Felbamate but not phenytoin or gabapentin reduces glutamate release by blocking presynaptic NMDA receptors in the entorhinal cortex.

Abstract

We have shown that a number of anticonvulsant drugs can reduce glutamate release at synapses in the rat entorhinal cortex (EC) in vitro. We have also shown that presynaptic NMDA receptors (NMDAr) tonically facilitate glutamate release at these synapses. In the present study we determined whether, phenytoin, gabapentin and felbamate may reduce glutamate release by blocking the presynaptic NMDAr. Whole cell patch clamp recordings of spontaneous excitatory postsynaptic currents (sEPSCs) were used as a monitor of presynaptic glutamate release. Postsynaptic NMDAr were blocked with internal dialysis with an NMDAr channel blocker. The antagonist, 2-AP5, reduced the frequency of sEPSCs by blocking the presynaptic facilitatory NMDAr, but did not occlude a reduction in sEPSC frequency by gabapentin or phenytoin. Felbamate also reduced sEPSC frequency, but this effect was occluded by prior application of 2-AP5. Thus, whilst all three drugs can reduce glutamate release, only the action of felbamate seems to be due to interaction with presynaptic NMDAr.

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Felbamate but not phenytoin or gabapentin reduces glutamate
release by blocking presynaptic NMDA receptors in the entorhinal
cortex
Jian Yang, Caroline Wetterstrand, and Roland S.G. Jones⁎
Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY,
UK.
Summary
We have shown that a number of anticonvulsant drugs can reduce glutamate release at synapses in
the rat entorhinal cortex (EC) in vitro. We have also shown that presynaptic NMDA receptors
(NMDAr) tonically facilitate glutamate release at these synapses. In the present study we determined
whether, phenytoin, gabapentin and felbamate may reduce glutamate release by blocking the
presynaptic NMDAr. Whole cell patch clamp recordings of spontaneous excitatory postsynaptic
currents (sEPSCs) were used as a monitor of presynaptic glutamate release. Postsynaptic NMDAr
were blocked with internal dialysis with an NMDAr channel blocker. The antagonist, 2-AP5, reduced
the frequency of sEPSCs by blocking the presynaptic facilitatory NMDAr, but did not occlude a
reduction in sEPSC frequency by gabapentin or phenytoin. Felbamate also reduced sEPSC frequency,
but this effect was occluded by prior application of 2-AP5. Thus, whilst all three drugs can reduce
glutamate release, only the action of felbamate seems to be due to interaction with presynaptic
NMDAr.
Keywords
Entorhinal cortex; Presynaptic NMDA receptors; Glutamate release; Phenytoin; Felbamate;
Gabapentin
Introduction
Experiments in this laboratory have shown that the anticonvulsant drugs phenytoin,
lamotrigine, gabapentin, pregabalin and valproate can reduce the spontaneous release of
glutamate from excitatory terminals in the rat entorhinal cortex (EC) in vitro (Cunningham and
Jones, 2000; Cunningham et al., 2000, 2003, 2004). The effect of valproate alone appeared to
depend on blockade of voltage-gated sodium channels (VGSC; Cunningham et al., 2003),
whereas the reduction in release elicited by the other drugs was independent of any action on
sodium channels. Gabapentin and pregabalin appeared to act partly via an action on voltage-
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⁎Corresponding author. Tel.: +44 1225 383935; fax: +44 1225 386114. r.s.g.jones@bath.ac.uk.
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gated calcium channels (VGCC), and partly via an unknown mechanism (Cunningham et al.,
2004).
We have also previously shown that glutamate release in the EC is tonically facilitated by
ambient glutamate acting via presynaptic NMDA autoreceptors (Berretta and Jones, 1996a;
Woodhall et al., 2001; Yang et al., 2006), an effect confirmed in visual cortex (Li and Han,
2007). Recently, it has been suggested that gabapentin may interact with presynaptic NMDA
receptors (NMDAr) in the hippocampus (Suarez et al., 2005). Furthermore, pregabilin has been
suggested to reduce GABA and, possibly, glutamate release in hippocampal cultures via an
interaction with presynaptic NMDAr (Micheva et al., 2006). There is some evidence, albeit
scant, to suggest that phenytoin may block NMDAr (e.g. Wamil and McLean, 1993; Naskar
et al., 2002), and that it may reduce NMDAr-stimulated monoamine transmitter release (Sethy
and Sage, 1992; Brown et al., 1994). Thus, we have considered the possibility that the reduction
of glutamate release by anticonvulsants in the EC may depend on blockade of presynaptic
facilitatory NMDAr.
