European Journal of Neuroscience
European Journal of Neuroscience, Vol. 34, pp. 615–621, 2011
doi:10.1111/j.1460-9568.2011.07780.x
NEUROSYSTEMS
Larger cortical motor maps after seizures Amy K. Henderson,1,2 Michael A. Galic,1,3 Karim Fouad,4 Richard H. Dyck,1,2,5 Quentin J. Pittman1,3 and G. Campbell Teskey1,2,3,5 1
Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Dr. N.W. Calgary, Alberta, Canada, T2N 4N1 Department of Psychology, University of Calgary, Alberta, Canada 3 Department of Physiology & Pharmacology, University of Calgary, Alberta, Canada 4 Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, Alberta, Canada 5 Department of Cell Biology and Anatomy, University of Calgary, Alberta, Canada 2
Keywords: cortex, epilepsy, Long Evans Hooded rat, movement representation, plasticity
Abstract Expansion of motor maps occurs in both clinical populations with epilepsy and in experimental models of epilepsy when the frontal lobes are involved. We have previously shown that the forelimb area of the motor cortex undergoes extensive enlargement after seizures, although the extent to which many movement representation areas are altered is not clear. Here we hypothesize that movement representations in addition to the forelimb area will be enlarged after cortical seizures. To test our hypotheses, Long Evans Hooded rats received 20 sessions of callosal (or sham) kindling, and then were subjected to intracortical microstimulation to map several movement representations including the jaw, neck, forelimb, hindlimb, trunk and tail. We found significantly larger total map areas of several movement representations, including movements that could be evoked more posterior than they are in control rats. We also show the presence of more multiple movement sites and lower movement thresholds in kindled rats, suggesting that movements not only overlap and share cortical territory after seizures, but become present in formerly non-responsive sites as they become detectable with our intracortical microstimulation methodology. In summary, several motor map areas become larger after seizures, which may contribute to the interictal motor disturbances that have been documented in patients with epilepsy.
Introduction Motor maps are a representation of the organization of movement in the neocortex. They are composed of cortical regions arranged in a mosaic pattern that are devoted to controlling movements of specific body parts (Kaas, 1997). Motor map organization can be altered by cortical injury (Nudo & Milliken, 1996), motor skill learning (Kleim et al., 1998), tactile experience (Keller et al., 1996), high-frequency electrical stimulation resulting in long-term potentiation (Flynn et al., 2004; Monfils et al., 2004) and seizures (Teskey et al., 2002). Some individuals with epilepsy have atypically organized motor maps. Movement evoked outside the classic motor strip (Uematsu et al., 1992), abnormal bilateral brain activity (Stoeckel et al., 2002) and interhemispheric asymmetry in inhibition (Hamer et al., 2005) have all been described in individuals with epilepsy. Similarly, we have shown that the forelimb area of the rat can significantly enlarge following electrically induced seizures elicited by stimulation of the corpus callosum, amygdala (Teskey et al., 2002) and hippocampus (van Rooyen et al., 2006), and following chemically induced seizures (Young et al., 2009), as long as the epileptiform discharges involve the motor cortex. Motor map enlargement is probably mediated, in part, by enhancement of synaptic efficacy because after seizures, map size is highly correlated with larger evoked potentials in the Correspondence: Dr. G. Campbell Teskey, as above. E-mail:
[email protected] Received 9 May 2011, accepted 26 May 2011
sensorimotor neocortex elicited by single-pulse electrical stimulation to the corpus callosum (Teskey et al., 2002). Despite numerous studies showing enlargement of the forelimb region after seizures, the extent to which other movement representations are influenced by prior seizures has not been examined. Knowledge of how the entire motor cortex is changing after seizures may be important not only for the clinicians who treat the sub-population of patients with epilepsy who have abnormal motor maps, but also to provide a foundation for the eventual target of preventing motor map reorganization in patients with epilepsy. Here we tested the hypothesis that several movement representations in addition to the forelimb area would be enlarged after seizures, and would be accompanied by lower movement thresholds in the motor cortex.
