Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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Tallent, Melanie K. and George R. Siggins. Somatostatin acts in CA1 and CA3 to reduce hippocampal epileptiform activity. Although the peptide somatostatin (SST) has been speculated to function in temporal lobe epilepsy, its exact role is unclear, as in vivo studies have suggested both pro- and anticonvulsant properties. We have shown previously that SST has multiple inhibitory cellular actions in the CA1 region of the hippocampus, suggesting that in this region SST should have antiepileptic actions. To directly assess the effect of SST on epileptiform activity, we studied two in vitro models of epilepsy in the rat hippocampal slice preparation using extracellular and intracellular recording techniques. In one, GABA-mediated neurotransmission was inhibited by superfusion of the GABAA receptor antagonist bicuculline. In the second, we superfused Mg2+-free artificial cerebrospinal fluid to remove the Mg2+ block of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We show here that SST markedly reduces the intensity of evoked epileptiform afterdischarges and the frequency of spontaneous bursts in both CA1 and CA3. SST appears to act additively in the two regions to suppress the transmission of epileptiform events through the hippocampus. We further examined SST's actions in CA3 and found that SST dramatically reduced the frequency of paroxysmal depolarizing shifts (PDSs) recorded intracellularly in current clamp, as well as increasing the threshold for evoking "giant" excitatory postsynaptic currents (EPSCs), large polysynaptically mediated EPSCs that are the voltage-clamp correlate of PDSs. We also examined the actions of SST on pharmacologically isolated EPSCs generated at both mossy fiber (MF) and associational/commissural (A/C) synapses. SST appears to act specifically to reduce recurrent excitation between CA3 neurons because it depresses A/C- but not MF-evoked EPSCs. SST also increased paired-pulse facilitation of A/C EPSCs, suggesting a presynaptic site of action. Reciprocal activation of CA3 neurons through A/C fibers is critical for generation of epileptiform activity in hippocampus. Thus SST reduces feedforward excitation in rat hippocampus, acting to "brake" hyperexcitation. This is a function unique from that described for other hippocampal neuropeptides, which affect more standard neurotransmission. Our results suggest that SST receptors could be a unique, selective clinical target for treatment of limbic seizures.
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INTRODUCTION |
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Epilepsy is a disease afflicting ~1% of the
U. S. population (Annegers 1994). This disease
state is characterized by abnormal hyperexcitation, which can include
several different brain regions. Temporal lobe epilepsy (TLE) involves
the hippocampus (Sloviter 1994
), and treatment for
intractable forms of this disorder sometimes involves surgical removal
of hippocampal tissue (Sloviter 1994
). In such tissue
removed from humans with TLE, neurons that contain the neuropeptide
somatostatin (SST) are selectively lost in the hilus of the dentate
gyrus (de Lanerolle et al. 1989
; Mathern et al.
1995
; Robbins et al. 1991
). Similar results have
been found in several animal models of epilepsy (Mitchell et al.
1995
; Schwarzer et al. 1995
; Sloviter
1987
), where SSTergic neuron loss can extend beyond the hilus
to the rest of the hippocampus (Lahtinen et al. 1993
;
Manfridi et al. 1991
). The loss of hippocampal neurons
is preceded in animal models by increased SST release (Manfridi
et al. 1991
; Mitchell et al. 1995
;
Vezzani et al. 1992
), suggesting that an early
consequence of hippocampal seizures is activation of SSTergic neurons.
The function of these SST neurons and the consequences of their loss
are unknown. However, the majority of SST interneurons make inhibitory
synapses onto primary hippocampal neurons (Freund and Buzsaki
1996
; Leranth et al. 1990
; Milner and
Bacon 1989
), suggesting they are involved in inhibitory
processes. Thus the death of SST interneurons in early stages of
epilepsy may contribute to subsequent abnormal hippocampal hyperexcitability.
In vivo studies examining the effect of SST on seizures induced in rats
have produced conflicting results. Early studies using cysteamine to
deplete SST suggested a facilitory role for SST, as cysteamine had
anticonvulsant actions when injected intraperitoneally (Higuchi
et al. 1983; Perlin et al. 1987
). Results with
intracerebroventricular injection of anti-SST antibody were similar
(Higuchi et al. 1983
). However, later studies suggested
an antiepileptic role for SST in the hippocampus. Thus an anti-SST
antibody perfused directly into the hippocampus enhanced the rate of
picrotoxin-induced (Mazarati and Telegdy 1992
) or
kindling-induced (Monno et al. 1993
) seizures in rats.
Also SST or its analogs perfused intrahippocampally reduced seizures
induced by picrotoxin (Mazarati and Telegdy 1992
) or kainate (Perez et al. 1995
).
