A grasp-related deficit in tactile discrimination following dorsal column lesion in the rat

Brain Research Bulletin, Vol. 54, No. 2, pp. 237–242, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter

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A grasp-related deficit in tactile discrimination following dorsal column lesion in the rat Mark Ballermann, John McKenna and Ian Q. Whishaw* Department of Psychology and Neuroscience, University of Lethbridge, Lethbridge, Alberta, Canada [Received 14 August 2000; Revised 2 January 2001; Accepted 3 January 2001] ABSTRACT: The dorsal columns of the spinal cord are a major source of haptic (sense of active touch) and proprioceptive input to the brainstem and sensory-motor cortex. Following injury in primates, there are impairments in two-point discrimination, direction of movement across the skin, and frequency of vibration, and qualitative control of the digits, but simple spatial discriminations recover. In the rat there are qualitative deficits in paw control in skilled reaching, but no sensory deficits have been reported. Because recent investigations of sensory control suggest that sensory functions may be related to specific actions, the present study investigated whether the dorsal columns contribute to hapsis during food grasping in the rat. Adult female Long-Evans rats were trained to reach with a single forepaw for a piece of uncooked pasta or for equivalent sized but tactually different nonfood items. One group was given lesions of the dorsal column ipsilateral to their preferred paw, while the second group served as a control. Postlesion, both groups were tested for skilled reaching success and force application as well as adhesive dot removal and forepaw placing. Performance levels on these tests were normal. Nevertheless, the rats with dorsal column lesions were unable to discriminate a food item from a tactually distinctive nonfood item as part of the reaching act, suggesting that the dorsal columns are important for on-line tactile discriminations, or “haptic actions,” which contribute to the normal performance of grasping actions © 2001 Elsevier Science Inc..

ganglia [5,27], and dorsal column of the spinal cord [11] contribute to skilled movement performance and success. Although there are similarities in skilled forelimb use between rodents, carnivores, and primates, rodents display striking differences in sensory control. Whereas primates use vision to guide reaches, rats use olfaction to locate and identify a food object [26] and hapsis (sense of touch during the grasping movement) to position the paw and grasp [2]. Because rats use olfaction and hapsis rather than vision to guide their reaching movements, it is likely that the neural systems controlling their reaching are organized differently than those of primates. For example, whereas primate sensory and motor cortex are topographically distinct, sensory and motor cortexes partially overlap in the rat [6]. Given the rat’s dependence on hapsis for skilled reaching, it is surprising that only qualitative changes in skilled reaching follow the presumptive loss of hapsis due to dorsal column lesions [11]. In their studies on visual function in humans and primates, Milner and Goodale [12] review a broad range of evidence that suggests a division of labor in visual processing between coding for perception and coding for action. Following this line of reasoning, it seems possible that there might be a similar division of labor in the somatosensory system. Thus, in order to detect somatosensory deficits following dorsal column lesions in the rat, it might be necessary to examine sensory contributions during relevant motor acts. The purpose of the present study was to reexamine whether somatosensory information contributes to skilled reaching in the rats by embedding a test of hapsis in a reaching task. Following dorsal column lesions, the rats were trained to reach through a slot and distinguish, on the basis of hapsis, a piece of pasta from a texturally distinct, nonfood item. The rats were also given tests of tactile sensitivity and a test for spontaneous forelimb placing.

KEY WORDS: Skilled reaching, Tactile discrimination, Dorsal column, Spinal cord, Grasping force, Pasta reaching, Adhesive dot removal, Rat motor system, Somatosensation, Hapsis, Haptic searching.

