Vol. 286, Issue 3, 1412-1419, September 1998
Modulation of Epileptiform Activity by Adenosine A1
Receptor-Mediated Mechanisms in the Juvenile Rat
Hippocampus1
Virginia
Tancredi,
Margherita
D'Antuono,
Astrid
Nehlig and
Massimo
Avoli
Dipartimento di Neuroscienze, Università degli Studi di Roma
"Tor Vergata," 00173 Rome, Italy (V.T., M.D.);
INSERM U398, 67091 Strasbourg Cedex, France (A.N.) and
Research Group on Cell Biology of
Excitable Tissue, Montreal Neurological Institute and Departments of
Neurology and Neurosurgery, and Physiology, McGill University,
Montreal, Quebec, H3A 2B4 Canada (M.A.)
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Abstract |
The modulatory role played by purinergic mechanisms on the epileptiform
discharges induced by 4-aminopyridine (4AP, 50 µM) in juvenile (10 to
25-day-old) rat hippocampal slices was studied with field potential
recordings in the CA3 stratum radiatum. 4AP-induced activity consisted
of interictal and ictal discharges along with isolated 
-aminobutyric
acid-mediated potentials. The adenosine analogues 2-Cl-adenosine
(10-200 µM) and N-ethylcarboxamido-adenosine (5-10 µM), the A1
receptor agonist N6-(L2-phenylisopropyl)-adenosine (2-10
µM), and the adenosine uptake inhibitor dipyridamole (1-40 µM)
reduced and eventually abolished interictal and ictal discharges with
IC50 values that were larger for ictal discharges as
compared to interictal activity. These purinergic agents did not modify
the rate of occurrence of the -aminobutyric acidmediated
potentials recorded during application of excitatory amino acid
receptor antagonists. The changes induced by 2-Cl-adenosine,
N6-(L2-phenylisopropyl)-adenosine, or dypiridamole were
reversed by caffeine (500 µM) or 8-cyclopentyl-1,3-dipropylxantine
(100 µM). However, these adenosine receptor antagonists did not alter the epileptiform discharges induced by 4AP. The depressant effects induced by N6-(L2-phenylisopropyl)-adenosine on the
epileptiform activity were maintained in the presence of barium (2 mM),
which blocks adenosine postsynaptic actions. These results demonstrate
that activation of adenosine A1 receptors in the juvenile rat
hippocampus leads to an anticonvulsant action that can be ascribed to a
decreased release of glutamate from CA3 pyramidal cell terminals. We
also propose that during the first weeks of postnatal life endogenous adenosine does not activate A1 receptors to a degree to control the
ability of hippocampal neurons to generate epileptiform activity in the
4AP model.
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Introduction |
The
depressant action exerted by adenosine on cortical neuron excitability
is mainly mediated through the activation of A1 receptors, and results
from presynaptic (i.e., reduction of transmitter release
from excitatory terminals) and postsynaptic (i.e., membrane hyperpolarization, increase in firing adaptation and enhancement of the
slow afterhyperpolarization) actions (Gerber et al., 1989
; Greene and Haas, 1985; Haas and Greene, 1984; Lupica et al.,
1992; Scanziani et al., 1992; Thompson et al.,
1992). Adenosine induces anticonvulsant effects both in vivo
and in vitro models of epileptiform discharge (Ault and
Wang, 1986; Barraco et al., 1984; Dunwiddie, 1980; see for
review Dragunow, 1988). Furthermore adenosine concentration rises
substantially during and following seizures in animals (Dragunow, 1988)
and in humans (During and Spencer, 1992), thus suggesting that
purinergic mechanisms may play a role in epileptogenesis and/or in the
arrest of seizures.
