In Vivo Intracellular Analysis of Granule Cell Axon Reorganization in Epileptic Rats

Paul S. Buckmaster1 and F. Edward Dudek2

 1Department of Comparative Medicine, Stanford University School of Medicine, Stanford, California 94305-5410; and  2Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523


    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Buckmaster, Paul S. and F. Edward Dudek. In vivo intracellular analysis of granule cell axon reorganization in epileptic rats. In vivo intracellular recording and labeling in kainate-induced epileptic rats was used to address questions about granule cell axon reorganization in temporal lobe epilepsy. Individually labeled granule cells were reconstructed three dimensionally and in their entirety. Compared with controls, granule cells in epileptic rats had longer average axon length per cell; the difference was significant in all strata of the dentate gyrus including the hilus. In epileptic rats, at least one-third of the granule cells extended an aberrant axon collateral into the molecular layer. Axon projections into the molecular layer had an average summed length of 1 mm per cell and spanned 600 µm of the septotemporal axis of the hippocampus---a distance within the normal span of granule cell axon collaterals. These findings in vivo confirm results from previous in vitro studies. Surprisingly, 12% of the granule cells in epileptic rats, and none in controls, extended a basal dendrite into the hilus, providing another route for recurrent excitation. Consistent with recurrent excitation, many granule cells (56%) in epileptic rats displayed a long-latency depolarization superimposed on a normal inhibitory postsynaptic potential. These findings demonstrate changes, occurring at the single-cell level after an epileptogenic hippocampal injury, that could result in novel, local, recurrent circuits.


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Temporal lobe epilepsy is one of the most common types of epilepsy and one of the most difficult to treat effectively with medication (Engel et al. 1997). Electroencephalography and response to surgical therapy suggest that structures of the mesial temporal lobe generate the complex partial seizures of temporal lobe epilepsy (Engel et al. 1997; Falconner et al. 1964). The hippocampal formation, a prominent component of the mesial temporal lobe, has been hypothesized to be involved in epileptogenesis by several different mechanisms (Babb et al. 1989; Buhl et al. 1996; de Lanerolle et al. 1989; During et al. 1995; Mody and Heinemann 1987; Peterson and Ribak 1989; Sloviter 1987). Considerable attention has focused on the dentate gyrus, in part because previous studies have provided evidence that it normally has a high seizure threshold and might act like a "gate," preventing seizure propagation into the hippocampus (e.g., Fricke and Prince 1984; Lothman et al. 1991, 1992; Schweitzer et al. 1992). The dentate gyrus is a good candidate for involvement in chronic epileptogenesis because of the neuropathological changes it displays in patients with temporal lobe epilepsy (de Lanerolle et al. 1989; Margerison and Corsellis 1966) and because of its remarkable potential for plasticity in response to injury (Nadler et al. 1980; Parent et al. 1997).

Tauck and Nadler (1985) proposed that seizures result from positive feedback through aberrant excitatory recurrent axon collaterals between granule cells. Granule cell axon reorganization has been demonstrated in tissue from patients (Babb et al. 1991; de Lanerolle et al. 1989; Houser et al. 1990; Sutula et al. 1989) and in experimental models of temporal lobe epilepsy (Buckmaster and Dudek 1997b; Cronin and Dudek 1988; Kotti et al. 1997; Mello et al. 1993; Nadler et al. 1980; Okazaki et al. 1995; Represa et al. 1993; Sutula et al. 1988). Most previous studies used staining techniques that label many or all granule cell axons; however, several have used intracellular labeling of individual granule cells (Franck et al. 1995; Isokawa et al. 1993; Sutula et al. 1998). These intracellular labeling studies employed the hippocampal slice preparation, limiting observations to that part of the axon arbor within a 400- to 500-µm-thick slice.