INTRODUCTION Since Peterson’s [17] seminal description of skilled reaching in the rat, the species has been used for a wide range of studies on neural control [27], recovery from nervous system injury [13,18,34], and evolution of skilled movements [29,32]. Tasks have been developed in which animals reach through slots, onto shelves of different heights, down onto staircases, onto moving conveyor belts or turntables, or reach for moving prey [8,24,32]. The movements and neural control of rat skilled forelimb movements have been found to be very similar to those used by carnivores [1] and primates [9]. A variety of neural structures including motor cortex [3,4,7,15,28], the pyramidal tract [31], rubrospinal tract [30], basal

MATERIALS AND METHODS Subjects Twelve adult, female, Long-Evans rats (250 g), housed together in wire mesh cages in a room maintained at a temperature of approximately 22°C and on a 12:12 h light-dark cycle, with lights on at 0730h were used. Animals were on a restricted food schedule that reduced their body weight to 95% of normal body weight. The

* Address for correspondence: Dr. Ian Q. Whishaw, Department of Psychology and Neuroscience, University of Lethbridge, 4401 University Dr., Lethbridge AB, T1K 3M4 Canada. Fax: ⫹1-403-329-2555; E-mail: [email protected]

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238 University of Lethbridge Animal Welfare Committee approved animal use procedures in this study. Surgery Eight of the 12 animals received unilateral lesions of the dorsal columns [11]. The animals were injected with 45 mg/kg of sodium pentobarbital (administered i.p.), with Isoflurane (inhaled) supplemented, for anesthesia. A sagittal incision was made in the nape of the neck, and the C1 and C2 vertebrae were exposed through blunt dissection [11]. The medial part of the dorsal arch of C1 was removed with a drill, and the dorsal column was cut using a sharp No. 11 razor blade. The transections were made on the ipsilateral side to the preferred paw for reaching. The incision was closed using surgical staples. Reaching Task The animals reached for pieces of pasta from a clear Plexiglas box with dimensions of 12 cm wide by 40 cm deep by 40 cm high. In the center of the front wall of the box was a slot (2 cm wide, 30 cm high) through which animals could reach to grasp pieces of pasta. In front of the slot was a 4-cm high shelf (3.5 cm deep and 10 cm wide). An open-ended frame (5 cm high, 5 cm wide, and 3 cm deep) was on top of the shelf, centered in front of the slot. Two holes (diameter ⫽ 2.3 mm) were located on each wall, the floor, and the roof of the frame, 2 cm away from the front of the box. The holes located on the shelf and roof of the frame were aligned with the edges of the slot. The holes in the walls of the frame were 1.5 and 3.0 cm above the shelf. Food and nonfood target items were placed into the holes so that they protruded 25 mm into the center of the frame. The food items consisted of uncooked pieces of spaghetti (1.6 mm diameter). The serrated nonfood target was the serrated portion of a drill bit (1.6 mm diameter; 25 mm length). The smooth nonfood target was a metal rod with a diameter of 1.6 mm (25 mm length). The nonfood target items were attached to a short segment of pasta via a nylon cable tie pulled tight around both items. Either nonfood target could be retrieved if similar force was applied. Because each object breaks proximally (at the tie for the nonfood item), the force required to break each discriminanda is equal. Presurgery, animals were given 15 training sessions where they were allowed to reach for pieces of spaghetti. Once an animal displayed a paw preference, the pasta was placed in the contralateral hole to preferred limb [25]. Reaching was filmed with a high-speed camera (Peak Performance Technologies, Englewood, CO, USA). The animals were filmed 1 day before their surgery, as well as 7, 11, 15, and 19 days following surgery. On each test day, the animals were given 10 trials, and success scores were obtained. An attempt was scored when the animal advanced its forepaw through the slot and a success was scored if it obtained the pasta. If the rat missed the pasta and withdrew its paw, the reach was scored as a failure. Results were summarized as number of attempts per success. Force Measurement To measure forces exerted by the limb when breaking free the pasta, pasta items were held in place by a metal collar that was also attached to the post of a force transducer (DX-300, Bokam Engineering, Santa Ana, CA, USA). Two thumbscrews attached the collar to both the pasta and the force transducer. A PC Computer using Windaq acquisition software (DataQ Instruments, Akron, OH, USA), configured to sample in three dimensions (fore-aft, lateral, and vertical) captured voltage data at 240 Hz/channel. The animals were filmed while they reached for pasta in the force