The anticonvulsant action of adenosine in young animals remains as yet
unestablished (but see Psarropoulou et al., 1990). Therefore, we used the in vitro hippocampal slice
preparation to investigate whether modulating the efficacy of
purinergic mechanisms may influence the epileptiform activity disclosed
by 4AP in the juvenile rat hippocampus (Avoli et al., 1993,
1996; Fueta and Avoli, 1992). We report that adenosine and its
analogues (including the adenosine uptake inhibitor dipyridamole)
reduce interictal and ictal discharges recorded in the CA3 subfield of
hippocampal slices from 10 to 25-day-old rats through an action on A1
receptors that are presumably located on presynaptic excitatory
terminals. Our findings also indicate that 4AP-induced epileptiform
discharges in juvenile hippocampus are not influenced by adenosine
receptor antagonists. These results have been published in abstract
form (Tancredi et al., 1994).
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Methods |
Sprague-Dawley or Wistar rat pups aged 10 to 25 days postnatally
were decapitated under ether or halothane anesthesia. The brain was
quickly removed from the skull and placed in cold (1-3°C) ACSF. The
hippocampi were dissected and cut transversely in slices (450-550
µm) using either a McIllwain tissue chopper or a vibratome. Slices
were then transferred to a tissue chamber, where they were maintained
at an interface between oxygenated ACSF and humidified atmosphere
gassed with 95%O2/5%CO2, at a temperature of
33 ± 1°C. The ACSF composition was (in mM): NaCl 124, KCl 2, KH2PO4 1.25, MgSO4 1, CaCl2 2, NaHCO3 26 and glucose 10 (pH 7.4).
Extracellular field potential recordings were made in the stratum
radiatum of the CA3 subfield with microelectrodes that were filled with
either 2M NaCl (resistance 4-8 M
) or ACSF (resistance 1-3 M).
Signals were fed to a high impedance DC amplifier and were displayed on
an oscilloscope and on a Gould pen chart recorder.
Drugs were added to the perfusing ACSF. Concentrations given through
the paper represent the presumptive peak concentration in the bath.
DPCPX was purchased from Research Biochemicals Inc.; CNQX and CPP from
Tocris Cookson; 4AP, 2-Cl-adenosine, L-PIA, NECA and dypiridamole from
Sigma Chemical Co. (St. Louis, MO) and the caffeine from Merck (Rahway,
NJ). With the exception of DPCPX, all substances could readily be
dissolved in water. DPCPX was dissolved in 0.05 ml DMSO and then into
water to form a 2 mM stock solution. The maximal DMSO concentration
(v/v) in the perfusing medium was 0.01%. In three experiments, DMSO
alone did not influence the synchronous epileptiform activity induced
by 4AP.
Slices were continuously perfused at a rate of 0.5 to 1 ml/min which
allowed for complete exchange of the medium in the tissue chamber in
less than 5 min. Preliminary experiments were performed to establish
the minimal drug concentrations required to influence the synchronous
epileptiform activity induced by 4AP in juvenile rat hippocampal
slices. In the course of these studies we noticed that prolonged (>30
min) application of adenosine agonists made spreading depression-like
episodes appear in most cases. Because the occurrence of spreading
depression-like episodes interferes with hippocampal tissue
excitability (Psarropoulou and Avoli, 1993; Avoli et al.,
1996), adenosine agonists were applied for periods of less than 25 min,
unless otherwise indicated. Drug application was then followed by
washout with control medium for at least 45 min. Hippocampal slices
were never treated with more than three different concentrations of any
given drug.
The rate of occurrence, the duration and/or the amplitude of the
different types of synchronous activity induced by 4AP under control
conditions were assessed for periods >5 min before adding ACSF
containing any of the tested compounds. These measurements were later
repeated before drug washout (i.e., at a time when the
compounds had presumably reached peak concentration in the tissue).
Throughout our report measurements are expressed as mean ± S.D.
and n indicates the number of slices used for any given
pharmacological protocol. Statistical analysis was performed using
either the paired or the unpaired t test. In addition a
standard one-way analysis of variance was used to establish whether the
concentrations of adenosine agonists required to abolish 4AP-induced
epileptiform discharges varied as a function of the slice postnatal
age. The computer software InStat was used for all statistical
comparisons. Data were considered significantly different if P < .05.
