Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evans, D. Ieuan,
Roland S. G. Jones, and
Gavin Woodhall.
Differential Actions of PKA and PKC in the Regulation of
Glutamate Release by Group III mGluRs in the Entorhinal Cortex.
J. Neurophysiol. 85: 571-579, 2001.
In a previous study we showed that
activation of a presynaptically located metabotropic glutamate receptor
(mGluR) with pharmacological properties of mGluR4a causes a
facilitation of glutamate release in layer V of the rat entorhinal
cortex (EC) in vitro. In the present study we have begun to investigate
the intracellular coupling linking the receptor to transmitter release.
We recorded spontaneous -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptor-mediated excitatory postsynaptic currents (EPSCs)
in the whole cell configuration of the patch-clamp technique, from
visually identified neurons in layer V. Bath application of the protein
kinase A (PKA) activator, forskolin, resulted in a marked facilitation
of EPSC frequency, similar to that seen with the mGluR4a specific
agonist, ACPT-1. Preincubation of slices with the PKA inhibitor H-89
abolished the effect of ACPT-1, as did preincubation with the adenylate cyclase inhibitor, SQ22536. Activation of protein kinase C (PKC) using
phorbol 12 myristate 13-acetate (PMA) did not affect sEPSC frequency;
however, it did abolish the facilitatory effect of ACPT-1 on glutamate
release. A robust enhancement of EPSC frequency was seen in response to
bath application of the specific PKC inhibitor, GF 109203X. Both H-89
and the group III mGluR antagonist
(RS)-
-cyclopropyl-4-phosphonophenylglycine (CPPG) abolished
the effects of GF 109203X. These data suggest that in layer V of the
EC, presynaptic group III mGluRs facilitate release via a positive
coupling to adenylate cyclase and subsequent activation of PKA. We have
also demonstrated that the PKC system tonically depresses transmitter
release onto layer V cells of the EC and that an interaction between
mGluR4a, PKA, and PKC may exist at these synapses.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neurotransmitter release is
governed by many complex and interacting factors in the presynaptic
terminal. One powerful means of controlling release is via receptors
located on the terminals, which can be acted on either by the same
transmitter released from those terminals (autoreceptors), or by a
different transmitter (heteroreceptors). There are an increasing number
of examples of both types of receptor at central synapses, and these
can be either ionotropic or metabotropic in nature (see
Manahan-Vaughan et al. 1999; Miller 1998
;
Starke et al. 1989
). In this laboratory we have been
studying the physiology and pharmacology of synaptic function in the
entorhinal cortex (EC), particularly with respect to epileptogenesis.
One of the main focuses of this work is to examine how
transmitter release at glutamate and GABA synapses is modulated by
presynaptic receptors.
We have previously shown that glutamate release is tonically
facilitated via presynaptic N-methyl-D-aspartate
(NMDA) autoreceptors in both layer II and layer V of the EC
(Berretta and Jones 1996; Woodhall and Jones
1999
). Current studies have also shown that spontaneous GABA
release onto layer V neurons is inhibited by presynaptic
GABAB autoreceptors, a control that may also be
tonically active (Wood and Jones 1999
; Wood et
al. 1999
). In addition, we have recently examined the role of
presynaptic metabotropic glutamate receptors (mGluRs). The mGluRs are a
group of G-protein-coupled receptors that have been shown to modulate
synaptic transmission via intracellular signaling pathways. The mGluRs
have been subdivided into three groups based on their amino acid
sequence homology, pharmacology, and intracellular coupling (for review
see Conn and Pin 1997
). Group III mGluRs (mGluRs 4, 6, 7, and 8) have been reported to depress glutamatergic transmission in a
number of brain areas (Baskys and Malenka 1991
;
Davies and Watkins 1982
; Dube and Marshall
1997
; Forsythe and Clements 1990
; Jin and
Daw 1998
; Trombley and Westbrook 1992
), and this
has been suggested to be through a negative coupling to adenylate
cyclase (Okamoto et al. 1994
; Saugstad et al.
1997
; Tanabe et al. 1993
), leading to inhibition
of calcium currents in presynaptic terminals (Glaum and Miller
1995
; Takahashi et al. 1996
). Another report has
implicated G-protein
sub-units in group III mGluR-mediated
inhibition of glutamate release (O'Connor et al. 1999
).
