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Long–Evans rats have a larger cortical topographic representation of movement than Fischer-344 rats: A microstimulation study of motor cortex in naı̈ve and skilled reaching-trained rats
Brain Research Bulletin, Vol. 59, No. 3, pp. 197–203, 2002 Copyright © 2002 Elsevier Science Inc. All rights reserved. 0361-9230/02/$–see front matter
PII: S0361-9230(02)00865-1
Long–Evans rats have a larger cortical topographic representation of movement than Fischer-344 rats: A microstimulation study of motor cortex in na¨ıve and skilled reaching-trained rats Penny M. VandenBerg, Theresa M. Hogg, Jeffrey A. Kleim and Ian Q. Whishaw∗ Department of Psychology and Neuroscience, Canadian Centre for Behavioural Neuroscience, University of Lethbridge, Lethbridge, Alta., Canada [Received 2 January 2002; Revised 26 June 2002; Accepted 7 July 2002] INTRODUCTION
ABSTRACT: Intracortical microstimulation of the frontal cortex evokes movements in the contralateral limbs, paws, and digits of placental mammals including the laboratory rat. The topographic representation of movement in the rat consists of a rostral forelimb area (RFA), a caudal forelimb area (CFA), and a hind limb area (HLA). The size of these representations can vary between individual animals and the proportional representation of the body parts within regions can also change as a function of experience. To date, there have been no investigations of strain differences in the cortical map of rats, and this was the objective of the present investigation. The effect of cortical stimulation was compared in young male Long–Evans rats and Fischer-344 rats. The overall size of the motor cortex representation was greater in Long–Evans rats compared to Fischer-344 rats and the threshold required to elicit a movement was higher in the Fischer-344 rats. An additional set of animals were trained in a skilled reaching task to rule out the possibility that experiential differences in the groups could account for the result and to examine the relationship between the differences in topography of cortical movement representations and motor performance. The Long–Evans rats were quantitatively and qualitatively better in skilled reaching than the Fischer-344 rats. Also, Long–Evans rats exhibited a relatively larger area of the topographic representation and lower thresholds for eliciting movement in the contralateral forelimb. This is the first study to describe pronounced strain-related differences in the microstimulation-topographic map of the motor cortex. The results are discussed in relation to using strain differences as a way of examining the behavioral, the physiological, and the anatomical organization of the motor system. © 2002 Elsevier Science Inc. All rights reserved.
Intracortical microstimulation of the frontal cortex evokes movements in the contralateral limbs, paws, and digits of many placental mammals including the laboratory rat [3,4,6,9,13,17,19, 20,23,25,26,30,34,36,38,39,41]. The topographic representation of limb movement in the rat can be subdivided into a number of subregions, including a rostral forelimb area (RFA), a caudal forelimb area (CFA), and a hind limb area (HLA) [25,30]. The size of the topographic representation can vary between individual animals. In addition, the proportional representation of the body parts within these regions can also change as a function of experience [19–41]. Intracortical microstimulation studies have used a variety of rat strains. Most cortical mapping studies have used Long–Evans [11–36,39,40], Wistar [23,40], or Sprague–Dawley rat strains [17,20]. These studies have indicated that the organization of motor cortex in the various strains is quite similar with respect to the presence of the three motor regions and accordingly there has been no reason to expect that motor cortex organization would vary as a function of rat strain in any important way. Nevertheless, these studies have been conducted over quite a long time period since the first study by Hall and Lindholm [20] and successive studies have been accompanied by many methodological changes. Important methodological differences are related to the tip diameter of stimulating electrodes, current intensity, and the level of resolution of the mapping grid. Thus, other than confirming the presence of an electrical-stimulation sensitive motor cortex, the studies are difficult to compare. Because there have been no explicit studies of strain differences, this was the purpose of the present experiment. In this study, we compared young male Long–Evans rats to young male Fischer-344 rats. Long–Evans rats have been extensively used in our research. To date, there have been no microstimulation studies of the motor cortex of Fischer-344 rats [7]. Because we found a number of differences between the Long–Evans strain and the Fischer-344
KEY WORDS: Corticospinal stimulation and movement, Fischer-344 motor cortex, Long–Evans rat motor cortex, Microstimulation of motor cortex, Motor cortex, Rat motor cortex, Skilled reaching, Strain differences in motor cortex, Stain differences in skilled reaching.
∗ Address for correspondence: Dr. Ian Q. Whishaw, Department of Psychology and Neuroscience, University of Lethbridge, 4401 University Drive, Lethbridge, Alta., Canada T1K 3M4. Fax: +1-403-329-2775; E-mail: [email protected]
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strain, we trained additional groups of rats in a skilled reaching task in which the rats reached through an aperture to obtain single pieces of food. The quantitative and qualitative aspects of reaching performance were compared in the two groups, and then the hemisphere contralateral to the preferred reaching paw was mapped.
