Department of Anatomy and Neurobiology, University of California, Irvine, California 92697-1280
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ABSTRACT |
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Ross, Stephen T. and Ivan Soltesz. Selective Depolarization of Interneurons in the Early Posttraumatic Dentate Gyrus: Involvement of the Na+/K+-ATPase. J. Neurophysiol. 83: 2916-2930, 2000. Interneurons innervating dentate granule cells are potent regulators of the entorhino-hippocampal interplay. Traumatic brain injury, a leading cause of death and disability among young adults, is frequently associated with rapid neuropathological changes, seizures, and short-term memory deficits both in humans and experimental animals, indicating significant posttraumatic perturbations of hippocampal circuits. To determine the pathophysiological alterations that affect the posttraumatic functions of dentate neuronal networks within the important early (hours to days) posttraumatic period, whole cell patch-clamp recordings were performed from granule cells and interneurons situated in the granule cell layer of the dentate gyrus of head-injured and age-matched, sham-operated control rats. The data show that a single pressure wave-transient delivered to the neocortex of rats (mimicking moderate concussive head trauma) resulted in a characteristic (~10 mV), transient (<4 days), selective depolarizing shift in the resting membrane potential of dentate interneurons, but not in neighboring granule cells. The depolarization was not associated with significant changes in action potential characteristics or input resistance, and persisted in the presence of antagonists of ionotropic and metabotropic glutamate, and GABAA and muscarinic receptors, as well as blockers of voltage-dependent sodium channels and of the h-current. The differential action of the cardiac glycosides oubain and stophanthidin on interneurons from control versus head-injured rats indicated that the depolarization of interneurons was related to the trauma-induced decrease in the activity of the electrogenic Na+/K+-ATPase. In contrast, the Na+/K+-ATPase activity in granule cells did not change. Intracellular injection of Na+, Ca2+-chelator and ATP, as well as ATP alone, abolished the difference between the resting membrane potentials of control and injured interneurons. The selective posttraumatic depolarization increased spontaneous firing in interneurons, enhanced the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in granule cells, and augmented the efficacy of depolarizing inputs to discharge interneurons. These results demonstrate that mechanical neurotrauma delivered to a remote site has highly selective effects on different cell types even within the same cell layer, and that the electrogenic Na+-pump plays a role in setting the excitability of hippocampal interneuronal networks after injury.
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INTRODUCTION |
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Head injury affects over a million people each
year in the United States, and leads to neurological disturbances
including seizures and memory deficits (Annegers 1980;
Binder 1986
; Goldstein 1990
; Grahm
et al. 1990
; Gualtieri and Cox 1991
;
Harrison and Dijkers 1992
; LeRoux and Grady
1995
; Rempel-Clower et al. 1996
; Salazar
1992
; Salazar et al. 1985
). Postmortem
examinations of head-injured patients often reveal selective damage to
the dentate gyrus ("end-folium sclerosis") (Bruton
1988
; Margerison and Corsellis 1966
). Fluid
percussion injury (FPI), involving the delivery of a single pressure
wave-transient to the neocortex, replicates various histological,
behavioral and cognitive consequences of human head trauma
(Dixon et al. 1989
; Lowenstein et al.
1992
; Lyeth et al. 1988
; McIntosh et al.
1989
). Because inhibitory interneurons synapsing on principal
cells play a central role in the regulation of the input-output
functions of the hippocampus (Buckmaster and Schwartzkroin
1995
; Buhl et al. 1994
, 1995
;
Buzsáki et al. 1983
; Halasy and Somogyi
1993
; Han et al. 1993
; Miles et al.
1996
; Mott et al. 1997
; Soltesz et al.
1995
), the perturbation of dentate interneuronal networks is
likely to be an important factor in the development of posttraumatic
pathological states.
Previous morphological results (Toth et al. 1997) showed
that the interneurons in the dentate granule cell layer are
instantaneously affected by the trauma, as seen with the posttraumatic
staining of these neurons by the Gallyas silver stain (Gallyas
et al. 1992a
,b
). However, in spite of the injury, these
interneurons all survived the trauma, as determined by
immunocytochemical markers months after the impact. Curiously, the
labeling of interneurons by the Gallyas stain was highly selective,
because granule cells in the same layer were not labeled (see Fig.
6B in Toth et al. 1997
). The present study
was designed to determine the functional correlates of the
histologically detected rapid perturbation of interneurons in the
dentate gyrus. The results reveal a posttraumatic mechanism that
selectively depolarizes interneurons, but not granule cells, leading to
an effective enhancement of the efficacy of excitatory inputs to
interneurons in the early posttraumatic period. Therefore, neurotrauma
can lead to surprisingly selective, cell type-dependent functional
alterations in the dentate gyrus.
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METHODS |
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Lateral fluid percussion injury
The lateral fluid percussion technique was carried out as
described previously (Dixon et al. 1989;
Lowenstein et al. 1992
; McIntosh et al.
1989
; Toth et al. 1997
). Briefly, young adult [postnatal day 20-25 (P20-P25)] Wistar rats
were anesthetized with pentobarbital sodium (Nembutal, 65 mg/kg ip;
adequate anesthesia was repeatedly ascertained by the lack of the
ocular reflex and by the absence of withdrawal response to a pinch of
the hindlimb), placed in a stereotaxic frame, and the scalp was
sagittally incised. A 2-mm hole was trephined to the skull at
3 mm
from bregma, 2.0 mm lateral from the sagittal suture. Two steel screws
were placed immediately rostral to bregma and caudal to lambda. A
Luer-Loc syringe hub with a 2.6-mm inside diameter was placed over the exposed dura and bonded to the skull with cyanoacrylate adhesive. Dental acrylic was poured around the injury tube and skull screws and
allowed to harden, and the scalp was sutured. Bacitracin was applied to
the wound, and the animal was returned to its home cage. A day later,
the rats were anesthetized with halothane in a glass chamber. After the
animal was anesthetized (surgical level of anesthesia was ascertained
as described above), it was removed from the anesthetizing chamber, and
immediately connected to the injury device (see below). The
establishment of the connection to the device took 5 s, and the
actual injury (release of the pendulum) took another 5 s,
therefore the animal was fully anesthetized at the time of injury, even
though the halothane anesthesia was not actively administered at the
time of injury. All animals were immediately ventilated with room air.
The animals were injured by a moderate (2.0-2.2 atm) impact
(Toth et al. 1997
). The moderate level of injury were
selected to produce neuronal degeneration in the dentate gyrus similar
to those reported previously (Toth et al. 1997
).
