Exp Brain Res (1992) 88:341-344
Experimental BrainResearch © Springer-Verlag1992
Expansion of stimulus-evoked metabolic activity in monkey somatosensory cortex after peripheral denervation Rebecca A. Code, Don E. Eslin, and Sharon L. Juliano Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA Received June 8, 1991 / Accepted October 23, 1991
Summary. The 2-deoxy-glucose (2DG) technique was used to study changes in stimulus-evoked metabolic activity in the somatosensory cortex o f the squirrel monkey Saimiri sciureus after unilateral digit amputation. Two to 52 weeks after digit 2 on the left hand was removed, a somatic stimulus was applied to digit 3 bilaterally. In area 3b corresponding to the deafferented side of the brain, the area of stimulus-evoked metabolic activity was greater than that on the opposite, control side o f the brain within the same animal. The extent of the topographic projection map of 2 D G label in area 3b on the deafferented side o f the brain was 1.92 to 4.75 times greater than that on the control side. There was no difference, however, in the topographical area o f stimulus-evoked metabolic activity between the left and right somatosensory cortices in a normal, unoperated animal. These data suggest that the changes in functional organization observed using electrophysiological recordings in somatosensory cortex after peripheral denervation may have a metabolic substrate. Key words: 2-deoxy-glucose - Autoradiography - Cortical plasticity - Topographic maps - M o n k e y
Killackey 1987). The mechanisms underlying these changes in cortical maps, however, remain to be clearly elucidated. One means o f studying changes in functional activity patterns in the somatosensory cortex is by observing alterations in the underlying metabolic activity levels using 2-deoxy glucose (2DG). The 2 D G method provides a powerful means of visualizing topographic changes as they occur in the cerebral cortex after peripheral injuries. A previous study demonstrated changes in stimulus-evoked metabolic activity in the somatosensory cortex of the cat after unilateral digit amputation (DA) followed by bilateral stimulation o f the adjacent digit (Jutiano et al. 1991). In particular, the topographic projection map o f stimulus-evoked metabolic activity increases on the deafferented side o f the somatosensory cortex compared to that in the opposite, control side within the same animal. We wished to demonstrate whether a comparable expansion of the digit 3 representation, as visualized by a greater area of 2 D G uptake, also occurs in the somatosensory cortex o f the squirrel monkey. Such data would suggest that the mammalian somatosensory cortex follows similar rules of reorganization after peripheral insult, possibly using changes in metabolic activity as cues.
Introduction
Material and methods
It is well established that the mammalian somatosensory cortex is capable o f functional reorganization after peripheral insult. Electrophysiological mapping studies have shown that deafferented regions of cortex become responsive to novel inputs following peripheral nerve transection (c.f. Merzenich et al. 1983), digit amputation (Kelahan et al. 1981; Merzenich et al. 1984) or spinal cord transection (McKinley and Smith 1990). In addition, it has been shown that the structural organization of neonatal somatosensory cortex is disrupted following digit amputation or forepaw removal (Dawson and
Data were obtained from 4 male squirrel monkeys (Saimiri sciureus) approximately 3-4 years old, weighing from 850-1100 g. Two animals were anesthetized with isoflurane and the second digit of the left hand was amputated at the metacarpophalangeal joint by a licensed veterinarian using sterile technique. Guidelines for maintaining proper levels of surgical anesthesia and postoperative care as stated in the Animal Welfare Act were strictly followed. A third animal came to our animal care facility missing the distal 2 phalanges of the second digit unilaterally; it did not undergo surgery but was housed for at least 1 year before the terminal 2DG experiment. The survival period of this animal is referred to as "> 52 weeks". The fourth animal with the normal complement of digits served as an unoperated control. After surviving 2, 7 or > 52 weeks, the animal was prepared for a 2DG experiment as described previously (Juliano et al. 1990).
Offprint requests to: S.L. Juliano
342 Briefly, the animal was anesthetized with halothane (2.5-3 %), the trachea was intubated and a cannula inserted in the femoral vein. After administration of gallamine triethiodide, the animal was maintained on an artificial respirator and his heart rate, body temperature and expired CO2 levels were monitored and kept within normal limits. The anesthetic was changed from halothane to nitrous oxide (70%) and oxygen (30%) because halothane is known to reduce cortical 2DG uptake (Shapiro et al. 1978). The animal's hands were positioned on soft pads in preparation for somatic stimulation which was applied to the distal tip of the third digit bilaterally. For the animal missing the distal 2 phalanges of digit 2 on one hand, the stimulus was applied to the center of the glabrous surface of the distal phalanx of digit 3 bilaterally. One and a half hr after the termination of halothane anesthesia, the somatic stimulation began; the stimuli consisted of intermittent vertical displacements of 8-25 Hz driven by a Ling stimulator with a rounded probe having a diameter of 6 ram. The stimulation was started 5 rain prior to an intravenous injection of 2DG (100 gCi/kg) and continued for 45 rain. At the end of this time, the animal received an overdose of sodium pentobarbital (35 mg/kg) and was quickly perfused intracardially with a 0.9% saline rinse followed by 4% paraformaldehyde/4% sucrose in 0.1 M phosphate buffer. The brain was immediately removed from the skull, blocked, flattened between 2 glass slides, frozen in Freon 22, and stored at - 7 0 ° C. Later, 30 gm thick tangential sections were cut roughly parallel to the cortical surface from the pia to the white matter in a cryostat. Every other section was saved for 2DG autoradiography; these sections were exposed to x-ray film (Kodak SB5) with I~C standards for 7-10 days. After autoradiographic film development, these same sections were stained for Nissl substance. The other series of adjacent sections was processed for cytochrome oxidase (CO) histochemistry to evaluate baseline metabolic activity (Wong-Riley 1979).