food and then consumed it, the reach was scored as a “hit.” For each test, a hit percent score was obtained using the following formula: Hit percent =
Number of hits × 100. Number of reaches
Reaching Movement Analysis MATERIAL AND METHODS Animals Seventeen Long–Evans rats approximately 4–6 months of age (400–486 g) and 14 male Fischer-344 rats approximately 4–6 months of age (300–400 g) were used. Ten Long–Evans and seven Fischer-344 rats were housed in standard wire mesh cages and allowed access to food and water ad lib. Seven Long–Evans rats and seven Fischer-344 rats were placed on a reduced diet until animals reached 90% of adult body weight for approximately 1 month prior to being trained on a skilled reaching task. Sixteen hours prior to electrophysiological mapping, all animals were food deprived to ensure effective levels of anesthesia. Skilled Reaching Test Prior to single pellet reach training, animals were subjected to 4 days of pretraining. The pretraining box consisted of a 20 cm × 28 cm × 26 cm plexiglass cage with a wire mesh bottom that did not allow animals to retrieve dropped food pellets (90 mg Rodent Chow food pellets, Bioserve Inc., Frenchtown, NJ). The cage had a 5.4 cm × 20 cm × 0.5 cm tray for holding pellets directly outside of the vertical bars at the front of the cage. The rats could reach through the bars of the cage with one or both paws in order to grasp pellets, and had to maintain their grip of the pellets in order to transfer the pellet to their mouth. On the first day, the animals were encouraged to reach through the bars to retrieve pellets. The subsequent days provided opportunity to establish limb preference and for the animals to practice pellet retrieval. After 4 days, all animals were proficient in the ability to retrieve and eat pellets from the tray. Single pellet boxes for training and evaluating skilled reaching were made of clear Plexiglas (25 cm × 35 cm × 30 cm high) so that the rats could be filmed from any perspective [43,45]. Five centimeters from the side of each front wall was a 1-cm wide slit that extended from the floor to a height of 15 cm. On the outside of the wall, in front of the slit, mounted 3 cm above the floor, was a 2-cm wide by 4-cm long shelf. Food pellets were placed in one of two small indentions on the floor of the shelf. The indentations were 2 cm away from the inside wall of the box and were centered on the edges of the slit through which the rats reached. Once the rats began reaching for food, food was placed in the indentation contralateral to the limb with which the rat used for reached. Training was administered in such a way that, when a rat made a successful reach, a short pause preceded presentation of the next food pellet, during which another food pellet was dropped into the back of the box. This was done to ensure that a rat left the food aperture after each reach and repositioned itself at the food aperture for the next food pellet. Video records of reaching performance were made with a Sony Video 8 CCD VII portable camera with a shutter speed of 1000th of a second. A two arm Nikon Inc. MII cold light source provided illumination for high shutter speed filming. Frame-by-frame analysis at 30–60 frames per second was provided by a Sony Video 8 recorder or through a computer-based frame grabber [43]. Reaching performance was scored by counting misses and successful reaches for each limb. If a rat made a reaching movement in which a paw was inserted through the bars/aperture of the cage, the movement was scored as a “reach.” If the rat obtained a piece of
Movements were analyzed using a conceptual framework derived from Eshkol–Wachmann Movement Notation (EWMN) [14]. In brief, EWMN is designed to express relations and changes of relation between the parts of the body. The body is treated as a system of articulated axes (i.e., body and limb segments). A limb is any part of the body that either lies between two joints or has a joint and a free extremity. These are imagined as straight lines (axes), of a constant length, which move with one end fixed to the center of a sphere. An important feature of EWMN is that the same movements can be notated in several polar coordinate systems. The coordinates of each system are determined with reference to the environment, to the animal’s body midline axis, and to the next proximal or distal limb or body segment. By transforming the description of the same behavior from one coordinate system to the next, invariances in that behavior may emerge in some coordinate systems but not in others. Thus, the behavior may be invariant in relation to some or all of the following: the animal’s longitudinal axis, gravity, or bodywise in relation to the next proximal or distal segment. On the basis of descriptions obtained from EWMN, rating scales of movements were derived. Three reaches for each limb by each rat were rated for qualitative features of the movement [46]. Ten component movements of a reach were rated. (1) Digits to the midline. Using mainly the upper arm, the reaching limb is lifted from the floor so that the tips of the digits are aligned with the midline of the body. (2) Digits flexed. As the limb is lifted, the digits are flexed and the paw is supinated and the wrists partially flexed. (3) Elbow-in. Using an upper arm movement, the elbow is adducted to the midline while the tips of the digits retain their alignment with the midline of the body. (4) Advance. The limb is advanced directly through the slot toward the food target. (5) Digits extend. During the advance, the digits extend so that the digit tips are pointing toward the target. (6) Arpeggio. When the paw is over the target, the paw pronates from digit 5 (the outer digit) through to digit 2, and at the same time the digits open. (7) Grasp. The digits close and flex over the food, with the paw remaining in place, and the wrist is slightly extended to lift the food. (8) Supination I. As the paw is withdrawn, the paw supinates by almost 90◦ . (9) Supination II. Once the paw is withdrawn from the slot to the mouth, the paw further supinates by about 45◦ to place the food in the mouth. (10) Release. The mouth contacts the paw and the paw opens to release the food. Each of the movements was rated on a 2-point scale. If the movement was performed normally, a score of “1” was given. If the movement was abnormal, a score of “0” was given. In cases where there was some ambiguity concerning the occurrence of a movement, a score of 0.5 was given. Reaching Posture On each reach, the posture used by the rat was rated on a 2-point scale [29]. If a rat supported itself on the contralateral-to-reaching forelimb and its diagonal hind limb as the reach was initiated, a score of “1” was given. If a rat failed to use this supporting posture, a score of “0” was given. Misses Three missed reaches from each rat were examined and the cause of the misses were tabulated as either short reach, long reach, displaced reach, or inappropriate grasp.
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Electrophysiological Mapping Standard intracortical microstimulation techniques were used to generate detailed maps of forelimb and hind limb representations within one hemisphere of the motor cortex [25]. Prior to surgery, animals were anesthetized with ketamine hydrochloride (70 mg/kg, i.p.) and xylazine (5 mg/kg, i.m.), receiving acepromazine (0.02 mg/kg, i.p.) and ketamine (20 mg/kg, i.p.) as needed. The level of anesthesia was assessed by monitoring breathing and heart rate. A craniotomy was performed over the motor cortex contralateral to the paw used in the reaching task. A small puncture was made in the cisterna magna to reduce edema prior to retraction of the dura. The exposed cortex was then covered with warm silicone oil. A glass electrode (controlled by a hydraulic microdrive) was used to make systematic penetrations at a depth of approximately 1550 µm (corresponding to cortical layer V), with an interpenetration distance of 375 µm. Stimulation consisted of 13,200 µs cathodal pulses delivered at 350 Hz from an electrically isolated stimulation circuit. Animals were maintained in a prone position with the limb supported. At each stimulation site, the minimal threshold required to elicit a movement was recorded and sites where no movement was detected at ≤60 µA were recorded as nonresponsive. When a series of nonresponsive sites were encountered, previous response sites were revisited to check for decreases in responsiveness. Forelimb movements were classified as either distal (wrist/digit) or proximal (elbow/shoulder). Representational maps were generated from the pattern of electrode penetrations. The CFA and RFA were defined by surrounding representations of nonforelimb such as vibrissae, head/neck, or nonresponse sites which represented the borders of these motor areas. HLA was defined by borders of nonhind limb response sites such as trunk, forelimb, and by nonresponse sites. An image analysis program (CANVAS v. 3.5) was used to calculate the extent of the RFA, CFA, and HLA [36].
