The innervation of the mystacial pad of the rat as revealed by PGP 9.5 immunofluorescence

THE JOURNAL OF COMPARATIVE NEUROLOGY 3373366-38.5 (1993)

The Innervation of the Mystacial Pad of the Rat as Revealed by PGP 9.5

Immunofluorescence

FRANK L. RICE, ERIK KINNMAN, H h A N ALDSKOGIUS, OLLE JOHANSSON, AND JAN ARVIDSSON

Department of Anatomy, Cell Biology, and Neurobiology, Albany Medical College, Albany, New York 12208 (F.L.R.); Department of Anatomy, University of California, San Francisco, California 94143 (E.K.); Department of Anatomy (H.A., J.A.) and Experimental Dermatology

Unit, Department of Histology and Neurobiology (O.J.), Karolinska Institute, Stockholm, Sweden S-104 01

ABSTRACT The innervation of the mystacial pad in the rat was investigated with the aid of antihuman

protein gene product (PGP) 9.5 immunofluorescence. PGP 9.5 is ubiquitin carboxyl-terminal hydrolase, which is distributed throughout neuronal cytoplasm. This technique revealed all previously known innervation as well as a wide variety of small-caliber axons and some endings of large-caliber afferents that had not been observed before. Newly revealed innervation affiliated with vibrissal-follicle sinus complexes included 1) fine-caliber, radially oriented processes in the epidermal rete ridge collar; 2) a loose network of fine-caliber, circumferentially arrayed processes in the centrifugal part of the mesenchymal sheath at the level of the ring sinus; 3) a loose haphazard network of fine-caliber and medium-caliber processes in the mesenchymal sheath and among the trabeculae of the cavernous sinus; 4) a loose network of circumferentially arrayed processes within the mesenchymal sheath of the cavernous sinus and in close proximity to the basement membrane; 5 ) a dense network of reticular-like endings provided by large-caliber afferents to the mesenchymal sheath in the upper part of the cavernous sinus; and 6) fine-caliber innervation to the dermal papilla at the base of all vibrissal shafts. In the intervibrissal skin, a dense distribution of fine-caliber individual and clustered profiles was detected in the epidermis. In addition to previously known innervation, Merkel endings were consistently observed in the epidermis at the mouths of guard hairs, loose networks of fine-caliber axons were found around the necks of occasional guard hairs, and fine-caliber profiles were frequently affiliated with vellus hairs. Vascular profiles were heavily innervated throughout the dermis. Axons and motor end plates of the facial nerve innervation to papillary muscles also were labeled. Transection of the infraorbital nerve eliminated all but the facial nerve innervation. Unilateral removal of the superior cervical ganglion eliminated the innervation to the dermal papillae but caused no other noticeable reduction. PGP 9.5-like immunofluorescence was also moderately expressed in apparent Schwann cells, in Merkel cells only in the external root sheath of vibrissal follicles, and in apparent dendritic and/or Langerhans cells usually located in the epidermis and occasionally in the follicles. PGP 9.5-like immunofluorescence persisted in highly vacuolated profiles along the usual courses of medium to large-caliber axons 2 weeks after nerve transection. The possible functional role of the newly discovered innervation is considered along with that of previously identified afferents.

8 1 19!1:? Wiley-Lisa. Inr.

Key words: vibrissae, sensory endings, Merkel cells, dendritic cells

Accepted June 18.1993 Address reprint requests to Dr Frank L. Rice, Department of Anatomy.

Cell Biology, and Neurobiology. Albany Medical College, 47 New Scotland Avenue. Albany, NY 12208

c 1993 WILEY-LISS, INC.

