Somatotopic Representation of Hindlimb Skin in Cat Dorsal Horn PAUL

B. BROWN

AND

JANNON

L. FUCHS

Neurological Unit, Boston State Hospital, and Department Harvard Medical School, Boston, Massachusetts 02115

investigations have shown detailed somatotopic organization of several regions of the somatosensory system. Wall (33) indicated that the somatotopic map of the dorsal horn of cats has a rostrocaudal and a mediolateral gradient of projection. He reported that distal hindlimb projected medially in the dorsal horn and proximal hindlimb projected laterally. Recently, Bryan et al. (5) confirmed the existence of a soma to topi c map for cells of origin of the spinocervical tract. They reported that embryologically ventral skin projected medially in the dorsal horn and embryologically dorsal skin projected laterallv. We have studied the somatotopy of the hindlimb region in more detail. Our data are adequate to treat the following questions: a) What is the somatotopic map of the dorsal horn? b) Are there multiple representations, as there are in the somatosensory cortex, suggesting functionally separate groups of cells? c) Are there segmental discontinuities, as there are in the dorsal columns (34)? d) How are the dorsal horn dermatomes related to the dorsal root dermatomes? e) What anatomical correlates exist for the somatotopic organization of the dorsal horn? NEUROPHYSIOLOGICAL

METHODS

Thirty-one adult cats of both sexes were used. A larger yield of single-unit data was obtained from males than from females, so males were selected for study in most of the later experiments. The animals were anesthetized with halothane and mounted in an animal holder. They were decerebrated at the midcollicular level and spinalized at T,,-L,. The anesthetic was then Received for publication

March 1, 1974.

of Neurology,

discontinued, artificial respiration was initiated, and paralysis was induced using gallamine triethiodide (Flaxedil, Davis and Geck). Further details of the surgical preparation have been presented elsewhere (32). The hair on the leg, hip, and tail was clipped, and in most cases a mild depilatory was applied. Recordings were made in the dorsal horn of segments L,-S,. The spinal segment to be explored was tentatively identified by anatomical criteria (11). The identification was then verified physiologically by placing the associated dorsal root on an Ag-AgCl hook electrode and determining the dermatome for light touch, which was compared with the schemes of Kuhn (14), Hekmatpanah (9), Ekholm (6), and Reid (21). For further verification the dermatomes of nearby dorsal roots were sometimes determined. Standard microelectrode-recording techniques were employed, using commercial stainless steel electrodes (Frederick Haer) with tip diameters less than 1 pm and exposed lengths less than 5 pm. A custom-made preamplifier (3) was used, which permitted deposition of ferric ions for histological determination of recording sites, using the method of Green (7). Isolated units were identified as postsynaptic neurons according to previously developed criteria (32). Only well-isolated single units were used in this study. Receptive fields (RFs) were outlined on the skin with indelible colored ink and drawn to scale at the end of the experiment. Electrode tracks were reconstructed from Nissl-stained serial paraffin sections cut at ZO30 pm from formalin-fixed material. Two marks were made in each track where more than one unit was isolated, allowing interpolation or extrapolation based on micrometer readings when necessary for localization of recording sites. In tracks where only one cell was isolated ions were usually deposited at the recording site. The boundary between laminae II and III could not be distinguished clearly, so these laminae 1

