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Author's personal copy Behavioural Brain Research 202 (2009) 138–141
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Short communication
Functional mapping of the motor cortex of the rat using transdural electrical stimulation Erich Talamoni Fonoff a,b,∗,1 , Jose Francisco Pereira Jr. b , Leonardo Valente Camargo a , Camila Squarzoni Dale a , Rosana Lima Pagano a,c , Gerson Ballester b , Manoel Jacobsen Teixeira a a
Teaching and Research Institute, Hospital Sírio-Libanês, Rua Coronel Nicolau dos Santos 69, Bela Vista, 01308-060, São Paulo, SP, Brazil Division of Functional Neurosurgery, Institute of Psychiatry, Hospital das Clínicas, Department of Neurology, School of Medicine, University of São Paulo, Rua Dr. Ovídio Pires de Campos 785, 01060-970, São Paulo, SP, Brazil c Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Avenue Professor Lineu Prestes 1524, Butantã, 05508-900, São Paulo, SP, Brazil b
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Article history: Received 3 December 2008 Received in revised form 7 March 2009 Accepted 12 March 2009 Available online 24 March 2009 Keywords: Motor cortex Transdural electrical stimulation Rat Electrophysiology Functional mapping Evoked motor response
a b s t r a c t Motor cortex stimulation oriented by functional cortical mapping is used mainly for treating otherwise intractable neurological disorders, however, its mechanism of action remains elusive. Herein, we present a new method for functional mapping of the rat motor cortex using non-invasive transdural electrical stimulation. This method allows a non-invasive mapping of the surface of the neocortex providing a differentiation of representative motor areas. This study may facilitate further investigation about the mechanisms mediating the effects of electrical stimulation, possibly benefiting patients who do not respond to this neuromodulation therapy. © 2009 Elsevier B.V. All rights reserved.
Motor cortex stimulation (MCS) has become a promising technique for the treatment of otherwise intractable central nervous system (CNS) disorders, including chronic pain and Parkinson’s disease. However, the mechanisms underlying the clinical effects of MCS are still uncertain. Experimental investigation about the neurophysiology of MCS has been recently initiated. Nevertheless, the animal models of cortical stimulation used in such experiments differ from the human setting, because in animal models the electrodes are implanted deep into the cerebral cortex next to the cell bodies of pyramidal neurons in layer V [7,18,19]. Transdural stimulation is less invasive than intracortical or subdural stimulation, once there is no penetration in the cerebral cortex, thus avoiding cortical tissue damage or possible complications such as post-operative meningitis, hemorrhage, tissue necrosis, and meningocortical scars. Results obtained with MCS seem to depend on the site of stimulation topographically related to the body segment affected by pain [2]. To our knowledge, the topography of the analgesic effects of MCS it has not been studied in animal models. Despite its small dimensions, the rat cerebral cortex possesses fine topography
∗ Corresponding author. Tel.: +55 11 31551234; fax: +55 11 31550494. E-mail address:
[email protected] (E.T. Fonoff). 1 Tel.: +55 11 32372031; Fax: +55 11 32372031. 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.03.018
and functions which are analogous to those found in non-human primates [16] and also in humans [1,4,6,9,12]. Experimental studies using intracortical microstimulation defined specific areas for different parts of body segments in Sprague-Dawley rats [10,15]. Although the topography of the rat motor cortex is well known, the maps available have been performed using intracortical stimulation and may not be directly applied in MCS studies using transdural stimulation. In recent studies aiming at the investigation of MCS effects in rats, the functional topography of motor cortex has not been taken into account [13,14]. The present work sought to provide a functional map of the motor cortex of rat based on transdural electrical stimulation in order to facilitate future investigation of the neurophysiology of MCS with the same protocols used in human subjects, or any studies involving plasticity of the motor cortex. Male Wistar rats weighing 180–200 g were used throughout this study (n = 20). Animals were maintained under controlled temperature (22 ± 2 ◦ C) and light cycle (12/12 h), with free access to water and food. All the procedures were in accordance with the guidelines for animal experimentation [20], and the study protocol was approved by the institutional review boards of the Hospital das Clínicas, University of São Paulo, and of the Teaching and Research Institute, Hospital Sírio-Libanês, both in Sao Paulo, Brazil. Animals were initially anesthetized with ketamine hydrochloride (0.5 mg/kg i.m.)/xylazine (2.3 mg/kg i.m.) and with local scalp
Author's personal copy E.T. Fonoff et al. / Behavioural Brain Research 202 (2009) 138–141
injections of 2% lidocaine (100 l/animal), and supplementary doses of ketamine were administered as needed. The rats were then placed in a stereotaxic apparatus (David Kopf Instruments, CA, USA), and a median axial incision was made in the scalp, exposing the dura mater after a unilateral rectangular craniotomy (8-mm long and 4.5-mm wide) with the bregma as the reference point. A stainless steel bipolar electrode, with a 100-m diameter pole and 200 m between each tip, was fixed to the skull so that functional mapping was initiated in the antero-medial region of the craniotomy. The electrode was serially positioned in contact with the dura mater, distancing 250 m from each other in all directions, covering the entire exposed area. The cathode was always positioned anterior to the anode pole (parallel to the sagittal plane). Stimulation was performed through the dura mater, mimicking the epidural stimulation adopted by the MCS protocols in humans. The impedance over de dura mater was measured routinely before each stimulation session (978 ± 23 ), which was different from the impedance on the cortical surface (835 ± 16 ) when the dura was removed. Stimulation consisted in 1-s trains of 10-s biphasic cathodal pulses delivered at 100 Hz generated by an electrical stimulator (S8800 – Grass Instruments, Quincy, EUA), attached to a current isolator (SIU5 – Grass Instruments, Quincy, EUA), as shown in Fig. 1. Evoked movements by the electrical stimulation were observed while the animals were maintained in a prone position and the limbs in a fixed position. At each site, the electric current was gradually increased until a movement could be detected, indicating the threshold current. Five similar trails were performed in order to determine the average current for the motor threshold for each site. If no movement was detected with stimulation reaching 850 A, the site was considered as non-responsive. Motor responses corresponding to each coordinate in the motor cortex were filled out into a spreadsheet table. These grids of coordinates referenced by the midline and bregma, corresponding to each tested animal, were compiled to compose probabilistic values for each point of the motor cortex included in the mapping procedure. Results are
