The Section on Neurobiology, directed by Phillip Nelson, studies those mechanisms by which input to the nervous system from the environment influences nervous system development. In particular, we focus on the influence of neural electrical and synaptic activity in modulating synaptic circuits during development. We have identified some of the cell-biologic processes, including protein kinase and neurotrophin action, that affect the strength of synapses in vitro and in vivo.

Protein Kinase C and Protein Kinase A Mediate Activity-Dependent Modulation of Synaptic Efficacy
Nelson, Jia, Li, Yang, Lanuza
Previous work with an in vitro synaptic system (see Figure 1) had shown that protein kinase C (PKC) activation is necessary and sufficient to produce a reduction in synaptic efficacy at the neuromuscular junction. This was primarily a postsynaptic phenomenon that involved a loss of acetylocholine receptors (AChR) from the high-density receptor clusters at the neuromuscular synapse. Collaborative in vivo experiments show that PKC plays a vital role in the intact animal. Transition from the polyneuronal to mononeuronal innervation is blocked by PKC blockers and accelerated by PKC activators during the middle stages of neuromuscular junction development. PKC-independent processes become evident late in the maturation process. We have used PKC theta knock-out animals in experiments both in vivo and in vitro. Mice in which the theta isoform of PKC was knocked out show a delay in the synapse elimination that occurs at the neuromuscular junction, although loss of multiple innervation eventually does occur. When synapses form in vitro between nerve and muscle from PKC theta knock-out animals, stimulation of PKC no longer produces synapse loss. When a PKC-activating phorbol ester such as PMA is placed in the center, synaptic chamber of our three-compartment system, we see loss of synapses as expected if PKC acted in the muscle. Similarly, a PKC blocker is effective if applied only in the center, synaptic compartment. Also consistent with a muscle locus of PKC action are the results of experiments in which PKC-deficient muscle was combined with normal nerve in the side neuronal chambers. These preparations showed a marked decrement in the synapse elimination produced by PKC activation. More surprising were results with normal muscle and PKC theta knock-out nerve. These preparations also showed a marked deficit in PKC-induced synapse loss, entirely comparable to that shown with the muscle knock-out/normal nerve combination. This suggests that presynaptic PKC function must be combined with postsynaptic PKC to produce synapse loss.

Figure 23

Illustration of the compartmental neuromuscular tissue system.
a. Diagram of the teflon insert with stimulating electrodes and the neural (side compartments) and myotube (center compartment) cultures. Cal 100 µm. b. Neurites coming from the two side compartments are stained with an antibody against choline aceyltransferase to show the bilateral cholinergic synaptic investment of a group of myotubes.

Protein kinase A (PKA) also probably has both pre- and postsynaptic action. PKA mediates the stabilization and strengthening of stimulated inputs, and we have shown that postsynaptic injection of PKI, an inhibitor of PKA, in conjunction with electrical activation of the synapse, results in a major loss of synaptic connectivity (Figure 24).

Figure 24

PKA activation prevents stimulation-related synapse down-regulation. a. Left two bars are from a control group with single inputs, stimulated for 2 h (n = 5). There was no significant difference before and after stimulation (*p > 0.4). Right two bars are for EPPs recorded in 10 experiments before and after treatment with 1 µM H-89 in the center chamber plus electrical stimulation for 2 h (n = 10, **p < 0.025). å before the treatment, after the treatment. b. Intracellular recording of EPPs were taken after PKI injection (5Hz. 30 msec ca. 1 nA for 30 minutes) but before and after a 20-minute period without (left) or with (right) 5 Hz. neural stimulation. For both data sets, n = 4; p < 0.005. c. Cyclic-AMP prevents the heterosynaptic loss of synapse strength produced by unilateral stimulation of bilaterally innervated myotubes. EPPs were measured before and after 2 h of stimulation with and without cAMP (2 mM) in the center chamber. For the unstimulated inputs, db cAMP produces a significant block of EPP decrement (p < 0.004) å stimulation only, no cAMP (n =10), _: stimulation with cAMP in the center chamber, the cAMP was then washed out and the EPPs remeasured (n = 5). d) Calcitonin gene-related peptide (CGRP) increases cellular cAMP level and leads to the activation of PKA in muscle. CGRP (1µM) reduced the functional synapse loss produced by unilateral electrical stimulation (treatment and stimulation were for 20 h), control n =14, CGRP n =15, p < 0.02.

When a PKA blocker, H-89, is applied to the side chamber only, we also see a major activity-dependent loss of synapses that is attributable to a decrease in the probability of release of neurotransmitter. Such sensitivity to stimulation takes 20 to 30 minutes to develop, which we interpret as the time for some PKA-dependent material to be transported from the cell body to the synapse, where it is needed for maintaining transmitter output.

Neurotrophic Action on Neuromuscular Synapse Structure
Yang, Nelson
We have examined the possibility that the Glia Derived Neurotrophic Factor (GDNF) may have an effect on synapse stabilization. Others have shown that GDNF released from muscle can affect presynaptic function. We have tested whether there may be some effect of GDNF on muscle function, specifically on the acetycholine receptor (AChR). We find that GDNF treatment of muscle, even in the absence of nerve but also in innervated fibers, increases the rate at which AChR is inserted into receptor clusters. GDNF treatment does not affect the rate of loss of receptors from the clusters. We have examined some of the cell biological mechanisms by which GDNF is coupled to receptor disposition.

We feel that our results identify some of the critical postsynaptic events mediating Hebbian plasticity; therefore, we will focus in the future on possible presynaptic mechanisms.