314
Brain Research, 568 (1991) 314-318 © 1991 Elsevier Science Publishers B.V. All rights reserved 0006-8993/911503.50
BRES 24987
Nerve injury-induced depletion of tachykinin immunoreactivity in the somatosensory cortex of adult squirrel monkeys Catherine G. Cusick Department of Anatomy and Neurosciences Trammg Program, Tulane Untverstty School of Medzcme, New Orleans, LA 70112 (U.S A )
(Accepted 17 September 1991) Key words. Nerve injury, Tachykmin; Somatosensory cortex
Cortical immunoreactw~tyfor tachykinin neuromodulators was evaluated 5-21 days after median and ulnar nerve transecUons in adult squirrel monkeys. Contralateral to the deafferentatlons, immunoreactive cells were reduced by about 20-40% in layer IV of the hand representation compared to the face region in area 3b. Slmdar deafferentations have been shown to alter the pattern of neuronal actlvlty m the somatosensory cortex. Unresponsive regions are produced that with time become reactwated by receptwe fields served by intact nerves. The immunocytochemical results reported here suggest that the presence or pattern of somatosensory input regulates content of tachyklnm neuropeptides m intrinsic cortical neurons.
Area 3b of squirrel monkeys contains an orderly map of low threshold inputs from the contralateral hand. The fifth digit is represented near the central sulcus and the first is represented laterally, near the face area. The finger tips are located anteriorly, and the palm posteriorly 14'1s. Immediately after median nerve transection, much of area 3b activated by that nerve's peripheral territory, on the radial half of the palm and digits 1-3, becomes unresponsive 12. Within 3 weeks of injury, however, this unresponsive cortex becomes reactivated from low threshold receptive fields innervated by intact nerves 12,13. Studies of cortical effects of peripheral deafferentation do not distinguish cortical and subcortical contributions to somatosensory map reorganization. However, a number of studies show that cortical responsiveness is altered following peripheral nerve injury, and reductions in cortical y-aminobutyric acid (GABA) or glutamic acid decarboxylase immunoreactivity have been reported 5m' 22. These findings suggest that peripheral nerve injury produces a downregulation within cortical inhibitory circuits. Cortical cells which are immunoreactive for tachykinin neuropeptides are non-pyramidal and appear to be intrinsic neurons 9. The present experiments were undertaken to examine the possibility that local circuit neurons in cortex alter their expression of neuromodulatory peptides during the time period in which map reorganization takes place. Five adult squirrel monkeys (Saimirt sciureus) were
deeply anesthetized with ketamine hydrochloride, and both the median and ulnar nerves were exposed in the middle of the right forearm, ligated with fine suture and transected. Several millimeters of the distal segments were removed as a further precaution against regeneration, and the skin was sutured. Since together the median and ulnar nerves innervate most of the glabrous surface of the hand, this procedure produced a large peripheral deafferentation that interrupted the projections to most of the cortical low threshold map 12A4"18. The proximodistal level of the injuries spared innervation of the long flexor muscles to the hand, and the animals used both hands as soon as they recovered from anesthesia. At times ranging from 5 days to 3 weeks after injury, the monkeys were euthanized with Nembutal (100 mg/ kg, i.v.) and perfused with fixative (4% paraformaldehyde, 0-0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). One animal was studied at each interval of 5, 9 and 21 days post-injury, and two animals were studied at 10 days. Anterior parietal cortex containing the primary somatosensory area was dissected away from the rest of the brain (Fig. 1), cryoprotected in 30% sucrose in buffer, and cut in the parasagittal plane at a thickness of 20 #m. Sections at 200/zm intervals were treated for localization of tachykinin-like immunoreactivity using a monoclonal antibody raised against the carboxyl terminus of substance P (Sera Labs) 1A1, and standard immunocytochemical procedures (avidin-biotin complex,
Correspondence C.G. Cuslck, Department of Anatomy and Neurosclence Tralmng Program, Tulane Umverslty School of Me&cme. New Orleans, LA 70112, U S.A
315 Vectastain kit) 8. Imidazole (0.01 M, p H 7.6) was ineluded in the diaminobenzidine reaction to enhance the staining intensity 17. Control sections were incubated with supernatant from a non-secreting m y e l o m a cell line or with the primary antibody preabsorbed with 5 - 5 0 / ~ M substance P. Tachykinin-immunoreactive neurons were charted throughout the cortical laminae using a light microscope with a drawing tube. Laminar and areal positions were determined by carefully aligning the immunostained sections with adjacent series stained with Cresyl violet or treated for cytochrome oxidase histochemistry 23. The normal location of the hand representations in squirrel m o n k e y anterior parietal cortex can be identified based on certain anatomical features (Fig. 1). First, the