Increased Excitability and Inward Rectification in Layer V Cortical Pyramidal Neurons in the Epileptic Mutant Mouse Stargazer

Eric Di Pasquale, Karl D. Keegan, and Jeffrey L. Noebels

Developmental Neurogenetics Laboratory, Department of Neurology, Section of Neurophysiology, Baylor College of Medicine, Houston, Texas 77030

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Di Pasquale, Eric, Karl D. Keegan, and Jeffrey L. Noebels. Increased excitability and inward rectification in layer V cortical pyramidal neurons in the epileptic mutant mouse stargazer. J. Neurophysiol. 77: 621-631, 1997. The excitability of layer V cortical pyramidal neurons was studied in vitro in the single-locus mutant mouse stargazer (stg), a genetic model of spike wave epilepsy. Field recordings in neocortical slices from mutant mice bathed in artificial cerebrospinal fluid revealed spontaneous synchronous network discharges that were never present in wild-type slices. Intracellular and whole cell recordings from stg/stg neurons in deep layers showed spontaneous giant depolarizing excitatory postsynaptic potentials generating bursts of action potentials, and a 78% reduction in the afterburst hyperpolarization. Whole cell recordings revealed gene-linked differences in active membrane properties in two types of regular spiking neurons. Single action potential rise and decay times were reduced, and the rheobase current was decreased by 68% in mutant cells. Plots of spike frequency-current relationships revealed that the gain of this relation was augmented by 29% in the mutant. Comparisons of visually identified pyramidal neuron firing properties in both genotypes revealed no difference in single action potential afterhyperpolarization. Voltage-clamp recordings showed an approximately threefold amplitude increase in a cesium-sensitive inward rectifier. No cell density or soma size differences were observed in the layer V pyramidal neuron population between the two genotypes. These results demonstrate an autonomous increase in cortical network excitability in a genetic epilepsy model. This defect could lower the threshold for aberrant thalamocortical spike wave oscillations in vivo, and may contribute to the mechanism of one form of inherited absence epilepsy.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The spike and wave pattern of cortical synchronization is a distinctive electrographic feature of generalized absence seizures, a major form of inherited epilepsy. Current hypotheses regarding the generation of this aberrant synchronous discharge center on abnormal synaptic control of intrinsic rhythmic membrane firing properties underlying normal thalamocortical oscillations (Steriade et al. 1993). The reciprocal connections between pyramidal neurons in deeper layers of the neocortex, cortical relay neurons in the thalamus, and their respective interneurons including those in the reticular thalamic nuclei are believed to form the elementary pacemaking circuit altered by the epileptogenic defect.

Several general possibilities, expressed at one or more sites within the cortex or thalamus, may contribute to instability within this oscillating loop. Aberrant rhythmic behavior could be favored by 1) intrinsic excitability shifts,including enhanced persistent inward sodium currents or low-threshold calcium currents that control membrane rhythmicity and postinhibitory rebound excitation in the principal cells (Alonso and Llinas 1989; Coulter et al. 1989; Guyon et al. 1993; Huguenard and Prince 1992, 1994; Silva et al. 1991; Steriade et al. 1990; Tsakiridou et al. 1995); 2) functional alterations in the strength of recurrent inhibitory synaptic inputs, in particular those of the reticulothalamic group (Crunelli and Leresch 1991; Hosford et al. 1992; Liu et al. 1991; but see Knight and Bowery 1992); or 3) abnormal extrinsic control of the thalamocortical network from ascending afferent pathways by neuromodulatory actions of transmitters such as noradrenaline and acetylcholine (Buzsaki et al. 1988; McCormick 1992; Noebels 1984). The existence of multiple spike wave gene loci (Noebels 1995) and the heterogeneity of the intervening mechanisms responsible for the spike wave seizure trait (Qiao and Noebels 1991) predict that any one or more of these distinct sites may be a potential target of different gene mutations.