RESULTS Movement Representations Representative cortical motor maps for one rat in each treatment group are shown in Fig. 1. In general, the maps of the Long–Evans rats were larger than those of the Fischer-344 rats. Further, many of the Fischer-344 rats failed to exhibit an HLA. Means and standard errors for the area representation of RFA, CFA, and HLA are illustrated in Fig. 2. An ANOVA gave a significant effect of rat strain [F (1,26) = 19.9, p < 0.001] but no significant effect of reaching training [F (1,26) = 0.92, p > 0.05]. The overall map sizes of the Long–Evans rats were larger than those of the Fischer-344 rats for both the untrained and the trained groups. There was a significant effect of cortical area [F (2,52) = 2.88, p < 0.001]. In both the Long–Evans and Fischer-344 rats, the CFA was larger than the RFA and the HLA. A CFA was found in all of the rats. An RFA was obtained in all 17 Long–Evans rats and in all but 2 (both untrained) of 13 Fischer-344 rats. An HLA was found in 3 of 10 untrained Long–Evans rats and in all 7 trained Long–Evans rats. An HLA was found in 5 of 6 untrained Fischer-344 rats, but in only 1 of the 7 trained Fischer-344 rats. Skilled Reaching Success on the skilled reaching task is illustrated in Fig. 3A. There was a significant rat strain difference in hit percent scores [F (1,12) = 20.5, p < 0.0001]. There was also a significant effect of reaching training days [F (9,127) = 4.26, p = 0.002] and a significant rat strain by reach-training days interaction [F (9,126) = 2.10, p = 0.03]. Both the Long–Evans and the Fischer-344 rats improved over the 10-day testing period, but the improvement of the Fischer-344 rats was much slower than that of the Long–Evans rats, and even at the end of the training period, the Fischer-344 rats were inferior to the Long–Evans rats.
FIG. 1. Representative maps of motor cortex from Long–Evans (A) and Fischer-344 (B) rats showing digit, wrist, elbow/shoulder, and hind limb areas. Maps from untrained rats (top). Maps from rats trained in skilled reaching (bottom). Note: maps from the Long–Evans rats are larger than those from the Fischer-344 rats. Abbreviations: rfa, rostral forelimb area; cfa, caudal forelimb area; ha, hind limb area.
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VANDENBERG ET AL. Average qualitative movement scores on 10 movements comprising a reach are illustrated in Fig. 3B. Average qualitative movement scores on 10 movements comprising a reach. There was a significant group effect [F (1,12) = 21.8, p = 0.001], with the Fischer-344 rats obtaining poorer scores than the Long–Evans rats. Follow-up tests on individual components gave significant differences for all 10 components (p < 0.05). When the Fischer-344 rats lifted the limb, they usually did not bring the digits to the midline of the body, as did the Long–Evans rats. They also did not bring the elbow to the midline, and what elbow-in movement did occur was generally accompanied by an ipsilateral shift of the body (Fig. 4). They did advance the paw toward the food pellet, and opened their digits as they did so, but the trajectory toward the pellet made by the paw was quite low. Surprisingly, even though the trajectory of the limb was low, the Fischer-344 rats raised their body to an exaggerated degree as they advanced the forelimb (Fig. 4). Most misses made by the Fischer-344 rats were because they knocked the food from the shelf with their digits. When the Fischer-344 rats did grasp food, they frequently caught the food between the second and third digits, again because the grasp was short. The arpeggio movement with which the paw was pronated over the food, and the grasp of the food pellet were also abnormal. Once the rat contacted the food, rather than fully pronating the paw, it flexed the paw medially, closing the digits as it did so (Fig. 5). When the paw was withdrawn through the slot, it was not supinated to 90◦ , and when food was brought to the mouth there was again less supination in the Fischer-344 rats than in the Long–Evans rats (Fig. 5). In short, the Fischer-344 rats were impaired on all 10 movements, but most severe impairments were in aiming the paw, in pronation, and grasping. Map Changes as a Function of Skilled Reaching Training
FIG. 2. Mean area (mm2 ) of movement representations of RFA, CFA, and HLA of Long–Evans rats and Fischer-344 rats. Rats were untrained (A). Rats were trained in a skilled reaching task and the motor cortex contralateral to the preferred paw was mapped (B). Note: in both untrained and trained conditions, the maps, particularly the CFA, are larger in Long–Evans than in Fischer-344 rats.
Measures of the percentage of the CFA occupied by distal forelimb representations revealed a significant effect of rat strain [F (1,26) = 42.3, p < 0.001] and reaching experience [F (1,26) = 4.16, p = 0.05] but no rat strain by reach-training interaction [F (1,26) = 0.29, p = 0.21]. As shown in Fig. 6, although the area of distal representations was larger in the Long–Evans rats, both the Long–Evans and the Fischer-344 rats displayed an increase in
FIG. 3. Skilled reaching performance (mean and standard error) in Long–Evans and Fischer-344 rats. Hit percent (number of food pellets retrieved as a function of the number of reaches) over 10 days of training (A). Average qualitative movement scores per movement on 10 movements comprising a reach (B). Note: Long–Evans rats are superior to Fischer-344 rats on both quantitative and qualitative measures of skilled reaching.
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FIG. 4. Examples of three differences between Fischer-344 rats and Long–Evans rats during transport of the limb to the food: the elbow is not adducted completely to the midline of the body, the paw has a low trajectory to the food so that the digits often hit the food and knock it away, and the body is raised and shifted contralaterally to an exaggerated degree.