low threshold inputs from the hand project just lateral to and extend approximately 3 m m from the shallow central sulcus. Second, the lateral extent of the hand area can be determined in parasagittal sections, since area 3b widens abruptly at the face-hand area border (Fig. 1) 4'ts. In order to compare the effects of nerve injury in deprived vs non-deprived cortical regions, the pattern of tachykinin immunoreactivity in the area 3b hand region contralateral to the nerve injuries was compared to that in the ipsilateral hand area. Since neuropeptide content could be affected ipsilateral to the injuries, the ipsilateral and contralateral face regions were studied as control, unaffected representations• The tachykinin-immunoreactivity in control (face) regions of squirrel m o n k e y anterior parietal areas was similar to that shown previously in macaques 9. The immunoreactive neurons exhibited non-pyramidal morphologies. Intensely stained neurons were distributed throughout the cellular layers of cortex and were especially prominent in layers II/III and VI. Populations of moderately stained cells were also present in all layers, but formed a noticeable band in layer IV. The tachykinin-positive fiber plexus was densest superficially in layers I/II, and extended throughout the cortical layers in area 3b. Numbers of immunostained neurons were c o m p a r e d in the face and hand regions both contralateral and ipsilateral to the nerve injuries. The sections from the hand cortex were all within 1 m m of the central sulcus, and thus occupied cortex predominantly activated from the ulnar nerve. Sections from the face region exhibited both the greater length of layer IV and were more than 3 m m from the central sulcus. Contralaterally, plots of labeled cells in layer IV (Fig. 1A,B) showed a greater density in the face compared to the hand area. Because cells in layer IV of area 3b are positioned at earlier stages of cortical circuitry by their proximity to thalamic afferents, quantification of immunostaining was focused on this layer.
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Fig. 1. A: the location of the representations of the body surface in areas 3b and i on a dorsal view of a squirrel monkey brain. The face and hand regions are located on the smooth surface of the brain lateraUy, the trunk and limbs are represented within the shallow central sulcus (CS), and the foot is located medaally. LS, lateral sulcus. In sagittal sections, the transition from the hand to the face representation shows an abrupt increase in the length of area 3b. The approximate locations of the sections shown in B and C are indicated on the drawing by arrows. B: camera luclda drawing of tachykinin-immunoreactive cells in the face area, and C, through the hand area. Layer IV is outlined, and the contained labeled cells are shown with larger dots for emphasis. Note the greater density of lmmunoreactive layer IV cells m the face region.
316
A 120"
Comparison of Tachykinin-Like Immunoreactivity in Face vs. Hand Region [ ] face
100" 80'
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60'
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Fig 2. Comparisons of tachykinm-labeled cells through layer IV of area 3b in the face (control) vs hand (deprived) regions contralateral to median and ulnar deafferentations. A' for 5 sections m each region, cells were counted along a constant length of layer IV, equal to the anteroposterior extent of area 3b in the medial one half of the hand represention. B: the percent depletion of cells m the hand compared to the face region xs shown Each of the differences ~s significant by a t-test. Bars equal one standard deviatmn from the mean.
Counts of labeled cells through equal lengths of layer IV were taken from 5 pairs of sections through the h a n d and face areas, both contralateral and ipsilateral to the nerve injuries. Analysis of variance ( A N O V A ) showed statistical differences b e t w e e n the deprived hand and (1) the non-deprived hand region, (2) the face region ipsilateral to the deprived area, and (3) the contralateral face region. N o differences were shown between counts from the two face regions, nor the ipsilateral face and hand regions. Focusing on within-hemisphere comparisons contralateral to the nerve injuries, fewer cells were labeled in the hand c o m p a r e d to the face region, and the differences were statistically significant (t-test, P < 0.05) in each of the 5 cases. T h e differences of the counts ranged from about 20 to 40% (Fig. 2B). The differences were maintained at postinjury times from 5 to 21 days, and the d a t a did not suggest t i m e - d e p e n d e n t changes within the p e r i o d studied. The depletion of tachykinin immunoreactivity was also
Fig. 3. Photomicrographs of tachykinin xmmunoreactivity m layer IV (indicated by bars) of somatosensory area 3b following median and ulnar nerve injury A and B are from two different control regmns, the ipsllateral hand area and contralateral face re.on, respectively. C is from the deprived hand representaUon. Note more numerous labeled cells and fibers in both A and B compared to C. Bar (A-C) = 200 pm.