The stargazer mutant mouse (stg, chromosome 15, recessive) shows generalized nonconvulsive spike wave seizures with behavioral arrest that are blocked by ethosuximide and resemble the clinical phenotype of generalized absence epilepsy (Noebels et al. 1990). The seizures consist of 1- to 10-s episodes of bilaterally synchronous and symmetric spike wave activity (6 per s) in cortex and thalamus. The seizures begin developmentally on postnatal day (P) 16-18, and occur at a high rate (~125 per h) during the waking state. To determine whether the stg mutant gene error directly alters the excitability of thalamocortical networks, and to localize where in this circuit the functional defect may be expressed, we examined the behavior of stg cortical neurons in hemisected brain slices with the use of field, intracellular, and whole cell current-clamp recordings. A region of parietal cortex showing maximal electroencephalographic (EEG) seizure discharge amplitude was examined. Layer V neurons were selected because of their reported role in generating synchronous cortical network oscillations (Silva et al. 1991). Here we describe the presence of cellular excitability defects in the isolated mutant neocortex, including an increased inward rectifier conductance, that may contribute to inherited spike wave epileptogenesis.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Slice recording methods

Naive adult (2-3 mo) and young (P18-20) homozygous stg (n = 28, B6C3Fe-stg/stg) and wild-type (n = 52, B6C3Fe-+/+) mice were killed by cervical dislocation and the brain was placed in cold (4°C) oxygenated artificial cerebrospinal fluid (aCSF) composed of (in mM) 124 NaCl, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 dextrose, pH adjusted to 7.4 with NaOH. In Mg2+-free experiments, MgSO4 was omitted. Serial oblique coronal slices (350-400 µm) oriented to preserve thalamocortical connectivity (Agmon and Connors 1991) were cut (Vibroslicer, Campden Instruments), incubated for >= 1 h at 25°C, and transferred to an interface-type recording chamber maintained at 32 ± 1°C. Standard electrophysiological techniques were used. Electrodes were pulled with the use of a horizontal puller (Flaming/Brown P-87) and filled with 3 M potassium acetate and 0.05 M KCl (70-110 MOmega ) for intracellular recording. NaCl (2 M) (5-10 MOmega ) was used to fill pipettes for field recording. To analyze layer V pyramidal cell properties, blind and visualized whole cell current-clamp recordings were obtained from coronal slices of temporoparietal cortex at the level of the habenular nucleus. These slices did not include thalamocortical connectivity. The procedure described previously was followed, except that the slices were submerged in the bathing solution.

Blind whole cell recordings

Most of the intrinsic properties were studied with the use of the blind whole cell recording technique (Blanton et al. 1989) (9 mice from each genotype). Electrodes (4-7 MOmega ) were pulled with the use of a two-step program and filled with (in mM) 130 potassium gluconate, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 MgCl2, 1 CaCl2, and 2 Na2ATP, pH adjusted to 7.4 with KOH. The microelectrodes were visually positioned under oblique lighting. Offset between the reference electrode and the pipette was zeroed when touching the recording chamber superfusate. Output bandwidth was 30 kHz for current-clamp experiments. The bridge was balanced after membrane breakthrough and monitored throughout the recording. The indifferent electrode was a Ag/AgCl wire. Liquid junction potentials between the perfusion and pipette solutions ranged from 9 to 10 mV. Resting membrane potential (Vr) values given in the text were corrected. Series resistances were typically <25 MOmega , and were compensated with the use of the bridge balance. Neurons were accepted for analysis if they had a gigaohm seal (typically 2-3 GOmega ), a stable Vr, and an overshooting action potential. Input membrane resistance was estimated from a 500-ms, 100-pA hyperpolarizing current pulse. Rise time, decay time, and half-width were measured for each of five single action potentials evoked by brief depolarizing current pulses (2-5 ms) in each cell. These values were then averaged for comparison between the two genotypes. For action potentials exhibiting a depolarizing afterpotential, the decay time measurements were performed at the end of the fast repolarizing phase before the depolarizing afterpotential was evoked.

The membrane responses to a series of 1-s negative current pulses (in 100-pA increments) were recorded. In the mutant cells, there was evidence of depolarizing "sag" during large hyperpolarizing responses. The voltage response was measured at the peak (150 ms from the onset of the pulse) and at the end of the 1 s hyperpolarizing current pulse. The intensity of the current pulse (1 s in duration, 0.5 Hz) that generated an action potential in 50% of the current injections was defined as rheobase. To study repetitive firing properties, depolarizing current pulses (1 s in duration, 0.16 Hz) were applied in 10- to 20-pA increments from 90 pA to the current that produced a maximum of 20 spikes/s. For each depolarizing step, the number of evoked action potentials was counted and plotted against current amplitude. The frequency-current (f-I) relationships were then graphed (Sigma Plot, Jandel Scientific). The mean firing frequency (Hz/nA) was calculated from the slope of a regression line obtained from each f-I plot. Membrane time constants were calculated from the membrane response to a 1-s, 100-pA hyperpolarizing pulse. An exponential curve fitting algorithm (pClamp, Axon Instruments) was applied to the first 50 ms of the response. In all instances, the best fit was achieved with the use of a single exponential.

