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Low-frequency stimulation reverses kindling-induced neocortical motor map expansion
Neuroscience 153 (2008) 300 –307
LOW-FREQUENCY STIMULATION REVERSES KINDLING-INDUCED NEOCORTICAL MOTOR MAP EXPANSION L. J. OZEN, N. A. YOUNG, Y. KOSHIMORI AND G. C. TESKEY*
representations in the sensorimotor neocortex. Moreover, expansion of the neocortical forelimb area has been reported in the absence of seizures following long-term potentiation (LTP) of layer V in Long-Evans (Monfils et al., 2004) and epileptogenic prone “FAST” rats (Flynn et al., 2004), suggesting that changes in synaptic efficacy may be necessary for changes in motor map size (Teskey et al., 2005). Low-frequency stimulation (LFS) can induce a persistent long-term depression (LTD), a weakening of synaptic efficacy (Christie et al., 1994; Bi and Poo, 1998). LFS administered to the corpus callosum induces neocortical LTD and leads to a decrease in typical neocortical motor map size (Teskey et al., 2007). LFS also reduces seizure susceptibility indicating that it may be a useful treatment for epilepsy. In amygdala-kindled rats, LFS-induced LTD has been shown to increase afterdischarge (AD) threshold, impede seizure development and seizure progression when administered to the seizure focus (Gaito 1981; Velisek et al., 2002; López-Meraz et al., 2004; Goodman et al., 2005; Carrington et al., 2007; Ghorbani et al., 2007) or when delivered to a distant brain structure (Yang et al., 2006; Zhu-Ge et al., 2007). Clinical reports have shown that LFS delivered to the epileptic neocortex through subdural electrodes suppressed ictal and interictal discharges in individuals with seizure disorders (Kinoshita et al., 2005; Yamamoto et al., 2002, 2006). Less invasive low-frequency repetitive transcranial magnetic stimulation has also proven effective in reducing epileptic muscle jerks (Rossi et al., 2004; Misawa et al., 2005), interictal spiking (Joo et al., 2007), and seizure frequency (Tergau et al., 1999) in individuals with epilepsy. We sought to investigate whether LFS delivered to the corpus callosum could reverse seizure-induced neocortical motor map expansion. To that end, seizures were elicited through indwelling electrodes in the hippocampus, as it is the brain structure with the lowest seizure threshold in mammals (Corcoran and Moshe, 2005) and most commonly acts as the seizure focus in human adults (Engel, 2005; Engel and Pedley, 1998). Rats underwent kindling stimulation to the right ventral hippocampus until 30 neocortical seizures were recorded. A parallel group received sham kindling stimulation. Rats then received either 20 sessions of LFS delivered to the corpus callosum or sham LFS stimulation. High-resolution intracortical microstimulation (ICMS) was subsequently performed on the forelimb area in the left hemisphere in all rats. The forelimb area was chosen because it is a convenient site with clear borders that yields a quantifiable area and because it shows plastic responses to a variety of treatments and
Behavioural Neuroscience Research Group, Department of Psychology, and Epilepsy and Brain Circuits, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada T2N 1N4
Abstract—Repeated application of low-frequency stimulation can interrupt the development and progression of seizures. Low-frequency stimulation applied to the corpus callosum can also induce long-term depression in the neocortex of awake freely moving rats as well as reduce the size of neocortical movement representations (motor maps). We have previously shown that seizures induced through electrical stimulation of the corpus callosum, amygdala or hippocampus can expand the topographical expression of neocortical motor maps. The purpose of the present study was to determine if low-frequency stimulation administered to the corpus callosum could reverse the expansion of neocortical motor maps induced by seizures propagating from the hippocampus. Adult Long-Evans hooded rats were electrically stimulated in the right ventral hippocampus, twice daily until 30 neocortical seizures were recorded. Subsequently, low-frequency stimulation was administered to the corpus callosum once daily for 20 sessions. High-resolution intracortical microstimulation was then utilized to derive forelimb-movement representations in the left (un-implanted) sensorimotor neocortex. Our results show that hippocampal seizures result in expanded motor maps and that subsequent low-frequency application can reduce the size of the expanded motor maps. Low-frequency stimulation may be an effective treatment for reversing seizure-induced reorganization of brain function. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: long-term depression, neocortical plasticity, forelimb movement representations, seizures, hippocampus, epilepsy.
A number of clinical reports have established that some people with epilepsy have atypically organized neocortical motor maps (Uematsu et al., 1992; Urasaki et al., 1994; Lado et al., 2002; Branco et al., 2003; Chlebus et al., 2004; Labyt et al., 2007), suggesting that repeated seizure activity may alter the topography of neocortical movement representations. Seizure-induced atypical motor map configurations have also been observed in animal models. Rats electrically kindled through the corpus callosum, amygdala (Teskey et al., 2002) and hippocampus (van Rooyen et al., 2006) have expanded forelimb movement *Corresponding author. Tel: ⫹1-403-220-4962; fax: ⫹1-403-282-8249. E-mail address: [email protected] (G. C. Teskey). Abbreviations: AD, afterdischarge; EEG, electroencephalogram; ICMS, intracortical microstimulation; LFS, low-frequency stimulation; LTD, long term depression; LTP, long-term potentiation; NMDA, Nmethyl-D-aspartate.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.01.051
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conditions including seizures (Teskey et al., 2002; van Rooyen et al., 2006), electrical stimulation (Monfils et al., 2005; Teskey et al., 2007), and behavioral training (Kleim et al., 1998, 2004).
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Twenty-seven Long-Evans hooded rats weighing 295– 454 g at the time of electrode-implantation surgery were used in this study. Rats were obtained from the University of Calgary Breeding Colonies. They were housed individually in clear plastic cages in a colony room that was maintained on a 12-h light/dark cycle, with lights on at 7:00 am. All experimental procedures were conducted in the light phase. Rats received Laboratory Diet 5001 (PMI Nutrition International, Brentwood, MO, USA) and water ad libitum. The rats were housed and handled according to the Canadian Council on Animal Care guidelines. All experiments were designed to minimize the number of rats used and their suffering, and conformed to international guidelines on the ethical use of animals as specified by the Canadian Council for Animal Care. Ethical protocol was also reviewed and passed by the local Life and Environmental Sciences Committee.
stimulation consisted of a 1-s train of 60-Hz biphasic rectangular wave pulses, 1 ms in duration and separated by 1 ms. The current commenced at 50 A and was increased in 50 A increments every 60 s until an AD of ⱖ4 s was recorded on the electroencephalogram (EEG) from the stimulating hippocampal electrode. Kindling stimulation was subsequently delivered through the hippocampal electrode at an intensity 100 A greater than the initial AD threshold, twice daily, Monday through Friday between 9:00 am–10:00 am and 3:00 pm– 4:00 pm, until 30 ADs were recorded in the sensorimotor neocortex. The sham-kindled⫹LFS group and the sham-kindled⫹sham-LFS group were treated the same as the rats that were kindled except they did not receive any kindling stimulation. EEG was recorded from the hippocampal and neocortical electrodes, and seizure behaviors were observed, recorded, and scored according to a five-stage seizure scale (Racine, 1972). The durations of the ADs recorded from the hippocampal and neocortical electrodes were also measured. Commencing on the day following the last kindling session, LFS was administered to the corpus callosum once daily for 15 min (900 pulses) Monday through Friday for 20 sessions. LFS consisted of single biphasic square wave pulses that were 200 s in duration, at a pulse intensity of 1000 A, base to peak, and a frequency of 1 Hz. The kindled⫹sham-LFS group and the shamkindled⫹sham-LFS group were treated in the same manner, except they did not receive LFS.