To investigate this possibility we monitored glutamate release in rat EC slices by recording
spontaneous excitatory postsynaptic currents (sEPSCs) mediated by AMPA receptors
(AMPAr) using whole-cell voltage clamp recordings. When postsynaptic NMDAr are blocked
by inclusion of MK-801 in the patch pipette, competitive NMDAr antagonists reduce the
frequency (but not amplitude or kinetics) of sEPSCs, by blocking the tonic presynaptic
facilitation of release (Berretta and Jones, 1996a; Woodhall et al., 2001; Yang et al., 2006; Li
and Han, 2007). Thus, we determined whether the reduction in frequency of sEPSCs by
phenytoin and gabapentin (Cunningham et al., 2000, 2004) could be occluded by prior blockade
of presynaptic NMDAr. We compared the effects of these two drugs to those of felbamate, a
second-generation anticonvulsant. Felbamate has consistently been shown to block NMDAr-
mediated currents (e.g. Harty and Rogawski, 2000; Kuo et al., 2004), so could also potentially
alter glutamate release via an action at presynaptic NMDAr.
The results suggest that neither the VGSC-independent reduction of glutamate release by
phenytoin, nor the VGCC-independent effect of gabapentin, are due to blockade of the
presynaptic NMDAr. However, felbamate does appear to reduce release via presynaptic
NMDAr blockade, and this could be a factor in its anticonvulsant effect.
Methods
Combined entorhinal-hippocampal slices were prepared from male Wistar rats, as previously
described (Jones and Heinemann, 1988). Rats were killed by cervical dislocation. They were
decapitated and 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, and
stored in ACSF bubbled with 95% O2/5% CO2, at room temperature. Following recovery for
at least 1 h, individual slices were transferred to a recording chamber mounted on the stage of
a Zeiss Axioskop FS microscope. The chamber was perfused (2 ml/min) with oxygenated
ACSF (pH 7.4) at 30–32 °C. The ACSF contained (in mM): NaCl (126), KCl (3.25),
NaH2PO4 (1.25), NaHCO3 (24), MgSO4 (2), CaCl2 (2), and D-glucose (10). Neurones were
visualized using differential interference contrast optics and an infrared video camera.
Patch pipettes (1–4 MΩ) were pulled from borosilicate glass on a Flaming/Brown
microelectrode puller. Pipettes were filled with a solution containing (in mM): Cs-gluconate
(100), HEPES (40), QX-314 (1), EGTA (0.6), NaCl (4), MgCl2 (5), TEA-Cl (1), ATP-Na (4),
GTP-Na (0.3), MK-801 (1). The solution was adjusted to 275 mOsmol by dilution, and set to
pH 7.3 with CsOH. Whole cell voltage clamp recordings were made from neurones in layer V
of the medial division of the EC, using an Axopatch 200B amplifier. Using this pipette solution
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and with membrane potential clamped at −60 mV, neurones displayed sEPSCs. The open
channel blocker, MK-801, was included in the patch pipette in order to block postsynaptic
NMDAr in the recorded neurone. To facilitate this blockade, neurones were depolarised to
−10 mV for 10 s at intervals (every 20 s) during a 10 min period following breakthrough to
whole-cell access. Using this approach NMDAr mediated EPSCs are rapidly abolished.
