Sex Differences in Cortical Plasticity and Behavior Following Anterior Cortical Kindling in Rats

G.C. Teskey, J.E. Hutchinson and B. Kolb1

Behavioural Neuroscience Research Group, Department of Psychology, University of Calgary, Calgary, Alberta, Canada T2N 1N4 and , 1 Department of Psychology and Neuroscience, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This experiment examined the effect of electrical kindling on the morphology of frontal (Fr1) neocortical layer III pyramidal cell dendrites in both male and female rats. Repeated elicitation of afterdischarge resulted in an increase in the severity of the behavioural seizures and an increase in afterdischarge duration, frequency and amplitude in all rats. The late component of the transcallosal evoked responses also increased following both 7 and 25 kindling sessions in male rats and following 25 kindling sessions in female rats. Analysis of the Golgi–Cox impregnated pyramidal cell dendrites indicated a significant decrease in the amount of apical and basilar dendritic spine density, length and branching in female rats following 7 days, but not 25 days, of kindling. Male rats had significantly lower apical and basilar dendritic spine density and branching measures following 25 days, but not 7 days, of kindling, as well as significantly lower apical and basilar dendritic length following 7 days of kindling. The differential gender effect suggests that males and females recruit similar plastic mechanisms although at different times in response to electrical kindling.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Kindling refers to a phenomenon in which the repeated elicitation of epileptiform afterdischarge (AD) results in the progressive intensification and trans-synaptic propagation of the AD (Goddard et al., 1969Go). This growth and spread of the AD normally occurs in association with progressively more seizure activity, through defined stages (Racine, 1972Go). The resulting persistent state of heightened seizure susceptibility (Dennison et al., 1995Go) is correlated with very long-lasting increases in synaptic efficacy (deJonge and Racine, 1987Go; Cain, 1989Go; Racine et al., 1991Go, 1995Go) and unit activity (Stasheff and Wilson, 1990Go, Teskey and Racine, 1993Go). Because kindling is an extremely robust example of plasticity in the mammalian central nervous system, we would expect neuronal morphological correlates to the enhanced electrophysiological and behavioural measures.

Several previous studies have demonstrated kindling-induced morphological alterations at the dendritic and synaptic level in the hippocampus (Geinisman et al., 1988Go, 1990Go, 1992Go; Sutula, 1990Go; Hawrylak et al., 1993Go; Hovorka et al., 1997Go; Armitage et al., 1998Go; Jiang et al., 1998Go) and amygdala (Nishizuka et al., 1991Go; Okada et al., 1993Go). However, there has been a lack of evidence of dendritic alterations in the neocortex following kindling. In the single documented attempt, Racine and coworkers found no changes in dendritic branching or spine density in the anterior cortex of cortically kindled rats (Racine et al., 1975Go). This is somewhat surprising because several different types of manipulations have been shown to alter dendritic morphology in the rat anterior neocortex. For instance, enriched environments (Greenough et al., 1973Go; Sirevaag and Greenough, 1988Go), training (Greenough et al., 1985Go; Withers and Greenough, 1989Go), gonadal hormone manipulation (Kolb and Stewart, 1991Go; Stewart and Kolb, 1994Go), noradrenaline depletion (Kolb and Sutherland, 1992Go), amphetamine sensitization (Robinson and Kolb, 1997Go) and lesions (Kolb and Gibb, 1991aGo,bGo) have all been shown to affect some measures of cortical dendritic morphology.

In this experiment we kindled adult rats in the medial frontal cortex. We chose this area because the transcallosal pathway is easily identifiable, kindles and shows synaptic potentiation effects (Racine et al., 1995Go). We also chose to examine sex differences in kindling in this area because its dendritic morphology shows a sexual dimorphism at the structural level (Kolb and Stewart, 1991Go), and because a differential plastic response to midline frontal lesions (Kolb and Whishaw, 1991Go) and gonadal hormones (Kolb and Stewart, 1995Go) has been reported. We measured neocortical evoked potentials prior to and following either 7 or 25 days of stimulation to confirm synaptic potentiation and then processed the brains for Golgi– Cox staining. A separate group of female animals had their estrous cycle followed to examine the effect of cycling female hormones on the evoked potential measures. The branching, length and spine density of both the apical and basal dendrites of pyramidal cells in frontal cortex (Fr1) layer III (Zilles, 1985Go) were analyzed.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Subjects

Nineteen male rats weighing 370–560 g and 20 sibling female rats weighing 210–290 g at the time of surgery were used. All rats were of the Long–Evans hooded variety and were obtained from the University of Calgary Breeding Colonies. Animals were housed individually in clear plastic cages in a colony room that was maintained on a 12 h on/12 h off light cycle. All experimentation was conducted in the light phase. Rats were maintained on Lab Diet no. 5001 (PMI Feeds Inc, St Louis, MO) and water ad libitum and were handled and maintained according to the Canadian Council of Animal Care guidelines.

Surgical Procedure

Twisted-wire bipolar stimulating and recording electrodes were prepared from Teflon-coated, stainless steel wire, 178 µm in diameter (A-M Systems, Everett, WA). Uninsulated ends of the electrodes were connected to gold-plated male amphenol pins. The two poles of the electrodes were separated by 1.0 mm. Animals were anesthetised with 58.83 mg/kg ketamine (85%) and xylazine (15%) at 0.5 ml/kg, injected i.m. Lidocaine 2% (Austin, Joliette, QA), a local anesthetic, was administered s.c at the incision site.

