Ca 2 1 y calmodulin-dependent protein kinase II phosphorylation of the presynaptic protein synapsin I is persistently increased during long-term potentiation ( hippocampus y synaptic plasticity )

Content Provider	Semantic Scholar
Author	Nayak, Abinash Moore, Charles I. Browning, Michael D.
Copyright Year	1996
Abstract	Long-term potentiation (LTP) is an increase in synaptic responsiveness thought to be involved in mammalian learning and memory. The localization (presynaptic andyor postsynaptic) of changes underlying LTP has been difficult to resolve with current electrophysiological techniques. Using a biochemical approach, we have addressed this issue and attempted to identify specific molecular mechanisms that may underlie LTP. We utilized a novel multipleelectrode stimulator to produce LTP in a substantial portion of the synapses in a hippocampal CA1 minislice and tested the effects of such stimulation on the presynaptic protein synapsin I. LTP-inducing stimulation produced a long-lasting 6-fold increase in the phosphorylation of synapsin I at its Ca21ycalmodulin-dependent protein kinase II (CaM kinase II) sites without affecting synapsin I levels. This effect was fully blocked by either the N-methyl-D-aspartate receptor antagonist D(2)-2-amino-5-phosphonopentanoic acid (APV) or the CaM kinase II inhibitor KN-62. Our results indicate that LTP expression is accompanied by persistent changes in presynaptic phosphorylation, and specifically that presynaptic CaM kinase II activity and synapsin I phosphorylation may be involved in LTP expression. Synaptic plasticity has been extensively studied in the mammalian hippocampal formation. Long-term potentiation (LTP) is one form of plasticity that has attracted considerable attention as a possible cellular substrate for learning and memory. LTP was first described by Bliss and Lomo (1), who demonstrated that a relatively brief burst of high-frequency stimulation (HFS) produces a long-lasting enhancement of synaptic responses in the mammalian hippocampus. Although presynaptic (2–4) andyor postsynaptic (5–8) changes have been proposed to underlie LTP, the exact localization of these changes remains highly controversial. This has been due in part to the difficulty in resolving presynaptic and postsynaptic events electrophysiologically (9). In this study, we have utilized a biochemical approach to address this issue and attempted to identify molecular mechanisms that may underlie LTP. Numerous studies suggest that protein phosphorylation (specifically by protein kinase C and Ca21ycalmodulindependent protein kinase II [CaM kinase II)] plays a critical role in the induction and possibly the maintenance of LTP (10–12). However, identification of the specific phosphoproteins involved in LTP has been problematic. Synapsin I is a phosphoprotein that has a number of features which make it a likely candidate to mediate persistent presynaptic plasticity (13–15). It is found exclusively in neuronal presynaptic terminals, where it associates with synaptic vesicles (16–18). Considerable evidence suggests that synapsin I plays a key role in neurotransmitter release by regulating the availability of synaptic vesicles for exocytosis (19–24) (however, see ref. 25). According to this model, synapsin I crosslinks synaptic vesicles to the cytoskeleton in a phosphorylation state-dependent manner, thereby limiting the ability of vesicles to move to active zones for exocytosis. Phosphorylation of synapsin I by CaM kinase II reduces its affinity for synaptic vesicles 10-fold, thus removing its constraint on the availability of vesicles for release. We have previously tested the hypothesis that synapsin I plays a role in LTP by examining the effects of pharmacological LTP-inducing, treatments [e.g., b-adrenergic agonists (26) and protein kinase C activators (27)] on synapsin I phosphorylation. Bath application of isoproterenol (14) or phorbol dibutyrate (28) produces a dose-dependent increase in the phosphorylation of synapsin I at its CaM kinase II sites. Furthermore, isoproterenol’s ability to induce synapsin I phosphorylation and its ability to generate LTP-like potentiation share the same brain region specificity (action on dentate gyrus and not CA1) (29). Such data support a role for synapsin I and its phosphorylation in LTP. There are, however, significant limitations to this pharmacological approach. The LTP-like potentiation produced by pharmacological treatments may not be identical to LTP induced by classical electrophysiological techniques (29–32). In addition, the observed changes in synapsin I phosphorylation may be due to non-LTP effects of drug treatment, rather than purely LTP-related mechanisms. Hence, here we have been particularly interested in studying synapsin I phosphorylation following electrophysiologically induced LTP. The typical LTP stimulation paradigm involves delivering HFS to area CA1 through a single stimulating electrode. Using this method, we have been unsuccessful at detecting any LTPassociated changes in synapsin I phosphorylation. We have been concerned that such stimulation may not have induced LTP in a sufficient fraction of synapses for any changes in synapsin I phosphorylation to be detectable. Indeed, studies by two different groups suggest that single electrode stimulation produces LTP in only a limited number of synapses even within subregions of the hippocampal slice (33, 34). In the present study, we have designed a new stimulating electrode array, called the rake, and developed a stimulation paradigm to deliver LTP to more synapses in the slice. We also used the CA1 minislice preparation to remove from our analyses the large unstimulated hippocampal areas (dentate The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: APV, D(2)-2-amino-5-phosphonopentanoic acid; CaM kinase II, Ca21ycalmodulin-dependent protein kinase II; EPSP, excitatory postsynaptic potential; HFS, high-frequency stimulation; LTP, long-term potentiation; NMDA, N-methyl-D-aspartate; s-HFS, simultaneous HFS. *Present address: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139. †To whom reprint requests should be addressed.
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