The Journal
of Neuroscience.
May
1990.
10(5):
1429-l
443
Age-Dependent Differences in Reorganization of Primary Somatosensory Cortex Following Low Thoracic (T,J Spinal Cord Transection in Cats P. A. McKinley’,’
and
J. L. Smith’
‘Department of Kinesiology and Occupational Therapy,
and Brain Research Institute, UCLA, McGill University, Montreal, Quebec,
The organization of primary somatosensory cortex was examined in chronic spinal cats that had sustained cord transection at T,, at 3 ages: 2 and 6 weeks of age, and as adults. Five months to 1 yr following transection, the deprived cortex was mapped electrophysiologically (multiunit recordings). The topographical organization found at each age was compared to that present in normal adults to study effects of developmental age on the ability of the somatosensory system to adjust to changes in afferent input. Cortical responses to deprivation of somatosensory input were age dependent. In animals cord transected at 2 weeks of age, the remaining somatic afferent input excited both its normal cortical area and the area normally reserved for the hindlimb. This resulted in 2 somatotopic maps of the rostra1 trunk and forelimb. In contrast, in cats spinalized at 6 weeks of age, there was only 1 map for the remaining somatosensory input that was distributed across the mediolateral axis of the primary somatosensory cortex. As a result, the remaining somatosensory input was shifted medially from its normal position and was narrower with respect to the rostrocaudal area driven by light tactile input. The amount of cortex that each body region could excite was essentially the same as in normal animals. In adults, a third response was observed; regions normally devoted to forelimb and trunk appeared to be unchanged, and the region previously serving the hindlimb responded only to a limited extent, and only to tactile stimulation of the trunk. In all cases, however, some sites in the cortex could be excited by parts of the body that in normal animals were served by cortical regions from 3 to 10 mm away, a distance much in excess of the maximum extent of reported thalamocortical overlap. We suggest that the various patterns of cortical organization observed at different ages reflect different developmental processes that are active at the time of transection. Further, we hypothesize that often, in major denervations
Rcwved Oct. 21. 1988: revised Sept. 28, 1989; accepted Nov. 8. 1989. We thank Drs. Michael Merxnich, William Jcnkms, and Scott Chandler for their help In settmg up the mltzd cxpcnmcnts. and Drs. Nina Bradley, Carol Gullam. Gall Koshland. and Ms. Grace C‘aneta In the collection ofdata. We also thank Dr. Robert Dykcs for thoughtful criticism of the manuscript. Tlus work was supported in part by NIH grant NS19864 and an Acadcmx Senate Grant to J. 1.. S. and by MRC grant MA10069 to P. McK. Correspondence should bc addressed to P. A. McKinlq. Ph.D.. McGill LJnivcrslt). School of Physlcal and Occupational Therapy. 3654 Drummond Ave.. Montreal, Quebec. Canada H3G IY5. Copyright c 1990 Society for Neuroscience 0270-6474/90/05 1429.15$02.00/O
Los Angeles, California Canada H3G lY5
90024,
and 5chool
of Physical
such as spinal cord transection, a significant component of the reorganization occurs at synaptic levels below the cortex in young animals. Topographical reorganization of the somatosensory cortex foollowing peripheral denervation or amputation has been reported to occur in a number of mammalian species: raccoon (Rasmusson, 1982; Doetsch and Kelahan. 1983; Kclahan and Doetsch, 1984), monkey (Wall et al., 1982. 1983; Mcr/cnich et al., 1983a, b), rat (Wall and Cusick. 1984). and cat (Kalaska and Pomeranz, 1979; Metzler and Marks. I97Y; Franck. 1980: McKinley et al., 1985, 1987). Although most data concern the results of amputation of 1 or 2 digits or partial denervation of a limb segment (see Wall and Kaas, 1985, for review). it has been suggested that cortical capacity to respond to nerve injury may have a distance limit related to the amount of overlap of thalamocortical axon terminal arbors (Mer;lenich ct al.. 1983a. b). Most of the changes reported in the studies cited above have occurred across cortical distances within the reported range of thalamocortical overlap. However, as the amount of thalamocortical overlap in cat has been estimated to be 1-I .5 mm in both the rostrocaudal and mediolateral directions (Landry and Deschenes, 198 1; Landry et al., 1986). this mechanism does not offer a satisfactory explanation for cortical reorganization over greater distances such as those reported in young kittens (Kalaska and Pomeranz, 1979; McKinley ct al.. 1987). Although there have been numerous reports on the gcncral age dependent effects of lesions within the central nervous systern (Finger and Stein, 1982, for review). reports concerning the reorganizing capacity ofthe cortex are inconsistent with respect to the magnitude of the response. While some reports indicate an increase in reorganizational response in younger animals (Kalaska and Pomeranz, 1979; Waite, 1984; McKinley et al.. I987), others report no difference (Carson et al.. I98 I: Kelahan et al., l981), while others find a reduction in response (Wall and Cusick, 1986). These contradictory results may be due to the use of different species that are at various stages of devclopment at birth. For example, if the reorganixational responses can be described as a function of the developmental growth process, then we might expect a variety of responses that arc reflective of particular developmental processes active during the period of the injury. We have chosen to use the cord transected cat (T,,) as a model for studying the capacity of the cortex to respond to a large magnitude of deprivation, and we have selected 3 age groups