Two bipolar electrodes were chronically implanted according to the stereotaxic coordinates of Swanson (Swanson, 1992Go). The stimulating electrode was implanted 1.0 mm anterior to bregma on the midline in the callosal white matter. The recording electrode was implanted 1.0 mm anterior to bregma and 4.0 mm lateral to midline in the frontal neocortex. Electrophysiological monitoring was performed during the surgery so that the dorsal–ventral placements could be adjusted for maximal evoked response amplitude.

Gold-plated male amphenol pins connected to the electrodes were inserted into a nine-pin McIntyre connector plug, adhered to the skull with four or five stainless steel screws and dental cement. One of the stainless steel screws served as the ground reference. Experimental procedures commenced no earlier than 7 days after surgical implantation.

Experimentation

Male and female rats were divided into three age-matched groups: those receiving 25 kindling sessions, seven kindling sessions and the electrodeimplanted, non-kindled control.

Baseline input-output (I/O) measures were conducted for two consecutive days prior to kindling. This was accomplished by administering pulses of increasing intensity to the callosal white matter and recording the resultant evoked potentials from the frontal neocortical electrode. The stimulation pulses consisted of biphasic rectangular waves, 200 µs in duration and separated by 200 µs. Ten stimulation pulses were delivered at each of the ascending logarithmic intensities (10, 32, 46, 68, 100, 147, 215, 316, 464, 681 and 1000 µA) at a frequency of 0.1 Hz. Stimulation voltages were computer generated, then converted to an amperage via a current constant current and isolation unit (World Precision Instruments, Sarasota FL). The recorded signals were filtered, at half amplitude, below 1 Hz and above 100 Hz and then amplified 1000 or 2000 times (Grass Neurodata Acquisition System Model 12). The analog signals were digitized at a sampling rate of 5 points/ms and the averaged evoked potential, at each intensity, was stored to computer hard disk (Datawave, CO). Only animals that had stable baseline I/Os were used for experimentation. All I/O measurements were conducted while the animals were immobile. This was necessary because ongoing behaviour can dramatically effect the size and shape of the evoked potentials (Teskey and Valentine, 1998Go).

Following the second I/O, an afterdischarge threshold (ADT) was determined for each animal in the kindling groups. The ADTs were defined as the weakest current required to induce an AD. The current delivered commenced at 100 µA, increasing in steps of 50 µA and was delivered at 30 s intervals until an AD of at least 4 s or longer was recorded. The EEG signal was filtered as described above.

Once-daily kindling stimulation was delivered through the electrode positioned in the callosal white matter. Stimulation consisted of a 1 s train of 60 Hz biphasic rectangular wave pulses, 1 ms in duration and separated by 1 ms, at an intensity 100 µA greater than ADT levels. A paper record of the resultant AD was obtained from both electrodes and the seizure behaviours were noted.

A single follow-up I/O was obtained 24 h after the last kindling session.

Analysis of Electrophysiological Recordings

The AD from the neocortical recording electrode was scored for duration, frequency and amplitude. The seizure behaviours were monitored and scored on a five-point scale (Racine, 1972Go) as well as tonic turning and twisting of the head and torso (Racine, 1975Go).

Evoked potentials obtained before and after neocortical kindling were measured for change in the area of the EPSP late component. The late component was designated as the surface negative component occurring between 12 and 40 ms after stimulation. The late component area score was calculated by summing all the difference values (every 200 µs) from the two evoked potentials, between the two crossing points (12–40 ms), and then dividing by the amount of amplification (Teskey and Valentine, 1998Go). Only potentials evoked with 681 µA were statistically analyzed. Refer to Figure 3AGo for an illustration of the evoked potentials and late component area.



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Figure 3. (A) Representative examples of the neocortical evoked response before and after 25 days of kindling. The responses were evoked with 200 µs biphasic square-wave pulses at an intensity of 681 µA applied to the corpus callosum and recorded in the neocortex for 80 ms. The representative waveforms are averages of 10 sweeps taken from a single animal. The arrow indicates the late component area, bounded by the two curves. Negative current is displayed in the up orientation. (B) The effect of 7 and 25 days of kindling, in male and female rats, on mean (± SEM) late component area. * indicates significantly different, at the 0.05 level, from same-sex sham controls. Male rats had significantly larger late component areas after both 7 and 25 days of kindling as compared to non-kindled controls. Female rats had a significantly larger late component area after 25 days of kindling as compared to non-kindled controls.

 
Perfusion and Histology

One day after the last I/O session, the animals were given a lethal dose of sodium pentobarbital and intracardially perfused with 0.9% saline. The brains were removed and immersed whole in 40 ml of Golgi–Cox solution (Glaser and Van der Loos, 1981Go). The solution was changed after 2 days and the brains remained immersed for at least an additional 20 days before being placed in a 30% sucrose solution for 2 days, cut on a vibratome at 200 µm, and developed using a procedure outlined by Gibb and Kolb (Gibb and Kolb, 1998Go).

Analysis of Dendritic Branching, Length and Spine Density

Layer III pyramidal cells in the frontal neocortex (Fr1), at the electrode tip site in the ipsilateral and in the unimplanted contralateral hemispheres were traced using a camera lucida at 250x under blind conditions. Dendritic trees had to maintain the following criteria to be included in the data analysis: (i) the cells had to be well impregnated and in full view, unblocked by blood vessels, astrocytes or clustering of dendrites from other cells; (ii) the apical and basilar arborizations had to appear intact and visible in the plane of section. Cells were chosen by locating area Fr1 (Zilles, 1985Go) and then by drawing each cell in the section that maintained the above criteria. Following drawing of the cells, each branch segment was counted and summarized by branch order according to the methods of Coleman and Riesen (Coleman and Riesen, 1968Go); as such, basilar dendrites were determined to be first order if the branch originated from the cell body increasing in order with every bifurcation. The branches were determined for the apical dendrites such that branches originating from the primary apical dendrite were first order. Five cells were drawn in each hemisphere of each rat. The mean of the measurements on ten cells per rat was used for statistical analyses. Apical and basilar branches were separated for the statistical analysis since basilar dendrites are believed to receive input from proximal neurons, whereas apical dendrites may receive input from more distal neurons.