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Results |
General features of the 4AP-induced synchronous activity.
Three types of spontaneous, synchronous activity were typically
recorded in the CA3 stratum radiatum of juvenile rat hippocampal slices
during application of 4AP (50 µM) (cf., Avoli et
al., 1993, 1996; Fueta and Avoli, 1992). These field potentials
consisted of: 1) interictal-like (thereafter called interictal)
positive-going discharges that lasted 0.2 to 1.2 sec and occurred at
0.2 to 1.4 Hz (arrows in fig. 1A, control); ictal-like (thereafter
termed ictal) discharges that had duration = 3 to 20 sec, were
made of trains of high-frequency (up to 22 Hz) positive potentials and occurred at intervals of 30 to 250 sec (continuous line in fig. 1A,
control) and 2) large-amplitude (up to 8 mV), negative-going potentials
that repeated every 20 to 45 sec (asterisk in fig. 1A, control). These
negative-going potentials often preceded the onset of the ictal
discharge.
As reported in previous studies (Avoli et al., 1993, 1996;
Fueta and Avoli, 1992), none of these spontaneous, synchronous activities was influenced by the antagonist of the NMDA receptor CPP
(10 µM, n = 3), although both ictal and interictal
events were abolished by application of the non-NMDA receptor
antagonist CNQX (10 µM, n = 3). By contrast, the
negative-going potentials continued to occur during concomitant
application of CPP and CNQX (10 µM for each of these compounds;
n = 10), but were reduced and eventually blocked by the
GABAA receptor antagonist bicuculline methiodide (10 µM;
n = 4). We have also shown that these negative-going potentials disappear during activation of mu opioid
receptors (Avoli et al., 1996; Barbarosie et al.,
1994). Hence these events represent synchronous, GABA-mediated
potentials.
Adenosine receptor agonists.
Application of 2-Cl-Adenosine
(10-200 µM, n = 18) reduced and eventually abolished
all epileptiform activities induced by 4AP (figs.
1 and 2).
By contrast GABA-mediated field potentials continued to occur and at
times (two of six slices studied with 200 µM of 2-Cl-adenosine) they
increased in amplitude and rate of occurrence. However, these changes
were not statistically significant. The effects induced by
2-Cl-adenosine developed over time. At first they were characterized by
a progressive reduction of the rate of occurrence and of the amplitude
of interictal discharges (fig. 1A, 8-min sample). Later, the intervals
between ictal events increased, although interictal activity decreased
further (fig. 1A, 14-min sample). Finally, both types of epileptiform
discharge disappeared (fig. 1A, 19-min sample). The results obtained in the experiment shown in figure 1A are plotted in the time histograms of
figure 1B.
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Fig. 1.
Effects induced by 2-Cl-adenosine on the synchronous
activity induced by 4AP in the CA3 subfield. A, Recordings were
obtained under control conditions and at different times of perfusion
with medium containing 200 µM 2-Cl-adenosine. Note that the
synchronous activity recorded in control consists of interictal and
ictal discharges (arrows and continuous line, respectively) and
large-amplitude, negative-going potentials (asterisk). Note that over
time 2-Cl-adenosine abolishes both types of epileptiform activity,
although negative-going synchronous field potentials continue to occur.
B, Time histogram of the rate of occurrence of interictal and ictal
discharges before and during application of 200 µM 2-Cl-adenosine.
These data were obtained from the experiment illustrated in A.
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Fig. 2.
A, 2-Cl-adenosine (200 µM) abolishes interictal
and ictal epileptiform discharges, although negative-going synchronous
field potentials continues to occur. These effects are reversed by
application of caffeine (500 µM). B, Dose-response curves of the
effects induced by 2-Cl-adenosine on ictal and interictal discharges
induced by 4AP. The % reduction values used for these plots were
obtained from 9 and 17 hippocampal slices, respectively.
