Spatial Analysis of the Nociceptive Withdrawal Response in the Hindlimb of Spinalized Rats _______________________ A Project Presented to the Faculty

of the Undergraduate College of Science and Mathematics James Madison University _______________________ in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science _______________________ by Craig Edison Archibald Esquivel May 2010

Accepted by the faculty of the Department of Biology, James Madison University, in partial fulfillment of the requirements for the Degree of Bachelor of Science. FACULTY COMMITTEE: HONORS PROGRAM APPROVAL:

Project Advisor: Corey L. Cleland, Ph.D., Associate Professor, Biology

Dr. Barry Falk, Ph.D., Director, Honors Program

Reader: Jim Dendinger, Ph.D., Associate Professor, Biology

Reader: Cheryl P. Talley, Ph.D. Associate Professor, Neuroscience Department of Psychology Virginia State University

Table of Contents List of Figures Acknowledgements Abstract Introduction Methodology Results Discussion Works Cited 3 4 5 6 13 24 36 40

List of Figures 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Tracking Points of the Left Hindlimb Rat Suspension Apparatus Cloth Sling Illumination Setup Stimulation Points Video Frame Sequence of Initial Movement Direction of Movement in Three Separate Dimensions Direction of Movement in Two Dimensions Average Response Direction (Caudal/Rostral & Lateral/Medial) Rat Coordinate Plane Caudal/Rostral & Lateral/Medial) Average Response Direction (Dorsal/Ventral & Caudal/Rostral) Rat Coordinate Plane ( Dorsal/Ventral & Caudal/Rostral ) Average Response Speed Video Frame Sequence of Second Movement Frequency of Movement 14 16 17 18 20 25 26 27 28 29 30 30 31 33 34

Acknowledgements

I would like to thank: Dr. Cleland for his help in guiding this research Dr. Dendinger and Dr. Talley for their efforts in reviewing this paper Lindsey Wyatt for her assistance in designing experiments The Jeffress Foundation (CLC) for funding

Abstract
The purpose of this study was to determine the behavior of the nociceptive withdrawal response (NWR) in the hindlimb of a spinalized rat when presented with a noxious heat stimulus to the foot. There are two competing schools of thought regarding the transformation of spatial aspects of sensory information in the NWR and this study focused on determining which would apply to the hindlimb. The first describes the NWR as continuous, meaning that each stimulation location will be directly linked to a specific withdrawal location. The other describes the NWR as categorical, meaning that the withdrawal reflex will consistently be in one or more preferred directions regardless of stimulus location. A heat stimulus was applied to the foot at eight location and movement of the the foot was recorded using two, high-speed video cameras. Offline analysis resulted in a three-dimensional trajectory of the foot. The results showed a withdrawal response with two distinct components: an initial movement and a secondary movement. The initial movement was consistent and in the rostralmedial direction with no dorsal/ventral component and the response direction was not dependent on stimulus location. However, the results showed that when heat stimuli were applied to the plantar surface of the foot, the response speed weakened as the stimulus location moved into the stimulus. The secondary movement was not as consistent as the initial movement, both in the frequency of occurrence and in direction (both rostral and caudal movements occurred) , but was predominantly in the rostral-medial direction with a dorsal component. The secondary movement was also not dependent on stimulus location. In conclusion, these results support the categorical hypothesis of the NWR of the foot of spinalized rats.