A Sholl analysis (Sholl, 1956Go) of ring intersections was used to estimate dendritic length. The number of intersections of dendrites with a series of concentric spheres at 20 µm intervals from the center of the cell body was counted for each cell. Total dendritic length (in µm) can be estimated by multiplying the number of intersections by 20.

Spine density was measured from one apical dendritic branch in the terminal tuft, one secondary apical branch beginning ~50% of the distance between the cell body and terminal tuft, and one from the secondary branch proximal to the cell body for one basilar branch, following the procedure of Woolley et al. (Woolley et al., 1990Go). Spine density measures were made from a segment between 10 and 50 µm in length. The dendrite was traced (1000x) using a camera lucida drawing tube and the exact length of the dendritic segment calculated by placing a thread along the drawing and then measuring the thread length. Spine density was expressed as the number of spines per 10 µm. No attempt was made to correct for spines hidden beneath or above the dendritic segment; therefore the spine density values are likely to underestimate the actual density of the dendritic spines.

Estrous Cycle and Evoked Potentials

Ten additional female Long–Evans hooded rats were cared for and underwent evoked potential and kindling experimentation as described above. They also had their estrous cycle monitored by daily vaginal epithelial smears. Vaginal smears were taken for 1 week prior to electrode implantation, for an additional 1 week following electrode implantation, as well as during pre-kindling I/O determination, 7 days of kindling, and for 8 days during post-kindling I/O determination. Vaginal smears were taken by inserting an eye dropper 7 mm into the vagina and repeatedly flushing with 1 ml of warmed saline. The suspended cells were flushed onto a glass slide and the slide placed onto a slide warmer and desiccated. Slides were stained according to the method of Shorr (Shorr, 1941Go) for 5 min, and then dehydrated in ethanol. The slides were then rinsed in xylene twice, coverslipped and left to dry for several hours. The phase of the cycle was determined by microscopic examination of the cells. The pre-kindled evoked potential maximal amplitude evoked with 681 µA and the post-kindling late component potentials evoked with 681 µA were compared with respect to proestrus, estrus, metestrus and diestrus.

Statistical Testing

Statistical significance was determined with a between-subjects t-test for the ADTs, with a two-factor mixed design analysis of variance (ANOVA) for AD duration, frequency amplitude and behavioural convulsions, and with a one-factor design ANOVA with Fisher's least significant difference post hoc tests for the maximal evoked potential, late component potentiation and dendritic branching, length and spine density measures. All testing was done with an a priori alpha level of 5%.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Afterdischarge Threshold

The mean ADT and associated standard errors for the male and female rats were 216 ± 22.5 and 207 ± 19.5 µA respectively and they were not significantly different from each other.

Afterdischarge Measures

The AD duration, frequency and amplitude all increased with repeated stimulations in both male and female rats (Fig. 1Go).



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Figure 1. Effect of daily kindling stimulation on the mean (± SEM) afterdischarge duration (A), frequency (B) and amplitude (C) over 25 days in male (diamonds) and female (squares) rats. Afterdischarges were elicited from the corpus callosum and recorded in both the corpus callosum and neocortex. Both sexes showed increases in afterdischarge duration and frequency over 25 days of kindling.

 
The 7 day kindling group displayed an initial combined AD duration of 9.33 ± 0.55 s (SEM), which increased to 14.40 ± 1.23 s following the seventh stimulation (data not shown). The 25 day kindling group displayed a comparable initial combined AD duration of 10.03 ± 0.42 s which progressively increased to 25.33 ± 11.90 s following the 25th stimulation (Fig. 1AGo). An ANOVA on the AD duration (Sex x Time) revealed a significant main effect of Time for both the 7 day kindling group [F(6,60) = 3.33, P = 0.0067] and the 25 day kindling group [F(24,240) = 12.49, P < 0.0001]. There was no significant main effect of Sex or a Sex x Time interaction in either the 7 or 25 day kindling groups.

The initial combined AD frequency for the 7 day kindling group was 2.00 ± 0.3 Hz which increased to 3.67 ± 0.33 Hz after the seventh stimulation (data not shown). In the 25 day kindling group, the initial combined AD frequency was 2.6 ± 0.4 Hz which increased to asymptotic levels of 5.55 Hz around stimulation day 15 (Fig. 1BGo). An ANOVA on the AD frequency (Sex x Time) showed a significant main effect of Time for both the 7 day kindling group [F(6,60) = 12.69, P < 0.0001] and the 25 day kindling group [F(24,240) = 16.30, P < 0.0001]. There was no significant main effect of Sex or a Sex x Time interaction in either the 7 or 25 day kindling groups.

The AD amplitude increased slightly with repeated stimulation for both male and female rats. The AD amplitude was also larger, for the first 7 kindling days, in females as compared to males (Fig. 1CGo). An ANOVA of the AD amplitude (Sex x Time) displayed a significant main effect of Time for both the 7 day kindling group [F(6,60) = 4.95, P = 0.0004] and the 25 day kindling group [F(24,240) = 1.69, P = 0.027]. The 7 day kindling group also had a significant main effect of Sex [F(6,60) = 8.38, P = 0.016]. There was no significant main effect of Sex x Time interaction for either the 7 or 25 day kindling groups. There was also no significant main effect of Sex for the 25 day kindling group.