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2

P. B. BROWN

AND

J. L.

FUCHS

normal approximation to binomial distribution: N = 78, P = 0.002). In 48 of these 78 cases, we were able to distinguish clearly which RF of the pair was centered on skin which was embryologically more dorsal. The embryologically dorsal surface corresponds roughly to the lateral surface of the leg and the dorsal surface of the foot. In 71% of these cases, the RF of the more lateral neuron was closer to the dorsal axial line more dorsal): P = (i.e., embryologically 0.003. This is in good accord with Byran et al. (5). There was no clear organization RESULTS of the -ML axis in terms of pre--and postReceptive fields (RFs) of 253 dorsal horn axial representation. In only 7% of 37 cell units were mapped. Al though single-unit pairs the more medial cell had the more recordings were obtained in all six dorsal preaxial RF location (P = 0.26). The ML horn laminae, most were in laminae IVgradient also does not reflect the dermaVI. The distribution of histologically local- tomal trajectory, since the trajectory reized recording sites was: lamina I = 8 units; verses its proximodistal progressio’n around II and III = 19; IV = 56; V = 71; VI = 48. L,, whereas no reversal occurs in the ML Units in deeper laminae or white matter axis of the dorsal horn (Fig. 1). were not included in this study. A total Since there appeared to be no difference of 243 (96.0%) of the cells had continuous in RF positions for cells in the same track, RFs located entirely on the ipsilateral skin we tested for the presence of a “neutral surface. One (0.4%) had a two-part ipsilataxis” analogous to the columns reported era1 RF. Seven (2.8%) had RFs overlapping for SI cortex (19). Along an axis for which the midline of the body. Two (0.8%) units there is no somatotopic organization, the had strictly contralateral RFs. This last distance between the- RF centers for two class was probably underrepresented be- cells should be independent of the distance cause the contralateral body surface was between the cells. We tested all cell pairs, not always examined. within a given animal, differing in depth but having nearly the same AP and ML Somatotopic erg-anixation locations (AAP < 0.1 segment, AML < Figure 1 shows schematic dorsal views 0.05). Of the 43 cell pairs which met these of segments L5 through S1, with figurine criteria, 22 were found in the same lamina and 21 were in different laminae. The drawings of RFs located at the ML and AP coordinates where the units were re- Mann-Whitney U test revealed significantly corded. Where more than one cell was re- larger RF differences for the cells in differcorded in a track, their RFs are drawn on ent laminae than for cells in the same the same figurine. Each drawing is based on lamina (P = 0.003). However, we could data from two cats. not discern any consistent somatotopic graCells with RF centers on the proximal dient. Of the 38 cell pairs for which such a comparison could be made, 24 (63%) exlimb were generally located in the lateral dorsal horn, and neurons with distal RFs hibited a proximodistal shift with increastended to be located more medially. This is ing depth, and 14 (37%) displayed a rethe ML gradient of projection reported by verse shift (P = 0.07). In 20 of these 38 cell Wall (33). In order to determine how con- pairs, the RF of the deeper cell had the more preaxial location (P = 0.44). sistently this mapping principle applied, we examined all cell pairs recorded at the Figure 2 is a summary map of the somasame AP level within a given animal and totopic organization of segments LB-&. The differing in ML coordinate by at least 0.10. relative areas of tissue devoted to the variIn 69y0 of the cell pairs the more medial ous portions of the limb vary considerably neuron had the more distal RF (two-tailed from one segment to another, although most

were treated as a single category. Recording sites were tabulated according to a) anteroposterior (AP) coordinate (segment, number, and segmental coordinate, where anterior end = 0.0 and posterior end = 1.0) using marks placed at the ends of the dorsal root entry zone to indicate anterior and posterior limits; b) mediolateral (ML) coordinate (medial edge of dorsal horn = 0.0 and lateral edge of dorsal horn = 1.0); and c) according to laminar location (I-VI). Distances between cells could thus be calculated as the differences in their AP, ML, and laminar coordinates.

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HINDLIMB

MAP

OF

CAT

DORSAL

HORN

3

L6

Each drawing is a dorsal view of the left FIG. 1. Somatotopic organization in the horizontal plane. Figurine drawings represent single-unit RFs placed at the, AP dorsal horn: left = lateral; top = rostral. and ML coordinates corresponding to the recording locus of the cell. Each drawing is a composite from two animals.

of the limb is represented in each segment, from Lg. to S1. In L5 and S1, the foot is represented primarily in the most medial portion, whereas the foot and toe regions dominate the maps of L6 and L7 and the proximal limb is represented only in the most lateral portion of the dorsal horn in these segments. The total area of the dorsal horn in which RF centers fall within a

given unit area of skin is greatest for the toes and foot and least for the calf. Segmental organization Figure 3 illustrates the superimposed RFs of 30-50 neurons in each of the segments from L4 through S2. Uniform stippling was applied to each RF until sufficient stippling had been accumulated to indicate the full

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P. B. BROWN

4

\

Lateral

AND

J. L.

FUCHS

the dorsal horn, we tested for discontinuities along the AP axis at segmental boundaries. In the region of a disjunctive shift, the relation of inter-RF distance to intercell distance should be different from the relation obtained in a region with a smooth

Thigh

Toes Hin .:::: .:;:.. :,:_..’ %‘::;& ..:.< ‘.$. :, .

y;‘1 ,::;.. i .::,:. .:T$ :;; ::.. _. ..‘..‘..

