The Journal of physiology
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The Journal of physiology · Jul 2015
Astrocyte VAMP3 vesicles undergo Ca2+ -independent cycling and modulate glutamate transporter trafficking.
Mouse cortical astrocytes express VAMP3 but not VAMP2. VAMP3 vesicles undergo Ca(2+) -independent exo- and endocytotic cycling at the plasma membrane. VAMP3 vesicle traffic regulates the recycling of plasma membrane glutamate transporters. cAMP modulates VAMP3 vesicle cycling and glutamate uptake. ⋯ Previous studies suggest that small synaptic-like vesicles in astrocytes carry vesicle-associated vSNARE proteins, VAMP3 (cellubrevin) and VAMP2 (synaptobrevin 2), both contributing to the Ca(2+) -regulated exocytosis of gliotransmitters, thereby modulating brain information processing. Here, using cortical astrocytes taken from VAMP2 and VAMP3 knock-out mice, we find that astrocytes express only VAMP3. The morphology and function of VAMP3 vesicles were studied in cultured astrocytes at single vesicle level with stimulated emission depletion (STED) and total internal reflection fluorescence (TIRF) microscopies. We show that VAMP3 antibodies label small diameter (∼80 nm) vesicles and that VAMP3 vesicles undergo Ca(2+) -independent exo-endocytosis. We also show that this pathway modulates the surface expression of plasma membrane glutamate transporters and the glutamate uptake by astrocytes. Finally, using pharmacological and optogenetic tools, we provide evidence suggesting that the cytosolic cAMP level influences astrocytic VAMP3 vesicle trafficking and glutamate transport. Our results suggest a new role for VAMP3 vesicles in astrocytes.
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The Journal of physiology · Jul 2015
Impairment of adenylyl cyclase-mediated glutamatergic synaptic plasticity in the periaqueductal grey in a rat model of neuropathic pain.
Long-lasting neuropathic pain has been attributed to elevated neuronal plasticity changes in spinal, peripheral and cortical levels. Here, we found that reduced neuronal plasticity in the ventrolateral periaqueductal grey (vlPAG), a midbrain region important for initiating descending pain inhibition, may also contribute to neuropathic pain. Forskolin- and isoproterenol (isoprenaline)-elicited EPSC potentiation was impaired in the vlPAG of a rat model of neuropathic pain induced by spinal nerve injury. Down-regulation of adenylyl cyclase-cAMP- PKA signalling, due to impaired adenylyl cyclase, but not phosphodiesterase, in glutamatergic terminals may contribute to the hypofunction of excitatory synaptic plasticity in the vlPAG of neuropathic rats and the subsequent descending pain inhibition, ultimately leading to long-lasting neuropathic pain. Our results suggest that drugs that activate adenylyl cyclase in the vlPAG have the potential for relieving neuropathic pain. ⋯ Neuropathic pain has been attributed to nerve injury-induced elevation of peripheral neuronal discharges and spinal excitatory synaptic plasticity while little is known about the contribution of neuroplasticity changes in the brainstem. Here, we examined synaptic plasticity changes in the ventrolateral (vl) periaqueductal grey (PAG), a crucial midbrain region for initiating descending pain inhibition, in spinal nerve ligation (SNL)-induced neuropathic rats. In vlPAG slices of sham-operated rats, forskolin, an adenylyl cyclase (AC) activator, produced long-lasting enhancement of EPSCs. This is a presynaptic effect since forskolin decreased the paired-pulse ratio and failure rate of EPSCs, and increased the frequency, but not the amplitude, of miniature EPSCs. Forskolin-induced EPSC potentiation was mimicked by a β-adrenergic agonist (isoproterenol (isoprenaline)), and prevented by an AC inhibitor (SQ 22536) and a cAMP-dependent protein kinase (PKA) inhibitor (H89), but not by a phosphodiesterase (PDE) inhibitor (Ro 20-1724) or an A1 -adenosine antagonist (DPCPX). Both forskolin- and isoproterenol-induced EPSC potentiation was impaired in PAG slices of SNL rats. The SNL group had lower AC, but not PDE, activity in PAG synaptosomes than the sham group. Conversely, IPSCs in vlPAG slices were not different between SNL and sham groups. Intra-vlPAG microinjection of forskolin alleviated SNL-induced mechanical allodynia in rats. These results suggest that SNL leads to inadequate descending pain inhibition resulting from impaired glutamatergic synaptic plasticity mediated by the AC-cAMP-PKA signalling cascade, possibly due to AC down-regulation in the PAG, leading to long-term neuropathic pain.
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The Journal of physiology · Jul 2015
Protein kinase C bidirectionally modulates Ih and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel surface expression in hippocampal pyramidal neurons.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, particularly that of the HCN1 isoform, are enriched in the distal dendrites of hippocampal CA1 pyramidal neurons; these channels have physiological functions with respect to decreasing neuronal excitability. In the present study, we aimed to investigate phosphorylation as a mechanism controlling Ih amplitude and HCN1 surface expression in hippocampal principal neurons under normal physiological conditions. Tyrosine phosphorylation decreased Ih amplitude at maximal activation (maximal Ih ), without altering HCN1 surface expression, in two classes of hippocampal principal neurons. Inhibition of serine/threonine protein phosphatases 1 and 2A decreased maximal Ih and HCN1 surface expression in hippocampal principal neurons. Protein kinase C (PKC) activation irreversibly diminished Ih and HCN1 surface expression, whereas PKC inhibition augmented Ih and HCN1 surface expression. PKC activation increased HCN1 channel phosphorylation. These results demonstrate the novel finding of a phosphorylation mechanism, dependent on PKC activity, which bidirectionally modulates Ih amplitude and HCN1channel surface expression in hippocampal principal neurons under normal physiological conditions. ⋯ Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels attenuate excitability in hippocampal pyramidal neurons. Loss of HCN channel-mediated current (Ih ), particularly that mediated by the HCN1 isoform, occurs with the development of epilepsy. Previously, we showed that, following pilocarpine-induced status epilepticus, there are two independent changes in HCN function in dendrites: decreased Ih amplitude associated with a loss of HCN1 surface expression and a hyperpolarizing shift in voltage-dependence of activation (gating). The hyperpolarizing shift in gating was attributed to decreased phosphorylation as a result of a loss of p38 mitogen-activated protein kinase activity and increased calcineurin activity; however, the mechanisms controlling Ih amplitude and HCN1 surface expression under epileptic or normal physiological conditions are poorly understood. We aimed to investigate phosphorylation as a mechanism regulating Ih amplitude and HCN1 surface expression (i.e. as is the case for HCN gating) in hippocampal principal neurons under normal physiological conditions. We discovered that inhibition of either tyrosine phosphatases or the serine/threonine protein phosphatases 1 and 2A decreased Ih at maximal activation in hippocampal CA1 pyramidal dendrites and pyramidal-like principal neuron somata from naïve rats. Furthermore, we found that inhibition of PP1/PP2A decreased HCN1 surface expression, whereas tyrosine phosphatase inhibition did not. Protein kinase C (PKC) activation reduced Ih amplitude and HCN1 surface expression, whereas PKC inhibition produced the opposite effect. Inhibition of protein phosphatases 1 and 2A and activation of PKC increased the serine phosphorylation state of the HCN1 protein. The effect of PKC activation on Ih was irreversible. These results indicate that PKC bidirectionally modulates Ih amplitude and HCN1 surface expression in hippocampal principal neurons.