Seizures

Cortical stimulation evoked a stimulation-bound forced motor response in all animals. The animals would roll to one side and display a tonic extension of one or both forelimbs, and an open mouth. Both male and female rats displayed seizure behaviors on the first kindling day. The initial short clonic or focal seizure consisted of mouth movements and forelimb clonus. The focal seizures became more intense with repeated stimulations, with a tonic component developing and eventually a late clonic component that generalized into a limbic-type stage 5 seizure (Fig. 2Go). An ANOVA revealed a significant main effect of Time [F(24,240) = 3.72, P < 0.0001] for the 25 day kindling group. There was no significant main effect of Sex or Sex x Time interaction for the 25 day kindling group, though the main effect of Sex approached statistical significance (P = 0.08). There was no significant main effects of Sex, Time or Sex x Time interaction for the 7 day kindling group.



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Figure 2. Effect of daily kindling stimulation to the corpus callosum on the mean (± SEM) seizure stage over 25 days in male (diamonds) and female (squares) rats. Both sexes showed increases in seizure severity over 25 days of kindling.

 
Evoked Potentials and Kindling-induced Potentiation

Brief stimulation of the corpus callosum gave rise to a stable and reliable waveform composed of an EPSP/IPSP sequence that has been described previously (Racine et al., 1995Go; Chapman et al., 1998Go; Teskey and Valentine, 1998Go). The thresholds for the evoked potentials ranged from 68 to 215 µA and reached maximum levels at 1000 µA. In most animals, the late component of the evoked potential was not clearly present in the prekindling responses but was reliably present after 7 days of kindling (Fig. 3AGo). A comparison of the field potentials and I/O curves before and after 7 and 25 kindling stimulations showed that the largest changes in the late component area were found to be evoked by 484 and 681 µA of current. There were slight decreases in the late component area for both male and female sham groups over 7 and 25 days. In the female 7 day kindling group there was an increase in the late component area in three of the six animals, whereas the other three animals in the group displayed a reduction in the late component area, which accounts for the both the lack of potentiation and large error variance in that group (Fig. 3BGo).

An ANOVA revealed a main effect of kindling treatment [F(5,26) = 3.81, P = 0.01]. The increase in the late component of the evoked response was observed following both 7 days (P = 0.01) and 25 days (P = 0.03) of kindling in male rats and following 25 days (P = 0.007), but not 7 days, of kindling in female rats.

Estrous Cycle and Evoked Potentials

Estrous cycle, as determined by vaginal smear, was not related to evoked potential maximal amplitude in pre-kindled animals. The mean ± SD of evoked potential maximal amplitude, in millivolts, for proestrus, estrous, metestrus and diestrus days was 1.6 ± 0.45, 1.52 ± 0.32, 1.56 ± 0.41 and 1.64 ± 0.41 respectively. An ANOVA showed a nonsignificant difference between the evoked potential maximal amplitude and cycle days. Late component area in the post kindling group was also unrelated to estrous cycle. The mean ± SD of late component area for proestrus, estrous, metestrus and diestrus days was 26.1 ± 2.55, 25.25 ± 3.2, 23.46 ± 3.41 and 24.98 ± 2.76 respectively. An ANOVA showed a nonsignificant difference between the late component area and cycle days.

Seven days of kindling increased the late component in all of the additionally kindled female animals. When the late component data from the additional 7 day kindled females was added to the first group of 7 day kindled females, the mean late component area was positive (7.9 standardized units) but still nonsignificantly different from sham controls.

Dendritic Branching

The general result was that the dendritic branching was decreased in the kindled rats (Fig. 4Go). This was, however, time and sex dependent (Fig. 5Go). Females with 7 days of stimulation had a significant reduction in dendritic branching and length relative to shams and males with 7 days of stimulation. In contrast males with 25 days of stimulation were significantly reduced relative to sham and females with 25 days of stimulation. Thus, although both male and female kindled rats showed a decrease in dendritic arborization, the females showed a drop earlier than the males and then apparently regrew the lost dendrites.



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Figure 4. Camera lucida drawings of representative layer III pyramidal neurons and spines in Zilles' area Fr1 from (A) a female sham and (B) a female following 7 days of kindling. Neurons from kindled brains show fewer dendritic branches, shorter dendrites and reduced terminal tip spine density as compared with non-kindled controls. Arrows indicate the dendritic branch from where the spines were drawn.

 


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Figure 5. Summary of the effects of 7 and 25 days of kindling in male and female rats on the number of apical and basilar dendritic branches, for layer III pyramidal cells in Zilles' area Fr1. Male rats showed significantly fewer apical and basilar dendritic branches after 25 days, but not 7 days, of kindling, whereas female rats showed significantly fewer apical and basilar dendritic branches after 7 days, but not 25 days, of kindling. Histobars represent the mean (± SEM) and * indicates significantly different, at the 0.05 level, from same-sex sham controls.

 
Separate ANOVAs were performed on the apical and basilar fields for both the dendritic branching measure, as well as the Sholl analysis. An ANOVA on dendritic branching data showed a main effect of kindling for both the apical [F(5,24) = 9.35, P < 0.001] and basilar fields [F(5,24) = 7.76, P < 0.001]. There was a significant decrease in apical and basilar dendritic branching in the male 25 day (P < 0.05) and female 7 day (P < 0.05) kindling groups, but the male 7 day and female 25 measures were not significantly different, as compared to same-sex sham controls.