Visualized whole cell recordings

To study single action potential afterhyperpolarization (AHP) and the inward rectifier conductance, layer V pyramidal neurons were individually identified by their morphological characteristics under video microscopy (Axioskop, Zeiss; Dage/Hamamatsu C2400 camera) as described by Stuart et al. (1993). Slices were prepared from P18 to P20 according to the procedure described above. Experiments were conducted at room temperature. Pipettes were pulled with the use of a vertical puller and polished (Narishige, PP-83 and Narishige microforge), then filled with (in mM) 145 KMeSO4, 2 Na2ATP, 2 HEPES, and 0.1 EGTA, to minimize alterations of the Ca2+-activated AHP (adapted from Zhang et al. 1994). In all experiments signals were amplified (Axopatch 1D, Axon Instruments), digitized (Neurocorder, Neurodata Instruments), and stored on tape for subsequent analysis (Digidata 1200/pClamp 6.1, Axon Instruments). A stimulator (Winston Electronics) was used for current-clamp protocols. Voltage-clamp protocols were run with the use of pClamp 6. Signals were filtered (5 kHz, -3 dB) and the series resistance (15-25 MOmega ) was compensated by >= 50%. The inward rectifier current amplitude was measured by subtracting instantaneous current from steady-state current. Leak was not subtracted. Cesium chloride (CsCl, 3 mM, Sigma) was used to block the inward rectifier. During CsCl superfusion, Vr was monitored, and the effect of CsCl on resting values was taken after stabilization. Neuron capacitance was estimated by compensation of the whole cell capacitance after breakthrough. All values are presented as means ± SE. For each parameter the statistical differences between both genotypes were tested with the use of the nonparametric Mann-Whitney U test (Sigma Stat, Jandel Scientific). Statistical significance was obtained for P < 0.05.

Immunostaining and morphological analysis

Deep layer pyramidal neurons from 2-mo-old stg and wild-type cortex were visualized with the use of immunocytochemistry with an antibody raised against potassium channel Kv2.1 (Shab-related subfamily 2, KCNB1) antigen that shows prominent staining of large pyramidal cell somas and apical dendrites (Maletic-Savatic et al. 1995). Sections (30 µm thick) were stained following the procedure described in Maletic-Savatic et al. (1995), and were visualized by light microscopy at ×40 magnification (Zeiss, Axioskop). The surface area of deep layer cortical pyramidal somas was measured by outlining the circumference in digitized images (IMAGE TOOL, UTSA). Statistical analysis was performed with the use of the Student's t-test.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Spontaneous synchronous discharges in stg neocortex in vitro

Extracellular recordings in aCSF revealed the presence of very low-frequency (<1 per min) abnormal spontaneous network discharges with an irregular rhythm in all layers of mutant thalamocortical slices (12 of 16 mice, Fig. 1A) but not in wild-type slices (0 of 37 mice). Field potentials in the stg/stg cortex had amplitudes ranging from 0.1 to 1 mV, with larger amplitudes measured in the deeper cortical layers. Discharge durations ranged from 160 to 270 ms. In slices from both genotypes, no spontaneous network discharge activity could be detected in thalamic nuclei, or within hippocampal granule cell and pyramidal cell layers. The cortical discharges in the mutant slices arose independently of thalamic inputs, because they persisted after surgical isolation of the cortex from the remainder of the slice.


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FIG. 1. Spontaneous discharges in stargazer (stg) mutant neocortex bathed in standard and Mg2+-free artificial cerebrospinal fluid (aCSF). A: discontinuous traces show examples of field recordings from layer V of neocortex in an adult stg/stg thalamocortical slice in standard aCSF. The network discharges ranged from 0.1-1 mV in amplitude and occurred irregularly at a frequency of 0.1-1 per min. No discharges were ever seen in wild-type (+/+) slices in aCSF. B: intracellular recording from layer V neuron showing a representative giant excitatory postsynaptic potential in aCSF. Note the lack of a prominent afterburst hyperpolarization. C and D: intracellular recordings of giant excitatory postsynaptic potentials during network discharges in 0-Mg2+ bath solution reveals reduction of afterburst hyperpolarization in mutant neurons (D) compared with the wild type (C). Action potentials are truncated in B-D. Calibration: 0.5 mV (A); 10 mV (B-D).