apparent in the fiber plexus m layer IV and the supragranular layers (Fig. 3C). H o w e v e r , the staining in the most p r o m i n e n t focus of stained fibers, in layers I and II, did not a p p e a r r e d u c e d in the hand c o m p a r e d to the
317 face region. Ipsilateral to the deafferentation, the density of the fiber plexuses in the cortical hand region was comparable to that in the face area (Fig. 3A,B). Thus, tachykinin-immunoreactive cells and fibers in m o n k e y primary somatosensory cortex are depleted following peripheral nerve injury. It is not possible to identify with certainty which specific immunoreactive neuropeptide has been depleted, because the antibody used recognizes the canonical sequence of the tachykinins t~. However, the content of the other tachykinins found in the mammalian nervous system, i.e. neurokinin A (substance K) and neurokinin B, is relatively much lower in cerebral cortex than substance P. Assuming that decreased immunoreactivity indicates a corresponding loss of peptide, it appears likely that at least substance P is downregulated following injury. The changes may be compared to the effects of visual deprivation by monocular tetrodotoxin injection on tachykinin immunoreactivity in the primary visual cortex of macaque monkeys 7. However, the 80% depletion of cells in layer IV of the visual cortex appears substantially greater than in the present study. Differences in staining depletion are probably not attributable to differences in times after the onset of sensory deprivation, since the visual deprivation effects were studied at 10 and 15 days. The deprivation of tachykininergic cells in layer IV may have been more effective in the visual cortex than in the present experiments. Ocular dominance bands appear anatomically and physiologically discrete in layer IV of Old World monkeys. In contrast, immediate shifts in cortical receptive fields following nerve injury suggest
convergence of inputs from different peripheral nerves onto layer IV of the somatosensory cortex 1°'15'2°. In the present experiments, the radial nerve to the dorsal surface of the hand was intact. Furthermore, complete cortical reactivation by the radial nerve following median and ulnar nerve transection in squirrel monkeys occurs at two months postinjury 6. Thus, not only might the deafferentation have been 'incomplete' cortically, the ongoing recovery of activation by the somatosensory cortex 3'19 could influence the tachykinin content of cortical neurons. Since the types of the injuries performed in this study do not produce a major degree of peripheral ganglion cell death, the changes shown in peptide neuromoduiators are likely to be due to a loss of activity or pattern of sensory input. Depletion of tachykiuin immunoreactivity is well-established in the dorsal horn following peripheral nerve injury, and this is restored to normal levels within several weeks 16. Although intrinsic neurons of the dorsal horn may contribute to the return of immunoreactivity, the time course of the restoration corresponds to the expected return of function following nerve regeneration. Whether cortical depletion of tachykinin immunoreactivity is reversible at longer times after injury, or following nerve regeneration, remains to be explored.
1 Cuello, A.C., Galfr6, G. and Mdstein, C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. Natl. Acad Sct., U.S.A., 76 (1979) 3532-3536. 2 Cusick, C.G., Early changes in tachykinm-like lmmunoreactivlty m primary somatosensory cortex of adult squirrel monkeys following nerve injuries, Soc. Neurosct. Abstr., 16 (1990) 630. 3 Cusick, C.G., Wall, J.T., Whmng, J.H. and Wiley, R.G., Temporal progression of cortical reorganization following nerve injury, Brain Research, 537 (1990) 355-358. 4 Cuslck, C.G., Wall, J.T. and Kaas, J.H., Representations of the face, teeth and oral cavity in areas 3b and I of somatosensory cortex in sqmrrel monkeys, Brain Research, 370 (1986) 359-364. 5 Dykes, R.W. and Lamour, Y., An electrophysiologlcal laminar analysis of single somatosensory neurons in partially deafferented rat hindlimb granular cortex subsequent to transection of the sciatic nerve, Brain Research, 449 (1988) 1-17. 6 Garraghty, EE., Clower, R.D., LaChica, E.A. and Kaas, J.H., Thalamic and cortical reorgamzatlon after chromc transection of the median and ulnar nerves m adult monkeys, Soc. Neurosci. Abstr., 16 (1990) 831. 7 Hendry, S.H.C., Jones, E G. and Burstem, N., Actiwty-dependent regulation of tachykmin-like immunoreactavity in neurons of monkey visual cortex, J Neurosct., 8 (1988) 1225-1238. 8 Hsu, S.M., Rame, L. and Fanger, H , Use of awdin-biotinperoxidase complex (ABC) in lmmunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J Histochem Cytochem., 29 (1981) 577-580.