An anticonvulsant action is supported by cellular studies of SST
effects in CA1 of hippocampal slices. Here SST hyperpolarizes pyramidal
neurons (HPNs) (Pittman and Siggins 1981) by augmenting K+ currents (Moore et al. 1988
;
Schweitzer et al. 1988
, 1990
), resulting in an
inhibition of firing. SST also depresses glutamatergic excitatory postsynaptic currents (EPSCs) in CA1 (Tallent and Siggins
1997
), probably through presynaptic inhibition of glutamate
release (Boehm and Betz 1997
). These results indicate
concerted inhibitory actions for SST at the cellular level in CA1; we
have suggested that this peptide may act as a homeostatic regulator to
depress abnormal excitation (Tallent and Siggins 1997
).
The actions of SST in the rest of the hippocampus remain uncharacterized.
The rat hippocampal slice has been used extensively to study the
cellular basis of seizure-like events (Schwartzkroin and Prince
1978, 1980
; Traub and Wong 1982
). This
preparation maintains much intact circuitry, exhibits epileptiform
activity in response to various pharmacological manipulations
(Mody et al. 1987
; Schwartzkroin and Prince
1978
; Traub et al. 1994
), and offers the
advantage that direct effects of exogenous SST can be examined.
Therefore we used rat hippocampal slices to study the actions of SST on seizure-like events and to characterize SST actions in CA3, a region
critical for the generation of seizure-like events. These are the first
studies to directly address the action of SST on epileptiform events in
vitro. A preliminary report of some of our findings has been published
in abstract form (Tallent and Siggins 1998
).
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METHODS |
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Slice preparation
Hippocampal slices were prepared as described previously
(Pittman and Siggins 1981; Schweitzer et al.
1993
). Briefly, male Sprague-Dawley rats (100-200 g) were
anesthetized with halothane (4%) and decapitated, and their
hippocampal formation was removed rapidly. We cut transverse slices of
350-µm thickness on a McIlwian brain slicer and placed them in
ice-cold (1-3°C) artificial cerebrospinal fluid (ACSF), gassed with
95% O2-5% CO2 (carbogen), of the following composition (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 1.5 MgSO4 · 7H2O, 2 CaCl2 · 2H2O, 24 NaHCO3, and 10 glucose. After ~30 min of incubation with
their upper surfaces exposed to warmed, humidified carbogen, the slices
were submerged completely and continuously superfused with ACSF at a
constant rate (2-4 ml/min) for the remainder of the experiment. The
inner chamber had a total volume of 1 ml; at the superfusion rates
used, 90% replacement of the chamber solution could be obtained within
1-1.5 min. Drugs were added to the bath from stock solutions at known
concentrations. We obtained SST from BaChem (Torrance, CA),
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from Tocris Cookson (St.
Louis, MO), 2-amino-5-phosphonovaleric acid (APV) from Research
Biochemicals International (Natick, MA), and all other chemicals from
Sigma (St. Louis, MO). SST was superfused at 1 µM final concentration
unless otherwise indicated. We maintained the bath temperature constant
at 31°C during testing; for studies examining epileptiform activity
in bicuculline or in the absence of external Mg2+, we
increased the temperature to 33-34°C.
Local application of SST to CA1 and CA3
For local application of SST, we oriented the slices such that flow through the chamber assisted in limiting diffusion of SST from CA3 to CA1 and vice versa (see Fig. 2A). A glass "puffer" pipette (~20-µm tip diam) was filled with 100 µM SST and lowered close to the surface of the slice. SST was ejected using 1-3 psi pressure. SST application to CA1 involved placing the puffer pipette within 2-4 mm of the recording electrode and applying SST for 10 s. In CA3, we placed the pipette in the stratum radiatum (SR) and applied SST for 20 s. To help ensure that SST reached the neurons from which events were recorded, we placed the recording electrode at a depth of only 50-60 µm into the slice. Three experiments conducted with 0.01% Fast Green in the pipette indicated that ~65-80% of CA3 was perfused; these studies also showed that the Fast Green (and thus probably also SST) was dispersed within a few seconds.
Extracellular recording
We recorded population spikes (PSs) by conventional means in the
CA1 or CA3 pyramidal layer using glass extracellular pipettes (1-3
M tip resistance, filled with 3 M NaCl) connected to an Axon
Instruments Axoclamp 2B or an NPI SEC-10L amplifier. PSs were filtered
at 3-10 kHz, digitized, averaged (2-3), stored and analyzed using
pClamp software (Axon Instruments, Foster City, CA) and a personal
computer. We evoked CA1 PSs with a bipolar metal stimulating electrode
(0.1-0.2-mm tip diam) placed in the SR; we evoked CA3 PSs by
stimulating just outside the granule cell layer in the inner blade of
the dentate for mossy fiber (MF) responses or at the CA1/CA3 border in
the SR for associational/commissural (A/C) responses (Salin et
al. 1996
). Stimuli of 0.01- to 0.05-ms duration were applied at
0.16-0.2 Hz during data acquisition. We measured PSs from the peak
negativity to peak positivity using three or more stimulus intensities
to generate input-output (I-O) curves. For normal PSs, intensities were
threshold for a consistent PS, half-maximal, and maximal. For evoked
bursts, we generally gave five stimulations from threshold to maximal;
the maximal stimulus intensity refers to the intensity that generates
the greatest number of afterdischarges and the largest burst envelope rather than the maximal initial PS. To maintain stable baseline PSs, we
continually delivered SR stimuli at 0.03 Hz.