Intracellular recordings were obtained from 22 wild-type and 13 mutant neurons located in, or bordering, neocortical layer V. These neurons showed electrophysiological properties similar to those described for regular-spiking neurons in these layers (Connors et al. 1982; McCormick et al. 1985). Spontaneous firing of the neurons was not observed on or immediately after impalement. Mean values for Vr (+/+ -68 ± 7 mV, n = 19; stg/stg -70 ± 8 mV, n = 13) and input resistance (Rin) (+/+ 62 ± 24 MOmega , n = 14; stg/stg 61 ± 18 MOmega , n = 13) did not significantly differ between wild-type and mutant neurons. In the mutant, intracellular recordings showed a giant depolarization during the spontaneous field discharge (Fig. 1B). This depolarization had a mean amplitude of 22.8 ± 9.1 mV (n = 13) and mean duration of 281 ± 126 ms (n = 13; range 120-460 ms). During simultaneous intra- and extracellular recordings, no giant excitatory postsynaptic potentials were observed to occur other than those arising concurrently with the spontaneous field potential discharge (data not shown).

Mutant cortical network excitability in magnesium-free aCSF

The rate of spontaneous abnormal discharges in mutant cortical neurons in aCSF was low (0.1-1 per min), thus making their bursting characteristics difficult to study. To analyze firing properties in mutant neurons during synchronous network bursting, we perfused the slices with a Mg2+-free aCSF. This solution induces frequent (2 per min) repetitive discharges without impairing synaptic inhibition, in part from the removal of Mg2+ blockade of the N-methyl-D-aspartate-receptor-gated ion channel (Nowak et al. 1984; Sutor and Hablitz 1989).

In 0 mM extracellular Mg2+ concentration, periodic field discharges and associated giant excitatory postsynaptic potentials were observed in the cortex of both +/+ and stg/stg slices. In field recordings, the most obvious difference between the two genotypes was a slightly less regular discharge rhythm in the mutant cortex associated with more pronounced afterdischarge activity. In intracellular recordings, there were no significant differences between the depolarizing component of the giant excitatory postsynaptic potentials recorded in +/+ (amplitude 31.1 ± 9 mV, duration 389 ± 114 ms, n = 14) and stg/stg (amplitude 21.9 ± 6.3 mV, duration 440 ± 158 ms, n = 12) neurons. However, the zero-Mg2+-induced afterburst hyperpolarization in the mutant neurons was significantly decreased by 77%, from 6.9 ± 1.1 mV to 1.5 ± 0.7 mV in +/+ and stg/stg neurons, respectively (P < 0.001) (Fig. 1, C and D, Table 1).

 
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TABLE 1. Comparison of electrophysiological properties in adult +/+ and stg/stg in deep layer cortical neurons

Cortical deep layer cell subtypes in stg neocortex defined by whole cell recordings

To determine whether the spontaneous discharges and reduced AHP might be due to intrinsic membrane alterations, we used whole cell recordings to analyze firing properties in deep cortical neurons. Whole cell recordings were obtained from 32 wild-type (9 adult and 6 P18-20 mice, from 1-4 neurons per mouse) and 33 mutant neurons (9 adult and 3 P18-20 mice, from 1-6 neurons per mouse) (with both filling solutions). Mean values for Vr and Rin were not statistically different between the two genotypes (see Table 1).

Cortical neurons were classified by the characteristics of their repetitive firing patterns during injection of depolarizing 1-s intracellular current pulses (potassium gluconate/10 mM EGTA filling solution). Two distinct groups were identified in both genotypes, and (following the nomenclature of Chagnac-Amitai and Connors 1995) were referred to as RS1 cells (+/+, 4 of 20; stg/stg, 5 of 22) and RS2 cells (+/+, 16 of 20; stg/stg, 17 of 22). No obvious differences in these firing patterns were found between the two genotypes. No cells showing intrinsic bursting properties were identified in either mutant or wild-type slices. In physiological aCSF, four stg/stg neurons (from 4 different brains) obtained from coronal cortical slices exhibited intermittent spontaneous discharge activity. Of these cells, one was classified as RS1 and three were classified as RS2 cells. Thus both cell subtypes are apparently activated and participate in the aberrant spontaneous network discharges.

