Min-Xu Li

Protein kinase C-mediated changes in synaptic efficacy at the neuromuscular junction in vitro: The role of postsynaptic acetylcholine receptors†

Activation of a mouse in vitro neuromuscular synapse produces a reduction in synaptic efficacy which is greater for nonactivated than for activated inputs to the myotubes. This has been shown to require thrombin and thrombin receptor activation and to involve a protein kinase C (PKC)‐mediated step. We show in the present work that phorbol ester activation of PKC produces physiological loss of synapses in a time‐ and dose‐related manner. We observe, using quantitative imaging methods, a parallel loss of acetylcholine receptors (AChR) from synaptically functional neurite‐associated receptor aggregates in nerve‐muscle cocultures. Biochemical measurements of total AChR show that PKC activation reduces both AChR stability (increases receptor loss) and receptor insertion into the surface membrane. Taken together, the data suggest that PKC activation decreases the stability of AChR aggregates in the muscle surface membrane. We conclude that PKC plays a crucial role in activity‐dependent synapse reduction and does so, at least in part, by altering AChR stability. J. Neurosci. Res. 61:616–625, 2000. Published 2000 Wiley‐Liss, Inc.

The thrombin receptor mediates functional activity-dependent neuromuscular synapse reduction via protein kinase C activation in vitro†

Activity-dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down-regulation of synapse effectiveness. We further demonstrate that a thrombin receptor-activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP-evoked synapse decrement is unaffected by a specific protein kinase A blocker, H-89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity-dependent synapse reduction expressed in our system.

Opposing actions of protein kinase A and C mediate Hebbian synaptic plasticity

A compartmental nerve–muscle tissue culture system expresses Hebbian activity-dependent synapse modulation. Protein kinase C (PKC) mediates a heterosynaptic loss of efficacy, and we now show that protein kinase A (PKA) is involved in homosynaptic stabilization. Both work through postsynaptic changes in the acetylcholine receptor (AChR) as measured electrophysiologically and by imaging techniques.

The Role of the Theta Isoform of Protein Kinase C (PKC) in Activity-Dependent Synapse Elimination: Evidence from the PKC Theta Knock-Out Mouse In Vivo and In Vitro

PKC plays a critical role in competitive activity-dependent synapse modification at the neuromuscular synapse in vitro and in vivo. This action involves a reduction of the strength of inactive inputs to muscle cells that are activated by other inputs. A decrease of postsynaptic responsiveness and a loss of postsynaptic acetyl choline receptors account for the heterosynaptic loss in vitro. The loss is not seen in preparations in which PKC has been blocked pharmacologically. Here, we show that the loss does not occur in in vitro preparations made from animals genetically modified to lack the theta isoform of PKC. Synapse elimination in the newborn period in vivo is delayed but is eventually expressed in knock-out animals. PKC-dependent synapse reduction is suppressed in heterologous cultures combining normal nerve and PKC theta-deficient muscle, as might be expected from the postsynaptic locus of the changes that underlie the activity-dependent plasticity. Preparations in which PKC theta-deficient neurons innervated normal muscle also exhibited a marked deficit in PKC-deficient synapse reduction. The presynaptic action of PKC theta implied by this observation is blocked by TTX, and we propose that activity-related synapse strengthening is decreased by presynaptic PKC theta. Thus, PKC theta in both presynaptic and postsynaptic elements plays a critical role in activity-dependent synapse modulation and loss. We provide a model for activity-dependent synapse loss incorporating these findings.

Protein Kinases and Hebbian Function

The Hebb synapse, in which the strength of synapses is affected by activity in presynaptic and postsynaptic nerve cells, is a widely used model for developmental and learning-related neuroplasticity. Presynaptic and postsynaptic firing that is correlated in time is postulated to increase synaptic strength while activity in presynaptic and postsynaptic neurons that is not correlated results in weakening. The authors describe a cell biologic, mechanistic model for activity-dependent modification of synapse strength that selectively weakens inactive inputs to activated targets. Differentially localized protein kinase A and protein kinase C molecules are activated by spike and synaptic activity. Subsequent kinase-specific phosphorylation and stabilization or destabilization of synaptic receptors are molecular and cell biologic substrates of the Hebb synapse. NEUROSCIENTIST 9(2): 110–116, 2003