Introduction The ability to detect pain is advantageous in animals because it allows them to detect stimuli that could result in damage to body tissue. The ability to detect pain is, however, only advantageous if the animal can then elicit a response that will stop the damage to tissue or remove the risk (Bear et al., 2006). One way in which organisms can do this is to move toward the stimulus, which may occur because the organism wishes to neutralize the cause of stimulation. For example, Little Blue herons use their bill to peck at and remove mosquitoes from their legs and upper body (Edman et al., 1984). In scorpions, when a light touch is applied, the scorpion vigorously moves its stinger and pedipalps toward the point of stimulation (Palka and Babu, 1967). However, another way in which organisms can stop the damage to tissue or remove the risk is to either move its body part or entire body away from the stimulus. For example, when a puff of air is blown on a scorpion, it responds by pulling in its pedipalps towards its mouth while erecting the tail and then scuttling backwards (Palka and Babu, 1967). In skinks, when a heat stimulus is applied to the tail, it frequently moves its tail towards the stimulus (Tam et al., 2005). When rats are exposed to the odor of a predator such as a cat, the rat will walk directly backwards or will turn around and rush away (Dielenberg and McGregor, 2001). Similarly, heat stimuli applied to the base of the tail of long tailed grass lizards caused them to run away from the stimulus (Del Toro et al. 2006) Pain is perceived through somatic nociceptors, which are sensory nerve endings that detect stimuli that could result in damage to body tissue (Cleland and Gebhart, 1997). Nociceptors can be activated by multiple types of stimuli such as strong mechanical stimulation, extremes in temperature, oxygen deprivation, and exposure to harmful chemicals. Nociceptors
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then transmit signals ultimately to the brain which may then trigger pain perceptions (Bear et al., 2006). Two types of nociceptors are A mechanoheat and c-fiber polymodal nociceptors (Leem, 1993), which are activated by noxious heat above 41C or cold below 5C. Recent findings have suggested that there may be at least two different types of thermal nociceptors that respond to heat, one type which is activated at 41C and another that is activated at 53C (Nagy and Rang, 1999). The neural substrates of motor control in mammals can be discussed in two parts: one part is the spinal cord, which has the ability to command and control coordinated muscle contraction, and another part is the brain, which has the ability to command voluntary movements and control the motor programs in the spinal cord. There are four major descending pathways from the brain to the spinal cord that influence movements. These pathways originate in the vestibular nuclei, the brain stem reticular formation, the red nucleus, and the cerebral cortex. These pathways can be grouped into the lateral column pathways (red nucleus, central canal) and the ventromedial column pathways (ventromedial cortex, reticular). The lateral pathways primarily control voluntary movement of the distal musculature whereas the ventromedial pathways primarily control and regulate posture, locomotion, and spinal reflexes (Bear et al., 2006). The spinal cord consists of an inner core of gray matter surrounded by a layer of white matter. Grey matter contains cell bodies whereas white matter contains axons which connect the spinal cord to the brain and other parts of the spinal cord and periphery. Within the gray matter, the tissue is further divided into the dorsal horn, intermediate zone, and ventral horn (Barr and Kiernan, 2004). Most of the second-order sensory neurons that reside in the spinal cord, those
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that receive sensory input from primary afferents, are located within the dorsal horn. There are many types of reflexes that the spinal cord can mediate, but one major type that is used to mediate an escape response is the nociceptive withdrawal reflex (NWR) (Bear et al., 2006). Nociceptive Withdrawal Response After being stimulated by a noxious stimulus, nociceptive sensory neurons can transmit signals that enter the spinal cord and are widely branched, and thus can activate interneurons in many different segments of the spinal cord. These interneurons can then excite motoneurons causing the limb to be withdrawn from the aversive stimulus. This response is called the nociceptive withdrawal response and can be stimulated by tissue-damaging or potentially tissuedamaging sensory stimuli. Creed et al. (1932) showed that mammals are still able to elicit a response in the muscles below a spinal transection , thus showing that the NWR was mediated by the spinal cord in spinalized animals. The NWR can also be evoked by non-nociceptors and there may be a convergence onto common interneurons in the reflex pathway (Hultborn, 2006). Multiple types of nociceptors such as mechanical and thermal can also evoke the NWR (Schouenborg and Kalliomaki 1992; Cleland and Bauer 2002). For the NWR to function correctly, it is necessary for the central nervous system (CNS) to transform nociceptive information into spatially and temporally coordinated activation of muscles (Bizzi et al., 1991). The way in which the CNS responds and transforms spatial aspects of the sensory information has given rise to multiple hypotheses as to how the CNS transforms the sensory information into a response.

Continuous and Categorical Patterns of Interpretation of the Nociceptive Withdrawal Response There are two competing hypotheses regarding the transformation of spatial aspects of sensory information in the NWR. The first describes the NWR as continuous, which was first defined by Lewis and Kristan (1998) to mean that each stimulation location corresponded to a withdrawal response that was characterized as directly away from the stimulus location. This definition was later revised, and expanded, by Cleland and Bauer (2002) who defined it as a response where each stimulation location was directly linked to a specific withdrawal location. In other words, as the stimulus location changed, the direction of withdrawal changed in order to better match the stimulus location. This could be beneficial to the animal since it would increase the chances of the response being directed away from the stimulus, thus leading to less tissue damage. The other hypothesis describes the NWR as categorical, meaning that the withdrawal reflex will consistently be in one or more preferred directions regardless of stimulus location (Lewis and Kristan, 1998). In other words, although the stimulus location may change, the withdrawal directions will not change as much. This type of reaction to pain could be beneficial to the animal due to reduced time needed to process the stimulus, thus leading to a faster response. One of the first researchers to report on the NWR was Sherrington (1910) who reported on the NWR in cats and dogs whose spinal cord had been transected. In his experiments, Sherrington electrically stimulated nerves and visually measured the changes in muscle length or force that occurred after stimulation. Sherringtons studies (1910) showed that generally flexor muscles throughout the hindlimb , which moved the limb towards the body, contracted, whereas
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the extensor muscles that straightened the limb and opposed gravity relaxed. This general pattern occurred irrespective of where the stimulus, thus supporting the categorical hypothesis. However, Sherrington also pointed out that the patterns of muscle activation and relaxation varied somewhat with stimulus location, which he termed local sign. The idea of local sign is consistent with the continuous organization of the NWR. Therefore Sherringtons work described both the categorical and continuous aspects of the NWR. Later, Schouenborg and Kalliomaki (1990) studied the NWR in anesthetized rats by monitoring electrical activity in the muscles of the leg. In response to mechanical stimuli, they found that the area that showed the most electrical activity corresponded to the most sensitive area of the nocireceptive field. Using the data that showed that the electrical activity changed to account for the most sensitive area, Schouenborg and Kalliomaki inferred that the movement also changed to account for the most sensitive area. In 1992, Schouenborg and Kalliomaki repeated the experiment using spinalized rats and reported similar results as the study in 1990. Schuouenborg and Kalliomaki (1992), using their data on electrical activity, tried to translate the electrical activity into a model of movement. Thus they reported that when certain portions of skin were stimulated by aversive stimuli, the muscles that withdrew the skin away from the stimulus corresponded to the most sensitive area of the nocireceptive field, which would be consistent with the continuous definition of the NWR. The major limitation of these studies was that the researchers were measuring the activity of muscles but trying to predict the movement of muscles. Such extrapolation is difficult because of the complexity of the biomechanics of the limb.