Important questions persist about granule cell axon reorganization. What proportion of granule cells extends axon collaterals into the molecular layer of the dentate gyrus? How long are the axon collaterals that invade the molecular layer, and how selective is that invasion? Do granule cell axon collaterals project to distant septotemporal regions of the dentate gyrus or do they remain close to the parent cell body? The kainate-treated rat is a widely accepted experimental model that replicates clinical and neuropathological features of temporal lobe epilepsy (Ben-Ari 1985; Hellier et al. 1998; Nadler 1981; Pisa et al. 1980). The present study used an in vivo intracellular approach to label granule cells in kainate-induced epileptic rats, examine their complete axon arbors, and record electrophysiological responses.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals

Male Sprague-Dawley rats (200-250 g; Harlan, Houston, TX) were treated with kainic acid (Sigma Chemical, St. Louis, MO) dissolved in 0.9% NaCl. Kainate was administered in doses of 5 mg/kg ip at 1-h intervals until the rat experienced >= 3.5 h of recurrent seizures. The average cumulative dose was 30 mg/kg. Age-matched control rats received a similar volume of vehicle. After recovering from acute seizures, animals were observed >= 6 h/wk for spontaneous motor seizures of grade 3 or greater on the Racine scale (forelimb clonus ± rearing ± falling) (Racine 1972). The average period between kainate treatment and electrophysiological recording and perfusion was 143 days (range 99-207).

Electrophysiology

Rats were anesthetized with urethan (1.2 g/kg ip) and placed in a stereotaxic frame with the nose bar set at -3.0 mm. Urethan was used because, unlike other drugs (e.g., barbiturates) (Nicoll et al. 1975), it appears to have little or no direct effect on GABAergic inhibition in the rat dentate gyrus (Shirasaka and Wasterlain 1995). Body temperature was maintained with a heating pad, and cerebrospinal fluid was drained from the cisterna magna to improve stability. Holes were drilled through the skull, and electrodes were directed toward the dentate gyrus and angular bundle at the following coordinates (relative to bregma): -4.6 mm posterior and 2.2 mm lateral for the recording electrode and -8.3 posterior and 3.6 mm lateral for the stimulating electrode. Electrode depths were determined by optimizing field potential responses to stimulation. A bipolar concentric stimulating electrode (SNEX-100, Rhodes Medical Instruments, Tujunga, CA) activated perforant pathway fibers. A glass micropipette filled with 0.9% NaCl and broken to an inner diameter of ~15 µm was used to measure the depth of the granule cell layer and to determine the stimulus threshold for a population spike (T). Then a sharp intracellular electrode, measuring 80-150 MOmega in vivo and filled with 1 M potassium acetate and 2% biocytin, was lowered toward the septal end of the dentate gyrus where granule cells were impaled.

Membrane and field potentials were amplified (Axoprobe 1A, Axon Instruments, Foster City, CA), observed on-line, and stored on video tape (Neuro-corder, Neuro Data Instruments, Delaware Water Gap, PA) and on computer (pCLAMP, Axon Instruments) for off-line analysis. Resting membrane potential was determined by subtracting the extracellular voltage measured when the electrode was withdrawn from the cell from the resting intracellular voltage. Membrane time constant was measured from responses to 0.1- to 0.5-nA hyperpolarizing current pulses. Action potential duration and amplitude were measured from responses to depolarizing current injections just large enough to evoke one action potential. Action potential duration was defined as the time between the onset of the action potential and the return of the membrane potential to the onset value. Action potential amplitude was measured from the onset value to the peak.

Perforant pathway fibers were activated at 0.1 Hz by 150-µs constant-current pulses. Stimulus intensity was standardized to the threshold (T) of the population spike; the average value of T was 1.09 mA (range 0.08-4.80). Stimulus intensity for inhibitory postsynaptic potential (IPSP) analysis was 1.5 × T. Measurements of fast IPSPs were made at a 20-ms latency after the stimulus; slow IPSPs were measured at a 150-ms latency. IPSP reversal potentials were determined by altering the membrane potential with DC current to change the amplitude and polarity of postsynaptic potentials. IPSP amplitude was plotted against the prestimulus membrane potential, and the data were fit with a least-squares regression line to determine the IPSP reversal potential. IPSP conductance was calculated by subtracting the cell's input conductance before stimulation from its conductance during the IPSP. Conductance was the slope of I-V curves, where I was DC holding current and V was membrane potential. Input resistance was the reciprocal of input conductance. Field potential responses, recorded after the electrode was withdrawn from the cell, were subtracted from the intracellularly recorded stimulus-evoked responses.

Morphology

After obtaining intracellular recordings, cells were labeled with 2% biocytin (Sigma) by passing 300-ms pulses of 0.4- to 1.0-nA hyperpolarizing current for an average of 8 min. However, cells began being labeled with biocytin even before the onset of the labeling procedure because they were impaled and current was passed for a period while they stabilized and while electrophysiological recordings were obtained. The rat then was perfused through the ascending aorta with 300 ml of 0.37% sodium sulfide and 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brain was removed, postfixed overnight, and cryoprotected in 30% sucrose in 0.1 M PB. The hippocampus was isolated, straightened, frozen, and sectioned (nominally at 60 µm) perpendicular to the septotemporal axis.

