somatosensory cortex - Europe PMC

Proc. Nail. Acad. Sci. USA Vol. 88, pp. 780-784, February 1991 Neurobiology

Cholinergic depletion prevents expansion of topographic maps in somatosensory cortex (neural plasticity/2-deoxyglucose/metabolic activity/acetylcholine/cortical column)

SHARON L. JULIANO*, WU MA, AND DON ESLIN Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814

Communicated by Mortimer Mishkin, October 23, 1990

tions in adults show dramatic rearrangements of cortical topographic maps in a number of species (see refs. 15 and 16 for reviews). For example, deafferentation by cutting nerves or by amputation generates expansions in the cortical representation of intact skin surfaces. Not yet known is the mechanism that produces such plastic changes; this study investigates the possibility that availability of ACh in the neocortex is a necessary ingredient of such rearrangements. Two questions regarding neuronal plasticity in the somatosensory cortex are addressed in the present study. (i) Do cortical metabolic maps of stimulus-evoked activity reflect previously discovered electrophysiological somatic map rearrangements? (ii) What is the role of ACh in forming metabolic activity maps in the somatosensory cortex, and does it participate in neocortical map rearrangements following deafferentation/amputation?

Although the role of acetylcholine in processABSTRACT ing stimuli in the cerebral cortex is becoming defined, the impact of cholinergic activity on the character of cortical maps remadns unclear. In the somatosensory cortex, topographic maps appear capable of lifelong modifications in response to alterations in the periphery. One factor proposed to influence this adaptational ability is the presence of acetylcholine in the cortex. The studies presented here, using the 2-deoxyglucose technique, demonstrate that the unilateral removal of a digit in cats, followed by stimulation of an adjacent digit, produces a pattern of metabolic activity in the somatosensory cortex that is dramatically expanded when compared with the opposite (normal) hemisphere. In contrast, experiments in which the somatosensory cortex was depleted of acetylcholine and the animal received a similar amputation led not to patterns of expanded metabolic activity, but rather to reductions in the evoked metabolic distribution. These studies implicate acetylcholine in normal map formation and in the maintenance of the capacity of cortical maps to adapt to changes in the periphery.

MATERIALS AND METHODS Seven adult female cats were anesthetized with halothane anesthesia (1-2%) and received a unilateral amputation of digit 2 on the forepaw at the metacarpal phalangeal joint under aseptic conditions. The animals were divided into two groups: the first group of three cats received a digit amputation (DA) alone; the second group of three cats received the DA plus a unilateral lesion ofthe contralateral basal forebrain (BF) that depleted the neocortex of ACh (Fig. 1). The lesions were made according to published protocols (3). Briefly, N-methyl-DL-aspartic acid (5% dissolved in sodium phosphate buffer at pH 7.0) was injected with a Hamilton syringe into an opening made in the skull at two sets of coordinates: (i) 15.0 mm A, 4.0 mm L, and 7.0 mm D (a total of 5 pl); (ii) 16 mm A, 7 mm L, and 8.0 mm D (a total of 3 pl). Regions affected by the neurotoxin included the horizontal and vertical limbs of the nucleus of the diagonal band of Broca, the substantia innominata, and ventrolateral portions of the globus pallidus. An example of a typical lesion site is shown in Fig. la. The lesions did not extend into the thalamus; examination of the ventrobasal nuclei of the thalamus indicated no differences in cell structure, density, or size in the lesioned versus normal hemispheres. Depletion of ACh in the somatosensory cortex was ascertained by acetylcholinesterase (AChE) histochemistry (17, 18) (Fig. 1 b and c). Although AChE histochemistry does not directly measure the presence of ACh, this procedure can adequately demonstrate ACh depletion (e.g., see ref. 19). The amount of depletion was quantified by counting the AChE-positive fibers in the normal and depleted hemispheres. A grid of 100 over three sites in layers II-IV in a vertical ILM2 wasandplaced the number of fibers was tallied. In a seventh array, animal, the BF lesion did not lead to ACh depletion and