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To surmount the limitations of inferring movement from muscle activity, Cleland and Bauer (2002) visually noted the direction of movement of the NWR in response to heat stimuli. Their results showed that the NWR of the tail may not fit into either the continuous or categorical definition of the NWR, but rather a hybrid continuous-categorical movement strategy. Cleland and Bauer found that the withdrawal responses in the tail had two spatial components: one directly away from the stimulus and the other in the ventral direction. However, the dependence on stimulus location was weak. In order to overcome the limitations of only focusing on one point on the tail, Bence et al. (2009) used less restrained spinalized rats and looked at the complete movement trajectory of the tail NWR movement. The tail of the rats was stimulated using heat stimuli from a laser and movement of the entire tail was recorded using video. Results showed that responses were directed ventral-laterally and were independent of stimulus location, which corresponded with the results reported by Cleland and Bauer (2002). Although these results helped to clarify the movement of the NWR of the tail, they did not necessarily relate to other parts of the body, specifically the limbs. Most recently, Davis (2008) focused on the NWR of the hindlimb of spinalized rats using thermal stimuli of regions skin just above the ankle. With stimuli applied to eight locations circumscribing the ankle, Davis found that the ankle tended to move inwards, forwards, and upwards (medial, rostral, and dorsal) irrespective of the stimulus location, which supported the categorical hypothesis; however, the major limitation of this research was that the responses were substantially weaker than the responses when the foot was stimulated, making accurate measurement difficult (Davis, 2008).
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Specific Aims In order to further explore the movement of the foot during the NWR, the goal of this study in spinalized rats was to determine the direction and speed of the response of the foot to stimuli applied to different locations on the foot. More specifically, the thermal stimuli was applied to four points on the plantar surface of the foot, one point on the left side of the foot, one point on the right side of the foot, and one point on the dorsal surface of the foot. The response was quantified by analyzing high-speed video to determine the trajectory of the foot movements in three dimensions. Based on the previous research in the tail of spinalized rats (Cleland & Bauer, 2002; Bence et al., 2009) and in the foot of spinalized rats (Davis, 2008) which supported the categorical hypothesis of the NWR, the hypothesis of this study was that the results reported in this study would also support the categorical hypothesis of the NWR.

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Methodology A. Animal Selection and Care Adult male rats (mean 88.222.2 SD days and mean 323.841.7 SD g) were bred at James Madison University from rats originally obtained from Harlan Laboratories (Indianapolis, IN). Rats were given unrestricted food and water and were housed in 18x10x8 polypropylene cages in a room with a 12-hour light/dark cycle. After the experiment was finished, rats were euthanized with an overdose of sodium pentobarbital (200 mg/kg i.p.), approved by the Panel on Euthanasia of the Veterinary Medical Association (Report of the AVMA, 2007). The experimental protocol was approved by the James Madison University Institutional Animal Care and Use Committee and conformed to federal requirements (National Council Guide, 1996) and the guidelines of the International Association for the Study of Pain (Zimmerman, 1983).

B.