FIG. 6. Area and threshold of the distal forelimb representation in untrained and trained Long–Evans and Fischer-344 rats. Area of the distal forelimb as a percent of total forelimb (A). Threshold for eliciting movements in the forelimb area (B). Note: the percent of distal area was higher and the threshold lower in Long–Evans as compared to Fischer-344 rats. Nevertheless, both groups of rats displayed increased distal representation as a function of training in the skilled reaching task.
the percentage of distal forelimb representations, relative to the overall forelimb area, as a function of training. Measures of the threshold for eliciting movements in the forelimb area gave a significant effect of rat strain [F (1,26) = 31.1, p < 0.001] but no significant effect of reaching training [F (1,26) = 1.65, p = 0.2]. There was also a significant interaction of strain by area [F (2,52) = 17.8, p < 0.001] with the threshold in the HLA of the Fischer-344 rats being higher than those of RFA and the CFA. FIG. 5. Examples of the two differences between Fischer-344 rats and Long–Evans rats in grasping and withdrawal. As the Fischer-344 rats grasp the food, they flex the paw medially, whereas the Long–Evans rats extend the paw at the wrist to lift the food from the surface of the shelf. During withdrawal, the Fischer-344 rats fail to supinate the paw to withdraw it through the slot but rather continue to drag the food back through the slot.
DISCUSSION The present experiment compared the microstimulation motor map of young Long–Evans rats to young Fischer-344 rats. Long–Evans rats had significantly larger motor representations than Fischer-344 rats. Also, thresholds required eliciting movement
202 responses were significantly lower for Long–Evans rats. Furthermore, both strains showed an expansion of distal representations following skilled forelimb training. This demonstrates that although there were differences in the total area of the motor map, both strains exhibited the capacity for motor learning-dependent functional plasticity. The Long–Evans rats were, however, superior reachers and again had larger topographic representation of motor cortex than the Fischer-344 rats. This is the first study to describe a strain-related difference in the topography of movement representations in the motor cortex. We found a significantly larger area of motor cortex elicited limb movements in Long–Evans rats than in Fischer-344 rats. The difference was especially pronounced in the CFA. Also, Fischer-344 rats required significantly higher thresholds to elicit a movement response in the RFA and CFA. It is unlikely that the strain differences are due simply to methodological differences, such as the effects of anesthesia or the intensity of electrical stimulation used. The levels of anesthesia were carefully monitored in both strains and mapping was only conducted, when movements were elicited by the lowest intensity of stimulation. Also, the same standards of respiration and heart rate for each group were used to determine the anesthetic level of the rat. According to these criteria, all animals were at the same anesthetic level at the time of mapping. In addition, low intensity stimulation did elicit movements in both strains, but the area from which this stimulation elicited movements in the Long–Evans rats was simply larger than that in the Fischer-344 rats. Thus, the strain differences could be due to structural/connection difference in the neurons of the motor system in the two strains. A potential explanation for the differences between the two strains is that they may have had different experiences prior to mapping [1,8,23,24,33]. The Long–Evans rats were raised in our animal colony whereas the Fischer-344 rats were purchased from a commercial supplier. In addition, previous work has shown that motor experience can change the topography of movement representations within the CFA [12,15,16,18]. Rats trained to perform skilled forelimb movements have a greater proportion of distal movement representations within the CFA [12,15,16,18]. To minimize the possibility that the strain differences were due to experiential differences, addition groups of rats were trained on a skilled reaching task, on the expectation that this training would require use of the motor cortex contralateral to the reaching limb [24]. The Long–Evans group was superior to the Fischer-344 group both in the number of successful reaches that they made and qualitatively in the way that they reached. At the completion of the training period, maps of the motor cortex of the Long–Evans strain were still larger than those of the Fischer-344 strain. Thus, it seems unlikely that the map-size difference is simply related to experiential differences in the two groups. In addition, experiential differences typically affect the relative size of distal or proximal movement zones within motor cortex (a result obtained with both groups of trained rats) and not the overall size of the forelimb motor representation [24]. Motor map topography within the motor cortex has been linked to the ability level of animals in various motor skill tasks. Increases in distal forelimb representation within human, primate, and rat motor representations have been observed following the acquisition of a specific motor skill requiring movements of digits and wrist [24,25,33,36]. It is, therefore, tempting to conclude that the relation between cortical topography and motor skill performance is causal. It is possible, however, that both the small maps of the Fischer-344 rats and their poor skilled movement performance are secondary effects of some other strain-related differences in other neural structures [34]. Nevertheless, a conclusion that the size of the motor map and skilled reaching performance would be consistent with the many studies that have shown a relation between cortical regions and performance.
VANDENBERG ET AL. Another finding that is novel to the present study is that rat strains can display qualitative differences in the way that they reach. The Fischer-344 rats displayed abnormal qualitative features of reaching that are found in rats with motor cortex [5,42,44], pyramidal tract [46], or dorsal column lesions [27]. Therefore, their reaching performance appears to be abnormal relative to Long–Evans rats. At present, we have not investigated whether other aspects of the motor system, including the cortical field of pyramidal cells, the red nucleus, somatosensory cortex, or the afferent or efferent projections of the areas are normal. In addition, models of intrinsic connections of the motor cortex propose that pyramidal neurons within different representational zones are connected via long, horizontal excitatory connections [2]. These connected cells may form functional clusters that elicit movements from similar combinations of muscles [10,21]. Strain-related differences in the structure and neuronal responsiveness of the cortex may be responsible for the difference in size of representational area and level of threshold. It is possible that Fischer-344 have smaller motor representational areas because of strain-related differences in neuronal structure, arbor, or connectivity. There has been only one previous investigation of strain differences in skilled reaching in rats. Nikkhah et al. [32] compared five rat strains on the staircase test, which similar to the present reaching task, evaluates skilled forelimb use. There was a large strain-dependent difference on the acquisition of the motor skill and a difference in final levels of performance of the task. An out-bred Sprague–Dawley rat strain was particularly skilled at the staircase test. Unfortunately none of the other strains included Long–Evans or Fischer-344 rats. Thus, the finding that there are strain differences in reaching success is not novel to the present study. However, novel to the present experiment is the finding that combined behavioral and electrophysiological methods could usefully address the potential neural differences provided by different laboratory rat strains. SUMMARY The present study shows that there are dramatic differences in the topography of motor cortex organization between two rat strains. The choice of strains used in the present study was opportunistic, and so it would be useful to determine whether there are also differences between other strains. The present finding also suggests that strain comparisons could be usefully pursued in understanding the cellular organization, connectivity, and function of the motor system of rodents. This is especially true because the size of the strain differences observed in the present study are generally larger than the differences produced by other manipulations such as enriched housing or enhanced training/learning experiences. ACKNOWLEDGEMENTS
This work was supported by grants from Natural Sciences and Engineering Research Council of Canada, the Canadian Institute of Health Research, the Alberta Heritage Foundation for Medical Research, and the Canadian Stroke Network.