Serial sections of the septal 75% of the hippocampus containing the labeled cell were mounted on gelatin-coated slides and exposed to 0.3-0.5% hydrogen peroxide and 10% ethanol in 0.1 M PB for 1 h and then 0.3% Triton X-100 in 0.1 M PB for 1 h before incubating in ABC solution (1:150, Vector Laboratories, Burlingame, CA) in 0.1 M PB with 0.1% Triton X-100 for 2-4 h at room temperature or overnight at 4°C. After thorough washing in PB and 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer (TB, pH 7.6), sections were exposed to 0.04% diaminobenzidine (Sigma) and 0.3% NiCl for 3 min. Hydrogen peroxide was added to result in a 0.0025% solution, and sections reacted for 0.5-1.0 h. The reaction was stopped in washes of 0.1 M TB, and the sections were dehydrated and coverslipped.

Axon arbors of 26 granule cells were drawn with a camera lucida, and two-dimensional axon lengths were measured from the drawings. This subgroup of our total sample of 64 labeled granule cells included the best cells used for axon reconstruction and measurement. These 26 cells used for quantitative analysis appeared to be completely labeled because their axons were darkly labeled, even at the extremes of the arbor. Cells with lightly labeled axons and cells in hippocampi with more than four labeled cells were excluded because overlapping axon collaterals might impede accurate axon reconstruction and measurement.

Granule cell axon arbors of 4 of the 26 cells (2 in kainate-induced epileptic rats and 2 in controls) were reconstructed three dimensionally with a Neurolucida system (MicroBrightField, Colchester, VT; Fig. 3). The complexity of intermingled axon arbors in preparations with more than one labeled cell precluded the use of the Neurolucida system in many cases. However, axon lengths measured from the two-dimensional camera-lucida representations were adjusted for three dimensionality by a correction factor (1.25), which was determined using values from the cells analyzed both ways. Axon lengths were adjusted for tissue shrinkage, which occurred during processing, by correction factors determined by measuring the distance between stereotaxically located fast green dye injections. Fast green dye (1%) was ejected iontophoretically from a field potential electrode, using 20 µA DC current for 10 min. In the transverse plane, tissue shrank to 94% of its original size (correction factor = 1.06); along the septotemporal axis it shrank to 51% (correction factor = 1.96). Because some hippocampi contained multiple labeled granule cells, average axon length per cell was estimated by dividing the summed length of all axon collaterals by the number of labeled cells. This would not affect the results, which were averaged and plotted by experimental group. The number of labeled somata was verified by determining the number of mossy fibers that projected through the CA3 field, there being only one mossy fiber in CA3 for each labeled granule cell. The dendritic trees of granule cells were reconstructed three dimensionally with the Neurolucida system.

Sections of the temporal 25% of the hippocampus were mounted on gelatin-coated slides and processed by a modified Timm staining protocol (Babb et al. 1991). Sections were placed in a solution consisting of 180 ml of 50% gum arabic, 30 ml of 2.25 M citrate buffer, 45 ml of 0.5 M hydroquinone, and 50 ml of 0.04% silver lactate. Sections were developed for ~70 min in darkness at room temperature, then they were rinsed in water, lightly counterstained with cresyl violet, dehydrated, and coverslipped.


    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Animals

Thirty rats were used in this study: 18 treated with kainic acid and 12 age-matched vehicle-treated controls. All of the kainate-treated rats displayed spontaneous, recurrent, motor seizures, which first were observed 70 ± 9 days after treatment (mean ± SE; range 25-168). The average seizure frequency over the period between the first observed seizure and use in an experiment was 0.34 ± 0.04 seizures/h (range 0.11-0.62). No control rats were observed to have a seizure. The rats used in this study came from the same pool of identically treated animals that were used in previously published studies (Buckmaster and Dudek 1997a,b; Hellier et al. 1998; Patrylo and Dudek 1998; Wuarin and Dudek 1996).

