The Journal of Neurophysiology Vol. 80 No. 5 November 1998, pp. 2797-2800
Copyright ©1998 by the American Physiological Society

RAPID COMMUNICATION

Effects of PKA and PKC on Miniature Excitatory Postsynaptic Currents in CA1 Pyramidal Cells

Reed C. Carroll¹, Roger A. Nicoll^{2, 3}, and Robert C. Malenka^{1, 2}

¹ Department of Psychiatry, ² Department of Physiology, and ³ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Carroll, Reed C., Roger A. Nicoll, and Robert C. Malenka. Effects of PKA and PKC on miniature excitatory postsynaptic currents in CA1 pyramidal cells. J. Neurophysiol. 80: 2797-2800, 1998. Protein kinases play an important role in controlling synaptic strength at excitatory synapses on CA1 pyramidal cells. We examined the effects of activating cAMP-dependent protein kinase or protein kinase C (PKC) on the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) with perforated patch recording techniques. Both forskolin and phorbol-12,13-dibutryate (PDBu) caused a large increase in mEPSC frequency, but only PDBu increased mEPSC amplitude, an effect that was not observed when standard whole cell recording was performed. These results support biochemical observations indicating that PKC, similar to calcium/calmodulin-dependent protein kinase II, has an important role in controlling synaptic strength via modulation of AMPA receptor function, potentially through the direct phosphorylation of the GluR1 subunit.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Protein phosphorylation is thought to be a ubiquitous and important mechanism for controlling the function of neurotransmitter gated ion channels (Smart 1997). Control of glutamate receptor function by protein kinases and phosphatases is of particular interest because of the potential importance of glutamate receptor modulation in various forms of synaptic plasticity (Nicoll and Malenka 1995; Roche et al. 1994; Soderling et al. 1994). Three major serine/threonine protein kinases, calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and cAMP-dependent protein kinase (PKA), which are implicated in N-methyl-D-aspartate receptor-dependent long-term potentiation, have also been shown to phosphorylate the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 on its intracellular C terminus. Specifically PKA phosphorylates serine 845, whereas PKC and CaMKII both appear to phosphorylate the same residue, serine 831 (Barria et al. 1997; Mammen et al. 1997; Roche et al. 1996).

By using expression systems or cultured hippocampal cells, all three kinases were found to potentiate the responses to exogenously applied AMPA receptor agonists (Greengard et al. 1991; McGlade-McCulloh et al. 1993; Raymond et al. 1993; Roche et al. 1996; Wang et al. 1991, 1994). However, these results were not uniformly replicated when the effects of manipulating protein kinase activity on synaptic responses in situ in hippocampal slices were examined. For example activation of PKA with forskolin or cAMP analogs enhances the frequency, but not the amplitude, of miniature excitatory postsynaptic currents (mEPSCs) in CA1 pyramidal cells, suggesting a purely presynaptic locus of action (Chavez-Noriega and Stevens 1994). Activation of PKC with phorbol esters has similar effects (Parfitt and Madison 1993). This latter result is particularly surprising because loading cells with CaMKII enhances mEPSC amplitude (Lledo et al. 1995), and CaMKII and PKC presumably phosphorylate the same residue on GluR1. Because of these apparent discrepancies we reexamined the effects of activation of PKA and PKC on the amplitude and frequency of mEPSCs in hippocampal CA1 pyramidal cells. The one important difference in our approach is the use of perforated patch rather than standard whole recording techniques as was done in previous studies. This should prevent the occurrence of "washout" that may have abolished postsynaptic effects in the previous studies.

	METHODS

Abstract Introduction Methods Results Discussion References

Standard techniques were used to prepare hippocampal slices from Sprague Dawley rats (age 15-21 days). Animals were completely anesthetized with halothane before sacrifice. Slices (400 µm) were allowed to recover for a minimum of 1.5 h before being transferred to a submerged recording chamber where they were superfused with artificial cerebrospinal fluid (ACSF) maintained at 24-26°C and saturated with 95% O₂-5% CO₂. Our standard ACSF for these experiments contained (in mM) 119 NaCl, 2.5 KCl, 2.5 CaCl₂, 1.3 MgSO₄, 1.0 NaH₂ PO₄, 26.2 NaHCO₃, 11 glucose, 0.1 picrotoxin, and 1.5 µM tetrodotoxin (pH 7.4). Perforated patch recordings (Rae et al. 1991) were made with pipettes (2-3 M) filled with a solution containing (in mM) 130 CsMeSO₃, 8.0 NaCl, 10 HEPES, 0.2 EGTA (pH 7.2 with CsOH, ~280 mosmol). Amphotericin B (0.6 mg/ml, Sigma) dissolved in dimethyl sulfoxide (DMSO, 0.6% final concentration) was added to this solution, triturated, and used to backfill pipettes. Experiments were begun only after the access resistance stabilized (typically 15-35 M). Cells were voltage clamped at 60 mV without correction for the liquid junction potential. Responses were amplified, low-pass filtered at 1 kHz, and collected on videotape for posthoc analysis.

