RAPID COMMUNICATION
Effects of PKA and PKC on Miniature Excitatory Postsynaptic
Currents in CA1 Pyramidal Cells
REED C. CARROLL, 1 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
INTRODUCTION
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 cAMPdependent protein kinase (PKA), which are implicated in
N-methyl-D-aspartate receptor-dependent long-term potentiation, have also been shown to phosphorylate the a-amino3-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
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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 (ChavezNoriega 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
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
mm) 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 24–267C 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 mM tetrodotoxin (pH 7.4). Perforated patch recordings (Rae et al. 1991) were made with pipettes
(2–3 MV ) filled with a solution containing (in mM) 130
CsMeSO3 , 8.0 NaCl, 10 HEPES, 0.2 EGTA (pH 7.2 with CsOH,
Ç280 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 MV ). Cells were voltage clamped at 060 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 02 pA. Each
putative mEPSC that was detected by the software was accepted
or rejected visually based on whether its general shape was as
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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.
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R. C. CARROLL, R. A. NICOLL, AND R. C. MALENKA
expected for synaptic events. All errors represented are SEs. Forskolin (Sigma), 4b-phorbol-12,13-dibutryate (RBI), and 4a-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 mM, 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
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 mM)
caused a rapid and dramatic increase in the frequency of
mEPSCs (Figs. 1A and 3A; n Å 6). However, despite using
perforated patch recording and waiting until the increase in
mEPSC frequency stabilized to analyze the mEPSCs in detail
( Ç20 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).
We next examined the effects on mEPSCs of activating
PKC with the phorbol ester, 4b-phorbol-12,13-dibutyrate
(PDBu, 10 mM). 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 {
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DISCUSSION
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 estermediated 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)
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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.
8%) was also observed in each of the recorded cells (Figs.
2B and 3B; n Å 6). This effect was specific because application of the inactive phorbol 4a-phorbol-12-myristate 13-acetate (4a-PMA, 10 mM) had no effect on either the frequency
or amplitude of mEPSCs (n Å 5: Fig. 3, A and B).
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 ( 05 { 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 Å
5). In contrast PDBu caused both an increase in mEPSC
frequency and amplitude (n Å 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.
KINASE MODULATION OF HIPPOCAMPAL
MEPSCS
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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.
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
ú2 h after the activator was applied and may not have
sufficiently developed in the time course ( Ç20–30 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.
FIG . 3. Summary of effects of PDBu and forskolin on mEPSC frequency
(A) and amplitude (B).
This work was supported by grants from the National Institutes of Health
(R. C. Malenka, R. A. Nicoll), and the Human Frontier Science program
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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
2800
R. C. CARROLL, R. A. NICOLL, AND R. C. MALENKA
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.
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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