Age-matched, sham-operated control animals were treated the same way,
including the connection to the fluid percussion injury device, but the
pendulum was not released. The animals recovered fully from
anesthesia within 10-15 min, and their subsequent behavior, such as
feeding and grooming, were normal. After various survival periods (see
RESULTS), the animals were euthanized after deep halothane
anesthesia by decapitation for slice physiology (see Slice
preparation).
The fluid percussion device (Department of Biomedical Engineering,
Virgina Commonwealth University, Richmond, VA) was identical to that
used by several other laboratories (Cortez et al. 1989; Coulter et al. 1996
; Dixon et al. 1989
;
Lowenstein et al. 1992
; McIntosh et al.
1989
; Povlishok et al. 1994
; Prasad et
al. 1994
), as well as by us (Toth et al. 1997
).
Briefly, the device consisted of a Plexiglas cylinder reservoir 60 cm
long and 4.5 cm diam. At one end of the cylinder, a rubber-covered
Plexiglas piston was mounted on O rings. The opposite end of the
cylinder had an 8-cm-long metal housing that contained a transducer.
Fitted at the end of the metal housing was a 5-mm metal tube with a
2-mm inner diameter that terminated in a male Luer-Loc fitting. This metal fitting was then directly connected to the female fitting that
had been chronically implanted (i.e., there was no pliable, e.g.,
plastic, tubing between the injury device and the implanted hub). The
entire system was filled with saline. The injury was produced by a
metal pendulum that strikes the piston of the injury device. The
resulting pressure pulse was recorded extracranially by a transducer
and expressed in atmospheres pressure. This injury device injected a
small volume of saline into the closed cranial cavity and produced a
brief (20 ms) displacement and deformation of brain tissue. The
magnitude of injury was controlled by varying the height from which the
pendulum was released (in these experiments, it was 12.5-13.5°,
which produced 2.0-2.2 atm pressure waves). It has been shown that
monitoring of blood pressures and arterial blood gases during and after
fluid percussion injury showed no evidence of significant
cardiorespiratory compromise in the injured animals (Lowenstein
et al. 1992
). The delivery of the pressure pulse was associated
with brief (<120-200 s), transient traumatic unconsciousness (as
assessed by the duration of the suppression of the withdrawal reflex).
Although the injured animals in this study, subjected only to moderate
impacts, did not exhibit any obvious lasting behavioral deficit, fluid
percussion head injury, especially at higher impact forces, can lead to
detectable motor and memory deficits lasting for days (Lyeth et
al. 1988
; McIntosh et al. 1989
;
Povlishock et al. 1994
). Before and during each
experiment, great care was taken to ensure that no air bubble was
trapped or formed in the device. Furthermore, in each experiment the
pressure wave was closely examined on the oscilloscope for any sign of a jagged rising edge (which would indicate the presence of air bubbles
in the system), and the amplitude of the oscilloscope reading of the
pressure wave was recorded.
Slice preparation
Brain slices were prepared as previously described (Otis
and Mody 1992; Soltesz and Mody 1994
;
Staley et al. 1992
). The fluid percussion-injured and
the sham-operated, age-matched rats were anesthetized with halothane.
Anesthetized rats were decapitated, and the brains were removed and
cooled in 4°C oxygenated (95% O2-5%
CO2) artificial cerebral spinal fluid (ACSF)
composed of (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3,
2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose.
Horizontal brain slices (Staley et al. 1992
) (300 µm
for visualized patch clamp) were prepared with a vibratome tissue
sectioner (Lancer Series 1000 or Leica VT1000S) from the midsection of
the hippocampus (for rationale and details, see Toth et al.
1997
). This procedure yielded about six slices. The brain
slices were sagittally bisected into two hemispheric components, and
the ipsilateral slices were incubated submerged in 32°C ACSF for
1 h in a holding chamber (the contralateral slices were not examined during these experiments).
Electrophysiology
Individual slices were transferred to a recording chamber
(Hollrigel et al. 1996, 1998
;
Soltesz et al. 1995
) perfused with oxygenated ACSF,
either with or without any of the following drugs (as specified in
RESULTS): 10 µM bicuculline methiodide (BMI), 10 µM
2-amino-5-phosphovaleric acid (APV), 5 µM
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 1 µM tetrodotoxin (TTX),
100 µM ZD-7288, 30 µM oubain and 30 to 100 µM strophanthidin, 1 µM atropine, and 500 µM (RS)-
-methyl-4-carboxyphenylglycine (MCPG). As described elsewhere (Senatorov et al. 1997
),
strophanthidin was first dissolved in dimethyl sulfoxide (0.5% DMSO in
stock solution). The brain slices were stabilized with platinum wire weights. The temperature of the perfusion solution was maintained at
34°C. As described before (Toth et al. 1997
), for
evoked responses, constant-current stimuli (0.1-2.0 mA; 20-200 µs)
were applied at 0.1 Hz through a bipolar 90-µm tungsten-stimulating
electrode placed in the perforant path, just at the fissure at the
junction of the dorsal blade and the crest. For these experiments
involving evoked responses from interneurons, the recorded interneurons were all situated at approximately the same distance form the stimulating electrode (~150 µm away from the point in the granule cell layer that was directly beneath the point of stimulation at the
fissure), always in the direction of the tip of the dorsal blade. All
salts were obtained from Fluka. APV, CNQX, MCPG, and ZD-7288 were
purchased from Tocris; atropine, oubain, and strophanthidin were
obtained from Sigma; BMI from Research Biochemicals International; and
TTX was obtained from Calbiochem.
Patch pipettes were pulled from borosilicate (KG-33) glass capillary
tubing (1.5 mm OD; Garner Glass) with a Narishige PP-830 two-stage
electrode puller. The basic pipette solutions consisted of (in mM) 140 K- or Cs-gluconate, 2 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES); in some experiments, the intracellular solution also
included 20 Na+-gluconate (replacing 20 K-gluconate), 0.2 EGTA and/or 4 ATP, or biocytin (0.3%), as specified
in RESULTS. Infrared video-microscopy-aided visualized
techniques were used (Axioscope FS, Zeiss) (Stuart et al.
1993). Recordings were obtained with either a NeuroData two-channel intracellular amplifier, or an Axopatch-200B amplifier (Axon Instruments), and digitized at 88 kHz (Neurocorder, NeuroData) before being stored in PCM form on videotape. The series resistance was
monitored throughout the recordings, and the data were rejected if it
significantly increased. Perforated patch-clamp recordings were carried
out as described by Kyrozis and Reichling (1995)
, and
the pipettes contained (in mM) 140 KCl, 2 MgCl2,
and 10 HEPES. As described previously (Hollrigel et al.
1998
), pipette-tips were filled with this solution, then
back-filled with one containing 10-20 µg/ml gramicidin (prepared
from a 5 mg/ml stock solution in DMSO).