IMMCNOFLUORESCENCE OF RAT MYSTACIAL PAD 367

The trigeminal system in the rat has become one of the most important models for studying the structure, function, development, and plasticity of the mammalian somatosensory system (see Simons and Carvell, '89; Lichtenstein et al., '90; Killackey et al., '90; Rhoades et al., '90; Woolsey, '90; Snow and Wilson, '91; Diamond et al., '92 for recent articles and reviews). Of particular interest, the mystacial pad on the snout of the rat is endowed with rows of long, densely innervated tactile hairs referred to as vibrissae (Vincent, '13; Andres, '66; Rice et al., '86a), which have morphologically distinct barrel or columnar-like correlates in the brainstem, thalamus, and cerebral cortex (Welker and Woolsey, '74; Belford and Killackey, '79; Killackey and Belford, '79; Arvidsson, '82; Rice, '85; Sugitani et al., '90; Ma, '91 ). Although considerable attention has been focused on the central connectivity, the innervation of the mystacial pad at the peripheral end of the system has still only been partially elucidated.

Each vibrissa emanates from a large spindle-shaped structure (Fig. l), which has recently been designated the follicle-sinus complex (F-SC) (Rice et a1.,'86a). This designa- tion recognizes the dual composition consisting of the epidermally derived follicle at the core, which is surrounded by a dermally derived, encapsulated blood sinus (Vincent, '13; Melaragno and Montagna, '53; Rice et al., '86a). Components of both tissue derivatives are innervated. Between the rows of F-SCs, numerous small guard hairs and smaller vellus or down hairs arise from simple hair follicles.

The vibrissal F-SCs and the intervibrissal fur receive a dense sensory innervation from the infraorbital branch of the maxillary division of the trigeminal nerve (Tello, '23; Dorfl, '85; Renehan and Munger, '86; Rice et al., '86a; Rice and Munger, '86). Each F-SC is also looped by a sling-like papillary muscle, which is innervated by branches of the facial nerve (Dorfl, '82, '85). The papillary muscles protract the vibrissa, which in some species can occur rapidly and rythmically during an exploratory behavior referred to as whisking (Welker, '64; Wineski, '85; Carvell et al., '911.

Previous assessments of mystacial pad innervation have used reduced silver light microscopic techniques and electron microscopy (Tello, '23; Andres, '66; Patrizzi and Munger, '66; Yohro, '77; Biemesderfer et al., '78; Renehan and Munger, '86; Rice et al., '86a; Rice and Munger, '86). These procedures have revealed several types of sensory endings with highly specific distributions within vibrissal F-SCs and intervibrissal follicles. However, a set of branched lanceolate endings was described in only one of the EM studies of rat vibrissae (Andres, '66) and was not observed by light microscopy (Rice et al. '86a). Also, examination of terminal nerve fascicles in semithin sections revealed far more innervation by A6 and C fibers than was seen by reduced-silver techniques or EM (Klein et al., '88).

Recently, immunocytochemistry with a polyclonal antibody against human neuronal protein gene product (PGP 9.5) has been shown to be effective in rats and other species for revealing the innervation in skin and other peripheral organs, including numerous small-caliber fibers not seen previously (Bradbury and Thompson '85; Gulbenkian et a1.,,'87; Wilson et al., '88; Lauweryns and Van Ranst, '88; Edvinsson et al., '89; Karanth et al., '90; Ramieri et al., '90; Wang et al., '90; Gronblad et al., '91; Rydh et al., '91). PGP 9.5 has been identified as ubiquitin carboxyl-terminal hydrolase, which is distributed throughout neuronal cytoplasm

(Jackson and Thompson, '81; Doran et al., '83; Thompson et al., '83; Day and Thompson, '87; Wilkenson et al. '89).

In this study we have used anti-PGP 9.5 immunofluorescence to examine the innervation of the mystacial pad in the rat. In addition to displaying all previously known innervation, this technique revealed additional, numerous, smaller- caliber axons and their terminations in the epidermis, follicles, and vasculature. Also, some nonargyrophilic endings from medium-caliber and large-caliber axons were labeled. Thus the innervation of the mystacial pad is even more complex than was previously demonstrated.