1430
McKinley
and
Smith
* Age-Dependent
Cortical
Reorganization
in Cat
(2 week, 6 week, and adult) to distinguish the effect of developmental influences on this response. The 2-week-old spinal kitten was chosen as a representative of the early postnatal developing animal because the somatosensory system is develoned. but asvects of it are in various stages of maturation. Electrophysiologically, the somatosensory cortex is approaching maturity, asboth responselatency (Grossman, 1955; Bruce and Tatton, 1980) and synaptic configuration (Voeller et al., 1963) are reaching the adult state. However, capacity to code and signal information from the peripheral organs is not well developed until 2 months of age(Connor et al., 1984; Ferrington et al., 1984). Likewise, the synaptic effectivenessfrom primary sensorycortex to the motor cortex remains low until about 8 weekspostnatal (Bruce and Tatton, 1980). In contrast, an animal cordotomized at 6 weeksof age is in a more mature state of development that may more closely reflect the adult situation, and yet when compared to the adult, may still be capableof a more robust responseto developmental processes.Our results demonstrate that central responsesto spinal cord transection are age dependent and the topographical reorganization at the cortical level may reflect different stepsof development at the time of injury. Preliminary reports of some of these findings have been published (McKinley et al., 1985, 1987). *
I
Materials and Methods Experimental design Three age groups of laboratory-raised cats were chosen for this study: juveniles of 2 weeks (n = 6) and 6 weeks (n = 6) of age, and adults (n = 6). Control groups of normal adult cats (n = 4) and 6-week-old kittens (n = 4) were also studied. All experimental cats were allowed to recover from spinal surgery for a period of at least 4 months, thus insuring that the most rapid and largest aspects of reorganization were completed (Merzenich et al., 1983a, b; Kelahan and Doetsch, 1984), and that the cats that had received cord transections as kittens had an adult-size brain. Although the age at which the cord transection had occurred was generally unknown to the primary investigator at the time of the experiment, all researchers in the laboratory were involved in animal care, and the morphological signs of the surgery were self-evident; thus, it was not feasible to do a blind mapping study. To prevent bias during the mapping procedures of the experimental animals, 1 investigator manipulated the electrode while another located and recorded receptive fields in the periphery, and a third recorded the data. Subject preparation Surgical procedure and postoperative care. Both the surgical procedure and postoperative care were routine procedures that have been described previously (Smith et al., 1982; Bradley and Smith, 1988; McKinley and Kruger, 1988). All operations were performed under general anesthesia and aseptic conditions using ketamine (25 mg/kg) and Acepromazine (0.5 mg/kg) for 2- and 6-week-old kittens, and nembutal(35 mg/kg IP) for adults. A laminectomy was performed between T,, and T,,. After topical application of Xylocaine (2%) on the area of the spinal cord to be lesioned, a hook was used to elevate the cord slightly, and it was severed at the segmental level ofT,,. The completeness of the transection was verified by lifting the hook through the lesioned area and observation of cord retraction. Gelfoam was packed between rostra1 and caudal segments and the wound was sutured. Figure 1.4 illustrates the somatic region which is spinal cord isolated by the lesion. For 1 week following surgery, antibiotics were administered prophylactically. Unweaned kittens were placed with the nursing mother immediately after they recovered from the effects of anesthesia. The hindlimbs were massaged daily, cleaned, and the digit, ankle, knee, and hip joints were moved through their range of motion to prevent stiffness. To initiate reflex evacuation of the urinary bladder, light tactile stimulation was applied to the perigenital area twice daily: manual expression of the bladder was used only when tactile stimulation was ineffective. Once weaned, kittens were housed by litters in spacious cages (3 x 6 x 6 ft) constructed of chain link fence, in which shredded paper covered the floor to prevent decubitus ulcers from forming. Similar