Several lines of evidence suggest that the cholinergic projection from the basal forebrain plays an important role in cortical physiology and plasticity. For example, in 1986, Bear and Singer (1) demonstrated that depletion of cortical acetylcholine (ACh) following lesions of the basal forebrain disrupts ocular dominance plasticity in kitten area 17. Interestingly, this effect required the concurrent depletion of cortical norepinephrine. Cortical ACh depletion has also been shown to cause a reduction in the cortical response to sensory stimulation, measured both electrophysiologically (2) and metabolically (3, 4). For example, Sato et al. (2) found that lesions of the basal forebrain, which depleted the visual cortex of ACh, led to neuronal responses in visual cortex that were sluggish and depressed. Previous studies in cat somatosensory cortex found that ACh depletion by basal forebrain lesions, or by the pharmacologic antagonism of ACh through topical applications of atropine, caused the stimulus-evoked metabolic pattern to be reduced in dimension and intensity in the hemisphere ipsilateral to the depletion or application of atropine (3). On the other hand, iontophoretic application of exogenous ACh can augment the responses to peripheral stimulation in visual, somatosensory, and auditory cortex (5-11). In these sensory cortical regions, the pairing of ACh with appropriate stimuli usually enhances cortical responses. In some cases, the augmentation substantially outlasts presentation of the stimulus, causing a long-lasting potentiation of neuronal responsivity (11-14). In the somatosensory cortex, topographic maps of the body surface appear capable of remodeling and demonstrating plastic changes throughout adulthood. Studies that evaluate the cortical response to various peripheral manipula-

Abbreviations: DA, digit amputation; BF, basal forebrain; 2DG, 2-deoxyglucose; NE, norepinephrine; ACh, acetylcholine; AChE, acetylcholinesterase. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 780

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Proc. Natl. Acad. Sci. USA 88 (1991)

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FIG. 1. (a) Camera lucida drawings of a typical BF lesion. Regions affected by the neurotoxin included the nucleus of the diagonal band of Broca (dbv, dbh), the substantia innominata (Si), and ventrolateral portions of the globus pallidus (GPU). (b) Normal disposition of AChE' fibers in somatosensory cortex as demonstrated by AChE histochemistry. (c) AChE' fibers found in the hemisphere ipsilateral to the BF lesion. Cortical layers are indicated with roman numerals. CA, caudate nucleus; PU, putamen; ic, internal capsule; ac, anterior commissure; VA, ventral anterior nucleus; OT, optic tract. [Bar (for b and c) = 200 u.m.]

served as a control for the effects of surgery and/or lesion alone. Two additional animals received unilateral BF lesions and no DAs. For all animals with ACh depletion, the tabulations of AChE fibers indicated at least a 73% reduction of AChE-positive fibers in the lesioned hemisphere. Each animal was injected 2, 4, or 7 weeks after surgery with 2-deoxy-D-[1-14C]glucose (2DG) (10 pCi/100 g; 50-60 mCi/ mmol; 1 Ci = 37 GBq) and received a somatic stimulus delivered equally to both forepaws. The stimulus consisted of intermittent vertical displacements (15 Hz; amplitude, 0.25 mm) to the ventral surface of digit 3 (i.e., the digit adjacent to the amputation). After 45 min of stimulation, each animal received an overdose of pentobarbital and was processed according to published protocols (20-22). An additional animal was not operated on before the 2DG experiment and received equal stimulation to digit 3 bilaterally. Digitized maps of metabolic activity were constructed through the somatosensory cortex of both hemispheres (Fig. 2 a and b). The maps were made with a video-based image analysis system (20). Autoradiographs were digitized for their optical density values, which were converted to 14C mea-

surements. The outer and inner boundaries of the somatosensory cortex were then outlined with a digitizing tablet. The outer boundary corresponded to the location of layer I and the inner boundary corresponded to the border between layer VI and the white matter. Specific morphologic features were also entered for use as points of reference (e.g., fundus of the coronal sulcus). The average 14C value in the tangential dimension was determined for each autoradiograph by obtaining the average optical density value across layers 11-V.