Data analysis Optical density measurements. Autoradiographic film images of tissue sections were displayed on a TV monitor using a video camera system. The intensity of 2DG label in unstimulated cortex adjacent to the somatosensory cortex (henceforth referred to as background) was measured in each tissue section on both sides of the brain. With the aid of a computer-enhanced image analysis system (Tommerdahl et at. 1985), the mean background optical density (OD) level was obtained by measuring the OD value within a 2.25 sq mm box, drawn 1.5 mm caudal to the lateral end of the central sulcus. Values were measured 3 times and averaged. Within the region corresponding to the digit 3 representation in area 3b, the OD of 2DG label in a box, 500 lam on a side, was measured 3 times and averaged to obtain the mean OD value of stimulusevoked metabolic activity. The difference between this value and the mean background OD value was determined for each tissue section on each side of the brain and expressed as the mean per cent difference of 2DG label {[(mean OD of stimulus-evoked label mean OD background tabeI)/mean OD background label] x 100%}. Only animals whose stimulus-evoked 2DG label was at least t5% above background cortical activity were included in this study.
jected area of 2DG labeling on the surface of area 3b. A master transparency was drawn for the deafferented side of the brain and the opposite, unoperated side which served as an intra-animal control. The size of the topographic projection map of 2DG label from each master transparency was measured with a computerized digitizing tablet and Sigma Scan software (Jandel Scientific, Inc.). Area measurements were performed in a single-blindfashion, which prevented the experimental identification of the transparency. The total area of the topographic projection of 2DG label was measured from both the deafferented and control sides of the brain at every survival time and compared.
Results 2DG autoradiography F o l l o w i n g u n i l a t e r a l digit 2 a m p u t a t i o n ( D A ) a n d s o m a t ic s t i m u l a t i o n o f digit 3 bilaterally, there was a n e x p a n sion o f the region o f s t i m u l u s - e v o k e d m e t a b o l i c activity in area 3b o n the deafferented side o f the b r a i n relative to t h a t o n the c o n t r o l side. F i g u r e 1 is a p h o t o m i c r o g r a p h t a k e n directly f r o m the a u t o r a d i o g r a p h i c film o f images o f tissue sections o b t a i n e d f r o m a n a n i m a l t h a t survived 2 weeks after D A . A n area o f increased optical d e n s i t y r e p r e s e n t i n g increased m e t a b o l i c u p t a k e i n the digit 3 r e p r e s e n t a t i o n o f area 3b is a p p a r e n t o n b o t h sides o f the b r a i n . T h e area o f i n t e n s e 2 D G label o n the deafferented side, however, is larger t h a n t h a t o n the c o n t r o l side o f the b r a i n . The t o p o g r a p h i c p r o j e c t i o n m a p , which represents the total a b o v e - b a c k g r o u n d label, is also greater in the deafferented h e m i s p h e r e t h a n o n the opposite, c o n t r o l side. F i g u r e 2 shows t h a t the o u t l i n e o f the t o p o g r a p h i c
Area measurements. Regions of stimulus-evoked activity were measured in cortical area 3b, rostral to the central sulcus. This site has been defined electrophysiologically by other investigators as the location of the representation of digit 3 (Sur et al. 1982). For each tissue section on each side of the brain, the region of area 3b displaying 2DG label with an OD value of at least 2 standard deviations greater than the mean background OD was traced onto a transparency. In order to obtain a topographical projection map of the stimulus-evoked metabolic activity across the surface of area 3b, the transparencies were stacked on top of each other and aligned along the central and lateral sulci. The areas of 2DG labeling within each tangential section from one side of the brain were outlined onto a master transparency which then represented the total pro-
Fig. 1. Two weeks after unilateral digit 2 amputation and bilateral stimulation of digit 3. The area of intense 2DG label (arrows) on the deafferented (dealT) side of the brain is greater than that on the control side. Photomicrographs were taken directly from autoradiographic images. The photographic image of the control side was reversed to aid comparison of 2DG label between the 2 sides of the brain. In both panels, rostral is toward the left; lateral is down. Scale bar applies to both panels. CS, central sulcus
343
control
Table 1. Area of stimulus-evoked metabolic activity (sq. mm)"
deaff
p
2 wks
A
Ratio of total area
Survival
Side of brain
(weeks)
control
deafferented
( deafferented/control)
2 7 > 52
1.43 1.82 3.02
2.76 8.65 9.99
1.92 4.75 3.30
left
right
right~left
3.99
0.90
Unoperated 4.41
a Areas of the topographical maps of 2DG label projected from tangential sections onto the surface of area 3b after unilateral amputation of digit 2. Areas represent regions of increased metabolic activity, evoked by bilateral stimulation of digit 3, at least 15% above adjacent, unstimulated cortex brain is almost twice that on the control side. At longer survival times, the topographical extent of 2 D G label expands even farther on the deafferented side to 4.75 times that on the control side at 7 weeks survival and to 3.3 times at > 52 weeks survival. The areas of 2 D G activity evoked by bilateral stimulation of digit 3 in the unoperated animal are similar in both the left and right hemispheres.