Timm stain

All rats in this study displayed dark Timm staining in the hilus and in stratum lucidum of CA3. Control rats showed very little Timm staining in the molecular layer of the dentate gyrus (Fig. 1A), except at the temporal pole of the hippocampus as reported previously (Buckmaster and Dudek 1997b; Cavazos et al. 1992). In contrast, all of the kainate-induced epileptic rats displayed dark Timm staining in the inner third of the molecular layer (Fig. 1B). This finding confirmed that granule cell axon reorganization occurred in all of the kainate-induced epileptic rats in this study. This finding also suggests that there was hilar neuron loss in kainate-induced epileptic rats because aberrant Timm staining is correlated with hilar neuron loss (Babb et al. 1991; Buckmaster and Dudek 1997b; Masukawa et al. 1995).



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Fig. 1. Timm stain of the dentate gyrus. Extensive Timm staining is evident in the inner third of the molecular layer (m) in the kainate-induced epileptic rat (B) but not in the control (A). Sections from similar levels of the septotemporal axis of the hippocampal formation were counterstained lightly with cresyl violet. g, granule cell layer; h, hilus. Scale bar = 50 µm.

Granule cells

MULTIPLE LABELING. The total number of labeled granule cells was 31 and 33 for the control and kainate-induced epileptic groups, respectively. The number of labeled granule cells per hippocampus ranged from one to six, and the average was similar in the control (2.8 cells) and kainate-induced epileptic groups (2.5 cells). Multiple labeling occurred because repeated penetrations with biocytin-containing recording electrodes were made to obtain electrophysiological recordings. In addition, multiple-labeled cell bodies were closely apposed in some cases, suggesting that some multiple filled cells resulted from electrode-induced injury (Claiborne et al. 1990) or from gap junction connections between granule cells (MacVicar and Dudek 1982).

AXONS. Granule cells were more likely to project axon collaterals into the granule cell layer and molecular layer of the dentate gyrus in kainate-induced epileptic rats versus controls. In all cases, granule cells extended a primary axon from the cell body, through the hilus, and into stratum lucidum of CA3. Granule cell axon arbors were examined in 11 hippocampi of 11 control rats. In five control hippocampi (45%) at least one axon collateral entered the granule cell layer, and in one control hippocampus (9%), one axon collateral entered the molecular layer. Granule cell axon arbors were examined in 12 hippocampi of 10 kainate-induced epileptic rats. In 11 of these hippocampi (92%) at least one axon collateral entered the granule cell layer and molecular layer (Figs. 2 and 3). Differences between the control and kainate-induced epileptic groups, with respect to the proportion of hippocampi with an axon collateral entering the granule cell layer or molecular layer, were significant (P < 0.025 and P < 0.005, respectively, chi 2 test).



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Fig. 2. Axon collaterals of granule cells project into the molecular layer of the dentate gyrus in kainate-induced epileptic rats. A: 2 granule cells were labeled with biocytin. Part of an axon collateral extended along the inner molecular layer (m; down-arrow ). B: multiple labeled granule cells in an adjacent section had axon collaterals that projected from the hilus (h), through the granule cell layer (g), and into the inner molecular layer (down-arrow ). Scale bars = 50 µm.



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Fig. 3. Granule cells in control (A and B) and kainate-induced epileptic rats (C and D). Granule cell axon collaterals in kainate-induced epileptic rats projected into the granule cell layer (g) and the inner molecular layer (m; down-arrow ). Cell shown in C had a basal dendrite in the hilus (h; black-down-triangle ), and part of it is evident in the photographic montage inset of one section. Neurolucida cell reconstructions have been rotated slightly from the plane of the photographs. Scale bars = 100 µm.

It would be useful to know the proportion of granule cells that project an axon collateral into the molecular layer of the dentate gyrus. In the control group, only 1/31 cells (3%) extended an axon collateral into the molecular layer. In the kainate-induced epileptic group, the minimum number of granule cells that extended an axon collateral into the molecular layer was 11/32 (34%), assuming that only one labeled cell accounted for all of the axon in the molecular layer in hippocampi with multiple labeled cells. The maximum number of granule cells that extended an axon collateral into the molecular layer was 31/32 (97%), assuming that all the labeled cells extended axon collaterals into the molecular layer in hippocampi with multiple labeled cells. Granule cells in kainate-induced epileptic rats, therefore, were 11-32 times more likely than granule cells in control rats to extend an axon collateral into the molecular layer. The difference was significant, even using the most conservative estimate (P < 0.005, chi 2 test).

