Departamento de Fisiología, Biofísica y
Neurociencias, Centro de Investigación y de Estudios
Avanzados del Instituto Politécnico Nacional, Mexico
D.F. 07000, Mexico
 |
INTRODUCTION |
Glutamic acid
decarboxylase (GAD) and GABA are normally present in the excitatory
granule cells of the dentate gyrus (DG) (Sandler and Smith
1991
; Sloviter et al. 1996
). They transiently overexpress GAD mRNA and GAD after seizures (Lehmann et al.
1996
; Schwarzer and Sperk 1995
), suggesting that
they can synthesize GABA de novo, enabling them to utilize it for fast
neurotransmission. Supporting this, glutamate receptors antagonists
(GluRAs) uncover a fast bicuculline-sensitive inhibitory postsynaptic
potential (IPSP) in CA3 pyramidal cells (PC) on DG stimulation in
kindled animals (Gutiérrez and Heinemann 1997
).
However, a permanent state of epilepsy in humans and animal models of
chronic epilepsy induces sprouting of mossy fibers
(MF) (Babb et al. 1991
; Cavazos et al.
1991
; Qiao and Noebels 1993
) and of inhibitory
interneurons (Davenport et al. 1990
) that can innervate
PC in CA3, and which can underlie aberrant inhibitory responses. Since
these changes may obscure the correct identification of the origin of
the synaptic responses of PC to DG stimulation, it was important to
test whether the fast IPSP in CA3 was provoked by stimulation of
rearranged neuronal processes that accompany kindling epilepsy, or to
the sole occurrence of a seizure, which is unlikely to induce sprouting (Cavazos et al. 1991
). Therefore this work analyzes the
synaptic responses of CA3 PC to DG stimulation, in kindled epileptic
animals with and without seizures, and in nonepileptic animals,
immediately after an acutely pentylenetetrazol (PTZ)-induced seizure.
 |
METHODS |
Adult Wistar rats were implanted in the amygdala (Paxinos
and Watson 1997
) for daily kindling stimulation (1-s
train at 60 Hz; 0.1-ms pulse duration;
500 µA) until five seizures
were evoked. Acute seizures were induced with a single administration
of PTZ (70 mg/kg ip). Intracellular recordings of CA3 PC were obtained in vitro, from the following groups of animals: kindled animals (n = 19), 1) 24 h and 2) 1 mo after the last seizure; 3) 24 h after one seizure
(n = 8), kindled after 1 mo, during which no seizures
were provoked and 4) 2 h after a single PTZ-induced
seizure (n = 10). Combined entorhinal
cortex-hippocampus slices (400 µm) were cut and transferred to an
interface recording chamber and constantly perfused with oxygenated
artificial cerebrospinal fluid (ACSF) at 35°C containing (in mM) 124 NaCl, 3 KCl, 1.25 NaH2PO4, 1.8 or 2.4 MgSO4, 1.6 or 0.8 CaCl2, 26 NaHCO3, and 10 glucose, pH 7.35. The drugs used were diluted in the ACSF; namely
(DL)-2-amino-5-phosphonovaleric acid (5APV; 30 µM;
Tocris); 6-nitro-7-sulfamoylbenzo(f)-quinolaxine-2,3-dione (NBQX; 10 µM; Tocris), and bicuculline methiodide (20 µM; Sigma); halothane
(10 µM); L(+)-2-amino-4-phosphonobutyric acid
(L-AP4; 10 µM; Tocris). Intracellular activity of PC was
recorded with microelectrodes filled with 2 M potassium acetate (70-90
M
) using an AxoClamp 2B amplifier. Acquisition and off-line analysis
were done with the program pClamp6 (Axon Instruments). Stimulation (pulse of 0.1 ms) was delivered over the intersection of the blades of
the granular layer of the DG, with a bipolar glass-insulated platinum
wire (50 µm) electrode, at an intensity that evoked 70% of the
excitatory postsynaptic potential (EPSP) amplitude needed to reach
threshold for evoking action potentials.
 |
RESULTS |
DG stimulation evoked EPSP/IPSP sequences in PC (Fig.
1A), which were blocked by
perfusion of the GluRAs, NBQX, and 5APV, in 50 of 50 PC, confirming
that IPSPs were disynaptically mediated (Fig. 1, A and
B1, and C, top; Fig.
2A). However, in PC from rats in which seizures were induced, GluRAs blocked the EPSP and isolated a
fast IPSP (Fig. 1, B2 and B3, and C,
bottom; Fig. 2B) as follows: 1) in
freshly kindled rats, 30 of 30 cells; 2) in preparations recorded a month after completion of the kindling process, 4 of 20 cells; 3) after a kindled seizure, 1 mo after completion of the initial kindling process, in 16 of 19 cells; and 4)
1 h after a single PTZ-induced seizure, in 14 of 20 cells. Neither
the resting membrane potential nor the input resistance of the cells
(
64 ± 3 mV; 33.8 ± 1.7 M
; mean ± SE;
n = 50) was altered by the presence of seizures (Fig.
1C). The reversal potential for the seizure-induced IPSP was
67.5 ± 0.3 mV (n = 35; Fig. 1C). The
onset latency of the DG-evoked EPSP, measured from the beginning of the
stimulus artifact to the beginning of the rising phase, was 4.2 ± 0.7 ms (n = 33) and of the pharmacologically isolated
IPSP was 4.8 ± 0.5 ms (n = 27; mean ± SD;
Fig. 2C). They were not statistically different. Perfusion
of the high Mg2+-low Ca2+
medium in control preparations blocked the IPSP but not the EPSP, whose
latency was not affected (Fig. 2D, top). This
also confirms that the control IPSP is disynaptically mediated and, as
expected, its apparent kinetics are different from those of the
postseizure fast IPSP (Fig. 1, A, B2, and B3),
since it was preceded by the monosynaptic EPSP. However, in postseizure
preparations, the low Ca2+ medium did not block
the pharmacologically isolated IPSP nor alter its onset latency
(n = 4; Fig. 2, C and D,
bottom). The DG-evoked IPSP could be blocked by bicuculline
(Fig. 3A), it was not altered
by halothane (n = 5; Fig. 3B), and it was
reversibly depressed by the mGluR agonist L-AP4 by around
70% (n = 7; Fig. 3C).