Sedation and Marking Twenty-four hours before each experiment, the rat weight and date of birth were

recorded. The rat was then placed in a sealed acrylic cylinder connected to a gas source (Oxygen (100%) mixed with halothane (1-5%)) and with an activated charcoal scavenger to absorb excess halothane. After one to two minutes, or until the rat appeared sedated but breathing normally, the rat was removed from the acrylic cylinder and given an intra-peritoneal (IP) injection of a sodium pentobarbital(150 mg/kg ;Sigma-Aldrich Laboratories, St. Louis, MO)-saline solution

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Lateral View

Plantar View

6 3 1 2 5

Lateral View
View from Camera B

Plantar View
View from Carmera A

3 1 2

6 5

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Figure 1. Tracking Points of the Left Hindlimb. This diagram shows each of the six tracking points represented by a black dot. The points labeled by numbers 1-4 on the left panel are on the lateral side of the foot and the points labeled by numbers 5 and 6 on the right panel are on the plantar surface of the foot. The arrows point to the two tracking points that were used as the center of movement.

Under anesthesia, the rats left leg was shaved closely with an electric razor (Oster #40 blade) up to and including the thigh, and its upper back was shaved up to 2 cm above the shoulder blades and 4 cm below. The left leg was then marked for later tracking with two, 1 mm black dots (Sharpie, 0.2 mm) on the outward-facing side of the foot. The plantar surface of the foot parallel to the two previous dots was also marked with another two 1 mm black dots. These points labeled by numbers 1 and 2 in Figure 1 were directly facing camera B and the points labeled by numbers 5 and 6 in Figure 1 were directly facing camera A . Another one 1 mm black dot was marked in the cavity of the ankle facing outwards and another one 1 mm black dot was marked on the lower thigh 1.5 cm above the knee facing outwards (Figure 1). These points are labeled by the numbers 3 and 4 in Figure 1 and directly face camera B The tracking points that were used to track the center of movement for this report are marked with arrows. C. Spinalization

Directly after the markings were applied to the left leg and while the rat was anesthetized, the rats T8-T9 thoracic spinal segments were surgically exposed by laminectomy and the dura was opened. Two drops of a local anesthetic (4.4 x10-2 4% lidocaine hydrochloride, Roxane Laboratories, Columbus, OH) were applied to the surface of the spinal cord to block injuryevoked action potentials that would occur during transection and potentially alter spinal cord processing. Five minutes were allowed to pass before the spinal cord was severed using scissors
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and scrapers and the spinalization was visually confirmed. The incision was closed using surgical staples and the rat was allowed to recover for 24 hours in a cage with fully accessible food and water. This extended period of time was allowed in order for the pentobarbital to wear off and for the rat to recover from surgery. D. Experimental Apparatus Design

Scaffolding

Suspension Apparatus

Copper-Foam Plate

B A
Laser

Figure 2. Rat Suspension Apparatus. This diagram represents the suspension apparatus that held the rat. Two copper plates with foam attached to them were placed on either side of the rat to limit body movement. The tail was constrained with an acrylic tube to further limit body movement. Camera A is shown facing the rear of the rat. Camera B is shown facing the left, lateral side of the rat. The laser is shown below the rat.

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The experiment was performed on a lab-created pipe scaffolding that suspended an apparatus that held the rat in place. The suspended apparatus consisted of one 20 cm rod that connected the apparatus perpendicularly to the platform, another 20 cm pole that held a cloth sling (not shown in Figure 2), a clamp that was parallel to the top of the platform, and one arm made up of interconnected rods on each side of the apparatus that held a copper plate with 3 cm thick foam. The purpose of the copper plates with foam was to limit body movement. The apparatus also included flexible rubber tubing, 15 cm long that had a 1.5 cm opening that could accommodate the tail and also a hard plastic tube, 15 cm long that had a 2 cm opening that the flexible rubber tubing could fit into. Its purpose was to minimize backward movement.
Right Side (Facing away from Camera B)

Left Side (Facing towards Camera B)

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Figure 3. Cloth Sling. This diagram shows the left and right side of the cloth sling that was used to hold the rat. The semi-circles at the corners depict the cutouts for each hind limb. The horizontal dotted line that runs along the top of the sling depicts a sewed seam that was placed over a rod. The vertical dotted line depicts the seam that was closed using safety pins to prevent the rat from moving its head out of the sling.

The cloth sling was made of opaque cotton fabric with a large opening for each leg to allow for free range of movement and a restricted opening at the front that allowed enough room for the rat to breathe but not move its head through. The sling also had a loop that ran the length of it that allowed it to be slipped onto the suspended apparatus.

