Calcineurin and Its Role in Synaptic Transmission.

Calcineurin (CaN) is a serine/threonine phosphatase widely expressed in different cell types and structures including neurons and synapses. The most studied role of CaN is its involvement in the functioning of postsynaptic structures of central synapses. The role of CaN in the presynaptic structures of central and peripheral synapses is less understood, although it has generated a considerable interest and is a subject of a growing number of studies. The regulatory role of CaN in synaptic vesicle endocytosis in the synapse terminals is actively studied. In recent years, new targets of CaN have been identified and its role in the regulation of enzymes and neurotransmitter secretion in peripheral neuromuscular junctions has been revealed. CaN is the only phosphatase that requires calcium and calmodulin for activation. In this review, we present details of CaN molecular structure and give a detailed description of possible mechanisms of CaN activation involving calcium, enzymes, and endogenous and exogenous inhibitors. Known and newly discovered CaN targets at pre- and postsynaptic levels are described. CaN activity in synaptic structures is discussed in terms of functional involvement of this phosphatase in synaptic transmission and neurotransmitter release.

CaMKII Is Involved in the Choline-Induced Downregulation of Acetylcholine Release in Mouse Motor Synapses.

We investigated the involvement of calcium-dependent enzymes, protein kinase C (PKC) and calcium-calmodulin-dependent protein kinase II (CaMKII), in the signaling pathway triggered by the activation of presynaptic alpha7-type nicotinic acetylcholine receptors by exogenous choline, leading to downregulation of the evoked acetylcholine (ACh) release in mouse motor synapses. Blockade of PKC with chelerythrine neither changed the evoked release of ACh by itself nor prevented the inhibitory effect of choline. The CaMKII blocker KN-62 did not affect synaptic activity but fully prevented the choline-induced downregulation of ACh release.

Postsynaptic Potentiation in Mouse Motor Synapses Induced by ATP Accumulation in Synaptic Cleft.

In mouse motor synapses, a non-selective purinoceptor antagonist suramin increased the quantum content of endplate potentials (EPP) without changing the time course of synaptic potentials. An ectonucleotidase inhibitor ARL 67156 had no effect on the amplitude and quantum content of EPP and miniature endplate potentials (mEPP) evoked by single stimuli, but significantly prolonged their duration. Long-term high-frequency stimulation of the nerve in the presence of ARL 67156 persistently increased the amplitude and duration of EPP during the train of impulses, but did not change their quantum content. ATP-γ-S, a non-hydrolyzed ATP analogue, significantly increased the amplitudes and prolonged the rising and falling phases of EPP and mEPP. The ATP-induced postsynaptic potentiation in neuromuscular transmission can result from the increase in ATP content and its longer presence in the synaptic cleft.

The mechanism of choline-mediated inhibition of acetylcholine release in mouse motor synapses.

The mechanism of action of tonically applied choline, the agonist of α7 nicotinic acetylcholine receptors (nAChRs), to the spontaneous and evoked release of a neurotransmitter in mouse motor synapses in diaphragm neuromuscular preparations using intracellular microelectrode recordings of miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) was studied. Exogenous choline was shown to exhibit a presynaptic inhibitory effect on the amplitude and quantal content of EPPs for the activity of neuromuscular junction evoked by single and rhythmic stimuli. This effect was inhibited either by antagonists of α7-nAChRs, such as methyllycaconitine and α-cobratoxin, or by blocking SK-type calcium-activated potassium (KCa) channels with apamin or blocking intraterminal ryanodine receptors with ryanodine. A hypothesis was put forward that choline in mouse motoneuron nerve terminals can activate presynaptic α7-nAChRs, followed by the release of the stored calcium through ryanodine receptors and activation of SK-type KCa channels, resulting in sustained decay of the quantal content of the evoked neurotransmitter release.

Multidirectional effects of calmodulin kinase II on transmitter release in mature and newly formed mouse motor synapses.

Calmodulin inhibitor W-7 did not cause changes in the quantal content of postsynaptic end-plate potentials (EPP) in newly formed synapses, but prevented facilitation of acetylcholine secretion induced by L-type Ca(2+)channels blocker nitrendipine. CaMKII inhibitor KN-62 produced similar effect and suppressed the increase in EPP quantal content caused by blockade of L-type Ca(2+)channels. Phosphatase PP2A inhibitor okadaic acid significantly facilitated secretion in newly formed synapses; the effect was completely blocked by KN-62. In mature synapses, okadaic acid had no effect on transmitter secretion. KN-62 increased EPP quantal content. We hypothesize that CaMKII produced different effects on acetylcholine secretion in mature and immature synapses depending on specificity of calcium signaling and PP2A phosphatase activity.

Involvement of basal and calcium-activated protein kinase C in neurotransmitter secretion in mouse motor synapses.