Granule cell axon arbors were longer in kainate-induced epileptic rats versus controls. Granule cell axon lengths were measured from a subset of the labeled granule cells in this study. Only preparations with the fewest number of labeled cells per hippocampus and the most clearly labeled axon arbors were used. Measurements of average axon arbor length were made of 13 granule cells in six hippocampi of six control rats and of 13 granule cells in seven hippocampi of six kainate-induced epileptic rats. Only axon collaterals within the dentate gyrus molecular layer, granule cell layer, and hilar region were measured. For this analysis, the hilar region was defined by its border with the granule cell layer and by a line connecting the tips of the granule cell layer. The combined (i.e., molecular layer + granule cell layer + hilar region) average length of axon per granule cell was 7.86 ± 0.74 (SE) mm (range 6.16-11.18) in controls and 15.75 ± 1.64 mm (range 8.89-20.04) in kainate-induced epileptic rats. Kainate-induced epileptic rats, therefore, had twice as much axon length per granule cell as controls (P < 0.002, t-test). These values of axon length are longer than those reported previously for normal and kainate-treated rats (Claiborne et al. 1986; Sutula et al. 1998) and epileptic humans (Isokawa et al. 1993), but those studies used the hippocampal slice preparation, which amputates axon collaterals.

Granule cell axon arbors in kainate-induced epileptic rats were longer than controls in all the strata of the dentate gyrus (Fig. 4A). The average distribution of axon length in the molecular layer, granule cell layer, and hilar region, respectively, was 0, 3, and 97% for controls and 8, 7, and 85% for kainate-induced epileptic rats. However, the septotemporal extent of the granule cell axon arbors was not significantly different between control and kainate-induced epileptic rats (t-test; Fig. 4B). The average septotemporal length of the granule cell axon arbors was 0.89 ± 0.08 mm (range 0.60-1.32) in controls and 1.05 ± 0.09 mm (range 0.60-1.56) in kainate-induced epileptic rats. In kainate-induced epileptic rats, the average septotemporal span of axon collaterals in the molecular layer was 0.61 ± 0.14 mm (range 0-1.56), similar to that reported previously (Sutula et al. 1998). In the control group, only one granule cell extended an axon collateral into the molecular layer, and the short projection was confined to the section containing the labeled cell body. This cell was not included in the quantitative analysis of axon arbors (e.g., Fig. 4), because it was in a hippocampus with five labeled cells.



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Fig. 4. Average axon lengths of granule cells in control and kainate-induced epileptic rats. A: granule cell axons in kainate-induced epileptic rats were longer than controls in all strata of the dentate gyrus. Error bars indicate SE (*P < 0.008, t-test). Hilar region was defined by its border with the granule cell layer and by a straight line between the tips of the granule cell layer. See METHODS for a description of axon length measurements. B: septotemporal span of granule cell axon arbors was not significantly different in control and kainate-induced epileptic rats (t-test). Values represent mean axon length per granule cell per section (± SE). Inset: schematic of a straightened rat hippocampus with the approximate location of labeled granule cells indicated (*).

DENDRITES. Granule cells in kainate-induced epileptic rats were significantly more likely to have a basal dendrite than those in controls (Figs. 3C and 5). In the control group, 0/31 labeled granule cells had a basal dendrite compared with 4/33 granule cells (12%) in the kainate-induced epileptic group (P < 0.05, chi 2 test). One of the basal dendrites branched, the other three did not. All of the basal dendrites were covered with spines (Fig. 5A, inset). In cells that had them, the average length of the basal dendrite was 0.23 mm or 6% of the total dendritic length per cell. The average total length of dendrites per cell for those cells with a basal dendrite (3.90 ± 0.22 mm) was not significantly different from that of the cells without basal dendrites in kainate-induced epileptic rats (3.56 ± 0.51 mm) or in controls (3.60 ± 0.10 mm) (t-test). These dendritic length results are similar to those reported previously for granule cells in normal rats (mean = 3.22-3.66 mm) (Claiborne et al. 1990; Desmond and Levy 1982).



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Fig. 5. Basal dendrites in the hilus from granule cells in kainate-induced epileptic rats. A: 1 granule cell in this study had a basal dendrite (down-arrow ) that branched. Neurolucida cell reconstruction has been rotated slightly from the plane of the photograph to better illustrate the basal dendrite. Photographic montage shows the cell's axon (black-down-triangle ), which lacks spines and is smaller in diameter than the basal dendrite (arrow). B: granule cell with an unbranched basal dendrite. m, molecular layer; g, granule cell layer; h, hilus. Scale bars = 50 µm; 25 µm for inset.