Figure 4. Illumination Setup. This diagram depicts the illumination setup that consisted of five fiber optic light sources and two CFL light sources. The fiber optic light sources are depicted as boxes with two fiber optic tubes attached to them. The CFL light sources are depicted as rods with black cylinders attached to them. Each CFL light source directly faces either Camera A or Camera B.
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Uniform illumination for video recording was provided by five, two-tube fiber optic light sources (Dolan-Jenner Industries, Boxborough, MA). Four light sources were placed on the side of the rat where camera B was located and they were setup in a line parallel with camera B. Each flexible light was used in an alternating up-down pattern so that on each light source one light was focused on the lowest part of the foot and the other light was focused slightly higher. This arrangement provided uniform light on the left side of the left foot, including areas where the leg might move. Another light source was placed directly beside camera A facing the plantar surface of the left foot. One arm was focused on the lowermost part of the foot and the other was focused slightly higher. This provided uniform light on the plantar surface of the left foot, including areas where the leg might move. Two 42 watt compact fluorescent lamps (CFL) were used with thin white cotton cloth covering each light in order to disperse light uniformly. The purpose of these lamps was to provide a bright background for each camera which would increase the contrast of the black dots. One CFL light was attached to the platform and directly faced camera A, with the left leg in between. Another CFL light was setup directly facing camera B, with the left leg in between. Stimulation

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E.

Stimulation

Figure 5. Stimulation Points. This diagram shows the stimulation points on the left foot. Each stimulation point is marked by an X while some tracking points have been shown as black dots. The stimulation points are differentiated using the letters A-H; however, these letters were not marked on the foot.

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Eight total points on the left foot and leg were stimulated (referred to as points A through H). Points A through E lined up with the middle of the left foot. Point A was located 1 cm below the heel. Point B was located directly between the two black dots on the plantar surface of the left foot. Point C was located in the cavity 3mm below the black dot closest to the digits. Point D was located on the pad directly below Point C. Point E was located on the skin of the distal phalange of the middle digit. Point F was located directly between the black dots on the plantar surface of the left foot; however, it was on the inwards-facing side of the foot. Point G was also located directly between the black dots on the plantar surface of the left foot; however, it was on the top of the foot. Point G was located directly between the black dots on the side of the left foot facing camera B. Each point was stimulated with a 15 watt, 980nm fiber-coupled laser diode (BWtek, Wilmington, DE), which was operated by a foot pedal. Each point was stimulated at 6 watts until there was a response visually. Stimuli that evoked responses in less than 1.5 seconds may result in tissue damage, while stimuli that evoked responses in over 3 seconds sometimes evoked a weak response. Therefore the intensity of the stimulus (range 5.0 -7.5 watts) was adjusted to obtain a latency of 1.5-3 seconds. The circular stimulus area measured 1 mm in diameter.

F.

Camera Setup, Data Collection and Software Movement was recorded by two, frame synchronized, high-speed video cameras

(RedLake imaging, San Diego, CA) at 250 fps with a shutter speed of 1/1000s. Each camera was positioned 25 cm from the left leg with its aperture fully opened to allow the maximum amount of light. At the beginning of every experiment a frame from each camera was taken with a ruler

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included next to the foot for calibration. Video recordings were saved 200ms before and after the onset of movement. The video recordings were analyzed using ProAnalyst (XCitex, Cambridge, MA). The onset of movement was visually determined and each tracking point was marked 50 frames before the onset of movement. The points were then tracked through the movement until the end of the recording. Of the points that were tracked, only the data from the point closest to the heel on the plantar surface and the middle point on the later surface were used to approximate the center of mass of the foot. Other markers were not analyzed for this report. The tracked image data was then analyzed using custom routines written in MATLAB software (Mathworks, Natick, MA). Using MATLAB, the two dimensional data from each camera was combined to create a three dimensional trajectory of movement of the foot. A 1 mm threshold from the origin was used to determine direction and speed of the initial movement. The initial direction of movement was defined by the vector from the origin to the first point that crossed the 1 mm threshold. The speed of movement was determined by measuring the distance from the first point that crossed the 1 mm threshold and the point directly previous. To determine whether the response direction or speed was based on stimulus location, statistics were computed using SPSS (non-circular) and Orion (circular). G. Experimental Protocol Twenty-four hours after spinalization, and immediately prior to the experiment the rats bladder was manually emptied. The rat was then placed in the cloth sling, with the left and right leg hanging outside the sling, and then the sling was placed in the experimental apparatus.
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Surgical gauze was placed underneath the rats lower body to absorb any excess urine. The foam attached to the copper on the right side of the body was placed directly against the body from the rats tail to front limbs while allowing the rat to breathe normally. The foam attached to the copper on the left side of the body was placed directly against the body from the rats left hip to the front limbs, making sure not to obstruct the movement of the left leg or left hip while allowing the rat to breath normally. The tail was then placed in the rubber tubing and the rubber tubing was then slid inside the plastic tubing to reinforce it. The plastic tubing was held up with the clamp attached to the experimental apparatus. The right leg was then moved parallel to the body and kept in that position using white tape attached to the copper and foam. This was done to limit the obstruction of camera Bs view. The rat was then allowed to acclimatize for five minutes. Three minutes passed between each stimulation and a total of eight stimulations (corresponding to points A through H) made up one set of trials. A total of five sets of trials were done following the same sequence of points for each rat.