The bin width for each tangential value is variable but measures =50 ,um. The resulting tangential histogram from each section is displayed as a vertical line of pixels on the video monitor; the bin height, which reflects the "'C concentration, is represented by pixel intensity. The lines are stacked horizontally and are aligned with respect to a selected point of reference. The normal hemisphere served as an internal control for the treated hemisphere (i.e., digit amputation or digit amputation plus BF lesion). To facilitate the comparison between normal and treated hemispheres, both hemispheres were cut together during the sectioning of the brains. Therefore, both halves of the brain were treated

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Proc. Natl. Acad. Sci. USA 88 (1991)

FIG. 2. Digitized maps of activity in a normal hemisphere (a) and in the hemisphere contralateral to amputation of digit 2 (b). Medial is up, and rostral is to the left. Evident in a is the normal pattern of 2DG uptake after stimulation to digit 3: a broken strip of activity running in a rostrocaudal direction. The scale was expanded in the rostrocaudal dimension to facilitate visualization of details. In the hemisphere contralateral to DA (b), the strip of activity is more extensive and tends to fill in the broken region on the normal side. (c and 6) Individual autoradiographs that produced the maps shown above. (c) Activity evoked in the normal hemisphere, seen as a single patch of 2DG uptake (arrow). (d) Multiple patches of activity (arrows) evoked in the hemisphere contralateral to the DA. The position ofeach section on the unfolded maps is also indicated with arrows. Autoradiographs were photographed directly from the film and are not digitized. CS, fundus of the coronal sulcus. (Bar = 1 mm.)

equally in all phases of tissue processing and data analysis, including digitization and image analysis.

RESULTS In experiments involving DA alone, the digitized maps demonstrate that, as in previous studies (3, 20), stimulation to a single digit produces individual patches of 2DG uptake that form strips of increased activity. When the cortex is "unfolded" and viewed in two dimensions, the strips of activity extend through the somatosensory cortex running roughly rostrocaudally. In the somatosensory cortex contralateral to the DA, stimulation to digit 3 causes the metabolic pattern to be substantially expanded 2, 4, or 7 weeks after surgery (Table 1). In all of these cases, the size of the pattern contralateral to the DA is at least twice that of the normal distribution. Evaluation of individual autoradiographic sections indicates that in the hemisphere contralateral to DA, evoked activity occurs in patches residing in the same cortical laminae as the patches of activity found in the normal hemisphere (Fig. 2 c and d). The density values of individual patches were obtained by expressing the density in the patch as a percent above the optical density values of the white matter. Patches were identified as sites that corresponded to increased metabolic activity levels at least 50% above white matter values. There were no appreciable differences from side to side in the amount of 2DG uptake (as measured by optical density) or within the individual patches. The mean patch width for all animals was 694 um on the left (normal) and 629 tm on the right (contralateral to DA); these values are not statistically different. The individual patches in the DA animals were approximately the same size as in the normal animals, but they were more numerous. Therefore, it appears that deafferentation by DA leads to expansion in the representation of body parts as revealed by maps of metabolic uptake.

In experiments in which the animals received DA plus a lesion of the BF, the hemisphere receiving input from the normal forepaw displayed the usual strip-like, stimulusevoked activity (Fig. 3a). In the ACh-depleted hemisphere contralateral to the DA, the labeling pattern differed from that in the animals with DA alone. The ACh-depleted animals displayed activity that was reduced in dimension, by at least 40%, from the opposite, control hemisphere (Fig. 3b; Table 1). The reduction can also be seen in the individual sections that produced the metabolic maps. The BF-lesioned hemisphere demonstrates individual patches of 2DG uptake of similar dimension and density to those on the normal side (Fig. 3 c and d), but they were less numerous. Therefore, the distribution of activity evoked in the ACh-depleted hemisphere did not demonstrate the expanded pattern evident in the metabolic maps seen after DA alone. In fact, this pattern was similar to the distribution of evoked activity seen in a previous study ofanimals receiving unilateral BF lesions with no DA. In the earlier experiments, the metabolic activity evoked in the ACh-depleted hemisphere was reduced in dimension and intensity (3). Information from one such animal is included in Table 1, which demonstrates that the BF lesion alone leads to measurements similar to those of animals with BF lesions plus DA. For the animal in which the lesion was misplaced slightly lateral and posterior to the intended location, and did not deplete the somatosensory cortex of ACh, the metabolic pattern was not reduced, as in those hemispheres with a BF lesion. In this animal, the 2DG uptake on the side of the lesion was expanded to 1.69 times the normal pattern, indicating that the surgical procedure alone does not produce a reduction in metabolic activity (Table 1, Control lesion). An additional experiment conducted on a normal animal receiving the same stimulus to each forepaw indicated that the

Neurobiology: Juliano et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

Table 1. Areas of evoked metabolic uptake obtained from digitized maps Animal L R R/L DA CJE 18