We evoked epileptiform activity in CA1 and CA3 by stimulating in the SR
near the CA1/CA3 border. Bicuculline (15 µM) or Mg2+-free
ACSF (0 Mg2+) was superfused for 30 min before the
experiment was initiated. We quantified events using coastline burst
analysis (CBI) (Korn et al. 1987
), which is a measure of
the "length" of the outline of the burst waveform. The CBI is
calculated by summing the distance between successive points in the
digitized burst and subtracting from it an equal duration of a
nonbursting recording. This is a sensitive measure of burst intensity
and is a useful parameter for determining drug effects (Korn et
al. 1987
).
Intracellular recording
We used discontinuous single-electrode voltage-clamp (switching
frequency 3-4 kHz) or current-clamp techniques with sharp intracellular micropipettes (3 M KCl, 50-80 M) as described
previously (Tallent and Siggins 1997
). To block
GABA-mediated inhibitory postsynaptic currents (IPSCs), 10-15 µM
bicuculline was included in the bath and, when a GABAB
component was apparent, 1 µM CGP 55845A. A/C EPSCs were evoked in CA3
HPNs by stimulating in SR near the CA1/CA3 border. To isolate
N-methyl-D-aspartate (NMDA) and
(R,S)-
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor-mediated EPSCs, we superfused either the NMDA receptor antagonist APV (30 µM) or the AMPA/KA receptor antagonist CNQX (20 µM). We generated MF EPSCs by stimulating in the hilus of
the dentate proximal to the inner blade. Because MF EPSCs can be
difficult to isolate, we took several precautions to avoid A/C
contamination (Claiborne et al. 1993
; Williams
and Johnston 1991
). Thus recordings were done in ACSF
containing 7 mM Mg2+/4 mM Ca2+ and 30 µM APV
to block polysynaptic A/C events (Williams and Johnston
1991
). We also analyzed only EPSCs with a latency of <4 ms, a
rise time <3 ms, and a smooth rising and falling phase (Claiborne et al. 1993
; Williams and Johnston
1991
). Two traces were averaged for each stimulus intensity. We
measured both the amplitude and the area (time integral) of the PSCs
using Clampfit software (Axon Instruments).
To study paired-pulse (P-P) modulation of A/C-generated NMDA EPSCs in CA3, we evoked EPSCs with paired stimuli applied to the SR at interstimulus intervals (ISIs) of 100-300 ms using a half-maximal stimulus intensity. The degree of modulation was calculated by dividing the amplitude of the second EPSC by the first. After bath superfusion of SST, the amplitude of the first EPSC was normalized to the control amplitude by increasing the stimulus intensity.
Statistical analysis
We performed statistics using two-way ANOVA for repeated measures (Crunch Software, Crunch Software Corporation) with Newman-Keuls post hoc test when appropriate. Data are reported as means ± SE and considered statistically significant at P < 0.05.
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RESULTS |
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SST effects in CA1
BATH SST SUPERFUSION ATTENUATES EPILEPTIFORM AFTERDISCHARGES
RECORDED IN BICUCULLINE.
When PSs were elicited in CA1 in normal ACSF (no drugs, 1.5 mM
Mg2+), superfusion of 1 µM SST significantly inhibited
the PS at all three stimulation intensities [F(1,7) = 11.90; P < 0.05]. To determine whether SST could
reduce epileptiform activity in CA1, we examined two in vitro epilepsy
models. In the first, bicuculline was added to the bath. Bicuculline
blocks synaptic inhibition mediated by GABAA receptors,
causing the slice to become hyperexcited. Figure 1A shows a normal PS recorded
in CA1. After addition of 15 µM bicuculline, a previously submaximal,
single stimulus now elicited a burst envelope containing multiple PSs
superimposed on an underlying field excitatory postsynaptic potential
(EPSP), reflecting abnormal, recurrent firing of CA1 HPNs
(Williamson and Wheal 1992). To determine more precisely
the action of SST on these epileptiform bursts, we applied five
different stimulation intensities to generate I-O curves. As measured
by CBI, 1 µM SST significantly and reversibly reduced the intensity
of the afterdischarges [F(1,6) = 16.0, P < 0.05; n = 7; Fig. 1A]. SST often completely
suppressed the afterdischarges at the lowest stimulus intensity,
although there was no statistically significant effect of intensity on
SST inhibition (P > 0.1). SST did not usually affect
the number of afterdischarges. In one slice, spontaneous bursts were
recorded in the absence of any stimulation; SST completely blocked
these spontaneous bursts (not shown).