9 Jones, E.G., DeFelipe, J., Hendry, S.H.C. and Maggio, J.E., A study of tachykmin-immunoreactive neurons in monkey cerebral cortex, J. Neurosci., 8 (1988) 1206-1224. 10 Kaas, J.H., Merzenieh, M.M. and Killackey, H.E, The reorganization of somatosensory cortex following peripheral nerve damage m adult and developing mammals, Annu Rev. Neurosci., 6 (1983) 325-356. 11 Maggio, J.E., Tachykimns, Annu. Rev. Neurosct., 11 (1988) 13-28. 12 Merzenich, M.M., Kaas, J.H., Wall, J , Nelson, R.J., Sur, M. and Felleman, D., Topographic reorganization of somatosensory cortical areas 3b and 1 in adult monkeys following restricted deafferentatlon, Neuroscience, 8 (1983a) 33-55. 13 Merzemch, M.M., Kaas, J.H., Wall, J.T., Sur, M., Nelson, R.J. and Felleman, D.J., Progression of change following median nerve section m the cortical representation of the hand m areas 3b and 1 m adult owl and squirrel monkeys, Neurosctence, 10 (1983b) 639-665. 14 Merzenich, M M, Nelson, R.J., Kaas, J.H., Stryker, M.P., Jenkins, W.M., Zook, J.M., Cynader, M.S. and Schoppmann, A., Variability in hand surface representations in areas 3b and 1 in adult owl and squirrel monkeys, J. Comp. Neurol., 258 (1987) 281-296. 15 Metzler, J. and Marks, ES., Functional changes in cat somatic sensory-motor cortex during short-term reversible epidural blocks, Brain Research, 177 (1979) 379-383. 16 Rhoades, R.W., Chiaia, N.L., Hess, P.R. and Miller, M.W.,
Acknowledgements. I thank Louis Lucas for help with statistics and graphics, and Nathaniel Lawson for assistance in surgery. The results have been reported previously in abstract form2.
318
17
18
19
20
Effect of neonatal infraorbital nerve transection on substance Pand leueine enkephalin-like immunoreactivities in trigermnai subnucleus caudalis of the rat, J. Neurosci., 8 (1988) 2234-2247. Straus, W., Imidazole increases the sensitivity of the cytochemical reaction for peroxidase with diaminobenzidine at a neutral pH, J. Histochem. Cytochem., 30 (1982) 491-493. Sur, M., Nelson, R.J. and Kaas, J.H., Representation of the body surface in cortical areas 3b and 1 of squirrel monkeys: comparisons with other primates, J. Comp. Neurol., 211 (1982) 177-192. Wall, J.T., Variable organization m cortical maps of the skin as an indication of the lifelong adaptive capacities of circuits m the mammalian brain, Trends Neurosci., 11 (1988) 549-557. Wall, J.T. and Cuslck, C.G., Cutaneous responsiveness m primary somatosensory (S-I) tundpaw cortex before and after par-
tlal hindpaw deafferentatlon m adult rats, J. Neurosc~., 4 (1984) 1499-1515. 21 Warren, R., Tremblay, N. and Dykes, R.W., Quantltauve study of glutamic acid decarboxylase-immunoreactive neurons and cytochrome oxldase activity in normal and partially deafferented rat hmdlimb somatosensory cortex, J Comp Neurol., 288 (1989) 583-592. 22 Welker, E., Sonano, E. and Van der Loos, H., Plasticaty in the barrel cortex of the adult mouse' effects of peripheral deprwatlon on GAD-lmmunoreactwlty, Exp. Bram Res., 74 (1989a) 441-452. 23 Wong-Raley, M.T T , Changes m the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochermstry, Brain Research, 171 (1979) 11-28.