In common with reports from other brain areas, we found that activation
of group III mGluRs with the agonist,
L(+)-2-amino-4-phosphonobutyric acid (L-AP4)
reduced glutamate release from terminals in layer II of the EC
(Evans et al. 2000). However, in complete contrast, the
same agonist caused a powerful facilitation of glutamate release in
layer V (Evans et al. 2000
). This may suggest that the
response in layer V is due to a different receptor subtype or a
receptor with a different intracellular coupling system to those
previously described. Since further pharmacological analysis confirmed
that the facilitation of release was mediated by group III receptors, and indicated that it was likely to be the result of activation of
mGluR4a, we think the latter explanation to be the most likely. In the
present study we have begun to define intracellular signaling mechanisms that may underlie the facilitatory effect of mGluR4a on
glutamate release by looking at the effect of activation or inhibition
of protein kinases. Some of this work has been presented in abstract
form (Evans et al. 1999
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hippocampal-EC slices were prepared from male Wistar rats
(50-110 g) as previously described (Jones and Heinemann
1988). In brief, rats were anesthetized with an intramuscular
injection of ketamine (120 mg/kg) plus xylazine (8 mg/kg) and
decapitated. The brain was rapidly removed and immersed in oxygenated
artificial cerebrospinal fluid (ACSF) chilled to 4°C. Slices (450 µm) were cut using a vibroslice (Campden Instruments) and stored in
ACSF continuously bubbled with 95% O2-5%
CO2, maintained at room temperature. Following a
recovery period of at least 1 h, individual slices were
transferred to a recording chamber mounted on the stage of an Olympus
upright microscope (BX50WI). The chamber was continuously perfused with
oxygenated ACSF at 30-32°C at a flow rate of approximately 2 ml/min.
The ACSF contained the following (in mM): 126 NaCl, 4 KCl, 1.25 NaH2PO4, 24 NaHCO3, 2 MgSO4, 2.5 CaCl2, and 10 D-glucose. The solution
was continuously bubbled with 95% O2-5%
CO2 to maintain a pH of 7.4. Neurons were
visualized using differential interference contrast optics and an
infrared video camera.
Patch-clamp electrodes were pulled from borosilicate glass (1.2 mm OD,
0.69 ID; Clark Electromedical) and had open tip resistances of 4-5
M. They were filled with a solution containing the following (in
mM): 130 Cs-methanesulphonate, 10 HEPES, 5 QX-314, 0.5 EGTA, 1 NaCl,
0.34 CaCl2, 1 MK801, 4 ATP, and 0.4 GTP. The
solution was adjusted to 290 mOsmol with sucrose and to pH 7.3 with
CsOH. Whole cell voltage-clamp recordings were made using an Axopatch 200B amplifier (Axon Instruments), and neurons were clamped at
60 mV.
Signals were filtered at 2 kHz and digitized at 20 kHz. Access
resistance was monitored at regular intervals and cells rejected if
this parameter changed by more than 15%. Data were recorded directly
to computer hard disk using Axoscope software (Axon Instruments). Under
these experimental conditions, EC neurons within layer V exhibited
excitatory postsynaptic currents (EPSCs), mediated by spontaneous
release of glutamate acting at
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors
(see Berretta and Jones 1996
). Analysis of spontaneous
events was carried out off-line using Minianalysis software
(Synaptosoft). EPSCs were detected automatically using a
threshold-crossing algorithm, and their frequency, amplitude, and
kinetic parameters analyzed. Two hundred randomly selected events were
analyzed for each cell under each condition. The nonparametric Kolmogorov-Smirnoff (KS) test was used to assess the significance of
shifts in cumulative probability distributions of inter-event interval
(IEI) and event amplitude (Van de Kloot 1991
). For
comparison of amplitude distributions, histograms were constructed with
events categorized into 2-pA bins. Kinetic analyses (rise and decay
times) were performed using Mini-Anal software. Two hundred events in each condition (control or drug application) were individually fitted,
and these data were pooled for a number of different cells. All error
values stated in the text refer to standard error of the mean (SE).
All salts used in preparation of ACSF were Analar grade and purchased
from Merck/BDH. Drugs used were
(1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-1),
phorbol 12 myristate 13-acetate (PMA), forskolin, and
2-[1-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF
109203X), all obtained from Tocris Cookson.
