, University of Milano
5Microscopy and Image Analysis Consortium, University Milano-Bicocca, Monza, Italy.
Abstract
Stressful life events impact on memory and cognition and are known to precipitate mood/anxiety disorders. It is increasingly recognized that stress and its neurochemical and endocrine mediators induce changes in glutamate synapse morphology and transmission, and this in turn modify mental states. Recent neuroimaging and histopathological studies on psychiatric patients have shown morphometrical and functional modifications in brain areas with glutamate predominance. Converging lines of evidence showed that repeated exposure to different stressful events represents a risk factor for neuropsychiatric diseases. Preclinical studies showed that the exposure of rodents to stress produces many concomitant findings observed in human pathology. Indeed, in rats stress induces dendritic remodeling in select brain areas, probably due to neuronal atrophy, associated with hyperactivation of excitatory amino acid transmission [1].
We have shown previously that acute footshock (FS)-stress induces a rapid up-regulation of depolarization-evoked glutamate release in prefrontal/frontal cortex (P/FC), by increasing circulating corticosterone (CORT) level, stimulation of glucocorticoid receptor (GR) by CORT and rapid accumulation of presynaptic SNARE complexes in synaptic membranes [2]. Previous chronic treatment with different antidepressants prevents the up-regulation of glutamate release in P/FC, by a mechanism that must be downstream of SNARE complex accumulation.
Aim of this work was to investigate wheter changes in synaptic vesicle pools are involved in the effect of acute stress on glutamate release. Rats were chronically (2 weeks) treated with vehicle or desipramine (a tricyclic antidepressant) and then subjected to a standard Footshock (FS)-stress protocol [2]. Immediately after FS-stress, P/FC was dissected and synaptosomes were purified on Percoll gradients; glutamate release evoked by 15 mM K+ or hypertonic sucrose was measured. Release evoked by sucrose (250-500 mM) mobilizes the readily releasable pool (RRP) of glutamate vesicles and allows measuring the size of this pool. Moreover, synaptic vesicles docked on the presynaptic membrane were visualized with EM and counted. Finally, changes in mobilization of FM1-43-labeled vesicles, induced by incubation of synaptosomes with 10 µM CORT for 10 min, were measured by Total Internal Reflection Fluorescence Microscopy (TIRFM).
Depolarization-evoked release of glutamate was increased in vehicle-treated FS-stressed rats (as shown previously, [2]). RRP size, measured with hypertonic sucrose, was increased 2-fold in P/FC synaptosomes from stressed rats (both in vehicle and desipramine-treated). Therefore, desipramine was able to prevent the effect of stress on depolarization-evoked release but not on hypertonic sucrose-evoked release. In line with the RRP measurement, EM showed a higher number of synaptic vesicles docked to the membrane in stressed rats. Results of TIRFM experiments showed that, like FS-stress, incubation of synaptosomes with 10 µM CORT increased the number of synaptic vesicles bound to the membrane.
Overall, this work showed that acute stress increases depolarization-evoked glutamate release by increasing the RRP of vesicles. Similar to acute stress, in vitro application of CORT also induced an increase in the pool of vesicles bound to presynaptic membrane, suggesting a mechanism mediated by a GR present in synaptic terminals.
[1] Sanacora, G., Zarate, C. A., Krystal J. H., Manji, H. K., 2008 Targeting the glutamatergic system to develop novel, improved therapeutics for mood disorders. Nature Review Drug Discovery 7, 426-437.
[2] Musazzi et al. (2010) Acute stress increases depolarization-evoked glutamate release and presynaptic SNARE complex accumulation in prefrontal/frontal cortex. The dampening action of antidepressants. PloS ONE 5(1): e8566. doi:10.1371/journal.pone.0008566.
We have shown previously that acute footshock (FS)-stress induces a rapid up-regulation of depolarization-evoked glutamate release in prefrontal/frontal cortex (P/FC), by increasing circulating corticosterone (CORT) level, stimulation of glucocorticoid receptor (GR) by CORT and rapid accumulation of presynaptic SNARE complexes in synaptic membranes [2]. Previous chronic treatment with different antidepressants prevents the up-regulation of glutamate release in P/FC, by a mechanism that must be downstream of SNARE complex accumulation.
Aim of this work was to investigate wheter changes in synaptic vesicle pools are involved in the effect of acute stress on glutamate release. Rats were chronically (2 weeks) treated with vehicle or desipramine (a tricyclic antidepressant) and then subjected to a standard Footshock (FS)-stress protocol [2]. Immediately after FS-stress, P/FC was dissected and synaptosomes were purified on Percoll gradients; glutamate release evoked by 15 mM K+ or hypertonic sucrose was measured. Release evoked by sucrose (250-500 mM) mobilizes the readily releasable pool (RRP) of glutamate vesicles and allows measuring the size of this pool. Moreover, synaptic vesicles docked on the presynaptic membrane were visualized with EM and counted. Finally, changes in mobilization of FM1-43-labeled vesicles, induced by incubation of synaptosomes with 10 µM CORT for 10 min, were measured by Total Internal Reflection Fluorescence Microscopy (TIRFM).
Depolarization-evoked release of glutamate was increased in vehicle-treated FS-stressed rats (as shown previously, [2]). RRP size, measured with hypertonic sucrose, was increased 2-fold in P/FC synaptosomes from stressed rats (both in vehicle and desipramine-treated). Therefore, desipramine was able to prevent the effect of stress on depolarization-evoked release but not on hypertonic sucrose-evoked release. In line with the RRP measurement, EM showed a higher number of synaptic vesicles docked to the membrane in stressed rats. Results of TIRFM experiments showed that, like FS-stress, incubation of synaptosomes with 10 µM CORT increased the number of synaptic vesicles bound to the membrane.
Overall, this work showed that acute stress increases depolarization-evoked glutamate release by increasing the RRP of vesicles. Similar to acute stress, in vitro application of CORT also induced an increase in the pool of vesicles bound to presynaptic membrane, suggesting a mechanism mediated by a GR present in synaptic terminals.
[1] Sanacora, G., Zarate, C. A., Krystal J. H., Manji, H. K., 2008 Targeting the glutamatergic system to develop novel, improved therapeutics for mood disorders. Nature Review Drug Discovery 7, 426-437.
[2] Musazzi et al. (2010) Acute stress increases depolarization-evoked glutamate release and presynaptic SNARE complex accumulation in prefrontal/frontal cortex. The dampening action of antidepressants. PloS ONE 5(1): e8566. doi:10.1371/journal.pone.0008566.