housing was provided for adults. This configuration encouraged social interaction, visual and tactile stimulation, and spontaneous play. Lesion con&nation. At the termination of the mapping experiments, animals were deeply anesthetized and perfused intracardially with 0.9% saline followed bv 10% buffered formalin. After Derfusion. the sninal cord was remove2 and processed using a modified Bielschowsky iechnique counterstained wiih trichrome tdconfirm the completeness of the spinal cord transection. The brains were removed, photographed, and placed in 30% sucrose formalin until they sank. Parasagittal sections of 28 pm were cut on the freezing microtome and stained with thionin for histological studies. Mapping procedure Animal preparation. The recording procedures were similar to those developed by Merzenich and colleagues (Merzenich et al., 1978, 1983a). Following a preanesthetic injection (s.q.) of atropine sulfate (0.1 mg/ kg), ketamine was administered (28 mg/kg i.m.) and maintained at surgical levels throughout both surgical and recording procedures with subsequent 0.1 ml i.m. doses of ketamine. Injections, i.p., of saline (35 ml/hr) were given to prevent dehydration. Rectal temperature was monitored and kept at 37°C throughout the experiment by placing the animal on a cushioned heating pad. The cranium was exposed, and a metal bar was attached to the skull with dental acrylic contralateral to the hemisphere to be mapped and then clamped to a stereotaxic frame so that the animal’s head was rigidly stabilized. A craniotomy was performed exposing the entire anterior cortex from a site posterior to the ansate sulcus to a point rostra1 to the cruciate sulcus. A cotton dam was constructed around the exposed area, saline placed in the well created by the dam and the craniotomy, and the dura was reflected while viewing through a surgical microscope (Zeiss). The saline was then removed, and the exposed cortex was covered with silicone (Accumetrics). A high resolution photograph of the brain surface was taken with high contrast film (Panex), developed and enlarged (25-30 x). The surface vasculature provided a reference system used to guide electrode penetrations during the experimental recordings. These locations were recorded on the photograph. Recording apparatus. Multiunit neuronal activity was recorded with paralene-coated tungsten microelectrodes (Microproduct) having impedances of between 1.5 and 2.0 MQ (at 1 kHz), attached to a handdriven triaxial microdrive (KopQ mounted on the stereotaxic frame. All signals were referenced to a ground attached to the animal, amplified 10,000 x (Dagan), passed through a band-pass filter (loo-10 kHz), displayed on a storage oscilloscope (Tektronix), and monitored through an audiomonitor (Grass). Mapping protocol. During the recording sessions, the microelectrode was lowered to the pial surface and contact with the pial surface was detected using the audiomonitor. Penetrations were made approximately normal to the cortical surface, except in the posterior bank of the coronal sulcus and the anterior wall of the medial and lateral branches of the ansate sulcus, where penetrations were parallel to the cortical surface. A grid-like array of penetrations was spaced at 200-300 Frn intervals rostrocaudally and in mediolateral rows spaced 300-700 pm apart across the area of primary sensory cortex that represented the hindlimb, trunk, and proximal forelimb. Mapping in the rostrocaudal direction was terminated when 3 penetrations in a row did not respond to light tactile input. For illustration purposes, only the first nonresponsive penetration is shown at these borders. The cortex normally devoted to forepaw and wrist was not routinely mapped in detail. At each “in depth” (parallel) penetration near the bank of the sulci, the microelectrode was advanced initially to a depth of 400 pm and then advanced in 200 pm increments to a depth of 1600 pm below the surface. At each recording site, attempts were made to evoke responses to somatic stimuli delivered to the trunk, forelimb, and face, using a handheld probe, brush, or bristle. In areas where there was a response, identification and characterization of receptive fields (RFs) proceeded as described below. On the crown of the gyrus, the electrode was quickly advanced to a depth of 600-800 Frn, where attempts were made to evoke responses to tactile stimuli. If no response was elicited, the electrode was moved up or down 200 pm and the body was stimulated again. When a response was observed, it was recorded and the depth was noted. Electrolytic lesions (10 PA, 10 set) were occasionally placed at recording sites for verification of penetration depth. Where no evoked responses were observed, efforts were made to stimulate the entire body before the electrode was moved to another site. When no response could be obtained,
The Journal
of Neuroscience,
May
1990.
lO(5)
1431
midline
D.
C. NORMAL
CAT
SPINALIZED
AT 2 WEEKS
H midline
Imm
midline r ”
,runk RFs
C
i)
t
e
w dimple
; I
F. SPINALIZED
the penetration (NCR).