7 wks
B
Cytochrome oxidase histochemistry Tissue f r o m all experimental animals and the unoperated control was processed for CO histochemistry to evaluate steady-state metabolic activity levels within a hemisphere. N o differences were evident in the levels of CO activity between the stimulated regions o f somatosensory cortex and the unstimulated adjacent cortex. In addition, no differences were observed between the deafferented and the opposite sides o f the brain at any survival time, or between the left and right sides o f the brain in the unoperated control. Finally, no differences in CO activity levels were noted between experimental animals and the unoperated control.
• 52 wks
C left
right
Discussion D
unop
--
r
4-J
Fig. 2A-D. Line drawings of topographical projection maps of
stimulus-evoked metabolic activity from the control and deafferented sides of the brain 2 (A), 7 (B) and > 52 (C) weeks after unilateral digit 2 amputation. The left and right sides of the brain from the unoperated control animal are depicted in panel D. For ease of comparison, the drawings have been oriented so that rostral is left and lateral is down for all panels. Scale bar applies to all panels. CS, central sulcus; LS, lateral sulcus projection o f 2 D G label in the hemisphere related to the deafferentation covers a larger extent of area 3b than that on the control side at every survival time. Areal measurements o f the topographical projections are displayed in Table 1. Two weeks after DA, the area o f stimulusevoked metabolic activity on the deafferented side of the
D a t a f r o m the present study indicate that digit a m p u t a tion and subsequent somatic stimulation of the adjacent digit results in an expansion in the area of stimulusevoked metabolic activity in the somatosensory cortex. At every survival time, the size of the topographic projection of 2 D G labeling in area 3b on the deafferented side of the brain increased relative to that in the opposite control side. These results are consistent with those from a similar study in cat, which also showed an expansion in the topographic m a p of 2 D G label in the deafferented somatosensory cortex 2, 4, and 7 weeks after unilateral digit a m p u t a t i o n (Juliano et al. 1991). One experimental animal in the present study did not undergo surgical digit amputation but was missing the distal 2 phalanges on one hand u p o n arrival at our animal care facility and thus precluded determination of the exact time of periph-
344
eral loss. A similar increase in the area of 2DG label on the deafferented side of the brain relative to the control side, however, was also observed. This implies that (1) the mechanisms underlying the expansion of stimulusevoked metabolic activity are similar regardless of the method of digit removal, and (2) the effects of digit removal on 2DG maps in the somatosensory cortex occur relatively quickly and are long-lasting (> 52 weeks). This suggests that the response of the adult primate brain to digit removal may be a constant feature of somatosensory cortex, at least in terms of its effect on stimulusevoked metabolic activity, and that the adult primate brain has a long-term capacity for cortical reorganization after peripheral trauma. Although a longer period of time following deafferentation appears to correlate with a greater amount of cortical territory involved, it is not clear whether the magnitude of the cortical rearrangements can be directly related to the time after deafferentation. The animal sustaining a > 52 week survival had a smaller cortical change than the animal surviving for 7 weeks, but this could be related to the fact that the former animal was lacking only the distal 2 phalanges of digit 2, and not the whole digit, as were the other experimental animals. The numbers of animals involved in the present study are too small to draw definitive conclusions on this matter, although it appears that longer survival times predict the involvement of larger cortical territories. Using electrophysiological techniques, other investigators have demonstrated cortical plasticity in the topographic maps of functional activity in the somatosensory cortex after various peripheral perturbations in the adult (for reviews, see Merzenich et al. 1988; Wall 1988; Kaas 1991). Results from the present study show similar rearrangements in the topographic projection maps of 2DG activity after digit amputation. The 2DG method is a unique means by which to visualize plastic changes in somatosensory cortex following deafferentation, since it allows appreciation of the entire cortical territory involved in processing the input from a locus on the periphery. In this regard, it is interesting that the distances involved in the cortical reorganization shown here match previously reported "limits" of cortical change (Merzenich et al. 1988; Kaas 1991). These limits appear to relate to the size of thalamocortical afferent terminations, i.e., 1 to 2 mm (Garraghty et al. 1989). A recent study, however, investigating long-term cortical reorganization after massive deafferentation suggests that the amount of cortex capable of change may be much greater than previously thought possible (Pons et al. 1991). Perhaps longer survival times in the present study would have elicited a greater expanse of stimulus-evoked metabolic activity. Future studies will hopefully determine the mechanisms involved in such cortical reorganizations. It is likely that the changes in stimulus-evoked metabolic activity shown here underlie and are necessary for the dynamic changes in cortical functional activity observed with electrophysiological recording. These studies will aid in the understanding of the central nervous system's response to injury in the adult. The capacity for reorganization of the somatosensory
