Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro Oanh H. Ho,* Jary Y. Delgado and Thomas J. O’Dell* *Department of Physiology and  Interdepartmental PhD Program for Neuroscience, David Geffen School of Medicine at UCLA, Los Angeles, California, USA Abstract The acute hippocampal slice preparation has been widely used to study the cellular mechanisms underlying activity- dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although protein phosphorylation has a key role in LTP and LTD, little is known about how protein phosphorylation might be altered in hippocampal slices maintained in vitro. To begin to address this issue, we examined the effects of slicing and in vitro maintenance on phosphorylation of six proteins involved in LTP and/or LTD. We found that AMPA receptor (AMPAR) glutamate receptor 1 (GluR1) subunits are persistently dephosphorylated in slices maintained in vitro for up to 8 h. a calcium/calmodulin-dependent kinase II (aCamKII) was also strongly dephosphorylated during the first 3 h in vitro but thereafter recovered to near control levels. In contrast, phos- phorylation of the extracellular signal-regulated kinase ERK2, the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2), and Src family kinases was significantly, but transiently, increased. Electrophysiological experiments revealed that the induction of LTD by low-frequency synaptic stimulation was sensitive to time in vitro. These findings indicate that phos- phorylation of proteins involved in N-methyl-D-aspartate (NMDA) receptor-dependent forms of synaptic plasticity is altered in hippocampal slices and suggest that some of these changes can significantly influence the induction of LTD. Keywords: brain slices, hippocampus, long-term depression, long-term potentiation, protein phosphorylation. J. Neurochem. (2004) 91, 1344–1357. Since its development more than 50 years ago by Henry McIlwain, the acute in vitro brain slice preparation has been extensively used in cellular, biochemical, and molecular studies of neuronal physiology (Collingridge 1995). Although brain slices provide a convenient and powerful way to study the physiology of neurons and synapses in the CNS, the techniques used to prepare and maintain brain slices in vitro can have significant effects on cell physiology. For instance, soon after tissue slicing there is a precipitous drop in cellular levels of glycogen, phosphocreatine, and ATP (McIlwain et al. 1951; Whittingham et al. 1984; Feig and Lipton 1990), a near complete loss of dendritic microtubules (Burgoyne et al. 1982; Fiala et al. 2003), and a dramatic increase in cAMP and cGMP levels (Whittingham et al. 1984). Although many of these changes recover with time, slices also undergo more persistent alterations such as a decrease in the glutamate receptor 1 (GluR1) and GluR3 subunits of AMPA-type glutamate receptors (Taubenfeld et al. 2002) and increases in mRNA levels for several transcription factors, including c-fos and zif268 (Zhou et al. 1995; Taubenfeld et al. 2002). Hippocampal slices also have more synapses than perfusion-fixed tissue, suggesting that a considerable amount of structural reorganization occurs in brain slices (Kirov et al. 1999). Received May 25, 2004; revised manuscript received August 11, 2004; accepted August 17, 2004. Address correspondence and reprint requests to Dr Thomas J. O’Dell, Department of Physiology, David Geffen School of Medicine at UCLA, 53–231 Center for the Health Sciences, Box 951751, Los Angeles, CA 90095, USA. E-mail: todell@mednet.ucla.edu Abbreviations used: ACSF, artificial cerebrospinal fluid; AMPAR, AMPA receptor; aCamKII, a calcium/calmodulin-dependent kinase II; APV, 2-amino-5-phosphonovaleric acid; ERK, extracellular signal- regulated kinase; fEPSP, field excitatory post-synaptic potential; GluR, glutamate receptor; LFS, low-frequency stimulation; LTD, long-term depression; LTP, long-term potentiation; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; NMDA, N-methyl-D-aspartate; Pyk2, proline-rich tyrosine kinase 2. Journal of Neurochemistry , 2004, 91, 1344–1357 doi:10.1111/j.1471-4159.2004.02815.x 1344 Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357