AT 6 WEEKS
,’
“;;/
ansate
.** paw ( digit
SPINALIZED
was classified as a nonresponsive cutaneous response
Stimulus presentation and classification Initially, to determine the RF, the researcher lightly touched the hair with the fingers. The entire surface of the animal was stimulated in this manner until a response that was clearly related to the touch was located. Then the researcher used a hand-held nonconductive glass probe and delicately touched the hairs in the area producing responses, until the RF was well delineated. This area was then drawn on a cartoon of the body part and logged in a record book. These neurons were classified as responding to light tactile stimuli. Neurons that responded to more intense stimulation only, such as light scratching, were defined as highthreshold neurons but were still considered to be responsive to cutaneous stimulation. In contrast, if the neurons responded only to tapping or joint movement, they were classified as deep and were not included as part of the population responsive to tactile stimulation.
Preparation of a somatosensory map After the experiment was finished, an enlargement of the cortical photograph was made by projecting a transparency copy of the photograph
AS ADULTS
Figure I. Comparison of reorganization maps in individual cats cord transected at 2 or 6 weeks of age or as adults. A, Region of afferent input isolated to the spinal cord after cord transection at Tlz is shaded. B, Region of somatosensory cortex shown in the maps below. C, Map of primary somatosensory cortex in normal cat. D-F, Cortical maps of individual chronic spinal cats cord transected at different ages: D, 2 weeks; E, 6 weeks; F, adult. Striped lines indicate areas of no cutaneous response. Dashed lines indicate borders of mapped/unmapped cortex. Abbreviations: ank, ankle; ch, chest; sh, shoulder. Forelimb includes forearm, paw, and digits.
against a wall and marking the numbered penetration sites on graph paper. Then, figurines of the cat marked with the appropriate RF location were placed over the corresponding cortical site. From this information a general somatotopic map was drawn with boundary lines placed midway between regions representing separate limb segments or regions of the trunk. Patterns of topographical organization and orientation were also noted, as they could be easily seen with respect to cortical placement on these enlarged maps.
Results General age-dependent responses Figure 1 illustrates the major differences in somatotopic maps in chronic cord transected animals of different agesand compares them to that of a normal adult cat. While the maps illustrated in Figure 1, C-F are not composites, but taken from individual animals, they are typical of the response of the majority of animals in each age group. Animals cord transected at 2 weeks of age demonstrated the most robust response; the entire expanse of deprived cortex was excited by input arising from
1432 McKinley and Smith * Age-Dependent Cortical Reorganlzat~on in Cat
Row F
Sh
FI lmm
P
x .q-.’ 0. . x* . .* .* .* ‘.:x. . .I
l
I-, :‘i,)‘(’ .? I’cnctrat~on map of primary sensory cortex in 1 animal cord transected at 2 weeks of age. C‘losc~d c~/wlccs reprcscnt s~tcs Ilght tactllc stimuli: .\“s represent sites with N. I)., and B. B. Stanfield (1985) Occipital cortical neurons with transient pyramidal tract axons extend and maintain collaterals to subcortical but not lntracortical targets. Brain Res. 336: 326-333. O’Leary. I>. I)., and B. B. Stanfield (1986) A transient pyramidal projcctlon from the visual cortex in the hamster and its removal by sclcctivc collateral elimination. Dev. Brain Res. 27: 87-99. Rasmusson. D. D. (I 982) Reorganization of raccoon somatosensory cortex following removal of the fifth digit. J. Comp. Neurol. 205; 313-326. Rasmusson. D. D., and R. W. Dykes (1988) Long-term enhancement of evoked potentials in cat somatosensory cortex produced by coactl\atlon of the basal forebrain and cutaneous receptors. Exp. Brain Res. 7(1: 276-286. Rasmusson. D. D., and D. M. Nance (1986) Nonoverlapping thalamocortical projections for separate forepaw digits before and after cortical reorganization in the raccoon. Brain Res. Bull. 16: 399406. Kubcl. E. (I 97 I) A comparison ofsomatotopic organization in sensory ncocortex of newborn kittens and adult cats. J. Comp. Neural. 143: 447-480. Schclbcl. M.. and A. B. Scheibel (1978) The development of somatosensor! thalamus in mammals. In Handbook c$‘Sensor~~ Ph~?~iolop, Vol. IX, Ikvclopmcnt of’Sm.sorv S~xtrms, H. Autrium, R. Jung, W. R. Lowenstein, et al.. eds.. pp. 239-277, Springer, New York. Schult/. W.. R. Wicscndanger, B. Hess, A. Ruffieux, and M. Wicsendanger (lY81) The somatotopy of the gracile nucleus in cats with agencsls of a hindfoot. Exp. Brain Res. 43: 413-418. Stegel.