N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulphonamide HCl (H-89),
4,9
,12
,13
,20-pentahydroxytiglia-1,6-dien-3-one (4
-phorbol), and
7
-acetoxy-6
-hydroxy-8,13-epoxy-labd-14-en-11-one (1,9-dideoxyforskolin) were obtained from Sigma, and dizocilpine maleate (MK801) and
(9-tetrahydro-2-furanyl)-9H-purin-6amine (SQ22536) from RBI.
Unless otherwise stated, all drugs were applied by inclusion in the
bath perfusion medium.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results reported here are based on recordings obtained from 87 neurons in layer V. With MK-801 (1 mM) in the patch pipette solution to
block postsynaptic NMDA receptors, spontaneous AMPA receptor-mediated
excitatory postsynaptic currents (sEPSCs) were recorded as inward
currents in neurons voltage clamped at 60 mV. sEPSCs had a mean
frequency of 2.4 Hz.
Forskolin mimics activation of presynaptic mGluRs
We have previously shown that bath application of the specific
group III mGluR agonists, L-AP4 and ACPT-1, resulted in a
facilitation of both spontaneous (Fig. 1)
and activity-independent glutamate release onto layer V neurons, and
that the receptors responsible were located presynaptically
(Evans et al. 2000). At the concentration used (20 µM), ACPT-1 has been reported to act specifically at mGluR4a
(Acher et al. 1997
). Group III mGluRs have been shown to
couple to adenylate cyclase (AC) and cAMP (Tanabe et al.
1993
), so our first approach was to determine whether
cAMP-dependent protein kinase A (PKA) could also modulate glutamate
release onto layer V neurons. Bath application of the PKA
activator, forskolin (10 µM; n = 12), resulted in a
marked facilitation of EPSC frequency similar to that seen with ACPT-1.
An example of this effect in one neuron is shown by the current traces
in Fig. 2A. In pooled data
from 12 neurons, the mean frequency was 2.42 ± 0.07 (SE) Hz
before, and 4.74 ± 0.14 Hz when measured after exposure to forskolin for 20 min. Figure 2B shows pooled cumulative
probability data for IEIs from all 12 neurons. There is a clear
leftward shift in the cumulative probability curve, demonstrating the
increase in event frequency, and this change was shown to be
statistically significant (P < 0.001, KS). Analysis of
the kinetics of sEPSCs in the presence of forskolin revealed no
significant change in rise time, although the decay time was increased,
and this change reached statistical significance (Fig. 2C
and Table 1). These data suggest that PKA
activation may have an effect on the postsynaptic cell. However, in
support of a presynaptic locus for the effect of forskolin are the
amplitude distribution data (Fig. 2D), which show that there
is no shift in the distribution of event amplitudes on application of
forskolin, while sEPSC frequency is increased. Mean event amplitude was
11.20 ± 0.19 pA under control conditions and 11.91 ± 0.22 pA on bath application of forskolin (P > 0.05, KS).
These data strongly suggest that the effects of forskolin are largely
confined to the presynaptic terminal.
|
|
|
1,9-Dideoxyforskolin does not affect EPSC frequency
To control for nonspecific effects of forskolin, we used the
forskolin analogue 1,9-dideoxyforskolin (50 µM), which does not activate adenylate cyclase (Seamon et al. 1983). In five
neurons tested, the mean frequency of events was 2.17 ± 0.07 Hz
under control conditions and remained unchanged at 1.67 ± 0.04 Hz
on bath application of 1,9-dideoxyforskolin (P > 0.05). These data suggest that the enhancement of EPSC frequency seen
on application of forskolin was indeed due to activation of PKA.
H-89 prevents the facilitatory effect of ACPT-1
Having established that the facilitatory effect of mGluR4a
activation was mimicked by PKA activation we investigated whether the
effect on glutamate release that we observed in response to ACPT-1 was
due to activation of PKA. Thus we incubated slices for at least 30 min
in ACSF containing the specific PKA inhibitor, H-89 (Chijiwa et
al. 1990) at 20 µM, prior to challenging with ACPT-1 (20 µM). H-89 was also perfused at a similar concentration during the
ACPT-1 application. In the presence of H-89, bath application of ACPT-1
did not cause a significant change in EPSC frequency. Figure
3A shows sample traces from
one cell before, and during, bath application of ACPT-1 in the presence
of H-89. In seven neurons, the mean frequency was 2.18 ± 0.14 Hz
in H-89 alone, and 2.12 ± 0.11 Hz during application of ACPT-1.