ELECTROPHYSIOLOGY. Intrinsic electrophysiological properties of granule cells were not significantly different in control and kainate-induced epileptic rats. Both groups displayed strongly negative resting membrane potentials, overshooting action potentials, and similar input resistances and membrane time constants (Table 1; Fig. 6A). These results are similar to previously published values for rat granule cells recorded in vivo (Buckmaster and Schwartzkroin 1995).


                              
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Table 1. Electrophysiological properties of granule cells in control and epileptic kainate-treated rats



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Fig. 6. Electrophysiological responses of a granule cell in a kainate-induced epileptic rat. A: intrinsic electrophysiological responses to current injection were normal in kainate-induced epileptic rats. B: perforant pathway stimulation evoked fast and slow inhibitory postsynaptic potentials (IPSPs) that were measured at different membrane potentials by injecting DC current into the cell. These responses and others were used to estimate IPSP conductances and reversal potentials. RMP, resting membrane potential. C: reversal potentials of fast and slow IPSPs were estimated by least-squares regression lines through plots of IPSP amplitude vs. prestimulus membrane potential. For this cell, the reversal potentials were -74 and -99 mV for the fast and slow IPSPs, respectively.

IPSP responses of granule cells to perforant pathway stimulation were similar in control and most kainate-induced epileptic rats (Table 1; Fig. 6, B and C). The reversal potentials of fast and slow IPSPs tended to be more depolarized and the conductances tended to be larger in kainate-induced epileptic rats compared with controls, but the differences were not significant (t-test). However, clearly evident long-latency depolarizations were superimposed on the IPSPs of five of nine granule cells (56%) in kainate-induced epileptic rats and in none (0/9) in controls (P < 0.01, chi 2 test, Fig. 7). The average latency to the first peak of the late depolarization was 103 ± 14 (SE) ms (range 69-135). The variability and range of latencies was large between cells, but in a given cell, latencies were relatively regular. Only in one case did the long-latency depolarization trigger an action potential and that required depolarizing DC current injection. Long-latency depolarizations sometimes had multiple peaks and sometimes failed to occur in cells that displayed them, suggesting a polysynaptic mechanism.



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Fig. 7. Long-latency depolarizations superimposed on the IPSPs of some granule cells in kainate-induced epileptic rats. Three examples of granule cells (A-C) in which perforant pathway stimulation (1.5 × threshold) evoked depolarizations (*) that sometimes failed to occur (bottom trace of each set). Action potentials have been clipped.

One granule cell in a kainate-induced epileptic rat responded to perforant pathway stimulation with bursts of action potentials and no evidence of an IPSP (Fig. 8A). The initial burst of <= 26 action potentials was followed by zero to five shorter bursts, each consisting of two to six action potentials. This cell also displayed spontaneous bursts of action potentials. Intrinsic electrophysiological properties of this cell were within the range of other granule cells in control and kainate-induced epileptic rats (Fig. 8B). The dentate gyrus that contained this granule cell generated field potential responses to perforant pathway stimulation that consisted of multiple population spikes (Fig. 8C1), indicating dramatic hyperexcitability compared with other kainate-induced epileptic rats (Fig. 8C2) and controls. A series of sections from this hippocampus was processed for parvalbumin immunoreactivity, and the results were presented in another paper where it was estimated that 83% of the parvalbumin-immunoreactive neurons in this dentate gyrus were missing (Buckmaster and Dudek 1997b). In most rats, parvalbumin-immunoreactive interneurons in the dentate gyrus survive kainate treatment and provide inhibitory input to the somatic region of granule cells (Best et al. 1993; Buckmaster and Dudek 1997b; Magloczky and Freund 1993; Nitsch et al. 1990).



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Fig. 8. Repetitive bursts of action potentials to perforant pathway stimulation in a granule cell of a kainate-induced epileptic rat with apparent loss of inhibition in the dentate gyrus. A: perforant pathway stimulation evoked a burst of action potentials followed by afterdischarges with no evidence of an IPSP. B: responses to current injection revealed normal intrinsic electrophysiological properties. C: dentate gyrus field potential response to perforant pathway stimulation from this rat consisted of multiple population spikes indicating marked hyperexcitability (1). The typical response of most kainate-induced epileptic rats consisted of only 1 population spike (2).


