THE JOURNAL OF COMPARATIVE NEUROLOGY 314:237-256 (1991)

Somatotopic Organization of Hindlimb Skin Sensory Inputs to the Dorsal Horn of Hatchling Chicks (GaZZus g. domesticus) C. JEFFERY WOODBURY AND SHERYL A. SCOTT Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York 11794

ABSTRACT The somatotopic organization of skin sensory nerve projections to the lumbosacral dorsal horn of hatchling chickens was determined with the aid of transganglionic transport of horseradish peroxidase (HRP) processed with tetramethylbenzidine histochemistry. A total of eight hindlimb nerves were studied, five of which were purely cutaneous. When combined, the innervation fields of these nerves covered most of the hindlimb surface, allowing a nearly complete somatotopic map of the hindlimb to be generated. This report describes a novel pattern of cutaneous nerve projections to the dorsal horn. Unlike other vertebrates, cutaneous nerves of chickens formed two separate, somatotopically organized projections across the mediolateral axis of the dorsal horn; when serially reconstructed and superimposed, these projections produced two nonoverlapping somatotopic maps of the skin surface lying side by side. Each of these separate maps was nearly identical to the other in overall topology. These two separate maps appear to represent distinct modalities of sensory information, as projections composing the medial map were preferentially labeled by choleragenoid-HRP, whereas those composing the lateral map were preferentially labeled by wheat germ agglutinin-HRP. In mammals, these HRP ligands selectively label the central projections of myelinated and unmyelinated cutaneous afferents, respectively. The present study, therefore, strongly supports the cytoarchitectonic findings of Brinkman and Martin (Brain Res. 56:43-62, '73) that lamina I11 lies medial, rather than ventral, to lamina I1 in the chicken dorsal horn. Further, the present studies also suggest that laminae I1 and I11 of chickens are homologous to the homonymous laminae in the dorsal horn of mammals. Key words: somatosensory system, avian, spinal cord, cutaneous nerves, transganglionic HRP

transport

Cutaneous primary afferents project somatotopically across the horizontal plane of the dorsal horn (DH) of the spinal gray matter, so that the topological relationships of sensory fiber endings in the skin are retained through the pattern of their central projections. The somatotopic map of the skin surface that results has been particularly well characterized in the DH of mammals through both electrophysiological (Wall, '60; Pomeranz et al., '68; Bryan et al., '73; Brown and Fuchs, '75; Brown et al., '80; Light and Durkovic, '84; Schouenborg, '84; Wilson et al., '86; Pubols et al., '89) and neuroanatomical studies (Koerber and Brown, '80, '82; Fitzgerald and Swett, '83; Ygge and Grant, '83; Molander and Grant, '85, '86; Swett and Woolf, '85; Fitzgerald, '87; Woolf and Fitzgerald, '86; Florence et al., '89; Ritz et al., '89; Brown et al., '91). These studies have demonstrated that the DH projections of both myelinated and unmyelinated primary afferents are somatotopically organized. The somatotopic maps in the DH of mammals as O

1991 WILEY-LISS, INC.

diverse as carnivores and rodents are markedly similar (Molander and Grant, '85, '86; Swett and Woolf, '85; Pubols et al., '89), suggesting that the developmental mechanisms guiding their formation are shared among mammals and perhaps all tetrapods (e.g., Bryan et al., '73). The DH can be divided into six dorsoventrally stacked laminae on cytoarchitectonic grounds (e.g., mammals: Rexed, '52, '54; Martin and Fisher, '68; Marsh, '72; Ralston, '79; Molander et al., '84, '89; reptiles: Cruce, '79; Kusuma et al., '79; birds: Leonard and Cohen, '75a; but see Brinkman and Martin, '73; Martin, '79). That these cytoarchitectonically defined laminae approximate functional subdivisions of the DH has been well established through Accepted September 6, 1991 C. Jeffery Woodhury's present address is Department of Ornithology, American Museum of Natural History, 79th Street and Central Park West, New York, NY 10024.

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physiological studies in mammals (Wall, '67; Christensen and Perl, '70; Price and Mayer, '74; Menetrey et al., '77; Fitzgerald and Wall, '80; Light and Durkovic, '84; but see Cervero et al., '77; Woolf and Fitzgerald, '83; Cook et al., '87; see also Necker, '83, '85a,b, for pigeons). This is perhaps best illustrated by the finding that individually labeled, physiologically identified skin sensory afferents terminate within specific laminae in a modality-dependent manner (Brown et al., '77, '78; Light and Perl, '79b; Brown, '81; Sugiura et al., '86; Woolf, '87; Shortland et al., '89); in general, myelinated afferents, both large (Ap) and small diameter (AS), that innervate low-threshold mechanoreceptors terminate predominantly within laminae 111-IV(Brown et al., '77, '78; Light and Perl, '79b; Woolf, '87; Shortland et al., '891, with fewer additional AP terminations in laminae V andVI (Brown, '81; see also Culberson et al., '88; Sonty et al., '89). In contrast, A6 myelinated fibers that innervate high-threshold mechanoreceptors terminate within laminae I and V (Light and Perl, '79b). Further, unmyelinated (C) cutaneous afferents, most of which respond only to noxious stimuli (Bessou and Perl, '69; Beitel and Dubner, '76; Kumazawa and Perl, '77; Shea and Perl, ' 8 3 , terminate predominantly within laminae 1-11(Sugiura et al.,'86). Of the complete spectrum of cutaneous primary afferents, most appear to project somatotopically within their respective laminae (but see Light and Perl, '79b, for AS HTMRs). In marked contrast to mammals, few studies have addressed the anatomical and physiological organization of cutaneous inputs to the DH of birds (Leonard and Cohen, '75b; Holloway et al., '80; Necker, '83, '85a,b; Necker and Schermuly, '85; Wild, '851, despite the relative ease with which avian embryos can be experimentally manipulated to address developmental questions (e.g., Lance-Jones and Landmesser, '80; Le Douarin, '80; see also Davis et al., '89). As in mammals and reptiles, the DH of birds can be divided on cytoarchitectonic grounds into discrete laminae (Brinkman and Martin, '73; Leonard and Cohen, '75a; Martin, '79). The lamination pattern in the pigeon DH is similar to the majority of vertebrates (Rexed, '52; see also Kusuma et al., '79) in that laminae I-V lie in register in the dorsoventral plane (Leonard and Cohen, '75a). In contrast, the lamination pattern in the chicken DH is notably differentrather than being stacked dorsoventrally, superficial laminae in the chicken DH lie side by side, with lamina 111 medial, rather than ventral, to lamina I1 (Brinkman and Martin '73; Martin, '79; but see Leonard and Cohen, '75a). This pattern is supported by neuronal birthdating (Martin and Brinkman, '70; Martin, '72), degeneration (Lee and Martin, '71), immunohistochemical (Lavalley and Ho, '83; New and Mudge, '86; Du et al., '87; Du and Dubois, '881, lectin-binding (Scott et al., '89), and neuroanatomical studies (Ohmori et al., '87). The present report describes the somatotopic organization of the central projections of hindlimb cutaneous nerves in the DH of hatchling chickens on the basis of transganglionic transport of horseradish peroxidase (HRP). Here, a novel pattern of cutaneous central projections is described that results in the formation of two somatotopic maps of the hindlimb skin, lying side by side, across the perimeter of the superficial DH. These separate maps are composed of distinct classes of primary afferents as evidenced by differences in central labeling patterns of HRP-ligand conjugates (below) and electrophysiological properties of the two projections (Woodbury, '89, '92). Combined, these studies strongly support the unusual cytoarchitectonic lamination

C.J. WOODBURY AND S.A. SCOTT SSI

2

3

SYNSACRAL N E R V E S 4 5 6 7

8

910

Fig. 1. Cutaneous nerves of the chicken hindlimb. The innervation fields and central projections were studied for each of the eight nerves indicated. In rostrocaudal sequence, these are the N. cutaneous femoralis lateralis (CFL), the N. cutaneous femoralis medialis (CFM), the N. cutaneous suralis lateralis (CSL),the N. fibularis profundus (FPro), the N. fibularis superficialis (FSup), the N. parafibularis (PFib), the N. cutaneous suralis medialis (CSM), and the N. cutaneous femoralis caudalis (CFC). All except the three fibular branches (e.g., FPro, FSup, PFib) were purely cutaneous nerves.

scheme proposed by Brinkman and Martin ('731, and suggest that cutaneous afferents in chickens segregate to specific laminae in a modality-dependent manner similar to that in mammals. This work sets the stage for future developmental studies of somatotopy and lamina-specific terminations in an embryonic system that is extremely amenable to experimental manipulation. Some of these findings have been reported in abstract form (Woodbury, '87).

