Increase In mIPSC Frequency Research Articles

Protein kinase A-mediated enhancement of miniature IPSC frequency by noradrenaline in rat cerebellar stellate cells.

1. Cellular mechanisms underlying the enhancement by noradrenaline (NA) of inhibitory postsynaptic currents (IPSCs) were studied at inhibitory synapses in the molecular layer of the cerebellum. IPSCs were obtained from stellate cells in rat cerebellar slices using tight-seal whole-cell recording. 2. Miniature IPSCs (mIPSCs) were recorded in the presence of tetrodotoxin (TTX; 100 nM). NA (10 microM) markedly increased the frequency of mIPSCs, but did not alter their mean amplitude. Bath application of the inhibitor of adenylyl cyclase 9-(tetrahydro-2'-furyl) adenine (SQ 22,536; 300 microM), of the wide spectrum protein kinase inhibitor staurosporine (1 microM), and of the Rp-diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS; 500 microM), a specific inhibitor of cAMP-dependent protein kinase (PKA), inhibited the mIPSC frequency increase induced by NA. 3. The increase in mIPSC frequency was not attenuated by Cd2+ (100 microM), a blocker of voltage dependent calcium channels. However, after a 12-15 min pre-incubation in Ca(2+)-free saline, the effect of NA on mIPSCs was markedly inhibited. If Ca2+ ions were readmitted in the presence of NA, enhancement of the mIPSC frequency was largely restored. 4. Application of the membrane permeant analogue of cAMP, 8-Br-cAMP (1 mM), together with the inhibitor of cAMP phosphodiesterase, 3-isobutyl-1-methylxanthine (IBMX; 100 microM), caused a frequency increase of mIPSCs. Forskolin also mimicked the stimulatory effect of NA on mIPSC frequency. The effects of both 8-Br-cAMP and forskolin persisted in Ca(2+)-free saline, suggesting that the modulation of transmitter release does not require Ca2+ influx. 5. On the whole, the results indicate that the potentiation of mIPSC frequency by NA is mediated through the sequential activation of adenylyl cyclase and protein kinase A (PKA), and that PKA modulates the vesicle release mechanism rather than Ca2+ influx. The lack of effect of NA after prolonged incubation in Ca(2+)-free solution may be due to an inhibition of adenylyl cyclase by a gradual lowering of the cytosolic presynaptic Ca2+ concentration.

Presynaptic enhancement of inhibitory synaptic transmission by protein kinases A and C in the rat hippocampus in vitro

The protein kinase C activator phorbol 12,13-dibutyrate (0.5 microM, PDBu) and the protein kinase A activator forskolin (20 microM) each increased evoked monosynaptic inhibitory postsynaptic current (IPSC) amplitude, without affecting its reversal potential, and increased the frequency of miniature IPSCs (mIPSCs), without affecting their amplitude or kinetics, as assessed with whole-cell recording form CA3 pyramidal cells in hippocampal slice cultures. The effects of forskolin and PDBu on both evoked IPSC amplitude and mIPSC frequency were additive and were antagonized by inhibitors of protein kinases A and C, respectively. The kinase activator-induced increases in mIPSC frequency were quantitatively comparable to the increases in evoked IPSC amplitude. The increases in mIPSC frequency were not attenuated by the voltage-dependent calcium channel blocker Cd2+ (100 microM). We conclude that stimulation of protein kinases A and C potentiates hippocampal inhibitory synaptic transmission through independent presynaptic mechanisms of action. Kinase-induced potentiation of spontaneous release does not require modulation of axon terminal Ca2+ channels. This mechanism may also contribute substantially to the potentiation of evoked release.

Calcium channels responsible for potassium-induced transmitter release at rat cerebellar synapses.

The effects of calcium channel blockers on potassium-induced transmitter release were studied in thin slices of cerebellum from neonatal rats using whole-cell patch clamp methods. Miniature inhibitory postsynaptic currents (mIPSCs) mediated by gamma-aminobutyric acid (GABA) were recorded from deep cerebellar nuclear neurones in the presence of tetrodotoxin. The frequency of mIPSCs was reproducibly increased by a brief application of high-potassium solution. In the presence of the L-type Ca2+ channel blocker nicardipine (10 microM), the potassium-induced increase in mIPSC frequency was suppressed by 49%. Neither the mean amplitude nor the time course of mIPSCs was affected by the blocker. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTX, 3 microM) had no effect on the frequency of potassium-induced mIPSCs. The P-type Ca2+ channel blocker omega-Aga-IVA (200 nM) suppressed the potassium-induced increase in mIPSC frequency by 83% without affecting the mean amplitude or time course of mIPSCs. Comparing these data with previous studies of neurally evoked transmission, it is concluded that the Ca2+ channel subtypes responsible for potassium-induced transmitter release may be different from those mediating fast synaptic transmission.