Dendritic Length

Like dendritic branching, the dendritic length showed a decrease in the kindled brains that was sex and time dependent (Fig. 6Go). In contrast to the branching measure, both the males and females showed a decline in dendritic length after 7 days of kindling, and the decline was larger in the females than the males. By 25 days the females had completely reversed their dendritic length loss, whereas the males had not, although the males did show a significant reversal.



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Figure 6. Summary of the effects of 7 and 25 days of kindling in male and female rats on the length of apical and basilar dendrites, using Sholl analysis (20 µm/ring), for layer III pyramidal cells in Zilles' area Fr1. Male rats showed significantly shorter apical and basilar dendrites after 7 days, and shorter basilar dendrites after 25 days of kindling. Female rats showed significantly shorter dendrites after 7 days, but not 25 days, of kindling. Histobars represent the mean (± SEM) and * indicates significantly different, at the 0.05 level, from same-sex sham controls.

 
An ANOVA on the apical dendrite length revealed a significant main effect [F(5,24) = 11.02, P = 0.0001]. Post hoc comparisons showed significantly decreased apical dendritic length in both the male (P < 0.05) and female (P < 0.05) 7 day kindling groups, but not the male and female 25 day kindling groups as compared to same sex sham controls (Fig. 5AGo).

An ANOVA on the basilar dendrite length showed a significant main effect [F(5,24) = 12.85, P = 0.0001]. Post hoc comparisons showed significantly decreased basilar dendritic length in the male 7 day kindling group (P < 0.05) and male 25 day kindling group (P < 0.05), as well as in the female 7 day kindling group (P < 0.05), but not the female 25 day kindling group, as compared to same sex sham controls (Fig. 5BGo).

Dendritic Spine Density

The spine density changes were parallel to the dendritic branching results. Both male and female rats showed a decrease in spine density but the spine changes were time dependent (Fig. 7Go). Females showed a drop in spine density after 7 days of kindling and this reversed to control levels by 25 days. In contrast, males showed no change at 7 days, but a significant drop by day 25.



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Figure 7. Summary of the effects of 7 and 25 days of kindling in male and female rats on the number of apical and basilar terminal tip spine density, per 10 µm length, for layer III pyramidal cells in Zilles' area Fr1. Male rats showed significantly reduced apical and basilar terminal tip spine density after 25 days, but not 7 days, of kindling. Female rats showed significantly reduced apical and basilar terminal tip spine density after 7 days, but not 25 days, of kindling. Histobars represent the mean (±SEM) and * indicates significantly different, at the 0.05 level, from same-sex sham controls.

 
An ANOVA on the apical spine density was significant [F(5,24) = 104.15, P = 0.0001]. Post hoc comparisons showed that the apical spine density for both the male 25 day and female 7 day kindling groups had significantly (P <0.05) reduced spine density, whereas the male 7 day and female 25 day kindling groups did not, as compared to same-sex sham controls (Fig. 7AGo).

Similar results were obtained for the basilar dendrite spine density. An ANOVA on the basilar spine density was significant [F(5,24) = 32.86, P = 0.0001]. Post hoc comparisons showed that the basilar spine density for both the male 25 day and female 7 day kindling groups had significantly (P < 0.05) reduced spine density, whereas the male 7 day and female 25 day kindling groups did not, as compared to same sex sham controls (Fig. 7BGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present results have shown that the repeated elicitation of AD in the anterior neocortex of male and female rats leads to progressively enhanced AD duration, frequency and amplitude, and more severe behavioral seizures, as well as synaptic potentiation of the evoked potential late component, and that this last observation was not related to estrous cycle. In association with the changes in the electrographic and behavioral measures, we also observed sexand time-specific decreases in apical and basilar dendritic branching, length and spine density measures of Golgi–Cox impregnated layer III pyramidal cells from neocortical area Fr1.

Kindling and Kindling-induced Potentiation

The kindling and kindling-induced potentiation results confirm previous observations and extend them to female rats. Our observations of ADTs in the 200 µA range, a stimulation bound forced motor response, initial short clonic or focal seizures which, with repeated stimulations, developed into generalized limbic-type seizures, were in general agreement with those made previously (Racine et al., 1975Go, 1995Go; Burnham, 1978Go; Seidel and Corcoran, 1986Go). However, our observations differed from those made by Seidel and Corcoran (Seidel and Corcoran, 1986Go) in two respects. We did not observe any failures to elicit an AD and we observed the seizure development to be relatively stable. These differences may have been the result of the location of our stimulating electrode in the corpus callosum on the midline, rather than in the neocortical deep layers, 2.5 mm lateral from midline, as used by Seidel and Corcoran (Seidel and Corcoran, 1986Go). This is the first report of kindling-induced potentiation of the neocortical evoked late component in female rats.

Sex Differences in Kindling and Kindling-induced Potentiation

Female rats displayed three differences in their kindling results compared to males. Female rats had slightly larger AD amplitudes over 7 days of kindling, more severe seizures and no potentiation of the late component after 7 days of kindling.

The larger amplitude AD observed in the female rats may simply be due to minor variation in electrode placement. On the other hand, the larger amplitude AD may be the result of an equal number of cells packed into a thinner neocortex (Juraska, 1990Go). It is possible that a more densely packed neocortex could generate a larger signal over the fixed size of the bipolar electrode. A comparative current source density analysis is needed to answer this question.