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SST SUPERFUSION DECREASED AFTERDISCHARGES RECORDED IN
MG2+-FREE ACSF.
When Mg2+ is removed from the superfusate, the
Mg2+ block of NMDA receptors is relieved and epileptiform
events can be evoked (Mody et al. 1987; Traub et
al. 1994
). These evoked events are similar in shape and
intensity to those elicited in bicuculline (Fig. 1B, top).
SST reduced the intensity of these evoked afterdischarges [F(1,6) = 32.29, P < 0.005; Fig. 1B,
bottom]. As with bicuculline, SST effects appeared larger
at the lower stimulation intensities, although there was no
statistically significant effect of stimulus intensity on the degree of
SST inhibition (P > 0.1). Figure 1B (top) shows afterdischarges from a representative cell
elicited at an intermediate stimulus intensity of 160 µA. SST
reversibly reduced the intensity of the afterdischarge.
SST effects in CA3
SST SUPERFUSION REDUCES EPILEPTIFORM EVENTS RECORDED IN
BICUCULLINE.
We evoked PSs in CA3 HPNs by stimulating either the MF or the A/C
pathways (see METHODS). In contrast to our observations in
CA1, 1 µM SST superfused for 10 min did not affect the amplitude of
MF [F(1,4) = 0.473, P > 0.5;
n = 5] or A/C PSs [F(1,4) = 1.78; P > 0.1; n = 5] recorded in normal
ACSF. After addition of 15 µM bicuculline to the bath, single,
previously submaximal stimuli produced epileptiform afterdischarges.
These events were similar to those evoked in CA1 in bicuculline,
although they were larger in amplitude and sometimes longer in duration
(
190 ms compared with a maximum duration of 96 ms in CA1). SST (4 min) reduced the intensity of representative afterdischarges (Fig.
2A) evoked by an intermediate
stimulation intensity (stimulus 4 in Fig. 2B). SST
significantly attenuated the intensity of the afterdischarges as
determined by CBI [F(1,6) = 12.2, P < 0.05; n = 7; Fig. 2A]. In contrast to CA1,
in CA3 a significant interaction of stimulus intensity and SST
inhibition was observed, with a greater inhibition at the lower two
stimulus intensities [F(4,24) = 6.75, P < 0.001].
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SST REDUCES EPILEPTIFORM EVENTS INDUCED IN MG2+-FREE ACSF. In the absence of extracellular Mg2+, epileptiform events could be evoked at previously submaximal stimulus intensities. The representative afterdischarges in Fig. 2B show that SST (1 µM, 4 min) can completely block the epileptiform events evoked at a threshold stimulus intensity. I-O curves generated from 6 slices show significant reduction of burst intensity by SST [F(1,5) = 34.43, P < 0.005], with a significant interaction between stimulus intensity and SST inhibition [F(4,20) = 5.76, P < 0.005]. SST also significantly reduced the frequency of spontaneous bursting recorded in CA3 [F(2,10) = 38.8, P < 0.0001; Fig. 2C], as in CA1, from 0.28 ± 0.04 Hz (range 0.17 to 0.45 Hz) to 0.08 ± 0.02 Hz (0-0.15 Hz; n = 7), representing a 70% reduction in rate of bursting. In two of the slices, SST completely suppressed the spontaneous activity. After washout of SST, bursting frequency returned to 0.27 ± 0.04 Hz (range 0.18-0.42 Hz). A higher concentration of SST (5 µM) reduced the frequency of spontaneous bursts by 80 ± 4% (n = 4) while 100 nM SST caused a 60 ± 7% decrease (n = 6). SST did not appear to consistently affect the shape or intensity of the spontaneous bursts.
Effects of locally applied SST
CA1 epileptiform events require input from CA3
(Schwartzkroin and Prince 1978). Stimulating near the
CA1/CA3 border evokes firing in CA1 both by directly activating
Schaeffer collaterals (SCs) and by indirectly activating SCs by
exciting CA3 neurons through stimulation of A/C fibers. Thus
transecting SC input from CA3 with a knife cut greatly curtailed
epileptiform events evoked in CA1 (not shown). Therefore, although SST
is known to act directly on CA1 HPNs (Moore et al. 1988
;
Pittman and Siggins 1981
; Schweitzer et al.
1993
), it also could act in CA3 to reduce CA1-recorded afterdischarges. SST effects on evoked epileptiform events appear more
robust in CA1 than CA3, especially at higher stimulation intensities,
suggesting that SST may act additiveley in CA1 and CA3 to reduce
CA1-recorded events. To resolve more discretely the SST site of action,
we examined epileptiform events evoked in bicuculline with SST applied
locally either to CA1 near the recording electrode or to CA3 (Fig.