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

This study describes morphological changes that occur in granule cells after epileptogenic hippocampal injury and examines intrinsic and synaptic electrophysiological properties of granule cells in epileptic rats.

Chronic morphological changes of granule cells after epileptogenic hippocampal injury

The morphological changes of granule cells in kainate-induced epileptic rats are summarized in Fig. 9. The biggest change was in mean axon length, which was twice that of controls, corroborating a previous in vitro study (Sutula et al. 1998). Dentate granule cells are not the only cell type to sprout axon collaterals in response to epileptogenic treatments. Axon sprouting has been reported for inhibitory interneurons in the dentate gyrus (Davenport et al. 1990; Mathern et al. 1995), CA3 pyramidal neurons (McKinney et al. 1997), CA1 pyramidal neurons (Perez et al. 1996), and layer V pyramidal neurons in neocortex (Salin et al. 1995), suggesting that this might be a general response of neurons to injury. The present study focused on granule cells in the septal end of the dentate gyrus; results from the temporal dentate gyrus might be different.



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Fig. 9. Schematic of the morphological changes of granule cells that occur in epileptic rats. Thick lines are dendrites; thin lines are axons. Solid lines are normal structures; dashed lines are new structures found in epileptic animals. Note the basal dendrite and the new axon collaterals that extend through the hilus (h), granule cell layer (g), and inner-third of the molecular layer (m). Despite axon sprouting, the septotemporal span of the axon arbor remains confined to a 1-mm-wide lamella, which is one-tenth the septotemporal length of the rat dentate gyrus. Therefore monosynaptic effects of circuit changes are restricted to the local vicinity of the cell.

Quantitatively, the largest morphological change was in axon length, but the most dramatic difference was in the distribution of axon collaterals in epileptic versus control rats. At least one-third of the granule cells in epileptic rats had axon projections into the molecular layer (compared with 1 of 31 cells in controls), and the averaged summed length of these projections was 1 mm per cell. Previous ultrastructural analyses have shown that these aberrant axon projections form synaptic contacts with other granule cells (Babb et al. 1991; Franck et al. 1995; Okazaki et al. 1995; Represa et al. 1993), and there is evidence that they could synapse with inhibitory interneurons, too (Kotti et al. 1997; Sloviter 1992).

Despite this dramatic change in circuitry, new axon collaterals respected most of the boundaries of normal granule cell axons. Along the longitudinal axis of the hippocampus, the axon arbors in epileptic rats remained confined to a relatively thin lamella of the dentate gyrus and did not extend to distant septotemporal regions. The septotemporal span of the granule cell axon arbor within the dentate gyrus in control and epileptic rats was ~1 mm, whereas the length of the rat hippocampus is 10 mm (Amaral and Witter 1989). In the transverse plane of the hippocampus, axon collaterals that entered the molecular layer remained in the inner third and did not extend into the middle or outer thirds. These findings corroborate previous reports (Franck et al. 1995; Isokawa et al. 1993; Represa et al. 1993; Sutula et al. 1998; but see Okazaki et al. 1995) and suggest that there are predictable rules of granule cell axon growth after epileptogenic injury.

In addition to changes in axon structure, there were changes in dendrites. Granule cells in adult rats do not normally have basal dendrites (Claiborne et al. 1990; Seress and Pokorny 1981). We were surprised to find that they occurred in 12% of the granule cells in kainate-induced epileptic rats. Recently, it was reported that basal dendrites occur in a similar proportion of granule cells in another experimental model of temporal lobe epilepsy (Spigelman et al. 1998). Granule cells in immature rats have basal dendrites (Seress and Pokorny 1981), suggesting the recent generation of granule cells with basal dendrites in adult epileptic rats (Parent et al. 1997). Basal dendrites normally occur in some granule cells in nonepileptic adult humans (Seress and Mrzljak 1987), but they are reported to be more common in patients with temporal lobe epilepsy (Franck et al. 1995; but see Von Campe et al. 1997). It is not known presently if aberrant basal dendrites of granule cells in epileptic tissue receive synaptic input from neighboring granule cells.