MATERIALS AND METHODS General Newly hatched white leghorn chicks (Gallus g. domesticus, 2-5 days posthatching) of either sex were used in these

studies. These were hatched from eggs (Westbrook Farms, Bohemia, NY) in a forced draft incubator maintained at 3840°C at 98% relative humidity. Prior to surgery, hatchlings were housed in brooders at the above temperature and given food and water ad libitum. Studies of the central projections of cutaneous nerves involved: 1) whole nerve electrophysiological recordings to determine the peripheral innervation field of each nerve; and 2) transganglionic transport of HRP to label the central projections of the same nerve. The hindlimb nerves studied are illustrated in Figure 1. In general, nomenclature follows

SOMATOTOPY OF CUTANEOUS INPUTS TO CHICKEN DH Nonina Anatomica Avium (Baumel et al., ’79); however, nomenclature for nerves not described in the above follows Appleton (’28).

Innervation field recording Hatchlings were anesthetized with subcutaneous injections of ketamine and xylazine (approximately 100 and 10 mg/kg, respectively). Birds were placed on a “hammock” formed from two parallel lengths of plastic tubing suspended above the table surface through which warm water (40°C) was circulated to maintain body temperature. The hindlimb was extended and stabilized, down feathers were clipped, and an incision was made in the skin under aseptic conditions to expose an underlying cutaneous nerve. Care was taken to keep the incision well outside the innervation field of the nerve (as determined in preliminary experiments). To expose nerve trunks innervating skin on the distal hindlimb, overlying muscle tissue occasionally had to be incised as well. The exposed nerve and muscle tissue was covered with avian Ringer’s solution (150 mM NaC1,3 mM KC1, 3 mM CaCl,, 1 mM MgCl,, 0.1 mM Hepes buffer, pH 7.4). The exposed nerve was freed of loose connective tissue and blood vessels and transected, and its distal cut end aspirated into a polyethylene suction electrode whose diameter closely matched that of the nerve. Evoked activity was amplified (Grass Instruments, model p15, bandwidth 0.1-10 kHz) and relayed through an audio monitor. To determine the location of the nerve’s cutaneous innervation field, the surface of the hindlimb was gently stimulated with a fine camel hair brush or single bristle to activate only low-threshold mechanoreceptors. The boundaries of the innervation fields were drawn on the skin with ink and subsequently redrawn onto a schematic of the hindlimb. In a few birds, multiple nerves (up to 5/bird) were recorded ipsilaterally to determine the amount of overlap between adjacent innervation fields; the latter birds were not included in subsequent studies of central projections. To check for motor fibers, brief trains of electrical stimuli were given to nerves in a few birds.

Central labeling After innervation field recording, the proximal cut end of the nerve was aspirated into a close-fitting polyethylene suction pipette. The distal tip of this pipette was glued onto exposed epineurium and surrounding muscle tissue with cyanoacrylate adhesive (Histoacryl). The pipette was severed to leave a “cuff’ enclosing the nerve, which was then filled with distilled water (e.g., Koerber and Brown, ’80). The water was changed 2-3 times in 15-20 minutes and replaced with 25% HRP (Boehringer-Mannheim) with 1% lysolecithin in distilled water (Frank et al., ’80);to facilitate exposure, the cut face of the nerve was teased apart with a tungsten needle after each fluid change. In a few experiments, wheat germ agglutinin (WGAI-HRPLO.01-20% (w/v) in distilled water, Sigma Chemicals, St. Louis, MO] or choleragenoid (B)-HRP LO.01-1% (w/v) in distilled water, List Biologicals, Campbell, CAI was used in place of native HRP. The open (distal) end of the cuff was capped with “5-minute’’ epoxy, the surgical field flooded with Ringer’s solution, and the incised skin closed with sutures. Precautions were taken during surgery to guard against HRP leakage. Thus, all cuffs were checked for capillary refilling through their base, and spillage of HRP solutions (if any) was rinsed thoroughly. While all cuffs were found to be intact at the end of the survival period, leakage was

239

verified histologically in one case by the presence of punctate labeling in motoneurons following the application of HRP to a purely cutaneous nerve. All cases in which leakage was verified or suspected were excluded from further study. In preliminary experiments, the central projections of labeled nerves were restricted almost exclusively to the ipsilateral DH. In a few nerves, a small number of labeled fibers crossed to the contralateral gray matter via the posterior commissure; however, the latter fibers were extremely sparse and restricted predominantly to deeper DH regions. Therefore, nerves were labeled bilaterally in some birds. After 12-96 hours [generally 48 for the cutaneous femoralis lateralis (CFL) and longer for more distal nerves], the birds were deeply anesthetized, heparinized, and perfused with Ringer’s solution followed by 100 ml of fixative [1% paraformaldehyde, 1.25%glutaraldehyde, 5% sucrose in 0.1 M phosphate buffer at pH 7.4 (PB),4”CI.The spinal column was removed and blocked into two to three pieces. Excess bone, cartilage, and meninges were removed, leaving the dorsal root ganglia (DRGs) attached. These spinal cord/ DRG pieces were embedded together in gelatidalbumin (30% albumin, 3% gelatin in distilled water), which was hardened in 4% paraformaldehyde, 20% sucrose in PB for 12-14 hours at 4°C. Serial frozen sections (50 pm) were collected in PB and washed 2 1hour at room temperature before histochemical processing. Axonally transported HRP was visualized by the tetramethyl benzidine (TMB) method of Mesulam (’78), with the stabilization step omitted (Mesulam and Brushart, ’79). Treated sections were washed in acetate buffer (pH 3.31, mounted from gelatin/alcohol mounting medium (0.1% gelatin, 40% ethanol) onto gelatidalum-coated slides and air-dried. Sections were then counterstained with 0.01% neutral red (pH 4.8) and dehydrated rapidly through graded ethanols.