S.
( 1056)
h’onpururtwtnc
.Slu/istrc~s,f2v
McGraw-HIII. New York. Skoglund. S. (I 969) Growth and differentiation on the central nervous system. Annu. Rev.
the Behavioral
with Physiol.
Scr~nces,
special emphasis 31: 19-42.
of Neuroscience.
May
1990,
fO(5)
1443
Smith. J. L., L. A. Smith, R. F. Zernicke. and M.
of Neuroscience.
May
1990.
10(5):
1429-l
443
Age-Dependent Differences in Reorganization of Primary Somatosensory Cortex Following Low Thoracic (T,J Spinal Cord Transection in Cats P. A. McKinley’,’
and
J. L. Smith’
‘Department of Kinesiology and Occupational Therapy,
and Brain Research Institute, UCLA, McGill University, Montreal, Quebec,
The organization of primary somatosensory cortex was examined in chronic spinal cats that had sustained cord transection at T,, at 3 ages: 2 and 6 weeks of age, and as adults. Five months to 1 yr following transection, the deprived cortex was mapped electrophysiologically (multiunit recordings). The topographical organization found at each age was compared to that present in normal adults to study effects of developmental age on the ability of the somatosensory system to adjust to changes in afferent input. Cortical responses to deprivation of somatosensory input were age dependent. In animals cord transected at 2 weeks of age, the remaining somatic afferent input excited both its normal cortical area and the area normally reserved for the hindlimb. This resulted in 2 somatotopic maps of the rostra1 trunk and forelimb. In contrast, in cats spinalized at 6 weeks of age, there was only 1 map for the remaining somatosensory input that was distributed across the mediolateral axis of the primary somatosensory cortex. As a result, the remaining somatosensory input was shifted medially from its normal position and was narrower with respect to the rostrocaudal area driven by light tactile input. The amount of cortex that each body region could excite was essentially the same as in normal animals. In adults, a third response was observed; regions normally devoted to forelimb and trunk appeared to be unchanged, and the region previously serving the hindlimb responded only to a limited extent, and only to tactile stimulation of the trunk. In all cases, however, some sites in the cortex could be excited by parts of the body that in normal animals were served by cortical regions from 3 to 10 mm away, a distance much in excess of the maximum extent of reported thalamocortical overlap. We suggest that the various patterns of cortical organization observed at different ages reflect different developmental processes that are active at the time of transection. Further, we hypothesize that often, in major denervations
Rcwved Oct. 21. 1988: revised Sept. 28, 1989; accepted Nov. 8. 1989. We thank Drs. Michael Merxnich, William Jcnkms, and Scott Chandler for their help In settmg up the mltzd cxpcnmcnts. and Drs. Nina Bradley, Carol Gullam. Gall Koshland. and Ms. Grace C‘aneta In the collection ofdata. We also thank Dr. Robert Dykcs for thoughtful criticism of the manuscript. Tlus work was supported in part by NIH grant NS19864 and an Acadcmx Senate Grant to J. 1.. S. and by MRC grant MA10069 to P. McK. Correspondence should bc addressed to P. A. McKinlq. Ph.D.. McGill LJnivcrslt). School of Physlcal and Occupational Therapy. 3654 Drummond Ave.. Montreal, Quebec. Canada H3G IY5. Copyright c 1990 Society for Neuroscience 0270-6474/90/05 1429.15$02.00/O
Los Angeles, California Canada H3G lY5
90024,
and 5chool
of Physical
such as spinal cord transection, a significant component of the reorganization occurs at synaptic levels below the cortex in young animals. Topographical reorganization of the somatosensory cortex foollowing peripheral denervation or amputation has been reported to occur in a number of mammalian species: raccoon (Rasmusson, 1982; Doetsch and Kelahan. 1983; Kclahan and Doetsch, 1984), monkey (Wall et al., 1982. 1983; Mcr/cnich et al., 1983a, b), rat (Wall and Cusick. 1984). and cat (Kalaska and Pomeranz, 1979; Metzler and Marks. I97Y; Franck. 1980: McKinley et al., 1985, 1987). Although most data concern the results of amputation of 1 or 2 digits or partial denervation of a limb segment (see Wall and Kaas, 1985, for review). it has been suggested that cortical capacity to respond to nerve injury may have a distance limit related to the amount of overlap of thalamocortical axon terminal arbors (Mer;lenich ct al.. 1983a. b). Most of the changes reported in the studies cited above have occurred across cortical distances within the reported range of thalamocortical overlap. However, as the amount of thalamocortical overlap in cat has been estimated to be 1-I .5 mm in both the rostrocaudal and mediolateral directions (Landry and Deschenes, 198 1; Landry et al., 1986). this mechanism does not offer a satisfactory explanation for cortical reorganization over greater distances such as those reported in young kittens (Kalaska and Pomeranz, 1979; McKinley ct al.. 1987). Although there have been numerous reports on the gcncral age dependent effects of lesions within the central nervous systern (Finger and Stein, 1982, for review). reports