3A).
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LOCAL SST REDUCES PSS IN CA1. We first determined whether puffer application of SST close to the CA1 recording electrode could reduce PSs recorded in normal ACSF. A 10-s application of SST significantly reduced the amplitude of the PS [F(1,4) = 13.2, P < 0.05, Fig. 3B; n = 5]. There was no significant interaction between stimulus intensity and SST's effect (P > 0.5). Complete recovery took 5-10 min in four of the five slices; one slice took 20 min to return to control values. Puffer application of ACSF alone close to the recording electrode did not significantly affect mean CA1 PS amplitudes [F(1,2) = 0.07, P > 0.5; n = 3; Fig. 3B], nor did a 20-s application of SST to the CA3 [F(1,3) = 0.014, P > 0.5, n = 4, Fig. 3B].
LOCAL SST REDUCES BICUCULLINE-INDUCED EPILEPTIFORM EVENTS IN CA1. To determine whether application of SST just to CA1 was sufficient to reduce the intensity of afterdischarges recorded in bicuculline, we locally applied 100 µM SST near the recording electrode and evoked burst afterdischarges at three different stimulation intensities. SST robustly inhibited the epileptiform event across the range of intensities [F(1,4) = 25.8, P < 0.01; Fig. 3C]. Recovery occurred after 6-13 min of washout. As with bath superfusion, at the lowest stimulation intensity local SST sometimes completely blocked the afterdischarge (Fig. 3C, top). However, the mean decrease in CBI at the minimal stimulation intensity was only to 42 ± 8.1% of control with local SST (Fig. 3C, bottom) compared with 19.8 ± 10% of control with bath perfusion (Fig. 1A, bottom). Although it is unclear whether a maximal equilibrium concentration of SST is reaching the relevant neuronal populations with short puffer application (even with a high SST concentration in the pipette), this difference in effect also could be due to additive actions of SST in both CA3 and CA1 with bath perfusion. Puffer application of ACSF alone to CA1 (n = 3; Fig. 3B, bottom) did not significantly alter the intensity of epileptiform bursts [F(1,2) = 0.197, P > 0.5].
LOCAL SST IN CA3 REDUCES EPILEPTIFORM EVENTS RECORDED IN CA1. To determine if direct actions of SST in CA3 alone could influence epileptiform events recorded in CA1, we locally applied 100 µM SST to CA3 positioned "downstream" from CA1 to limit possible diffusion of SST to the CA1 (Fig. 3A). A 20-s application of SST to CA3 caused a modest but significant reduction of epileptiform intensity recorded in CA1 [F(1,4) = 39.4, P < 0.005, n = 5; Fig. 3C], with no significant effect of stimulus intensity (P > 0.1). Recovery occurred in 4-8 min in three slices and in 15-20 min in the other two. With the same pressure and pipettes, the degree of inhibition was less than that produced by local application of SST to CA1 (Fig. 3C, bottom). The representative recording illustrated in Fig. 3C (top) shows that even at the lowest stimulation intensity a complete reduction of the epileptiform event did not occur. ACSF alone applied to CA3 did not effect CA1 epileptiform events (n = 3; Fig. 3C, bottom). Thus SST applied to CA3 can depress epileptiform events recorded in CA1, although to a lesser degree than local CA1 or bath application.
SST effects on intracellularly recorded CA3 events
SST REDUCES PAROXYSMAL DEPOLARIZING SHIFTS AND GIANT EPSCS IN CA3
NEURONS.
Paroxysmal depolarizing shifts (PDSs) recorded in HPNs in current-clamp
mode are the intracellular correlate of spontaneous epileptiform bursts
recorded extracellularly (Johnston and Brown 1984;
Traub et al. 1994
). These events are driven
polysynaptically (Wong and Traub 1983
) and consist of a
large EPSP underlying a burst of action potentials. The PDS contains
both NMDA and AMPA/KA glutamate receptor-mediated components when
generated in Mg2+-free ACSF (Mody et al.
1987
; Traub et al. 1994
) or bicuculline (Dingledine et al. 1986
). In our intracellular studies,
SST reduced the frequency of CA3 PDSs recorded in Mg2+-free
ACSF (Fig. 4A) from a control
rate of 0.22 ± 0.02 Hz to 0.09 ± 0.02 Hz (n = 4), with recovery on washout (0.26 ± 0.04 Hz). Although
individual bursts were quite variable, we observed no significant
alteration of their shape or size by SST.
|
SST EFFECTS ON ISOLATED CA3 EPSCS.