Segmental boundaries The axial skeleton of birds exhibits pronounced interspecific variability in the numbers of vertebrae present (e.g., Baumel, ’79), as well as in the number of vertebrae contributing to the formation of the synsacrum. As consensus is lacking on the identity and homologies of synsacral vertebrae (e.g., Raikow, ’85), we have adopted the term synsacral (SS) to refer to all spinal nerves originating within this structure (Breazile and Yasuda, ’79; BubiefiWaluszewska, ’85). Hence, the subcostal nerve, formerly T7, originates within the synsacrum and is thus the first synsacral nerve (SS1, segment 22). The present study includes observations on the chicken DH between T4 and SS13 (T4-LS12, segments 14-33). The midpoint of each spinal cord segment was determined ipsilaterally for every labeled nerve by locating the median section of a transverse series through each dorsal root. For an experiment to be included in further analyses, the following criteria had to be satisfied: 1) heavily labeled neurons were contained within a subset of ipsilateral DRGs; 2) labeled fibers ascended the dorsal funiculus to the rostralmost tissue section, and ascended the dorsolateral funiculus (including Lissauer’s tract) for a variable number of segments; and 3) dense patches of label in the superficial DH did not fade away gradually but instead disappeared rather abruptly along a rostrocaudal series. To provide an estimate of the amount of interanimal variability, three

240

Fig. 2. Schematic transverse section of the chicken dorsal horn illustrates the method used to generate a dorsal view of a nerve’s central projections by “unrolling” the superficial dorsal horn (i.e., laminae I11 and 11); medial is to the left. The locations and widths of projections to each lamina (cross-hatching) were measured from camera lucida drawings with a digitizing tablet. Measurements followed an arc that traversed mediolaterally across the middle of each lamina (dotted line), beginning at the medial edge of lamina I11 (solid arrow) and ending at the “lateral” edge of lamina I1 (note the ventral position of the latter due to the parasagittal orientation of lamina 11). Such measurements were obtained from alternate serial sections throughout the rostrocaudal extent of a nerve’s central projection zone. These digitized measurements were normalized and plotted in serial order, aligning the medial and lateral borders of lamina 111 and I1 in vertical register. The projections to laminae 111 and I1 in individual sections therefore appear as a single, albeit interrupted, horizontal line in these dorsal views (see Fig. 11). DF, dorsal funiculus; DR, dorsal root; LF, lateral funiculus; solid arrow, medial edge of lamina 111; open arrow, inflection (lamina II/III border); double arrowheads, lateral edge of lamina 11.

C.J. WOODBURY AND S.A. SCOTT

3 v

3Y

Fig. 3. Peripheral innervation fields to light touch stimuli of the eight hindlimb nerves included in this study. Overlap between adjacent innervation fields is not represented. Refer to legend of Figure I for the names of these nerves.

Nerve innervation fields

Figures 1 and 3 represent the typical arrangement for hindlimb nerves and their peripheral innervation fields, respectively. The CFL and cutaneous femoralis medialis (CFM) are derived from the lumbar (or crural) plexus and innervated skin on the anterolateral thigh and anteromedial shank (crus), respectively. The remaining hindlimb skin was innervated by nerves derived from the sacral (or ischiadic) plexus; in rostrocaudal order, these were the N. cases satisfying the above criteria were obtained for each cutaneous suralis lateralis (CSL), N. fibularis profundus nerve, and analyzed as described below. (FPro), N. fibularis superficialis (FSup), N. parafibularis Sections were viewed at 1 0 0 with ~ brightfield and polar- (PFib), N. cutaneous suralis medialis (CSM),and N. cutaneized light microscopy and every fourth section was drawn ous femoralis caudalis (CFC). [Although not included in with the aid of a camera lucida. The location and mediolat- studies of central projections, the tibia1 nerve, when reera1 extent of labeling in laminae I1 and I11 (Brinkman and corded at midshank levels, also had a small cutaneous Martin, ’73; Martin, ’79) was then digitized from these innervation field on the rostromedial aspect of the ankle tracings, as described in Figure 2. Digitized data were (intertarsal) joint-not shown in Fig. 3.1 Because the purely normalized and serially plotted to generate a dorsal view cutaneous nerves that innervated foot and toe skin of map of the nerve’s central projection zone (e.g., Koerber hatchlings were extremely small and difficult to isolate, the and Brown, ’80, ’82). Normalization gave laminae I1 and I11 sensory innervation of this skin was studied by recording equal and constant widths; in actuality, however, the width from three larger mixed nerve trunks at midshank levels, of lamina I1 generally exceeded that of lamina 111, and the the FPro, FSup, PFib. These three were the only nerves widths of both laminae varied along the rostrocaudal axis. A studied that elicited visible muscle contractions in the summary (composite)somatotopic map of the hindlimb was hindlimb when stimulated electrically, and gave rise to produced by superimposing a representative dorsal view dense HRP labeling in motoneurons (below); all other from all eight nerves after each was first normalized in the nerves studied, therefore, were purely cutaneous. rostrocaudal axis. In general, the location, size, and shape of peripheral innervation fields were consistent from animal to animal. Further, a high degree of bilateral symmetry was seen RESULTS when innervation fields of homonymous nerves were The goal of these experiments was to describe the somato- mapped on both hindlimbs of an individual bird. The topic organization of the central projections of hindlimb innervation field of each nerve occupied an uninterrupted cutaneous nerves in the DH of hatchling chickens. Because region of skin, although some patchiness was occasionally nothing was known about the peripheral innervation fields evident along the border. Here, for example, light touch of of these nerves, the first step was to construct a map of their some feather follicles along the border of the innervation field elicited a response in the nerve even though the same tactile innervation fields on the hindlimb.

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SOMATOTOPY OF CUTANEOUS INPUTS TO CHICKEN DH TABLE 1. DRGs Containing Labeled Neurons Following HRP Application to Hindlimb Nerves Segmental level

T6

ss1

ss2

553

ss4

+ ++ ++ + + +

+++

+++ ++ +++

+ + ++ +++ ++ +++ ++ ++ + ++ +++

ss5

SS6

ss7

++

++ ++ ++ ++

SS8

ss9

SSIO

SSIl

~~

CFL

+

CFM CSL

+

FPro

+

FSup PFib

+

+ +

+++ +++

++ ++ ++ + + + + + + +

+ + + +

+++ +++ +++

+ +

+ + ++ + + + + +

CSM CFC

+ +

+

++ ++

++ ++ + + ++ ++ + + +

+ + + ++ ++ ++

++ ++

+++ +++

+++ ++

++ ++ ++

+++ +++ ++ ++ ++

+++ +++ +++ ++ t

+++ +++

+

+ ++ ++ ++ ++ +++ +++ +++ ++ ++ ++

+

+t

++

t

+ +

++ +

++ ++ ++ ++ ++

+++

+++ +++ ++

+++ +++

+ + + +

+ ++

+ + ++ ++

++ +++ +++

+++

+ ++

+ ++

+

'CFC, N. cutaneous femoralis caudalis; CFL, N. cutaneous fernoralis lateralis; CFM, N. cutaneous femoralis medialis; CSL, N. cutaneous suralis lateralis; CSM, N. cutaneous suralis medialis; FPro, N. fibularis profundus; FSup, N. fibularis superficialis; PFib, N. parafibularis; SS, synsacral; T, thoracic. +, Lightly labeled DRG s 10 neuronsiDRG (counted in alternate 50 km sections); + + , Moderately labeled DRG labeled neurons frequently restricted in location within the ganglion; + + +, Heavily labeled DRG: labeled neurons distributed throughout the ganglion

stimulus applied to an intervening follicle did not. While these borders thus appeared to be somewhat crenulated, this could also reflect an artifact of inadvertent nerve damage. While not illustrated in Figure 3, contiguous innervation fields overlapped slightly (1-3 mm) at their borders. This overlap was especially evident when the innervation fields of multiple nerves (up to 5 ) were mapped in the same leg, and appeared to be genuine as opposed to an interdigitation of patchy borders (above); in one case, for example, extremely light touch of a single feather follicle elicited a response in three different nerves. The locations of these innervation fields were correlated with the rostrocaudal position of their nerves within the plexes. In rostrocaudal order, innervation fields shifted from proximal to distal primarily along the preaxial surface of the hindlimb, followed by a distal to proximal shift primarily along the postaxial surface of the hindlimb, reflecting the segmental innervation of the hindlimb (Scott, '82).