Electrode implantation
ICMS
Prior to surgery, twisted-wire bipolar stimulating and recording electrodes were constructed from Teflon-coated stainless steel wire that was 178 m in diameter (A-M Systems, Everett, WA, USA). Uninsulated ends of the electrodes were connected to gold-plated male amphenol pins, and the two poles of the electrodes were separated by 1.0 mm. Rats were anesthetized with 1.0 ml/kg via an i.m. injection of 85.0/15.0 mg/ml ketamine/xylazine. Supplemental injections (0.05 ml) were administered as required. Lidocaine (2%) was administered s.c. at the incision site. Three bipolar electrodes were permanently implanted in the right hemisphere of each rat according to the stereotaxic coordinates of Swanson (1992). One electrode, used to deliver LFS, was implanted in the callosal white matter (1.0 mm anterior to bregma, 0.5 mm lateral to midline) to a depth that maximized the callosal– neocortical evoked response. The second electrode, used to record neocortical ADs, was implanted so that one pole was in layer III and the other in layer V of the right frontal neocortex (1.0 mm anterior to bregma, 4.0 mm lateral to midline, 1.5 mm from brain surface). The third electrode, used to deliver the hippocampal kindling stimulation, was implanted in the ventral hippocampus (4.5 mm posterior to bregma, 4.5 mm lateral to midline and 7.0 mm from brain surface). The amphenol pins connected to the electrodes were subsequently inserted into a nine-pin McIntyre connector plug (Molino and McIntyre, 1972) (Ginder Science, Ottawa, ON, Canada), which was adhered to the skull with dental cement and anchored with five stainless steel screws. One of the five screws served as a ground electrode. The skull surface to the left of midline and anterior to bregma was left free of screws and dental cement in order to perform a craniotomy during the ICMS procedure. Subsequent experimental procedures commenced no earlier than 7 days after surgery.
Standard ICMS techniques (Nudo et al., 1990; Kleim et al., 1998; Teskey et al., 2007) were used to generate detailed maps of the sensorimotor neocortical forelimb regions 1 to 9 days following the final LFS session. Twenty-four hours prior to surgery, rats were food deprived but had free access to water. Rats initially received i.p. injections of ketamine (100 mg/kg) and xylazine (5 mg/kg). Supplemental injections of ketamine alone (25 mg/kg), or a cocktail of both ketamine (17 mg/kg) and xylazine (2 mg/kg) were delivered i.p. as required throughout the surgical procedure to maintain a constant level of anesthesia, as indicated by breathing rate, vibrissae whisking, and a foot reflex in response to a gentle pinch. A 7⫻5-mm craniotomy exposed the sensorimotor neocortex of the left hemisphere. The window extended 4 mm anterior and 3 mm posterior from bregma and from midline to 5 mm lateral of midline. With an 18-gauge needle, a small puncture was made in the cisterna magna to reduce pressure due to edema. Dura was carefully removed and 37.4 °C silicone fluid (Factor II, Inc., Lakeside, AZ, USA) was used to cover the neocortical surface. A 32⫻ digital image of the exposed portion of the brain was captured by using a Stemi 2000-C stereomicroscope (Carl Zeiss, Thornwood, NY, USA), digital camera (Canon Canada Inc., Mississauga, ON, Canada) and displayed on a computer. A grid composed of 500 m squares was overlaid on the digital image. Penetration points were chosen at the intersections of the grid lines and at a central point in the middle of each square (interpenetration distance of 353 m), except when located over a blood vessel. Microelectrodes made from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL, USA) on a micropipette puller (Kopf, Tujunga, CA, USA) were filled with 3.5 M NaCl, and beveled at a 30° angle to yield a 3-m tip diameter with impedance values between 1.0 and 1.5 M⍀. Penetrations of the neocortex by the glass electrode were guided by a microdrive (Narishige, Tokyo, Japan) to a depth of 1550 m from the cortical surface, corresponding to the cell-body region of neocortical layer V. Electrical stimulation was delivered via an isolated stimulator (A-M Systems, Carlsborg, WA, USA) and consisted of 13 monophasic cathodal pulses, each 200 s in duration, delivered at a frequency of 333 Hz, repeated every second. Rats were maintained in a prone position, with the limb contralateral to the stimulation side being supported by placing one
EXPERIMENTAL PROCEDURES Rats
Stimulation The rats were divided into four groups: sham-kindle⫹sham-LFS (n⫽7), sham-kindle⫹LFS (n⫽9), kindle⫹sham-LFS (n⫽5), and kindle⫹LFS (n⫽6). On experimental day 1, a hippocampal AD threshold was determined for each rat in the kindle⫹sham-LFS and kindle⫹LFS groups. AD thresholds were defined as the weakest current necessary to induce an AD. Current was delivered through the electrode placed in the ventral hippocampus. The
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finger below the elbow joint and elevating the forelimb. This allowed visual detection of all possible forelimb (digit, wrist, elbow, or shoulder) movements. At each penetration site, the minimal threshold required to elicit a movement was recorded, and a color-coded dot was placed on the digital image. Penetration sites that failed to elicit a movement at any current intensity, up to the maximum of 60 A, were defined as non-responsive. To determine movement threshold, current intensity began at 0 A and was rapidly increased until a movement was detected, and then decreased until the movement was no longer present. No more than 10 trains of pulses were delivered to a single site to determine movement. The border of the forelimb motor map was defined first and was characterized by any non-forelimb movement that included head and neck, jaw, vibrissa, trunk, tail, hind limb, or non-responsive sites. The more central map points were then determined in an effort to reduce the likelihood of ICMS shifting the border points of the map (Nudo et al., 1990). The level of anesthesia was also assessed by revisiting positive-response forelimb sites to check for changes in movement thresholds as mapping progressed. Following the mapping session, rats were humanely killed with intracardial injection of 0.35 ml of 240 mg/ml sodium pentobarbital. The brains were subsequently removed and electrode placement was verified.
Movement-representation analysis Canvas (version 9.0.1) imaging software (ACD Systems Inc., Miami, FL, USA) was used to calculate the aerial extent of both the caudal and rostral forelimb areas. Because kindled rats did not display a clear border between the two forelimb areas, both areas were included in all analyses for all four groups. The proportion of distal and proximal movements that occupied the total forelimb area was calculated. The mean stimulation threshold for each movement category was also calculated.
Statistical analysis Two-tailed between subjects t-tests were used to compare the following measurements between kindle⫹sham-LFS and kindle⫹LFS groups: AD thresholds, AD durations of the first hippocampal kindling session, AD durations of the 45th hippocampal kindling session, AD durations of the first neocortical seizure, AD durations of the 30th neocortical seizure, and the number of generalized seizures. One-tailed between subjects t-tests were used to assess the progression of hippocampal AD duration between sessions 1 and 45 of kindling, the progression of AD duration between 1 and 30 neocortical seizure days, and seizure stage between sessions 1 and 45 of kindling. A one-way analysis of variance (ANOVA) was used to compare the ICMS ketamine dosage, xylazine dosage, current intensities for movement thresholds, and total forelimb area movement representations between the four groups. A post hoc Tukey’s honestly significant difference test was used to assess the specific differences in mean motor map area between the four groups. A one-tailed between subjects t-test was used to compare the distal forelimb areas between kindle⫹sham-LFS and kindle⫹LFS groups. SPSS 15.0 for windows was used to analyze the data.