Brain Research, 568 (1991) 314-318 © 1991 Elsevier Science Publishers B.V. All rights reserved 0006-8993/911503.50
BRES 24987
Nerve injury-induced depletion of tachykinin immunoreactivity in the somatosensory cortex of adult squirrel monkeys Catherine G. Cusick Department of Anatomy and Neurosciences Trammg Program, Tulane Untverstty School of Medzcme, New Orleans, LA 70112 (U.S A )
(Accepted 17 September 1991) Key words. Nerve injury, Tachykmin; Somatosensory cortex
Cortical immunoreactw~tyfor tachykinin neuromodulators was evaluated 5-21 days after median and ulnar nerve transecUons in adult squirrel monkeys. Contralateral to the deafferentatlons, immunoreactive cells were reduced by about 20-40% in layer IV of the hand representation compared to the face region in area 3b. Slmdar deafferentations have been shown to alter the pattern of neuronal actlvlty m the somatosensory cortex. Unresponsive regions are produced that with time become reactwated by receptwe fields served by intact nerves. The immunocytochemical results reported here suggest that the presence or pattern of somatosensory input regulates content of tachyklnm neuropeptides m intrinsic cortical neurons.
Area 3b of squirrel monkeys contains an orderly map of low threshold inputs from the contralateral hand. The fifth digit is represented near the central sulcus and the first is represented laterally, near the face area. The finger tips are located anteriorly, and the palm posteriorly 14'1s. Immediately after median nerve transection, much of area 3b activated by that nerve's peripheral territory, on the radial half of the palm and digits 1-3, becomes unresponsive 12. Within 3 weeks of injury, however, this unresponsive cortex becomes reactivated from low threshold receptive fields innervated by intact nerves 12,13. Studies of cortical effects of peripheral deafferentation do not distinguish cortical and subcortical contributions to somatosensory map reorganization. However, a number of studies show that cortical responsiveness is altered following peripheral nerve injury, and reductions in cortical y-aminobutyric acid (GABA) or glutamic acid decarboxylase immunoreactivity have been reported 5m' 22. These findings suggest that peripheral nerve injury produces a downregulation within cortical inhibitory circuits. Cortical cells which are immunoreactive for tachykinin neuropeptides are non-pyramidal and appear to be intrinsic neurons 9. The present experiments were undertaken to examine the possibility that local circuit neurons in cortex alter their expression of neuromodulatory peptides during the time period in which map reorganization takes place. Five adult squirrel monkeys (Saimirt sciureus) were
deeply anesthetized with ketamine hydrochloride, and both the median and ulnar nerves were exposed in the middle of the right forearm, ligated with fine suture and transected. Several millimeters of the distal segments were removed as a further precaution against regeneration, and the skin was sutured. Since together the median and ulnar nerves innervate most of the glabrous surface of the hand, this procedure produced a large peripheral deafferentation that interrupted the projections to most of the cortical low threshold map 12A4"18. The proximodistal level of the injuries spared innervation of the long flexor muscles to the hand, and the animals used both hands as soon as they recovered from anesthesia. At times ranging from 5 days to 3 weeks after injury, the monkeys were euthanized with Nembutal (100 mg/ kg, i.v.) and perfused with fixative (4% paraformaldehyde, 0-0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). One animal was studied at each interval of 5, 9 and 21 days post-injury, and two animals were studied at 10 days. Anterior parietal cortex containing the primary somatosensory area was dissected away from the rest of the brain (Fig. 1), cryoprotected in 30% sucrose in buffer, and cut in the parasagittal plane at a thickness of 20 #m. Sections at 200/zm intervals were treated for localization of tachykinin-like immunoreactivity using a monoclonal antibody raised against the carboxyl terminus of substance P (Sera Labs) 1A1, and standard immunocytochemical procedures (avidin-biotin complex,
Correspondence C.G. Cuslck, Department of Anatomy and Neurosclence Tralmng Program, Tulane Umverslty School of Me&cme. New Orleans, LA 70112, U S.A