Analysis
Recordings of spontaneous inhibitory postsynaptic currents
(sIPSCs) were filtered at 3 kHz before digitization at 20 kHz by a
personal computer for analysis using Strathclyde Electrophysiology Software (courtesy of Dr. J. Dempster) and Synapse software (courtesy of Dr. Y. De Koninck). Data for the analysis of action potentials were
digitized at 50 kHz and not filtered. Detection of individual sIPSCs
and spontaneous excitatory postsynaptic currents (sEPSCs) was performed
with a software trigger previously described (Otis and Mody
1992; Soltesz et al. 1995
). All of the detected
events were analyzed and any noise that spuriously met trigger
specifications was rejected. A least-squares Simplex based algorithm
was used to fit the ensemble averages of EPSCs and IPSCs with the sum
of two (1 rising and 1 decaying) exponentials (Otis and Mody
1992
; Soltesz and Mody 1995
)
[I(t) =
A *
e
t/
r + Ae
t/
d, where A
is a constant,
r and
d are the rise and decay time constants].
Statistical analyses were performed with SPSS for Windows or SigmaPlot,
with a level of significance of P
0.05. Data are
presented as means ± SE.
Histology
To visualize recorded cells filled with biocytin, slices were
processed as whole mounts (Claiborne et al. 1986;
Hollrigel and Soltesz 1997
). Briefly, after
allowing diffusion of the tracer 20-60 min in the recording chamber,
the slice was fixed in a solution of 4% paraformaldehyde, and 0.5%
glutaraldehyde overnight at 4°C and washed thoroughly before it was
reacted with 3,3'-diaminobenzidine tetrahydrochloride (concentration,
0.015%) and 0.006% H2O2.
The reacted slices were cleared in an ascending series of glycerol (Claiborne et al. 1986
) and coverslipped before
the cells were reconstructed with a camera lucida.
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RESULTS |
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Electrophysiological correlates of histochemically detected injury
What are the functional correlates of the posttraumatic labeling
of interneurons within the granule cell layer with an injury marker
such as the Gallyas stain (Toth et al. 1997)?
Interneurons in the granule cell layer from fluid percussion-injured
animals appeared to be able to fire action potentials in a manner
comparable with their control counterparts, and the intact
morphological appearance also indicated that the interneuronal membrane
integrity was preserved after the impact (Fig.
1; unless specifically noted otherwise,
the experiments were conducted 1-4 h after injury). In addition, there
was no difference in the firing properties and action potential
characteristics [action potential maximum rate of rise: 160.5 ± 22.3 mV/ms and 173.6 ± 13.3 mV/ms, mean ± SE; half-width:
0.94 ± 0.04 ms and 1.03 ± 0.05 ms; amplitude of fast
afterhyperpolarization:
11.3 ± 1.4 mV and
13.5 ± 0.7 mV; the frequency adaptation ratio, defined as the ratio of the frequency of the last 2 spikes of the train to the frequency of the 1st
2 spikes, see Mott et al. (1997)
: 0.49 ± 0.07 and
0.43 ± 0.05; n = 7 and n = 7],
the input resistance [308.7 ± 46.3 M
, n = 12;
vs. 347.2 ± 29.2 M
, n = 13; measured with
small hyperpolarizing pulses; for full current-voltage (I-V)
curves, see Figs. 4 and 6], and the series resistance (8.3 ± 0.4 M
and 8.8 ± 0.7 M
) between the interneurons recorded from
control and head-injured animals, respectively (the input resistance
was determined using small, <5 mV, 500 ms-long hyperpolarizing pulses
from
60 mV, to minimize the influence of the h-current on the voltage
responses; for considerations of the role of
Ih, see Possible mechanisms of
interneuronal depolarization). In addition, we found no difference in the amplitude of interneuronal action potentials between control and
FPI interneurons [74.3 ± 2.5 mV and 75.7 ± 2.1 mV,
respectively, measured from the inflection point visible on the rising
phase of the voltage change giving rise to an action potential during an intracellular current pulse, as recorded with the Axopatch 200B
amplifier; the similar amplitude of the action potentials in control
and FPI interneurons was also verified with the Neurodata (current
clamp) amplifier; data not shown].
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Unlike the lack of alterations in the above-described intrinsic
electrophysiological parameters or in morphology, the resting membrane
potential was found to be significantly depolarized in interneurons
from the injured animals, compared with controls (control: 64.9 ± 1.3 mV; n = 12; FPI:
52.2 ± 1.6;
n = 13; Fig. 2; in the
case of spontaneously firing interneurons, membrane potential was
determined during transient epochs free of action potentials). In
addition, the depolarization was associated with a sevenfold increase
in the frequency of rest firing in interneurons (0.2 ± 0.2 Hz,
n = 7 to 1.5 ± 0.9 Hz, n = 8;
Fig. 2A). In contrast to the interneurons, neighboring
granule cells did not show a comparable depolarizing shift in membrane
potential (Fig. 2C; control:
78.0 ± 1.5 mV,
n = 8; FPI:
78.3 ± 1.0, n = 8),
indicating that the depolarization was highly cell-type specific even
within the same cell layer. The interneuron-specific posttraumatic
physiological alteration is in excellent agreement with the
histochemical labeling of the interneurons in the granule cell layer,
but not of the dentate granule cells themselves, by the Gallyas injury
stain following the delivery of a pressure wave-transient to the
neocortex (Lowenstein et al. 1992
; Toth et al.
1997
).
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To determine that the observed interneuronal depolarization is not
simply associated with the whole cell recording itself, gramicidin-based perforated patch-clamp recordings (Hollrigel et
al. 1998; Kyrozis and Reichling 1995
) were
performed, which fully verified the whole cell data regarding the
existence of a posttraumatic depolarization in interneurons in the
dentate gyrus (control:
63.5 ± 4.3 mV, n = 3;
FPI:
52.1 ± 3.6 mV, n = 3). Furthermore,
cell-attached recordings also indicated increased spontaneous firing of
interneurons after fluid percussion injury (Fig. 2B;
control: 0.2 ± 0.1 Hz, n = 3; FPI: 2.6 ± 1.3, n = 4). The increased spontaneous firing rate of
interneurons was not due to alterations in the frequency of sEPSCs
following trauma, because no posttraumatic change could be observed in
the frequency, amplitude or kinetics of sEPSCs recorded from
interneurons in the granule cell layer (Fig.
3; control: n = 4; FPI:
n = 4), indicating that the depolarization itself plays
a major role in enhancing tonic firing in interneurons after impact. In
conclusion, these whole cell, perforated and cell-attached patch-clamp
recordings clearly determined the existence of a selective
posttraumatic depolarizing shift of the resting membrane potential in
dentate interneurons situated in the granule cell layer.