Blocker of presynaptic protein kinase C isoforms, GF109203X, reduced quantal content of single and rhythmic evoked end-plate potentials. The increase in quantal content of single potentials under the effect of 4- aminopyridine was neutralized by 75% under the effect of L-type Ca(2+)-channel blocker nitrendipine and completely returned to the control level after protein kinase C inhibition with chelerythrine. Neither nitrendipine, nor GF109203X affected the potentiating effect of tetraethylammonium on quantal content of end-plate potentials. Thus, we discovered basal activity of presynaptic protein kinase C under normal conditions that is aimed at the maintenance of quantal content of evoked release. It has been concluded that 4-aminipyridine, but not tetraethylammonium, triggers Ca(2+) entry into the terminal, which activates protein kinase C and enhanced the evoked acetylcholine release.

Facilitation of neurotransmitter release in mouse motor synapses in different modes of protein kinase C activation.

Protein kinase C blocker chelerythrine prevented the increase in quantal content of single and rhythmic evoked end-plate potentials after disinhibition of L-type Ca(2+)-channels with paxillin. Phorbol ester increased quantal content of single end-plate potentials and changed rhythmic activity of mouse motor synapses. The effects of phorbol ester were to a great extent neutralized by L-type Ca(2+)-channel blocker nitrendipine and were completely abolished by K(+)-channel blocker 4-aminopyridine. Thus, we discovered different facilitations of transmission after protein kinase C activation with calcium current through L-type channels and with phorbol ester.

Role of stored calcium in the regulation of neurotransmitter quantum size.

Release of stored calcium ions during activation of ryanodine receptors with ryanodine or caffeine elevates the mean amplitude of spontaneous miniature end-plate potentials. Blockade of these receptors with selective antagonists abolishes this effect. Preliminary loading of the motor nerve terminals with intracellular calcium buffer EGTA-AM, but not with BAPTA-AM, also completely prevented the ryanodine-induced increment of miniature end-plate potential amplitude probably induced by the release of stored calcium. Vesamicol, a selective blocker of acetylcholine transport into vesicles, prevented the ryanodine-induced increment of the mean amplitude of miniature end-plate potentials. This increment was 2-fold more pronounced after preliminary blockade of protein kinase C with chelerythrine and was completely abolished by blockade of protein kinase A with H-89.

Mechanism of inhibition of acetylcholine secretion in newly formed mouse synapses involving Ca2+-dependent kinases and voltage-gated K+-channels.

Nifedipine, a blocker of L-type Ca(2+)-channels, increased quantal content of endplate potentials in newly formed nerve-muscle synapses, while R 24571 (calmodulin inhibitor) and KN 62 (inhibitor of calmodulin-dependent kinase II) did not change it. KN 62 suppressed the increase in quantal content of endplate potentials evoked by nifedipine. Similar to nifedipine, chelerythrine and bisindolylmaleimide I (blockers of protein kinase C) increased quantal content of endplate potentials. In the presence of chelerythrine, nifedipine lost its ability to facilitate secretion of neurotransmitter. 4-Aminopyridine, a blocker of voltage-gated potassium channels, increased quantal content of endplate potentials. In the presence of chelerythrine, 4-aminopyridine induced no additional increase in the quantal content of endplate potentials. In its turn, chelerythrine induced no extra facilitation of secretion in the presence of 4-aminopyridine. It is hypothesized that Ca(2+)-dependent inhibition of secretion results from suppression of calmodulin-dependent kinase II and activation of protein kinase C, which potentiates the work of voltage-gated K(+)-channels.

Effect of fast and slow calcium buffers on induced secretion of neurotransmitter.

Loading of mouse motor nerve terminals with EGTA-AM, but not with BAPTA-AM, inhibited the release of the neurotransmitter in response to stimulation of the nerve with rare (0.3 Hz) "single" pulses. During rhythmic stimulation with short (50 EPP) high-frequency (20 Hz) series, BAPTA-AM buffer modified burst pattern in a dose-dependent manner: it replaced the phase of initial facilitation by persistent depression of secretion and decreased its plateau level at the end of the burst. In contrast, loading of the nerve terminals with EGTA-AM buffer produced no effect on the phase of initial facilitation, but decreased the plateau level to the same degree as BAPTA-AM did. Probably, the different effects of both buffers on secretion of neurotransmitter reflect peculiarities of involvement of fast and slow Ca(2+)signals of motor terminals in single and rhythmic release of the neurotransmitter.

Facilitation of acetylcholine secretion in mouse motor synapses caused by calcium release from depots upon activation of L-type calcium channels.

Pharmacological disinhibition of L-type Ca(2+) channels by two ways (with agonist S(-) BAY K 8644 and iberiotoxin, a Ca(2+)-activated BK-type K(+)-channel blocker) increases quantal content of evoked end-plate potentials, which was completely prevented by ryanodine (2 microM) blockade of ryanodine receptors. We conclude that increased quantal secretion of the transmitter induced by L-type Ca(2+) channel functioning requires activation of ryanodine receptors and calcium release from depots in motor terminals in mice.

[Suppression of mediator secretion in murine neogenic motor synapses with the participation of L-type Ca(2+)-channels and ryanodine receptors].