2.30

4.85*

2.11

3.65

8.60*

2.36

2.57

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3.42

1.68

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0.48

3.36

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0.97

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0.52

(2-wk survival) CJE 13

(4-wk survival) CJE 24

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(4-wk survival) CJE 26 (4-wk survival) CJE 32

(4-wk survival)

Control lesion

CJE 19

(4-wk survival)

2.21

Presented are areas of metabolic uptake, measured from digitized maps and evoked by stimulation to digit 3. Areas are expressed in mm2 and represent regions of increased activity at least 50% above white matter values, averaged across cortical layers 1I-V. Values for both hemispheres are indicated: left (L) and right (R) (the left hemisphere is always the normal hemisphere) and the ratio of the right, treated, hemisphere to the left hemisphere (R/L) is also shown. Five categories of experiment are shown: DA, animals with a unilateral DA; DA + BF lesion, animals with a unilateral DA plus a contralateral lesion to the BF that depleted the somatosensory cortex of ACh; Control lesion, animal with a BF lesion that did not produce ACh depletion; Normal, animal with no prior surgery; BF lesion, animal that had a BF lesion, but no DA. *Contralateral DA. tContralateral DA/ACh depleted. tContralateral DA/no depletion.

§ACh depleted. stimulus-evoked metabolic patterns of each hemisphere are of nearly identical size (Table 1, Normal). The possibility that the BF lesion caused a global reduction in 2DG uptake, rather than the activity levels being specifically related to somatic stimulation, was investigated in two ways. (i) Activity levels were measured in regions of somatosensory cortex that did not receive specific somatic stimulation. Background values were measured by expressing regions of somatosensory cortex that were not specifically stimulated as a percent of white matter values. There were no differences in the background activity levels between hemispheres. (ii) We also evaluated the density and distribution of cytochrome oxidase activity in sections adjacent to the 2DG autoradiographs. There were no changes in this measure of metabolic activity when comparing the normal and AChdepleted hemispheres. Therefore, in the animals depleted of ACh, the activity evoked by somatic stimulation was reduced, whereas global levels of background activity were not changed from the opposite hemisphere.

DISCUSSION These studies provide direct evidence for the rearrangement of maps in the neocortex of adult animals. Although previous studies identified the necessity of cholinergic (and noradrenergic) innervation for plasticity to occur in neonates (1), the present results suggest that cholinergic innervation is a re-

783

quirement for such changes in adults as well. Numerous examples suggest the significance of ACh in the normal functioning of sensory cortical regions in adults, since cholinergic depletion of these areas results in alterations of neuronal properties in response to stimulation (2-4). It now appears that ACh is an important factor in the evolution of the reorganizations of topographic maps that have been observed in the somatosensory cortex of adult animals. It is true that we cannot automatically assume a congruency between maps derived electrophysiologically and those obtained with metabolic mapping; they do not necessarily demonstrate equal phenomena. The correspondence between the changes observed in the present study, however, and those found electrophysiologically in previous studies after amputation/ deafferentation (15, 16) suggests that similar mechanisms cause the map changes in both experimental situations. The metabolic maps may therefore be a direct visualization of observations revealed during electrophysiologic experiments. In an earlier study by Bear and Singer (1), depletion of both ACh and norepinephrine (NE) seemed necessary to prevent ocular dominance changes in kittens. The reason why both noradrenergic and cholinergic depletion was not required to prevent the map expansions seen in the present experiments is not clear. One possibility is that there may be differences in the response of adult versus neonatal cortex. Young animals may be more susceptible to the influence of NE or less susceptible to ACh. Others (23) have demonstrated that neuronal responses to specific substances are more effective in young versus adult animals. It is also possible that somatosensory cortical areas may have different requirements from visual cortical areas. Another idea that may account for this discrepancy is that ACh and NE may work together to regulate levels of neuronal activity. Under certain circumstances, both substances may be necessary to achieve threshold levels of neural responsivity, while under other conditions, one neurotransmitter alone may be sufficient. An earlier study examining the somatosensory cortex of rats found that depletion of NE caused an expansion of stimulusevoked metabolic activity (24). A follow-up study revealed that depletion of NE was sufficient to prevent plastic changes normally present in the rat barrel field (25). The data presented here and in earlier studies indicate that, in the somatosensory cortex, ACh depletion results in a reduction of stimulus-evoked activity (3, 4). These results suggest that ACh and NE may work together in an as yet undetermined fashion to regulate activity levels in neocortex. It is also possible that our basal forebrain lesions (i) damaged other fiber systems that innervate the cortex or (ii) caused a reduction in overall cortical responsivity. On the first point, although we cannot unequivocably rule out the chance that other damage resulted from our lesions, it is not likely that the specific deficit found here would result from the loss of a different neurotransmitter. It is also unlikely that the thalamus was damaged since our histological examination of the ventrobasal complex indicated that the cytoarchitecture of this nucleus on the side of the N-methyl-DL-aspartic acid injection was not different from that of the opposite, unlesioned side. On the second point, our measurements of background cortical activity (both by 2DG uptake and by cytochrome oxidase activity) appear to indicate that normal, ongoing baseline activity levels occur in the BF lesioned hemispheres. Other studies also indicate that, after BF lesions, metabolic activity levels in cortex that is not specifically stimulated become normal after a period of several days (26, 27). Spontaneous neural activity was also found to be normal in BF lesioned rats (28). In the neocortex, ACh enhances neuronal responses by a voltage-sensitive mechanism that permits an increased response to a threshold level of incoming activity (e.g., stimulation) (29-33). In the ACh-depleted situation, the threshold