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Results From the data that was analyzed it was possible to determine that there were two characteristic movements that made up the NWR in response to a heat stimulus applied to the surface of the foot. The initial movement tended to be almost purely rostral-medial, or forwards and inwards, with no dorsal, or upwards, component. This initial movement occurred in the majority of the trials of all six experiments. The second movement, that followed a pause after the first movement, did not occur in all trials, however when it did occur it was rostral-medial with a distinct dorsal component. Therefore, the following results are presented in two parts: the first section focuses on the initial rostral-medial movement and the second section presents those instances when a second movement followed the initial movement.

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Section One: Results from the initial movement The sequence of video frames in Figure 6 was taken from one trial and shows a typical response of the left hindlimb in response to a heat stimulus applied to plantar surface of the foot. In the second frame the foot moves rostrally and in the fourth frame the foot moves medially. These two frames, although captured by two different cameras, occurred at the same point in time thus showing a rostral-medial movement.

Figure 6. Video Frame Sequence of Initial Movement. Frames 1 and 2 shows the view of the plantar surface of the left foot from camera A. Frames 3 and 4 show the view of the lateral surface of the left foot from camera B. Frames 1 and 3 show the original position of the foot at 200ms (marked by a red point) before the onset of movement. Frame 2 shows the initial
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movement (marked by a green triangle) in the lateral-medial and dorsal-ventral directions. Frame 4 shows the initial movement (marked by a green triangle) in the caudal-rostral and dorsalventral directions.

distance (mm)

4 0 -4 4 0 -4

lateral-medial

distance (mm) distance (mm)

caudal-rostral

dorsal-ventral
0

200 time (ms)

400

Figure 7. Direction of Movement in Three Separate Dimensions. The plots represent the movement of the center of the foot over time. The top panel represents the movement in the lateral-medial directions. The middle panel represents the movement in the caudal-rostral direction. The bottom panel represent the movement in the dorsal-ventral direction. The dotted line represents the onset of movement. The movement of the tracked points in two-dimensions was then combined to calculate a three-dimensional trajectory of movement of the paw. Figure 7 shows a typical response of the left hindlimb in response to an aversive heat stimulus applied to the plantar surface of the foot (same as Figure 6). The onset of movement is indicated by the dotted line.
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The trajectory of the approximate center of mass of the foot in 3-dimensional rat coordinates is shown in Figure 7. The initial movement shown in Figure 7 moves medially and rostrally with no dorsal-ventral component.

caudal - rostral (mm)

-2

-4 -4 4 -2 0 2 lateral - medial (mm) 4

caudal - rostral (mm)

-2

-4 -4 -2 0 2 dorsal - ventral (mm) 4

Figure 8. Direction of Movement in Two Dimensions. The plots represent the movement of the foot over time in two dimensions. The top panel represents the movement in the caudal-rostal and lateral-medial directions. The bottom panel represents the movement in the caudal-rostal and dorsal-ventral directions. Time(4ms) is represented as the distance between
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points. The 1mm diameter circles represent the approximate movement threshold used to measure direction and speed of the initial response .

Figure 8 shows the trajectory of movement in the caudal-rostral/lateral-medial (top panel) and caudal-rostral/dorsal-ventral (bottom panel) planes (same trial as Figs 6 and 7 In the top panel, the initial movement is in the rostral-medial direction. In the bottom panel, the movement is almost purely rostral with no dorsal-ventral component. The first point to exceed the 1mm threshold was used to calculate the direction and speed of the initial response. In order to determine if the response direction depended on stimulus location, the data from all six experiments was analyzed.

Response direction (caudal-rostral/lateral-medial)

80

60

40

20

0 1 A 2 B 3 C 4 D 5 E 6 F 7 G 8 H

Stimulus location

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Figure 9. Average Response Direction (Caudal/Rostral & Lateral/Medial).This graph represents the average response direction in the caudal-rostral and lateral-medial plane for each stimulus location. The averages are based on six experiments. Stimulus locations 1-8 correspond to stimulus locations A-H. (P> 0.05 ANOVA)

+90

+/-180

-90

Figure 10. Rat Coordinate Plane in Two Dimensions (Caudal/Rostral & Lateral/Medial). This diagram represents the rat coordinate plane in the lateral-medial and caudal-rostral directions.

Figure 9 shows the direction of movement at each stimulus location using data from all six experiments. The direction of movement did not depend on stimulus location (P= 0.05, ANOVA). The mean direction was mostly rostral (64.40 2.63 SD).