cortex after peripheral insult may subserve improvements in sensory abilities that could lead to behavioral recovery. Understanding the mechanisms underlying the observations detailed here, i.e., an increase in the cortical territory involved in processing innocuous stimuli, may eventually lead to the development of therapies to improve recovery after CNS damage. Acknowledgments. We thank Dr. Malcolm Carpenter for his generous gift of 4 squirrel monkeys. The use of Dr. Rosemary Borke's computer-aided, digitizing measurement system is greatly appreciated. We are grateful to Dr. Peter Strick for his helpful comments and suggestions. This research was supported by PHS grant NS24014 to SLJ. References Dawson DR, Killackey HP (1987) The organization and mutability of the forepaw and hindpaw representations in the somatosensory cortex of the neonatal rat. J Comp Neurol 256:246-256 Garraghty PE, Pons TP, Sur M, Kaas JH (1989) The arbors of axons terminating in middle cortical layers of somatosensory area 3b in owl monkeys. Somatosens Mot Res 6:401---411 Juliano SL, Friedman DP, Eslin DE (1990) Corticocortical connections predict patches of stimulus-evoked metabolic activity in monkey somatosensory cortex. J Comp NeuroI 298:23-39 Juliano SL, Ma W, Eslin D (1991) Cholinergic depletion prevents expansion of topographic maps in somatosensory cortex. Proc Natl Acad Sci USA 88:780784 Kaas JH (1991) Plasticity of sensory and motor maps in adult mammals. Ann Rev Neurosci 14:137-167 Kelahan AM, Ray RH, Carson LV, Massey CE, Doetsch GS (1981) Functional reorganization •fadu•t raccoon somatosensory cerebral cortex following neonatal digit amputation. Brain Res 223:152-159 McKinley PA, Smith JL (1990) Age-dependent differences in reorganization of primary somatosensory cortex following low thoracic (T12) spinal cord transection in cats. J Neurosci 10:1429-1443 Merzenich MM, Kaas JH, Wall J, Nelson R J, Sur M, Felleman D (1983) Topographic reorganization of somatosensory cortical areas 3b and 1 in adult monkeys following restricted deafferentation. Neuroscience 8: 33-55 Merzenich MM, Nelson R J, Stryker MP, Cynader M, Schoppmann A, Zook JM (1984) Somatosensory cortical map changes following digit amputation in adult monkeys. J Comp Neurol 224: 591-605 Merzenich MM, Recanzone G, Jenkins WM, Allard TT, Nudo RJ (1988) Cortical representational plasticity. In: Rakic P, Singer W (eds) Neurobiology of neocortex. Wiley & Sons, New York, pp 41-67 Pons TP, Garraghty PE, Ommaya AK, Kaas JH, Taub E, Mishkin M (1991) Massive cortical reorganization after sensory deafferentation in adult macaques. Science 252:1857-1860 Shapiro HM, Greenberg JH, Reivich M, Ashmead G, Sokoloff L (1978) Local cerebral glucose uptake in awake and halothaneanesthetized primates. Anesthesiology 48:97-103 Sur M, Nelson RJ, Kaas JH (1982) Representations of the body surface in cortical areas 3b and 1 of squirrel monkeys: comparisons with other primates. J Comp Neurot 211 : 177-192 Tommerdahl M, Baker R, Whitsel BL, Juliano SL (1985) A method for reconstructing patterns of somatosensory cerebral cortical activity. Biomed Sci Instrum 21:93-98 Wall JT (1988) Variable reorganization in cortical maps of the skin as an indication of the lifelong adaptive capacities of circuits in the mammalian brain. Trends Neurosci 11 : 549-557 Wong-Riley MTT (1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res 171:11-28
Experimental BrainResearch © Springer-Verlag1992
Expansion of stimulus-evoked metabolic activity in monkey somatosensory cortex after peripheral denervation Rebecca A. Code, Don E. Eslin, and Sharon L. Juliano Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA Received June 8, 1991 / Accepted October 23, 1991
Summary. The 2-deoxy-glucose (2DG) technique was used to study changes in stimulus-evoked metabolic activity in the somatosensory cortex o f the squirrel monkey Saimiri sciureus after unilateral digit amputation. Two to 52 weeks after digit 2 on the left hand was removed, a somatic stimulus was applied to digit 3 bilaterally. In area 3b corresponding to the deafferented side of the brain, the area of stimulus-evoked metabolic activity was greater than that on the opposite, control side o f the brain within the same animal. The extent of the topographic projection map of 2 D G label in area 3b on the deafferented side o f the brain was 1.92 to 4.75 times greater than that on the control side. There was no difference, however, in the topographical area o f stimulus-evoked metabolic activity between the left and right somatosensory cortices in a normal, unoperated animal. These data suggest that the changes in functional organization observed using electrophysiological recordings in somatosensory cortex after peripheral denervation may have a metabolic substrate. Key words: 2-deoxy-glucose - Autoradiography - Cortical plasticity - Topographic maps - M o n k e y