.

L4 Perineum

L

I

Lat. -

2. organization plane. FIG.

Summary of the

Med.

1 1

t

._

L5

..:.:’ ...’ (’(”

diagram of the somatotopic dorsal horn in the horizontal

2 extent of the dorsal horn dermatome and the relative densities of representation of the different parts of the limb. The progression of segmental representation agrees with the dermatomal trajectory of the dorsal roots, as described by Werner and Whitsel (35) for the monkey. The trajectory from rostra1 to caudal segments proceeds from the hip and trunk (LV3, not shown, and L,), down the anterolateral leg to the anterior ankle (L5 and LG), across the dorsal foot to the medial toes (L7 and S,), and up the posterior leg (L7 and S,) to the perineum and tail (S, and S,). Each dorsal horn dermatome is similar in location and size to the dorsal root dermatome of the Same segment (6, 99 14-P 2 I>* However, quantitative comparisons cannot be made because of methodological differences. Gross electrode recordings were used in most of the dorsal root studies, and the contribution due to interanimal variation varies as a function of the number of animals studied. In the lumbar fasciculus gracilis, which provides most of the myelinated cutaneous input to the dorsal horn, discontinuities of representation are observed as an exploring microelectrode crosses successive bands of somatotopically organized fibers (34). Although our data are inadequate to test for similar discontinuities in the ML axis of

L6

l

c p::: y.: ,

Y

L7

,I:::;: y.:.:..! 8:: ..Z.’

s 1

ti

.:.:.:. :: :s :3 :..:.$&s. ::’ .:. v0 ‘/, “)

3

FIG. 3. Dorsal stippling indicates in our sample. superimposed.

horn dermatomes. Density of relative density of representation In each drawing, 30-50 RFs are

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HINDLIMB

MAP

OF

To obtain enough cell mapping function. pairs with similar ML positions (AML < O.lO), cell pairs consisting of units from different animals were included. We compared 94 such pairs within the middle half of every segment with 241 pairs straddling intersegmental boundaries and located within -F- 0.25 segment of the boundary. The two groups were compared using analysis of variance, with inter-RF distance as the dependent variable and intercell distance as the independent variable (F test). No significant differences were found in a) variances about the calculated regressions; b) slopes of regressions; or c) elevations of regressions (P > 0.25 for all three estimators). DISCUSSION

Somatotopic

organization

The topology of the tactile representation along the AP axis is largely determined by the dermatomes of the dorsal roots: each dorsal horn dermatome closely corresponds to the dermatome of its dorsal root. Previous descriptions of the mediolateral gradient as distoproximal (33) and as ventrodorsal (5) apply equally well to the map. The scale factor for the cutaneous projection varies with the RF position on the hindlimb. The foot and to& project to a disproportionately large area, as reported for other portions of the somatosensory CNS (e.g., 8, 12, 14-16, 22, 37-39). These relative enlargements of the distal limb projection areas have been suggested to mirror relative peripheral innervation densities. (number of afferent axons innervating a unit skin area). Quantitative data to demonstrate this are apparently lacking (26), althoug,h Wall (33) claims that innervation density is greater for distal than for proximal hindlimb skin. Some data compatible with this assumption are available. Werner and Whitsel (34) report that, in squirrel monkeys, the number of cutaneous afferent fibers in the fasciculus gracilis observed for different portions of the hindlimb were: hip-knee = 38; kneeankle-foot = 173 (calculated ankle = 59; from their Table 1). Some further data have been provided by L. M. and B. H. Pubols (personal communication). In stud-

CAT

DORSAL

HORN

5

ies of mechanoreceptive fibers innervating glabrous skin of raccoons and squirrel monkeys, they have encountered more units with RFs on the digits (242 in raccoons, 56 in monkeys) than on the palm (106 in raccoons, 44 in monkeys). Discounting possible sampling bias, these data are in accord with the generalization that distal skin innervation density exceeds that in proximal skin. The relative representation of a limb part in a dorsal horn segment may be correlated with its relative representation in that segment’s dorsal root, but there is insufficient data in the literature to test this hypothesis quantitatively. There appears to be a single representation of the hindlimb in the dorsal horn, as surmised by Bryan et al. (5), at least for all cells isolated with our microelectrodes. G. Werner has pointed out (personal communication) that our map is similar in its ’ organization of the hindlimb skin projection to that observed in SI cortex. This is most evident if Fig. 2 of this paper is compared with Werner and Whitsel’s Fig.- 7

(35).