Labeling in dorsal root ganglia The application of HRP to any particular cutaneous nerve labeled neuronal somata within a subset of ipsilateral dorsal root ganglia. In general, application of HRP to the same nerve in different animals consistently labeled the same subset, as seen in Table 1; further, certain DRGs of this subset were consistently more heavily labeled than others (i.e., contained more labeled neurons). As illustrated by Table 1, the segmental locations of a nerve's heavily labeled DRGs reflected the rostrocaudal position of that nerve within the plexus. The heavily labeled DRGs occupied consecutive spinal segments. However, following the application of HRP to some nerves, a small number of labeled neurons could be found in DRGs that extended a variable number of segments from the heavily labeled subset. These lightly labeled DRGs usually contained fewer than five labeled neurons (as counted in alternate sections) and were generally located rostra1 rather than caudal to the main subset. In some cases

(e.g., CSM, CFC), these lightly labeled DRGs were separated from the heavily labeled DRGs by unlabeled ganglia; however, discontinuous sets of labeled DRGs may be more apparent than real as only alternate tissue sections were examined. Most labeled neuronal somata in lightly labeled DRGs were filled with a dense TMB reaction product, comparable to that seen in somata from the main subset of labeled DRGs. Therefore, this widespread labeling of DRG neurons did not appear to result from the leakage and subsequent uptake of HRP by remote sensory fibers. All sizes of sensory neurons were found within the labeled subpopulation, and were generally intermixed throughout DRGs, as noted previously (e.g., Honig, '82). Although found throughout the DRG, however, large neurons appeared to outnumber smaller neurons along the ventrolateral perimeter of some ganglia, reminiscent of the condition in early development (e.g., Hamburger and LeviMontalcini, '49).In many cases, labeled neurons regardless of size were restricted in location within the DRG. For example, when SS1 (T7) had a significant contribution to the CFL, labeled neurons tended to be restricted to its caudal pole. When such segmental DRG polarity was seen in other cases, labeled neurons were similarly restricted to the caudal pole of the rostralmost DRG of the main subset, or vice versa. Further, additional clustering of labeled neurons was evident in either dorsal or ventral regions of the DRG in a few cases. Although detailed topographical studies of labeled neurons within DRGs were not conducted, these limited observations suggest that sensory ganglia may be both segmentally and somatotopicallyorganized, as suggested by others (e.g., Kausz and Rethelyi, '85).

Labeling in DH General observations. In transverse sections of the chicken spinal cord, a prominent inflection can be seen slightly medial to the apex of the DH. This inflection extends the length of the cord and is frequently the site where a large fiber bundle penetrates the DH. The superfi-

242

Fig. 4. Brightfield photomicrographs of the central projections of the CFL (A) and CFM (B)at the SS2 (LS1) midroot; midline is to the right. Note that the projections of the two nerves occupy different

CJ. WOODBURY AND S.A. SCOTT

portions of laminae I1 and 111. Solid arrow, medial edge of lamina 111; open arrow, inflection (lamina II/III border); double arrowheads, lateral edge of lamina 11. Scale bar = 200 pm.

cial DH is thus effectively divided into a large lateral and a lamina at certain segmental levels (e.g., Fig. 10). The smaller medial region, corresponding to laminae 1-11 and density of labeling was often highly variable within dif111, respectively, of Brinkman and Martin ('73; see also ferent regions of the CPZ, as described below. Martin, '79). Transganglionic transport of HRP revealed that cutaneCentral projections of individual nerves ous nerves formed two dense projections across the mediolateral axis of the superficial DH separated by the DH The somatotopic organization of the CPZs of individual inflection (e.g., Figs. 4,6, 7, lo), a medial projection within nerves is described below. Only projections to laminae I1 lamina I11 and a lateral projection within lamina 11. A and I11 are described, as somatotopy in other laminae was slightly more diffuse projection was also seen deep to and neither obvious (lamina I) nor easily determined (laminae centered between the projections in laminae I1 and 111, IV-V) . primarily within lamina IV but extending into lamina V as Rostra1 cutaneous nerves CFL. The CFL is the rostralmost hindlimb cutaneous well. Application of HRP to mixed nerves labeled additional projections, including dense terminations in the nucleus of nerve derived from the lumbar plexus and innervates skin the dorsolateral funiculus (nDLF; e.g., Fig. 7), as well as on the rostrolateral thigh and knee (Fig. 3).The CPZ of this more diffuse terminations medially in lamina VI and ven- nerve is divided rostrocaudally into two subzones in each trally in the intermediate zone of the gray matter (lamina lamina. The rostral subzone formed the primary CPZ, VII). In all nerves studied, dense projections were also seen occupying a lateral position between T4 and SS3 (T&LS2), in lamina I, although these were frequently not aligned as seen in Figure 4A. The caudal subzone was far less dense with the projections in lamina 11, especially for mixed and located laterally near SS7-8 (LS6-7), as seen in Figure 5 nerves (but see Fig. 7). Each of the dense projections to (see also Fig. 11). laminae I1 and I11 appeared to be composed of radially CFM. The CFM runs along the medial surface of the arranged patches (e.g., Figs. 4 , 6 , 7, 101,similar to previous thigh and innervates skin on the medial thigh and anteromeobservations in mammals (e.g., Ygge and Grant, '83). dial shank (Fig. 3). The CPZ of this nerve, like the CFL Serial reconstructions showed that these two separate (above), was divided rostrocaudally into two subzones in projections occupied corresponding locations in the medio- each lamina. The rostral subzone formed the primary CPZ lateral plane of laminae I1 and 111, respectively, and paral- and occupied a medial position in both laminae I1 and I11 leled one another throughout their entire rostrocaudal between T5 and SS4 (T5-LS3), as seen in Figure 4B (see extent, that is, any medial or lateral shift in one projection also Fig. 11).The caudal subzone was an extremely faint was mirrored by a similar shift in the other. In general, the projection near SS8 (LS7) (not shown in Fig. 11).Thus, projections to lamina I11 appeared to have a slightly greater although the CFM and CFL projected to similar segmental rostrocaudal extent than those to lamina 11. Projections to levels, their primary CPZs occupied mediolaterally separate lamina IV had the greatest rostrocaudal extent, usually regions of each lamina. This point is illustrated in Figure 4. extending one to two segments beyond the rostrocaudal CSL. The CSL is the rostralmost cutaneous nerve limits of lamina I11 projections; these far-ranging lamina IV derived from the sacral plexus (Fig. 1)and innervates skin on the lateral shank (Fig. 3). The CPZ of this nerve was projections, however, were generally sparse. While the peripheral innervation fields of the nerves divided rostrocaudally into two subzones in each lamina, studied occupied continuous areas of skin (above), the similar to the CFL and CFM (above). The rostral subzone central projection zones (CPZs) of many nerves were discon- formed the primary CPZ and occupied a lateral position in tinuous within each lamina. For example, many nerves each lamina between SS2 and 5 (LS1-4), slightly caudal to formed projections in widely disjunct locations, separated the projections of the CFL. As seen in Figure 6, the caudal rostrocaudally by up to five segments. Interestingly, a few subzone was formed by far less dense projections near SS8 nerves also formed two separate projections within each (LS7).

SOMATOTOPY OF CUTANEOUS INPUTS TO CHICKEN DH

243

ss I

T6

T5

Fig. 5. Camera lucida drawings through the central projection zone of the CFL; midline is to the left. Each drawing represents the midroot of the segment indicated. The primary projection zone lies between T5 and SS3 (LSZ), and an additional diffuse projection lies caudally near SS8 (LS7). In this and all subsequent figures showing camera lucida

drawings, the rostralmost section is at lower left and caudalmost section at upper right. Note also that the nucleus of the dorsolateral funiculus (arrows, T6-SS3) is devoid of labeling while surrounded by labeled fibers. Scale bar = 400 pm.