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Possible mechanisms of interneuronal depolarization
What is the mechanism underlying the approximately 10-mV
posttraumatic depolarizing shift in interneuronal resting membrane potential? First, the depolarization in interneurons in fluid percussion-injured animals could not be accounted for by activation of
TTX-sensitive voltage-gated Na+ channels,
N-methyl-D-aspartate (NMDA),
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or
kainate receptors, or GABAA receptor channels, because the depolarization persisted even in the presence of TTX (1 µM), APV (10 µM), CNQX (5 µM), and bicuculline (10 µM; control:
58.7 ± 1.8 mV, n = 6; FPI:
51.2 ± 1.7 mV, n = 6). The fact that granule cells displayed no
posttraumatic changes in their resting membrane potential also argued
against a persistent elevation of glutamate in the granule cell layer
after impact. Furthermore, as discussed above, there was no significant
increase in the input resistance of interneurons from injured versus
control brains (see also below), indicating that closure of
K+ channels (e.g., mechanosensitive channels) is
not a major factor in the depolarization following trauma. Furthermore,
atropine (1 µM) and MCPG (500 µM) caused only a small,
nonsignificant hyperpolarization (0.6 ± 1.2 mV; n = 3) of the depolarized resting membrane potential of FPI interneurons,
indicating that a post-FPI blockade of the m-type
K+ current by acetylcholine, and tonic activation
of metabotropic glutamate receptors, do not underlie the interneuronal
depolarization following FPI.
The possible involvement of an Ih
current [which is known to influence the resting membrane potential in
the depolarizing direction by a few millivolts in some cells (e.g.,
Maccaferri and McBain 1996; Soltesz et al.
1991
; for a review, see Pape 1996
)] could be
excluded, because the depolarized resting membrane potential in dentate
interneurons from fluid percussion-injured animals was not
significantly hyperpolarized by switching the perfusate to a medium
containing the Ih channel blocker
ZD-7288 (100 µM) (BoSmith et al. 1993
;
Gasparini and DiFrancesco 1997
; Harris and Constanti 1995
; Maccaferri and McBain
1996
) (n = 5;
Vm before ZD-7288 in FPI interneurons:
54.0 ± 4.8 mV; in the presence of ZD-7288:
53.4 ± 4.1 mV; the effective blockade of the Ih
current by ZD-7288 within <5 min was verified by the disappearance of the depolarizing "sag" from the voltage responses to
hyperpolarizing current pulses in the same interneurons; not shown).
Furthermore, interneurons in FPI animals remained significantly more
depolarized compared with interneurons from control animals even when
slices were bathed in and continuously perfused with ZD-7288
(Vm in the presence of APV, CNQX, TTX,
bicuculline, and ZD-7288: in control interneurons:
51.5 ± 2.1 mV, n = 8; in FPI:
43.6 ± 2.3 mV,
n = 8; the reason for the relatively depolarized
membrane potential of both control and FPI interneurons after prolonged
incubation of slices in the presence of these drugs was not
investigated, because the important point here was that statistically
significant depolarization could be observed in the FPI interneurons in
the presence of ZD-7288; the Vm in the
prolonged, >45 min, presence of ZD-7288, and APV, CNQX, TTX, and
bicuculline, was ~7 mV more depolarized in both control and FPI cells
than in the same drugs without ZD-7288; see above for
Vm values in APV, CNQX, TTX, and bicuculline only; note that blockade of
Ih would be expected to hyperpolarize,
and not depolarize, Vm).
A recent report suggested that the Cs+-sensitive
inward-rectifier K+ current is down-regulated in
glial cells after FPI (D'Ambrosio et al. 1999), as
measured by the diminished blocking action of Cs+
on the inward currents evoked with voltage steps to membrane potentials
more hyperpolarized than EK (in the
presence of ZD-7288 to eliminate the action of extracellular
Cs+ on Ih).
Because the inward rectifier can conduct a small outward current above
EK, which can contribute to the
resting membrane potential in some cells (Hille 1993
),
we proceeded to determine whether a similar posttraumatic decrease of
these channels also occurs in our interneurons, possibly contributing
to the post-FPI depolarization seen in interneurons. However, when
I-V curves were constructed (in voltage clamp in the
presence of ZD-7288, APV, CNQX, bicuculline, and TTX), no effect of
extracellular Cs+ (1 mM) could be seen on the
inward current even at very hyperpolarized membrane potentials (e.g.,
at
120 mV, where the inward rectifier should be active), suggesting
the low level of expression of the classical anomalous
K+-dependent inward rectifier channels by these
dentate interneurons (Fig. 4;
n = 4 in both groups; 5-15 min was
allowed for Cs+ to exert any possible effect,
because in separate experiments we determined that
Cs+ fully blocked the
Ih current in our slices in <2.5 min,
n = 3). Consequently, a down-regulation of the outward
current carried by the inward rectifier is unlikely to underlie the
post-FPI depolarization; indeed, Cs+ did not
cause hyperpolarization of the Vm of
FPI interneurons, in fact, Cs+ caused a
nonsignificant depolarization of Vm in
both control and FPI interneurons (depolarization in control: 1.0 ± 1.6 mV; n = 4; in FPI: 2.7 ± 0.8 mV;
n = 4). These experiments also confirmed that there was
no significant change in the input resistance (e.g., Fig.
4A), even when measured with strong hyperpolarizing or
depolarizing voltage steps (note that the more depolarized
Vm of FPI cells was present in these
cells as well, although the rightward shift of the I-V curve
in Fig. 4A is difficult to discern due to the necessarily
large scale on the x-axis).
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Involvement of the Na+-pump
The Na+/K+-ATPase, an
electrogenic pump ubiquitously expressed in most tissues (Glynn
1993; Lees 1991
; Thomas 1972
), is
known to contribute to the resting membrane potential of most cells by
~5-15 mV (see DISCUSSION), i.e., in the range of the
interneuron-specific depolarization observed here (the electrogenicity
of the Na+/K+-ATPase arises
from the fact that the pump exports three Na+
ions from the intracellular to the extracellular space, but imports only two K+ ions, during each pump cycle). Apart
from the similarity of the absolute value of the observed posttraumatic
depolarization in interneurons and the hyperpolarization of the resting
membrane potential by the electrogenic
Na+/K+-ATPase, recent
studies from cultured cortical cells also suggested that the
Na+/K+-ATPase can be
down-regulated after mechanical injury (Tavalin et al.
1995
, 1997
). Furthermore, the stretch-induced
depolarization of cultured cortical cells shared other similarities
with the posttraumatic interneuronal depolarization reported here, for example, the lack of prominent changes in input resistance and action
potential characteristics, as well as the inability of ionotropic
glutamate receptor antagonists to reverse the depolarization after
induction (Tavalin et al. 1995
, 1997
).