Intrinsic membrane properties in stg/stg cortical neurons

Multiple characteristics of single action potentials were altered in the mutant and are summarized in Table 1. These include significant decreases in rise time (15.0%), decay time (13.5%), and half-width (12.3%) compared with the wild type. Action potential narrowing in stg/stg neurons is illustrated in Fig. 2, A-C. No statistical differences were found in the amplitude of single action potential overshoot. Rheobase current for stg/stg neurons was significantly decreased by 68% (histogram in Fig. 2F), from 80 ± 17 pA (+/+, Fig. 2D) to 25 ± 8 pA (stg/stg, Fig. 2E, Table 1). Despite this striking decrease in firing threshold, no significant differences in Vr, Rin, or membrane time constant were found between the pooled values of these parameters in the two genotypes (summarized in Table 1). No correlations were found when comparing single action potential rise time, decay time, and half-width with the membrane Rin.


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FIG. 2. Altered action potential parameters in stg/stg deep cortical neurons. Action potential rise time, decay time, and half-width are decreased in stg/stg deep cortical neurons. A-C: spikes from wild-type (A) and mutant (B) neurons are triggered by a 2-ms depolarizing current pulse; fast sweeps of A and B are superimposed in C. D-F: spike firing threshold for a 1-s depolarizing current injection (I rheobase) in +/+ neuron (D) is significantly lowered by an average of 68% in mutant neurons (E). Histogram in F: data (mean ± SE) from both genotypes.

The relationship between firing rate and injected current (f-I plot) in stg/stg and +/+ neurons is shown in Fig. 3. The mean interspike interval was characterized by a single slope. The slopes were averaged in each genotype (+/+, 154.4 ± 24.5 Hz/nA, n = 10; stg/stg, 217.7 ± 24.1 Hz/nA, n = 11, Fig. 3A) and statistical comparison of the mean slope values revealed a significant increase of 29% in stg/stg cells (Fig. 3B, Table 1).


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FIG. 3. Increased gain in repetitive firing in stg/stg cortical neurons. A: plots of frequency-current (f-I) relationship in 2 representative cortical neurons from mutant (bullet ) and wild-type (black-square) slices. Straight lines: regression lines drawn by least-squares fit. B: histogram summarizes the mean gain (Hz/nA) of the firing patterns obtained by averaging the calculated slopes of the regression lines for firing frequency (mean ± SE). The mean gain in wild-type neurons was 154 Hz/nA, compared with 217 Hz/nA in mutant neurons. Mutant neurons required ~30% less injected current than wild-type cells to attain a sustained discharge.

Afterpotentials following short depolarizations in visually identified pyramidal neurons

Because the observed decrement in network burst-induced AHP (Fig. 1D), and the increased f-I relationship (Fig. 3) might result in part from a membrane repolarization defect in mutant neurons, we examined afterpotentials in stg/stg and +/+ cells. These data were obtained only from visually identified deep cortical pyramidal neurons. Somatic recordings were obtained with the use of a KMeSO4/0.1 mM EGTA filling solution. Short current pulses (2-5 ms, +/+, n = 7; stg/stg, n = 6) were used to evoke single action potentials. No qualitative differences in the repolarization of the action potential were found between the two genotypes. Thus most of the action potentials exhibited a depolarizing afterpotential on the falling phase, the amplitude of which could be modified by varying the membrane potential (Fig. 4, A and B). The depolarizing afterpotential was always followed by a slow decay toward resting potential. No fast AHP was observed, and slow AHPs ranged from 0 to 3 mV in both genotypes without significant amplitude differences.


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FIG. 4. Afterhyperpolarizations (AHPs) following short depolarizations in identified stg deep layer pyramidal neurons. The stg pyramidal cell shown in A and B was recorded under visualized control with pipettes containing KMeSO4 and 0.1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). A: neuron was depolarized from rest (-65 mV) with a 3-ms depolarizing step (510 pA, top trace in A). Note the lack of fast AHP when the action potential is evoked from rest. B: same cell is depolarized to -56 mV with the use of direct current injection, and the action potential is evoked with a 300-pA depolarizing current step (top trace). The AHP is enhanced when the action potential is triggered from a depolarized membrane potential. Similar single action potential AHP behavior was observed in +/+.