BALLERMANN, MCKENNA AND WHISHAW transducer to determine where the animals grasped the pasta. The force transducer was calibrated using known weights hung at varying distances from the collar. Force magnitude and direction was calculated using the voltage data from the force transducer and the distance measurements from the video record. The animals were given 15 trials where the force direction and duration were measured. The data points examined included the forces in all three directions at the breaking point (peak force level), as well as the time elapsed from the first deviation from 0 N to the peak force. Haptic Discrimination For the haptic discrimination [2] task, animals were presented with two horizontal stimuli (one above the other), a 25-mm piece of spaghetti, and the serrated nonfood target. The two targets were placed in random positions for each trial. Animals were given 50 trials per day for 7 consecutive days, beginning on day 20 following surgery. Following this training they were given 50 probe trials on a single day. On the probe trial, the animals were tested with a smooth nonfood item in place of the serrated nonfood item. Performance was scored as correct responses and errors defined as follows: 1. Correct discrimination—the animal contacted the nonfood stimulus, and subsequently chose the food stimulus. 2. Error—the animal broke off the nonfood target item. 3. Percentage success—The number of correct discriminations divided by the total number of trials on which an animal contacted a nonfood target (correct plus errors), multiplied by 100%. Control for Use of Olfactory Cues Food and nonfood cues were presented in close proximity to each other to prevent the animals from discriminating between the objects based on their olfactory cues alone. The nonfood items were also stored with the food items to cause them to absorb any odor given off by the food item. In addition, animals were given a probe trial where only the texture of the nonfood item was changed. This suggests that animals were not using olfactory cues as their accuracy in discriminating between the items was reduced by the change in texture, whereas olfactory cues remained the same (see Results). If the animals use olfaction to make the discrimination, they should choose the food item without contacting the nonfood item (in significantly greater than 50% of trials), and do this regardless of how the texture of the nonfood item changes. Animals in this study did not choose the food item without contacting the nonfood item in significantly more than 50% of the trials (see Results). Chance levels in haptic discrimination depend on the animals’ tendency to switch their grasp from one discriminanda to the other spontaneously. If animals switch on all trials, chance lies at 50%, but if they always break the first stimuli they contact, then chance lies at 0%. Chance levels (including the effects of olfactory cues, temperature, and other variables) could be defined as the level of the probe trial. Vertical Paw Placing Vertical paw placing was measured in a Plexiglas cylinder that was 20 cm in diameter and 30 cm in height. The animals were placed in the cylinder for a single 5-min trial and videotaped from below. Forelimb contacts with the wall were counted to determine if there was an asymmetry in limb use [21].

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Adhesive Dot Removal The latency to contact and remove circular adhesive dots (diameter ⫽ 0.8 cm) from the radial aspect of the forelimbs was measured in animals tested singly in their home cage [22]. The animals were removed from their cage, the dots were applied, and the animals were returned to the cage. The time taken to contact and remove each dot using the mouth was recorded. The animals were given three trials per day for 3 days. Histology Following testing, the animals were deeply anesthetized with Euthansol and were perfused by intraventricular injection of 200 ml of 0.9% saline, followed by 150 ml of 4% formaldehyde. The brains and spinal cords were removed and cryo-protected by placing them in a solution of 30% sucrose and 4% formaldehyde. The spinal cord was cut into 60-␮m coronal sections using a cyrostat. Sections were stained with cresyl violet to determine the location of the lesion. Statistical Analysis Results were examined using ANOVA [33] and Statview statistical software (Abacus Concepts, Berkeley, CA, USA). The alpha value is set at p ⫽ 0.05. RESULTS Histology Histological examination of the spinal cord revealed that the lesion successfully interrupted the dorsal column, while leaving the dorsal horns intact (Fig. 1). In all but one of the rats the dorsal portion of the dorsal column was partially or completely interrupted, in two of these animals the dorsal column was completely sectioned. In addition, three of these animals had a small amount of damage extending into the dorsal portion of the pyramidal tract. These animals were not significantly impaired when compared to other lesioned animals. In only one rat was the dorsal column spared by the lesion, and this animal displayed no deficit on the haptic discrimination task (see below). Reaching Success and Reaching Force The dorsal column lesion animals did not make significantly more attempts per successful retrieval on any day of testing [F(1,10) ⫽ .426, p ⫽n.s.; Fig. 2]. Forces applied to a single piece of pasta were calculated in the fore-aft, lateral, and vertical directions (Fig. 3A). There was no significant difference in the time taken by the two groups to break the pasta once the pasta had been grasped [F(1,10) ⫽ 0.03, p ⫽ n.s.; Fig. 3B). Forces applied to the pasta in the three dimensions were compared at the point immediately before the pasta broke. Force direction did not change as a result of the lesion [F(2,20) ⫽ 1.6, p ⫽ n.s.).

