Synaptic Plasticity: Spatio-Temporal Analysis of Actin Dynamics

Abstract

Synaptic Plasticity: Spatio-Temporal Analysis of Actin Dynamics Olga Kochan 1 * and Robert S. Zucker** *Molecular & Cell Biology and Psychology Major, College of Letters and Science, **Department of Molecular & Cell Biology, University of California, Berkeley Key words: Synaptic plasticity, synaptic transmission, cAMP dependent enhancement, actin, HCN channels ABSTRACT: Glutamatergic synapses are highly modifiable, making them key targets in processes such as learning and memory. In crayfish glutamatergic neuromuscular junctions, hyperpolarization and cyclic nucleotide-activated (HCN) channels and actin cytoskeleton dynamics are critical intermediate factors in hormonal modulation of glutamatergic synapses which lead to cAMP (3’-5’-cyclic adenosine monophosphate)-dependent enhancement of synaptic transmission. Although models have been proposed, there has been a lack of experimental evidence on the relationship between HCN channels and the integrity of the actin cytoskeleton during cAMP-dependent enhancement. The specific goal of this study is to test the sequence of activation of the aforementioned mediators in synaptic enhancement via precisely controlled pharmacological experiments. At glutamatergic neuromuscular junctions of crayfish limb muscles, HCN channel activator, lamotrigine (50 µM), enhanced synaptic transmission about 20%. This enhancement was completely blocked with actin depolymerizer, latrunculin B (3 µM). These results support previous models of the temporal arrangement of events leading to synaptic enhancement, specifically that changes in actin cytoskeleton follow HCN channel activation. Concurrently, we are also using a spatio-temporal marker called phalloidin, a toxin which binds actin filaments, to further test the hypothesis that activation of HCNCs precedes actin cytoskeleton polymerization. This allows for manipulation of HCN channels and visualization of actin that could propose the associated molecular mechanisms. Preliminary evidence suggests actin reorganization. INTRODUCTION Synaptic plasticity underlies adaptability of the nervous system to changing circumstances and is involved in learning and memory. One such modifiability includes cAMP-dependent enhancement of synaptic transmission. At crayfish neuromuscular junctions (NMJ), the number of glutamate-containing synaptic vesicles available for release by action potentials at the exciter is increased by the circulating neurohormone serotonin (1) acting in part by production of the presynaptic second messenger cAMP (2). This cAMP-dependent enhancement of transmission, which can also be produced directly by stimulating adenylyl cyclase with forskolin, involves the activation of presynaptic hyperpolarization and cyclic nucleotide-activated channels (HCNCs) by cAMP as well as the integrity of the actin cytoskeleton (3). In past studies, it was noted that any of three HCNC blockers (ZD 7288, DK-AH 269, or Cs+) or depolymerization of actin by cytochalasin D, latrunculin B, or swinholide A, greatly reduce the forskolin-induced enhancement of transmission (4). A schematic model of the steps involved in induction of serotonergic (cAMP- dependent) enhancement of transmission has been proposed (4). From the model, it is evident that both HCN channels and actin cytoskeleton dynamics are required to mediate synaptic enhancement; however, the relationship between HCNCs and actin still remains elusive and merely hypothesized (Figure 1). Therefore, in this study, we set out to determine the sequential relationship between the actin cytoskeleton and HCN channels in supporting synaptic plasticity and transmission. We used the crayfish NMJ to study the implicated mediators of synaptic enhancement. This model offers many advantages such as availability, suitability for electrophysiological recording and molecular manipulations, as well as physiological and biochemical properties nearly identical to those of mammalian brain synapse. We investigated the mechanism by which changes in HCN channel activation and actin cytoskeleton dynamics result in synaptic enhancement via two main experimental protocols. The first experimental technique manipulates HCN channels 8-CPT Jasplakinolide Lat B Figure 1: This previously published figure (4) depicts a schematic of the steps involved in induction of serotonergic (cAMP-dependent) enhancement of transmission, tetanic activation of LTF (long term facilitation), and synaptic tagging, which are all mechanisms of synaptic plasticity. cAMP enhances transmission by activating Epac and HCN channels, the latter acting via an actin dependent step. The temporal relationship between HCN channels and actin was still indefinite and needed to be further investigated, leading to the experiments in the present study. Corresponding author: University of California, Department of Molecular and Cell Biology, 111 LSA, Berkeley, Ca 94720. Phone number: (925) 286-8491. E-mail: olga54@berkeley.edu.