Foot and toe skin. As mentioned previously,the cutaneous nerve branches that innervate skin on the foot and toes of hatchlings are extremely delicate. We therefore chose to study the central projections of these afferents by applying HRP to their axons in the large nerve trunks (e.g., FPro, FSup, and PFib) at midshank levels. In all cases, the density of labeling in laminae I1 and I11 from these nerves

was less than that seen after labeling purely cutaneous nerves, presumably reflecting HRP dilution (e.g., longer transport distances). Projections in laminae I1 and 111were taken to represent the cutaneous components of the mixed nerves, but may overrepresent their actual cutaneous component as noncutaneous afferents might also project t o these laminae (see Discussion); at present, nothing is

244

Fig. 6 . Brightfield photomicrographs of the central projections of the CSL at the SS3 (LS2) midroot (A), and midway between SS7 and SS8 (LSG-7) (B);midline is to the right. Like the CFL and CFM, the CSL projects to two disjunct regions in the rostrocaudal axis. The

CJ. WOODBURY AND S.A. SCOTT

density of labeling in the primary projection zone (A) is far greater than that in the caudal projection zone (B). Solid arrow, medial edge of lamina 111; open arrow, inflection (lamina IIiIII border); double arrowheads, lateral edge of lamina 11. Scale bar = 200 km.

quickly, and formed only a minor projection along the medial edge of each lamina by SS6-7 (e.g., Fig. 11). FSup. The cutaneous component of the FSup innervates skin along the anterior ankle and tarsometatarsus, foot, and parts of toes 3 and 4 (Fig. 3). The CPZ of this nerve was undivided and located between SS3 and 7 (LS2-6) (e.g., Fig. 11). The projections occupied lateral positions in each lamina rostrally, but caudally they expanded medially to occupy nearly the entire extent of each lamina near SS6. Where the FSup projected medially in each lamina, these projections were caudal to those of the FPro. PFib. The cutaneous component of the PFib innervates skin along the posterolateral aspects of the tarsometatarsus, foot, and toe 4 (Fig. 3 ) . The CPZ of this nerve is divided into three separate subzones within each lamina (e.g., Fig. 11); rostrally, between SS3 and 4 (LS2-4), there are two mediolaterally separate projections in each lamina, whereas the primary CPZ is caudal to these two, forming a single Fig. 7. Brightfield photomicrograph of the central projection of the projection within each lamina between SS5 and 8 (LS4-7). FPro at the SS4 midroot; midline is to the right. Solid arrow, medial edge of lamina 111; open arrow, inflection (lamina IIIIII border); double This primary CPZ approaches the medial edge of each arrowheads, lateral edge of lamina 11. Projections to laminae I1 and 111 lamina caudal to the projections of both the FPro and FSup both lie medially within these laminae (lamina I1 is rotated, such that it (e.g., Fig. 11). is oriented parasagittally). Label ventral to the double arrowheads lies Caudal cutaneous nerves outside lamina 11. Note the dense projections to both the nucleus of the CSM. The CSM innervates an elongated, oval patch of dorsolateral funiculus (asterisk) and lamina I; the latter are aligned skin oriented proximodistally along the posterior aspect of radially with the wedge-shaped projection in lamina I1 (long arrow). Note further that a large, unlabeled fiber bundle penetrates the dorsal the shank (Fig. 3 ) . As seen in Figure 8, the CPZ of this nerve is again divided into three separate subzones within horn at the inflection in this section, thus separating the lateral edge of lamina 111 from the medial edge of lamina 11. Scale bar = 200 +m. each lamina. Rostrally, there are two mediolaterally separate subzones between SS3 and 4 (LS2-31, shown also in Figure 9. However, the primary CPZ, illustrated in Figure 8 known about the central projections of purely motor nerves (see also Fig. l l ) , is a single caudal subzone between SS7 and 9 (LS6-8). in chickens. CFC. The CFC arises in the sacral plexus caudal to the FPro. The cutaneous component of the FPro innervates skin on the medial tarsometatarsus and foot, including toes tibia1 nerve trunk and innervates a large area of skin 1, 2 , and the medial aspect of toe 3 (Fig. 3 ) . As seen in extending caudally from midthigh to nearly the cloaca, and Figure 7, the CPZ of this nerve was undivided and occupied ventrally to the abdominal midline. As shown in Figures 10 a position along the medial edge of each lamina between and 11, the CPZ of this nerve was divided into three SS4 and 7 (LS3-6). Between SS4 and 5, projections ex- separate subzones within each lamina, two mediolaterally panded laterally to occupy more than half of each lamina. separate rostral subzones between SS2 and 4 (LS1-3) (Fig. Further caudally, however, these projections tapered 10A) and a single large caudal subzone between SS8 and 12

245

SOMATOTOPY OF CUTANEOUS INPUTS TO CHICKEN DH

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Fig. 8. Camera lucida drawings through the central projection zone of the CSM, midline is to the left. The central projection zone of the CSM is split in the rostrocaudal axis. In the rostra1 zone (SS2-4, or LS1-3), two projections are formed in the mediolateral axis of each

lamina (e.g., SS314); in the caudal zone (SS7-9, or LS6-81, the projections lie at the medial edge of each lamina between SS8 and SS9 midroots. Scale bar = 400 km.

(LS7-11) (Figs. 10B-D). The latter is the primary CPZ of this nerve and it occupies nearly the entire mediolateral extent of each lamina near SS9 (LS8), as seen in Figure 1OC. The primary CPZ of this nerve is caudal to that of the CSM.

Intermittence of central projections The relative density of labeling often varied dramatically between alternate serial sections, resulting in an apparent periodicity, or intermittence, in certain portions of the

246

Fig. 9. Camera lucida drawings through the rostra1 projection zone (SS2-4) of the CSM to show intermittence of central projections; midline is to the left. Series represented rostrocaudally from bottom to top, left to right; the first (lower left) in this series of alternate 50 pm

CPZs of most nerves studied, for any given nerve, these regions appeared to be consistent from animal to animal. Although not readily apparent in dorsal view maps (Fig. 111, an example of this intermittence can be seen in Figure 9, which illustrates the alternate waxing and waning of the two mediolaterallyseparate projections of the CSM seen within each lamina. This intermit-

CJ. WOODBURY AND S.A. SCOTT

sections was three sections caudal to the SS2 midroot (shown in Fig. 8). Note the alternate waxing and waning of each of the dual projections in each lamina throughout the rostrocaudal axis. Scale bar = 400 pm.

tence usually appeared to be most pronounced near CPZ borders or within smaller subzones (e.g.,Fig. 11).

Somatotopic map Dorsal views were superimposed to generate a somatotopic map of hindlimb skin, shown in Figure 12; for

247

SOMATOTOPY OF CUTANEOUS INPUTS TO CHICKEN DH

Fig. 10. Brightfield photomicrographs of the central projections of the CFC nerve at different segmental levels: midway between roots SS2 and 3 (A); at the SS8 midroot (B);at the SS9 midroot (C); and at the SSl0 midroot (D); midline is to the right in all sections. The central projection zone of the CFC is split in the rostrocaudal axis. Note the two mediolaterally separate projections in each lamina in the rostra1 projection zone (between SS2 and 3; A). In the primary projection zone, the projections are restricted laterally at the SS8 midroot (B), but

expand medially further caudally to occupy nearly the entire mediolateral axis of each lamina by the SS9 midroot ( C ) .Further caudally, these projections contract to occupy an intermediate region of each lamina by the SSlO midroot (D). In D, remnants of the formerly widespread projections can be seen at the medial edge of lamina 11. Solid arrow, medial edge of lamina 111;open arrow, inflection (lamina IIiIII border); double arrowheads, lateral edge of lamina 11. Scale bar = 200 km.

illustrative purposes, only dense labeling within the primary CPZs was represented. Further, fairly extensive central overlap appeared to occur in some regions of the map, especially between nerves with contiguous peripheral innervation fields (not shown in Fig. 12); however, the real overlap might actually be much less due to both variation in density as well as the intermittence of central projections within certain regions of the CPZs (above). Two separate somatotopic maps of the hindlimb skin across the superficial DH of chickens are clearly revealed by this composite summary. These two maps lie on either side of the inflection in the DH (open arrow in Fig. 121, the medial map in lamina I11 and the lateral map in lamina I1 of Brinkman and Martin ('73) and Martin ('79). Further, each of these maps is markedly similar in overall topology to the maps of the hindlimb in the DH of mammals (e.g., Koerber and Brown, '82; Molander and Grant, '86; see Discussion).