Figure 3B illustrates that there is no shift in the
cumulative probability curve for IEIs (P > 0.5, KS).
Since slices were incubated in H-89 prior to recording, it was not
possible to compare directly the frequency of sEPSCs before and
during H-89 application. However, in all cells, the frequency of
sEPSCs recorded in slices incubated in H-89 was very similar to control
values obtained in layer V neurons in previous experiments.
|
SQ 22536 prevents the facilitatory effect of ACPT-1
Modulation of adenylate cyclase activity would be a prerequisite
in a potential transduction pathway between mGluR4a and PKA. Therefore
we investigated the effect of blocking adenylate cyclase with the
specific adenylate cyclase inhibitor, SQ22536 (50 µM) (Fabbri
et al. 1991) on the response to ACPT-1. In seven neurons, in
slices preincubated (30 min) with SQ22536, the facilitation of release
by ACPT-1 was abolished. Figure
4A shows raw data traces from
one neuron. Mean frequency was 2.25 ± 0.09 Hz in SQ22536 alone,
and 1.78 ± 0.10 Hz in the presence of ACPT-1. The cumulative probability plots in Fig. 4B show pooled data from seven
neurons, illustrating that there is no difference in IEI distribution
under control conditions, and during application of ACPT-1, when
SQ22536 was present (P > 0.1, KS). Again, although it
was not possible to compare directly the frequency of sEPSCs before and
after application of SQ22536, the mean frequency (2.25 ± 0.09 Hz)
was similar to that observed under control conditions in other
experiments.
|
Protein kinase C (PKC) activation does not facilitate release
Thus the data strongly indicated that PKA was involved in the
facilitatory response to ACPT-1 at synapses in layer V. A second serine/threonine kinase, PKC, has been shown to have effects at synapses at which PKA is also modulatory (Capogna et al.
1995; Gubitz et al. 1996
; Malenka et al.
1987
). To investigate whether activation of the PKC system
could also modulate the release of glutamate onto layer V cells, we
used the diacylglycerol (DAG) mimetic phorbol ester, PMA. In six
neurons, application of PMA (500 nM) did not result in any significant
change in sEPSC frequency. Figure
5A shows raw data traces from
one neuron. Mean sEPSC frequency was 4.80 ± 0.09 Hz under control
conditions, and 5.03 ± 0.09 Hz (P > 0.5, KS) on
bath application of PMA. The cumulative probability plots in Fig.
5B show pooled data from six neurons, illustrating that
there is no difference in IEI distribution under control conditions,
and during application of PMA. We did not observe the marked
enhancement of EPSC frequency seen in other preparations (e.g.,
Carrol et al. 1998
), in any of our cells, even at high concentrations of PMA. In nine neurons, bath application of PMA (10 µM) resulted in a very small decrease in sEPSC frequency from 2.20 ± 0.08 Hz to 1.85 ± 0.07 Hz (P < 0.01, KS). Figure 6A shows raw
data traces from one neuron. The cumulative probability plots in Fig.
6B show pooled data from nine neurons, illustrating that there is a rightward shift in IEI distribution during application of
PMA, reflecting a decrease in sEPSC frequency. As a negative control,
we also bath applied the biologically inactive analogue, 4-
phorbol
(10 µM), which had no effect on sEPSC frequency (control 2.43 ± 0.07 Hz, 4-
phorbol 2.50 ± 0.06 Hz, n = 5, P > 0.05, KS).
|
|
When ACPT-1 was applied after perfusion with PMA (data not shown), it
failed to cause a change in mean sEPSC frequency (control in PMA,
2.62 ± 0.06 Hz; ACPT-1, 2.06 ± 0.04 Hz, n = 6, P > 0.05, KS), suggesting a potential inhibitory
interaction between PKC and the group III mGluR or a downstream target.
We examined this possibility further using the specific PKC inhibitor,
GF 109203X (Falet and Rendu 1994).