Therefore we employed the cardiac glycosides oubain and strophanthidin,
specific blockers of the
Na+/K+-ATPase
(Gadsby and Nakao 1989
; Nakao and Gadsby
1986
; Priebe et al. 1996
; Senatorov and
Hu 1997
; Senatorov et al. 1997
), to determine
whether the Na+/K+-ATPase
activity is down-regulated in dentate interneurons after trauma and
thus contributes to the depolarized interneuronal resting membrane
potential. The
Na+/K+-ATPase blocker
oubain (30 µM, duration of application: 5 min, in the presence of
APV, CNQX, TTX, and bicuculline) depolarized control interneurons by
17.5 ± 1.5 mV (n = 7), indicating a significant contribution to the interneuronal resting membrane potential by the
electrogenic Na+/K+-ATPase
pump. However, the effects of oubain, applied at the same concentration
for the same amount of time, appeared different on interneurons from
fluid percussion-injured animals. Specifically, oubain (30 µM)
caused a significantly smaller depolarization in interneurons from
injured animals compared with controls (7.7 ± 1.3 mV,
n = 6; Fig.
5A). Importantly, not only did
oubain have a significantly different effect on interneurons from
injured versus control animals, but the
Na+/K+-ATPase blocker was
also able to completely abolish the difference between the resting
membrane potentials of interneurons from the experimental and the
control groups (Fig. 5B).
|
With sharp electrode recordings, after prolonged application of high
concentration of
Na+/K+-ATPase blockers, a
collapse of the cellular ion gradient can take place. However, whole
cell recording and TTX reportedly prevent the collapse of ion gradients
(Senatorov and Hu 1997; Senatorov et al.
1997
). Nevertheless, to further minimize the possibility that a
differential effect of oubain on the membrane potential of injured
versus control cells was due to a differential collapse of ion
gradients, we proceeded to repeat the oubain experiments under
conditions that decreased the likelihood of collapsing ion gradients
during applications of
Na+/K+-ATPase blockers.
Specifically, the less potent
Na+/K+-ATPase blocker
strophanthidin (30 µM) was applied for 20 s only (in the
presence of APV, CNQX, TTX, bicuculline, and ZD-7288). Therefore unlike
the previous experiments in which the more potent oubain (e.g.,
McCarren and Alger 1987
) was applied for 5 min and caused a depolarization that reached a steady-state, the brief application of strophanthidin would be expected to cause only a partial
block of the pump current, and the partial block would be expected to
be larger in the control. Indeed, as predicted from the oubain data,
the strophanthidin-sensitive current in the posttraumatic interneurons
was only 62.1% of control [control: 17.4 ± 3.6 pA;
n = 8; FPI: 10.8 ± 2.4 pA, n = 8;
the corresponding depolarization of the resting membrane potential,
measured in current clamp, was 10.1 ± 1.2 mV in the controls, and
6.3 ± 1.3 mV in the FPI animals (Fig.
6, A and B) in the
case of the strophantidin-experiments, partial recovery could be
obtained; not shown; the strophantidin-sensitive current and the
corresponding change in resting membrane potential were measured in the
same cells].
|
Unchanged pump-current in granule cells
Next, the reverse experiment was performed. If the posttraumatic depolarization is due to a malfunctioning Na+/K+-ATPase in interneurons, the Na+/K+-ATPase should be unchanged in those cells that are not depolarized after FPI, i.e., the granule cells. Indeed, the strophanthidin-sensitive current measured under identical conditions as described in the previous paragraph was not decreased in granule cells of the dentate gyrus from injured animals compared with control (Control: 11.5 ± 2.9 pA, n = 4; FPI: 13.2 ± 1.8 pA, n = 5), in agreement with the similar resting membrane potential of granule cells from injured and control animals.
Possible mechanisms of decreased Na+/K+-ATPase function in interneurons after impact
The lower pump activity in interneurons after trauma could
be due to a posttraumatic decrease in the number of functional pump
molecules expressed in the cell membrane, and/or to a decreased pump
rate. To distinguish between these two possibilities, the forward pump
turnover rate was strongly enhanced (Gadsby and Nakao 1989) by the inclusion of 20 mM Na+, 0.2 mM EGTA (because intracellular Ca2+ can
powerfully block the ATPase, see DISCUSSION), and 4 mM ATP in the recording pipette (in addition to K-gluconate, see
METHODS). Under these conditions (and in the presence of
TTX, APV, CNQX, and bicuculline), the resting membrane potential of
interneurons from injured and control animals became indistinguishable
(control:
67.5 ± 3.1 mV, n = 6; FPI:
65.8 ± 3.1 mV, n = 5). Interestingly, the
inclusion of 4 mM ATP in the pipette alone was able to abolish the
difference between the resting membrane potential of interneurons from
injured and age-matched control animals (Fig. 5B; control: n = 7; FPI: n = 6; it is important to
reemphasize here that the existence of the posttraumatic depolarization
was not simply due to an artifact of the whole cell recording per se,
because, as described above, the posttraumatic depolarization existed
also under perforated patch conditions, as well as in cell-attached mode as seen by the enhanced firing rate).
If it is the pump rate, and not the number of the pump molecules, which
is decreased after trauma, enhancement of the pump current by the
intracellular application of pump substrates should not only abolish
the difference between the resting membrane potentials (as shown
above), but it would also be expected to eradicate the difference
between the pump currents in interneurons from experimental and control
groups. Indeed, when the forward pump rate was enhanced (as described
above), the difference between the strophanthidin-sensitive current
(after complete blockade of the maximized pump activity with 100 µM
strophanthidin applied for 5 min, in the presence of TTX, APV, CNQX,
and bicuculline) in interneurons from control and fluid
percussion-injured animals was abolished (control: 47.5 ± 1.9 pA, n = 4; FPI: 42.7 ± 4.1 pA, n = 3; remarkably, the strophanthidin-sensitive current density shown in
Fig. 6C measured in interneurons of the dentate gyrus was in
the same order of magnitude as that measured in cardiac cells)
(Gadsby and Nakao 1989). The similarity of the pump
current (and of the resting membrane potential) recorded under
saturating conditions (i.e., when the forward pump rate is maximized
and the pump blocker is applied at a higher concentration for a longer
period of time) also indicate that the number of pump molecules remain
similar to controls in interneurons after impact, and that it is mainly
the pump rate that limits the
Na+/K+-ATPase function
after head trauma.