RESULTS Kindling The mean⫾S.E.M. AD threshold for hippocampal kindled rats was 163.64⫾45.27 A. There was no significant difference between AD thresholds in the kindle⫹sham-LFS and kindle⫹LFS groups (t(9)⫽⫺0.31, P⫽0.760). Fig. 1 shows mean⫾S.E.M. hippocampal AD durations, seizure stages, and neocortical AD durations ob-
Fig. 1. Progression of electrographic and behavioral seizures. (A) Mean⫾S.E.M. hippocampal AD durations. (B) Mean⫾S.E.M. seizure stages. (C) Mean⫾S.E.M. neocortical AD durations. Rats received hippocampal kindling until 30 neocortical seizures were recorded. Increases in both hippocampal and neocortical AD durations, as well as increases in seizure stages were observed over the course of kindling.
served over the course of kindling. There was no significant difference between the kindle⫹sham-LFS and kindle⫹LFS groups on the first session hippocampal AD durations (t(9)⫽⫺0.27, P⫽0.791) or the 45th session hippocampal AD durations (t(7)⫽0.52, P⫽0.620). There was also no significant difference between the kindle⫹shamLFS and kindle⫹LFS groups on the first session neocortical AD durations (t(9)⫽0.19, P⫽0.852) or the 30th session neocortical AD durations (t(8)⫽⫺1.29, P⫽0.235). All kindled rats displayed progressive increases in hippocampal and neocortical AD durations with repeated kindling stimulation. Specifically, rats had a mean⫾S.E.M. hippocampal AD duration of 30.27⫾2.31 s during session 1 of kindling which significantly (t(18)⫽⫺5.00, P⬍0.001) increased to 75.33⫾9.60 s by the 45th session. Mean neocortical AD durations also significantly (t(19)⫽⫺1.96, P⫽0.03) increased from 30.73⫾4.20 s during the first neocortical seizure, to 44.90⫾6.04 s by the 30th neocortical
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seizure. It took a mean of 23 days for kindled rats to display neocortical seizure activity. As kindling continued, hippocampal kindled rats demonstrated more severe seizures. During the first kindling session, 72.7% of rats displayed a stage 1 seizure, which is characterized by freezing and vibrissae twitching. By session 45, 81.8% of rats displayed a generalized seizure (stage 4 or 5). There was no significant difference (t(9)⫽ 1.49, P⬎0.170) in the number of generalized seizures between the kindle⫹sham-LFS group (25.80⫾1.69) and the kindle⫹LFS group (28.17⫾0.40). Kindled rats had an initial mean⫾S.E.M. seizure stage of 0.91⫾0.16, which significantly (t(17)⫽⫺7.10, P⬍0.001) increased to 4.00⫾ 0.39 by session 45. Effects of hippocampal kindling on neocortical forelimb movement representations The mean number of days between the last LFS or shamLFS session and the ICMS procedure was 2.8 days and the four groups did not significantly differ (F(3, 1)⫽1.449, P⫽0.255) from each other. During ICSM, the mean⫾ S.E.M. amounts of ketamine, as a function of body weight and duration of surgery, administered were 0.0264⫾
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0.0042 ml/kg/min for the sham-kindled⫹sham-LFS rats, 0.0128⫾0.0016 ml/kg/min for the sham-kindled⫹LFS rats, 0.0140⫾0.0009 ml/kg/min for the kindled⫹shamLFS rats, and 0.0145⫾0.0013 ml/kg/min for kindled⫹ LFS rats. These amounts did not differ significantly (F(1, 3)⫽1.218, P⫽0.326). The mean⫾S.E.M. amounts of xylazine, as a function of body weight and duration of surgery were 0.0053⫾0.0009 ml/kg/min for the sham-kindle⫹shamLFS rats, 0.0040⫾0.0007 ml/kg/min for sham-kindle⫹LFS rats, 0.0032⫾0.0005 ml/kg/min for kindle⫹sham-LFS rats, and 0.0052⫾0.0006 ml/kg/min for kindle⫹LFS rats. These amounts did not differ significantly (F(1, 3)⫽1.363, P⫽0.279). In addition, the mean⫾S.E.M. current intensities required to elicit forelimb motor responses (movement thresholds) were 27.53⫾1.44 A for the sham-kindled⫹sham-LFS rats, 22.04⫾1.42 A for the sham-kindled⫹LFS rats, 25.63⫾1.85 A for the kindled⫹ sham-LFS rats, and 23.51⫾2.10 A for the kindle⫹LFS rats. These current intensities did not differ significantly (F(1, 3)⫽2.151, P⫽0.121). There was an overall significant (F(1, 3)⫽11.654, P⬍0.001; see Fig. 2) main effect of treatment on total neocortical forelimb motor map area. Kindled⫹sham-LFS rats had significantly (t(3)⫽5.084, P⬍0.001) larger maps
Fig. 2. Forelimb area movement representations. Color-coded topography of motor maps is represented from a sham-kindled⫹sham-LFS rat, a sham-kindled⫹LFS rat, a kindle⫹sham-LFS rat and a kindle⫹LFS rat. The stimulating microelectrode was repeatedly lowered 1550 m from brain surface into the left motor cortex, and up to 60 A of stimulation was applied until either a movement was elicited or no response was observed. External map boundaries were defined as electrode penetrations that did not elicit forelimb movements. A-P refers to the direction of the anterior–posterior axis. M-L refers to the direction of the medial–lateral axis. Scale bars⫽1 mm.
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Fig. 3. Mean areas (mm2) of (A) total forelimb representations and (B) distal (wrist/digit) area proportions of proximal (elbow/shoulder) forelimb representations in the left sensorimotor neocortex of shamkindled⫹sham-LFS rats, sham-kindled⫹LFS rats, kindled⫹sham-LFS rats, and kindled⫹LFS rats. Kindle⫹sham-LFS rats had motor maps that were significantly larger than sham-kindle⫹sham-LFS rats. Kindle⫹LFS rats had maps that were significantly smaller than kindle⫹sham-LFS rats.
compared with sham-kindled⫹sham-LFS rats. On average, kindled⫹sham-LFS rats had maps that were 214% (9.556⫾1.374 mm2) that of rats that were shamkindled⫹sham-LFS (4.472⫾0.637 mm2). Kindled⫹LFS rats exhibited significantly (t(3)⫽3.456, P⫽0.030) smaller maps (6.101⫾0.667 mm2) compared with rats that received kindling⫹sham-LFS (9.556⫾1.374 mm2). The size of kindle⫹LFS maps was not statistically different (P⫽0.431) from that of sham-kindled⫹sham-LFS. Reduction in the size of kindled maps was due to a significant decrease in distal forelimb movement representations (t(9)⫽1.97, P⫽0.040). On average, kindled⫹LFS rats had neocortical motor maps that were 36.2% smaller in overall size than rats that received kindling⫹sham-LFS (see Fig. 3). Sham-kindle⫹LFS (3.546⫾0.208 mm2) rats had motor maps that were, on average, 21% smaller in size than sham-kindle⫹sham-LFS (4.472⫾0.637 mm2) rats, although this difference was not statistically significant (P⫽0.769).