We also observed that female rats had more severe seizures as compared to male rats. It has long been known that exogenously administered estrogen has a proconvulsant effect in rats (Werboff and Corcoran, 1961Go) and that female human epileptics are more prone to seizures and have more severe seizures during the time of high endogenous levels of circulating estrogen (Laidlaw, 1956Go). While our results did not quite reach statistical significance, they are in apparent agreement with those of Buterbaugh, who observed that the female hormone estradiol accelerated the rate of seizure acquisition to anterior neocortical kindling and advanced the onset of generalized seizures compared to rats without estradiol (Buterbaugh, 1989Go).

The failure of the female 7 day kindling group to show a kindling-induced potentiation effect may be due to greater inhibition in the female neocortex. The neocortex in the awake, freely moving rat has been shown to be highly resistant to potentiation effects. Several days of stimulations are usually required before potentiation phenomenon are first observed (Teskey and Valentine, 1998Go). Therefore it is possible that seven stimulations were insufficient to produce a reliable potentiation effect in the female rats. The stability of the evoked response measures examined in the additional female rats determined that a minimal and insignificant amount of variance was due to normal estrous cyclicity and that estrous cycle was not responsible for the failure to find late component potentiation in all animals. Furthermore, the additional 7 day kindled female animals all showed late component potentiation effects, which suggests that the failure to observe a significant late component potentiation effect in the original 7 day kindled female animals was probably due to some unknown factor in a subset of those animals.

Dendritic Morphology

It has been suggested that dendritic spines and a larger dendritic area serve to increase the available surface for synaptic contact, thereby permitting higher levels of neuronal connectivity (Ramon y Cajal, 1911Go). Thus, one might predict increased dendritic branching, length and spine density as the result of the increased electrographic and behavioral seizure severity and synaptic potentiation phenomena we observed. However, in the hippocampus, dendritic spines are known to regress and regrow in response to the onset and cessation of epileptic seizures (Scheibel et al., 1974Go; Isokawa and Levesque, 1991Go; Müller et al., 1993Go; Jiang et al., 1998Go). Other researchers have found decreases in the number of axospinous synapses in the hippocampus (Geinisman et al., 1988Go, 1990Go) and in the number of shaft and spine synapses in the amygdala following electrical kindling of those structures (Nishizuka et al., 1991Go; Okada et al., 1993Go). Thus, while the relationship between dendritic morphology and epileptic activity may jibe with common-sense expectations, our results are consistent with those from the hippocampus and amygdala.

In the only previously published study that investigated the effect of anterior neocortical kindling on the morphological characteristics of dendrites, Racine and co-workers examined cells in layers III and V in the immediate vicinity of the electrode tracks after 20 days of kindling in male rats (Racine et al., 1975Go). Racine measured the number of dendritic spines, and apical dendritic branching, and found no differences between kindled and control rats (Racine et al., 1975Go). Perhaps the apparent disparity in the results between the earlier study and this present experiment can be account for by methodological differences. We restricted our analysis to layer III, which receives direct callosal input, in contrast to layer V, which receives minimal direct callosal input. We were also careful to restrict our examination to area Fr1 regardless of the location of the electrodes because in our material the dendritic morphology can change widely from one neocortical area to the next. It also appears that our staining procedure was more sensitive because our branching was more extensive. Perhaps Racine et al.’s staining was incomplete, leading them to find no kindlingrelated changes (Racine et al., 1975Go). Finally, it is possible that more than 20 kindling stimulations are required, in male rats, before measurable changes occur in dendritic branching and spine density.

It may be the case that the increased excitatory drive associated with kindling is manifested at the synaptic ultrastructural level. While kindling may lead to decreased synapse number there is anatomical evidence that the surviving synapses may become more efficacious. Racine and Zaide examined the size of synaptic terminals 2 weeks after neocortical kindling (Racine and Zaide, 1978Go). They reported that kindled tissue showed larger synaptic terminals and more synaptic terminals that appeared to ‘wrap around’ dendritic spines. Geinisman et al. reported a 12% increase in the maximal profile length and a 19% increase in mean area of post-synaptic density (PSD) of the perforated synapses (Geinisman et al., 1990Go). Finally, Hovorka et al. reported a long-term increase in preand post-synaptic swelling and the size of the vesicles at or near the terminal zone (Hovorka et al., 1997Go). They interpret this redistribution of synaptic vesicles to the immediate vicinity of the synaptic cleft as a possible mechanism responsible for the increased excitability associated with kindling. This evidence suggests that, while overall number of synapses may be decreasing with kindling, the remaining synapses may be more efficacious.

Sex Differences in Dendritic Morphology

There is now considerable evidence that the dendritic organization of neocortical neurons is sexually dimorphic (Kolb and Stewart, 1991Go), that gonadal hormones influence cortical functional development (Clark and Goldman-Rakic, 1989Go) and that changes in dendritic organization after experience or injury are sexually dimorphic [for a review, see Kolb et al. (Kolb et al., 1998Go)]. In the current study, there were no differences between the cells in the male and female sham animals, but there was a clear sexually dimorphic effect of brain stimulation. These sexrelated changes correlate with the behavioral sex differences, thus lending support to the conclusion that the dendritic and spine changes reflect the effect of the brain stimulation on behavior. One surprising result is that because the pyramidal neurons in the female brain changed more rapidly than those in the male brain, the neurons in the female cortex appear to be more sensitive to the affect of brain stimulation than those in the male cortex. Previous studies have shown that the male, rather than the female, cortex changes faster and more extensively in response to environmental stimulation and injury [for a review, see Kolb et al. (Kolb et al., 1998Go)]. Evidently the plastic changes in response to brain stimulation leading to seizures is different to the changes in response to other forms of experience. Future studies will compare ovariectomized and ovariectomized plus estrogen-primed animals to identify the role that estrogen may play in brain-stimulation induced cortical plasticity.