Killackey 1987). The mechanisms underlying these changes in cortical maps, however, remain to be clearly elucidated. One means o f studying changes in functional activity patterns in the somatosensory cortex is by observing alterations in the underlying metabolic activity levels using 2-deoxy glucose (2DG). The 2 D G method provides a powerful means of visualizing topographic changes as they occur in the cerebral cortex after peripheral injuries. A previous study demonstrated changes in stimulus-evoked metabolic activity in the somatosensory cortex of the cat after unilateral digit amputation (DA) followed by bilateral stimulation o f the adjacent digit (Jutiano et al. 1991). In particular, the topographic projection map o f stimulus-evoked metabolic activity increases on the deafferented side o f the somatosensory cortex compared to that in the opposite, control side within the same animal. We wished to demonstrate whether a comparable expansion of the digit 3 representation, as visualized by a greater area of 2 D G uptake, also occurs in the somatosensory cortex o f the squirrel monkey. Such data would suggest that the mammalian somatosensory cortex follows similar rules of reorganization after peripheral insult, possibly using changes in metabolic activity as cues.
Introduction
Material and methods
It is well established that the mammalian somatosensory cortex is capable o f functional reorganization after peripheral insult. Electrophysiological mapping studies have shown that deafferented regions of cortex become responsive to novel inputs following peripheral nerve transection (c.f. Merzenich et al. 1983), digit amputation (Kelahan et al. 1981; Merzenich et al. 1984) or spinal cord transection (McKinley and Smith 1990). In addition, it has been shown that the structural organization of neonatal somatosensory cortex is disrupted following digit amputation or forepaw removal (Dawson and
Data were obtained from 4 male squirrel monkeys (Saimiri sciureus) approximately 3-4 years old, weighing from 850-1100 g. Two animals were anesthetized with isoflurane and the second digit of the left hand was amputated at the metacarpophalangeal joint by a licensed veterinarian using sterile technique. Guidelines for maintaining proper levels of surgical anesthesia and postoperative care as stated in the Animal Welfare Act were strictly followed. A third animal came to our animal care facility missing the distal 2 phalanges of the second digit unilaterally; it did not undergo surgery but was housed for at least 1 year before the terminal 2DG experiment. The survival period of this animal is referred to as "> 52 weeks". The fourth animal with the normal complement of digits served as an unoperated control. After surviving 2, 7 or > 52 weeks, the animal was prepared for a 2DG experiment as described previously (Juliano et al. 1990).
Offprint requests to: S.L. Juliano
342 Briefly, the animal was anesthetized with halothane (2.5-3 %), the trachea was intubated and a cannula inserted in the femoral vein. After administration of gallamine triethiodide, the animal was maintained on an artificial respirator and his heart rate, body temperature and expired CO2 levels were monitored and kept within normal limits. The anesthetic was changed from halothane to nitrous oxide (70%) and oxygen (30%) because halothane is known to reduce cortical 2DG uptake (Shapiro et al. 1978). The animal's hands were positioned on soft pads in preparation for somatic stimulation which was applied to the distal tip of the third digit bilaterally. For the animal missing the distal 2 phalanges of digit 2 on one hand, the stimulus was applied to the center of the glabrous surface of the distal phalanx of digit 3 bilaterally. One and a half hr after the termination of halothane anesthesia, the somatic stimulation began; the stimuli consisted of intermittent vertical displacements of 8-25 Hz driven by a Ling stimulator with a rounded probe having a diameter of 6 ram. The stimulation was started 5 rain prior to an intravenous injection of 2DG (100 gCi/kg) and continued for 45 rain. At the end of this time, the animal received an overdose of sodium pentobarbital (35 mg/kg) and was quickly perfused intracardially with a 0.9% saline rinse followed by 4% paraformaldehyde/4% sucrose in 0.1 M phosphate buffer. The brain was immediately removed from the skull, blocked, flattened between 2 glass slides, frozen in Freon 22, and stored at - 7 0 ° C. Later, 30 gm thick tangential sections were cut roughly parallel to the cortical surface from the pia to the white matter in a cryostat. Every other section was saved for 2DG autoradiography; these sections were exposed to x-ray film (Kodak SB5) with I~C standards for 7-10 days. After autoradiographic film development, these same sections were stained for Nissl substance. The other series of adjacent sections was processed for cytochrome oxidase (CO) histochemistry to evaluate baseline metabolic activity (Wong-Riley 1979).