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The effects exerted by 2-Cl-adenosine on the rate of occurrence of
interictal and ictal discharges were dose-dependent and had
IC50 of 21 µM (n = 17) and 59 µM
(n = 9) respectively (fig. 2B). As illustrated in
figure 2A, these effects were reversed by bath application of caffeine
(500 µM; n = 6). Similar findings were also obtained
with DPCPX (100 µM; n = 4) (not illustrated).
To establish further the action exerted by 2-Cl-adenosine on the
GABA-mediated field potentials we analyzed six slices during concomitant application of CPP (10 µM), CNQX (10 µM) and 4AP (50 µM). Under these experimental conditions negative-going field potentials were recorded in isolation. As illustrated in figure 3, further application of 2-Cl-adenosine
(200 µM) to these slices did not modify the rate of occurrence or the
amplitude of the GABA-mediated field potentials.
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Fig. 3.
2-Cl-adenosine does not modify the rate of occurrence
or the amplitude of the GABA-mediated, synchronous field potentials
recorded during application of 4AP and excitatory amino acid receptor
antagonists (CNQX and CPP). A, Field potential recordings obtained
under control conditions, during application and after washout of
2-Cl-adenosine (200 µM). B, Plot of the rate of occurrence (expressed
in Hz) and of the amplitude of the negative-going, GABA-mediated
synchronous field potentials analyzed in six slices under control
conditions (i.e., during concomitant application of CPP,
CNQX and 4AP) and during application of 2-Cl-adenosine (200 µM).
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Application of the specific agonist of the A1 receptor L-PIA (2-10
µM, n = 12) reduced the frequency of occurrence of
both interictal (IC50 = 5.6 µM, n = 12)
and ictal discharges (IC50 = 7.2 µM, n = 8) (fig. 4). High concentrations of L-PIA
(8-10 µM, n = 8) abolished all types of epileptiform
activity, without exerting any significant change on the GABA-mediated
field potentials, although a significant increase in their rate of
occurrence was seen with L-PIA doses ranging 3 to 5 µM. However, as
with 2-Cl-adenosine, L-PIA (10 µM, n = 4) did not
induce any change in the rate of occurrence or in the amplitude of the
isolated GABA-mediated potentials recorded during application of
excitatory amino acid receptor antagonists (not illustrated).
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Fig. 4.
Effects induced by L-PIA on 4AP-induced synchronous
epileptiform discharges. A, A high concentration of L-PIA (10 µM)
abolishes both types of epileptiform activity, without exerting any
significant change on the negative-going, GABA-mediated field
potential. These effects are reversed by the specific adenosine A1
receptor antagonist DPCPX (100 µM). B, Dose-response curves of the
effects induced by L-PIA on the rate of occurrence of interictal and
ictal discharges, as well as of the isolated negative-going
potentials.
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The effects induced by maximal concentrations of L-PIA (10 µM) were
reversed by application of the specific adenosine A1 receptor antagonist DPCPX (100 µM, n = 4) (fig. 4A).
Reappearance of the epileptiform activity during DPCPX application was
accompanied by a significant reduction in the amplitude of the
negative-going GABA-mediated, field potentials (from 3.4 ± 0.2 to
1.8 ± 0.1 mV, n = 4).
In six experiments we also analyzed the effects induced by NECA (5 and
10 µM) on the synchronous activity disclosed by 4AP. In four slices 5 µM of NECA abolished both interictal and ictal discharges, although
GABA-mediated field potentials continued to occur (not illustrated). In
the remaining two experiments ictal discharges continued to occur in
the presence of 5 µM of NECA, but disappeared when the concentration
of this compound was brought to 10 µM.