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Fig. 1.
A: control synaptic responses of a pyramidal cell (PC) to
dentate gyrus (DG) stimulation. B1: synaptic potentials were
blocked by glutamate receptor antagonists (GluRAs) in control
preparations and in the majority of cells from kindled nonseizing rats.
After an acute pentylenetetrazol (PTZ)-induced seizure
(B2) or a day after a rekindled seizure (B3), a
fast inhibitory postsynaptic potential (IPSP) was evident under GluRAs.
C: current-voltage (I-V) curves of a control PC
(top) and a PC recorded after seizures, under GluRAs
(bottom). The traces in the bottom panel show the
reversal potential of the postseizure IPSP. Calibration bars in
B also apply to A; those in C apply to
both panels. Dots signal the stimulus artifact and arrows the afferent
volley. Traces in A and B are averages of 10 responses. NBQX, 6-nitro-7-sulfamoylbenzo(f)-quinolaxine-2,3-dione;
5APV, (DL)-2-amino-5-phosphonovaleric acid.
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Fig. 2.
A: control responses to DG stimulation were blocked by
GluRAs. B: after seizures, GluRAs block the excitatory
postsynaptic potential (EPSP) and isolate a fast IPSP. C:
histogram showing the onset latencies of the control DG-evoked EPSP
(n = 33) and the postseizure pharmacologically isolated
IPSP (n = 27). D: perfusion of a low
Ca2+-high Mg2+ medium
blocks the polysynaptic IPSP in control preparations (top),
but not the postseizure DG-evoked fast IPSP (bottom),
without affecting the onset latencies. Calibration bars in B
also apply to A (10 mV for the EPSP; 5 mV for the IPSP).
Dots signal the stimulus artifact and the arrow the afferent volley.
All traces are averages of 10 responses.
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Fig. 3.
A: averaged (n = 10) synaptic responses
of a PC from a rekindled preparation before (1), during
perfusion of GluRAs (2), and during perfusion of
bicuculline (3). B: DG-evoked IPSP during
GluRAs perfusion, without (1) and during perfusion of
halothane (2). C: average traces showing
the pharmacologically isolated DG-evoked IPSP (1), and
the effect of the simultaneous perfusion of
L(+)-2-amino-4-phosphonobutyric acid (L-AP4;
2). Dots signal the stimulus artifact and arrows the
afferent volley.
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DISCUSSION |
-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA)/ kainate and
N-methyl-D-aspartate (NMDA) receptors
antagonists normally suppress synaptic responses in CA3 to stimulation
of the DG and associational-commissural inputs (Weisskopf and
Nicoll 1995
). Accordingly, synaptic responses were blocked in
all control PC recorded. However, stimulation of the DG induces fast
inhibitory transmission on PC after seizures. That this IPSP is not
mediated by the activation of sprouted interneurons and that it has a
MF origin is supported by the following. 1) Despite the
excitatory nature of the granule cells, they contain GAD and GABA
(Sandler and Smith 1991
; Sloviter et al.
1996
), and seizures transiently up-regulate its synthesis
(Schwarzer and Sperk 1995
). Indeed, a single seizure,
which is unlikely to induce sprouting within 2 h, induces the fast
IPSP. Conversely, 1 mo after the last kindled seizure, the fast IPSP
was rarely detected, despite the structural alterations that remain for
several months (Cavazos et al. 1991
), and rekindling the
animal 1 mo after seizure arrest reinduced the fast IPSP. 2)
The DG-evoked IPSP is monosynaptic. The onset latency of the control
EPSP coincides with previous reports (Brown and Johnston
1983
), and the latencies of the DG-evoked EPSP and IPSP are
similar, even after reducing the probability of neurotransmitter release. Also, the lack of effect of halothane discards involvement of
electrical synapses. 3) The DG-evoked IPSP is sensitive to the mGluR agonist L-AP4, which is indicative of inhibition
of MF transmission (Maccaferri et al. 1998
;
Manzoni et al. 1995
).
The induction of fast inhibition from DG to CA3 after a seizure,
besides the existing feed-forward inhibition (Buzsáki
1984
) and the inhibition from adjacent synapses (Vogt
and Nicoll 1999
), can further modulate MF-CA3 neurotransmission
after enhanced excitability. Interestingly, it coincides with postictal
depression and inhibition (Hong et al. 1979
; Post
et al. 1984
) that follows seizures, and it can be relevant in
the anterograde amnesia that appears in this period. Finally, the
present results provide electrophysiological evidence that supports the
hypothesis that the MF are able to release GABA, as previously
suggested (Gutiérrez and Heinemann 1997
;
Lehman et al. 1996
; Sandler and Smith
1991
; Sloviter et al. 1996
).
This work was supported by Consejo Nacional de Ciencia y
Tecnología Grant 29309-M.
Address for reprint requests: Dept. de
Fisiología, Biofísica y Neurociencias, Centro de
Investigación y de Estudios Avanzados del IPN, Apartado
Postal 14-740, Mexico D.F. 07000, Mexico (E-mail: grafael{at}fisio.cinvestav.mx).