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Response direction (caudal-rostral/ventral-dorsal)

100 90 80 70 60 50 40 30 20 10 0 1 A 2 B 3 C 4 D 5 E 6 F 7 G 8 H

Stimulus location

Figure 11. Average Response Direction (Dorsal/Ventral & Caudal/Rostral). This graph represents the average response direction in the caudal-rostral and dorsal-ventral directions at each stimulus location. The averages are based on six experiments. Stimulus locations 1-8 correspond to stimulus locations A-H. (P= >0.05, ANOVA)

90 180

-90

Figure 12. Rat Coordinate Plane ( Dorsal/Ventral & Caudal/Rostral ). This diagram represents the rat coordinate plane in the lateral-medial and dorsal-ventral directions.

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Figure 11 shows another view of the initial direction of movement, except now in the caudal-rostral and dorsal-ventral planes. Figure 11 shows the direction of movement at each stimulus location using data from all six experiments. Figure11 shows the direction of movement at each stimulus location using data from all six experiments. The direction of movement did not depend on stimulus location (P= >0.05, ANOVA). The mean direction was mostly rostral (75.66 3.48 SD).

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Response Speed (mm/s)

40

20

0 1 A 2 B 3 C 4 D 5 E 6 F 7 G 8 H

Stimulus location

Figure 13. Average Response Speed. This graph represents the average response speed at each stimulus location. The averages are based on six experiments. Stimulus locations 1-8 correspond to stimulus locations A-H. Each black dot on the plantar surface of the paw represents a stimulus location.

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Figure 13 shows the speed of response at each stimulus location using data from all six experiments. The direction of movement did not depend on stimulus location (P= 0.05 ANOVA). However, when only stimulus locations 1-5 were examined, there appeared to be a pattern (Figure 13). Stimulus locations 1-5 corresponded to the five stimulus locations on the plantar surface of the foot . The response speed decreased as the stimulus location moved from 1-5. The response speed at stimulus locations 1-5 did show a dependence on stimulus location (P = 0.01, Linear Regression). What this showed was that as the response moved into the stimulus, the response weakened (Figure 13).

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Section Two: Results from the second movement This sequence of video frames shown in Figure 14 was taken from one trial and shows a typical response of the left hindlimb in response to heat stimulus applied to plantar surface of the foot. In the second frame the foot moves medially and in the fourth frame the foot moves rostraldorsally. These two frames, although captured by two different cameras, occurred at the same point in time thus showing a rostral-medial-dorsal movement.

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Figure 14. Video Frame Sequence of Second Movement. Frames 1 and 2 shows the view of the plantar surface of the left foot from camera A. Frames 3 and 4 show the view of the lateral surface of the left foot from camera B. Frames 1 and 3 show the original position of the foot at 200 ms (marked by a red point) before the onset of movement. Frame 2 shows the second movement (marked by a green triangle) in the lateral-medial and dorsal-ventral directions. Frame 4 shows the initial movement (marked by a green triangle) in the caudal-rostral and dorsalventral directions.

100

80
frequency (%)

60

40

20

12 34567 8

1 234 5678

12 34567 8

12 34567 8

rostral (vs caudal)

medial (vs lateral)

dorsal oscillations (vs ventral) (vs no oscillations)

Figure 15. Frequency of Movement. This graph represents the frequency of movement in the rostral, medial, and dorsal directions along with the frequency of oscillations for each stimulus location. The frequency at each stimulus location is an average of the results of all six experiments. (P=0.38, 0.74, 0.50,0.09 ANOVA for rostral, medial, dorsal, and oscillations, respectively)

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Figure 15 shows frequency of movement (rostral, medial, dorsal, and oscillations, respectively) at each stimulus location using data from all six experiments. Figure 16 shows that the second movement was rostral about 84% (mean 84.34%) of the time and did not significantly depend on stimulus location (P=0.38, ANOVA). Similarly, neither medial, dorsal, nor oscillations significantly depended on stimulus location (P > 0.05, ANOVA). Taken together, these results show that although the secondary response varied in direction, the response direction did not significantly depend on stimulus location.

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Discussion The results have shown that when a heat stimulus is applied to the surface of the foot, there are two possible movements that can occur. The initial movement, which occurs in almost every instance where a heat stimulus is applied, tends to move the foot almost purely in the rostral and medial (forward and inward) directions with no dorsal or ventral (up or down) movement. The second movement does not occur as consistently as the initial movement, however when it does occur, the movement is stronger and in the dorsal, or upward, direction. For both the initial and secondary movement, however, the response direction in either case did not significantly depend on stimulus location. In terms of response speed, the speed was stronger when the stimulus was opposed to the rostral-medial response direction; for example, stimuli applied to the rostral (front) portion of the foot evoked weaker responses than stimuli applied to the caudal portion (heel) of the foot. Categorical versus Continuous Hypothesis of the NWR The main focus of this study was to determine whether the categorical or continuous hypothesis of the NWR would be most supported. The work done by Schouenborg and Kalliomaki (1992), in which reflex actions of cutaneous stimulation were assessed by EMG recording, provided support for the continuous hypothesis. In contrast, the findings from Sherrington (1910), Davis (2009) and those reported here, support the categorical hypothesis in that the response direction was independent of stimulus location. Further, both Sherrington (1910) and Davis (2009) found the preferred direction of response was toward the body.