DISCUSSION The main finding of this study is that following seizureinduced functional reorganization, expressed as a 214% expansion of forelimb motor maps, repeated application of LFS to the corpus callosum contracted the expanded motor maps to the size of control levels. Furthermore, the expansion and contraction of motor maps occurred via
distal representations that have previously been shown to exhibit extensive plasticity (Kleim et al., 1998; 2004). Additionally, this study is the first to demonstrate that map expansion persisted for at least 5 weeks following the cessation of seizure activity. We postulate that the neocortical forelimb area expands due to seizures that propagate from the hippocampus to the neocortex and induces a potentiation of layer V horizontal synapses. The subsequent contraction of the expanded map as a result of LFS application is likely due to a depotentiation of those synapses. The size of a motor map can be affected by various stimulation parameters such as current intensity, pulse train, stimulation frequency, as well as by anesthetics (Donoghue and Wise, 1982), thus the baseline map parameters reported in any experiment are dependent on the methods used. In the present study, the difference in map size between groups is not a result of differences in anesthetic levels, ICMS current intensity, or the number of days between the last stimulation session and ICMS procedure, as these measurements were not statistically different between the four treatment groups. It is not essential that ICMS be conducted immediately following the last experimental session as neocortical motor map reorganization has been shown to persist for at least 3 weeks (Teskey et al., 2002) and, as the current study has demonstrated, up to 5 weeks. Since the topography of movement representations in control rats in the present experiment is consistent with those of previous experiments using similar stimulation parameters (Gioanni and Lamarche, 1985; CastroAlamancos and Borrel, 1995; Kleim et al., 1998; Teskey et al., 2002; VandenBerg et al., 2002; Monfils et al., 2004; van Rooyen et al., 2006), we are confident that the seizureinduced map expansion and LFS-induced map contraction are due to functional reorganization and not confounded by anesthetics or stimulation parameters. Furthermore, since both the rostral and caudal forelimb regions are measured and combined in our analysis, the expansion and contraction of total forelimb area cannot be not due to a simple invasion and retreat of the caudal forelimb area into the rostral forelimb area. This study replicates earlier work showing that experimentally induced seizures result in expansion and reorganization of motor maps (Teskey et al., 2002; van Rooyen et al., 2006) akin to motor map expansion (Uematsu et al., 1992; Urasaki et al., 1994; Labyt et al., 2007) and reorganization (Lado et al., 2002; Branco et al., 2003; Chlebus et al., 2004) in the primary and sensory motor cortices in some individuals with epilepsy. Our laboratory has also previously shown that motor map expansion is related to polysynaptic potentiation in layer V. The magnitude of potentiation of the polysynaptic component of the callosal– neocortical evoked response is enhanced (Racine et al., 1995) as well as significantly and positively correlated with expansion of forelimb movement representations after kindling (Teskey et al., 2002) and LTP induction in LongEvans (Monfils et al., 2004) and epileptogenic-prone rats (Flynn et al., 2004). Movements are most easily elicited when the stimulating electrode is lowered into layer V of
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the sensorimotor neocortex during ICMS (Asanuma and Rosen, 1972), also suggesting that potentiated layer V connections may be the cause of motor map expansion. In contrast, Teskey et al. (2007) observed a decrease in the size of the monosynaptic and polysynaptic components of evoked potentials, as well as a reduction in motor map size in rats that only received LFS. We extend those observations by reporting that LFS is effective in reducing the size of expanded motor maps in rats that have experienced repeated seizures. While non-kindled rats that received LFS in this study did not exhibit statistically significantly smaller maps compared with controls, there was a 21% reduction in map size. The failure to find a significant reduction may be because rats in the present study were approximately three times older than those previously examined (Teskey et al., 2007) and plasticity has been shown to decline with age (Rosenzweig and Barnes, 2003). The anatomical and neurochemical mechanisms underlying map expansion and contraction are likely related to changes in glutamatergic and GABAergic transmission. Repeated neocortical seizures are associated with increases in the number of excitatory perforated synapses (Goertzen and Teskey, 2003) that are associated with 180% more N-methyl-D-aspartate (NMDA) and approximately 650% more AMPA channels compared with nonperforated synapses (Ganeshina et al., 2004). Following kindling of the hippocampus, glutamatergic transmission has been shown to be enhanced through increased presynaptic glutamate release (Geula et al., 1988; Jarvie et al., 1990), recruitment of previously dormant NMDA receptors (Mody and Heinemann, 1987; Mody et al., 1988), and enhanced NMDA-receptor activation (Behr et al., 2000). In contrast, LFS has been shown to be associated with a decrease in dendritic arborization (Monfils and Teskey, 2004), in the number of excitatory perforated synapses, and thus in glutamatergic activity, as well as an increase in inhibitory synapses (Teskey et al., 2007). Enhanced GABAergic activity can decrease motor map size by masking lateral excitatory connections (Jacobs and Donoghue, 1991) resulting in a decrease in the total neocortical area that is responsible for forelimb movements and a corresponding increase in the amount of neocortex that becomes unresponsive to ICMS. LFS is not limited to reversing functional brain reorganization. It is also a tool that appears to have anti-convulsant efficacy. After amygdala-kindled rats achieved a fullykindled state, LFS applied to the amygdala increased AD thresholds (Carrington et al., 2007) and LFS administered to the central piriform cortex resulted in a reduction of both seizure severity and duration (Zhu-Ge et al., 2007). Preemptive LFS administered to the amygdala of fully amygdala-kindled rats decreased the mean number of seizures elicited (Goodman et al., 2005). LFS also has a suppressive effect on seizures when administered during the kindling process. LFS applied to the central piriform cortex had the same suppressive effect on amygdaloid-kindled rats (Yang et al., 2006) and piriform-kindled rats (Ghorbani et al., 2007) when applied after each kindling session. Rat
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pups that received LFS to the amygdala following each amygdala-kindling session (Velisek et al., 2002) and adult rats that received LFS to the amygdala prior to each amygdala kindling session (Goodman et al., 2005) exhibited a mean decrease in the AD duration and seizure stage throughout kindling. López-Meraz et al. (2004) found that the application of LFS to the amygdala following each amygdaloid-kindling session decreased the likelihood that rats would reach a fully kindled state. LFS applied to the amygdala following each perforant path kindling session delayed kindling development, inhibited kindling-induced potentiation, and prevented the increase in early and late paired-pulse depression (Mohammad-Zadeh et al., 2007). Moreover, in humans with intractable mesial temporal lobe epilepsy, applying LFS through subdural electrodes suppressed ictal and interictal discharges (Kinoshita et al., 2005; Yamamoto et al., 2002, 2006).
CONCLUSION In summary, we have demonstrated that LFS reverses kindling-induced functional reorganization expressed as neocortical motor map expansion. Hippocampal kindling followed by LFS delivered to the corpus callosum resulted in a decrease in expanded forelimb movement representations so that the total neocortical forelimb area was equivalent to non-stimulated rats. As a result of kindling, the maps may have expanded due to kindling-induced synaptic potentiation, mediated by an increase in the number of excitatory synapses, an enhancement of glutamatergic transmission, and possibly a reduction of GABAergic inhibition. Following LFS, the maps may have subsequently contracted due to synaptic depotentiation, mediated by a loss of perforated synapses, decreased glutamatergic transmission, and an enhancement of GABAergic inhibition. Future studies are needed to examine the effects of callosal LFS on AD duration, threshold, seizure severity, and motor map size when administered immediately following the cessation of each hippocampal AD. LFS may also be used as a means to ameliorate inter-ictal behavioral deficits that often coincide with seizure disorders.