Fiber composition of the dual central projections If Brinkman and Martin's ('73) laminae I1 and I11 in chicks correspond to homonymous laminae in the DH of other vertebrates (e.g., Rexed, '52), one would expect that the two separate projections of cutaneous nerves revealed in the present studies represent the central projections of separate functional subsets of skin sensory afferents (e.g., Brown et al., '77, '78; Light and Perl, '79a,b; Sugiura et al., '86). In order to test this, choleragenoid (B)- and wheat germ agglutinin (WGA)-HRP conjugates were each applied separately to the CFL nerve. These markers have been shown in mammals to label preferentially the central projections of large-diameter myelinated and unmyelinated cutaneous afferents, respectively (Robertson and Arvidsson, '85; Robertson and Grant, '85; Swett and Woolf, '85; Fitzgerald, '87).

248

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111 is represented to the left of lamina 11,with the DH inflection (lamina 111-11 border) signified by the solid vertical line in the middle of each dorsal view. Segmental levels indicated to the right of each dorsal view represent dorsal root midpoints. The order of these cases from left to right corresponds to the order of cases from top to bottom in Table 1.

C.J. WOODBURY AND S.A. SCOTT

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Fig. 12. Somatotopic organization of the central projections of hindlimb nerves. This somatotopic map is a composite produced by superimposing representative dorsal views of the nerves used in this study. For illustrative purposes, only areas of dense projections within the primary central projection zones of each nerve were included. Further, overlap between projections from different nerves is not represented. Solid arrow, medial edge of lamina 111; open arrow, inflection (lamina II/III border); double arrowheads, lateral edge of lamina 11. Note the overall similarity of the separate maps in laminae 11 and 111. For abbreviations, see Figure 1legend.

In the chick, the pattern of DH labeling differed dramatically depending upon the HRP ligand used, as shown in Figure 13 (cf. Fig. 4A). Application of B-HRP to the CFL labeled fibers in the dorsal columns and gave rise to robust labeling in laminae I11 and IV (Fig. 13A); only faint labeling was found in the dorsolateral funiculus and appropriate region of lamina I1 (asterisk in Fig. 13A). In contrast, WGA-HRP applied to the same nerve labeled fibers in the dorsolateral funiculus and projections in lamina I1 (Fig. 13B), with only faint labeling in the dorsal columns and appropriate regions of laminae I11 and IV (asterisk in Fig. 13B). Curiously, WGA-HRP was a far less sensitive marker than B-HRP (note range of concentrations used in Materials and Methods). Nevertheless, the labeling patterns of these two HRP ligands were reciprocal, suggesting that the primary afferents that produce these two projections are largely from nonoverlapping subpopulations.

DISCUSSION Central projections and lamination The most striking finding of these studies was that application of HRP to cutaneous (and mixed) nerves in the chick labeled two separate somatotopically organized projec-

tions on opposite sides of a pronounced inflection in the DH. This is what one would expect if: 1) lamina I11 is medial rather than ventral to lamina 11, with the lamina 11-111 border marked by the DH inflection (Brinkman and Martin, '73; Martin, '79; but see Leonard and Cohen, '75a); and 2) these laminae in chickens are homologous to the homonymous laminae in mammals (e.g., Rexed, '52,'54). It has been well established in mammals that cutaneous afferent inputs are somatotopically organized in lamina I1 (Schouenborg, '84; Molander and Grant, '85; Swett and Woolf, '85; Molander and Grant, '86) and laminae 111-V (Koerber and Brown, '80, '82; Molander and Grant, '86; Woolf and Fitzgerald, '86). These presynaptic maps are stacked dorsoventrally (actually radially-see Koerber and Brown, '82), a consequence of the stacking pattern of their respective postsynaptic targets in laminae 11-V. Further, these presynaptic maps lie in register, that is, when HRP is applied to a cutaneous nerve in mammals, a single wedgeshaped projection is formed in the DH whose medial and lateral borders traverse laminar boundaries (Koerber and Brown, '80, '82; Molander and Grant, '86). This finding is in marked contrast to the dual projections in the DH of chicks, present from late embryonic stages (Woodbury, unpublished observations) through adulthood (Ohmori et al., '87; Woodbury, '89, '92). If laminae I1 and 111 of chickens are homologous to the homonymous laminae of mammals, one would predict that the two nonoverlapping maps in chickens represent distinct populations of skin sensory afferents. In mammals, myelinated fibers (both AD and AS) conveying information from low-threshold mechanoreceptors project almost exclusively into laminae 111-V, ventral to the lamina 11-111 border (Brown et al., '77, '78; Light and Perl, '79b; Brown, '81; but see Proshansky and Egger, '77; Light and Perl, '79b; (D-hairs); Woolf, '87; Shortland et al., '89). In marked contrast, unmyelinated (C) fibers, most of which are polymodal nociceptors (Bessou and Perl, '69; Kumazawa and Perl, '77; Shea and Perl, '85), project almost exclusively to lamina I and I1 (Sugiura et al., '86; Ralston and Ralston, '79). These distinct subpopulations of primary afferents can be labeled differentially by applying specific HRP ligands to cutaneous nerves. In particular, WGA-HRP preferentially labels projections to laminae I and I1 when applied to cutaneous afferents (Brushart and Mesulam, '80; Fitzgerald and Swett, '83; Molander and Grant, '85; Robertson and Arvidsson, '85; Robertson and Grant, '85; Swett and Woolf, '85; Fitzgerald, '87), whereas B-HRP preferentially labels projections to laminae I11 and IV (Robertson and Arvidsson, '85; Robertson and Grant, '85). In the present experiments, WGA-HRP and B-HRP produced distinct patterns of DH labeling, predominantly within laminae I1 and 111-IV,respectively. The laminar specificities of these HRP ligands in chickens are consistent with predictions based upon mammalian studies, suggesting that laminae I1 and I11 in chickens (Brinkman and Martin, '73) are homologs of laminae I1 and I11 in mammals (and pigeons) (e.g., Itexed, '52). This interpretation, of course, depends in part upon an assumption that WGA-HRP and B-HRP identify homologous afferent subpopulations in both chickens and mammals; recent physiological studies suggest that this is indeed the case (Woodbury, '89, '92). The present study, in conjunction with immunohistochemical, neuroanatomical, and physiological findings, suggests that the functional organization of the chicken DH may be similar overall to that of mammals (and pigeons,

251

SOMATOTOPY OF CUTANEOUS INPUTS TO CHICKEN DH

Fig. 13. Darkfield photomicrographs show the differential labeling of the dual central projections of the CFL by choleragenoid (B)-HRP (A), and wheat germ agglutinin (WGA)-HRP (B); midline is to the right for each section, taken near the SSl midroot. The labeling patterns of these two HRP ligands were reciprocal, that is, application of B-HRP to the cut end of the CFL preferentially labeled projections to laminae 111-IV,with only faint labeling of corresponding projections to lamina I1

(asterisk in A), whereas application of WGA-HRP to a transected CFL preferentially labeled projections in lamina I1 with only faint labeling of corresponding projections to lamina 111 (asterisk in B). Survival time was 48 hours in both cases. Solid (small) arrow, medial edge of lamina 111; open arrow, inflection (lamina II/III border); double arrowheads, lateral edge of lamina 11. Scale bar = 200 km.