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level of activity may no longer be effective in enhancing a neuronal response, as the cellular mechanisms initiated by ACh do not occur. As a result, the cortical representation in response to stimulation is reduced. The dynamic capacity of cortical maps to adjust to their environment may therefore require the presence of specific substances; ACh may be one such substance in adults as well as neonates. This work was supported by Public Health Service Grant NS24014 (S.L.J.). 1. Bear, M. F. & Singer, W. (1986) Nature (London) 320, 172-176. 2. Sato, H., Hata, Y., Hagihara, K. & Tsumoto, T. (1987) J. Neurophysiol. 58, 781-794. 3. Juliano, S. L., Ma, W., Bear, M. & Eslin, D. E. (1990) J. Comp. Neurol. 2917, 106-120. 4. Ma, W., Hohmann, C. F., Coyle, J. T. & Juliano, S. L. (1989) J. Comp. Neurol. 288, 414-427. 5. Donoghue, J. P. & Carrol, K. L. (1987) Brain Res. 408, 367-371. 6. Sato, H., Hata, Y., Masui, H. & Tsumoto, T. (1987) J. Neurophysiol. 58, 765-780. 7. Sillito, A. M. & Murphy, P. C. (1987) in Cerebral Cortex, eds. Jones, E. G. & Peters, A. (Plenum, New York), Vol. 6, pp. 161-185. 8. McKenna, T. M., Ashe, J. H., Hui, G. K. & Weinberger, N. M. (1988) Synapse 2, 54-68. 9. Metherate, R., Tremblay, N. & Dykes, R. W. (1988) J. Neurophysiol. 59, 1231-1252. 10. Metherate, R. & Weinberger, N. M. (1989) Brain Res. 480, 372-377. 11. Sillito, A. M. & Kemp, J. A. (1983) Brain Res. 289, 143-155. 12. Metherate, R., Tremblay, N. & Dykes, R. W. (1987) Neuroscience 22,73-81.

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FIG. 3. (a and b) Digitized maps of stimulus-evoked activity from an animal that received a DA of digit 2 and a lesion of the BF that depleted the somatosensory cortex of ACh. Pattern in the normal hemisphere (a) appears as the usual strip-like distribution of activity after stimulation to digit 3. Pattern in b (contralateral to DA and ACh depleted) is reduced compared with the normal pattern and does not display the expansion evident in animals with DA alone. Conventions are the same as in Fig. 2. (c and d) Individual autoradiographs that produced the digitized maps shown above. (c) Activity evoked in the normal hemisphere, consisting of a patch of 2DG uptake evoked by stimulation to the contralateral digit 3. (d) Metabolic activity evoked in the ACh-depleted hemisphere, contralateral to DA of digit 2. The patches of activity appear similar on the two sides of the brain, in contrast to the activity elicited in the animal with DA alone (Fig. 2). These autoradiographs are photographed directly from the film and are not digitized. CS, fundus of the coronal sulcus. (Bar = 1 mm.)

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