Recovery of the interneuronal depolarization after trauma
Next, experiments were carried out to determine the duration
of the depolarization of interneurons after trauma. The data described
above showed that the interneurons from injured animals were
depolarized for at least 1-4 h after impact. Experiments carried out 4 days after impact indicated that the difference between the resting
membrane potential of interneurons from control and fluid
percussion-injured animals became nonsignificant (Control: 60.6 ± 2.4 mV, n = 8; FPI:
57.5 ± 1.7, n = 8). Therefore there is a recovery of the
posttraumatic depolarization in dentate interneurons within days after impact.
Functional consequences of the posttraumatic depolarization of interneurons
Does the approximately 10 mV depolarization in interneurons enhance the ability of incoming depolarizing signals to discharge interneurons in the early posttraumatic dentate gyrus? To answer this question, the effect of perforant path stimulation was compared between interneurons from control and experimental rats. As shown in Fig. 7, A and B, the perforant path input (in the presence of 10 µM bicuculline) had a significantly increased efficacy in eliciting action potential discharges in interneurons from fluid percussion-injured rats compared with controls (control: n = 4; FPI: n = 4). Obviously, these latter results could be complicated by a potentially enhanced posttraumatic excitatory input [e.g., by a posttraumatic increase in evoked excitatory postsynaptic potentials (EPSPs); this issue will not be addressed in this paper]. To eliminate the influence of a potentially increased evoked EPSP amplitude on the latter results, short (10 ms) depolarizing current pulses ("artificial EPSPs") of increasing amplitude were injected from the resting membrane potential of the same interneurons as in Fig. 7, A and B. The data showed that interneurons in the posttraumatic dentate gyrus started to fire action potentials to the artificial EPSPs at significantly lower amplitude of intracellular current injection compared with controls (Fig. 7, C and D). Therefore the postimpact depolarization of interneurons enhances the efficacy of incoming excitatory signals to discharge interneurons.
|
A related issue is whether the increased tonic firing of interneurons
(see Fig. 2) results in a posttraumatic increase in the frequency of
spontaneous IPSCs in granule cells. As shown in Fig.
8, granule cells showed a significantly
enhanced frequency of sIPSCs (without a change in kinetics; note that
the mIPSC frequency actually decreases after FPI) (Toth et al.
1997; see DISCUSSION). The increased frequency was
also accompanied by an enhancement of the sIPSC amplitude (Fig.
8C), as would be expected from an increase in tonic firing
of the presynaptic interneurons leading to synchronized release of GABA
from multiple terminals belonging to the same interneuron (e.g.,
Buhl et al. 1995
) [the mIPSC amplitude does not
increase after FPI (Toth et al. 1997
)].
|
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DISCUSSION |
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Correlation of histological and electrophysiological data on the posttraumatic perturbation of interneurons
Previous data showed that dentate interneurons, unlike the granule
cells themselves, display histological signs of injury after FPI
(Toth et al. 1997). Although histological stains
(Gallyas et al. 1992a
,b
) are widely used to detect
neuronal injury, the nature of the functional changes in neurons
histologically labeled as "injured" has not been clarified. The
present results determined that physiological changes can be detected
in interneurons, but not in granule cells, in agreement with the
histological data. Second, the data show that the physiological
correlate of histological injury is a highly selective depolarization
of the interneuronal resting membrane potential. The good agreement
between the histological and electrophysiological data indicate that
histological staining techniques such as the Gallyas "dark neuron"
labeling method (Gallyas et al. 1992a
,b
) can be used to
map patterns of neuronal damage in a functionally meaningful manner.
The diversity of dentate interneuronal firing properties and
axodendritic processes (Mott et al. 1997) does not allow
the direct correlation of the recorded cells with immunocytochemically identified interneuronal groups. However, it has been shown that the
injured interneurons in the granule cell layer all survive FPI
(Toth et al. 1997
). Therefore it is unlikely that we
sampled a different subpopulation of interneurons in control and
injured animals. The similar morphological (e.g., Fig. 1) and (apart
from the resting membrane potential) electrophysiological properties of
control and FPI interneurons also support this conclusion.
Cell specificity of the posttraumatic depolarization
Depolarization due to stretch-induced down-regulation of the
Na+/K+-ATPase has been
demonstrated in cultured cortical cells plated on a deformable
substrate (Tavalin et al. 1995, 1997
),
indicating that the
Na+/K+-ATPase-linked
regulation of the membrane potential in response to mechanical forces
can take place in various neuronal cell types. How does the highly cell
type-specific depolarization of dentate interneurons arise after FPI?
Previous data showed that interneurons displayed histological signs of
selective injury after FPI even when the animals were cooled, prefixed,
and loaded with glutamate receptor antagonists before impact,
indicating that purely mechanical reasons may also contribute to the
initial pattern of selective injury (e.g., the simple fact that
interneurons in the granule cell layer have larger somata than the
granule cells may preferentially predispose interneurons to be
stretched and bent to a greater degree during the passage of the
pressure wave) (see Toth et al. 1997
for details). The
initial "mechanical" injury pattern is likely to be later modified
by "biological" factors (e.g., glutamatergic inputs, calcium-rises,
etc.) (Toth et al. 1997
). For example, interneurons in
the granule cell layer express highly
Ca2+-permeable AMPA and NMDA channels (Koh
et al. 1995
), and increases in intracellular
[Ca2+] can potently inhibit the
Na+/K+-ATPase
(Calabresi et al. 1995
; Fukuda and
Prince 1992a
,b
; Lees 1991
; Thompson and
Prince 1986
; for detailed discussions on the role of the pump,
see below). The specificity of the interneuronal depolarization may
also be influenced by the apparently higher expression of the
3
subunit of the
Na+/K+-ATPase by
interneurons compared with granule cells (Chauhan and Siegel
1996
), because differences in subunits can influence substrate sensitivity (Glynn 1993
; Jewell and Lingrel
1991
; Peng et al. 1997
). Furthermore, a
possibility is that the decreased pump activity in interneurons was
related to a posttraumatic decrease in the availability of pump
substrates (see below). Various forms of neuronal injury, including
head injury, compromise cellular energetic status (Ankarcrona et
al. 1995
; Beal et al. 1993
; Faden et al. 1989
; Retz and Coyle 1982
; Rothman and
Olney 1986
; Sullivan et al. 1998
), and
interneurons tend to fire at higher frequencies than granule cells
(Buzsáki et al. 1983
; Ylinen et al.
1995
), indicating that their energy requirements may also be
higher (indeed, the number of mitochondria is higher in interneuronal
somata compared with granule cells) (Freund and Buzsáki
1996
). Therefore a combination of these factors (cell size,
expression of Ca2+-permeable glutamate receptors,
higher energy requirements) may all play a role in the cell specificity
of the posttraumatic depolarization in dentate interneurons, but not in
neighboring granule cells.