Materials and methods Animals Sixteen adult male Long-Evans Hooded rats were used in this study (n = 7 control, n = 9 kindled). They were obtained from the University of Calgary breeding colonies, and were housed individually under standard laboratory conditions where food and water were always available. The colony room was maintained on a 12-h on ⁄ off cycle, with lights on at 07:00 h, and all experimentation was conducted during the light phase. All protocols were approved by
ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
616 A. K. Henderson et al. the University of Calgary animal care committee and were in accordance with the Canadian Council on Animal Care guidelines.
Implantation of chronic electrodes Teflon-coated stainless steel wire (178 lm in diameter, A-M Systems, Everett, WA, USA) was twisted to produce bipolar stimulating and recording electrodes. Gold-plated amphenol pins were connected to one end of the wires that were stripped of insulation, and the other ends were separated by 1.0 mm. During the surgical procedure rats were anaesthetized using isoflurane (3% induction and 1.5–2.5% maintenance), and anaesthesia levels were monitored by checking the response to a tail pinch. Lidocaine 2% was administered subcutaneously at the incision site to further minimize any discomfort. Two bipolar electrodes were permanently implanted into the right hemisphere according to the stereotaxic coordinates of Swanson (1992). One electrode was placed in the corpus callosum (1.0 mm anterior to bregma, 0.5 mm lateral to midline and lowered to a depth that optimized the evoked potential: 3 mm), and one in the sensorimotor neocortex (1.0 mm anterior to bregma, 4.0 mm lateral to midline and 1.5 mm ventral to the brain surface). The amphenol pins were then inserted into a nine-pin McIntyre connector plug (Ginder Science, Ottawa, ON, Canada) and secured to the skull with dental cement and five stainless steel screws. One of the screws served as the ground reference. The left hemisphere remained free of electrodes, screws and cement to permit the craniotomy required during the intracortical microstimulation (ICMS) procedure. Experimental procedures commenced 7 days after electrode implantation. All rats were implanted with chronic electrodes.
Kindling The kindling protocol was derived from Teskey et al. (2002). Briefly, after-discharge (AD) thresholds were determined for each rat assigned to the kindled group (n = 7) by finding the weakest current capable of producing an AD. Current was administered through the electrode tip placed in the corpus callosum. The stimulation consisted of a 1-s train of 60-Hz biphasic rectangular wave pulses, 1 ms in duration, separated by 1 ms. To determine the AD threshold, current intensity began at 50 lA and was increased by 50 lA every 60 s until an AD of ‡ 4 s was seen on the callosal or cortical electroencephalogram (EEG). Once this intensity was determined, daily stimulation was delivered at an intensity of 100 lA greater than the AD threshold for the duration of the kindling sessions. Stimulation was given once a day, 5 days per week, over 4 weeks. The control rats (n = 17) were placed in the recording chamber, but no stimulation was given at any time. EEG was recorded from the callosal and neocortical electrodes in kindled rats, where the duration of the longest AD recorded from either the callosal or the neocortical electrode was measured and recorded for each seizure elicited. Seizure behaviors were scored and recorded according to a five-stage seizure scale (Racine, 1972). To assess the extent of seizure spread throughout the motor cortex, we implanted two additional rats with a chronic stimulating electrode in the corpus callosum (1.0 mm anterior to bregma, 0.5 mm lateral to midline and lowered to a depth that optimized the evoked potential: 3 mm), one recording electrode at the most rostral point of a typical enlarged motor map that is found after seizures (5 mm anterior to Bregma, 1 mm lateral to midline and lowered to layer 5, 1.5 mm), one recording electrode at the most lateral point of an enlarged motor map (1 mm anterior to Bregma, 5 mm lateral to midline and lowered to 1.5 mm) and one recording electrode at the most posterior point of an
enlarged motor map ()3 mm posterior to Bregma, 1 mm lateral to midline and lowered to 1.5 mm from brain surface). All chronically implanted electrodes described above were located in the right hemisphere. Seizures were then elicited in an acute setting by passing current through the stimulating electrode and were recorded from each of the three recording electrodes. Signals were visualized with a Hitashi Digital storage Oscilloscope (VC-6023), were filtered below 0.1 Hz and above 0.3 kHz, and were amplified 5000 times (Grass QP511 Quad AC Amplifier).