Increased depolarizing sag and inward rectifier current in stg pyramidal neurons

Hyperpolarizing pulses 1 s in duration (in 100-pA increments) were used to further analyze membrane responses at potentials more negative than Vr. In current-clamp mode, 10 cells were recorded with the use of the blind patch-clamp technique (+/+, n = 5; stg/stg, n = 5; potassium gluconate/10 mM EGTA) and 8 were studied under visual control (+/+, n = 4; stg/stg, n = 4; KMeSO4/0.1 mM EGTA). Electrotonic responses (depolarizing sag) were observed at potentials more negative than rest in both genotypes (Fig. 5, A and D). The membrane potential reached an initial peak value and then decayed slowly toward resting potential until the end of the hyperpolarizing pulse. When the peak value was subtracted from the end value at very hyperpolarized potentials (+/+, -115 ± 9 mV; stg/stg, -114 ± 2 mV), the membrane potential difference was significantly increased in the mutant cortex, in both nonvisualized and visually identified neurons (Tables 1 and 2). In voltage-clamp mode, 13 visually identified pyramidal neurons were recorded(+/+, n = 7; stg/stg, n = 6; KMeSO4/0.1 mM EGTA). All cells were held at -70 mV and hyperpolarized during a 2-s pulse with 10-mV incremented steps (Fig. 5, B and E). An apparent increase in a slowly activating inward current (+/+, 76.8 ± 14.4 pA; stg/stg, 221 ± 44.2 pA) was measured (see METHODS), leading to a strong rectification of the current-voltage curve in stg/stg neurons at hyperpolarized potentials (Fig. 5, C and F, Table 2). When normalized to cell capacitance, a significant increase in current density was observed (Table 2). This current was reversibly blocked within 15 min of bath perfusion with 3 mM CsCl (not shown, n = 3 each genotype) and thus referred to here as the cesium-sensitive hyperpolarization-activated current. Current traces were well fitted with one exponential (Fig. 6A), the activation time constant appeared to be voltage dependent (Fig. 6B), and no significant differences were observed between values obtained from +/+ and stg/stg neurons (Table 2). The voltage dependence of the activation was determined from tail current relaxations that followed a family of hyperpolarizing voltage steps (Fig. 5, B and E). Normalized peak tail current amplitudes in both genotypes were well fitted with a single Boltzman function (Fig. 6C). In young mice (P18-20), half-activation did not differ between the two genotypes (Table 2), with a slope factor of 5.2 ± 0.5. Thus, at -80 mV [a potential within the voltage range of the cellular repolarization (AHP)], no significant difference in the average percentage of activation was measured (+/+, 15.6 ± 1.4%; stg/stg, 11.7 ± 3.3%; P > 0.3). When CsCl (3 mM) was bath applied in current-clamp mode at rest, a hyperpolarization of 2.9 ± 0.7 mV of the Vr was measured (+/+, n = 3; stg/stg, n = 2) without differences between the two genotypes.


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FIG. 5. Depolarizing sag and inward rectifier modifications in visually identified stg cortical layer V pyramidal neurons. A-C and D-F: current- and voltage-clamp recordings from wild-type and mutant pyramidal neurons, respectively. Cells were held at -70 mV in both clamp modes. Membrane responses to the application of an incremental series of 1-s hyperpolarizing steps in a +/+ (A) and stg/stg (D) layer V cortical neuron. Note the increased depolarizing sag in the stg/stg neuron. B and E: recordings from the same cells as in A and D in voltage-clamp mode with application of 10-mV incremented hyperpolarizing steps. Note the increased inward current in the stg/stg neuron. C and F: current-voltage plots of the cells in B and E, respectively. The rectification of current-voltage curves from each genotype is shown in comparisons of the instantaneous (square ) vs. the steady-state current (triangle ) in the current-voltage plot. Leakage current was subtracted from total membrane current records before plotting.

 
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TABLE 2. Comparison of membrane properties in visually identified layer V cortical pyramidal neurons in developing +/+ and stg/stg at postnatal day 18-20


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FIG. 6. Inward rectifier characteristics in a representative stg layer V cortical neuron. Same cell as shown in Fig. 5. A: current traces were fit with a single exponential. B: inward rectifier time constant of activation obtained from the exponential fit in A was voltage dependent. C: tail currents (shown in Fig. 5E) were used to generate the activation curve (V1/2 = -94 mV, slope = 5.2 mV) for this cell. The inward rectifier amplitude was normalized to the peak amplitude. Apart from the amplitude, no differences in inward rectifier characteristics were observed between the 2 genotypes.

Normal morphological appearance of stg deep layer cortical neurons

To determine whether any obvious morphological alterations such as soma size might contribute to the excitability differences measured in the mutant neurons we sampled, cortical sections corresponding to the regions analyzed were stained with an antibody for the delayed-rectifier-type K+ channel Kv2.1 and compared between genotypes. Immunocytochemistry with this antibody clearly outlined the soma and apical dendrite over an extensive distance in the deep pyramidal neurons. No cell density or soma size differences were observed in this cell population between the two genotypes (Fig. 7). Calculated mean soma surface area was 263.0 ± 34.0 µm2 in +/+ mice (n = 30) and 253.2 ± 47.1 µm2 in stg/stg mice (n = 30, P = 0.36). In addition, the intensity of the Kv2.1 antibody staining used as a marker did not reveal any discernible difference in the density of this particular K+ channel subtype on these cells, and its distribution throughout the remainder of the brain was not visibly altered between the two genotypes.