Possible functional effects of morphological changes in granule cells

Granule cell axon projections into the molecular layer and basal dendrites that extend into the hilus offer possible avenues for recurrent excitation that normally are rare or nonexistent. Previous studies provide evidence for recurrent excitation between granule cells in kainate-treated and kindled rats (Buckmaster and Dudek 1997a; Cronin et al. 1992; Golarai and Sutula 1996; Masukawa et al. 1992; Patrylo and Dudek 1998; Tauck and Nadler 1985; Wuarin and Dudek 1996). The finding of late depolarizations in granule cells of epileptic but not control rats is consistent with a recurrent excitatory mechanism. It is unlikely that these depolarizations are due to intrinsic electrophysiological properties of granule cells because those are similar in control and kainate-induced epileptic rats. However, we cannot exclude the possible contribution of differences in neuronal circuits outside of the dentate gyrus (e.g., Du et al. 1995) or alterations in receptor pharmacology of granule cells (e.g., Mody and Heinemann 1987). The late depolarizations in granule cells resemble responses of CA3 pyramidal neurons (McKinney et al. 1997) and layer V pyramidal neurons in neocortex (Prince and Tseng 1993) after axon sprouting was induced in those regions by epileptogenic treatments.

Whether there is enough recurrent excitation to lower seizure threshold will depend in part on inhibitory control mechanisms (Miles and Wong 1987). Previous studies suggest that recurrent inhibition is enhanced after granule cell axon reorganization (Buckmaster and Dudek 1997a,b; Milgram et al. 1991). In the present study, inhibition was quantified by evoking IPSPs with standardized stimuli and measuring their conductance during the fast and slow components. However, there are many different types of inhibition [e.g., feedforward vs. feedback, gamma -aminobutyric acid-A (GABAA)-receptor-mediated vs. GABAB-receptor-mediated vs. non-GABA-receptor-mediated, presynaptic vs. postsynaptic, etc.], and inhibition is a dynamic process making it difficult to assess comprehensively. Nevertheless, robust IPSPs in granule cells, found in all but one kainate-induced epileptic rat, demonstrated strong inhibition. Prominent IPSPs occur in principal cells in other experimental models after axon sprouting is induced by epileptogenic treatments (McKinney et al. 1997; Prince and Tseng 1993), and inhibition is intact in tissue from many patients with temporal lobe epilepsy (Colder et al. 1996; Franck et al. 1995; Uruno et al. 1995; Williamson et al. 1995). Granule cell axon elongation, especially in the hilus where inhibitory interneurons concentrate and where granule cells normally synapse preferentially with interneurons (Acsády et al. 1998), could provide a mechanism for increasing excitatory synaptic drive of interneurons, as has been shown in kindled rats (Buhl et al. 1996).

Epileptogenic injuries vary in severity, and the most severe injuries not only induce granule cell axon sprouting but also kill inhibitory interneurons. In those cases where a critical population of interneurons is missing, inhibition of granule cells will be compromised (Buckmaster and Dudek 1997b). The granule cell that responded to afferent stimulation with repetitive burst discharges and without an IPSP may demonstrate the effect of recurrent excitation between granule cells that is no longer masked by inhibition, because blockade of synaptic inhibition of normal granule cells does not result in repetitive burst discharges in response to afferent stimulation (Buckmaster and Dudek 1997a; Fricke and Prince 1984). In other regions of the brain, where epileptogenic treatments induce axon sprouting, pharmacological blockade of inhibition results in similar burst discharges to afferent stimulation (Hoffman et al. 1994; McKinney et al. 1997).

Summary

This study provides detailed data on the morphological changes of granule cells in kainate-induced epileptic rats. Measurements of the complete axon arbors of granule cells in vivo have confirmed results from previous in vitro studies. Changes in axonal and dendritic structure offer possibilities for the formation of novel, local, recurrent circuits within the dentate gyrus. Electrophysiological data are consistent with strong synaptic inhibition masking recurrent excitation between granule cells most of the time. These findings are similar to those from epileptogenic treatments in other brain regions and suggest that a general response of neurons to injury is to increase connectivity according to potentially predictable rules.


    ACKNOWLEDGMENTS

We are grateful to A. Kuhn for excellent technical assistance and Dr. Bret Smith for helpful comments on the manuscript.

This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-01778 and NS-16683. P. S. Buckmaster is a recipient of a Burroughs Wellcome Fund Career Award.


    FOOTNOTES

Address for reprint requests: P. S. Buckmaster, Dept. of Comparative Medicine, Stanford University School of Medicine, Stanford, CA 94305-5410.

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 soley to indicate this fact.

Received 3 September 1998; accepted in final form 23 October 1998.


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