ABSTRACT
Title
Synaptic action of acute behavioural stress and corticosterone: effect on the readily releasable pool of vesicles in prefrontal/frontal cortex
Authors
G. Treccani1,2, L. Musazzi1, M. Milanese3, A. Mallei1, C. Perego4, A. Corbelli5, A. Villa5, G. Racagni1, G. Bonanno3, M. Popoli1
1Center of Neuropharmacology – Dept. of Pharmacological Sciences, University of Milano
2Dept of Experimental and Clinical Pharmacology, University of Catania
3Dept of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genova
4Dept of Molecular Sciences Applied to Biosystems, University of Milano
5Microscopy and Image Analysis Consortium, University Milano-Bicocca
1Center of Neuropharmacology – Dept. of Pharmacological Sciences, University of Milano
2Dept of Experimental and Clinical Pharmacology, University of Catania
3Dept of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genova
4Dept of Molecular Sciences Applied to Biosystems, University of Milano
5Microscopy and Image Analysis Consortium, University Milano-Bicocca
Abstract
Accumulating evidence suggests that repeated exposure to different stressful events represents a risk factor for neuropsychiatric diseases. Acute and chronic stressors can have lasting consequences on the brain; indeed, stress has been shown to cause structural changes, such as dendritic atrophy and loss of dendritic spines in neuronal populations. Although different neurotransmitter and neuromodulatory systems in various brain regions have been shown to contribute to the stress response, several studies suggest a critical role of glutamatergic neurotransmission, in particular in the prefrontal cortex, in mediating some of the major effects of stress on psychopathology and cognition (Arnsten, 2009). Indeed, in previous studies we found that Footshock (FS)-stress induced a marked increase of circulating corticosterone (CORT) and a rapid (non genomic) increase of glutamate release from synaptosomes of prefrontal/frontal cortex (P/FC) via selective activation of glucocorticoid receptor and rapid accumulation of SNARE complexes in synaptic membranes. The increase of glutamate release was prevented by chronic antidepressant (AD) treatments (Musazzi et al., 2010). Therefore aim of the present work was to study if FS-stress induces a change in glutamate release by increasing the number of vesicles anchored to the presynaptic membrane and modifying the distribution of vesicles among the presynaptic pools, in particular if the readily releasable pool (RRP) is increased by stress or CORT.
Rats were chronically (2 weeks) treated with vehicle or desipramine (DMI) 10 mg/Kg and then subjected to a standard Footshock (FS)-stress protocol (Vollmayr and Henn, 2001). Immediately after FS-stress, P/FC was dissected and synaptosomes were purified on Percoll gradients. Glutamate release was measured in freshly purified synaptosomes by using superfusion technique (Bonanno et al., 2005). The number of docked vesicles in P/FC synaptosomes was measured using electron microscopy (EM). Changes in vesicle mobilization were measured in P/FC synaptosomes from control rats loaded with 4 mM FM1-43 and incubated or not with CORT (10 mM, 10 min) by Total Internal Reflection Fluorescence Microscopy (TIRFM) (Serulle et al., 2007).
We found that acute stress induced a marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex (P/FC). Intriguingly, chronic pretreatment with desipramine prevented the stress-induced increase of glutamate release. FS-stress also induced an increase in glutamate release evoked by hypertonic sucrose, which mobilizes the RRP. Chronic desipramine did not block the increase of glutamate evoked by hypertonic sucrose.
Since this effect on glutamate release suggests that FS-stress may increase the size of RRP, we measured the number of docked vesicles in P/FC synaptosomes of FS-stressed rats (vehicle- and DMI-treated), using EM. Preliminary results suggest that FS-stress increases the number of docked vesicles.
Furthermore, we investigated the effects of stress on synaptic vesicles mobilization with TIRFM. Since the effects of FS-stress on glutamate release are mediated by CORT, we incubated synaptosomes in vitro with CORT and visualized by TIRFM the changes induced by the hormone on mobilization of vesicles labeled with FM1-43. Preliminary experiments suggest that the incubation in vitro of P/FC synaptosomes with CORT increases the size of the RRP.
Together, these results suggest that acute stress upregulates glutamate release by increasing the size of RRP and that the dampening effect of antidepressants involves downstream mechanisms (probably affecting the priming of vesicles and the probability of release).
Arnsten (2009). Nature Rev Neurosci. 10, 410-422.
Musazzi et al. (2010) PLoS One. 5, e8566.
Vollmayr and Henn (2001) Brain Res Brain Res Protoc. 8, 1-7.
Bonanno et al. (2005) J Neurosci. 25, 3270-9.
Serulle et al. (2007) PNAS. 104, 1697-1702.
Rats were chronically (2 weeks) treated with vehicle or desipramine (DMI) 10 mg/Kg and then subjected to a standard Footshock (FS)-stress protocol (Vollmayr and Henn, 2001). Immediately after FS-stress, P/FC was dissected and synaptosomes were purified on Percoll gradients. Glutamate release was measured in freshly purified synaptosomes by using superfusion technique (Bonanno et al., 2005). The number of docked vesicles in P/FC synaptosomes was measured using electron microscopy (EM). Changes in vesicle mobilization were measured in P/FC synaptosomes from control rats loaded with 4 mM FM1-43 and incubated or not with CORT (10 mM, 10 min) by Total Internal Reflection Fluorescence Microscopy (TIRFM) (Serulle et al., 2007).
We found that acute stress induced a marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex (P/FC). Intriguingly, chronic pretreatment with desipramine prevented the stress-induced increase of glutamate release. FS-stress also induced an increase in glutamate release evoked by hypertonic sucrose, which mobilizes the RRP. Chronic desipramine did not block the increase of glutamate evoked by hypertonic sucrose.
Since this effect on glutamate release suggests that FS-stress may increase the size of RRP, we measured the number of docked vesicles in P/FC synaptosomes of FS-stressed rats (vehicle- and DMI-treated), using EM. Preliminary results suggest that FS-stress increases the number of docked vesicles.
Furthermore, we investigated the effects of stress on synaptic vesicles mobilization with TIRFM. Since the effects of FS-stress on glutamate release are mediated by CORT, we incubated synaptosomes in vitro with CORT and visualized by TIRFM the changes induced by the hormone on mobilization of vesicles labeled with FM1-43. Preliminary experiments suggest that the incubation in vitro of P/FC synaptosomes with CORT increases the size of the RRP.
Together, these results suggest that acute stress upregulates glutamate release by increasing the size of RRP and that the dampening effect of antidepressants involves downstream mechanisms (probably affecting the priming of vesicles and the probability of release).
Arnsten (2009). Nature Rev Neurosci. 10, 410-422.
Musazzi et al. (2010) PLoS One. 5, e8566.
Vollmayr and Henn (2001) Brain Res Brain Res Protoc. 8, 1-7.
Bonanno et al. (2005) J Neurosci. 25, 3270-9.
Serulle et al. (2007) PNAS. 104, 1697-1702.