Voltage-clamp whole-cell recordings were acquired with a Multiclamp 700A amplifier (Axon Devices, Sunnyvale, CA, USA)

Voltage-clamp whole-cell recordings were acquired with a Multiclamp 700A amplifier (Axon Devices, Sunnyvale, CA, USA). == Fig.1. regulate LTP expression. Keywords:calmodulin, rat hippocampus, NMDARs, PKC, synaptic plasticity == INTRODUCTION == Long-term potentiation (LTP) is one of the best-characterized forms of synaptic plasticity (Lisman, 1989;Alkon & Nelson, 1990;Bliss & Collingridge, 1993;Malenka & Nicoll, 1999;Hayashiet al., 2000;Malinowet al., 2000). At CA1 hippocampal excitatory synapses, two different classes of glutamate receptors are crucial for synaptic plasticity: ionotropic NMDA receptors (NMDARs) and metabotropic glutamate receptors (mGluRs). NMDAR activation triggers an influx of Ca2+into the dendritic spine, resulting in a series of Ca2+-dependent Rabbit Polyclonal to MADD events, e.g. Ca2+/CaM-dependent protein kinase II (CaMKII) activation, and ultimately AVX 13616 leading to the expression of LTP indicated by insertion of AMPA receptors (AMPARs) into the synapses. The activation of mGluRs, on the other hand, results in the initiation of the phospholipase C/diacylglycerol/protein kinase C (PLC/DAG/PKC) second messenger pathway. Although both NMDAR and mGluR signaling are crucial for LTP, the mechanism by which they crosstalk remains unresolved. Neurogranin (Ng), a neuron-specific, postsynaptic protein, may provide insight into such crosstalk. Ng is usually a known PKC substrate that is essential for LTP and potentiates synaptic strength in an NMDAR-dependent manner (Zhonget al., 2009). These effects are attributed to Ngs ability to bind and target CaM in the dendritic spines. Ng is the AVX 13616 most abundant CaM-binding protein postsynaptically under basal conditions (Represaet al., 1990;Gerendasyet al., 1994a;Gerendasyet al., 1994b;Watsonet al., 1994;Zhabotinskyet al., 2006). Since the amount of CaM in specified cell compartments is usually a limiting factor for the target activation (Zhabotinskyet al., 2006), Ng, through its regulated binding AVX 13616 to CaM, can modulate LTP expression. You will find two mechanisms that can regulate Ng-CaM binding and hence the local availability of free unbound CaM. First, CaM can dissociate, reversibly, from Ng when local Ca2+is usually increased, e.g. via NMDAR activation (Huanget al., 1993;Gerendasyet al., 1995). On the other hand, activation of mGluRs results in PKC-mediated phosphorylation of Ng at its serine 36 (S36) residue within the IQ-motif, rendering CaM incapable of rebinding to phosphorylated Ng (Ramakerset al., 1995). These two mechanisms can be simplified as follows: It has been shown that Ng-mediated potentiation is dependent on NMDAR activity as well as Ngs ability to release CaM upon demand (Zhonget al., 2009). However, the role of Ng phosphorylation–the crosstalk point between the two major classes of glutamate receptors in LTP–in synaptic function has not been previously explored. In this study, we investigated the role of Ng phosphorylation in synaptic function and plasticity in rat CA1 hippocampal neurons. We expressed Ng mutants with different phosphorylation properties and examined their effects on synaptic transmission and LTP expression. Our results show that phosphorylation of Ng plays an important role in determining the magnitude of LTP expression. We propose a novel AVX 13616 mechanism through which Ng phosphorylation fine-tunes synaptic plasticity. == MATERIALS AND METHODS == == Animals and hippocampal slice preparation == Young Sprague-Dawley rats (postnatal day 5 or 6) were purchased from Charles River Laboratories (Portage, MI, USA) and managed on a daily 12 h light: 12 h dark cycle. All biosafety procedures and animal care protocols were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee (IACUC). Hippocampal slices were prepared as explained previously (Gahwileret al., 1997). == DNA constructs and expression == GFP-tagged Ng mutants (Ng-SA and Ng-SD) were cloned from GFP-Ng plasmid as explained (Zhonget al., 2009) using the gene-tailor site-directed mutagenesis system (Invitrogen, Carlsbad, CA, USA). Mutations were made in the serine 36 residue to an alanine for Ng-SA and to an aspartate in Ng-SD. Constructs were re-cloned into pSinRep5 (Invitrogen) for Sindbis computer virus preparation. Recombinant plasmids have been verified by sequencing. After 27 days in culture, the recombinant gene was delivered into the slices. For the experiments shown inFig. 3, we used the biolistic delivery method (Loet al., 1994), which allowed us to deliver two plasmids bearing mammalian promoters. For expression of single proteins, we used the Sindbis computer virus expression system, which is a replication-deficient, low-toxicity and neuron-specific system (Malinow, 1999). == Fig.3. Non-phosphorylatable mutant of neurogranin does not interfere with AMPA receptor insertion. == The rectification index (RI) is usually calculated as the ratio of the amplitude of AMPAR-mediated responses at 60 mV over that at +40 mV. Endogenous receptors conduct currents at 60 and +40 mV, whereas recombinant receptors conduct currents only at unfavorable potentials. Therefore, delivery of the recombinant GluR1 homomeric receptors is usually accompanied by an increase in the RI (comparable results AVX 13616 were obtained for the control neurons.