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FIG. 7. Morphological appearance of stg deep layer cortical pyramidal neurons. Photomicrographs (×40 magnification) of +/+ and stg/stg pyramidal neurons in layer V stained immunocytochemically with anti-Kv2.1 antibody. Morphometric analysis showed no genotypic differences in pyramidal cell soma surface area within these layers.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Here we report that synchronous network discharges arise spontaneously in isolated neocortical slices from the epileptic mutant mouse stg in the absence of extracellular ionic changes or pharmacological modulation. The presence of synchronous discharges in the stg/stg cortex in vitro and their persistence after isolation from thalamic afferents indicates that a functionally autonomous hyperexcitability defect is expressed within mutant neocortical networks. In addition to spontaneous discharges, neurons within deeper cortical layers that are known to participate in cortical synchronization (Silva et al. 1991) express altered intrinsic active properties and a major threshold decrease favoring repetitive firing. A twofold enhancement of a hyperpolarization-activated, cesium-sensitive inward current could be partly responsible for the observed hyperexcitability. Although the existence of intrinsic membrane property changes in other cortical layers remains to be explored, these results provide the first evidence for a neocortical excitability defect within thalamocortical circuitry in a naturally occurring model of spike wave epilepsy.

Cortical excitability defects and spike wave epilepsy

Although in vitro hyperexcitability does not necessarily signify that generalized spike wave seizures are of cortical origin in the intact animal, the neocortex has long been proposed as a site for defects favoring spike wave epileptogenesis (for review see Gloor et al. 1990). Electrical stimulation of reticular (but not relay) thalamic nuclei produces spike wave synchronization, but only during altered cortical excitability states (Jasper and Droogleever-Fortuyn 1946). Steriade and Contreras (1995) recently found that a majority of thalamic relay neurons are actually hyperpolarized during in vivo cortical spike wave activity. In contrast, nucleus reticularis neurons are actively bursting, and Tsakiridou et al. (1995) have shown that low-threshold calcium currents in these cells are augmented in an inbred epileptic rat strain. Although the nucleus reticularis defect should favor rhythmic activity, it appears many weeks before the onset of spontaneous EEG seizures in that model, suggesting that subsequent excitability increases elsewhere in the network are required. Finally, the neocortex alone is less capable of sustaining spike wave activity, because seizures disappear in vivo when the cortex is isolated from thalamic inputs (Pelligrini et al. 1979; Vergnes et al. 1987) or when excitability in thalamic reticular nuclei is reduced (Avanzini et al. 1993). These data suggest that elevated cortical excitability during thalamocortical oscillations, rather than augmented reticulothalamic pacemaking alone, is essential to generate the abnormal synchronization pattern of the spike wave EEG rhythm.

Intrinsic hyperexcitability in stg deep cortical pyramidal neurons

The passive cell membrane properties in visually identified, deep layer regular spiking neocortical pyramidal neurons in this study are consistent with those previously described in other mouse strains (Agmon and Connors 1989) and other species (Chagnac-Amitai et al. 1990; Connors et al. 1982; Foehring et al. 1991; Mason and Larkman 1990; McCormick et al. 1985), and were not altered in the stg mutant. The average values for Vr, Rin, and membrane time constant showed little variability in both genotypes, suggesting that the neurons we sampled comprise one predominant cell population. Thus, regardless of the recording method used, the excitability defects observed in stg/stg cells were independent of the passive membrane properties measured in this study.

In stg/stg neurons, action potential half-width was shortened as a result of a more rapid depolarization and repolarization. Rise time was increased by 15%, consistent with the possibility of an increased Na+ channel density in the mutant cells, as described in seizure-susceptible El mice (Sashihara et al. 1992); an increase in Na+ flux per channel, as reported in epileptic tottering mutants (Willow et al. 1986); or potential upregulation of a beta 1 Na+ channel subunit (Patton et al. 1994). Although we have no direct evidence for Na+ channel rearrangements in stg/stg cells, their modification could explain the observed 68% decrease in the rheobase intensity, because membrane resistivity was not modified.