The use of intracellular MK-801 to block postsynaptic responses has been described in detail
by us previously (Berretta and Jones, 1996a; Woodhall et al., 2001; Yang et al., 2006). Fig. 1
shows an experiment that confirms the ability of MK-801 to block postsynaptic NMDAr. In
this case, EPSCs were evoked (eEPSC) by electrical stimulation in layer V of the lateral EC,
with MK-801 in the patch pipette, and at a holding potential of −60 mV (Fig. 1A). Blockade
of AMPA and GABA receptors left a small shallow eEPSC. When the holding potential was
changed to +40 mV (Fig. 1B), this was revealed as a large slow NMDAr mediated eEPSC. The
holding potential was then stepped repetitively from −60 to −10 mV (for just 5 min in this
case), and subsequent stimulation at +40 mV showed that the eEPSC was now abolished. This
approach has become a widely accepted means of selectively blocking postsynaptic NMDAr,
leaving presynaptic receptors intact, and has been used successfully by a number of other
groups in cortical, hippocampal and amygdala neurones (e.g. Sjostrom et al., 2003; Massey et
al., 2004; Samson and Pare, 2005; Bender et al., 2006; Jourdain et al., 2007; Li and Han,
2007).
Even when postsynaptic receptors are not blocked with MK-801, spontaneous events mediated
solely by NMDAr are very infrequent, and there is only a minor contribution of NMDAr to
the decay phase of sEPSCs (Berretta and Jones, 1996b). In these circumstances, sEPSCs are
abolished by bath application of NBQX and 2-AP5 (e.g. Stacey et al., 2002). Fig. 1C shows
recordings of sEPSCs in one neurone and confirms that with postsynaptic NMDAr blocked
with intracellular MK-801, addition of an AMPAr antagonist abolishes all spontaneous
currents. It should be stressed that all the recordings presented in this paper were conducted
with postsynaptic NMDAr blocked and under these experimental conditions, sEPSCs are
mediated by glutamate acting at AMPA receptors (Berretta and Jones, 1996b; Woodhall et al.,
2001). In other experiments in this laboratory (Chamberlain, S.E.L., Jones, R.S.G.) we have
shown that kainate receptors can mediate postsynaptic excitatory responses in the EC, but these
receptors are not activated by spontaneously released glutamate. Thus, sEPSCs in our
recordings are mediated solely by AMPAr.
The experimental protocols required patch clamp recordings for periods of up to 60 min, so
we had to consider the possibility that changes in sEPSCs might be complicated by run-down
of events. However, the inclusion of ATP-Na and GTP-Na largely precluded this, and we have
previously made similar long duration patch clamp recordings of sEPSCs in many studies
without any problems regarding stability (e.g. Berretta and Jones, 1996a,b; Cunningham et al.,
2000, 2003, 2004; Cunningham and Jones, 2000; Evans et al., 2001; Woodhall et al., 2001;
Stacey et al., 2002; Yang et al., 2006). Series resistance compensation was not employed in
the present experiments, but access resistance (10–30 MΩ) was monitored at regular intervals
throughout and neurones were discarded from analysis if it changed by more than ±10%. Liquid
junction potentials were estimated using the calculator of pClamp 8 software, and compensated
for in the holding potentials.
After gaining whole cell access, and allowing time for MK-801 to diffuse into the neurone and
block the postsynaptic NMDAr (with repeated depolarizations), the neurone was then allowed
to stabilize for a further control period of not less than 10 min after which sEPSCs were
recorded. 2-AP5 was then added to the perfusion medium for a period of 20 min before further
addition of an anticonvulsant and perfusion of both drugs for a further 20 min. In a second
series of experiments, the drugs were perfused in reverse order. sEPSCs were recorded
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throughout and analysis periods confined to the final 5 min of perfusion with one, and then
both drugs.
Data were recorded to computer hard disk using Axoscope software. Minianalysis
(Synaptosoft, U.S.A.) was used for analysis of sEPSCs off-line. sEPSCs were detected
automatically using a threshold-crossing algorithm. Threshold varied from neurone to neurone,
but was always maintained at a constant level in each individual recording. 200 sEPSCs were
sampled during a continuous recording period for each neurone under each condition. The
populations of events sampled were mixed, and represent both activity-dependent and activity-
independent miniature EPSCs (mEPSCs) from multiple terminals on the same neurones,
although the majority of events (around 60–70%) are mEPSCs (see Berretta and Jones,
1996b). No attempt was made to separate these in the present study, but we have previously
shown that presynaptic NMDAr can facilitate both forms of release (Berretta and Jones,
1996a,b; Woodhall et al., 2001), and also that phenytoin and gabapentin depress the frequency
of both sEPSCs, and mEPSCs recorded in the presence of TTX (Cunningham et al., 2000,
2004). To compare pooled data under control and drug conditions we determined mean inter-
event interval (IEI), the frequency derived from the IEI, amplitude, and rise and decay times
for sEPSCs in each cell. A paired t-test was used to compare mean amplitudes and frequencies,
rise and decay times, and the non-parametric Kolmogorov–Smirnoff (KS) test to assess the
significance of shifts in cumulative probability distributions of IEI. All error values stated in
the text refer to standard error of the mean.