GF 109203X facilitates glutamate release
In 13 layer V neurons, we found that inhibition of PKC resulted in a reliable enhancement of transmitter release. Bath application of GF 109203X (500 nM) caused a clear increase in sEPSC frequency. The mean frequency was 2.79 ± 0.03 Hz before and 3.46 ± 0.04 Hz during application of GF 109203X, which suggested that PKC was tonically active in suppressing activity at these synapses. Figure 7A shows sample raw traces from one neuron. Figure 7B is the cumulative probability plot of IEI (pooled data from 13 cells) showing a shift to the right, indicative of a significant (P < 0.001, KS) increase in frequency in the presence of GF 109203X. Analysis of the kinetics of sEPSCs in the presence of GF 109203X revealed no significant change in rise or decay times (Table 1), consistent with a presynaptic locus.
|
H-89 abolishes the effects of GF 109203X
If our hypothesis that PKC exerts tonic inhibition of glutamate release through the group III mGluR pathway is correct, then inhibition of AC or PKA activity might be expected to prevent the effect of GF 109203X and the subsequent "release" of these elements from inhibition. After 30 min incubation with the specific PKA inhibitor, H-89 (20 µM), subsequent bath application of the PKC inhibitor, GF 109203X, even at a relatively high concentration of 100 µM, no longer resulted in a facilitation of glutamate release. Figure 8A shows records from one cell showing that there was no increase in EPSC frequency when GF 109203X was applied in the presence of H-89. The mean EPSC frequency was 2.26 ± 0.10 Hz before and 2.25 ± 0.09 Hz during the application of GF 109203X. Figure 8B shows cumulative probability plots indicating that there was no detectable change in IEI distribution (pooled data from 5 cells, P > 0.5).
|
Facilitatory effects of forskolin and GF 109203X are additive
It seems clear that blockade of PKA prevents the facilitatory effect of PKC inhibition on glutamate release. We reasoned that if the effects of PKC inhibition are mediated via PKA and that PKC inhibits a receptor pathway that would otherwise be tonically active, then we might predict two testable hypotheses: 1) that the effects of PKA activation would be occluded to some extent by prior inhibition of PKC and 2) that effects of PKC inhibition would be abolished by application of the mGluR4a antagonist CPPG.
We attempted to occlude the effect of forskolin by prior application of GF 109203X. We applied forskolin (10 µM) after preequilibration with GF 109203X (500 nM). Bath application of the PKA activator forskolin resulted in an additional increase in sEPSC frequency above that observed with GF 109203X alone. Mean sEPSC frequency was 4.42 ± 0.09 Hz in the presence of GF 109203X and 6.23 ± 0.15 Hz after application of forskolin. Figure 9A shows traces from one neuron, and Fig. 9B shows IEI distributions where GF 109203X was applied alone or together with forskolin. There was clearly an increase in sEPSC frequency (P < 0.001, pooled data from 5 cells). Thus while blockade of PKA prevented the facilitatory effect of PKC inhibition on glutamate release, blockade of PKC did not prevent the facilitatory effect of PKA activation with forskolin. When these experiments were performed using a much larger concentration of GF 109203X (100 µM), we observed complete occlusion of the effect of forskolin (n = 5, data not shown). However, we feel that this result cannot be relied on because of potential nonspecific effects of GF 109203X at this concentration, and the fact that glutamate release may already be maximal prior to forskolin application.