L-type Ca(2+)-channel blockers, verapamil (5 microM) and nifedipine (10 microM), have increased the quantum composition of endplate potentials (EPP) and the level of induced rhythmic activity of neogenic synapses. L-type Ca(2+)-channel activator BAY 8644 (1 microM) has a decreased mediator secretion level. Nifedipine (10 microM) has not changed the frequency and amplitude of diminutive EPPs in the dormant state or during potassium depolarization. Blocking of the prejunctional ryanodine receptor with ryanodine (10 microM) led to an increase in the single EPP quantum composition that was qualitatively similar to nifedipine and verapamil, but more marked, and also caused the reinforcement of mediator release during the rhythmic EPP salvo. Ryanodine receptor activation with ryanodine (1 microM) resulted in reduction of the quantum composition of single and rhythmic EPPs. This effect was partially prevented with nifedipine (10 microM).

Effect of L-type calcium channel blockers on activity of newly formed synapses in mice.

Verapamil (5 microM), nifedipine (10 microM), and ryanodine (10 microM) potentiated rhythmic activity of newly formed synapses, while apamin produced no effect on this potentiation. Ryanodine (1 microM) suppressed synaptic activity, and this effect can be prevented with nifedipine. It was hypothesized that in newly formed synapses Ca2+ entry through L-type channels triggers the release of stored Ca2+, which inhibits secretion of the neurotransmitter.

Digoxin facilitates neuromuscular transmission in mouse diaphragm.

Low concentration of digoxin (3 nM) facilitated spontaneous and evoked release of neurotransmitter acetylcholine thereby increasing the frequency of miniature end-plate potentials, amplitude of single end-plate potentials, their quantum content and the plateau level in the bursts during stimulation of the phrenic nerve at rates of 4, 7, and 50 Hz. These effects were prevented by blockade of ryanodine receptors with ryanodine (10-20 microM).

Effect of PAR1 agonist on acetylcholine secretion in a newly formed neuromuscular synapse in mice.

Peptide agonist of PARI in a concentration of 10 microM significantly facilitated neuromuscular transmission in newly formed synapses in mice. The absence of changes in the amplitude of miniature end-plate potentials attests to presynaptic mechanism of the effect of PAR1 agonist. The effect of the peptide was blocked by protein kinase A inhibitor H89 (1 microM). Blockade of inositol-1,4,5-trisphosphate receptors with 2-amino-ethoxydiphenylborate (30 microM) did not prevent the effects of PARI agonist. Inhibition of protein kinase C with bisindolylmaleimide (1 microM) facilitated neuromuscular transmission in newly formed synapses. Protein kinase C inhibition was associated with reversal of the object of PARI agonist: transmission inhibition instead of facilitation.

Effect of nicotine on neuromuscular transmission in mouse motor synapses.

Nicotine (10 nM) inhibits rhythmic activity of the neuromuscular synapse in mice. This effect was prevented by alpha-cobratoxin and apamin. Hence, the effects of nicotine are realized via presynaptic neuronal nicotinic cholinoceptors and Ca(2+)-activated potassium channels.

Study of neurotrophic activity of thrombin on the model of regenerating mouse nerve.

Experiments demonstrated a dose-dependent facilitating effect of thrombin and peptide thrombin receptor agonist PAR1 (TRAP6) on regeneration of mouse peripheral nerve after its crushing. The maximum neurotrophic effect was observed at low concentrations of thrombin (10 nM) and TRAP6 (10 microM).

[Study of neurotrophic effects of yashaltin mud in experimental model of denervation-reinnervation syndrome of shank muscles in mice].

On the experimental model of denervation-reinnervation syndrome of mouse shank muscles neurotrophic effects of jashaltinsky medicinal mud were studied. In experimental mice therapeutic mud has been applied on the small back, tail and rib areas of the body 10 days after nerve crushing. On day 11 after nerve crushing a stable reproduction of a compound nerve action potential with constant amplitude was revealed in repetitive (50 Hz) nerve stimulation.

[Dual action of intracellularly released calcium on the quantal mediator secretion].

The mice diaphragm muscle and microelectrode technique were used to check the influence of ryanodine (0.5 mcM) on spontaneous and evoked mediator release under conditions of potassium depolarization (8-16 mM [K+]ex or rhythmic (4-100 Hz) stimulation of motor nerve terminals. Weak tonic calcium loading (by muscle exposition to 8 mM [K+]ex) caused a two-fold frequency increase if miniature and plate potentials (MEPPs), which was returned to the basal level by subsequent application of ryanodine. This inhibitory effect of ryanodine was blocked by apamin (500 nM) a blocker of K+(Ca)-channels. A greater calcium load of terminals (in solution with 16 mM [K+]ex) caused a 15-fold increase of MEPPs frequency. Subsequent ryanodine application caused an additional 2-3-fold increase of MEPPs frequency. During rhythmic activity of motor synapses, ryanodine was able to decrease the amplitude of EPP by 60% at plateau phase at short low frequency (4 Hz) of discharges and to increase the amplitude of EPP by 60-150% at high frequency (70-100 Hz) of discharges. It is concluded that rynodine induced calcium release from intraterminal Ca2+-stores can influence dual: excitatory or inhibitory, action on spontaneous and evoked mediator release, due to different intraterminal calcium loads and regimen of synaptic activity.