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(Accepted 12 January 2008) (Available online 8 February 2008)
LOW-FREQUENCY STIMULATION REVERSES KINDLING-INDUCED NEOCORTICAL MOTOR MAP EXPANSION L. J. OZEN, N. A. YOUNG, Y. KOSHIMORI AND G. C. TESKEY*
representations in the sensorimotor neocortex. Moreover, expansion of the neocortical forelimb area has been reported in the absence of seizures following long-term potentiation (LTP) of layer V in Long-Evans (Monfils et al., 2004) and epileptogenic prone “FAST” rats (Flynn et al., 2004), suggesting that changes in synaptic efficacy may be necessary for changes in motor map size (Teskey et al., 2005). Low-frequency stimulation (LFS) can induce a persistent long-term depression (LTD), a weakening of synaptic efficacy (Christie et al., 1994; Bi and Poo, 1998). LFS administered to the corpus callosum induces neocortical LTD and leads to a decrease in typical neocortical motor map size (Teskey et al., 2007). LFS also reduces seizure susceptibility indicating that it may be a useful treatment for epilepsy. In amygdala-kindled rats, LFS-induced LTD has been shown to increase afterdischarge (AD) threshold, impede seizure development and seizure progression when administered to the seizure focus (Gaito 1981; Velisek et al., 2002; López-Meraz et al., 2004; Goodman et al., 2005; Carrington et al., 2007; Ghorbani et al., 2007) or when delivered to a distant brain structure (Yang et al., 2006; Zhu-Ge et al., 2007). Clinical reports have shown that LFS delivered to the epileptic neocortex through subdural electrodes suppressed ictal and interictal discharges in individuals with seizure disorders (Kinoshita et al., 2005; Yamamoto et al., 2002, 2006). Less invasive low-frequency repetitive transcranial magnetic stimulation has also proven effective in reducing epileptic muscle jerks (Rossi et al., 2004; Misawa et al., 2005), interictal spiking (Joo et al., 2007), and seizure frequency (Tergau et al., 1999) in individuals with epilepsy. We sought to investigate whether LFS delivered to the corpus callosum could reverse seizure-induced neocortical motor map expansion. To that end, seizures were elicited through indwelling electrodes in the hippocampus, as it is the brain structure with the lowest seizure threshold in mammals (Corcoran and Moshe, 2005) and most commonly acts as the seizure focus in human adults (Engel, 2005; Engel and Pedley, 1998). Rats underwent kindling stimulation to the right ventral hippocampus until 30 neocortical seizures were recorded. A parallel group received sham kindling stimulation. Rats then received either 20 sessions of LFS delivered to the corpus callosum or sham LFS stimulation. High-resolution intracortical microstimulation (ICMS) was subsequently performed on the forelimb area in the left hemisphere in all rats. The forelimb area was chosen because it is a convenient site with clear borders that yields a quantifiable area and because it shows plastic responses to a variety of treatments and
Behavioural Neuroscience Research Group, Department of Psychology, and Epilepsy and Brain Circuits, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada T2N 1N4
Abstract—Repeated application of low-frequency stimulation can interrupt the development and progression of seizures. Low-frequency stimulation applied to the corpus callosum can also induce long-term depression in the neocortex of awake freely moving rats as well as reduce the size of neocortical movement representations (motor maps). We have previously shown that seizures induced through electrical stimulation of the corpus callosum, amygdala or hippocampus can expand the topographical expression of neocortical motor maps. The purpose of the present study was to determine if low-frequency stimulation administered to the corpus callosum could reverse the expansion of neocortical motor maps induced by seizures propagating from the hippocampus. Adult Long-Evans hooded rats were electrically stimulated in the right ventral hippocampus, twice daily until 30 neocortical seizures were recorded. Subsequently, low-frequency stimulation was administered to the corpus callosum once daily for 20 sessions. High-resolution intracortical microstimulation was then utilized to derive forelimb-movement representations in the left (un-implanted) sensorimotor neocortex. Our results show that hippocampal seizures result in expanded motor maps and that subsequent low-frequency application can reduce the size of the expanded motor maps. Low-frequency stimulation may be an effective treatment for reversing seizure-induced reorganization of brain function. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: long-term depression, neocortical plasticity, forelimb movement representations, seizures, hippocampus, epilepsy.
A number of clinical reports have established that some people with epilepsy have atypically organized neocortical motor maps (Uematsu et al., 1992; Urasaki et al., 1994; Lado et al., 2002; Branco et al., 2003; Chlebus et al., 2004; Labyt et al., 2007), suggesting that repeated seizure activity may alter the topography of neocortical movement representations. Seizure-induced atypical motor map configurations have also been observed in animal models. Rats electrically kindled through the corpus callosum, amygdala (Teskey et al., 2002) and hippocampus (van Rooyen et al., 2006) have expanded forelimb movement *Corresponding author. Tel: ⫹1-403-220-4962; fax: ⫹1-403-282-8249. E-mail address: [email protected] (G. C. Teskey). Abbreviations: AD, afterdischarge; EEG, electroencephalogram; ICMS, intracortical microstimulation; LFS, low-frequency stimulation; LTD, long term depression; LTP, long-term potentiation; NMDA, Nmethyl-D-aspartate.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.01.051
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conditions including seizures (Teskey et al., 2002; van Rooyen et al., 2006), electrical stimulation (Monfils et al., 2005; Teskey et al., 2007), and behavioral training (Kleim et al., 1998, 2004).
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Twenty-seven Long-Evans hooded rats weighing 295– 454 g at the time of electrode-implantation surgery were used in this study. Rats were obtained from the University of Calgary Breeding Colonies. They were housed individually in clear plastic cages in a colony room that was maintained on a 12-h light/dark cycle, with lights on at 7:00 am. All experimental procedures were conducted in the light phase. Rats received Laboratory Diet 5001 (PMI Nutrition International, Brentwood, MO, USA) and water ad libitum. The rats were housed and handled according to the Canadian Council on Animal Care guidelines. All experiments were designed to minimize the number of rats used and their suffering, and conformed to international guidelines on the ethical use of animals as specified by the Canadian Council for Animal Care. Ethical protocol was also reviewed and passed by the local Life and Environmental Sciences Committee.
stimulation consisted of a 1-s train of 60-Hz biphasic rectangular wave pulses, 1 ms in duration and separated by 1 ms. The current commenced at 50 A and was increased in 50 A increments every 60 s until an AD of ⱖ4 s was recorded on the electroencephalogram (EEG) from the stimulating hippocampal electrode. Kindling stimulation was subsequently delivered through the hippocampal electrode at an intensity 100 A greater than the initial AD threshold, twice daily, Monday through Friday between 9:00 am–10:00 am and 3:00 pm– 4:00 pm, until 30 ADs were recorded in the sensorimotor neocortex. The sham-kindled⫹LFS group and the sham-kindled⫹sham-LFS group were treated the same as the rats that were kindled except they did not receive any kindling stimulation. EEG was recorded from the hippocampal and neocortical electrodes, and seizure behaviors were observed, recorded, and scored according to a five-stage seizure scale (Racine, 1972). The durations of the ADs recorded from the hippocampal and neocortical electrodes were also measured. Commencing on the day following the last kindling session, LFS was administered to the corpus callosum once daily for 15 min (900 pulses) Monday through Friday for 20 sessions. LFS consisted of single biphasic square wave pulses that were 200 s in duration, at a pulse intensity of 1000 A, base to peak, and a frequency of 1 Hz. The kindled⫹sham-LFS group and the shamkindled⫹sham-LFS group were treated in the same manner, except they did not receive LFS.