Intracortical microstimulation Fourteen rats (n = 7 control, n = 7 kindled) underwent standard ICMS procedures to derive detailed maps of movement representations in the neocortex within 1 week after the last day of kindling or the control procedure (Nudo et al., 1990; Kleim et al., 1998; Teskey et al., 2007). Twelve hours prior to surgery, rats were food restricted, but had water available ad libitum. On the day of surgery, an intraperitoneal injection of ketamine (100 mg ⁄ kg) and xylazine (5 mg ⁄ kg) was given. Subsequent injections of ketamine (25 mg ⁄ kg) or a cocktail of both ketamine (17 mg ⁄ kg) and xylazine (2 mg ⁄ kg) were delivered as needed during the surgery to maintain a constant level of anesthesia, monitored by observing changes in breathing rate, vibrissae whisking and response to a pinch of the hindlimb or tail. The amount of anesthetic given throughout the surgical procedure was recorded and expressed as a function of body weight and duration of surgery (ml ⁄ kg ⁄ min) for each animal. A 9 · 5-mm craniotomy was performed to expose the sensorimotor neocortex of the left hemisphere. The exposed area extended 5 mm anterior and 4 mm posterior from bregma and 5 mm lateral to midline. A small puncture was made in the cisterna magna with an 18-gauge needle to reduce intracranial pressure. The dura was subsequently removed and a silicone fluid at 37 C (Factor II, Inc., Lakeside, AZ, USA) was used to bathe the cortical surface. A 32 · digital image of the brain surface was acquired with a Stemi 2000-C stereomicroscope (Zeiss) and a digital camera. The image was displayed on a microcomputer and a grid composed of 500-lm squares was superimposed on the image. Electrode penetration locations were chosen at both the intersections of the grid lines and at the center point of each square resulting in an interpenetration distance of 353 lm. Potential points located over a blood vessel were avoided. Borosilicate glass capillary microelectrodes were constructed using a micropipette puller (Kopf, Tujunga, CA, USA), bevelled at a 30 angle to form a 3-lm tip, and filled with 3.5 m NaCl, resulting in impedance values between 1.0 and 1.5 MX. A microdrive (Narishige, Tokyo, Japan) was used to guide the electrode to its penetration points and to a depth of 1550 lm from the brain surface, which corresponds to the cell-body region of neocortical layer V. Electrical pulses were delivered through an isolated stimulator (A-M Systems, Carlsborg, WA, USA) and consisted of 13 monophasic cathodal pulses, where each pulse was 200 ls in duration, and delivered at a frequency of 300 Hz, repeated every second which has been validated to be the optimal stimulation parameters in rats (Young et al., 2011). While the rat was in the prone position, the forelimb contralateral to the stimulation side was elevated by supporting it below the elbow joint. This allowed easy visual detection of all possible forelimb movements involving the digits, wrist, elbow and shoulder. The entire body was monitored at each point to also detect non-forelimb movements. The minimal threshold required to elicit a movement at each penetration site was recorded, and a color-coded marker was placed on the acquired digital image. In the cases where two
ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 34, 615–621
Motor map enlargement after kindling 617 movements were elicited at the same site (dual movement sites), the movement with the lowest threshold was recorded on the digital image. To be considered a dual movement site, the two movements must be evoked within ± 10 lA from each other. Non-responsive sites were defined as those where no movement was elicited at a current up to 60 lA. The determination of movement threshold began when the current was rapidly increased from 0 to 60 lA allowing the detection of movement, and then decreased until the movement was no longer detectable. This procedure was done quickly to ensure that no more than ten trains of pulses were delivered at a single penetration site. The entire border of the motor map encompassing several movement representations was defined prior to its interior mapping to reduce the likelihood of the ICMS procedure affecting the border points of the map (Nudo et al., 1990). These points were probed in an outward and clockwise fashion until a non-responsive point was detected. Subsequently, possible movements located outside of the entire border (such is the case for the hindlimb area in most rats) were examined. All border points consisted of non-responsive points. The level of anesthesia and thresholds to evoke movement were constantly monitored as described above as the mapping procedure progressed. After the motor map was generated, rats were killed with an overdose of sodium pentobarbital. Brains were removed, and placement of the electrodes was verified. ICMS was also performed on the two rats that were implanted with chronic stimulating and recording electrodes to assess seizure propagation across the cortex. After the craniotomy was performed under ketamine ⁄ xylazine anesthesia as described above, the rats were subsequently maintained on urethane anesthesia (2 g ⁄ 10 ml saline, 0.1 ml given every 20 min) for the duration of the recordings to permit seizure activity under anesthetic. The movement that was evoked at each site where an AD was recorded in the acute setting was recorded on the digital image.
