Selective Depolarization of Interneurons in the Early Posttraumatic Dentate Gyrus: Involvement of the Na+/K+-ATPase

Stephen T. Ross and Ivan Soltesz

Department of Anatomy and Neurobiology, University of California, Irvine, California 92697-1280


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-alpha -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/tau r + Ae-t/tau d, where A is a constant, tau r and tau 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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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega , n = 12; vs. 347.2 ± 29.2 MOmega , 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 MOmega and 8.8 ± 0.7 MOmega ) 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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Fig. 1. Interneurons in the dentate granule cell layer after fluid percussion injury display intrinsic physiological properties similar to controls (with the exception of the resting membrane potential, see Fig. 2), and their overall dendritic and axonal morphologies appear intact. A and C: representative voltage responses (top traces) in response to intracellularly injected current pulses (bottom traces) obtained from the cells shown in B and D from age-matched, sham-operated control and fluid percussion injured animals, respectively. Note that the interneuron from the injured animal was capable of firing action potentials similarly to its control counterpart (see RESULTS for details on the numerical values for the intrinsic electrophysiological properties such as action potential characteristics and input resistance). B and D: camera lucida drawings of the biocytin-injected interneurons from the control and fluid percussion injury (FPI) animals, respectively, are illustrated (these 2 representative examples are from a pool of n = 7 and n = 8 interneurons filled with biocytin in control and FPI animals). The similar morphological appearance of the interneurons from the control and FPI animals indicate that the interneuronal membrane integrity was preserved after the impact (indeed, all of these interneurons in the granule cell layer have been shown to survive the impact, as demonstrated using substance-P receptor immunostaining) (see Toth et al. 1997). GL, granule cell layer; Mol, molecular layer of the dentate gyrus.

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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Fig. 2. Posttraumatic enhancement of spontaneous firing and depolarization in interneurons. A: the resting membrane potential of an interneuron situated in the dentate granule cell layer (similar to those illustrated in the previous figure) is shown to be depolarized after FPI, compared with its control counterpart. Note that when the interneuron from the FPI animal was hyperpolarized by injection of current through the pipette from its resting membrane potential to -60 mV, no spontaneous firing could be seen, indicating that the interneuron was not firing simply due to a poorer recording condition or direct injury from the electrode. B: cell-attached recordings are shown from interneurons from a control and experimental rat. Note the enhanced spontaneous firing after trauma (for numerical values, see RESULTS). C: summary of data obtained from interneurons in control and FPI animals, similar to those shown in A and in the previous figure. Note that the figure also indicates that the neighboring granule cells were not depolarized after fluid percussion injury [note that depolarization is downward on the y-axis; these recordings were carried out in control artificial cerebrospinal fluid (ACSF), 1-4 h after impact]. As described in the text, similar results were obtained with gramicidin-based perforated patch-clamp recordings.

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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Fig. 3. Lack of posttraumatic modification in spontaneous excitatory postsynaptic currents (sEPSCs) in interneurons. A: representative sEPSC recordings are shown from interneurons from an age-matched, sham-operated control and an FPI animal. B: averages of sEPSCs show the lack of a difference between the kinetics of the sEPSCs in interneurons from control and experimental rats (the parameters for the 2 fitted curves are as follows: control: A = -30.6 pA; tau r = 0.36 ms; tau d = 1.9 ms; FPI: A = -34.9 pA; tau r = 0.27 ms; tau d = 2.1 ms; see equation in METHODS). C: summary data are presented showing the lack of posttraumatic alterations in sEPSC frequency, amplitude, or kinetics in interneurons after FPI.

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), alpha -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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Fig. 4. Lack of a posttraumatic change in input resistance and lack of down-regulation of the classical inward rectifier. A: current-voltage (I-V) curves are shown, constructed from experiments similar to the ones shown in the insets, for control and FPI interneurons. The cells were held at -60 mV in voltage clamp, in the presence of 2-amino-5-phosphovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), tetrodotoxin (TTX), and bicuculline, as well as ZD-7288 to block h-channels ("control" medium in B and C). The voltage step commands (every 5 mV) are not shown; the measurements of the steady-state currents plotted in A-C were taken just before the end of the voltage command steps. Note that, although it is difficult to observe due to the large scale on the x-axis, the FPI curve is shifted to the right, in agreement with the depolarized Vm of the interneurons after injury (control: -50.5 ± 2.1 mV; FPI: -43.7 ± 4.6 mV; these values are before Cs+ and are very similar to the values determined in a larger group of cells recorded under the same conditions in a different set of experiments: control: -51.5 ± 2.1, n = 8; FPI: -43.6 ± 2.3, n = 8; see RESULTS; as mentioned in RESULTS, the Vm of interneurons of both control and FPI animals was depolarized in the prolonged presence of these drugs with ZD-7288 compared with the same drugs without ZD-7288; the reason for this effect is not known). B and C: when the same cells (n = 4 in both) were switched to the same medium containing 1 mM Cs+, no blockade of the currents could be seen at hyperpolarized potential (unlike what was described in hippocampal glial cells) (D'Ambrosio et al. 1999). Therefore inward rectifying K+ channels (which can be blocked by extracellular Cs+) are not strongly expressed by these interneurons, and, consequently, they are unlikely to be down-regulated and cause the post-FPI depolarization.

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).