Electrode implantation
ICMS
Prior to surgery, twisted-wire bipolar stimulating and recording electrodes were constructed from Teflon-coated stainless steel wire that was 178 m in diameter (A-M Systems, Everett, WA, USA). Uninsulated ends of the electrodes were connected to gold-plated male amphenol pins, and the two poles of the electrodes were separated by 1.0 mm. Rats were anesthetized with 1.0 ml/kg via an i.m. injection of 85.0/15.0 mg/ml ketamine/xylazine. Supplemental injections (0.05 ml) were administered as required. Lidocaine (2%) was administered s.c. at the incision site. Three bipolar electrodes were permanently implanted in the right hemisphere of each rat according to the stereotaxic coordinates of Swanson (1992). One electrode, used to deliver LFS, was implanted in the callosal white matter (1.0 mm anterior to bregma, 0.5 mm lateral to midline) to a depth that maximized the callosal– neocortical evoked response. The second electrode, used to record neocortical ADs, was implanted so that one pole was in layer III and the other in layer V of the right frontal neocortex (1.0 mm anterior to bregma, 4.0 mm lateral to midline, 1.5 mm from brain surface). The third electrode, used to deliver the hippocampal kindling stimulation, was implanted in the ventral hippocampus (4.5 mm posterior to bregma, 4.5 mm lateral to midline and 7.0 mm from brain surface). The amphenol pins connected to the electrodes were subsequently inserted into a nine-pin McIntyre connector plug (Molino and McIntyre, 1972) (Ginder Science, Ottawa, ON, Canada), which was adhered to the skull with dental cement and anchored with five stainless steel screws. One of the five screws served as a ground electrode. The skull surface to the left of midline and anterior to bregma was left free of screws and dental cement in order to perform a craniotomy during the ICMS procedure. Subsequent experimental procedures commenced no earlier than 7 days after surgery.
Standard ICMS techniques (Nudo et al., 1990; Kleim et al., 1998; Teskey et al., 2007) were used to generate detailed maps of the sensorimotor neocortical forelimb regions 1 to 9 days following the final LFS session. Twenty-four hours prior to surgery, rats were food deprived but had free access to water. Rats initially received i.p. injections of ketamine (100 mg/kg) and xylazine (5 mg/kg). Supplemental injections of ketamine alone (25 mg/kg), or a cocktail of both ketamine (17 mg/kg) and xylazine (2 mg/kg) were delivered i.p. as required throughout the surgical procedure to maintain a constant level of anesthesia, as indicated by breathing rate, vibrissae whisking, and a foot reflex in response to a gentle pinch. A 7⫻5-mm craniotomy exposed the sensorimotor neocortex of the left hemisphere. The window extended 4 mm anterior and 3 mm posterior from bregma and from midline to 5 mm lateral of midline. With an 18-gauge needle, a small puncture was made in the cisterna magna to reduce pressure due to edema. Dura was carefully removed and 37.4 °C silicone fluid (Factor II, Inc., Lakeside, AZ, USA) was used to cover the neocortical surface. A 32⫻ digital image of the exposed portion of the brain was captured by using a Stemi 2000-C stereomicroscope (Carl Zeiss, Thornwood, NY, USA), digital camera (Canon Canada Inc., Mississauga, ON, Canada) and displayed on a computer. A grid composed of 500 m squares was overlaid on the digital image. Penetration points were chosen at the intersections of the grid lines and at a central point in the middle of each square (interpenetration distance of 353 m), except when located over a blood vessel. Microelectrodes made from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL, USA) on a micropipette puller (Kopf, Tujunga, CA, USA) were filled with 3.5 M NaCl, and beveled at a 30° angle to yield a 3-m tip diameter with impedance values between 1.0 and 1.5 M⍀. Penetrations of the neocortex by the glass electrode were guided by a microdrive (Narishige, Tokyo, Japan) to a depth of 1550 m from the cortical surface, corresponding to the cell-body region of neocortical layer V. Electrical stimulation was delivered via an isolated stimulator (A-M Systems, Carlsborg, WA, USA) and consisted of 13 monophasic cathodal pulses, each 200 s in duration, delivered at a frequency of 333 Hz, repeated every second. Rats were maintained in a prone position, with the limb contralateral to the stimulation side being supported by placing one
EXPERIMENTAL PROCEDURES Rats
Stimulation The rats were divided into four groups: sham-kindle⫹sham-LFS (n⫽7), sham-kindle⫹LFS (n⫽9), kindle⫹sham-LFS (n⫽5), and kindle⫹LFS (n⫽6). On experimental day 1, a hippocampal AD threshold was determined for each rat in the kindle⫹sham-LFS and kindle⫹LFS groups. AD thresholds were defined as the weakest current necessary to induce an AD. Current was delivered through the electrode placed in the ventral hippocampus. The
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finger below the elbow joint and elevating the forelimb. This allowed visual detection of all possible forelimb (digit, wrist, elbow, or shoulder) movements. At each penetration site, the minimal threshold required to elicit a movement was recorded, and a color-coded dot was placed on the digital image. Penetration sites that failed to elicit a movement at any current intensity, up to the maximum of 60 A, were defined as non-responsive. To determine movement threshold, current intensity began at 0 A and was rapidly increased until a movement was detected, and then decreased until the movement was no longer present. No more than 10 trains of pulses were delivered to a single site to determine movement. The border of the forelimb motor map was defined first and was characterized by any non-forelimb movement that included head and neck, jaw, vibrissa, trunk, tail, hind limb, or non-responsive sites. The more central map points were then determined in an effort to reduce the likelihood of ICMS shifting the border points of the map (Nudo et al., 1990). The level of anesthesia was also assessed by revisiting positive-response forelimb sites to check for changes in movement thresholds as mapping progressed. Following the mapping session, rats were humanely killed with intracardial injection of 0.35 ml of 240 mg/ml sodium pentobarbital. The brains were subsequently removed and electrode placement was verified.
Movement-representation analysis Canvas (version 9.0.1) imaging software (ACD Systems Inc., Miami, FL, USA) was used to calculate the aerial extent of both the caudal and rostral forelimb areas. Because kindled rats did not display a clear border between the two forelimb areas, both areas were included in all analyses for all four groups. The proportion of distal and proximal movements that occupied the total forelimb area was calculated. The mean stimulation threshold for each movement category was also calculated.
Statistical analysis Two-tailed between subjects t-tests were used to compare the following measurements between kindle⫹sham-LFS and kindle⫹LFS groups: AD thresholds, AD durations of the first hippocampal kindling session, AD durations of the 45th hippocampal kindling session, AD durations of the first neocortical seizure, AD durations of the 30th neocortical seizure, and the number of generalized seizures. One-tailed between subjects t-tests were used to assess the progression of hippocampal AD duration between sessions 1 and 45 of kindling, the progression of AD duration between 1 and 30 neocortical seizure days, and seizure stage between sessions 1 and 45 of kindling. A one-way analysis of variance (ANOVA) was used to compare the ICMS ketamine dosage, xylazine dosage, current intensities for movement thresholds, and total forelimb area movement representations between the four groups. A post hoc Tukey’s honestly significant difference test was used to assess the specific differences in mean motor map area between the four groups. A one-tailed between subjects t-test was used to compare the distal forelimb areas between kindle⫹sham-LFS and kindle⫹LFS groups. SPSS 15.0 for windows was used to analyze the data.