Movement representation analysis To calculate the area of neocortex devoted to each movement, Canvas imaging software (version 9.0.1; ACD Systems Inc., Miami, FL, USA) was used. All movement representation areas were analysed independently. A separate analysis was also done to compare the map areas of the caudal forelimb area (CFA), the rostral forelimb area (RFA) and the posterior forelimb area (PFA). In some rats, the RFA and PFA merge with the CFA so a determination of the boundaries of those areas was made on a case-by-case basis. The PFA was defined to include any forelimb movements that were evoked more posterior than )0.5 mm to Bregma based on the organization of control motor maps. The proportion of total map area comprising each movement type was calculated, as well as the percentage change in motor map size for each movement representation after kindling. Percentage change was calculated by subtracting the control movement area from the kindled movement area, dividing by the control movement area and multiplying by 100. For example, a 100% increase in map size would indicate a motor map that has doubled in size. Map areas are expressed in mm2.
sample sizes. In the cases where there were only two samples per movement type within a group, statistical analyses were not performed. Data are presented as mean ± SEM. The Statistical Package for the Social Sciences (version 15.0) was used to analyse the data.
Results Kindling and its effects on neocortical movement representations Kindled rats showed the expected increase in seizure stage (Fig. 1A) and increase in AD duration (Fig. 1B) over the 20 kindling sessions, and seizures were elicited after every stimulation session (clear evidence of both an AD and a behavioral seizure according to a fivestage seizure scale) (Racine, 1972). During the ICMS procedure, the total amounts of anesthetic used did not differ significantly between the groups. The amounts of ketamine administered was 0.0139 ± 0.0013 ml ⁄ kg ⁄ min for the controls and 0.0107 ± 0.0007 ml ⁄ kg ⁄ min for the kindled rats (t13 = 1.94, P > 0.05). Similarly, the amounts of xylazine administered were 0.0028 ± 0.0003 ml ⁄ kg ⁄ min for the control rats and 0.0022 ± 0.0003 ml ⁄ kg ⁄ min for kindled rats (t13 = 1.42, P > 0.05). To assess the extent of seizure spread throughout the motor cortex, seizures were elicited through the chronically implanted stimulating electrode and were recorded from each of the three recording electrodes in two rats. In both rats, an AD was recorded from all electrodes, and in fact, an AD was even recorded from the electrode that was placed furthest away from the stimulating electrode on the very first seizure. After this widespread seizure activity was confirmed by chronically implanted electrodes, we confirmed with ICMS that specific movements (jaw, neck, forelimb, hindlimb, tail and trunk) could be recorded from their respective locations across the cortex. ADs were recorded from each movement area, as shown in Fig. 2. Figure 3 shows representative neocortical motor maps from a control and a kindled rat, demonstrating the enlargement in total map area following kindling. During the ICMS procedure, evidence of a posterior region where forelimb movements could be found was
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B
Statistical analysis Independent Student’s t-tests were used to compare the differences in anesthetic dosage, motor map areas, the number of dual movement sites, and movement thresholds between the treatment and control groups. For the movement threshold analysis, some movement types are not typically present in control rats, and thus they have small
Fig. 1. Electrographic and behavioural seizure progression. (A) Seizure severity and (B) neocortical afterdischarge duration (AD; in lA) during the 20 callosal kindling sessions. Both the seizure severity and AD duration scores steadily increased over time.
ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 34, 615–621
618 A. K. Henderson et al. but unlike control rats, digit movements could also be evoked in kindled rats from this posterior location. Figure 4 displays the mean motor map areas for the jaw, neck, wrist, digit, elbow, shoulder, trunk, hindlimb and tail movement representations (Fig. 3A) the mean areas for the RFA, CFA and PFA (describing those movements which could be evoked more posterior to the CFA, Fig. 3B), the proportion of total map size contributed by each area (Fig. 3C), as well as the percentage change in each area in kindled rats compared with controls (Fig. 3D). There was a significant effect of treatment on total map size such that kindled rats had motor maps that were significantly larger (t12 = 6.17, P = 0.00002) than controls: 16.03 ± 1.37 vs. 5.60 ± 0.98 mm2, respectively. To demonstrate further that this effect is not dependent upon our levels of anesthetic, we performed an analysis of co-variance and found that the main effect of our treatment was still significant (F1,11 = 26.46, P < 0.0001). When each movement representation was examined independently, kindled rats showed a significantly larger area of the neocortex that was dedicated to movement of the jaw (t12 = 2.93, P = 0.006), wrist (t12 = 4.79, P = 0.0002), digit (t12 = 2.35, P = 0.018), trunk (t12 = 1.87, P = 0.042) and tail (t12 = 3.31, P = 0.003) compared with controls (Fig. 4A). Kindled rats showed significantly (t12 = 4.14, P = 0.001) more area devoted to forelimb movements in the CFA (8.45 ± 0.96 vs. 3.57 ± 0.69 mm2) compared with controls (Fig. 4B). However, representations in close proximity to the wrist including the neck, elbow, shoulder and hindlimb areas were not significantly different in area between kindled and control groups (all P > 0.05). The proportional area of each movement representation was calculated by dividing the map area of each movement representation by the total map area. Kindled rats had a greater proportion of total map area dedicated to movement of the jaw, wrist, digit, hindlimb and tail, while control rats showed greater proportions for movements of the neck, elbow, shoulder and trunk (Fig. 4C). The percentage change in area of each movement representation after kindling is shown in Fig. 4D. Percentage change values ranged from a 16% decrease (shoulder) to a 1012% increase (tail). The total number of dual movements and number of dual movements evoked within a standardized area were also calculated.
Fig. 2. Description of seizure propagation. A craniotomy was performed on the left hemisphere, and seizures were elicited under light urethane anesthesia. Colored squares show the location of the recording electrodes during the acute examination of seizure propagation. ADs were recorded at all locations. ICMS was subsequently used to confirm that the recording electrodes were located in specific areas devoted to movement of the jaw, neck, forelimb (elbow), hindlimb, trunk and tail. The black square represents the location where an AD could be recorded in the absence of evoked movement. Green circles show the location of the chronically implanted recording electrodes (these were implanted in the right hemisphere, but are shown here for comparison purposes). The top green circle located over the skull represents the location of the chronic stimulating electrode in the right hemisphere.
observed in some kindled rats, which has not previously been described (as seen in Fig. 3B). This area was located more posterior to the CFA, approximately 3 mm lateral from the midline and extended as far as 3 mm posterior to bregma. This region was found separate from the CFA in some rats, but merged into the CFA in others. Interestingly, in a case where these posterior movements were found to be separated from the CFA, five of the six wrist movements evoked in this area were flexion of the wrist, in contrast to the extension of the wrist that are typically found in the CFA and RFA. One control rat had three responsive forelimb points that could be evoked from this posterior location, which were wrist, elbow and shoulder. These movements were also found in the posterior location in kindled rats,
A
B
Fig. 3. Neocortical movement representations. Representative color-coded motor maps from a control (A) and a kindled rat (B). Note the merging of the rostral forelimb area (RFA) and caudal forelimb area (CFA) in B. We also document the presence of movement that can be evoked more posterior to the CFA (see arrow in B). Dual movement sites are not represented on the motor maps. In the case of a dual movement, the movement with the lowest threshold was placed on the digital image. Locations outside the entire border of the map were extensively probed for movement. A-P, direction of the anterior–posterior axis; M-L, the direction of the medial– lateral axis. The scale bar indicates 1 mm of neocortical space. The black vertical line on each motor map denotes the location of bregma. ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 34, 615–621
Motor map enlargement after kindling 619 A
C
B
D
Fig. 4. Kindling-induced motor map changes. (A) Neocortical motor map movement areas (mm2), (B) motor map areas of the RFA, CFA and ‘PFA’, (C) proportion of total map area contributed by each movement representation, and (D) percentage change in areas after kindling are presented. There was significantly more area dedicated to movement representations for the jaw, wrist, digit, trunk and tail in kindled rats compared with controls. The majority of this enlargement is located within the CFA. Due to lack of movement that can be evoked posterior to the CFA in control rats (it was found in only one rat), statistical analyses were not performed on these movements. An asterisk denotes areas that differ significantly (P < 0.05) from controls. Error bars indicate SEM.