Data analysis Optical density measurements. Autoradiographic film images of tissue sections were displayed on a TV monitor using a video camera system. The intensity of 2DG label in unstimulated cortex adjacent to the somatosensory cortex (henceforth referred to as background) was measured in each tissue section on both sides of the brain. With the aid of a computer-enhanced image analysis system (Tommerdahl et at. 1985), the mean background optical density (OD) level was obtained by measuring the OD value within a 2.25 sq mm box, drawn 1.5 mm caudal to the lateral end of the central sulcus. Values were measured 3 times and averaged. Within the region corresponding to the digit 3 representation in area 3b, the OD of 2DG label in a box, 500 lam on a side, was measured 3 times and averaged to obtain the mean OD value of stimulusevoked metabolic activity. The difference between this value and the mean background OD value was determined for each tissue section on each side of the brain and expressed as the mean per cent difference of 2DG label {[(mean OD of stimulus-evoked label mean OD background tabeI)/mean OD background label] x 100%}. Only animals whose stimulus-evoked 2DG label was at least t5% above background cortical activity were included in this study.
jected area of 2DG labeling on the surface of area 3b. A master transparency was drawn for the deafferented side of the brain and the opposite, unoperated side which served as an intra-animal control. The size of the topographic projection map of 2DG label from each master transparency was measured with a computerized digitizing tablet and Sigma Scan software (Jandel Scientific, Inc.). Area measurements were performed in a single-blindfashion, which prevented the experimental identification of the transparency. The total area of the topographic projection of 2DG label was measured from both the deafferented and control sides of the brain at every survival time and compared.
Results 2DG autoradiography F o l l o w i n g u n i l a t e r a l digit 2 a m p u t a t i o n ( D A ) a n d s o m a t ic s t i m u l a t i o n o f digit 3 bilaterally, there was a n e x p a n sion o f the region o f s t i m u l u s - e v o k e d m e t a b o l i c activity in area 3b o n the deafferented side o f the b r a i n relative to t h a t o n the c o n t r o l side. F i g u r e 1 is a p h o t o m i c r o g r a p h t a k e n directly f r o m the a u t o r a d i o g r a p h i c film o f images o f tissue sections o b t a i n e d f r o m a n a n i m a l t h a t survived 2 weeks after D A . A n area o f increased optical d e n s i t y r e p r e s e n t i n g increased m e t a b o l i c u p t a k e i n the digit 3 r e p r e s e n t a t i o n o f area 3b is a p p a r e n t o n b o t h sides o f the b r a i n . T h e area o f i n t e n s e 2 D G label o n the deafferented side, however, is larger t h a n t h a t o n the c o n t r o l side o f the b r a i n . The t o p o g r a p h i c p r o j e c t i o n m a p , which represents the total a b o v e - b a c k g r o u n d label, is also greater in the deafferented h e m i s p h e r e t h a n o n the opposite, c o n t r o l side. F i g u r e 2 shows t h a t the o u t l i n e o f the t o p o g r a p h i c
Area measurements. Regions of stimulus-evoked activity were measured in cortical area 3b, rostral to the central sulcus. This site has been defined electrophysiologically by other investigators as the location of the representation of digit 3 (Sur et al. 1982). For each tissue section on each side of the brain, the region of area 3b displaying 2DG label with an OD value of at least 2 standard deviations greater than the mean background OD was traced onto a transparency. In order to obtain a topographical projection map of the stimulus-evoked metabolic activity across the surface of area 3b, the transparencies were stacked on top of each other and aligned along the central and lateral sulci. The areas of 2DG labeling within each tangential section from one side of the brain were outlined onto a master transparency which then represented the total pro-
Fig. 1. Two weeks after unilateral digit 2 amputation and bilateral stimulation of digit 3. The area of intense 2DG label (arrows) on the deafferented (dealT) side of the brain is greater than that on the control side. Photomicrographs were taken directly from autoradiographic images. The photographic image of the control side was reversed to aid comparison of 2DG label between the 2 sides of the brain. In both panels, rostral is toward the left; lateral is down. Scale bar applies to both panels. CS, central sulcus
343
control
Table 1. Area of stimulus-evoked metabolic activity (sq. mm)"
deaff
p
2 wks
A
Ratio of total area
Survival
Side of brain
(weeks)
control
deafferented
( deafferented/control)
2 7 > 52
1.43 1.82 3.02
2.76 8.65 9.99
1.92 4.75 3.30
left
right
right~left
3.99
0.90
Unoperated 4.41
a Areas of the topographical maps of 2DG label projected from tangential sections onto the surface of area 3b after unilateral amputation of digit 2. Areas represent regions of increased metabolic activity, evoked by bilateral stimulation of digit 3, at least 15% above adjacent, unstimulated cortex brain is almost twice that on the control side. At longer survival times, the topographical extent of 2 D G label expands even farther on the deafferented side to 4.75 times that on the control side at 7 weeks survival and to 3.3 times at > 52 weeks survival. The areas of 2 D G activity evoked by bilateral stimulation of digit 3 in the unoperated animal are similar in both the left and right hemispheres.