Anatomical

correlations

OF DORSAL ROOTS. We can predict the segmental extent of projections from dorsal roots if some assumptions are made, based on an observation by Brown, Moraff, and Tapper (4): cells responding to single action potentials in type I afferents had no large regions of their RFs which were devoid of monosynaptic responses. If this finding can be generalized to all lowthreshold tactile input and to all dorsal horn cells responding to such input, then the following projection scheme applies: at least some afferents from a given skin region project to all areas of the dorsal horn representing that skin region. According to this projection scheme, if a dorsal horn dermatome overlaps a dorsal root dermatome, then that dorsal root projects monosynaptically to that dorsal horn segment. The fact that a dorsal horn dermatome is most similar to the dorsal root dermatome for the same segment suggests that the heaviest ,dorsal horn projection of a dorsal root is to the dorsal horn of its own segment. This is the case in all of thesanatomiPROJECTIONS

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6

P. B. BROWN

AND

cal degeneration studies we will review here (10, 17, 27-29). Table 1 shows agreement between the predicted projections of dorsal roots L4-S4 to dorsal horn segments L4-S2, and the observed distribution from degeneration studies. The observed AP distribution of dorsal root fibers (often over several segments) may reflect fiber sorting which distributes afferent collaterals to appropriate regions of the dorsal horn map. The findings of degeneration studies were also in accord with the ML organization of single units in the dorsal horn. When the L5 or LB dorsal root is cut (17, 27, 28), thus severing few fibers from the hip but many from the rest of the leg, there is much less degeneration in the lateral (hip) region of L7 than in the more medial regions. When dorsal roots S1 and S2 are cut, severing input from the hip as well as the leg, then substantial degeneration is found in lateral L7 dorsal horn (28). Sterling and Kuypers (29) reached similar conclusions from their degeneration study of the brachial cord: roots innervating distal and 1. Comparison of predicted and observed projections of dorsal roots L,-S, to dosal horn segments L4-S2

TABLE

Dorsal Horn Segment

Dorsal L,

L,

L4

+

L5

+

L6

+

L,

-

+

s,

-

+

s,

-

L,

+ -I+

+

+ +

-

-

?

+ +

?

?

-

-

+

? +

-

?

+

?

? +

+ +

?

? -

+

+ 3

?

3

+

?

+

+

-

?

-

+

+ -

?

+

+

S,

-

+ +

S,

?

+? +

+ +

?

+

+ +

S,

+ +

+

+ -I-

L,

+ ?

+

Root

+ +

-I-

+

Symbol in upper left of each matrix position indicates predicted projection or absence of projection, lower right symbol indicates observed projection or absence of projection. -I- , projection present; -, projection absent; ?, uncertain. Predictions are based on presence or absence of overlap of dorsal horn dermatome (present study) and dorsal root dermatome (14). Observations of projections are from degeneration studies (10, 17, 27, 28) in which dorsal roots were sectioned and terminal degeneration was examined,

J. L. FUCHS

postaxial forelimb distributed preferentially to the medial parts of laminae III and IV; roots innervating proximal and preaxial limb areas distributed more laterally. An anatomical indication of a ventrodorsal skin axis along the ML dorsal horn axis was reported by Szenthagothai and :Kiss (31). In both the trigeminal nerve and the CZ spinal nerve dorsal skin projected to lateral substantia gelatinosa (ventral, in the nucleus of V, which is rotated in its orientation). Ventral skin projected to medial substantia gelatinosa (dorsal, in the nucleus of v bur data are consistent with single-unit studies besides those of Wall (33) and Bryan et al. (5). Armett, Gray, and Palmer (1) noted that single units in the medial portion of LB dorsal horn responded to foot pad stimulation. On the basis of single-unit studies of the thoracic dorsal horn, Pomeranz, Wall, and Weber (18) concluded that dorsal skin projects laterally, and ventral skin projects medially in lamina IV. SORTING. Since there is considerable overlap among the dermatomes of the fiber bands in the fasciculus gracilis, fibers in different bands must frequently have similar RF locations, and therefore might project to similar terminal fields in dorsal horn. The absence of similar banding patterns in the degeneration of dorsal roots in the dorsal horn is compatible with a continuous (nonsegmental) ML somatotopic’ representation. A projection to a region without segmental &continuities inay-involve. sorting of fiber collaterals, similar to the sorting of fasbiculus gracilis fibers into a smooth somatotopic distribution by the time they reach cervical levels (36). An anatomical phenomenon has been described by Scheibel and Scheibel (23, 24) which may reflect ‘a sorting process of this kind: collaterals from different ML positions in the fasciculus gracilis of ten ag&re&ate in to “mi crobundles” as they Stream into the dorsal horn.