Control rats had a mean of 1.43 dual movements per rat while kindled rats had a mean of 11.17 dual movements per rat, which was significantly different (t11 = 5.97, P = 0.00009). When standardized for total map area, kindled rats still had more dual movements per mm2 (kindled: 0.70 ± 0.22 vs. control: 0.31 ± 0.35, t11 = 2.29, P = 0.04). Figure 5 shows the movement thresholds for control and kindled rats for each movement type (Fig. 5A) as well as for the RFA, CFA and PFA (Fig. 5B). Kindled rats had significantly lower movement thresholds for the jaw (t8 = 5.27, P = 0.0003), neck (t8 = 1.72; P = 0.06), wrist (t12 = 4.33, P = 0.0005) and elbow (t12 = 2.54, P = 0.01). Kindled rats also had lower movement thresholds in the RFA (t12 = 3.37, P = 0.006) and the CFA (t12 = 4.74, P = 0.005) compared with controls.
Discussion This is the first time a phenomenon of a widespread increase in several movement representations has been reported from rats subjected to A
repeated cortical seizures. Kindled seizures resulted in an increase in the total size of the motor map. Specifically, significant increases in area were found for movement representations of the jaw, wrist, digit, trunk and tail. This demonstrates that in addition to forelimb areas, other areas of the motor neocortex are enlarged following kindling. In a typical control motor map, jaw representations lie anterior and lateral to the CFA, elbow representations are dispersed within the CFA and the hindlimb area lies immediately posterior to the CFA. Thus, these areas essentially surround movements of the wrist and digit. The enlargement of the wrist and digit areas may come at the expense of these bordering areas. This suggests that wrist and digit movement representations may be ‘more plastic’ than their neighboring representations, and outcompete them for cortical space. It is possible that this adaptation allows the rat to learn skilled manipulations of the distal forelimb such as skilled reaching (Monfils & Teskey, 2004; Hodgson et al., 2005). We have confirmed that the expansion of the wrist area at the expense of the elbow area, for example, is not a result of subtle differences in the placement of our stimulating electrodes. ADs were B
Fig. 5. Average movement thresholds. (A) Average movement thresholds for each movement area in control and kindled rats. (B) Average movement thresholds in the RFA, CFA and ‘PFA’ in control and kindled rats. Overall, there was a decrease in specific movement thresholds in kindled rats compared with controls, which occurred in each area of the entire motor map. Numbers above the bars in A represent the sample size. Movement types such as digit, trunk, hindlimb and tail are relatively rare in control rats, and therefore these movements have a small sample size. Statistical analyses were not performed on sample sizes of two. Only one control rat had movement that could be evoked more posterior to the CFA (the ‘PFA’), and thus statistics were not performed on PFA thresholds. An asterisk denotes thresholds that differ significantly (P < 0.05) from controls. #P = 0.06. Error bars indicate SEM. ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 34, 615–621
620 A. K. Henderson et al. not only recorded chronically in three areas that border a typical enlarged motor map, but were also recorded in an acute setting in all areas tested. Therefore, seizures are propagating throughout the entire motor cortex, and the regional-specific expansion of movement areas are a property of the motor map itself and not due to characteristics of electrode placement. Further supporting this widespread propagation, we have previously shown that seizure activity can be recorded from as far away as the amygdala on the first stimulation session when stimulation is applied to the corpus callosum (Henderson et al., 2009). Furthermore, when seizures are induced in the hippocampus, there is a strong, positive correlation between motor map size and the number of ADs recorded in the motor neocortex (van Rooyen et al., 2006). There are at least two ways for movement representations to enlarge or occupy more space within the neocortex. First, movements may ‘fill in’ formerly non-responsive areas, or movements may overlap with each other so that more than one movement type can be evoked at the same location. These multiple movement sites have been documented in previous reports (Teskey et al., 2002) and were found in this study as well. Kindled rats showed