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PII: S0361-9230(02)00865-1
Long–Evans rats have a larger cortical topographic representation of movement than Fischer-344 rats: A microstimulation study of motor cortex in na¨ıve and skilled reaching-trained rats Penny M. VandenBerg, Theresa M. Hogg, Jeffrey A. Kleim and Ian Q. Whishaw∗ Department of Psychology and Neuroscience, Canadian Centre for Behavioural Neuroscience, University of Lethbridge, Lethbridge, Alta., Canada [Received 2 January 2002; Revised 26 June 2002; Accepted 7 July 2002] INTRODUCTION
ABSTRACT: Intracortical microstimulation of the frontal cortex evokes movements in the contralateral limbs, paws, and digits of placental mammals including the laboratory rat. The topographic representation of movement in the rat consists of a rostral forelimb area (RFA), a caudal forelimb area (CFA), and a hind limb area (HLA). The size of these representations can vary between individual animals and the proportional representation of the body parts within regions can also change as a function of experience. To date, there have been no investigations of strain differences in the cortical map of rats, and this was the objective of the present investigation. The effect of cortical stimulation was compared in young male Long–Evans rats and Fischer-344 rats. The overall size of the motor cortex representation was greater in Long–Evans rats compared to Fischer-344 rats and the threshold required to elicit a movement was higher in the Fischer-344 rats. An additional set of animals were trained in a skilled reaching task to rule out the possibility that experiential differences in the groups could account for the result and to examine the relationship between the differences in topography of cortical movement representations and motor performance. The Long–Evans rats were quantitatively and qualitatively better in skilled reaching than the Fischer-344 rats. Also, Long–Evans rats exhibited a relatively larger area of the topographic representation and lower thresholds for eliciting movement in the contralateral forelimb. This is the first study to describe pronounced strain-related differences in the microstimulation-topographic map of the motor cortex. The results are discussed in relation to using strain differences as a way of examining the behavioral, the physiological, and the anatomical organization of the motor system. © 2002 Elsevier Science Inc. All rights reserved.
Intracortical microstimulation of the frontal cortex evokes movements in the contralateral limbs, paws, and digits of many placental mammals including the laboratory rat [3,4,6,9,13,17,19, 20,23,25,26,30,34,36,38,39,41]. The topographic representation of limb movement in the rat can be subdivided into a number of subregions, including a rostral forelimb area (RFA), a caudal forelimb area (CFA), and a hind limb area (HLA) [25,30]. The size of the topographic representation can vary between individual animals. In addition, the proportional representation of the body parts within these regions can also change as a function of experience [19–41]. Intracortical microstimulation studies have used a variety of rat strains. Most cortical mapping studies have used Long–Evans [11–36,39,40], Wistar [23,40], or Sprague–Dawley rat strains [17,20]. These studies have indicated that the organization of motor cortex in the various strains is quite similar with respect to the presence of the three motor regions and accordingly there has been no reason to expect that motor cortex organization would vary as a function of rat strain in any important way. Nevertheless, these studies have been conducted over quite a long time period since the first study by Hall and Lindholm [20] and successive studies have been accompanied by many methodological changes. Important methodological differences are related to the tip diameter of stimulating electrodes, current intensity, and the level of resolution of the mapping grid. Thus, other than confirming the presence of an electrical-stimulation sensitive motor cortex, the studies are difficult to compare. Because there have been no explicit studies of strain differences, this was the purpose of the present experiment. In this study, we compared young male Long–Evans rats to young male Fischer-344 rats. Long–Evans rats have been extensively used in our research. To date, there have been no microstimulation studies of the motor cortex of Fischer-344 rats [7]. Because we found a number of differences between the Long–Evans strain and the Fischer-344
KEY WORDS: Corticospinal stimulation and movement, Fischer-344 motor cortex, Long–Evans rat motor cortex, Microstimulation of motor cortex, Motor cortex, Rat motor cortex, Skilled reaching, Strain differences in motor cortex, Stain differences in skilled reaching.
∗ Address for correspondence: Dr. Ian Q. Whishaw, Department of Psychology and Neuroscience, University of Lethbridge, 4401 University Drive, Lethbridge, Alta., Canada T1K 3M4. Fax: +1-403-329-2775; E-mail: [email protected]
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strain, we trained additional groups of rats in a skilled reaching task in which the rats reached through an aperture to obtain single pieces of food. The quantitative and qualitative aspects of reaching performance were compared in the two groups, and then the hemisphere contralateral to the preferred reaching paw was mapped.