e.g., Necker, '83, '85a,b), despite obvious differences in lamination patterns. For instance, substance P (Lavalley and Ho, '83; New and Mudge, '86; Du et al., '87; Du and Dubois, '88; Woodbury and Scott, unpublished observations), somatostatin (Lavalley and Ho, '83), and calcitonin gene-related peptide (New and Mudge, '86) are largely restricted in the chicken DH to laminae I and 11, similar to their localization in mammals (see review in Jessell and Dodd, '86) and that of substance P in pigeons (Davis and Cabot, '84); in the DRG, these neuropeptides are localized primarily within small diameter neurons (Barber et al., '79; Jessell and Dodd, '86; New and Mudge, '861, presumably C cells (e.g., Leah et al., '85). Lamina I1 (and small-diameter DRG neurons) of chickens and mammals also exhibit similar lectin-binding patterns (Mori, '86; Plenderleith et al., '88; Scott et al., '89), although this may reflect developmental as opposed to functional commonality (e.g., Dodd and Jessell, '86). In addition, the central projections of visceral nerves are also similar in chickens (Ohmori et al., '87) and mammals (e.g., Morgan et al., '81), and are found predominantly within lamina I and the base of the DH. Further, previous physiological studies of laminae IV-VI neurons of chickens revealed that most respond to inputs from low-threshold cutaneous mechanoreceptors (Holloway et al., '76, '80), similar to the response properties of neurons in homonymous laminae in the DH of mammals (Wall, '60, '67; Wagman and Price, '69; Willis et al., '73; Cervero et al., '77; Menetrey et al., '77; Light and Durkovic, '84) and pigeons (Necker, '83, '85a,b). Recent physiological studies of the segregation of cutaneous inputs to neurons in superficial DH laminae of adult chickens (Woodbury, '89, '92) provide additional support for a common organizational plan in the DH of birds and mammals.

In light of these findings, this side-by-side, as opposed to dorsoventral, arrangement of laminae I1 and I11 in chickens serves to illuminate earlier questions concerning subdivisions within the substantia gelatinosa of mammals (e.g., Szentagothai, '64; reviewed in Cervero and Iggo, '80; see also Martin and Brinkman, '70); the fact that lamina I1 and lamina I11 are both composed of "gelatinosal" neurons in chickens (Brinkman and Martin, '73; Martin, '79) underscores the separate and distinct nature of these two populations of DH neurons. The greater size of lamina I1 compared to lamina I11 in all species studied so far presumably reflects trophic interactions between gelatinosal neurons and their primary afferent inputs (see also Lee and Martin, '711, that is, lamina I1 is the primary recipient zone for unmyelinated cutaneous afferents, which generally outnumber myelinated afferents in cutaneous nerves (e.g., Ranson and Davenport, '31).

Somatotopic organization Innervation field topology. The topology of innervation fields of hindlimb nerves of chickens reported here is similar in most respects to the innervation patterns of mammalian hindlimbs (Koerber and Brown, '82; Swett and Woolf, '85); nerves of the lumbar plexus innervate rostra1 thigh and shank skin, and nerves of the sacral plexus innervate distal and caudal thigh skin (Fig. 3). Viewed according to the rostrocaudal position of their nerves within the plexes (or labeled DRG neurons-Table 1; see below), innervation fields generally followed an orderly progression along the hindlimb approximating that seen in the segmental innervation of the limb (Scott, '82; see also Dykes and Terzis, %1), that is, nerve innervation fields generally progressed proximodistally along the preaxial

252 surface of the hindlimb, and then distoproximally along the postaxial surface of the hindlimb, looping around the axial line of the hindlimb, as do dermatomes (Dykes and Terzis, '81; see also Scott, '82). Unlike dermatomes, however, only minor overlap occurred between adjacent innervation fields along common borders, as noted previously (e.g., Sherrington, 1898). The progression of innervation fields is perhaps most easily interpreted in light of the orientation of the embryonic hindlimb during the establishment of sensory innervation (see arguments by Patterson, 1889; Sherrington, 1893; see also Bryan et al., '73; Scott, '82). For example, when sensory fibers first contact hindlimb skin, presumptive toes 1-4 are arranged rostrocaudally (Scott, '82; see also Honig, '82). If tissues are innervated preferentially from nerves derived from the same segmental level (e.g., Cole et al., '68; see discussion in Patterson, 18891, the sensory innervation of toe 1 would be derived from more rostral segmental levels than that of caudal toes. During subsequent limb morphogenesis, distal hindlimb regions rotate about the axial line, bringing toe 1 medially and toe 4 laterally. As a result, the rostrocaudal segmental innervation now traverses the toes from medial (toe 1)to lateral (toe 4),the pattern seen in the present studies (e.g., Fig. 3). Labeling in dorsal root ganglia. Outside of the heavily labeled subset, DRGs at many segmental levels contained small numbers of labeled neurons following application of HRP to both cutaneous and mixed nerves. That mixed nerves exhibited the greatest segmental spread presumably reflects noncutaneous afferents (see also McLachan and Janig, '83). Widespread segmental input to cutaneous nerves, however, contrasts with the previous findings of far more restricted inputs (McLachan and Janig, '83; Molander and Grant, '86; see also Honig, '82; Scott, '82). One possible explanation for the present findings is that they reflect HRP leakage. However, where leakage was confirmed following application of HRP to a cutaneous nerve (cases not included), pale, punctate reaction product was found in a few motoneurons (and many more widely dispersed DRG neurons). In the cases reported here, however, most DRG neurons were filled with a dense reaction product, regardless of segmental location, arguing against leakage. Nevertheless, i f these scattered DRG neurons were both cutaneous and inadvertently labeled, their small numbers would appear to contribute little to the gross patterns of central projections in laminae I1 and I11 (e.g., relatively minor interanimal variation; Fig. 11). Alternatively, if all labeled DRG neurons in the present study had axons within the nerves cuffed, then the patterns of skin sensory innervation in chickens may be more diffuse than previously indicated (Honig, '82; Scott, '82; see also Smith, '83; Fitzgerald, '87 for mammals), with axons travelling great rostrocaudal distances within (and between) the hindlimb plexes before exiting via a cutaneous nerve; discrepancies between this and previous studies in chickens might be explained by incomplete labeling of DRGs (e.g., Scott, '82) as well as the use of a less sensitive chromagen (diaminobenzidineHonig, '82; Scott, '82). These questions warrant further study. Central projections of mixed nerves. The central projections of cutaneous and mixed nerves shared many similarities, although mixed nerves also projected to regions outside those of cutaneous nerves. One such region was the nucleus of the dorsolateral funiculus (nDLF), which re-