significantly more dual movement sites than control rats, and in all cases where more than one movement was found, they were located on the border between different movement representations. This provides strong evidence that movement representations overlap and share cortical territory. We also provide evidence of forelimb movement that can be evoked from more posterior locations in the motor map after kindling. These posterior forelimb movements are probably involved in reaching movements as well, but are only revealed after kindling lowers the movement thresholds into a detectable range using standard ICMS techniques. That is, the ‘PFA’ is not a separate cortical area per se, but the sustained increase in excitability induced by repeated seizures reveals the entire motor map that is present in all rats. It does this by lowering the movement thresholds into a detectable range, as we have shown in this study. ICMS parameters such as frequency, pulse train and current intensity are known to influence the characteristics and size of a motor map (Donoghue & Wise, 1982). However, the parameters used in this study and the organization of the motor maps from our controls are consistent with those used and found in previous mapping studies (Castro-Alamancos & Borrel, 1995; Kleim et al., 1998; Ozen et al., 2008). This suggests that the motor map enlargement seen in the kindled rats is due to the repeated seizures and not to ICMS parameters or anesthetic levels. The type of anesthetic used during ICMS and its quantity can have a profound impact on the results obtained with this methodology. Ketamine, which is an N-methyld-aspartate (NMDA) receptor antagonist (Harrison & Simmonds, 1985), has the potential to mask the enlargement of motor maps after seizures. However, blocking these receptors yet still maintaining the enlargement suggests that we may in fact be under-estimating the true size of the motor maps, which may be even larger if glutamatergic antagonists were not used. The mechanism of motor map alterations after seizures has received little attention, although it is possible that increases in cortical excitation mediated by alterations in excitatory and inhibitory receptors may play a role, as AMPA (2-amino-3-(5-methyl-3-oxo1,2-oxazol-4-yl)propanoic acid), NMDA and c-aminobutyric acid receptors, for example, are known to be critical for plasticity (Collingridge et al., 2004) and kindling (McNamara et al., 1990; Gavrilovici et al., 2006). Prior work has shown that kindling in the corpus callosum increased the number of excitatory perforated synapses in layer V (Henry et al., 2008), which have been shown to contain high quantities of AMPA and NMDA receptors (Ganeshina et al., 2004). Similarly, Ekonomou et al. (2001) have shown increased
mRNA expression of the GluR2 subunit in the motor cortex after pentylenetetrazol-induced kindling. Future studies investigating the mechanism of motor map alterations after seizures should focus on these receptors and their subunits. It has also been suggested that potentiation in layer V may contribute to motor map enlargement as well, as the degree of motor map enlargement is positively correlated with the magnitude of evoked potentials measured in layer V (Teskey et al., 2002). In summary, our data demonstrate that many functional areas of the rat motor cortex undergo reorganization following kindling. Knowledge of the mechanisms behind seizure-induced reorganization of the motor cortex could prove useful for investigators studying cortical plasticity, and for clinicians who manage patients with epilepsy that have abnormal motor maps (Lado et al., 2002; Labyt et al., 2007).
Acknowledgements This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC; K.F., R.H.D., Q.J.P., G.C.T.) and the Canadian Institutes of Health Research (CIHR; K.F., R.H.D, Q.J.P., G.C.T). A.K.H. was supported by an Alberta Innovates-Health Solutions (AIHS) Scholarship. M.A.G. is a Killam Scholar and was supported by scholarships from AIHS and CIHR. K.F. and Q.J.P. are AHFMR Medical Scientists. We would like to thank Dr S. Tsutsui, Dr K. Riazi and Lorenzo Bauce for technical assistance.
Abbreviations AD, after-discharge; AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; CFA, caudal forelimb area; GABA, gamma-aminobutyric acid; ICMS, intracortical microstimulation; NMDA, N-methyl-d-aspartic acid; PFA, posterior forelimb area; RFA, rostral forelimb area.
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