food and then consumed it, the reach was scored as a “hit.” For each test, a hit percent score was obtained using the following formula: Hit percent =
Number of hits × 100. Number of reaches
Reaching Movement Analysis MATERIAL AND METHODS Animals Seventeen Long–Evans rats approximately 4–6 months of age (400–486 g) and 14 male Fischer-344 rats approximately 4–6 months of age (300–400 g) were used. Ten Long–Evans and seven Fischer-344 rats were housed in standard wire mesh cages and allowed access to food and water ad lib. Seven Long–Evans rats and seven Fischer-344 rats were placed on a reduced diet until animals reached 90% of adult body weight for approximately 1 month prior to being trained on a skilled reaching task. Sixteen hours prior to electrophysiological mapping, all animals were food deprived to ensure effective levels of anesthesia. Skilled Reaching Test Prior to single pellet reach training, animals were subjected to 4 days of pretraining. The pretraining box consisted of a 20 cm × 28 cm × 26 cm plexiglass cage with a wire mesh bottom that did not allow animals to retrieve dropped food pellets (90 mg Rodent Chow food pellets, Bioserve Inc., Frenchtown, NJ). The cage had a 5.4 cm × 20 cm × 0.5 cm tray for holding pellets directly outside of the vertical bars at the front of the cage. The rats could reach through the bars of the cage with one or both paws in order to grasp pellets, and had to maintain their grip of the pellets in order to transfer the pellet to their mouth. On the first day, the animals were encouraged to reach through the bars to retrieve pellets. The subsequent days provided opportunity to establish limb preference and for the animals to practice pellet retrieval. After 4 days, all animals were proficient in the ability to retrieve and eat pellets from the tray. Single pellet boxes for training and evaluating skilled reaching were made of clear Plexiglas (25 cm × 35 cm × 30 cm high) so that the rats could be filmed from any perspective [43,45]. Five centimeters from the side of each front wall was a 1-cm wide slit that extended from the floor to a height of 15 cm. On the outside of the wall, in front of the slit, mounted 3 cm above the floor, was a 2-cm wide by 4-cm long shelf. Food pellets were placed in one of two small indentions on the floor of the shelf. The indentations were 2 cm away from the inside wall of the box and were centered on the edges of the slit through which the rats reached. Once the rats began reaching for food, food was placed in the indentation contralateral to the limb with which the rat used for reached. Training was administered in such a way that, when a rat made a successful reach, a short pause preceded presentation of the next food pellet, during which another food pellet was dropped into the back of the box. This was done to ensure that a rat left the food aperture after each reach and repositioned itself at the food aperture for the next food pellet. Video records of reaching performance were made with a Sony Video 8 CCD VII portable camera with a shutter speed of 1000th of a second. A two arm Nikon Inc. MII cold light source provided illumination for high shutter speed filming. Frame-by-frame analysis at 30–60 frames per second was provided by a Sony Video 8 recorder or through a computer-based frame grabber [43]. Reaching performance was scored by counting misses and successful reaches for each limb. If a rat made a reaching movement in which a paw was inserted through the bars/aperture of the cage, the movement was scored as a “reach.” If the rat obtained a piece of
Movements were analyzed using a conceptual framework derived from Eshkol–Wachmann Movement Notation (EWMN) [14]. In brief, EWMN is designed to express relations and changes of relation between the parts of the body. The body is treated as a system of articulated axes (i.e., body and limb segments). A limb is any part of the body that either lies between two joints or has a joint and a free extremity. These are imagined as straight lines (axes), of a constant length, which move with one end fixed to the center of a sphere. An important feature of EWMN is that the same movements can be notated in several polar coordinate systems. The coordinates of each system are determined with reference to the environment, to the animal’s body midline axis, and to the next proximal or distal limb or body segment. By transforming the description of the same behavior from one coordinate system to the next, invariances in that behavior may emerge in some coordinate systems but not in others. Thus, the behavior may be invariant in relation to some or all of the following: the animal’s longitudinal axis, gravity, or bodywise in relation to the next proximal or distal segment. On the basis of descriptions obtained from EWMN, rating scales of movements were derived. Three reaches for each limb by each rat were rated for qualitative features of the movement [46]. Ten component movements of a reach were rated. (1) Digits to the midline. Using mainly the upper arm, the reaching limb is lifted from the floor so that the tips of the digits are aligned with the midline of the body. (2) Digits flexed. As the limb is lifted, the digits are flexed and the paw is supinated and the wrists partially flexed. (3) Elbow-in. Using an upper arm movement, the elbow is adducted to the midline while the tips of the digits retain their alignment with the midline of the body. (4) Advance. The limb is advanced directly through the slot toward the food target. (5) Digits extend. During the advance, the digits extend so that the digit tips are pointing toward the target. (6) Arpeggio. When the paw is over the target, the paw pronates from digit 5 (the outer digit) through to digit 2, and at the same time the digits open. (7) Grasp. The digits close and flex over the food, with the paw remaining in place, and the wrist is slightly extended to lift the food. (8) Supination I. As the paw is withdrawn, the paw supinates by almost 90◦ . (9) Supination II. Once the paw is withdrawn from the slot to the mouth, the paw further supinates by about 45◦ to place the food in the mouth. (10) Release. The mouth contacts the paw and the paw opens to release the food. Each of the movements was rated on a 2-point scale. If the movement was performed normally, a score of “1” was given. If the movement was abnormal, a score of “0” was given. In cases where there was some ambiguity concerning the occurrence of a movement, a score of 0.5 was given. Reaching Posture On each reach, the posture used by the rat was rated on a 2-point scale [29]. If a rat supported itself on the contralateral-to-reaching forelimb and its diagonal hind limb as the reach was initiated, a score of “1” was given. If a rat failed to use this supporting posture, a score of “0” was given. Misses Three missed reaches from each rat were examined and the cause of the misses were tabulated as either short reach, long reach, displaced reach, or inappropriate grasp.
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Electrophysiological Mapping Standard intracortical microstimulation techniques were used to generate detailed maps of forelimb and hind limb representations within one hemisphere of the motor cortex [25]. Prior to surgery, animals were anesthetized with ketamine hydrochloride (70 mg/kg, i.p.) and xylazine (5 mg/kg, i.m.), receiving acepromazine (0.02 mg/kg, i.p.) and ketamine (20 mg/kg, i.p.) as needed. The level of anesthesia was assessed by monitoring breathing and heart rate. A craniotomy was performed over the motor cortex contralateral to the paw used in the reaching task. A small puncture was made in the cisterna magna to reduce edema prior to retraction of the dura. The exposed cortex was then covered with warm silicone oil. A glass electrode (controlled by a hydraulic microdrive) was used to make systematic penetrations at a depth of approximately 1550 µm (corresponding to cortical layer V), with an interpenetration distance of 375 µm. Stimulation consisted of 13,200 µs cathodal pulses delivered at 350 Hz from an electrically isolated stimulation circuit. Animals were maintained in a prone position with the limb supported. At each stimulation site, the minimal threshold required to elicit a movement was recorded and sites where no movement was detected at ≤60 µA were recorded as nonresponsive. When a series of nonresponsive sites were encountered, previous response sites were revisited to check for decreases in responsiveness. Forelimb movements were classified as either distal (wrist/digit) or proximal (elbow/shoulder). Representational maps were generated from the pattern of electrode penetrations. The CFA and RFA were defined by surrounding representations of nonforelimb such as vibrissae, head/neck, or nonresponse sites which represented the borders of these motor areas. HLA was defined by borders of nonhind limb response sites such as trunk, forelimb, and by nonresponse sites. An image analysis program (CANVAS v. 3.5) was used to calculate the extent of the RFA, CFA, and HLA [36].