315 Vectastain kit) 8. Imidazole (0.01 M, p H 7.6) was ineluded in the diaminobenzidine reaction to enhance the staining intensity 17. Control sections were incubated with supernatant from a non-secreting m y e l o m a cell line or with the primary antibody preabsorbed with 5 - 5 0 / ~ M substance P. Tachykinin-immunoreactive neurons were charted throughout the cortical laminae using a light microscope with a drawing tube. Laminar and areal positions were determined by carefully aligning the immunostained sections with adjacent series stained with Cresyl violet or treated for cytochrome oxidase histochemistry 23. The normal location of the hand representations in squirrel m o n k e y anterior parietal cortex can be identified based on certain anatomical features (Fig. 1). First, the low threshold inputs from the hand project just lateral to and extend approximately 3 m m from the shallow central sulcus. Second, the lateral extent of the hand area can be determined in parasagittal sections, since area 3b widens abruptly at the face-hand area border (Fig. 1) 4'ts. In order to compare the effects of nerve injury in deprived vs non-deprived cortical regions, the pattern of tachykinin immunoreactivity in the area 3b hand region contralateral to the nerve injuries was compared to that in the ipsilateral hand area. Since neuropeptide content could be affected ipsilateral to the injuries, the ipsilateral and contralateral face regions were studied as control, unaffected representations• The tachykinin-immunoreactivity in control (face) regions of squirrel m o n k e y anterior parietal areas was similar to that shown previously in macaques 9. The immunoreactive neurons exhibited non-pyramidal morphologies. Intensely stained neurons were distributed throughout the cellular layers of cortex and were especially prominent in layers II/III and VI. Populations of moderately stained cells were also present in all layers, but formed a noticeable band in layer IV. The tachykinin-positive fiber plexus was densest superficially in layers I/II, and extended throughout the cortical layers in area 3b. Numbers of immunostained neurons were c o m p a r e d in the face and hand regions both contralateral and ipsilateral to the nerve injuries. The sections from the hand cortex were all within 1 m m of the central sulcus, and thus occupied cortex predominantly activated from the ulnar nerve. Sections from the face region exhibited both the greater length of layer IV and were more than 3 m m from the central sulcus. Contralaterally, plots of labeled cells in layer IV (Fig. 1A,B) showed a greater density in the face compared to the hand area. Because cells in layer IV of area 3b are positioned at earlier stages of cortical circuitry by their proximity to thalamic afferents, quantification of immunostaining was focused on this layer.
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Fig. 1. A: the location of the representations of the body surface in areas 3b and i on a dorsal view of a squirrel monkey brain. The face and hand regions are located on the smooth surface of the brain lateraUy, the trunk and limbs are represented within the shallow central sulcus (CS), and the foot is located medaally. LS, lateral sulcus. In sagittal sections, the transition from the hand to the face representation shows an abrupt increase in the length of area 3b. The approximate locations of the sections shown in B and C are indicated on the drawing by arrows. B: camera luclda drawing of tachykinin-immunoreactive cells in the face area, and C, through the hand area. Layer IV is outlined, and the contained labeled cells are shown with larger dots for emphasis. Note the greater density of lmmunoreactive layer IV cells m the face region.
316
A 120"
Comparison of Tachykinin-Like Immunoreactivity in Face vs. Hand Region [ ] face
100" 80'
J
60'
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Fig 2. Comparisons of tachykinm-labeled cells through layer IV of area 3b in the face (control) vs hand (deprived) regions contralateral to median and ulnar deafferentations. A' for 5 sections m each region, cells were counted along a constant length of layer IV, equal to the anteroposterior extent of area 3b in the medial one half of the hand represention. B: the percent depletion of cells m the hand compared to the face region xs shown Each of the differences ~s significant by a t-test. Bars equal one standard deviatmn from the mean.
Counts of labeled cells through equal lengths of layer IV were taken from 5 pairs of sections through the h a n d and face areas, both contralateral and ipsilateral to the nerve injuries. Analysis of variance ( A N O V A ) showed statistical differences b e t w e e n the deprived hand and (1) the non-deprived hand region, (2) the face region ipsilateral to the deprived area, and (3) the contralateral face region. N o differences were shown between counts from the two face regions, nor the ipsilateral face and hand regions. Focusing on within-hemisphere comparisons contralateral to the nerve injuries, fewer cells were labeled in the hand c o m p a r e d to the face region, and the differences were statistically significant (t-test, P < 0.05) in each of the 5 cases. T h e differences of the counts ranged from about 20 to 40% (Fig. 2B). The differences were maintained at postinjury times from 5 to 21 days, and the d a t a did not suggest t i m e - d e p e n d e n t changes within the p e r i o d studied. The depletion of tachykinin immunoreactivity was also
Fig. 3. Photomicrographs of tachykinin xmmunoreactivity m layer IV (indicated by bars) of somatosensory area 3b following median and ulnar nerve injury A and B are from two different control regmns, the ipsllateral hand area and contralateral face re.on, respectively. C is from the deprived hand representaUon. Note more numerous labeled cells and fibers in both A and B compared to C. Bar (A-C) = 200 pm.