MATERIALS AND METHODS Ten young adult female rats (Sprague-Dawley, 200-250

g) were overdosed with chloral hydrate and perfused with Tyrode's solution at room temperature followed by a 4°C solution of 4% paraformaldehydei 14% saturated picric acid in 0.15 M phosphate-buffered saline (PBS) a t pH 6.9. Por- tions of mystacial pad containing caudal vibrissae from rows B, C, or D were removed and postfixed in the same fixative for 1-2 hours, rinsed several times in PBS, and cryoprotected by overnight infiltration in 104 sucrose in PBS. All postfixation and PBS solutions were at 4°C. Fourteen-micrometer-thick serial sections were cut by cryo- stat in longitudinal (perpendicular to the skin surface) or transverse (parallel to the skin surface) planes through the F-SCs. The sections were thawed directly onto slides coated with chrome-alumigelatin and air dried for not more than 2 hours.

For immunofluorescence, the sections were first soaked in 10% normal goat serum and 0.3% Triton X-100 in PBS for 1 hour, then incubated overnight at 4°C in rabbit antihuman PGP 9.5 antiserum (Ultraclone) at a 1:10,000 dilution in PBS with 4'1; fetal calf serum and 0.34 Triton X-100. Following the primary incubation, the sections were rinsed three times for 10 minutes each with PBS and incubated for 1 hour in rhodamine-conjugated goat antirab- bit IgG (Boehringer-Mannheim) at a 1:200 dilution in PBS with 0.3% Triton X-100. After the sections were rinsed three times for 10 minutes each with PBS, coverslips were mounted with glycerol.

Sections were viewed and photographed with a Nikon Microphot-FX equipped for rhodamine epifluorescence. The analysis was restricted to the innervation of the caudal three F-SCs from rows B, C, and D; the intervening intervibrissal fur; the blood vessels within the dermis; and the papillary muscles. Source fascicles and nerves in the dermis were also examined.

For controls, some sections from each mystacial pad were incubated without primary antibody. Also, PGP 9.5-like immunoreactivity (PGP 9.5-LIR) was assessed in sections from three mystacial pads in which the ipsilateral infraorbital nerve had been transected 10-14 days prior to sacrifice. These later results were compared to the results from normal, contralateral mystacial pads which had not been denervated. To check for possible sympathetic contributions, the superior cervical ganglion was removed on one side in two rats 10-14 days prior to sacrifice. Sections from the denervated mystacial pads were processed by the above- described procedure for PGP 9.5 immunofluorescence and were compared with contralateral and other normal mystacial pad sections.

Merkel cell Vibrissal shaft --- - - -- - - -_ -

Merkel ending, I l l I

I / I I ,Rete ridge collar

body

- - - - - --Inner conical body

Glassy membrane

- - - Ring sinus

_ _ - - _ _ - - Capillary network

--Meandering endings --- - - -

. . . . . , ;> Cavernous sinus

0 , 0 .

_ _ _ _ _ Dermal papilla

)\ Figure 1

IMMUNOFLUORESCENCE OF RAT MYSTA4CIAL PAD 369

RESULTS Basic structure of F-SCs

The basic structure of vibrissal F-SCs is shown schemati- cally in Figure 1. The epidermally derived follicle at the core is composed of two concentric layers: an internal root sheath, which is next to the hair shaft, and an adjacent external root sheath, which is surrounded by the basement membrane. Around the middle of the follicle, the basement membrane is especially thick and is referred to as the “glassy” membrane. At the mouth of the F-SC, where the epidermis and follicle meet, the epidermis forms a thickened rete ridge collar. The deep end of the follicle forms a bulb that contains a dermal core referred to as the dermal papilla.

The follicle is enveloped by a dermally derived, blood- filled sinus, which has an inner lining of loose connective tissue, referred to as the mesenchymal sheath, that sur- rounds the glassy membrane. The sinus is enclosed by a dense collagenous capsule that merges with the follicle at the neck and dermal papilla to seal off the apex and base of the sinus respectively. At the apex of the sinus, the mesenchymal sheath expands to form the inner conical body IICB).