RESULTS Movement Representations Representative cortical motor maps for one rat in each treatment group are shown in Fig. 1. In general, the maps of the Long–Evans rats were larger than those of the Fischer-344 rats. Further, many of the Fischer-344 rats failed to exhibit an HLA. Means and standard errors for the area representation of RFA, CFA, and HLA are illustrated in Fig. 2. An ANOVA gave a significant effect of rat strain [F (1,26) = 19.9, p < 0.001] but no significant effect of reaching training [F (1,26) = 0.92, p > 0.05]. The overall map sizes of the Long–Evans rats were larger than those of the Fischer-344 rats for both the untrained and the trained groups. There was a significant effect of cortical area [F (2,52) = 2.88, p < 0.001]. In both the Long–Evans and Fischer-344 rats, the CFA was larger than the RFA and the HLA. A CFA was found in all of the rats. An RFA was obtained in all 17 Long–Evans rats and in all but 2 (both untrained) of 13 Fischer-344 rats. An HLA was found in 3 of 10 untrained Long–Evans rats and in all 7 trained Long–Evans rats. An HLA was found in 5 of 6 untrained Fischer-344 rats, but in only 1 of the 7 trained Fischer-344 rats. Skilled Reaching Success on the skilled reaching task is illustrated in Fig. 3A. There was a significant rat strain difference in hit percent scores [F (1,12) = 20.5, p < 0.0001]. There was also a significant effect of reaching training days [F (9,127) = 4.26, p = 0.002] and a significant rat strain by reach-training days interaction [F (9,126) = 2.10, p = 0.03]. Both the Long–Evans and the Fischer-344 rats improved over the 10-day testing period, but the improvement of the Fischer-344 rats was much slower than that of the Long–Evans rats, and even at the end of the training period, the Fischer-344 rats were inferior to the Long–Evans rats.
FIG. 1. Representative maps of motor cortex from Long–Evans (A) and Fischer-344 (B) rats showing digit, wrist, elbow/shoulder, and hind limb areas. Maps from untrained rats (top). Maps from rats trained in skilled reaching (bottom). Note: maps from the Long–Evans rats are larger than those from the Fischer-344 rats. Abbreviations: rfa, rostral forelimb area; cfa, caudal forelimb area; ha, hind limb area.
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VANDENBERG ET AL. Average qualitative movement scores on 10 movements comprising a reach are illustrated in Fig. 3B. Average qualitative movement scores on 10 movements comprising a reach. There was a significant group effect [F (1,12) = 21.8, p = 0.001], with the Fischer-344 rats obtaining poorer scores than the Long–Evans rats. Follow-up tests on individual components gave significant differences for all 10 components (p < 0.05). When the Fischer-344 rats lifted the limb, they usually did not bring the digits to the midline of the body, as did the Long–Evans rats. They also did not bring the elbow to the midline, and what elbow-in movement did occur was generally accompanied by an ipsilateral shift of the body (Fig. 4). They did advance the paw toward the food pellet, and opened their digits as they did so, but the trajectory toward the pellet made by the paw was quite low. Surprisingly, even though the trajectory of the limb was low, the Fischer-344 rats raised their body to an exaggerated degree as they advanced the forelimb (Fig. 4). Most misses made by the Fischer-344 rats were because they knocked the food from the shelf with their digits. When the Fischer-344 rats did grasp food, they frequently caught the food between the second and third digits, again because the grasp was short. The arpeggio movement with which the paw was pronated over the food, and the grasp of the food pellet were also abnormal. Once the rat contacted the food, rather than fully pronating the paw, it flexed the paw medially, closing the digits as it did so (Fig. 5). When the paw was withdrawn through the slot, it was not supinated to 90◦ , and when food was brought to the mouth there was again less supination in the Fischer-344 rats than in the Long–Evans rats (Fig. 5). In short, the Fischer-344 rats were impaired on all 10 movements, but most severe impairments were in aiming the paw, in pronation, and grasping. Map Changes as a Function of Skilled Reaching Training
FIG. 2. Mean area (mm2 ) of movement representations of RFA, CFA, and HLA of Long–Evans rats and Fischer-344 rats. Rats were untrained (A). Rats were trained in a skilled reaching task and the motor cortex contralateral to the preferred paw was mapped (B). Note: in both untrained and trained conditions, the maps, particularly the CFA, are larger in Long–Evans than in Fischer-344 rats.
Measures of the percentage of the CFA occupied by distal forelimb representations revealed a significant effect of rat strain [F (1,26) = 42.3, p < 0.001] and reaching experience [F (1,26) = 4.16, p = 0.05] but no rat strain by reach-training interaction [F (1,26) = 0.29, p = 0.21]. As shown in Fig. 6, although the area of distal representations was larger in the Long–Evans rats, both the Long–Evans and the Fischer-344 rats displayed an increase in
FIG. 3. Skilled reaching performance (mean and standard error) in Long–Evans and Fischer-344 rats. Hit percent (number of food pellets retrieved as a function of the number of reaches) over 10 days of training (A). Average qualitative movement scores per movement on 10 movements comprising a reach (B). Note: Long–Evans rats are superior to Fischer-344 rats on both quantitative and qualitative measures of skilled reaching.
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FIG. 4. Examples of three differences between Fischer-344 rats and Long–Evans rats during transport of the limb to the food: the elbow is not adducted completely to the midline of the body, the paw has a low trajectory to the food so that the digits often hit the food and knock it away, and the body is raised and shifted contralaterally to an exaggerated degree.
FIG. 6. Area and threshold of the distal forelimb representation in untrained and trained Long–Evans and Fischer-344 rats. Area of the distal forelimb as a percent of total forelimb (A). Threshold for eliciting movements in the forelimb area (B). Note: the percent of distal area was higher and the threshold lower in Long–Evans as compared to Fischer-344 rats. Nevertheless, both groups of rats displayed increased distal representation as a function of training in the skilled reaching task.
the percentage of distal forelimb representations, relative to the overall forelimb area, as a function of training. Measures of the threshold for eliciting movements in the forelimb area gave a significant effect of rat strain [F (1,26) = 31.1, p < 0.001] but no significant effect of reaching training [F (1,26) = 1.65, p = 0.2]. There was also a significant interaction of strain by area [F (2,52) = 17.8, p < 0.001] with the threshold in the HLA of the Fischer-344 rats being higher than those of RFA and the CFA. FIG. 5. Examples of the two differences between Fischer-344 rats and Long–Evans rats in grasping and withdrawal. As the Fischer-344 rats grasp the food, they flex the paw medially, whereas the Long–Evans rats extend the paw at the wrist to lift the food from the surface of the shelf. During withdrawal, the Fischer-344 rats fail to supinate the paw to withdraw it through the slot but rather continue to drag the food back through the slot.