RESULTS Kindling The mean⫾S.E.M. AD threshold for hippocampal kindled rats was 163.64⫾45.27 A. There was no significant difference between AD thresholds in the kindle⫹sham-LFS and kindle⫹LFS groups (t(9)⫽⫺0.31, P⫽0.760). Fig. 1 shows mean⫾S.E.M. hippocampal AD durations, seizure stages, and neocortical AD durations ob-
Fig. 1. Progression of electrographic and behavioral seizures. (A) Mean⫾S.E.M. hippocampal AD durations. (B) Mean⫾S.E.M. seizure stages. (C) Mean⫾S.E.M. neocortical AD durations. Rats received hippocampal kindling until 30 neocortical seizures were recorded. Increases in both hippocampal and neocortical AD durations, as well as increases in seizure stages were observed over the course of kindling.
served over the course of kindling. There was no significant difference between the kindle⫹sham-LFS and kindle⫹LFS groups on the first session hippocampal AD durations (t(9)⫽⫺0.27, P⫽0.791) or the 45th session hippocampal AD durations (t(7)⫽0.52, P⫽0.620). There was also no significant difference between the kindle⫹shamLFS and kindle⫹LFS groups on the first session neocortical AD durations (t(9)⫽0.19, P⫽0.852) or the 30th session neocortical AD durations (t(8)⫽⫺1.29, P⫽0.235). All kindled rats displayed progressive increases in hippocampal and neocortical AD durations with repeated kindling stimulation. Specifically, rats had a mean⫾S.E.M. hippocampal AD duration of 30.27⫾2.31 s during session 1 of kindling which significantly (t(18)⫽⫺5.00, P⬍0.001) increased to 75.33⫾9.60 s by the 45th session. Mean neocortical AD durations also significantly (t(19)⫽⫺1.96, P⫽0.03) increased from 30.73⫾4.20 s during the first neocortical seizure, to 44.90⫾6.04 s by the 30th neocortical
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seizure. It took a mean of 23 days for kindled rats to display neocortical seizure activity. As kindling continued, hippocampal kindled rats demonstrated more severe seizures. During the first kindling session, 72.7% of rats displayed a stage 1 seizure, which is characterized by freezing and vibrissae twitching. By session 45, 81.8% of rats displayed a generalized seizure (stage 4 or 5). There was no significant difference (t(9)⫽ 1.49, P⬎0.170) in the number of generalized seizures between the kindle⫹sham-LFS group (25.80⫾1.69) and the kindle⫹LFS group (28.17⫾0.40). Kindled rats had an initial mean⫾S.E.M. seizure stage of 0.91⫾0.16, which significantly (t(17)⫽⫺7.10, P⬍0.001) increased to 4.00⫾ 0.39 by session 45. Effects of hippocampal kindling on neocortical forelimb movement representations The mean number of days between the last LFS or shamLFS session and the ICMS procedure was 2.8 days and the four groups did not significantly differ (F(3, 1)⫽1.449, P⫽0.255) from each other. During ICSM, the mean⫾ S.E.M. amounts of ketamine, as a function of body weight and duration of surgery, administered were 0.0264⫾
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0.0042 ml/kg/min for the sham-kindled⫹sham-LFS rats, 0.0128⫾0.0016 ml/kg/min for the sham-kindled⫹LFS rats, 0.0140⫾0.0009 ml/kg/min for the kindled⫹shamLFS rats, and 0.0145⫾0.0013 ml/kg/min for kindled⫹ LFS rats. These amounts did not differ significantly (F(1, 3)⫽1.218, P⫽0.326). The mean⫾S.E.M. amounts of xylazine, as a function of body weight and duration of surgery were 0.0053⫾0.0009 ml/kg/min for the sham-kindle⫹shamLFS rats, 0.0040⫾0.0007 ml/kg/min for sham-kindle⫹LFS rats, 0.0032⫾0.0005 ml/kg/min for kindle⫹sham-LFS rats, and 0.0052⫾0.0006 ml/kg/min for kindle⫹LFS rats. These amounts did not differ significantly (F(1, 3)⫽1.363, P⫽0.279). In addition, the mean⫾S.E.M. current intensities required to elicit forelimb motor responses (movement thresholds) were 27.53⫾1.44 A for the sham-kindled⫹sham-LFS rats, 22.04⫾1.42 A for the sham-kindled⫹LFS rats, 25.63⫾1.85 A for the kindled⫹ sham-LFS rats, and 23.51⫾2.10 A for the kindle⫹LFS rats. These current intensities did not differ significantly (F(1, 3)⫽2.151, P⫽0.121). There was an overall significant (F(1, 3)⫽11.654, P⬍0.001; see Fig. 2) main effect of treatment on total neocortical forelimb motor map area. Kindled⫹sham-LFS rats had significantly (t(3)⫽5.084, P⬍0.001) larger maps
Fig. 2. Forelimb area movement representations. Color-coded topography of motor maps is represented from a sham-kindled⫹sham-LFS rat, a sham-kindled⫹LFS rat, a kindle⫹sham-LFS rat and a kindle⫹LFS rat. The stimulating microelectrode was repeatedly lowered 1550 m from brain surface into the left motor cortex, and up to 60 A of stimulation was applied until either a movement was elicited or no response was observed. External map boundaries were defined as electrode penetrations that did not elicit forelimb movements. A-P refers to the direction of the anterior–posterior axis. M-L refers to the direction of the medial–lateral axis. Scale bars⫽1 mm.
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Fig. 3. Mean areas (mm2) of (A) total forelimb representations and (B) distal (wrist/digit) area proportions of proximal (elbow/shoulder) forelimb representations in the left sensorimotor neocortex of shamkindled⫹sham-LFS rats, sham-kindled⫹LFS rats, kindled⫹sham-LFS rats, and kindled⫹LFS rats. Kindle⫹sham-LFS rats had motor maps that were significantly larger than sham-kindle⫹sham-LFS rats. Kindle⫹LFS rats had maps that were significantly smaller than kindle⫹sham-LFS rats.
compared with sham-kindled⫹sham-LFS rats. On average, kindled⫹sham-LFS rats had maps that were 214% (9.556⫾1.374 mm2) that of rats that were shamkindled⫹sham-LFS (4.472⫾0.637 mm2). Kindled⫹LFS rats exhibited significantly (t(3)⫽3.456, P⫽0.030) smaller maps (6.101⫾0.667 mm2) compared with rats that received kindling⫹sham-LFS (9.556⫾1.374 mm2). The size of kindle⫹LFS maps was not statistically different (P⫽0.431) from that of sham-kindled⫹sham-LFS. Reduction in the size of kindled maps was due to a significant decrease in distal forelimb movement representations (t(9)⫽1.97, P⫽0.040). On average, kindled⫹LFS rats had neocortical motor maps that were 36.2% smaller in overall size than rats that received kindling⫹sham-LFS (see Fig. 3). Sham-kindle⫹LFS (3.546⫾0.208 mm2) rats had motor maps that were, on average, 21% smaller in size than sham-kindle⫹sham-LFS (4.472⫾0.637 mm2) rats, although this difference was not statistically significant (P⫽0.769).