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The difference in findings may be explained in two potential ways. First, Sherrington (1910) did report local sign, in which stimuli at different locations resulted in slightly different patterns of muscle activity, and in our study response speed did depend on stimulus location. Thus, it is possible there are simultaneously both a large categorical and a smaller continuous response. Second, Schouenborg and Kalliomaki (1990) did not directly study movement. It is possible that although muscles show different electrical activity at different stimulus locations, the end result of any combination of muscle activation in the hindlimb is movement in one specific direction. Relating Results to Previous Research in the Tail Previous research in the tail of a spinalized rat has also reported results that have supported the categorical hypothesis; however it was largely unknown whether these results could be related to movements in other parts of the body, specifically the limbs. More specifically, Cleland and Bauer (2002) described a withdrawal response with two spatial components: one large component in the ventral-lateral direction that was independent of stimulus locations and the other a smaller component dependent on stimulus location. Similar results were obtained by Weiss (2008) and Bence et al. (2009) using different measures of response (isometric force and movements, respectively). Relating Results to Previous Research in the Foot The major limitation of the study conducted by Davis (2008) was that the responses were substantially weaker than the responses of the foot to a heat stimulus and that the stimulus locations differed from those in this study. Previous research of the foot in response to heat
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stimuli applied just above the ankle was conducted by Davis (2008) who found that response direction was not dependent on stimulus location and that the predominant movement direction was rostral-medial-dorsal. As described above, Daviss results are fully consistent with the results of this study even though he stimulated at a much different location on the leg, which further supports the idea that the NWR direction is independent of stimulus location. Daviss results do not mention an initial movement without a dorsal-ventral component; however, what Davis describes as the initial movement of the foot is the same as what this study describes as the second movement of the foot. Daviss responses were much weaker than the responses found in this study and it is possible that Davis may have only been able to elicit the second movement due to the extremely small magnitude of the response overall. This idea is support is support by Schouenborg and Kalliomaki (1992), who reported that stimuli to foot evoked greater responses than stimuli to the leg. Relating Results to Intact Rats A study was conducted by Wyatt (2010) which used the exact same stimulation locations as this study; however, Wyatts study focused on the response direction in response to a heat stimulus in intact rats. By comparing the results reported in this study to those of Wyatt (2010), it may be possible to determine what role, if any, the brain plays in transforming the sensory information from nociceptors into reflex movement. The results of the NWR of the foot of intact rats differ significantly with the results of this study in spinalized rats. The results shown by Wyatt (2010) showed that there were at least three components to the response, with a second and main component highly dependent on stimulus location. Further, reflex responses in the intact rat were much faster than reported here. The discrepancy between Wyatts results and the
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results of this study may show that the NWR is not a purely spinal reflex and in intact rats, the brain plays a role in the spatial transformation of sensory data in the NWR. Implications in Future Research The consistency between components of movement in the tail and the foot show that in the future it may be possible to use the tail to acceptably model the movements of other parts of the body, specifically the limbs. The consistency between the response direction of the foot ,although different stimulation locations were used, means that in the future it may be possible to elicit the same NWR using other parts of the foot or leg. Each of the studies of the tail and the foot, including this one, have shown either a weak or lack of dependence of response direction on stimulus location which means that the categorical hypothesis has increasing support and future studies may support this as a widespread principle of motor organization.

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Works Cited AVMA Guidelines on Euthanasia (Formerly Report of the AVMA Panel on Euthanasia) June 2007. Barr ML and Kiernan JA. (2004). The Human Nervous System : An Anatomical Viewpoint. 6th Ed. Harper & Row. Bear MF, Connors BW, Paradiso MA. (2006). Neuroscience: Exploring The Brain. 3rd Ed. Lippincott Williams & Wilkins Bence N., Young GS, Tongen AL, Cleland CL. (2009). Kinematic tail withdrawal strategy of the nociceptive withdrawal response in spinalized rats. Society for Neuroscience. Abstract 39: 271.11 Bizzi E, Mussa-Ivaldi FA, Giszster S. (1991). Computations underlying the execution of movement: A biological perspective. Science 253:87-291 Clarke RW, Harris J. (2004). The organization of motor responses to noxious stimuli. Brain Research Reviews 46:163-172 Cleland CL, Bauer RE. (2002). Spatial transformations in the withdrawal response of the tail in intact and spinalized rats. Journal of Neuroscience.22:5265-70

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