Mechanisms underlying the posttraumatic depolarization
The precise origin of the resting membrane potential is not fully
understood for most cell types (Jones 1989; Koh
et al. 1992
; Storm 1990
), including the dentate
granule cells and interneurons (e.g., it is not known why granule cells
rest ~15 mV more hyperpolarized compared with interneurons in the
same cell layer). This lack of detailed understanding of how the
resting membrane potential is generated becomes an especially serious
limitation when the modulation of the resting membrane potential needs
to be investigated. Our results indicated that ionotropic or
metabotropic glutamate receptors, TTX-sensitive voltage-gated
Na+ channels, GABAA
receptors, acetylcholine-related m-current blockade, a decreased inward
rectifier K+ current, or enhanced
Ih action are all unlikely to underlie
the post-FPI depolarization.
The nature of posttraumatic changes in
[K+]o after mild to
moderate fluid percussion injury is not well understood. Although some
reports suggested that the immediate posttraumatic increases in
[K+]o return to control
levels in <5 min (Katayama et al. 1990; similar results
were obtained from cortical contusion injury, Nilsson et al.
1993
; see also Obrenovitch and Urenjak 1997
), a
small (<0.5 mM), but significant increase in
[K+]o has recently been
reported in the CA3 region 2 days after FPI (D'Ambrosio et al.
1999
). Although the 0.5-mM increase in
[K+]o would not be
expected to cause more than a few millivolt change in
Vm, it is possible that post-FPI
increases in extracellular K+ may contribute to
the observed 10-mV depolarization in interneurons. In fact, disturbance
of the Na+/K+-ATPase would
be expected to result in an increased extracellular [K+], in addition to causing a decrease in the
resting membrane potential related to its electrogenicity.
Based on our data, the following points can be considered. First,
intracellular injection of ATP abolished the difference between the
resting membrane potential of interneurons in control and FPI animals,
indicating that manipulation of the interneurons alone (i.e., not the
glial cells) could restore the Vm in
these cells to control levels. This result would not be expected if the
only reason for the depolarization of interneurons were due to increased [K+]o
related to a deficit in glial K+ uptake
(D'Ambrosio et al. 1999; Janigro et al.
1997
; Newman 1995
). Second, if the
[K+]o increase were to
occur in the presence of unchanged
Na+/K+-ATPase function,
increased [K+]o should
increase the pump rate in interneurons, whereas we found a decreased,
and not increased, pump rate in interneurons. Third, granule cells were
not depolarized, which argues against a uniform increase in
[K+]o. However, localized
increases in [K+]o in the
extracellular space immediately surrounding interneurons may occur,
perhaps as a direct result of the decreased interneuronal Na+/K+-ATPase. Fourth,
oubain and strophantidin abolished the difference between in
interneuronal Vm of control and FPI
animals, indicating that the
Na+/K+-ATPase pump is
involved in the mechanism causing the difference between resting
membrane potential of control and injured interneurons.
However, it is possible that other factors contributing to the
generation of the resting membrane potentials may also have changed as
a result of the injury. For example, although no change in input
resistance could be detected with either small pulses in current clamp,
or with I-V plots scanning 100 mV (from 120 to
20 mV;
Fig. 4), it cannot be excluded that a decrease in a K+ conductance took place simultaneously with an
increase in a nonselective "leak" conductance (Koh et al.
1992
). The possible involvement of ATP-sensitive
K+ channels is an interesting question in this
regard, because these channels, similarly to the Na+-pump,
couple the cell's metabolic activity to its physiology. A recent study
showed that some hippocampal interneurons express high levels of
ATP-sensitive K+ channels (Zawar et al.
1999
). Whether a decrease in the activity of these channels
contributes to the interneuronal depolarization is not known; however,
a posttraumatic drop in intracellular ATP levels would be expected to
enhance the activity of ATP-sensitive K+ channels
(Ashcroft and Gribble 1998
), leading to
hyperpolarization (and not to depolarization, as shown here).
Na+/K+-ATPase after trauma
The Na+/K+-ATPase
plays a crucial role in living cells in maintaining ion gradients and
resting membrane potential (Glynn 1993). Indeed, a large
fraction (~40%) of the resting mitochondrial respiration is used to
fuel the Na+/K+-ATPase
(Astrup et al. 1981
; Erecinska and Silver
1989
; Whittam 1962
). The involvement of the
Na+/K+-ATPase has been
implicated in both neurotrauma and epilepsy (Fukuda and Prince
1992a
,b
; Haglund and Schwartzkroin
1990
; Haglund et al. 1985
;
Tavalin et al. 1995
, 1997
;
Thompson and Prince 1986
). Inhibition of the
Na+/K+-ATPase in principal
cells enhances excitability (Haglund and Schwartzkroin
1990
; McCarren and Alger 1987
), and biochemical studies have also indicated disturbed
Na+/K+-ATPase function in
epilepsy (Anderson et al. 1994
; Brines et al.
1995
; Delgado-Escueta and Horan 1980
;
Grisar 1984
; Grisar et al. 1992
;
Nagy et al. 1990
).
Our results suggest that the number of pump molecules is unlikely
to have changed after FPI, because the inclusion of intracellular pump
substrates (with EGTA) restored both the pump function and the resting
membrane potential. The reason for the apparent decrease in pump rate,
however, is less clear. The most straightforward interpretation of
these experiments is that trauma resulted in a decrease in the
intracellular availability of these pump substrates, thereby
compromising pump function. Out of the four pump substrates (Na+, K+, ATP, and water),
intracellular Na+ and water, and extracellular
K+, would be expected to possibly increase, and
not decrease, after injury. Intracellular Ca2+
may have played a role in inhibiting the pump; however,
ATPi alone was also able to abolish the
difference between the resting membrane potential of control and FPI
interneurons. The rate-limiting role of intracellular ATP for
Na+-pump function is known; e.g., the
Na+/K+-ATPase activity
reportedly declines when [ATP]i <0.4 mM
(Dagani and Erecinska 1987) (for a review on
[ATP]i as a rate-limiting factor for the
Na+/K+-ATPase, see
Lees 1991
). Although the interpretation of these experiments as indicating a decrease in ATP levels seems plausible, it
should be pointed out that it is also possible that the primary alteration is in some other pathway that regulates enzyme activity (e.g., regulatory kinase, other 2nd messengers), and that
down-regulation may have been overcome by enhanced substrate
availability. Although these alternatives will need to be investigated
in the future, the present data strongly suggest that the decrease in
pump function is not due to an irreversible loss of the enzyme from
interneurons as a result of the traumatic injury. It should also be
noted that the smaller blocking effect of the glycosides on (the
depolarized) FPI interneurons was not due to a voltage dependency of
the pump current, because, as seen in Fig. 6A, the pump
current (i.e., the difference between the I-V curves in Fig.