apparent in the fiber plexus m layer IV and the supragranular layers (Fig. 3C). H o w e v e r , the staining in the most p r o m i n e n t focus of stained fibers, in layers I and II, did not a p p e a r r e d u c e d in the hand c o m p a r e d to the
317 face region. Ipsilateral to the deafferentation, the density of the fiber plexuses in the cortical hand region was comparable to that in the face area (Fig. 3A,B). Thus, tachykinin-immunoreactive cells and fibers in m o n k e y primary somatosensory cortex are depleted following peripheral nerve injury. It is not possible to identify with certainty which specific immunoreactive neuropeptide has been depleted, because the antibody used recognizes the canonical sequence of the tachykinins t~. However, the content of the other tachykinins found in the mammalian nervous system, i.e. neurokinin A (substance K) and neurokinin B, is relatively much lower in cerebral cortex than substance P. Assuming that decreased immunoreactivity indicates a corresponding loss of peptide, it appears likely that at least substance P is downregulated following injury. The changes may be compared to the effects of visual deprivation by monocular tetrodotoxin injection on tachykinin immunoreactivity in the primary visual cortex of macaque monkeys 7. However, the 80% depletion of cells in layer IV of the visual cortex appears substantially greater than in the present study. Differences in staining depletion are probably not attributable to differences in times after the onset of sensory deprivation, since the visual deprivation effects were studied at 10 and 15 days. The deprivation of tachykininergic cells in layer IV may have been more effective in the visual cortex than in the present experiments. Ocular dominance bands appear anatomically and physiologically discrete in layer IV of Old World monkeys. In contrast, immediate shifts in cortical receptive fields following nerve injury suggest
convergence of inputs from different peripheral nerves onto layer IV of the somatosensory cortex 1°'15'2°. In the present experiments, the radial nerve to the dorsal surface of the hand was intact. Furthermore, complete cortical reactivation by the radial nerve following median and ulnar nerve transection in squirrel monkeys occurs at two months postinjury 6. Thus, not only might the deafferentation have been 'incomplete' cortically, the ongoing recovery of activation by the somatosensory cortex 3'19 could influence the tachykinin content of cortical neurons. Since the types of the injuries performed in this study do not produce a major degree of peripheral ganglion cell death, the changes shown in peptide neuromoduiators are likely to be due to a loss of activity or pattern of sensory input. Depletion of tachykiuin immunoreactivity is well-established in the dorsal horn following peripheral nerve injury, and this is restored to normal levels within several weeks 16. Although intrinsic neurons of the dorsal horn may contribute to the return of immunoreactivity, the time course of the restoration corresponds to the expected return of function following nerve regeneration. Whether cortical depletion of tachykinin immunoreactivity is reversible at longer times after injury, or following nerve regeneration, remains to be explored.
1 Cuello, A.C., Galfr6, G. and Mdstein, C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. Natl. Acad Sct., U.S.A., 76 (1979) 3532-3536. 2 Cusick, C.G., Early changes in tachykinm-like lmmunoreactivlty m primary somatosensory cortex of adult squirrel monkeys following nerve injuries, Soc. Neurosct. Abstr., 16 (1990) 630. 3 Cusick, C.G., Wall, J.T., Whmng, J.H. and Wiley, R.G., Temporal progression of cortical reorganization following nerve injury, Brain Research, 537 (1990) 355-358. 4 Cuslck, C.G., Wall, J.T. and Kaas, J.H., Representations of the face, teeth and oral cavity in areas 3b and I of somatosensory cortex in sqmrrel monkeys, Brain Research, 370 (1986) 359-364. 5 Dykes, R.W. and Lamour, Y., An electrophysiologlcal laminar analysis of single somatosensory neurons in partially deafferented rat hindlimb granular cortex subsequent to transection of the sciatic nerve, Brain Research, 449 (1988) 1-17. 6 Garraghty, EE., Clower, R.D., LaChica, E.A. and Kaas, J.H., Thalamic and cortical reorgamzatlon after chromc transection of the median and ulnar nerves m adult monkeys, Soc. Neurosci. Abstr., 16 (1990) 831. 7 Hendry, S.H.C., Jones, E G. and Burstem, N., Actiwty-dependent regulation of tachykmin-like immunoreactavity in neurons of monkey visual cortex, J Neurosct., 8 (1988) 1225-1238. 8 Hsu, S.M., Rame, L. and Fanger, H , Use of awdin-biotinperoxidase complex (ABC) in lmmunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J Histochem Cytochem., 29 (1981) 577-580.