The sinus has two subdivisions. The upper half, which is referred to as the ring sinus, has an open lumen and contains a ring-like structure, the “ringwulst,” which is suspended from the circumference of the mesenchymal sheath. In the deep half or cavernous sinus, a lattice-work of trabeculae lines the capsule. Just below the interface with the ring sinus, the trabeculae completely span the lumen between the capsule and the mesenchymal sheath (Rice, ’93).

Innervation to each F-SC is provided by a single, relatively large deep vibrissal nerve (DVN), which penetrates the capsule at the level of the ring sinus, and by several superficial vibrissal nerves (SVNs), which converge on the mouth and neck of the F-SC (Fig. 1).

Specificity of labeling In intact mystacial pads, nearly all of the PGP 9.5-LIR

was extremely intense and localized throughout the cytoplasm of sensory and motor axons and their endings (Figs. 2-12). For simplification, profiles that express PGP 9.5-LIR will simply be referred to as “labeled.” Cross sections of infraorbital nerve branches revealed a wide range in the caliber of labeled axons (Fig. 2A). Close inspection of such cross sections also revealed faint PGP 9.5-LIR in the outer perimeter of myelin sheaths, suggesting that a PGP 9.5-like antigen may also be expressed at low levels within Schwann cell cytoplasm.

The specificity of labeling was confirmed by lack of fluorescence when the primary antibody was eliminated

Fig. 1. A schematic longitudinal cutaway drawing of a vibrissal follicle-sinus complex (F-SC) indicating the structural elements and the relationship of the innervation. The follicle is epidermal in origin, consists only of the internal and external root sheaths, and forms only the core of the F-SC. The basement or “glassy“ membrane separates the follicle from the surrounding sinus-related components, which are dermally derived. Most of the axons supplied by several superficial vibrissal nerves (SVNs) and one deep vibrissal nerve (DVN) terminate in the mesenchymal sheath or its apical enlargement, the inner conical body. Only the Merkel endings and some free nerve endings at the rete ridge cross the basement membrane to terminate in epidermal locations.

from the incubations. Also, 10-14 days after transection of the infraorbital nerve, PGP 9.5-LIR was eliminated from all apparent axonal endings (Figs. 13, 14) except for the motor endplates on the papillary muscles supplied by the facial nerve (Figs. 2D, 11B, 12). Although infraorbital nerve transections also eliminated label from all small-caliber axons, some residual PGP 9.5-LIR did remain in highly vacuolated, medium- and large-caliber profiles along the pathways normally traversed by medium- to large-caliber axons (Figs. 2C, 13). Presumably this expression is within reactive Schwann cells.

A moderate level of PGP 9.5-LIR was expressed in Merkel cells at the ring sinus level of vibrissal follicles (Figs. 3, 4D, 5B) but not in Merkel cells in the rete ridge collar or the epidermis (Figs. 4A,B, 10A). The PGP 9.5-LIR persisted in these nonneuronal cells after nerve transection (Fig. 13C) but was absent when the anti-PGP 9.5 primary antibody was omitted.

A moderate level of presumptive PGP 9.5-LIR was also present within highly branched cells that resemble nonneuronal Langerhans cells and/or dendritic cells described in previous studies (Shiohara et al., ’90; Tigelaar et al., ’90). These cells and their branches were entirely confined to the epidermis of the intervibrissal fur or occasionally superficial portions of the epidermally derived follicles (Fig. 4A,B,D). The branches of these cells were similar in caliber to presumptive free nerve endings (FNEs) but were more tortuous and less intensely labeled. These cells seemed to be more prevalent in the epidermis and follicles after denervation (Fig. 14).

Nonspecific fluorescence was fairly intense in the ringwulst and hair shafts (Fig. 6) and was moderate within the extracellular space between the cells of the vibrissal and intervibrissal hair follicles. This fluorescence persisted after nerve transection and incubations without primary antibody.