FIG. 1. Coronal sections taken through a representative dorsal column lesion. (A) Rostral 180 ␮m to the lesion site. (B) The lesion site. Dotted lines and arrows show primary site of the lesion. (C) Caudal 180 ␮m from the lesion site.

Vertical Paw Placing The percent usage of each paw (ipsilateral vs. contralateral) was compared in the vertical exploration task. Statistical tests showed no significant differences in the paw usage between groups (Fs ⬍ 2.7, p ⫽ n.s.).

Tactile Discrimination Measures Accuracy in haptic discrimination was compared in the two groups during the acquisition and probe tests (Fig. 4A). Dorsal column lesion animals performed at chance levels on the textured nonfood stimulus vs. 60% accuracy by the control rats [F(1,10) ⫽ 6.081, p ⬍ 0.05; Fig. 4B). When the animals were allowed to choose between pasta and smooth nonfood target on the final day of testing (Fig. 4C), control performance also fell to chance levels [F(1,10) ⫽ 0.029, p ⫽ n.s.; Fig. 4D).

Adhesive Dot Removal The order and latency of contact and removal with each dot was compared in both groups of animals. The dorsal column-injured animals did not contact or remove the dot from the contralateral forelimb first more often when compared to controls [Fs(1,10) ⬍ 1.67, p ⫽ n.s.], nor did they contact or remove the dot from the contralateral forelimb sooner than the ipsilateral forelimb, when compared to controls [Fs(1,10) ⬍ 1.216, p ⫽ n.s.).

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FIG. 2. (A) Success in the single pasta-reaching task was measured in intact and lesion groups. (B) The mean numbers of attempts per successful retrieval were counted before surgery (Preop) and on postsurgical days 7, 11, 15, and 19. Error bars represent standard errors of the mean. Note: No significant differences were found on any day of testing. A slight tendency for dorsal column-injured rats to take more attempts per retrieval was completely absent by postsurgery day 19.

DISCUSSION The present research demonstrates that damage to the dorsal columns, although sparing simple tactile sensation, and a number of motor aspects of reaching, including reaching success and force application, abolishes the rats’ ability to distinguish between a food item and a textured nonfood item that has been grasped. This finding confirms that the dorsal columns carry sensory information that is important for rat hapsis. Furthermore, the finding that sensory detection and a number of other aspects of reaching were spared by the dorsal column lesions, suggests that the dorsal columns may make a specific contribution to hapsis during grasping. The major deficit observed in the rats with dorsal columns is novel and consisted of an inability to discriminate a food from a nonfood item that is grasped. When the rats reached through a slot, they encountered either a piece of pasta or a serrated drill bit of similar size. The target objects were placed one above the other with the position of the items changing in random sequence. If the rat encountered a piece of pasta with its paw, the correct response

FIG. 3. (A) Pasta was placed in a force transducer that could measure forces in the fore-aft, lateral, and vertical directions over time. (B) Force duration from the contact with the pasta to the break was measured for both groups. (C) Force direction immediately before the break was compared between both intact and lesion groups. All bars represent the means ⫾ standard errors. Note: No significant differences were found in the duration nor in the direction of force applied.