7 wks
B
Cytochrome oxidase histochemistry Tissue f r o m all experimental animals and the unoperated control was processed for CO histochemistry to evaluate steady-state metabolic activity levels within a hemisphere. N o differences were evident in the levels of CO activity between the stimulated regions o f somatosensory cortex and the unstimulated adjacent cortex. In addition, no differences were observed between the deafferented and the opposite sides o f the brain at any survival time, or between the left and right sides o f the brain in the unoperated control. Finally, no differences in CO activity levels were noted between experimental animals and the unoperated control.
• 52 wks
C left
right
Discussion D
unop
--
r
4-J
Fig. 2A-D. Line drawings of topographical projection maps of
stimulus-evoked metabolic activity from the control and deafferented sides of the brain 2 (A), 7 (B) and > 52 (C) weeks after unilateral digit 2 amputation. The left and right sides of the brain from the unoperated control animal are depicted in panel D. For ease of comparison, the drawings have been oriented so that rostral is left and lateral is down for all panels. Scale bar applies to all panels. CS, central sulcus; LS, lateral sulcus projection o f 2 D G label in the hemisphere related to the deafferentation covers a larger extent of area 3b than that on the control side at every survival time. Areal measurements o f the topographical projections are displayed in Table 1. Two weeks after DA, the area o f stimulusevoked metabolic activity on the deafferented side of the
D a t a f r o m the present study indicate that digit a m p u t a tion and subsequent somatic stimulation of the adjacent digit results in an expansion in the area of stimulusevoked metabolic activity in the somatosensory cortex. At every survival time, the size of the topographic projection of 2 D G labeling in area 3b on the deafferented side of the brain increased relative to that in the opposite control side. These results are consistent with those from a similar study in cat, which also showed an expansion in the topographic m a p of 2 D G label in the deafferented somatosensory cortex 2, 4, and 7 weeks after unilateral digit a m p u t a t i o n (Juliano et al. 1991). One experimental animal in the present study did not undergo surgical digit amputation but was missing the distal 2 phalanges on one hand u p o n arrival at our animal care facility and thus precluded determination of the exact time of periph-
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eral loss. A similar increase in the area of 2DG label on the deafferented side of the brain relative to the control side, however, was also observed. This implies that (1) the mechanisms underlying the expansion of stimulusevoked metabolic activity are similar regardless of the method of digit removal, and (2) the effects of digit removal on 2DG maps in the somatosensory cortex occur relatively quickly and are long-lasting (> 52 weeks). This suggests that the response of the adult primate brain to digit removal may be a constant feature of somatosensory cortex, at least in terms of its effect on stimulusevoked metabolic activity, and that the adult primate brain has a long-term capacity for cortical reorganization after peripheral trauma. Although a longer period of time following deafferentation appears to correlate with a greater amount of cortical territory involved, it is not clear whether the magnitude of the cortical rearrangements can be directly related to the time after deafferentation. The animal sustaining a > 52 week survival had a smaller cortical change than the animal surviving for 7 weeks, but this could be related to the fact that the former animal was lacking only the distal 2 phalanges of digit 2, and not the whole digit, as were the other experimental animals. The numbers of animals involved in the present study are too small to draw definitive conclusions on this matter, although it appears that longer survival times predict the involvement of larger cortical territories. Using electrophysiological techniques, other investigators have demonstrated cortical plasticity in the topographic maps of functional activity in the somatosensory cortex after various peripheral perturbations in the adult (for reviews, see Merzenich et al. 1988; Wall 1988; Kaas 1991). Results from the present study show similar rearrangements in the topographic projection maps of 2DG activity after digit amputation. The 2DG method is a unique means by which to visualize plastic changes in somatosensory cortex following deafferentation, since it allows appreciation