concerning the reorganizing capacity ofthe cortex are inconsistent with respect to the magnitude of the response. While some reports indicate an increase in reorganizational response in younger animals (Kalaska and Pomeranz, 1979; Waite, 1984; McKinley et al.. I987), others report no difference (Carson et al.. I98 I: Kelahan et al., l981), while others find a reduction in response (Wall and Cusick, 1986). These contradictory results may be due to the use of different species that are at various stages of devclopment at birth. For example, if the reorganixational responses can be described as a function of the developmental growth process, then we might expect a variety of responses that arc reflective of particular developmental processes active during the period of the injury. We have chosen to use the cord transected cat (T,,) as a model for studying the capacity of the cortex to respond to a large magnitude of deprivation, and we have selected 3 age groups
1430
McKinley
and
Smith
* Age-Dependent
Cortical
Reorganization
in Cat
(2 week, 6 week, and adult) to distinguish the effect of developmental influences on this response. The 2-week-old spinal kitten was chosen as a representative of the early postnatal developing animal because the somatosensory system is develoned. but asvects of it are in various stages of maturation. Electrophysiologically, the somatosensory cortex is approaching maturity, asboth responselatency (Grossman, 1955; Bruce and Tatton, 1980) and synaptic configuration (Voeller et al., 1963) are reaching the adult state. However, capacity to code and signal information from the peripheral organs is not well developed until 2 months of age(Connor et al., 1984; Ferrington et al., 1984). Likewise, the synaptic effectivenessfrom primary sensorycortex to the motor cortex remains low until about 8 weekspostnatal (Bruce and Tatton, 1980). In contrast, an animal cordotomized at 6 weeksof age is in a more mature state of development that may more closely reflect the adult situation, and yet when compared to the adult, may still be capableof a more robust responseto developmental processes.Our results demonstrate that central responsesto spinal cord transection are age dependent and the topographical reorganization at the cortical level may reflect different stepsof development at the time of injury. Preliminary reports of some of these findings have been published (McKinley et al., 1985, 1987). *
I
Materials and Methods Experimental design Three age groups of laboratory-raised cats were chosen for this study: juveniles of 2 weeks (n = 6) and 6 weeks (n = 6) of age, and adults (n = 6). Control groups of normal adult cats (n = 4) and 6-week-old kittens (n = 4) were also studied. All experimental cats were allowed to recover from spinal surgery for a period of at least 4 months, thus insuring that the most rapid and largest aspects of reorganization were completed (Merzenich et al., 1983a, b; Kelahan and Doetsch, 1984), and that the cats that had received cord transections as kittens had an adult-size brain. Although the age at which the cord transection had occurred was generally unknown to the primary investigator at the time of the experiment, all researchers in the laboratory were involved in animal care, and the morphological signs of the surgery were self-evident; thus, it was not feasible to do a blind mapping study. To prevent bias during the mapping procedures of the experimental animals, 1 investigator manipulated the electrode while another located and recorded receptive fields in the periphery, and a third recorded the data. Subject preparation Surgical procedure and postoperative care. Both the surgical procedure and postoperative care were routine procedures that have been described previously (Smith et al., 1982; Bradley and Smith, 1988; McKinley and Kruger, 1988). All operations were performed under general anesthesia and aseptic conditions using ketamine (25 mg/kg) and Acepromazine (0.5 mg/kg) for 2- and 6-week-old kittens, and nembutal(35 mg/kg IP) for adults. A laminectomy was performed between T,, and T,,. After topical application of Xylocaine (2%) on the area of the spinal cord to be lesioned, a hook was used to elevate the cord slightly, and it was severed at the segmental level ofT,,. The completeness of the transection was verified by lifting the hook through the lesioned area and observation of cord retraction. Gelfoam was packed between rostra1 and caudal segments and the wound was sutured. Figure 1.4 illustrates the somatic region which is spinal cord isolated by the lesion. For 1 week following surgery, antibiotics were administered prophylactically. Unweaned kittens were placed with the nursing mother immediately after they recovered from the effects of anesthesia. The hindlimbs were massaged daily, cleaned, and the digit, ankle, knee, and hip joints were moved through their range of motion to prevent stiffness. To initiate reflex evacuation of the urinary bladder, light tactile stimulation was applied to the perigenital area twice daily: manual expression of the bladder was used only when tactile stimulation was ineffective. Once weaned, kittens were housed by litters in spacious cages (3 x 6 x 6 ft) constructed of chain link fence, in which shredded paper covered the floor to prevent decubitus ulcers from forming. Similar