The following drugs were used: 2-AP5 (D,L-2-amino-5-phosphonovalerate; Tocris, UK);
phenytoin sodium (Sigma, UK); gabapentin (1-(aminomethyl)cycloheaneacetic acid; a gift
from Pfizer, Global Research & Development, Ann Arbor, Michigan); felbamate (2-
phenyl-1,3-propanediol dicarbamate; Tocris, UK).
Results
Experiments were performed on a total of 21 neurones in layer V of the medial EC. sEPSCs
in control recordings had a mean IEI of 291 ± 44 ms (equating to a frequency of 5.3 ± 0.7 Hz),
an amplitude of 12.0 ± 0.9 pA, and rise (10–90%) and decay times (60%) of 1.9 ± 0.1 and
5.8 ± 0.9 ms, respectively.
Phenytoin
In three neurones, perfusion with 2-AP5 (50 μM) increased the mean IEI of sEPSCs from
211 ± 66 to 327 ± 117 ms (reflecting a decrease in frequency from 6.5 ± 2.9 to 4.2 ± 1.7 Hz,
P < 0.01), with no significant change in amplitude (10.2 ± 1.0 pA versus 12.2 ± 1.7 pA). The
decrease in frequency without change in amplitude or kinetics (see below) is strongly indicative
of a presynaptic effect, and, as we have previously demonstrated, reflects blockade of the tonic
facilitation of glutamate release via the presynaptic NMDA autoreceptor (Berretta and Jones,
1996a; Woodhall et al., 2001). In the same neurones, subsequent addition of phenytoin
(50 μM in the presence of 2-AP5) caused a further increase in IEI to 459 ± 175 ms (3.4 ± 1.6 Hz,
P < 0.05), again without change in amplitude. Fig. 2A shows examples of voltage clamp
recordings from one neurone, and cumulative probability analysis of IEI in pooled data from
all three neurones (Fig. 2B). There was a shift towards longer intervals in the presence of 2-
AP5, and a further shift to the right with the addition of phenytoin. On average, the frequency
of sEPSCs was reduced by −37 ± 8% by 2-AP5. With the addition of the anticonvulsant the
total reduction compared to control was −55 ± 3%. Comparing the frequency of events in
phenytoin plus 2-AP5 to 2-AP5 alone gave an average reduction of −21 ± 4%. sEPSC rise
times (10–90%) were 2.0 ± 0.4 ms in control, 2.1 ± 0.2 ms in the presence of 2-AP5, and
2.6 ± 0.6 ms with subsequent addition of phenytoin. Corresponding times to 60% decay were
5.1 ± 2.0, 5.6 ± 2.3 and 5.4 ± 1.4 ms, respectively.
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We conducted the reverse experiment in a further four neurones. Phenytoin, applied alone,
increased IEI from 163 ± 36 to 250 ± 43 ms, and subsequent perfusion with 2-AP5 resulted in
a further increase in IEI to 630 ± 258 ms. The respective changes in sEPSC frequency were
from 7.8 ± 2.5 to 4.6 ± 1.1 (P = 0.05) with phenytoin, and to 2.4 ± 0.8% (P < 0.01) on addition
of 2-AP5. Again, there were no changes in amplitude (not shown). The pooled cumulative
probability analysis for IEI data are illustrated in Fig. 2C, showing clearly the shift towards
longer intervals in phenytoin, and a further shift when 2-AP5 was added. The mean reductions
in sEPSC frequency were −37 ± 4% with phenytoin alone, and −71 ± 7% when both drugs
were present. This corresponds to a decrease in frequency of −47 ± 10% when comparing
phenytoin alone to phenytoin plus 2-AP5.