9 Jones, E.G., DeFelipe, J., Hendry, S.H.C. and Maggio, J.E., A study of tachykmin-immunoreactive neurons in monkey cerebral cortex, J. Neurosci., 8 (1988) 1206-1224. 10 Kaas, J.H., Merzenieh, M.M. and Killackey, H.E, The reorganization of somatosensory cortex following peripheral nerve damage m adult and developing mammals, Annu Rev. Neurosci., 6 (1983) 325-356. 11 Maggio, J.E., Tachykimns, Annu. Rev. Neurosct., 11 (1988) 13-28. 12 Merzenich, M.M., Kaas, J.H., Wall, J , Nelson, R.J., Sur, M. and Felleman, D., Topographic reorganization of somatosensory cortical areas 3b and 1 in adult monkeys following restricted deafferentatlon, Neuroscience, 8 (1983a) 33-55. 13 Merzemch, M.M., Kaas, J.H., Wall, J.T., Sur, M., Nelson, R.J. and Felleman, D.J., Progression of change following median nerve section m the cortical representation of the hand m areas 3b and 1 m adult owl and squirrel monkeys, Neurosctence, 10 (1983b) 639-665. 14 Merzenich, M M, Nelson, R.J., Kaas, J.H., Stryker, M.P., Jenkins, W.M., Zook, J.M., Cynader, M.S. and Schoppmann, A., Variability in hand surface representations in areas 3b and 1 in adult owl and squirrel monkeys, J. Comp. Neurol., 258 (1987) 281-296. 15 Metzler, J. and Marks, ES., Functional changes in cat somatic sensory-motor cortex during short-term reversible epidural blocks, Brain Research, 177 (1979) 379-383. 16 Rhoades, R.W., Chiaia, N.L., Hess, P.R. and Miller, M.W.,
Acknowledgements. I thank Louis Lucas for help with statistics and graphics, and Nathaniel Lawson for assistance in surgery. The results have been reported previously in abstract form2.
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Effect of neonatal infraorbital nerve transection on substance Pand leueine enkephalin-like immunoreactivities in trigermnai subnucleus caudalis of the rat, J. Neurosci., 8 (1988) 2234-2247. Straus, W., Imidazole increases the sensitivity of the cytochemical reaction for peroxidase with diaminobenzidine at a neutral pH, J. Histochem. Cytochem., 30 (1982) 491-493. Sur, M., Nelson, R.J. and Kaas, J.H., Representation of the body surface in cortical areas 3b and 1 of squirrel monkeys: comparisons with other primates, J. Comp. Neurol., 211 (1982) 177-192. Wall, J.T., Variable organization m cortical maps of the skin as an indication of the lifelong adaptive capacities of circuits m the mammalian brain, Trends Neurosci., 11 (1988) 549-557. Wall, J.T. and Cuslck, C.G., Cutaneous responsiveness m primary somatosensory (S-I) tundpaw cortex before and after par-
tlal hindpaw deafferentatlon m adult rats, J. Neurosc~., 4 (1984) 1499-1515. 21 Warren, R., Tremblay, N. and Dykes, R.W., Quantltauve study of glutamic acid decarboxylase-immunoreactive neurons and cytochrome oxldase activity in normal and partially deafferented rat hmdlimb somatosensory cortex, J Comp Neurol., 288 (1989) 583-592. 22 Welker, E., Sonano, E. and Van der Loos, H., Plasticaty in the barrel cortex of the adult mouse' effects of peripheral deprwatlon on GAD-lmmunoreactwlty, Exp. Bram Res., 74 (1989a) 441-452. 23 Wong-Raley, M.T T , Changes m the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochermstry, Brain Research, 171 (1979) 11-28.