was to grasp the pasta, break it from its anchor, and so retrieve it. If the rat encountered the serrated drill bit, the correct response was to release the drill bit, then reposition the paw to retrieve the pasta. Previous research has shown that control rats are able to make this discrimination [2]. These findings are replicated in the current study, as control rats also made this discrimination. Nevertheless, it was found that rats with dorsal column lesions did not distinguish between the pasta and the serrated drill bit. In their studies on visual function in humans and primates, Milner and Goodale [12] review a broad range of evidence that suggests a division of labor in visual processing between coding

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241 present study may in some ways be analogous to a deficit in “foveation” reported by Leonard et al. [10]. They observed that primates with a dorsal column lesion continued to use their affected paw for a variety of behaviors including holding the fur while grooming. The way that the animals shaped their digits to hold the fur was not normal, however, suggesting that haptic information conveyed by the dorsal columns is necessary for appropriate digit positioning. It is also possible to argue that the deficit observed in the rats in the present study is simply a motor deficit. This suggestion is weakened, however, when it is considered that the rats with dorsal columns were as successful as control rats in reaching for single pieces of pasta and could exert similar forces when breaking off pasta as the control rats. Furthermore, it is unlikely that the deficit was simply a deficit in releasing the incorrect target item once it was grasped. The rats quickly released the item once they attempted to eat it and found that it was a nonfood item (see also [11]). CONCLUSION

FIG. 4. (A) Animals chose between a food target and a serrated nonfood item during the acquisition phase of testing. (B) Control animals learned to make more correct discriminations during acquisition, whereas lesion animals did not discriminate between the two stimuli. Error bars represent standard errors of the mean. (C) During probe testing, the serrated nonfood item was replaced with the smooth nonfood item. (D) Performance of control subjects fell to chance levels when the nonfood target was exchanged.

for perception and coding for action. It is not known whether similar divisions of labor occur for other sensory modalities, but the findings of the present study suggest that a specific deficit in “hapsis for action” follows dorsal column lesions in the rat. That is, the animals with dorsal column lesions performed at control levels on a number of other tests of sensory function and motor function, but were not able to identify the items that they were grasping as food or nonfood based on the basis of their tactual properties. Thus, the sensory deficit appeared to be selective to the action of grasping. It would be interesting to determine whether this deficit is specifically related to the action of grasping or a general deficit in stereoagnosia (the ability to detect shape). We have considered whether the deficit could be due simply to impairment in sensory detection or a motor deficit. It seems unlikely that the deficit in food detection is simply a result of a sensory deficit. The rats with dorsal column lesions could locate and grasp a piece of pasta as successfully as the control rats. The rats could also detect a small stimulus that was attached to the radial aspect of the affected forelimb. In addition, there was no apparent deficit in limb use in an open field test in which the animals used their forelimbs to support their weight and explore the vertical surfaces of a container. The finding that the animals were able to respond to sensory stimuli with the affected limb is consistent with studies in primates that show that sensory detection survives dorsal column lesions in primates [23] and humans [14, 19,20]. Interestingly, Nudo et al. [16] have reported that primates with motor cortex lesions will examine a paw after reaching for a food item even though no food had been grasped. This result further suggests a role for motor cortex in sensory detection of grasped objects. The deficit in sensory detection observed in the rats in the

In conclusion, the present study demonstrates that rats do display a deficit in hapsis following lesions of the dorsal columns. This deficit was revealed in the action of grasping, but was not evident in sensitive tests of simple sensory or motor function. These results suggest that in order to detect sensory deficits following such injury in the rat it may be necessary to examine haptic use during motor acts. This study also lends support to the idea that sensory systems differentially code for discrimination and for action, with the added possibility that hapsis for action may be especially important for rats. Finally, the results of the present study allow for further studies on the contributions of the dorsal columns to somatosensation in the rat, as the methods described here could be used for two point discriminations, vibration and flutter [14,19,20], and other forms of sensory analysis. ACKNOWLEDGEMENTS

This research was supported by the Alberta Heritage Foundation for Medical Research Scholarships to M.B. and J.M., and by Medical Research Council and National Sciences and Engineering Research Council Grants to I.Q.W.

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