FIBER

TERMINAL

FIELDS

OF

b SINGLE

AFFERENTS.

From Fig. 2 it is evident that the projection of a small circular area *of skin -onto the dorsal horn map is several times longel” (in the rostrocaudal dimension).‘thafi it is wide (in the mediolateral dimension), if-the relative dimensions of ‘the drawing are taken

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HINDLIMB

MAP

OF

into account: the L,-S2 length is approxi-mately 30 mm, whereas the-dorsal horn is only about 1 mm wide. Golgi studies provide a possible anatomical correlate for such a distribution of primary afferent projections. The terminal arborizations of myelinated afferents are distributed in radial, or roughly sagittal, sheets (23-25). One afferent terminal field can be hundreds of microns long and only 20 pm wide (23), as we would expect from the dimensions of the dorsal horn map, if an individual afferent projected to most of the dorsal horn region where it is represented. These arborizations are found-predominantly in the substantia gelatinosa. Tightly coupled to these terminal arbors are similarly shaped dendritic trees of gelatinosal neurons, whose cell bodies are arranged in radial columns (20, 23-25, 30, 31). Undoubtedly the repeated collateralization of dorsal column afferents along their rostrocaudal curve further elongates their projection fields. In the absence of any detailed physiological data, Scheibel and Scheibel (23) formulated a first approximation of the process involved in the collection of afferent projections into postsynaptic RFs. They concluded that the shapes of afferent arbors which they had observed would result in a very precise segregation of projections from different skin areas in the ML axis of the dorsal horn and smearing of the body image on the AP axis, a conclusion which is supported by our data. They visualized the sagittal sheet structure of substantia gelatinosa neuropil as representing parallel strips of skin on the leg. Our mapping data suggests instead that each sagittal sheet probably represents a discrete, small area on the skin, which need not be elongated. BASIS FOR A NEUTRAL AXIS. A cell’s RF center should be determined by that portion of the substantia gelatinosa neuropil penetrated by its dendrites. Since the dorsal dendrites of laminae II-VI, and the lateral dendrites of lamina I, are roughly symmetrical about a dorso ventral line passing through the cell body, all cells directly under a point in the gelatinosa should have roughly similar RF centers. Therefore a roughly vertical (dorsoventral) orientation could constitute a “neutral ANATOMICAL

CAT

DORSAL

HORN

7

axis.” Since our “vertical cell pairs” (with similar AP and ML coordinates) may not exactly correspond to such a neutral axis, this may account for the statistical signs of variable RF position along the depth axis. SUMMARY

Single-unit exploration of the dorsal horn of segments L4-S2 of unanesthetized cats with the neuraxis transected at lower thoracic levels reveals a somototopic organization in the horizontal plane. The dorsal horn dermatomes correspond closely to the dermatomes of the corresponding dorsal roots, and the ML gradient is equally well described by two different projection schemes: a distoproximal gradient and a ventrodorsal one (5, 33). There is no evidence of segmental discontinuity of the map. As is the case in other nuclear regions of the CNS, the relative area devoted to projections from the foot is disproportionately large relative to the area devoted to skin regions of similar size which are located more proximally on the limb. From our data, and from the close correspondence to anatomical data obtained by others, we suggest that at least some cutaneous afferent fibers from a given skin area project directly to any dorsal horn region where that skin area is represented. This assumption, together with the organization of the dorsal horn map, yields a model which predicts a precise somatotopic organization of presynaptic neuropil in the substantia gelatinosa. ACKNOWLEDGMENTS

In addition to the personal communications cited in the text, we gratefully acknowledge the helpful comments of Drs. R. N. Bryan, M. D. Mann,

Somatotopic representation of hindlimb skin in cat dorsal horn.

Single-unit exploration of the dorsal horn of segments L4-S2 of unanesthetized cats with the neuraxis transected at lower thoracic levels reveals a so...
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