To more precisely determine SST's effects on synaptic transmission in
CA3, we recorded intracellularly from CA3 HPNs and evoked EPSCs by
stimulating either mossy or A/C fibers. MF EPSCs were isolated in the
presence of APV (30 µM), to block the NMDA neurotransmission largely
derived from A/C synapses (Claiborne et al. 1993).
Interestingly, monosynaptic MF EPSCs were insensitive to SST (Fig.
5A), even though SST induced
an outward current in four of the five cells (10-30 pA,
VH =
74 ± 1 mV). In contrast, SST
significantly reduced both the amplitude and area of both AMPA/KA and
NMDA EPSCs generated at A/C synapses (Fig. 5, B and
C). Digital subtraction of the SST-insensitive EPSC from the
control EPSC revealed that the SST-sensitive component of these
currents appeared to be largely polysynaptic (Fig. 5, B and
C, top) as suggested by the relatively long latency and slow rise-time of the waveforms (see Berry and Pentreath
1976
; Claiborne et al. 1993
). SST induced an
outward current in 6 of the 10 neurons from which A/C generated EPSCs
were recorded, ranging from 10-30 pA (VH =
75 ± 1 mV). Again, no correlation was found between the
SST-induced outward current and SST inhibition of the EPSCs.
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DISCUSSION |
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We have shown that SST has robust antiepileptic properties in both
CA1 and CA3 regions of the hippocampus. The cellular actions of SST in
CA1 have been well characterized. SST augments two types of
K+ currents in CA1 HPNs, the M current (Moore et al.
1988; Schweitzer et al. 1990
, 1993
) and a
voltage-insensitive leak current (Schweitzer et al.
1998
). SST also depresses glutamatergic EPSCs in CA1 HPNs (Tallent and Siggins 1997
), probably through a
presynaptic mechanism (Boehm and Betz 1997
). All these
actions are inhibitory in that they reduce the likelihood of a neuron
firing an action potential.
SST superfusion inhibits CA1 but not CA3 PSs recorded in normal ACSF
SST superfusion reduced normal PSs recorded in CA1, as previously
reported (Watson and Pittman 1988). These results
indicate that SST decreased the number of neurons that fired at a given stimulus strength. Because SST reduces the probability of neuronal firing by postsynaptic augmentation of K+ currents
(Moore et al. 1988
; Schweitzer et al.
1998
) and also reduces presynaptic glutamate release
(Boehm and Betz 1997
), the pre- and postsynaptic actions
of superfused SST synergistically act to reduce PSs. In contrast, SST
superfusion did not reduce normal PSs recorded in CA3 at either MF or
A/C synapses. Although the postsynaptic actions of SST on CA3 neurons
have not been characterized, in the present study, SST induced only
weak outward currents in CA3 neurons from which synaptic events were
recorded. For example, at
70-mV holding potentials, SST induced an
outward current of 10-30 pA compared with a mean of 50 pA reported for
CA1 neurons (Schweitzer et al. 1993
). Therefore SST
may have weaker postsynaptic effects in CA3 than in CA1, perhaps
accounting for the lack of SST actions on normal CA3 PSs. However, we
did not examine postsynaptic effects of SST in the depolarized range,
where its actions on the voltage-sensitive M current would be more
apparent (Moore et al. 1988
).
SST superfusion reduces epileptiform events in CA1
In the bicuculline "seizure" model, all synaptic inhibition
mediated by GABAA receptors is blocked, whereas excitatory
events are normal (Schwartzkroin and Prince 1980). In
normal extracellular K+ (3.5 mM), spontaneous epileptiform
"interictal" or "preictal" events do not generally occur in
CA1 or CA3 (McNamara 1994
). However, afterdischarges are
elicited at relatively low stimulus intensities. In the other model
used (Mg2+-free ACSF), the Mg2+ block of NMDA
receptors is removed, increasing the excitatory drive onto HPNs
(Traub et al. 1994
); inhibitory pathways are largely unaffected (Mody et al. 1987
; Traub et al.
1994
). Spontaneous bursting is generated in CA3 and transmitted
to CA1 via Shaeffer collaterals (Mody et al. 1987
). We
used these two different models to determine whether SST effects were
similar under these different conditions.
In CA1 in bicuculline, SST often completely suppressed the epileptiform
afterdischarges elicited at low stimulation intensities, thus shifting
the threshold for evoking an afterdischarge to a higher stimulus
strength. Inhibition of epileptiform events peaked within 2-4 min of
onset of SST perfusion and was reversible on washout. SST had similar
effects in CA1 in the Mg2+-free ACSF model. Here, the
spontaneous interictal or preictal bursts at the cellular level are
composed of synchronized bursting of neuronal populations (Traub
and Wong 1982). SST markedly decreased the frequency of this
spontaneous bursting in both CA1 and CA3 while not consistently
affecting the shape or intensity of the individual bursts.