mEPSC events were acquired (2 kHz) with Fetchex (Axon Instruments) and detected with software (generously provided by J. Steinbach, Washington University) that detected the fast rise time of synaptic events. Threshold for detection was set at 2 pA. Each putative mEPSC that was detected by the software was accepted or rejected visually based on whether its general shape was as expected for synaptic events. All errors represented are SEs. Forskolin (Sigma), 4-phorbol-12,13-dibutryate (RBI), and 4-phorbol-12-myritate,13-acetate (RBI) were dissolved in DMSO as stock solutions of 10 mM and were diluted in the ACSF immediately before application to the slice. In experiments with forskolin, 3-isobutyl-1-methyl xanthine (IBMX, 10 µM, Sigma) was also added. To eliminate confusion by nonspecific effects of IBMX on adenosine receptors, 8-cyclopentyl-1,3-dimethylxanthine (RBI), an A1 adenosine receptor antagonist, was also included throughout forskolin experiments.

	RESULTS

Abstract Introduction Methods Results Discussion References

In the first set of experiments we examined the consequences of activating PKA with the adenylyl cyclase activator forskolin on mEPSCs. In agreement with previous reports (Chavez-Noriega and Stevens 1994), forskolin (10 µM) caused a rapid and dramatic increase in the frequency of mEPSCs (Figs. 1A and 3A; n = 6). However, despite using perforated patch recording and waiting until the increase in mEPSC frequency stabilized to analyze the mEPSCs in detail (~20 min after forskolin addition), there was no significant change in the amplitude distribution of mEPSCs nor in the mean mEPSC amplitude (7 ± 4%, Figs. 1B and 3B).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Forskolin enhances miniature excitatory postsynaptic current (mEPSC) frequency but not amplitude. A: example of time course of changes in mEPSC frequency during bath application of forskolin. B: mEPSC amplitude distribution in this cell before and after forskolin.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Summary of effects of PDBu and forskolin on mEPSC frequency (A) and amplitude (B).

We next examined the effects on mEPSCs of activating PKC with the phorbol ester, 4-phorbol-12,13-dibutyrate (PDBu, 10 µM). Like forskolin, PDBu caused a dramatic increase in the frequency of mEPSCs (Figs. 2A and 3A). Surprisingly however, an increase in the cumulative amplitude distribution and mean amplitude of mEPSCs (34 ± 8%) was also observed in each of the recorded cells (Figs. 2B and 3B; n = 6). This effect was specific because application of the inactive phorbol 4-phorbol-12-myristate 13-acetate (4-PMA, 10 µM) had no effect on either the frequency or amplitude of mEPSCs (n = 5: Fig. 3, A and B).

View larger version (25K): [in this window] [in a new window]   FIG. 2. PDBu enhances mEPSC frequency and amplitude. A and B: effect of phorbol-12,13-dibutryate (PDBu) on mEPSC frequency and amplitude in a cell recorded with perforated patch technique. C and D: effect of PDBu on mEPSC frequency and amplitude in a cell recorded with whole cell techinque.

The observation that PKC activation increases mEPSC amplitude differs from a previous report (Parfitt and Madison 1993). To determine whether this could be attributed to the difference in recording technique (perforated patch vs. whole cell) we repeated this experiment with standard whole cell recording. Consistent with this hypothesis, PDBu had no significant effect on mEPSC amplitude (5 ± 5%) in cells recorded with standard whole cell techniques (Figs. 2D and 3B). PDBu did still dramatically increase mEPSC frequency, thus confirming that the drug was effective in these experiments (Figs. 2C and 3A).