of the entire cortical territory involved in processing the input from a locus on the periphery. In this regard, it is interesting that the distances involved in the cortical reorganization shown here match previously reported "limits" of cortical change (Merzenich et al. 1988; Kaas 1991). These limits appear to relate to the size of thalamocortical afferent terminations, i.e., 1 to 2 mm (Garraghty et al. 1989). A recent study, however, investigating long-term cortical reorganization after massive deafferentation suggests that the amount of cortex capable of change may be much greater than previously thought possible (Pons et al. 1991). Perhaps longer survival times in the present study would have elicited a greater expanse of stimulus-evoked metabolic activity. Future studies will hopefully determine the mechanisms involved in such cortical reorganizations. It is likely that the changes in stimulus-evoked metabolic activity shown here underlie and are necessary for the dynamic changes in cortical functional activity observed with electrophysiological recording. These studies will aid in the understanding of the central nervous system's response to injury in the adult. The capacity for reorganization of the somatosensory
cortex after peripheral insult may subserve improvements in sensory abilities that could lead to behavioral recovery. Understanding the mechanisms underlying the observations detailed here, i.e., an increase in the cortical territory involved in processing innocuous stimuli, may eventually lead to the development of therapies to improve recovery after CNS damage. Acknowledgments. We thank Dr. Malcolm Carpenter for his generous gift of 4 squirrel monkeys. The use of Dr. Rosemary Borke's computer-aided, digitizing measurement system is greatly appreciated. We are grateful to Dr. Peter Strick for his helpful comments and suggestions. This research was supported by PHS grant NS24014 to SLJ. References Dawson DR, Killackey HP (1987) The organization and mutability of the forepaw and hindpaw representations in the somatosensory cortex of the neonatal rat. J Comp Neurol 256:246-256 Garraghty PE, Pons TP, Sur M, Kaas JH (1989) The arbors of axons terminating in middle cortical layers of somatosensory area 3b in owl monkeys. Somatosens Mot Res 6:401---411 Juliano SL, Friedman DP, Eslin DE (1990) Corticocortical connections predict patches of stimulus-evoked metabolic activity in monkey somatosensory cortex. J Comp NeuroI 298:23-39 Juliano SL, Ma W, Eslin D (1991) Cholinergic depletion prevents expansion of topographic maps in somatosensory cortex. Proc Natl Acad Sci USA 88:780784 Kaas JH (1991) Plasticity of sensory and motor maps in adult mammals. Ann Rev Neurosci 14:137-167 Kelahan AM, Ray RH, Carson LV, Massey CE, Doetsch GS (1981) Functional reorganization •fadu•t raccoon somatosensory cerebral cortex following neonatal digit amputation. Brain Res 223:152-159 McKinley PA, Smith JL (1990) Age-dependent differences in reorganization of primary somatosensory cortex following low thoracic (T12) spinal cord transection in cats. J Neurosci 10:1429-1443 Merzenich MM, Kaas JH, Wall J, Nelson R J, Sur M, Felleman D (1983) Topographic reorganization of somatosensory cortical areas 3b and 1 in adult monkeys following restricted deafferentation. Neuroscience 8: 33-55 Merzenich MM, Nelson R J, Stryker MP, Cynader M, Schoppmann A, Zook JM (1984) Somatosensory cortical map changes following digit amputation in adult monkeys. J Comp Neurol 224: 591-605 Merzenich MM, Recanzone G, Jenkins WM, Allard TT, Nudo RJ (1988) Cortical representational plasticity. In: Rakic P, Singer W (eds) Neurobiology of neocortex. Wiley & Sons, New York, pp 41-67 Pons TP, Garraghty PE, Ommaya AK, Kaas JH, Taub E, Mishkin M (1991) Massive cortical reorganization after sensory deafferentation in adult macaques. Science 252:1857-1860 Shapiro HM, Greenberg JH, Reivich M, Ashmead G, Sokoloff L (1978) Local cerebral glucose uptake in awake and halothaneanesthetized primates. Anesthesiology 48:97-103 Sur M, Nelson RJ, Kaas JH (1982) Representations of the body surface in cortical areas 3b and 1 of squirrel monkeys: comparisons with other primates. J Comp Neurot 211 : 177-192 Tommerdahl M, Baker R, Whitsel BL, Juliano SL (1985) A method for reconstructing patterns of somatosensory cerebral cortical activity. Biomed Sci Instrum 21:93-98 Wall JT (1988) Variable reorganization in cortical maps of the skin as an indication of the lifelong adaptive capacities of circuits in the mammalian brain. Trends Neurosci 11 : 549-557 Wong-Riley MTT (1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res 171:11-28