Synchronization of interictal events may lead to ictal or seizure
episodes (Traynelis and Dingledine 1988
;
Williamson et al. 1995
). Thus suppression of interictal
event frequency by SST could have a protective effect, preventing the
onset of seizure activity by reducing the probability of synchronization.
SST superfusion reduces epileptiform events in CA3
In CA3, SST effectively reduced or blocked afterdischarges evoked
in both bicuculline and Mg2+-free ACSF. SST's actions in
CA3 under either condition were qualitatively similar to those in CA1
but were less intense, especially at higher stimulation intensities.
Thus in CA3, but not CA1, SST effects could be overcome by increasing
stimulation intensity. At the cellular level, this suggests that
glutamate release in CA3 is sufficient at more intense stimuli to
compensate for presynaptic inhibition and/or postsynaptic
hyperpolarization by SST. As noted above, SST's postsynaptic actions
in CA3 may be weaker than in CA1. Further there appears to be less
expression of SST receptors in CA3 than in CA1 (Leroux et al.
1993; Martin et al. 1991
).
More robust SST effects in CA1 also may reflect additive actions in CA3
and CA1. Generation of seizure-like events occurs in CA3, through
synchronization of CA3 HPN firing via feedforward recurrent excitatory
axon collaterals between these primary neurons (Traub and Wong
1982; Wong and Traub 1983
). Transecting SC input blocks the invasion of seizures from CA3 into CA1 (Schwartzkroin and Prince 1978
). Furthermore, stimulation of SC under
epileptiform conditions activates CA1 neurons both directly and
indirectly by firing CA3 neurons. Therefore with bath superfusion,
direct actions of SST in both CA1 and CA3 could markedly affect
epileptiform events recorded in CA1. We addressed this issue by
applying SST locally to either CA1 or CA3 while recording CA1 PSs or
afterdischarges evoked in bicuculline. SST applied locally near the
recording electrode robustly decreased both normal PSs and epileptiform afterdischarges. SST applied in CA3 also could significantly reduce CA1
afterdischarges, while having no effect on normal CA1 PSs. Thus SST can
have effects in CA1 when applied only to CA3, although its actions are
less pronounced than when applied locally to CA1 or in the superfusate.
SST therefore can act directly in both CA3 and CA1 to reduce the spread
of epileptiform events through the hippocampus.
SST selectively reduces excitatory transmission at A/C synapses
SST dramatically reduced the frequency of PDSs recorded
intracellularly in CA3 neurons. PDSs are generated when A/C synapses, which form recurrent excitatory connections between CA3 HPNs, are
unmasked (Traub et al. 1994). Recorded in voltage-clamp,
PDSs are seen as nongraded giant EPSCs (Ben-Ari and Gho
1988
) that contain both NMDA and AMPA/KA components
(Dingledine et al. 1986
; Mody et al.
1987
; Traub et al. 1994
). SST increased the
threshold for evoking giant EPSCs without affecting their basic shape,
suggesting that SST does not preferentially act on the NMDA or AMPA/KA
component. These results are consistent with a presynaptic action on
glutamate release. Interestingly, SST was ineffective in depressing
monosynaptic MF EPSCs, suggesting that SST may not act on primary
synaptic transmission between the dentate and CA3. However, SST did
depress both pharmacologically isolated NMDA and AMPA/KA
receptor-mediated EPSCs generated at A/C synapses, further supporting a
presynaptic site of action. We observed a greater effect of SST on
isolated NMDA EPSCs compared with AMPA/KA EPSCs, especially at the
highest stimulus intensity (Fig. 5, B and C,
bottom), that may reflect the recruitment of more
polysynaptic pathways (Crepel et al. 1997
). NMDA
receptors make a larger contribution at recurrent excitatory synapses
between HPNs in both CA1 and CA3 than at primary MF/CA3 (Zalutsky and Nicoll 1990
) or SC/CA1 (Deuchars
and Thomson 1996
) synapses. Furthermore, when isolated, NMDA
receptors are more efficient at generating polysynaptic transmission
than AMPA/KA receptors (Crepel et al. 1997
) and are
increased in epileptic hippocampus (Ashwood and Wheal
1986
; Mody and Heinemann 1987
; Turner and
Wheal 1991
). These results suggest a selective action for SST
in CA3 on the recurrent feedforward excitatory synapses that generate
epileptiform activity (see Boehm and Betz 1997
).
We further characterized SST actions on A/C EPSCs in CA3 by examining
P-P modulation of NMDA EPSCs, whereby facilitation or inhibition of the
second of a pair of EPSCs by the first, conditioning EPSC, is measured.
An inverse relationship exists between degree of facilitation and
glutamate release (Manabe et al. 1993; Schulz et
al. 1994
; Zucker 1973
). Thus an increase in P-P
facilitation by a drug suggests presynaptic inhibition of glutamate
release. SST enhanced P-P facilitation of A/C-generated NMDA EPSCs,
substantiating a presynaptic site of action in CA3. Nonetheless,
postsynaptic SST-induced outward currents also sometimes were observed.