Figure 3 shows a summary of these experiments. Forskolin caused a 13-fold increase in mEPSC frequency (from 0.3 to 4 Hz) but no significant change in amplitude (n = 5). In contrast PDBu caused both an increase in mEPSC frequency and amplitude (n = 6), whereas an inactive phorbol ester had no effect on either frequency or amplitude. Finally, when whole cell recording was used, PDBu caused an equivalent increase in mEPSC frequency to that observed when perforated patch recording was used, but no change in mEPSC amplitude. This result suggests that "wash-out" of some intracellular constituent that is required for the PKC modulation of AMPA receptor function occurs during whole cell recording. It also indicates that the increase in mEPSC amplitude caused by PDBu cannot be attributed to inaccurate measurements caused by superimposition of mEPSCs because the increase in mEPSC frequency in the two recording conditions was the same.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

We examined the consequences of activation of PKA and PKC on mEPSCs in CA1 pyramidal cells in hippocampal slices. Consistent with their documented presynaptic actions on transmitter release at CA1 synapses (Chavez-Noriega and Stevens 1994; Malenka et al. 1986), both forskolin and PDBu caused large increases in mEPSC frequency. However, only PDBu caused an increase in mEPSC amplitude. A previous study (Parfitt and Madison 1993) examining the effects of phorbol esters on mEPSCs in CA1 pyramidal cells did not observe this effect, but we directly demonstrated that this is likely due to the wash-out that occurs during whole cell recording.

It is possible that PDBu may be working through the activation of proteins containing phorbol-binding domains other than PKC. However, the vast majority of phorbol ester-mediated effects have been attributed to the actions of PKC, and it seems the most probable candidate for the actions observed here. Consistent with this conclusion, direct loading of cultured hippocampal neurons with the catalytic fragment of PKC was found, in some cultured hippocampal cells, to enhance mEPSC amplitude (Wang et al. 1994). Our finding that PDBu can increase mEPSC amplitude and earlier results in CA1 pyramidal cells demonstrating that postsynaptic increases in CAMKII activity can enhance synaptic strength (Lledo et al. 1995; Shirke and Malinow 1997) and mEPSC amplitude (Lledo et al. 1995) together suggest that PKC and CAMKII can similarly regulate the function of AMPA receptors through the phosphorylation of a postsynaptic target. Although the direct target of these kinases cannot be identified through these studies and could be one of multiple postsynaptic proteins, biochemical data indicating that CAMKII and PKC both phosphorylate the same residue, serine 831, on GluR1 make this a very likely candidate for this regulation.

The lack of effect of forskolin on mEPSC amplitude is likely due to one of two reasons. PKA in CA1 pyramidal cells may not have access to its postsynaptic target at synapses and thus even when active would not be able to modulate AMPA receptor function. Alternatively, if GluR1 is the target of PKA, its recognition site (serine 845) on endogenous synaptically localized GluR1 may already be fully phosphorylated. It was in fact reported that both serine 845 and serine 831 are basally phosphorylated in hippocampal slices (Mammen et al. 1997). In one study, an effect of forskolin was observed on mEPSC amplitude (Bolshakov et al. 1997); however, this effect was detected at time points >2 h after the activator was applied and may not have sufficiently developed in the time course (~20-30 min) of the experiments performed in this study.

Together with previous results, the present data demonstrate that both postsynaptic CaMKII and PKC can enhance AMPA receptor function. Other studies demonstrated that protein phosphatase inhibitors can also affect AMPA receptor function (Figurov et al. 1993; Wang et al. 1991, 1994; Wyllie and Nicoll 1994). Thus, as was pointed out previously (Mulkey et al. 1993; Roche et al. 1994), strong evidence is accumulating that postsynaptic regulation of protein kinase and protein phosphatase activity is an important mechanism that contributes to the activity-dependent bidirectional control of synaptic strength at hippocampal synapses, perhaps through the direct phosphorylation of AMPA receptors. Further work is required to delineate the detailed mechanisms by which this occurs.

	ACKNOWLEDGEMENTS

  This work was supported by grants from the National Institutes of Health (R. C. Malenka, R. A. Nicoll), and the Human Frontier Science program and an Investigator Award from the McKnight Endowment Fund for Neuroscience (R. C. Malenka). R. C. Carroll was supported by a National Research Service Award from The National Instutute of Neurological Disorders and Stroke.

	FOOTNOTES

  Address for reprint requests: R. Malenka, Dept. of Psychiatry, LPPI Box 0984, University of California, San Francisco, CA 94143.

  Received 12 June 1998; accepted in final form 6 August 1998.

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