    Notes
 
We thank R. Gibb, G. Gorny and M.H. Monfils for technical assistance. Supported by NSERC of Canada grants to G.C.T. and B.K., and by an AHFMR scholarship to J.E.H.

Address correspondence to Dr G.C. Teskey, Department of Psychology, University of Calgary, Calgary, Alberta, Canada T2N 1N4. Email: gteskey{at}ucalgary.ca.


    References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Armitage LL, Mohapel P, Jenkins EM, Hannesson DK, Corcoran ME (1998) Dissociation between mossy fiber sprouting and rapid kindling with low-frequency stimulation of the amygdala. Brain Res 781:37–44.[ISI][Medline]

Burnham WM (1978) Cortical and limbic kindling: similarities and differences. In: Limbic mechanisms (Livingston KE, Hornykiewicz O, eds), pp. 507–519. New York: Plenum.

Buterbaugh GG (1989) Estradiol replacement facilitates the acquisition of seizures kindled from the anterior neocortex in female rats. Epilepsy Res 4:207–215.[ISI][Medline]

Cain DP (1989) Long-term potentiation and kindling: how similar are the mechanisms? Trends Neurosci 12:6–10.[ISI][Medline]

Chapman CA, Trepel C, Ivanco TL, Froc DJ, Wilson K, Racine RJ (1998) Changes in field potentials and membrane currents in rat sensorimotor cortex following repeated tetanization of the corpus callosum in vivo. Cereb Cortex 8:730–742.[Abstract]

Clark AS, Goldman-Rakic PS (1989) Gonadal hormones influence the emergence of cortical function in nonhuman primates. Behav Neurosci 103:1287–1295.[ISI][Medline]

Coleman PD, Riesen AH (1968) Environmental effects on cortical dendritic fields: I Rearing in the dark. J Anat 102:363–374.[ISI][Medline]

deJonge M, Racine RJ (1987) The development and decay of kindlinginduced increases in paired-pulse depression in the dentate gyrus. Brain Res 412:318–328.[ISI][Medline]

Dennison Z, Teskey GC, Cain DP (1995) Persistence of kindling: effect of partial kindling, retention interval, kindling site and stimulation parameters. Epilepsy Res 21:171–182.[ISI][Medline]

Geinisman Y, Morrell F, deToledo-Morrell L (1988) Remodelling of synaptic architecture during hippocampal ‘kindling’. Proc Natl Acad Sci USA 85:3260–3264.[Abstract]

Geinisman Y, Morrell F, deToledo-Morrell L (1990) Increase in the relative proportion of perforated axospinous synapses following hippocampal kindling is specific for the synaptic field of stimulated axons. Brain Res 507:325–331.[ISI][Medline]

Geinisman Y, Morrell F, deToledo-Morrell L (1992) Increase in the number of axospinous synapses with segmented postsynaptic densities following hippocampal kindling. Brain Res 569:341–357.[ISI][Medline]

Gibb R, Kolb B (1998) A method for Golgi–Cox staining of vibratome cut tissue. J Neurosci Methods 79:1–4.[ISI][Medline]

Glaser EM, van der Loos H (1981) Analysis of thick brain sections by obverse–reverse computer microscopy: application of a new, high clarity, Golgi–Nissl stain. J Neurosci Methods 4:117–125.[ISI][Medline]

Goddard GV., McIntyre D, Leech C (1969) A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol 3:538–544.

Greenough WT, Volkmar FR, Juraska J (1973) Effects of rearing complexity on dendritic branching in frontolateral and temporal cortex of the rat. Exp Neurol 40:371–378.

Greenough WT, Larson JR, Withers GS (1985) Effects of unilateral and bilateral training in a reaching task on dendritic branching of neurons in the rat motor-sensory forelimb cortex. Behav Neural Biol 44:301–314.[ISI][Medline]

Hawrylak N, Chang F-LF, Greenough WT (1993) Astrocytic and synaptic response to kindling in hippocampal subfield CA1. II. Synaptogenesis and astrocytic process increases to in vivo kindling. Brain Res 603:309–316.[ISI][Medline]

Hovorka J, Langmeier M, Mares P, Koryntova C (1997) Synaptic vesicle size and shape profile in the kindling model of epileptogenesis. Epilepsy Res 28:225–231.[ISI][Medline]

Isokawa M, Levesque MF (1991) Increased NMDA responses and dendritic degeneration in human epileptic hippocampal neurons in slices. Neurosci Lett 132:212–216.[ISI][Medline]

Jiang M, Lee CL, Smith KL, Swann JW (1998) Spine loss and other persistent alterations of hippocampal pyramidal cell dendrites in a model of early-onset epilepsy. J Neurosci 18:8356–8368.[Abstract/Free Full Text]

Juraska J (1990) The structure of the cerebral cortex: Effects of gender and the environment. In: The cerebral cortex of the rat (Kolb B, Tees R, eds), pp. 483–506. Cambridge, MA: MIT Press.