It was clear from the experiments performed with these adenosine
agonists that the concentrations required to influence 4AP-induced epileptiform activities were higher that what previously shown in other
in vitro models of epileptiform discharge (e.g.,
Dunwiddie and Fredholm, 1984; Ault and Wang, 1986). We interpreted
these findings as due to the relatively short time of drug application used in our experiments to avoid the appearance of spreading
depression-like episodes (see "Methods"). Hence, we performed
experiments in which low concentrations of L-PIA (0.1-1 µM) were
applied for periods of 40 to 60 min, although limiting the analysis to
those slices where spreading depression-like episodes were not
recorded. No change in the patterns of epileptiform discharge was seen
with 0.1 to 0.8 µM L-PIA (n = 4), although a 20 to
30% decrease in interictal discharge rate of occurrence was induced by
1 µM L-PIA after approximately 55 min of perfusion (n = 2; not shown).
Effects induced by the adenosine uptake inhibitor
dipyridamole.
The adenosine uptake inhibitor dipyridamole (Bender
et al., 1980; Stafford, 1966) was tested in 13 slices at
concentrations ranging between 1 and 40 µM. Dipyridamole reduced in a
dose-dependent manner the frequency of occurrence of ictal
(IC50 = 27 µM, n = 10 slices) and
interictal (IC50 = 22 µM, n = 13 slices)
discharges and eventually abolished all epileptiform activity (fig. 5).
Reduction and/or blockade of interictal and ictal activity were
accompanied by an increase in the rate of occurrence of the
GABA-mediated field potentials (Fig. 5A
and B).
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Fig. 5.
Effects induced by the adenosine uptake inhibitor
dipyridamole. A, A maximal concentration of dipyridamole blocks both
ictal and interictal discharges, although increasing the rate of
occurrence of the negative-going field potentials. These effects are
antagonized by DPCPX (100 µM). Note that during DPCPX application
interictal and ictal discharge increase in amplitude and duration
respectively, although the negative-going field potentials are
apparently abolished. B, Dose-response curves of the changes induced by
dipyridamole on the rate of occurrence of interictal and ictal
discharges, as well as of the isolated negative-going potentials.
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The effects induced by dypiridamole were reversed by DPCPX (100 µM,
n = 4 slices) (fig. 5A). This antagonistic action was often accompanied by a long-lasting increase in the duration/amplitude of both interictal and ictal discharge and by a reduction in the amplitude of the GABA-mediated field potentials.
Age-dependency of the effects induced by 2Cl-adenosine and
dypiridamole.
The results obtained while lasting 2-Cl-adenosine
and dypiridamole on the 4AP-induced epileptiform discharges were also
segregated in two age-groups (10- to 15- and 16- to 25-day-old rats) to
establish whether the modulation of epileptiform activity exerted by
these two purinergic agents had age-dependent features. However, we were unable to observe any significant difference between these two
groups both in the IC50 value of the dose-response curves and in the maximal doses required for blocking 4AP-induced epileptiform discharges.
We also used the analysis of variance test to identify differences
among the concentrations of 2-Cl-adenosine that were able to abolish
the 4AP-induced epileptiform activity generated by slices of different
age. However, we were unable to detect any significant difference among
the concentrations of this adenosine agonist that were required to
block either interictal or ictal discharges in slices obtained from 11- to 23-day-old rats (n = 11).
Effects of adenosine receptor antagonists.
Application of
caffeine (500 µM) to 4AP-containing medium prolonged interictal (from
0.9 ± 0.2 to 1.3 ± 0.3 sec) and ictal (from 7.7 ± 3.2 to 10.5 ± 3.4 sec) discharges in three of seven experiments. In
the remaining four experiments caffeine did not induce any change in
the duration or in the rate of occurrence of both types of epileptiform
activity. However, in one experiment large amplitude (up to 11 mV),
long-lasting (40-190 sec) negative-going shifts resembling spreading
depression-like episodes appeared approximately 30 min after caffeine
was added to the medium. Caffeine did not significantly influence the
amplitude of the GABA-mediated field potentials (not shown).
Application of DPCPX (100 µM, n = 9) to medium
containing 4AP did not alter the patterns of spontaneous synchronous
activity including interictal and ictal discharges as well as
GABA-mediated field potentials (not shown). In three experiments,
further addition of L-PIA (10 µM) did not modify the synchronous
activity recorded in the presence of 4AP and DPCPX; hence, DPCPX
appeared to have exerted its antagonist action on the A1 receptor, in
spite of its inability to induce changes on the 4AP-induced activity.