Action potential decay time was significantly decreased by 13% in stg/stg neurons, suggesting the further possibility that changes in voltage-dependent K+ conductances may also be present. Deep layer cortical neurons are known to coexpress multiple potassium channel genes (Drewe et al. 1992; Sheng et al. 1994; Weiser et al. 1994), and several contribute to the shape of action potentials in pyramidal cells (Chagnac-Amitai and Connors 1995; Schwindt et al. 1988b). Modification of the repolarizing phase of the stg/stg action potential could contribute to the increase in mean firing rate (154 Hz/nA in the wild type vs. 217 Hz/nA in the mutant).

Possible contribution of the cesium-sensitive inward rectifier to enhanced pyramidal cell excitability in stg neurons

The increased current we described is activated on hyperpolarization, is cesium sensitive, and has a slow and voltage-dependent activation time constant resembling the mixed cationic inward rectifier Ih (Pape 1996). We found that the amplitude of this current was increased approximately threefold in stg neurons. Previous studies in cortical neurons (Schwindt et al. 1988a; Spain et al. 1987) have demonstrated that hyperpolarization-activated currents contribute to spike repolarization, spike frequency adaptation, and AHP magnitude and duration. Thus the properties we observed could be responsible, at least in part, for the lower rheobase and hyperexcitability in stg neurons.

Bath applications of 3 mM CsCl hyperpolarized the pyramidal neurons in both genotypes from rest. This response suggests that the current remains tonically activated at rest, which could favor increased excitability. This resting component in stg should have positively shifted (by a few mV) the mean resting potential in mutant neurons; however, this effect may have been masked by the range of resting potentials observed in both genotypes (+/+, -61/-90 mV; stg/stg, -60/-94 mV). We have not excluded the possibility that other currents responsible along with the inward rectifier for setting resting potential, e.g., the m current (Maccaferri et al. 1993), could also be modified in stg/stg. Spain et al. (1987) have proposed that Ih could modify membrane repolarization by reducing AHP amplitude and duration. We observed that after a zero-Mg2+ burst (Fig. 1, C and D), the resulting slow AHPs were decreased in the mutant. Although this reduction could reflect alterations in potassium currents and inhibitory synaptic inputs, the data suggest that the increased inward rectifier current in stg/stg may modify the intrinsic component of these AHPs, contributing to a hyperexcitable network.

Candidate gene mechanisms for the stg mutant cortical network excitability defect

The network bursting observed in stg/stg neocortex can be phenocopied by many convulsants, and the potential contribution of other molecular and synaptic mechanisms underlying the excitability defect remains to be determined. Currently available mouse linkage data make it possible to exclude loci for 8 sodium channel and 14 potassium channel genes as positional candidates for the intrinsic hyperexcitability changes produced by stg locus (Mouse Genome Database, The Jackson Laboratory). Although these genes may not be the primary target of the mutation, the expression levels and functional kinetics of their products are modulated by activity-dependent processes and seizures (Bosma et al. 1993; Drain et al. 1994; Esguerra et al. 1994; Hoger et al. 1991; Tsaur et al. 1992). We observed a striking increase in the inward rectifier current in P18-20 neurons, at a time when EEG studies show that spontaneous cortical spike wave discharges are brief (<2 s) and rare (<1 per h), preceding the appearance of frequent spike wave discharges by ~1 wk (Qiao and Noebels 1993), suggesting that it is unlikely that this alteration is secondary to a prolonged seizure history. The properties of inward rectifiers could, however, be indirectly modified by extracellular neuromodulators such as serotonin (Bobker and Williams 1989; Spain 1994), intracellular Mg2+ (Stanfield et al. 1994), or polyamines (Fakler et al. 1995; Lopatin et al. 1994). Interestingly, a spontaneous mutation in a gene for another inward rectifier channel, GIRK2, produces an epileptic phenotype in the weaver mutant mouse (Eisenberg and Messer 1989; Patil et al. 1995). Although the intervening steps and secondary cellular pleiomorphisms will become evident once the product of the stg gene is identified, our results suggest that the mutant locus produces multiple changes in deep layer pyramidal neuron firing properties that contribute to the stg cortical hyperexcitability phenotype.

    ACKNOWLEDGEMENTS

  We thank N. Lenn for immunocytochemical staining, J. Trimmer for the Kv2.1 antibody, and H. D. Shine for assistance with morphological analysis.

  This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-29709 and NS-11535. E. Di Pasquale acknowledges partial financial support from the Philippe Foundation.

    FOOTNOTES

  Address for reprint requests: J. L. Noebels, Dept. of Neurology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

  Received 11 July 1996; accepted in final form 23 September 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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