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presented using positive responses in the total number of animals tested, which means the proportion of animals that had specific evoked motor responses in each point of the cortex (Figs. 2 and 3). The proposed anesthetic medication provided adequate analgesia, suppressed the spontaneous movement and did not interfere with the evoked motor responses. Evoked movements from different body segments could be clearly observed, including whiskers, neck muscles, hind limb, fore limb, tail, and eye and eyelid (the latter two only in restricted areas of the cortex). The average ± SD of the motor thresholds was 750 ± 65 A. In 22.1% of the stimulated sites, simultaneous movements were observed in more than one body segment during the first stimulation. In these cases, a new sequence of electrical stimulation was performed using more graduated currents, inducing muscle contraction in a specific body segment obtained at lower current intensities. Figs. 2 and 3 illustrate the results obtained from the transdural electrical stimulation of the motor cortex of the right hemisphere of the Wistar rats. The map generated in this procedure reveals the cortical topography of the different body segments according to the stimulation coordinates. Fig. 3 shows the probability of evoked motor responses in different body segments (n = 20). The mean current necessary to elicit motor response in the present study was higher than in previous studies, probably because of the increased dissipation of energy on the dura mater and the superficial layers of cortex. The authors observed that the areas corresponding to whiskers, hind limb, fore limb and cervical muscles markedly agreed with the currently available cortical maps of the rat based on intracortical stimulation [1,4,6,7,9,17]. The area related to ocular motility was fairly restricted and located medially and anterior to the bregma. In the study by Leegaard et al. [9], the eyelid area was also located medially and with a strap shape. Brecht et al. [1] mapped the cortex using stimulus applied deep in the cerebral cortex and evidenced that the area responsible for eye movement was in the interhemispheric face of the cortex, next to the corpus callosum, therefore in an area underneath that
Fig. 1. Panel A shows a panoramic view of the animal positioning in the stereotaxic apparatus during functional cortical mapping with transdural stimulation. Panel B shows the zoomed view of the craniotomy. The dura mater is seen as the thin translucent membrane over the cortex.
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Fig. 2. Panel A shows the matrix representation of the functional mapping obtained by transdural electrical stimulation of the motor cortex in the right hemisphere. Each value corresponds to the percentage of animals that had evoked motor responses in each point of the matrix. The left lateral margin refers to the median line of the rat’s skull and the transverse line, to the bregma. E, eyes; FL, fore limb; HL, hind limb; N, neck muscles; T, tail; W, whiskers. Panel B shows the same map overlapped with a topographical model of the cortex of the rat, extracted from Krieg [8]. Point zero in the ruler represents the bregma in the antero-posterior axis of the cerebral cortex.
Fig. 3. Three-dimensional illustration of the cortical areas mapped with cortical electrical stimulation. The graph’s floor (0%) corresponds to the whole area exposed with the craniotomy, with the median line as starting point. The vertical axis (0–100%) shows the probability of evoking motor responses in each coordinate in the matrix. E, eyes; FL, fore limb; HL, hind limb; N, neck muscles; T, tail; W, whiskers.
found in the present study. As the stimulation was performed in the transdural compartment, the interhemispheric region could not be directly assessed, which may explain the disagreement between the findings in this study and the previous ones [1,9]. We could also delineate clearly the area related to the control of tail motility, which was not described with precision in previous [1,9] studies. This is considered important for studies involving nociception because one of the most frequently used methods evaluating the threshold is the tail flick test. Similar descriptions of cortical areas related to the proximal tail muscles were made in non-human primates (Macaca mulata) by Woosley et al. [17]; however, no mention of these areas were included in functional maps of rodents [11]. Previous studies also found cortical areas that elicited movement of more than one body segment, something pointed out as a possible cause of discrepant results between different studies [3,5,10]. In the present work, it was possible to isolate the movement of different body segments by gradually increasing the electric current. This map of motor cortex presents the probability of evoking motor responses on specific body segments according to the coordinates in the brain cortical surface of the rat. This method provides a map where functional areas corresponding to the body segments are undoubtedly located on the brain cortical surface according to the stereotactic coordinates, since 100% of the animals responded to the stimulation in particular sites. This work provides a functional map of the motor cortex of the Wistar rat using transdural electrical stimulation. The fact that this stimulation technique is similar to the one used in humans may facilitate future studies that aim at understanding the mechanisms
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