housing was provided for adults. This configuration encouraged social interaction, visual and tactile stimulation, and spontaneous play. Lesion con&nation. At the termination of the mapping experiments, animals were deeply anesthetized and perfused intracardially with 0.9% saline followed bv 10% buffered formalin. After Derfusion. the sninal cord was remove2 and processed using a modified Bielschowsky iechnique counterstained wiih trichrome tdconfirm the completeness of the spinal cord transection. The brains were removed, photographed, and placed in 30% sucrose formalin until they sank. Parasagittal sections of 28 pm were cut on the freezing microtome and stained with thionin for histological studies. Mapping procedure Animal preparation. The recording procedures were similar to those developed by Merzenich and colleagues (Merzenich et al., 1978, 1983a). Following a preanesthetic injection (s.q.) of atropine sulfate (0.1 mg/ kg), ketamine was administered (28 mg/kg i.m.) and maintained at surgical levels throughout both surgical and recording procedures with subsequent 0.1 ml i.m. doses of ketamine. Injections, i.p., of saline (35 ml/hr) were given to prevent dehydration. Rectal temperature was monitored and kept at 37°C throughout the experiment by placing the animal on a cushioned heating pad. The cranium was exposed, and a metal bar was attached to the skull with dental acrylic contralateral to the hemisphere to be mapped and then clamped to a stereotaxic frame so that the animal’s head was rigidly stabilized. A craniotomy was performed exposing the entire anterior cortex from a site posterior to the ansate sulcus to a point rostra1 to the cruciate sulcus. A cotton dam was constructed around the exposed area, saline placed in the well created by the dam and the craniotomy, and the dura was reflected while viewing through a surgical microscope (Zeiss). The saline was then removed, and the exposed cortex was covered with silicone (Accumetrics). A high resolution photograph of the brain surface was taken with high contrast film (Panex), developed and enlarged (25-30 x). The surface vasculature provided a reference system used to guide electrode penetrations during the experimental recordings. These locations were recorded on the photograph. Recording apparatus. Multiunit neuronal activity was recorded with paralene-coated tungsten microelectrodes (Microproduct) having impedances of between 1.5 and 2.0 MQ (at 1 kHz), attached to a handdriven triaxial microdrive (KopQ mounted on the stereotaxic frame. All signals were referenced to a ground attached to the animal, amplified 10,000 x (Dagan), passed through a band-pass filter (loo-10 kHz), displayed on a storage oscilloscope (Tektronix), and monitored through an audiomonitor (Grass). Mapping protocol. During the recording sessions, the microelectrode was lowered to the pial surface and contact with the pial surface was detected using the audiomonitor. Penetrations were made approximately normal to the cortical surface, except in the posterior bank of the coronal sulcus and the anterior wall of the medial and lateral branches of the ansate sulcus, where penetrations were parallel to the cortical surface. A grid-like array of penetrations was spaced at 200-300 Frn intervals rostrocaudally and in mediolateral rows spaced 300-700 pm apart across the area of primary sensory cortex that represented the hindlimb, trunk, and proximal forelimb. Mapping in the rostrocaudal direction was terminated when 3 penetrations in a row did not respond to light tactile input. For illustration purposes, only the first nonresponsive penetration is shown at these borders. The cortex normally devoted to forepaw and wrist was not routinely mapped in detail. At each “in depth” (parallel) penetration near the bank of the sulci, the microelectrode was advanced initially to a depth of 400 pm and then advanced in 200 pm increments to a depth of 1600 pm below the surface. At each recording site, attempts were made to evoke responses to somatic stimuli delivered to the trunk, forelimb, and face, using a handheld probe, brush, or bristle. In areas where there was a response, identification and characterization of receptive fields (RFs) proceeded as described below. On the crown of the gyrus, the electrode was quickly advanced to a depth of 600-800 Frn, where attempts were made to evoke responses to tactile stimuli. If no response was elicited, the electrode was moved up or down 200 pm and the body was stimulated again. When a response was observed, it was recorded and the depth was noted. Electrolytic lesions (10 PA, 10 set) were occasionally placed at recording sites for verification of penetration depth. Where no evoked responses were observed, efforts were made to stimulate the entire body before the electrode was moved to another site. When no response could be obtained,
The Journal
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1990.