|
CPPG reduces sEPSC frequency in the presence of GF 109203X
The data described so far suggest that the increased sEPSC
frequency seen after blockade of PKC by GF 109203X was related to
disinhibition of mGluR4a. We tested this scenario directly, by applying
the group III mGluR antagonist CPPG after sEPSC frequency had been
enhanced by prior application of GF 109203X. In six cells tested, bath
application of GF 109203X resulted in a facilitation of mean sEPSC
frequency from 2.42 ± 0.08 Hz under control conditions to
3.75 ± 0.10 Hz on application of GF 109203X (P < 0.01). In the same cells, subsequent bath application of the specific
group III mGluR antagonist CPPG (10 µM) in the presence of GF 109203X resulted in return of mean sEPSC frequency to a level that was statistically indistinguishable from control (3.04 ± 0.09 Hz, P > 0.01 compared with predrug control). Figure
10A
shows records from one cell showing that there was a reduction in EPSC
frequency when CPPG was applied in the presence of GF 109203X. Figure
10B shows the cumulative probability plots (pooled data from
6 neurons). We have previously reported that bath application of CPPG
alone does not affect mean sEPSC frequency in layer V of the EC
(Evans et al. 2000). Since the antagonist (CPPG) only
reduces sEPSC frequency when PKC is inhibited, these data would seem to
support our hypothesis that mGluR4a is under a tonic inhibitory
influence by PKC, and that blockade of PKC enhances spontaneous
neurotransmission via release of mGluR4a and/or PKA from this
inhibitory influence.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The principal finding of this study was that the increase in sEPSC frequency induced by the specific mGluR4a agonist, ACPT-1, was mimicked by modulation of protein kinase activity. Activation of PKA enhanced glutamate release, whereas activation of PKC had little effect. By contrast, inhibition of PKC enhanced glutamate release, and further experiments suggested that mGluR4a/PKA and PKC interact at synapses onto layer V neurons.
PKA activation has been reported to potentiate excitatory transmitter
release in the hippocampus (Chavez-Noriega and Stevens 1994; Hopkins and Johnston 1988
; Malenka
et al. 1986a
, 1987
; Weisskopf et al.
1994
). In layer V of the EC, the specific PKA activator forskolin caused a rapid increase in sEPSC frequency. The facilitation of release was similar to that we observed in response to ACPT-1 and
raised the possibility that mGluR4a, present at synapses in layer V of
the EC, may be positively coupled to activation of the cAMP cascade and
thus to activation of PKA. This was strongly supported by the
observation that the PKA inhibitor H-89, abolished the effects of
ACPT-1.
To confirm that mGluR4a activation was coupled via the cAMP cascade to PKA, we blocked the signaling system upstream of PKA using an adenylate cyclase inhibitor. This also abolished the effects of ACPT-1. It seems likely then that facilitation of glutamate release induced by activation of presynaptic mGluR4a is dependent on positive coupling to adenylate cyclase and subsequent activation of PKA.
Much of the previous literature concerning presynaptic mGluRs in
mammalian CNS has suggested that group II and group III receptors are
negatively coupled to adenylate cyclase, and that their activation leads to decreased formation of cAMP and a subsequent reduction in
transmitter release. However, evidence does exist for positive coupling
of mGluRs to adenylate cyclase (Cartmell et al. 1994; Gereau and Conn 1994
; Musgrave et al.
1994
; Schoepp and Johnson 1993
; Sortino
et al. 1996
; Winder and Conn 1995
).
Sciancalepore et al. (1995)
have described an increase
in spontaneous GABA release via a mGluR (probably group I) that was
also positively coupled to adenylate cyclase in the hippocampus of
immature rats. Of particular relevance to our current observations,
Zhang et al. (1999)
recently reported that activation of
a mGluR (analogous to group II) increased glutamate release at the
Drosophila neuromuscular junction, an action that occurred
via positive coupling to adenylate cyclase and activation of PKA. Our
evidence supports a similar effect of the mGluR4a receptor in layer V
of the EC. We could not directly assess the effects of PKA and AC
inhibition on baseline sEPSC frequency in our experiments, because of
the requirement for prolonged incubation in the inhibitors prior to
recording. However, at ~2 Hz, mean sEPSC frequency in these
experiments was indistinguishable from that under control conditions in
untreated slices. Hence our data indicate (albeit indirectly) that
baseline sEPSC frequency is not greatly affected by PKA or AC
inhibition, and it seems unlikely that the PKA system has a tonic
effect on glutamate release in this area. This is consistent with our
previous observation that the group III mGluR antagonist, CPPG, had no
effect on sEPSC frequency (Evans et al. 2000
).
Previous reports from cultured and native hippocampal cells
(Finch and Jackson 1990; Hori et al.
1999
; Malenka et al. 1986b
, 1987
;
Parfitt and Madison 1993
) have shown that activation of PKC with phorbol esters can result in a robust enhancement of transmitter release. We have also demonstrated that PKC can modulate transmitter release in layer V of the EC, but, surprisingly, we did not
find a marked facilitation of release in response to PKC activation.