Figure 6 schematizes possible sites of
SST action in CA3. SST receptors may be localized preferentially at A/C
synapses (Fig. 6, A and B), as suggested by the
relative paucity of SST binding in the stratum lucidum (Leroux
et al. 1993
), where MF synapses are located. SST released from
inhibitory terminals would diffuse across the synaptic cleft to
interact with postsynaptic receptors and to nearby glutamatergic
terminals to interact with presynaptic receptors (Fig. 6B).
Postsynaptic activation of K+ channels and presynaptic
inhibition of glutamate release by SST would synergistically reduce
excitability in the postsynaptic neuron.
|
A sparse network of recurrent excitatory connections exists between CA1
HPNs (Christian and Dudek 1988; Crepel et al.
1997
). These recurrent axon collaterals are unmasked by
bicuculline and generate the polysynaptic EPSCs sometimes observed in
bicuculline after a single SC stimulation, as they persist after
cutting afferent input from CA3 (Crepel et al. 1997
;
Gereau and Conn 1994
). In cells with clearly
distinguishable polysynaptic EPSCs, SST almost completely suppressed
the second component, while having little effect on the primary EPSC
(Tallent and Siggins 1997
). These results suggest that
SST may not significantly alter primary hippocampal synaptic
transmission along the major trisynaptic pathway but that aberrant or
recurrent excitation is needed for unmasking of SST-sensitive synapses
in both CA1 and CA3. This also is suggested by our findings that SST
has no effect on CA3 PSs elicited in normal ACSF but inhibits both
spontaneous and evoked epileptiform events recorded in
Mg2+-free ACSF and bicuculline. However, SST (both locally
applied and superfused) does depress normal PSs in CA1 as well as some apparent monosynaptic NMDA and AMPA/KA EPSCs (Tallent and
Siggins 1997
), suggesting that in CA1 SST also can affect
primary synaptic transmission. However, its CA1 effects appear to be
amplified when recurrent excitation is unmasked (Tallent and
Siggins 1997
).
Interestingly, postsynaptic effects of SST in CA1 are greatest when the
neuron depolarizes, because SST augments the outwardly rectifying
K+ M current (Moore et al. 1988;
Schweitzer et al. 1993
). Thus SST effects in
hyperexcited hippocampus may be increased both by unmasking of latent
SST-sensitive recurrent excitatory synapses and by depolarization of
HPNs during the PDS. Endogenous release of peptides appears to require
high-frequency activation of the peptidergic neuron (Hokfelt
1991
), such as might occur during an epileptiform burst (Vezzani et al. 1992
). Therefore in epileptic
hippocampus, conditions for SST release would be matched with increased
SST efficacy. This may be an especially important homeostatic mechanism
given the collapse of GABAergic inhibition during high-frequency firing (Le Beau and Alger 1998
). This apparent selective action
of SST on feedforward excitation is unique from that of other
neuropeptides, such as neuropeptide Y and the opioids, that have much
greater effects on standard hippocampal neurotransmisssion
(Capogna et al. 1993
; Klapstein and Colmers
1993
).
Recurrent feedforward excitation may be increased in animal models of
epilepsy. In addition to the MF sprouting that occurs in the dentate in
these models (de Lanerolle et al. 1989; Tauck and
Nadler 1985
), recent studies suggest that sprouting of local axon collaterals occurs in CA1 as well (Perez et al.
1996
). These collaterals appeared to form synaptic contacts
with HPN dendrites and also may have formed autapses (Perez et
al. 1996
). Autaptic EPSCs are SST sensitive in cultured
hippocampal neurons (Boehm and Betz 1997
).
Electrophysiological evidence of increased recurrent excitation in CA1
has been suggested by the appearance of prolonged synchronous
afterdischarges in isolated CA1 from kainate-treated rats (Meier
and Dudek 1996
; Meier et al. 1992
). The increase
of these SST-sensitive synapses in animal models of epilepsy and the
fact that SST receptors largely are spared or even upregulated in
epileptic tissue (Perez et al. 1995
; Piwko et al.
1996
; Robbins et al. 1991
) indicates that SST
receptors could be significant pharmacological targets for clinical
treatment of epileptic disorders.
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ACKNOWLEDGMENTS |
---|
We thank P. Schweitzer and S. G. Madamba for helpful comments, G. Martin for assistance with the artwork, and P. L. Herrling (Novartis Pharma) for the gift of various drugs. We also thank Dr. Ray Dingledine (Emory) for assistance with the coastline burst analysis.
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FOOTNOTES |
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Address for reprint requests: M. K. Tallent, Dept. of Neuropharmacology, CVN-12, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 August 1998; accepted in final form 14 December 1998.
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REFERENCES |
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