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Fig. 5. Posttraumatic interneuronal depolarization and its relationship to the Na+/K+-ATPase. A and B: when the external medium (see below) was switched (arrowheads in A) to a medium containing the Na+/K+-ATPase blocker oubain (30 µM) for 5 min, the difference between the resting membrane potentials of the interneurons from age-matched, sham-operated controls and FPI animals ("preoubain" in B) was abolished. In other words, oubain caused a larger steady-state depolarization in interneurons from control than from FPI animals. Note that these experiments also indicate that activation of TTX-sensitive voltage-gated Na+-channels, N-methyl-D-aspartate (NMDA), alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainate receptors, or GABAA receptor channels could not account for the difference in resting membrane potentials between interneurons from control and FPI animals, because these experiments were carried out in the presence of TTX (1 µM), APV (10 µM), CNQX (5 µM), and bicuculline (10 µM). The pair of bars on the right shows that inclusion of 4 mM ATP in the recording patch pipette abolished the difference between the interneuronal resting membrane potentials of the control and FPI groups (in the presence of TTX, APV, CNQX, and bicuculline). Similar results could also be obtained when 20 mM Na+, 0.2 mM EGTA, and 4 mM ATP were included in the recording patch pipette (see RESULTS). These data are consistent with the involvement of the Na+/K+-ATPase in the interneuronal depolarization.

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].



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Fig. 6. Brief application of the Na+/K+-ATPase blocker strophanthidin reveals a difference between the pump currents in interneurons from control and FPI animals. A: I-V relationship from 2 representative interneurons from control and FPI animals. Note that a short (20 s) application of the lower-potency strophanthidin (30 µM) caused a larger shift in the I-V curve in interneurons from control vs. experimental animals (in TTX, APV, CNQX, bicuculline, and ZD-7288). These data indicate that the differential depolarizing effect of oubain (shown in the previous figure) was not due to a differential collapse of the ion gradients (which, in any case, is extremely unlikely to occur in TTX and in whole cell patch-clamp mode) (see Senatorov et al. 1997). B: summary data of experiments similar to those shown in A illustrate that the strophanthidin-sensitive current density is smaller in FPI animals. C: in contrast, when the forward pump rate was enhanced by the inclusion of 20 mM Na+, 0.2 mM EGTA, and 4 mM ATP in the recording pipette (in addition to K-gluconate, and in the presence of TTX, APV, CNQX, and bicuculline), and a higher concentration of strophanthidin (100 µM) was applied for a longer period of time (5 min) to achieve a complete block of the pump, the difference between the strophanthidin-sensitive current in interneurons from control and FPI animals was abolished. These results are in agreement with the data shown in Fig. 4B (bars on the right) indicating that inclusion of ATP in the pipette (with or without 20 mM Na+ and 0.2 mM EGTA, see RESULTS) can abolish the difference between the membrane potential of interneurons in the granule cell layer of the traumatized vs. control dentate gyrus.

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.



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Fig. 7. Posttraumatic depolarizing shift enhances the efficacy of excitatory postsynaptic potentials (EPSPs) to discharge interneurons. A: representative EPSPs, evoked by stimulation of the perforant path (in the presence of 10 µM bicuculline), are shown from interneurons from a control and experimental animal, recorded at the resting membrane potential of the 2 cells indicated on the left. Stimulation intensity for the top traces: 0.4 mA; bottom traces: 1.0 mA. B: summary of data obtained from experiments similar to those shown in A. Note the lower stimulation threshold necessary to evoke firing in the interneurons from the head-injured animals. C and D: "artificial EPSPs" also show enhanced efficacy to evoke interneuronal firing after trauma. Specifically, in the same group of cells as those in A and B, depolarizing current pulses of increasing amplitude (duration: 10 ms; amplitudes: 20-180 pA) were injected from the resting membrane potential of the interneurons (indicated on the left in C). Note that these artificial EPSPs evoked firing at lower depolarizing current amplitudes in the interneurons from the FPI animals. These data show that the posttraumatic depolarization can indeed enhance the ability of incoming excitatory signals to discharge the 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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Fig. 8. Frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells of the dentate gyrus is enhanced after fluid percussion head trauma. A: representative traces of sIPSCs are shown from dentate granule cells (recorded at 0 mV, with Cs-gluconate-containing patch pipettes, in control ACSF), in age-matched, sham-operated control and fluid percussion-injured animals. B: cumulative probability plots of sIPSC inter-event intervals show that the frequency of the sIPSCs is significantly higher (i.e., the inter-event interval is lower; median inter-event interval in control: 101.3 ms; in FPI: 74.6 ms) in granule cells from fluid percussion injured animals compared with controls (the plots were constructed by pooling 150 sIPSCs from each of the n = 5 cells in both groups; for cumulative probability distributions, Kolmogorov-Smirnov test was used) (e.g., Chen et al. 1999). C: cumulative probability plots of the sIPSCs indicate a significantly enhanced sIPSC amplitude after trauma (median sIPSC amplitude in control: 24.8 pA; in FPI: 33.2 pA). D: normalized sIPSCs (150 sIPSCs from each of the n = 5 cells in both groups; the parameters for the 2 fitted curves are: control: tau r = 0.46 ms; tau d = 8.7 ms; FPI: tau r = 0.46 ms; tau d = 9.1 ms; see equation in METHODS) from interneurons from control and FPI animals are shown to illustrate the similar kinetics of the events.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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.


    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.


    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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ABSTRACT
INTRODUCTION
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DISCUSSION
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