Instead, we found that application of the phorbol ester, PMA, did not
significantly affect sEPSC frequency, and indeed reduced it at a higher
concentration. The demonstration that activation of PKC can influence
release does not necessarily show that PKC is the signal used by any
given physiological pathway. However, our subsequent studies showed
that inhibition of PKC with GF 109203X resulted in a facilitation of
glutamate release. This facilitation could be blocked by the specific
group III mGluR antagonist CPPG, and by the PKA antagonist H-89.
Furthermore, PKC activation prevented the facilitatory effects of
mGluR4a activation with APCT-1. Although we could not occlude the
effect of forskolin by prior application of GF 109203X, we conclude
that the increase in sEPSC frequency observed during blockade of PKC is
mediated by the mGluR4a/PKA pathway. These experiments indicate that
PKC may be tonically activated in glutamatergic terminals in the EC, having an overall dampening effect on spontaneous excitatory synaptic transmission.
Hence it seems likely that mGluR4a can increase spontaneous glutamate
release by activation of PKA, but this effect is not operating
tonically. In contrast, PKC appears to be tonically holding spontaneous
transmitter release in check. It is plausible that the lack of tonic
activity at mGluR4a receptors may therefore be related to inhibition of
mGluR4a or PKA by PKC. If PKC exerts a tonic inhibitory effect on the
PKA system, then facilitation of release may only occur after
significant activation of PKA via mGluR4a, or on release of either
element from inhibition exerted by the PKC system. In fact, there is
evidence that activation of PKC inhibits a variety of intracellular
pathways, including those involving adenylate cyclase (Jakobs et
al. 1985; Kassis et al. 1985
; Katada et
al. 1985
; Mukhopadhyay and Schumacher 1985
; Rebois and Patel 1985
; Sibley et al.
1984
). It is noteworthy that activation of PKC has been shown
to greatly reduce the presynaptic inhibitory effects of
group II mGluRs at cortico-striatal glutamate synapses (Swartz
et al. 1993
; Tyler and Lovinger 1995
). More
recently, Macek et al. (1999)
showed that group II and
group III mGluR-mediated inhibition of glutamate release is mediated by
negative coupling to adenylate cyclase at hippocampal synapses.
Activation of PKC suppressed this inhibitory mGluR effect, probably due
to uncoupling of the receptors from G-proteins.
Taken together, our results imply that the activity of mGluR4a is regulated by the level of PKC activity within the terminal. It is possible then, that PKC acts as a thresholding device, the activity of which may determine the overall effect of mGluR4a activity and hence the rate of spontaneous glutamate release. At the very least, PKC represents a potential nexus at which cross talk between mGluR4a and other receptor-effector systems may occur.
It remains to be determined at which point(s) between autoreceptor
activation and vesicle exocytosis the protein kinases act to modulate
synaptic transmission. As discussed above, PKC has recently been
reported to inhibit presynaptic group III mGluRs in hippocampus
(Macek et al. 1999). This is consistent with our observations, and with the hypothesis that blockade of PKC releases the
receptor from tonic inhibition. However, it is equally plausible that
PKA and PKC act at other elements of the exocytotic machinery. They
may, for example, phosphorylate synaptic proteins (e.g.,
-SNAP,
SNAP-25, both of which contain consensus sequences for phosphorylation
by both PKA and PKC), thereby altering their calcium sensitivity.
Alternatively, they may phosphorylate presynaptic Ca2+ channels (Gubitz et al.
1996
), or proteins that regulate release, such as synapsin. In
support of the latter hypothesis, there is evidence that synapsins are
prominent substrates for PKA (Hosaka et al. 1999
), and
that phosphorylation results in a facilitation of release
(Greengard et al. 1993
; Jovanovic et al.
2000
). Similarly, experiments on cerebellar neurons indicate
that PKA-dependent synaptic facilitation operates via direct modulation
of the release machinery (Chavis et al. 1998
;
Chen and Regehr 1997
; Trudeau et al.
1996
) and not on factors such as presynaptic
Ca2+ entry. Further experiments aimed at
elaboration of the mechanism of this presynaptic mGluR action are under
way in our laboratory.
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank The Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and the Taberner Trust for financial support.
![]() |
FOOTNOTES |
---|
Address for reprint requests: G. Woodhall (E-mail: g.l.woodhall{at}bristol.ac.uk).
Received 5 May 2000; accepted in final form 4 October 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|