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Stimulus presentation and classification Initially, to determine the RF, the researcher lightly touched the hair with the fingers. The entire surface of the animal was stimulated in this manner until a response that was clearly related to the touch was located. Then the researcher used a hand-held nonconductive glass probe and delicately touched the hairs in the area producing responses, until the RF was well delineated. This area was then drawn on a cartoon of the body part and logged in a record book. These neurons were classified as responding to light tactile stimuli. Neurons that responded to more intense stimulation only, such as light scratching, were defined as highthreshold neurons but were still considered to be responsive to cutaneous stimulation. In contrast, if the neurons responded only to tapping or joint movement, they were classified as deep and were not included as part of the population responsive to tactile stimulation.
Preparation of a somatosensory map After the experiment was finished, an enlargement of the cortical photograph was made by projecting a transparency copy of the photograph
AS ADULTS
Figure I. Comparison of reorganization maps in individual cats cord transected at 2 or 6 weeks of age or as adults. A, Region of afferent input isolated to the spinal cord after cord transection at Tlz is shaded. B, Region of somatosensory cortex shown in the maps below. C, Map of primary somatosensory cortex in normal cat. D-F, Cortical maps of individual chronic spinal cats cord transected at different ages: D, 2 weeks; E, 6 weeks; F, adult. Striped lines indicate areas of no cutaneous response. Dashed lines indicate borders of mapped/unmapped cortex. Abbreviations: ank, ankle; ch, chest; sh, shoulder. Forelimb includes forearm, paw, and digits.
against a wall and marking the numbered penetration sites on graph paper. Then, figurines of the cat marked with the appropriate RF location were placed over the corresponding cortical site. From this information a general somatotopic map was drawn with boundary lines placed midway between regions representing separate limb segments or regions of the trunk. Patterns of topographical organization and orientation were also noted, as they could be easily seen with respect to cortical placement on these enlarged maps.
Results General age-dependent responses Figure 1 illustrates the major differences in somatotopic maps in chronic cord transected animals of different agesand compares them to that of a normal adult cat. While the maps illustrated in Figure 1, C-F are not composites, but taken from individual animals, they are typical of the response of the majority of animals in each age group. Animals cord transected at 2 weeks of age demonstrated the most robust response; the entire expanse of deprived cortex was excited by input arising from
1432 McKinley and Smith * Age-Dependent Cortical Reorganlzat~on in Cat
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I-, :‘i,)‘(’ .? I’cnctrat~on map of primary sensory cortex in 1 animal cord transected at 2 weeks of age. C‘losc~d c~/wlccs reprcscnt s~tcs Ilght tactllc stimuli: .\“s represent sites with N. I)., and B. B. Stanfield (1985) Occipital cortical neurons with transient pyramidal tract axons extend and maintain collaterals to subcortical but not lntracortical targets. Brain Res. 336: 326-333. O’Leary. I>. I)., and B. B. Stanfield (1986) A transient pyramidal projcctlon from the visual cortex in the hamster and its removal by sclcctivc collateral elimination. Dev. Brain Res. 27: 87-99. Rasmusson. D. D. (I 982) Reorganization of raccoon somatosensory cortex following removal of the fifth digit. J. Comp. Neurol. 205; 313-326. Rasmusson. D. D., and R. W. Dykes (1988) Long-term enhancement of evoked potentials in cat somatosensory cortex produced by coactl\atlon of the basal forebrain and cutaneous receptors. Exp. Brain Res. 7(1: 276-286. Rasmusson. D. D., and D. M. Nance (1986) Nonoverlapping thalamocortical projections for separate forepaw digits before and after cortical reorganization in the raccoon. Brain Res. Bull. 16: 399406. Kubcl. E. (I 97 I) A comparison ofsomatotopic organization in sensory ncocortex of newborn kittens and adult cats. J. Comp. Neural. 143: 447-480. Schclbcl. M.. and A. B. Scheibel (1978) The development of somatosensor! thalamus in mammals. In Handbook c$‘Sensor~~ Ph~?~iolop, Vol. IX, Ikvclopmcnt of’Sm.sorv S~xtrms, H. Autrium, R. Jung, W. R. Lowenstein, et al.. eds.. pp. 239-277, Springer, New York. Schult/. W.. R. Wicscndanger, B. Hess, A. Ruffieux, and M. Wicsendanger (lY81) The somatotopy of the gracile nucleus in cats with agencsls of a hindfoot. Exp. Brain Res. 43: 413-418. Stegel.
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Smith. J. L., L. A. Smith, R. F. Zernicke. and M.