6A) was smaller in the FPI cells at all membrane potentials.
Secondary increases in
[Na+]i, perhaps also
related to the decreased pump function, may also take place. Although
we found no significant changes in action potentials in interneurons
from FPI animals, the action potential characteristics do not appear to
be overtly sensitive to partial/transient blockade of the
Na+/K+-ATPase
(Haglund and Schwartzkroin 1990; McCarren and
Alger 1987
; unpublished observation), and the
Na+/K+-ATPase activity is
significantly decreased, but not blocked, in interneurons after FPI
[the possibly preferential dendritic localization of the pump may also
influence the relationship between action potentials and
Na+/K+-ATPase activity
(Brines et al. 1995
)].
A final point on the pump function concerns the magnitude of the
contribution to the resting membrane potential by the
Na+/K+-ATPase. Although it
is widely believed that the pump contributes no more than a few
millivolts to the resting membrane potential in most cells, the actual
contribution strongly depends on the magnitude of the pump current and
the input resistance of the cells. In T lymphocytes of mice, 40-70 mV
of the Vm is reported to be produced
by the action of the
Na+/K+-ATPase
(Ishida and Chused 1993), and the resting membrane
potential of mast cells is set predominantly by the sodium pump
(Bronner et al. 1989
). In isolated vomeronasal neurons,
20-s application of oubain causes a 25-mV depolarization of
Vm, without a significant change in
input resistance (Trotier and Doving 1996
). Similar to
our data, in frog sympathetic neurons, the electrogenic pump was shown
to contribute to the resting membrane potential by ~10 mV
(Jones 1989
). Therefore it seems that the contribution
of the sodium pump to Vm can vary from
a few millivolts to ~10 mV, or perhaps even 10 s of millivolts,
depending on the cell type.
Functional implications
The data showed that the
Na+/K+-ATPase-related
depolarization of interneurons in the dentate gyrus enhanced the
spontaneous firing rates of interneurons, and resulted in a
posttraumatic augmentation of the frequency and amplitude of sIPSCs in
granule cells. The increased sIPSC frequency takes place in spite of
the posttraumatic decrease in the frequency of mIPSCs (most likely related to the loss of interneurons from the hilus) (Toth et al. 1997). Therefore the surviving interneurons, by increasing
their firing rates, can actually enhance the level of spontaneous
inhibition of granule cells, in spite of the decrease in the action
potential-independent GABA release.
Furthermore, the results also indicated that the posttraumatic
depolarization of interneurons can increase the efficacy of incoming
excitatory signals to discharge interneurons. Excitatory inputs to
hippocampal interneurons have recently been reported not to exhibit the
classical, direct forms of activity-dependent synaptic plasticity
(Maccaferri and McBain 1995; McBain and
Maccaferri 1997
; McMahon and Kauer 1997
),
perhaps as a result of the conspicuous absence of important
plasticity-related signaling pathways in hippocampal GABAergic cells
(Liu and Jones 1996
, 1997
; Sik et al. 1998
). The present data suggest that after neurotrauma,
interneurons can enhance the efficacy of their excitatory inputs;
however, this enhancement is qualitatively distinct from synaptic
plasticity associated with normal levels of activity, because the
posttraumatic depolarization nonselectively increases the ability of
depolarizing signals to discharge interneurons.
The increased tonic firing of interneurons may have disruptive and
deleterious effects for the normal functions of interneurons suggested
to involve precisely timed discharges of hippocampal interneuronal
networks, including their hypothesized participation in memory
processes (Buzsáki and Chrobak 1995;
Buzsáki et al. 1983
; Lisman and Idiart
1995
; Soltesz and Deschênes 1993
;
Traub et al. 1998
). Whether such disturbed interneuronal
firing patterns in the dentate gyrus described in this paper actually
contribute to the amnesia and memory problems that frequently occur
during the early posttraumatic period as part of the "postconcussion syndrome" (Binder 1986
; Lowenstein et al.
1992
) is not yet determined. In addition to memory problems,
posttraumatic seizures are also a well-known consequence of brain
injury, and the early (<1 wk) posttraumatic seizures have been
identified to be among the major risk factors for late seizures
(greater than weeks and months) (Asikainen et al.
1999
; Jennett 1975
; Jennett and
Lewin 1960
; Willmore 1997
). Pathophysiological
events during the first hours and days after head injury are
potentially significant determinants of the long-term outcome of
traumatic brain injury (e.g., Grady and Lam 1995
).
Recent results showed that the application of TTX onto the mechanically
injured neocortex is effective in preventing the long-term development
of posttraumatic hyperexcitability when applied shortly after injury,
but not when applied 11 days after trauma (Graber and Prince
1999
). Therefore it is conceivable that even relatively
transient posttraumatic changes (e.g., lasting a few days, such as the
interneuronal depolarization studied here) may have long-lasting
effects with regards to the long-term outcome.
Finally, it should be noted that the posttraumatic increase in
interneuronal firing may appear to be paradoxical, in light of
posttraumatic seizures. However, increased GABAergic synaptic transmission has been reported in several models of hyperexcitability, including kindling, drug-induced status epilepticus, and febrile seizures (Brooks-Kayal et al. 1998; Buhl et al.
1996
; Chen et al. 1999
; Nusser et al.
1998
; Otis et al. 1994
). Whether the enhanced inhibition in these paradigms is principally compensatory and antiepileptic, or actually contribute to neuronal synchronization of
the postsynaptic excitatory cells is not yet known (Cobb et al.
1995
; Walker and Kullmann 1999
). The
pathological plasticity of the GABAergic system exhibits a variety of
mechanisms, including increases in the number of postsynaptic
GABAA receptors (Buhl et al. 1996
;
Nusser et al. 1998
; Otis et al. 1994
),
alterations in subunit composition (Brooks-Kayal et al.
1998
; Buhl et al. 1996
), protein
kinase-A-dependent presynaptic enhancement of inhibition (Chen
et al. 1999
), and the selective,
Na+/K+-ATPase-related
depolarization of interneurons in neurotrauma (present study),
indicating that disease-specific therapeutic avenues may be explored in
the future.
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ACKNOWLEDGMENTS |
---|
We thank Dr. K. Kaila for comments on the manuscript and R. Zhu for expert technical assistance.
This work was financially supported by National Institute of Neurological Disorders and Stroke Grant NS-35916 and American Epilepsy Society Grant EFA-24106 to I. Soltesz. S. T. Ross was partially supported by National Institutes of Health Training Grant in Cellular and Molecular Neuroscience NS-07444.
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FOOTNOTES |
---|
Address reprint requests to I. Soltesz.
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 22 November 1999; accepted in final form 9 February 2000.
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REFERENCES |
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