Effects of barium on 4AP-induced synchronous activity.
Adenosine postsynaptic actions are blocked by extracellular application
of barium (Birnstiel et al., 1992; Thompson et
al., 1992). Therefore, we studied whether the depressant action of L-PIA on the epileptiform discharges elicited by 4AP was maintained in
the presence of barium (2 mM, n = 6). Addition of
barium to 4AP-containing medium increased the frequency of occurrence
and the duration of epileptiform discharges, and often disclosed large amplitude (8-20 mV) sustained shifts of positive polarity that lasted
5 to 20 sec and were associated with repetitive, positive-going population spikes (fig. 6A). These
effects were accompanied by the disappearance of the negative-going
GABA-mediated potentials. Despite these dramatic effects, further
application of L-PIA (10 µM) to medium containing 4AP and barium was
able in all cases to abolish the epileptiform discharges (fig. 6A).
Such an effect was associated with the reappearance of the
GABA-mediated synchronous potentials that occurred at a higher rate
than in control.
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Fig. 6.
The depressant effects exerted by L-PIA on the
4AP-induced epileptiform discharges are maintained in the presence of
barium (2 mM). A, Addition of barium to 4AP-containing medium enhances
epileptiform activity and makes negative-going field potentials
disappear. Despite these dramatic effects, L-PIA (10 µM) is still
capable of abolishing the epileptiform discharges and makes
negative-going synchronous field potentials reappear at a higher rate
than in control. B, Effects induced by barium (2 mM) on the
negative-going GABA-mediated field potentials recorded during
application of CPP and CNQX. Note that barium increases the rate of
occurrence of the negative-going field potentials.
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To better understand the changes induced by barium on the GABA-mediated
field potentials we added CPP and CNQX to 4AP-containing ACSF before
barium application (n = 9). As illustrated in figure 6B, barium increased in all experiments the rate of occurrence of the
synchronous GABA-mediated events recorded during blockade of excitatory
amino acid receptors.
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Discussion |
We have analyzed the anticonvulsant action exerted in
vitro by manipulation of the adenosine receptor functions in the
CA3 area of juvenile rat hippocampus. The CA3 subfield plays a key role
in seizure generation (Traub and Wong, 1983; Miles and Wong, 1986) and
displays a high binding density for the A1 receptor during early
postnatal development (Daval et al., 1991). Our findings indicate that purinergic activation: 1) results in a powerful, A1
receptor-mediated anticonvulsant effect on both interictal and ictal
discharges induced by 4AP; 2) does not influence the occurrence of
glutamatergic-independent, synchronous GABA-mediated potentials and 3)
is mainly exerted on presynaptic receptors that are located on
(recurrent) excitatory terminals. Moreover the lack of effects during
application of adenosine receptor antagonists suggests that during the
first 2 to 3 postnatal wk endogenous concentrations of adenosine may be
unable to control the generation of epileptiform activity, at least in
the 4AP model.
A1 receptor activation and synchronous activity induced by
4AP.
In line with previous in vivo and in
vitro studies (see for review Dragunow, 1988), 2-Cl-adenosine,
NECA and the A1 receptor agonist L-PIA were able to reduce and abolish
interictal and ictal discharges, that in this model are due to non-NMDA
receptor-activated mechanisms (Avoli et al., 1993, 1996;
Fueta and Avoli, 1992). We have also provided evidence indicating that
the effects induced by these adenosine agonists are blocked by both
caffeine and the A1 receptor antagonist DPCPX. Hence, as documented for
several models of epileptiform discharge purinergic mechanism exert
anticonvulsant effects in the juvenile rat hippocampus through a
mechanism that relates to the A1 receptor.