DISCUSSION The present experiment compared the microstimulation motor map of young Long–Evans rats to young Fischer-344 rats. Long–Evans rats had significantly larger motor representations than Fischer-344 rats. Also, thresholds required eliciting movement
202 responses were significantly lower for Long–Evans rats. Furthermore, both strains showed an expansion of distal representations following skilled forelimb training. This demonstrates that although there were differences in the total area of the motor map, both strains exhibited the capacity for motor learning-dependent functional plasticity. The Long–Evans rats were, however, superior reachers and again had larger topographic representation of motor cortex than the Fischer-344 rats. This is the first study to describe a strain-related difference in the topography of movement representations in the motor cortex. We found a significantly larger area of motor cortex elicited limb movements in Long–Evans rats than in Fischer-344 rats. The difference was especially pronounced in the CFA. Also, Fischer-344 rats required significantly higher thresholds to elicit a movement response in the RFA and CFA. It is unlikely that the strain differences are due simply to methodological differences, such as the effects of anesthesia or the intensity of electrical stimulation used. The levels of anesthesia were carefully monitored in both strains and mapping was only conducted, when movements were elicited by the lowest intensity of stimulation. Also, the same standards of respiration and heart rate for each group were used to determine the anesthetic level of the rat. According to these criteria, all animals were at the same anesthetic level at the time of mapping. In addition, low intensity stimulation did elicit movements in both strains, but the area from which this stimulation elicited movements in the Long–Evans rats was simply larger than that in the Fischer-344 rats. Thus, the strain differences could be due to structural/connection difference in the neurons of the motor system in the two strains. A potential explanation for the differences between the two strains is that they may have had different experiences prior to mapping [1,8,23,24,33]. The Long–Evans rats were raised in our animal colony whereas the Fischer-344 rats were purchased from a commercial supplier. In addition, previous work has shown that motor experience can change the topography of movement representations within the CFA [12,15,16,18]. Rats trained to perform skilled forelimb movements have a greater proportion of distal movement representations within the CFA [12,15,16,18]. To minimize the possibility that the strain differences were due to experiential differences, addition groups of rats were trained on a skilled reaching task, on the expectation that this training would require use of the motor cortex contralateral to the reaching limb [24]. The Long–Evans group was superior to the Fischer-344 group both in the number of successful reaches that they made and qualitatively in the way that they reached. At the completion of the training period, maps of the motor cortex of the Long–Evans strain were still larger than those of the Fischer-344 strain. Thus, it seems unlikely that the map-size difference is simply related to experiential differences in the two groups. In addition, experiential differences typically affect the relative size of distal or proximal movement zones within motor cortex (a result obtained with both groups of trained rats) and not the overall size of the forelimb motor representation [24]. Motor map topography within the motor cortex has been linked to the ability level of animals in various motor skill tasks. Increases in distal forelimb representation within human, primate, and rat motor representations have been observed following the acquisition of a specific motor skill requiring movements of digits and wrist [24,25,33,36]. It is, therefore, tempting to conclude that the relation between cortical topography and motor skill performance is causal. It is possible, however, that both the small maps of the Fischer-344 rats and their poor skilled movement performance are secondary effects of some other strain-related differences in other neural structures [34]. Nevertheless, a conclusion that the size of the motor map and skilled reaching performance would be consistent with the many studies that have shown a relation between cortical regions and performance.
VANDENBERG ET AL. Another finding that is novel to the present study is that rat strains can display qualitative differences in the way that they reach. The Fischer-344 rats displayed abnormal qualitative features of reaching that are found in rats with motor cortex [5,42,44], pyramidal tract [46], or dorsal column lesions [27]. Therefore, their reaching performance appears to be abnormal relative to Long–Evans rats. At present, we have not investigated whether other aspects of the motor system, including the cortical field of pyramidal cells, the red nucleus, somatosensory cortex, or the afferent or efferent projections of the areas are normal. In addition, models of intrinsic connections of the motor cortex propose that pyramidal neurons within different representational zones are connected via long, horizontal excitatory connections [2]. These connected cells may form functional clusters that elicit movements from similar combinations of muscles [10,21]. Strain-related differences in the structure and neuronal responsiveness of the cortex may be responsible for the difference in size of representational area and level of threshold. It is possible that Fischer-344 have smaller motor representational areas because of strain-related differences in neuronal structure, arbor, or connectivity. There has been only one previous investigation of strain differences in skilled reaching in rats. Nikkhah et al. [32] compared five rat strains on the staircase test, which similar to the present reaching task, evaluates skilled forelimb use. There was a large strain-dependent difference on the acquisition of the motor skill and a difference in final levels of performance of the task. An out-bred Sprague–Dawley rat strain was particularly skilled at the staircase test. Unfortunately none of the other strains included Long–Evans or Fischer-344 rats. Thus, the finding that there are strain differences in reaching success is not novel to the present study. However, novel to the present experiment is the finding that combined behavioral and electrophysiological methods could usefully address the potential neural differences provided by different laboratory rat strains. SUMMARY The present study shows that there are dramatic differences in the topography of motor cortex organization between two rat strains. The choice of strains used in the present study was opportunistic, and so it would be useful to determine whether there are also differences between other strains. The present finding also suggests that strain comparisons could be usefully pursued in understanding the cellular organization, connectivity, and function of the motor system of rodents. This is especially true because the size of the strain differences observed in the present study are generally larger than the differences produced by other manipulations such as enriched housing or enhanced training/learning experiences. ACKNOWLEDGEMENTS
This work was supported by grants from Natural Sciences and Engineering Research Council of Canada, the Canadian Institute of Health Research, the Alberta Heritage Foundation for Medical Research, and the Canadian Stroke Network.
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