C.J. WOODBURY AND S.A. SCOTT ceived a particularly robust projection from mixed nerves (e.g., Fig. 7) and only sparse projections from purely cutaneous nerves; in many sections, the nDLF was visibly devoid of projections while surrounded by labeled cutaneous fibers in the dorsolateral funiculus (e.g., Fig. 5). The identity of the dense nDLF projections from mixed nerves is unknown. Although not mentioned in previous studies of visceral and mixed nerves of the pudendal plexus in chickens (Ohmori et al., '871, these dense nDLF projections are unlikely to be hindlimb-specific as the nDLF is well developed in thoracic levels as well. Interestingly, nDLF neuropil in chickens is labeled by antibodies to substance P (Du and Dubois, '881, as in pigeons (Davis and Cabot, '84) and mammals (e.g., Ljungdahl et al., '78; Barber et al., '79; Seybold and Elde, '80). If primary afferent in origin (e.g., Davis and Cabot, '84), substance P-positive projections to nDLF may reflect muscle or sympathetic afferents in light of the distinctions between cutaneous and mixed nerves reported here. In mammals, the nDLF has been suggested to be a caudal continuation of the nucleus of the spinocervical tract (nSCT) (Gwyn and Waldron, '69). Unlike nSCT neurons, however, nDLF neurons exhibit little cutaneous sensibility (Giesler et al., '79; Menetrey et al., '80; Menetrey and Besson, '81).Indeed, the mammalian nDLF and nSCT, if serial homologs, contribute to fundamentally distinct CNS pathways (Menetrey et al., '82, '83; Menetrey and Basbaum, '87; see also Willis and Coggeshall, '78). In pigeons (Necker, '89) and mammals (Menetrey et al., '83; see also Menetrey et al., '82; Menetrey and Basbaum, '87), the nDLF projects heavily to the brainstem reticular formation; similar data for chickens are lacking. The projections of mixed nerves to laminae I1 and I11 were interpreted to represent the cutaneous components of these nerves. Whether noncutaneous primary afferents also project to these laminae in chickens is unknown, although visceral afferents apparently do not (Ohmori et al., '87). In mammals, laminae I1 and I11 do not appear to receive significant inputs from noncutaneous sources, such as visceral (Kuo and DeGroat, '85; Morgan et al., '811, articular (Craig et al., '88), and muscle (Craig and Mense, '83; Abrahams and Swett, '86; but see Molander and Grant, '87; Mense et al., '81). This may also be true in pigeons (Wild, '85), that is, injections of WGA-HRP into muscle labeled projections that were restricted predominantly to lamina I, although diffuse, sporadic labeling was occasionally seen in lamina I1 (Wild, '85).Whether the latter arose from leakage of WGA-HRP onto overlying skin (see Molander and Grant, '87; Craig et al., '88) or actually signals a genuine difference between birds and mammals needs clarification. Dorsal horn somatotopy. Each of the separate hindlimb maps demonstrated in laminae I1 and 111 of chickens were markedly similar in overall somatotopy to the hindlimb maps described in the DH of mammals (Koerber and Brown, '82; Molander and Grant, '86;Woolf and Fitzgerald, '86). For example, projections from distal hindlimb skin occupied a disproportionate area in each map and were bounded rostrally and caudally by projections from preaxial and postaxial skin surfaces, respectively. Each map also exhibits a clear segmental organization, in that the rostrocaudal sequence of cutaneous nerves in the hindlimb plexes (alternatively, their constituent DRGs, e.g., Table 1) is mirrored by the relative rostrocaudal positions of their primary CPZs within the map. The toes are sequentially represented in the rostrocaudal axis along the medial edge of the map, from most rostral (toe 1) to most caudal (toe 41,

SOMATOTOPY OF CUTANEOUS INPUTS TO CHICKEN DH as defined by the embryonic axis of the limb (e.g., Sherrington, 1893). Further, each map also exhibits a distoproximal as well as ventrodorsal gradient across its mediolateral axis (e.g., Bryan et al., '73; Brown and Fuchs, '75). For example, preaxial nerves that innervate distal hindlimb skin map medially (e.g., CFM, FPro) while those innervating proximal hindlimb skin map laterally (e.g., CFL, CSL). In addition, nerves innervating skin on the embryologically ventral surface of the hindlimb (e.g., CFM, CSM) map medially to nerves innervating skin on the embryologically dorsal surface (e.g., CFL, CSL). Interestingly, the CFL of pigeons also maps laterally in the DH (Wild, '85); thus, in all major respects, the same rules appear to guide the formation of somatotopic maps in birds and mammals (Bryan et al., '73; Brown and Fuchs, '75). A surprising feature of many of the central projections of these nerves was their fragmentation, many consisting of two to three separate projections to widespread regions of each lamina (e.g., Fig. 11).Split central projections have also been seen in mammals (Koerber and Brown, '82; Swett and Woolf, '85; Molander and Grant, '86; Woolf and Fitzgerald, '86), although to a lesser extent and in fewer hindlimb nerves than in chickens. In the present studies, rostral nerves exhibiting split projections (e.g., CFL, CFM, CSL) showed a greater density of labeling in their rostral subzones than in their caudal subzones (e.g., Figs. 5,6). In contrast, caudal nerves exhibiting split projections (e.g., PFib, CSM, CFC) showed similar density of labeling in both rostral and caudal subzones (c.f. Figs. 8, 10). This disparity in labeling density could reflect an actual difference in the density of fibers projecting to the caudal projection zones of rostral nerves; alternatively, it could be an artifact of the survival time allotted for HRP transport, that is, HRP may be transported faster in ascending than in descending branches of primary afferents (e.g., Brown, '81). These fragmented central projections provide evidence of major discontinuities in the hindlimb maps (e.g., Wilson et al., '86),that is, at one or more locations in the innervation fields of these nerves, primary afferents that innervate contiguous regions of skin project to noncontiguous areas in the central map. That some nerves exhibited three separate subzones centrally (e.g., PFib, CSM, CFC) suggests that there is probably more than a single discontinuity in the hindlimb map. One such discontinuity appears to correspond to the axial line in dorsal thigh; for example, innervation fields of the CFL and CFC are contiguous in the thigh while their central projections are both discontinuous rostrocaudally (Figs. 11, 12-compare with Koerber and Brown, '80, '82). This discontinuous representation of thigh skin along the lateral edge of the map in chickens contrasts with previous findings in mammals (Brown and Fuchs, '75; Wilson et al., '86; but see Brown et al., '80). Further, Wilson et al. ('86)described a line of discontinuity along the posteromedial aspect of the cat hindlimb approximating the axial line of the embryologically ventral surface of the hindlimb. Determining whether a similar line of discontinuity exists in the chicken hindlimb will require additional information from finer level neuroanatomical and/or physiological mapping studies. The overall similarity between the somatotopic maps in lamina I1 and lamina I11 of chickens suggests that the innervation fields of A and C fibers within a nerve may also be similar to one another. An overall similarity in the somatotopic maps in laminae I1 and I11 was also seen in the rat by Woolf and Fitzgerald ('86). Noting slight discrepan-

253

cies between the two maps, however, these authors suggested that "the terminals of A and C afferents in a given cutaneous nerve are not arranged in simple vertical columns containing high- and low-threshold information from a given skin area but rather as horizontal sheets lying on top of each other" (p. 529). The present findings in chickens of two nonoverlapping maps, formed by different subsets of primary afferents, clearly underscore the distinct nature of these two afferent sheets. Further, the nonoverlapping nature of these two separate maps implies that their formation is autonomous. Thus, it is clear that positional, nearest neighbor information, suggested to play a role in the formation of CNS maps (reviewed in Udin and Fawcett, '88),cannot account entirely for the formation of two maps from just a single set of hindlimb nerves; additional factors, such as fiber type and/or modality (e.g., Dodd and Jessell, '861, must also play a role. Continued studies in chickens, therefore, are likely to reveal new insights into the developmental mechanisms producing laminar-specific terminations, and indeed, DH lamination itself. Interestingly, the pronounced inflection in the DH of chickens, evident in the illustrations of earlier students (e.g., Clarke, 1859; Steida, 1869; Kolliker, '02; Ram6n y Cajal, '09; Keenan, '29; Matsushita, '68), along with the dual projection of cutaneous nerves, actually characterizes the DH of many diverse avian species; the phylogenetic significance of these findings in birds is discussed elsewhere (Woodbury, in preparation).

ACKNOWLEDGMENTS This work was submitted by C.J.W. in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the State University of New York at Stony Brook, NY. Throughout these studies, much thoughtful advice was provided by Drs. P.B. Brown, J.B. Cabot, C.E. Evinger, and L.M. Mendell, to whom we wish to extend our most sincere appreciation. This work was supported by an NIMH Predoctoral Fellowship and Sigma Xi grant-in-aid-of-research to C.J.W., and grants from NIH (NS16067) and NSF (BSN8518927) to S.A. Scott.

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