With all purinergic drugs used in this study the interictal discharges
were more sensitive than ictal events as demonstrated by the
IC50 of the dose-response curves obtained for
2-Cl-adenosine and L-PIA. Similar effects are also observed with
baclofen (Motalli et al., 1997) that like adenosine agonists
acts via a presynaptic mechanism. By contrast, we have reported that
several antiepileptic drugs that interfere with
Na+-dependent, repetitive action potential firing display
an action that is more evident on ictal discharges as compared with the interictal activity induced by 4AP in the juvenile hippocampus (Fueta
and Avoli, 1992). This difference underscores the role of presynaptic
versus postsynaptic mechanisms in modulating synchronous epileptiform
discharges.
We have also observed that reduction and/or blockade of epileptiform
activity by 2-Cl-adenosine and L-PIA is at times associated with an
increased rate in occurrence of the GABA-mediated synchronous potentials disclosed by 4AP in the hippocampus. However, these effects
were not seen when the purinergic compounds were tested on the
GABA-mediated synchronous potentials recorded during concomitant application of CNQX and CPP. Therefore, we interpret the facilitatory effects as due to the reduction of epileptiform activity rather than to
a direct action of adenosine on the GABA-mediated potential per se. Our
conclusion is in line with several studies of the cellular mechanisms
of adenosine which have shown that purinergic activation controls the
synaptic release of glutamic acid from excitatory terminals, although
it does not affect the release of GABA from inhibitory interneuron
terminals (Lupica et al., 1992; Thompson et al.,
1992; Yoon and Rothman 1991; Scanziani et al., 1992).
Adenosine agonist concentrations in the 4AP model.
The
concentrations of purinergic agents required to influence 4AP-induced
epileptiform activity in the juvenile rat hippocampus are larger than
those used by several investigators who have analyzed the effects of A1
adenosine agonists on synaptic transmission and epileptiform discharges
in the adult hippocampus (Dunwiddie, 1980; Dunwiddie and Fredholm,
1984; Lee et al., 1984; Ault and Wang, 1986). However, such
a difference is not likely to reflect the immaturity of the adenosine
A1 receptor whose density is already high in the CA3 area between
postnatal days 15 and 25. Moreover these receptors are already
functional since they are effectively coupled to G proteins (Daval
et al., 1991).
An alternative explanation for the high concentrations of adenosine
receptor agonists required to control 4AP-induced epileptiform activity
may reside in the experimental procedures used in our study. In the
majority of the experiments we used application times <25 min to avoid
the appearance of spreading depression-like episodes, that can modify
neuronal excitability and thus interfere with the effects induced by
the tested drugs. However, analysis of the changes induced by prolonged
applications of low L-PIA concentrations in the few slices where
spreading depression-like episodes did not occur, indicates that even
with this procedure concentrations <0.8 µM were unable to modify
4AP-induced epileptiform discharges, although 1 µM L-PIA only reduced
the rate of occurrence of interictal events without altering the
pattern of ictal activity.
The high concentrations of adenosine receptor agonists required to
influence 4AP-induced epileptiform activity in the juvenile hippocampus
may indeed relate to the model used in our study. In line with this
conclusion, we have reported that L-PIA reduces the interictal
discharges induced by 4AP in the adult hippocampus with an
IC50 = 8.75 µM (Barbarosie et al., 1994).
Previous studies have shown that 4AP reverses the inhibitory effects
induced by adenosine on cell firing (Perkins and Stone, 1980) and also
antagonizes the anticonvulsant effects of adenosine on epileptiform
activity in the hippocampal slice (Schubert and Lee, 1986). Besides
possible direct postsynaptic effects of 4AP on adenosine function, the larger doses of purinergic compounds required in our study may reflect
the increase in transmitter release that is exerted by 4AP at both
inhibitory and excitatory synaptic terminals in the hippocampus
(Perreault and Avoli, 1989, 1991). Previous in vitro studies
of the effects induced by purinergic compounds on epileptiform discharges were most often performed in models where GABAA
receptor antagonists were used (Dunwiddie, 1980; Dunwiddie and
Fredholm, 1984; Ault and Wang, 1986).