F-SC innervation Within each F-SC, labeled axons contributed unique sets

of endings to six locations: the rete ridge collar, the ICB, the neck of the sinus capsule, the ring sinus, the cavernous sinus, and the dermal papilla (Figs. 3-7). In general, SVNs provided all of the innervation to the rete ridge collar and most to the ICB (Figs. 3, 4), whereas the DVN provided innervation primarily to the level of the ring and cavernous sinuses (Figs. 3, 5-7). Sparse innervation to the dermal papilla appeared to enter with small blood vessels (Fig. 7D).

Rete ridge collar. In the rete ridge collar (Fig. l), several extremely fine, labeled, thread-like profiles ex- tended radially through most of the epidermis, reaching almost to stratum corneum (Fig. 4A,B). These were not seen previously with reduced-silver techniques (Rice et al., ’86a) and were presumably FNEs. They arose from medium- caliber SVN axonal profiles that could be A6 fibers and/or clusters of C-fibers.

Clusters of labeled disk-like profiles were located at the dermal-epidermal border of the rete ridge collar (Fig. 4A,B). These were sensory endings that had been shown previously to terminate in this location on Merkel cells in the stratum basalis (Rice et al., ’86a). The combination of the sensory ending and the Merkel cell has been referred to as the Merkel-neurite complex. The Merkel cells were not labeled. The source of the Merkel endings was medium- to large-caliber profiles, which were presumably AP or multiple A6 afferents.

370 F.L. RICE ET AL.

Fig. 2. A,B: Protein gene product ( P G P ) 9.5 immunofluorescencc of some branches of an intact infraorbital nerve cut in cross section (A. x450) and longitudinally (B , ~ 3 0 0 ) . Note the intense, uniform PGP 9.5-LIR filling the axons and faint label a t the perimeter of the myelin sheaths. C: PGP 9.5 immunofluorescence o f a branch of an infraorbital

nerve 2 weeks after tiansection at the infraorbital foramen. Note profiles have a persisting PGP 9.5-LIR but with a vacuolar appearance; ~ 3 0 0 . D: A labeled intact branch of the facial nerve from a mystacial pad with a transected infraorbital nerve; X300.

Znner conical body. The ICB was innervated by a dense circumferential array of many labeled medium-caliber profiles and more numerous fine-caliber profiles (Figs. 1, 3, 4C,D, 5A). This array was located in the inner portion of the ICB, near the glassy membrane, with the larger profiles generally distributed more centrifugally and the finer profiles more centripetally. Although a mixture of thin lanceolate endings from A6 axons and clusters of varicose endings from bundles of C fibers has been demonstrated in a serial EM study of the rat ICB (Mosconi et al., '93), these two types of endings could not be individually discerned at the light microscopic level. Potential variations in their distribution around the circumference of the ICB also could not be evaluated because of variations in section alignment.

Nearly all of the ICB innervation descended from the SVNs, but occasional labeled fine- or medium-caliber profiles were continuous between the level of the ring sinus and the ICB (Figs. 4C,D). These profiles appeared to be ascending from the DVN to the ICB, although the possibility could not be ruled out that some SVN afferents may be descending to the level of the ring sinus. Their particular contribution t o either the ICB or the ring sinus could not be discerned among the intermingled innervation.

Small bundles of fine-caliber profiles were oriented circumferentially in close proximity to and within the substance of the sinus capsule at the neck of the F-SC (Fig. 8A). The source is presumably from the SVNs, although this was not observed directly.

Neck of sinits capsule.

IMMUNOFLUORESCENCE OF RAT MYSTACIAL PAD 371

Fig. 3. Low-power ( ~ 2 0 0 ) PGP 9.5 immunofluorescence photomicrograph of a longitudinal section through an F-SC from the inner conical body (ICB) to the cavernous sinus (CS). Circumlerentially oriented innervation of the ICBs is shown between the broad solid arrows. Lanceolate endings and Merkel-neurite complexes at the level of the

ring sinus are indicated by the long arrows and the arrowheads, respectively. Reticular endings at the level of the cavernous sinus are indicated by broad open arrows. RS, ring sinus: RW, ringwulst: C, capsule; DVN, deep vibrissal nerve.