Rise times (control 1.5 ± 0.1 ms; phenytoin 2.1 ± 0.5 ms; phenytoin plus 2-AP5 1.8 ± 0.4 ms)
and decay times (5.3 ± 0.3 ms versus 5.4 ± 0.2 ms versus 5.3 ± 0.3 ms) were again unaltered.
Felbamate
In three neurones 2-AP5 (50 μM) again reduced glutamate release, reflected by an increase in
mean IEI (from 522 ± 154 to 950 ± 325 ms; frequency 2.1 ± 0.6 to 1.2 ± 0.3 Hz, P < 0.05) with
no obvious change in sEPSC amplitude (13.5 ± 3.1 pA versus 12.5 ± 1 pA). However,
subsequent addition of felbamate (100 μM), elicited no further change in IEI (883 ± 277 ms).
In fact, in two neurones, sEPSC frequency was slightly increased when felbamate was added,
although overall there was no marked change (1.3 ± 0.3 Hz, P > 0.1), and again amplitude
remained about the same (12.6 ± 1.2 Hz). Fig. 3A shows voltage clamp recordings from one
neurone and the cumulative probability analysis of pooled data are illustrated in Fig. 3B. sEPSC
rise times were unaltered at 2.0 ± 0.4 ms in control, 2.4 ± 0.3 ms in the presence of 2-AP5, and
2.5 ± 0.4 ms with the addition of felbamate. Corresponding times to 60% decay were 5.3 ± 1.8,
6.0 ± 1.2 and 5.8 ± 1.9 ms, respectively.
The reverse experiment was conducted in four neurones and gave very similar results.
Felbamate alone decreased EPSC frequency from 5.1 ± 1.1 to 2.5 ± 0.9 Hz (P < 0.01), IEI
increasing from 238 ± 67 ms versus 587 ± 193 ms. Now, addition of 2-AP5 failed to cause any
significant further change (2.6 ± 0.9 Hz; 528 ± 140 ms). The pooled cumulative probability
data for IEI from the four neurones can be seen in Fig. 3C. Mean amplitudes were essentially
the same throughout (11.2 ± 3.8 pA versus 10.7 ± 4.1 pA versus 11.2 ± 4.5 pA). Rise times
were also unaltered (control 2.2 ± 0.6 ms; felbamate 2.2 ± 0.7 ms; felbamate plus 2-AP5
2.4 ± 0.6 ms), as were decay times (4.8 ± 0.6 ms versus 4.7 ± 0.7 ms versus 4.4 ± 0.5 ms.
The normalised data supported the observation that the NMDA antagonist and the
anticonvulsant could occlude each other's effects. Thus, application of 2-AP5 reduced sEPSC
frequency by −48 ± 3%, and the percentage reduction in the presence of both 2-AP5 and
felbamate was virtually the same at −43 ± 1%. When the effect of felbamate was normalised
to the frequency in 2-AP5, there was a slight increase to +10 ± 5%. Application of felbamate
alone in the second set of experiments reduced frequency by −55 ± 9% and with addition of
2-AP5 the total reduction was −52 ± 8%. This reflected a change of +7 ± 12% when 2-AP5
was normalised to the frequency in felbamate alone.
Gabapentin
Gabapentin has been suggested to target presynaptic NMDAr (Suarez et al., 2005), so we
compared its effects to those of the other anticonvulsants. sEPSC frequency in three neurones
was significantly (P < 0.05) reduced from 6.8 ± 2 to 3.1 ± 1.4 Hz (IEI: 188 ± 72 ms versus
552 ± 290 ms) during perfusion with 2-AP5. Addition of gabapentin (25 μM) caused a further
reduction (P < 0.05) to 1.6 ± 0.4 Hz (693 ± 191 ms). Amplitudes were not significantly altered
throughout (11.5 ± 2.5 pA versus 12.2 ± 4.2 pA versus 11.8 ± 4.2 pA), as were rise times
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