Endogenous adenosine and anticonvulsant effects.
The ability
of the adenosine uptake blocker dipyridamole to reduce both interictal
and ictal discharges induced by 4AP indicates that increasing the brain
level of endogenous adenosine may represent an effective anticonvulsant
strategy. Moreover, as with 2-Cl-adenosine and L-PIA, dipyridamole
effects had IC50 values that were larger for ictal
discharges as compared to interictal activity and were mediated by
activation of the A1 receptor. As with the adenosine receptor agonists
the concentration of dipyridamole required to abolish epileptiform
discharges induced by 4AP were larger than what reported in a previous
study of epileptiform discharge induced by the GABAA
receptor antagonist bicuculline (Ault and Wang, 1986).
We have also shown that caffeine and the specific A1 receptor
antagonists DPCPX do not alter the pattern of synchronous activity induced by 4AP. Adenosine receptor antagonists possess proconvulsant activity in the adult rat hippocampus (Ault et al., 1987;
Alzheimer et al., 1989) suggesting that endogenous adenosine
may contribute to terminate the epileptic burst. Our negative findings
may reflect the low concentration of adenosine in the immature brain,
which between postnatal day 10 and 21 is 3- to 6-fold lower than in 2-mo-old rats (Aranda et al., 1989; Sarda et al.,
1989). A previous ontogenic study of adenosine functions has shown that
caffeine often fails in affecting the field EPSP recorded from slices
obtained from rats younger than 10 days (Psarropoulou et
al., 1990).
Pre- vs. postsynaptic sites of action.
Our study provides
evidence indicating that the anticonvulsant effects exerted by
purinergic agents on 4AP-induced epileptiform discharges rests mainly
on a presynaptic site of action. Accordingly, L-PIA maintains its
ability to decrease and eventually to abolish 4AP-induced epileptiform
discharges recorded in barium-containing ACSF. Barium at the
concentrations used in our study blocks the potassium-mediated
hyperpolarization induced by adenosine (Gerber et al., 1989;
Thompson et al., 1992) have reported that barium abolishes
this postsynaptic, potassium conductance without influencing the
ability of adenosine to inhibit isolated EPSPs.
We are therefore inclined to conclude that the effects induced by L-PIA
as well as by the other purinergic compounds tested in this study are
due to the ability of A1 receptor activation to decrease the release of
glutamate from CA3 pyramidal neuron terminals. A previous study
performed in the CA1 subfield of the adult rat hippocampus has shown
that epileptiform discharges induced by tetraethylammonium and barium
are still inhibited by adenosine (Birnstiel et al., 1992).
| |
Acknowledgments |
The authors thank Drs. A. Siniscalchi and M. Barbarosie for
performing some preliminary experiments.
| |
Footnotes |
Accepted for publication May 1, 1998.
Received for publication December 19, 1997.
1
This work was supported by a grant from the Consiglio
Nazionale della Ricerca (CNR) of Italy to VT, and grants from the
Medical Research Council (MRC) of Canada, the Hospital for Sick
Children Foundation and the Savoy Foundation to MA. V.T. and M.A.
were also supported in part by CNR-MRC travel grants.
Send reprint requests to: M. Avoli, 3801 University Street,
Montreal, Quebec, H3A 2B4 Canada.
| |
Abbreviations |
ACSF, artificial cerebrospinal fluid;
4AP, 4-aminopyridine;
CNQX, 6-cyano-7-nitro-quinoxaline-2,3 dione;
CPP, 3-3,(2-carboxypiperazine-4-yl)propyl-1-phosphonate;
DPCPX, 8-cyclopentyl-1,3-dipropylxantine;
L-PIA, N6-(L2-phenylisopropyl)-adenosine;
NECA, N-ethylcarboxamido-adenosine;
NMDA, N-methyl-D-aspartate;
DMSO, dimethyl sulfoxide;
GABA, -aminobutyric acid.
| |
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Abstract
Introduction