Kolb B, Gibb R (1991a) Environmental enrichment and cortical injury: Behavioural and anatomical consequences of frontal cortex lesions. Cereb Cortex 1:189–191.[Abstract]

Kolb B, Gibb R (1991b) Sparing of function after neonatal frontal lesions correlates with increased cortical dendritic branching: a possible mechanism for the Kennard effect. Behav Brain Res 43:51–56.[ISI][Medline]

Kolb B, Stewart J (1991) Sex-related differences in dendritic branching of cells in the prefrontal cortex of rats. J Neuroendocrinol 3:95–99.[ISI]

Kolb B, Stewart J (1995) Changes in neonatal gonadal hormonal environment prevent behavioural sparing and alter cortical morphogenesis after early frontal cortex lesions in male and female rats. Behav Neurosci 109:285–294.[ISI][Medline]

Kolb B, Sutherland RJ (1992) Noradrenaline depletion blocks behavioural sparing and alters cortical morphogenesis after neonatal frontal cortex damage in rats. J Neurosci 12:2321–2330.[Abstract]

Kolb B, Whishaw IQ (1991) Mechanisms underlying behavioural sparing after neonatal retrosplenial cingulate lesions in rats: spatial navigation, cortical architecture, and electroencephalographic activity. Brain Dysfunc 4:75–92.

Kolb B, Forgie M, Gibb R, Gorny G, Rowntree S (1998) Age, experience, and the changing brain. Neurosci Biobehav Rev 22:143–159.[ISI][Medline]

Laidlaw J (1956) Catamenial epilepsy. Lancet 271:1235–1237.[ISI]

Müller M, Gähwiler BH, Rietschin L, Thompson SM (1993) Reversible loss of dendritic spines and altered excitability after chronic epilepsy in hippocampal cultures. Proc Natl Acad Sci USA 90:257–261.[Abstract]

Nishizuka M, Okada R, Seki K, Arai Y, Iizuka R (1991) Loss of dendritic synapses in the medial amygdala associated with kindling. Brain Res 552:351–355.[ISI][Medline]

Okada R, Nishizuka M, Iizuka R, Arai Y (1993) Persistence of reorganized synaptic connectivity in the amygdala of kindled rats. Brain Res Bull 31:631–635.[ISI][Medline]

Racine RJ (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroenceph Clin Neurophysiol 32:281–294.[ISI][Medline]

Racine RJ (1975) Modification of seizure activity by electrical stimulation: cortical areas. Electroenceph Clin Neurophysiol 38:1–12.[ISI][Medline]

Racine RJ, Zaide J (1978) A further investigation into the mechanisms underlying the kindling phenomenon. In: Limbic Mechanisms, (Livingston K, Hornykiewicz O, eds), pp. 457–493. New York: Plenum.

Racine RJ, Tuff L, Zaide J (1975) Kindling, unit discharge patterns, and neural plasticity. Can J Neurol Sci 2:395–406.[Medline]

Racine RJ, Moore K-A, Evans C (1991) Kindling-induced potentiation in the piriform cortex. Brain Res 556:218–225.[ISI][Medline]

Racine RJ, Chapman CA, Teskey GC, Milgram NW (1995) Post-activation potentiation in the neocortex: III. Kindling-induced potentiation in the chronic preparation. Brain Res 702:77–86.[ISI][Medline]

Ramon y Cajal S (1911) Histologie du système nerveux de l’homme et des vertèbrès, vol. II. Paris: Moloine.

Robinson TE, Kolb B (1997) Persistent structural adaptations in nucleus accumbens and prefrontal cortex neurons produced by prior experience with amphetamine. J Neurosci 17:8491–8498.[Abstract/Free Full Text]

Scheibel T, Crandall PH, Scheibel AB (1974) The hippocampal dentate complex in temporal lobe epilepsy. Epilepsia 15:55–80.[ISI][Medline]

Seidel WT, Corcoran ME (1986) Relations between amygdaloid and anterior neocortical kindling. Brain Res 385:375–278.[ISI][Medline]

Sholl DA (1956) The organization of the cerebral cortex. London: Methuen.

Shorr E (1941) A new technique for staining vaginal smears: a single differential stain. Science 94:545.

Sirevaag AM, Greenough WT (1988) A multivariate statistical summary of synaptic plasticity measures in rats exposed to complex, social and individual environments. Brain Res 441:386–392.[ISI][Medline]

Stasheff SF, Wilson WA (1990) Increased ectopic action potential generation accompanies epileptogenesis in vitro. Neuroscience 111:144–150.

Stewart J, Kolb B (1994) Dendritic branching in cortical pyramidal cells in response to ovariectomy in adult female rats: suppression by neonatal exposure to testosterone. Brain Res 654:149–154.[ISI][Medline]

Sutula, T (1990) Experimental models of temporal lobe epilepsy: new insights from the study of kindling and synaptic reorganization. Epilepsia 31(Suppl. 3):S45–S54.

Swanson LW (1992) Brain maps: structure of the rat brain. New York: Elsevier.

Teskey GC, Racine RJ (1993) Increased spontaneous unit discharge rates following electrical kindling in the rat. Brain Res 624:11–18.[ISI][Medline]

Teskey GC, Valentine PA (1998) Post-activation potentiation in the neocortex of awake freely moving rats. Neurosci Biobehav Rev 22:195–207.[ISI][Medline]

Werboff J, Corcoran JB. (1961) Effects of sex hormone manipulation on audiogenic seizures. Am J Physiol 201:830–832.

Withers GS, Greenough WT (1989) Reaching training selectively alters dendritic branching in subpopulations of layer II–III pyramids in rat motor-somatosensory forelimb cortex. Neuropsychologia 27:61–69.[ISI][Medline]

Woolley CS, Gould E, Frankfurt M, McEwen BS (1990) Naturally occurring fluctuations in dendritic spine density on adult hippocampal neurons. J Neurosci 10:4035–4039.[Abstract]

Zilles K (1985) The cortex of the rat: a stereotaxic atlas. New York: Springer-Verlag.