DISCUSSION The main finding of this study is that following seizureinduced functional reorganization, expressed as a 214% expansion of forelimb motor maps, repeated application of LFS to the corpus callosum contracted the expanded motor maps to the size of control levels. Furthermore, the expansion and contraction of motor maps occurred via
distal representations that have previously been shown to exhibit extensive plasticity (Kleim et al., 1998; 2004). Additionally, this study is the first to demonstrate that map expansion persisted for at least 5 weeks following the cessation of seizure activity. We postulate that the neocortical forelimb area expands due to seizures that propagate from the hippocampus to the neocortex and induces a potentiation of layer V horizontal synapses. The subsequent contraction of the expanded map as a result of LFS application is likely due to a depotentiation of those synapses. The size of a motor map can be affected by various stimulation parameters such as current intensity, pulse train, stimulation frequency, as well as by anesthetics (Donoghue and Wise, 1982), thus the baseline map parameters reported in any experiment are dependent on the methods used. In the present study, the difference in map size between groups is not a result of differences in anesthetic levels, ICMS current intensity, or the number of days between the last stimulation session and ICMS procedure, as these measurements were not statistically different between the four treatment groups. It is not essential that ICMS be conducted immediately following the last experimental session as neocortical motor map reorganization has been shown to persist for at least 3 weeks (Teskey et al., 2002) and, as the current study has demonstrated, up to 5 weeks. Since the topography of movement representations in control rats in the present experiment is consistent with those of previous experiments using similar stimulation parameters (Gioanni and Lamarche, 1985; CastroAlamancos and Borrel, 1995; Kleim et al., 1998; Teskey et al., 2002; VandenBerg et al., 2002; Monfils et al., 2004; van Rooyen et al., 2006), we are confident that the seizureinduced map expansion and LFS-induced map contraction are due to functional reorganization and not confounded by anesthetics or stimulation parameters. Furthermore, since both the rostral and caudal forelimb regions are measured and combined in our analysis, the expansion and contraction of total forelimb area cannot be not due to a simple invasion and retreat of the caudal forelimb area into the rostral forelimb area. This study replicates earlier work showing that experimentally induced seizures result in expansion and reorganization of motor maps (Teskey et al., 2002; van Rooyen et al., 2006) akin to motor map expansion (Uematsu et al., 1992; Urasaki et al., 1994; Labyt et al., 2007) and reorganization (Lado et al., 2002; Branco et al., 2003; Chlebus et al., 2004) in the primary and sensory motor cortices in some individuals with epilepsy. Our laboratory has also previously shown that motor map expansion is related to polysynaptic potentiation in layer V. The magnitude of potentiation of the polysynaptic component of the callosal– neocortical evoked response is enhanced (Racine et al., 1995) as well as significantly and positively correlated with expansion of forelimb movement representations after kindling (Teskey et al., 2002) and LTP induction in LongEvans (Monfils et al., 2004) and epileptogenic-prone rats (Flynn et al., 2004). Movements are most easily elicited when the stimulating electrode is lowered into layer V of
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the sensorimotor neocortex during ICMS (Asanuma and Rosen, 1972), also suggesting that potentiated layer V connections may be the cause of motor map expansion. In contrast, Teskey et al. (2007) observed a decrease in the size of the monosynaptic and polysynaptic components of evoked potentials, as well as a reduction in motor map size in rats that only received LFS. We extend those observations by reporting that LFS is effective in reducing the size of expanded motor maps in rats that have experienced repeated seizures. While non-kindled rats that received LFS in this study did not exhibit statistically significantly smaller maps compared with controls, there was a 21% reduction in map size. The failure to find a significant reduction may be because rats in the present study were approximately three times older than those previously examined (Teskey et al., 2007) and plasticity has been shown to decline with age (Rosenzweig and Barnes, 2003). The anatomical and neurochemical mechanisms underlying map expansion and contraction are likely related to changes in glutamatergic and GABAergic transmission. Repeated neocortical seizures are associated with increases in the number of excitatory perforated synapses (Goertzen and Teskey, 2003) that are associated with 180% more N-methyl-D-aspartate (NMDA) and approximately 650% more AMPA channels compared with nonperforated synapses (Ganeshina et al., 2004). Following kindling of the hippocampus, glutamatergic transmission has been shown to be enhanced through increased presynaptic glutamate release (Geula et al., 1988; Jarvie et al., 1990), recruitment of previously dormant NMDA receptors (Mody and Heinemann, 1987; Mody et al., 1988), and enhanced NMDA-receptor activation (Behr et al., 2000). In contrast, LFS has been shown to be associated with a decrease in dendritic arborization (Monfils and Teskey, 2004), in the number of excitatory perforated synapses, and thus in glutamatergic activity, as well as an increase in inhibitory synapses (Teskey et al., 2007). Enhanced GABAergic activity can decrease motor map size by masking lateral excitatory connections (Jacobs and Donoghue, 1991) resulting in a decrease in the total neocortical area that is responsible for forelimb movements and a corresponding increase in the amount of neocortex that becomes unresponsive to ICMS. LFS is not limited to reversing functional brain reorganization. It is also a tool that appears to have anti-convulsant efficacy. After amygdala-kindled rats achieved a fullykindled state, LFS applied to the amygdala increased AD thresholds (Carrington et al., 2007) and LFS administered to the central piriform cortex resulted in a reduction of both seizure severity and duration (Zhu-Ge et al., 2007). Preemptive LFS administered to the amygdala of fully amygdala-kindled rats decreased the mean number of seizures elicited (Goodman et al., 2005). LFS also has a suppressive effect on seizures when administered during the kindling process. LFS applied to the central piriform cortex had the same suppressive effect on amygdaloid-kindled rats (Yang et al., 2006) and piriform-kindled rats (Ghorbani et al., 2007) when applied after each kindling session. Rat
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pups that received LFS to the amygdala following each amygdala-kindling session (Velisek et al., 2002) and adult rats that received LFS to the amygdala prior to each amygdala kindling session (Goodman et al., 2005) exhibited a mean decrease in the AD duration and seizure stage throughout kindling. López-Meraz et al. (2004) found that the application of LFS to the amygdala following each amygdaloid-kindling session decreased the likelihood that rats would reach a fully kindled state. LFS applied to the amygdala following each perforant path kindling session delayed kindling development, inhibited kindling-induced potentiation, and prevented the increase in early and late paired-pulse depression (Mohammad-Zadeh et al., 2007). Moreover, in humans with intractable mesial temporal lobe epilepsy, applying LFS through subdural electrodes suppressed ictal and interictal discharges (Kinoshita et al., 2005; Yamamoto et al., 2002, 2006).
CONCLUSION In summary, we have demonstrated that LFS reverses kindling-induced functional reorganization expressed as neocortical motor map expansion. Hippocampal kindling followed by LFS delivered to the corpus callosum resulted in a decrease in expanded forelimb movement representations so that the total neocortical forelimb area was equivalent to non-stimulated rats. As a result of kindling, the maps may have expanded due to kindling-induced synaptic potentiation, mediated by an increase in the number of excitatory synapses, an enhancement of glutamatergic transmission, and possibly a reduction of GABAergic inhibition. Following LFS, the maps may have subsequently contracted due to synaptic depotentiation, mediated by a loss of perforated synapses, decreased glutamatergic transmission, and an enhancement of GABAergic inhibition. Future studies are needed to examine the effects of callosal LFS on AD duration, threshold, seizure severity, and motor map size when administered immediately following the cessation of each hippocampal AD. LFS may also be used as a means to ameliorate inter-ictal behavioral deficits that often coincide with seizure disorders.
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(Accepted 12 January 2008) (Available online 8 February 2008)