Ring sinus. Three sets of labeled profiles arose from DVNs and distributed to the level of the ring sinus (Fig. 1). Two sets were large-caliber afferents that terminated as morphologically distinct lanceolate and Merkel endings, which had been observed in previous studies (Figs. 3, 5). The third set had not been described previously and consisted of a network of fine-caliber profiles (Fig. 5).

The lanceolate endings were located in the mesenchymal sheath adjacent to the glassy membrane and had a flattened blade-like appearance (Figs. 3, 5A). The PGP 9.5-LIR was very intense and fairly uniform throughout the endings. At most, a few lanceolate endings arose from the tip of each afferent like the tines of a fork. Individually they were oriented parallel to the vibrissal shaft. Collectively they formed a palisade around the circumference of the follicle (Fig. 3).

Merkel afferents penetrated the glassy membrane to terminate on Merkel cells in the external root sheath of the follicle. The Merkel-related labeling appeared as a regularly spaced array of numerous ovoid profiles oriented circumferentially within the external root sheath (Figs. 3, 5B). Groups of profiles were interconnected by labeled fine- caliber strands, indicating that single s o n s give rise to many endings (Fig. 5B). In contrast to label in lanceolate endings, the PGP 9.5-LIR was relatively faint throughout most of the ovoid profiles and intense only at one end or along one border. After transection of the infraorbital nerve, the faint labeling persisted in the ovoid profiles, whereas the more intense labeling along the edges and the labeling in the interconnecting strands was eliminated (Fig. 13C). Thus PGP-LIR appears to be expressed within the Merkel cells as well as within the endings at the level of the


Fig. 4. High-power ( x450) photomicrographs of longitudinal sections through the superficial end of F-SCs. A and B are from sections through the center and at the edge. respectively, of the rete ridge collar. Note the fine processes iarrowheadsi radiating through the epidermis and the Merkel endings (between broad open arrows) at the stratum basalis. The Merkel cells are not labeled. C and D are from sections through the edge and slightly off-center through the ICB, respectively.

Note the mixture of medium and fine-caliber profiles. Broad open arrow in C indicates afferents descending from an SVN. Circumferential endings span between the broad solid arrowheads in D. Note the location of the basement or "glassy" membrane (asterisks), Merkel- neurite complexes at the level of the ringsinus (arrowheads), occasional ascending DVN axons to the ICB (small arrows). and cells with dendritic-like processes (large arrows).

ring sinus, whereas only the endings are apparently labeled in the rete ridge collar.

The loose network of fine-caliber labeled profiles mean- dered around the circumference of the mesenchymal sheath

throughout the level of the ring sinus (Fig. 5). Some of these profiles continue into the lower portion of the ICB. Al- though they were not reported in previous studies, these profiles were apparently neuronal since they were elimi-


Fig. 5. Innervation in longitudinal sections paz ing the edge of the ICB and of the mesenchymal sheath (A) and follicle (BI a t the level of the ring sinus. A: Longitudinally oriented lanceolate endings (narrow arrows) in the mesenchymal sheath at the level of the ring sinus and dense concentration of circumferentially oriented profiles (between the broad arrows) at the level of the ICB. ~ 3 0 0 . Note sparse, fine caliher profiles (arrowheads) in the mesenchymal sheath centrifugal to the lanceolate endings. B: Circumferentially oriented Merkel-neurite complexes in the external root sheath of the follicle at the level of the ring sinus interconnected by fine caliber processes (small arrowheads). x 500). The glassy membrane [asterisks) is centrifugal to these endings. Note the loose plexus of fine-caliher profiles (large arrowheads) a t the upper portion of the mesenchymal sheath exterior to the glassy membrane.

Fig. 6. Reticular-like endings in slightly oblique section between the mesenchymal sheath and follicle a t the upper end of the cavernous sinus tCS) just below t.he ring sinus (RS). ~ 3 0 0 . Note the large caliber source axons (solid arrows) and the traheculae (open arrow) in the lumen of the cavernous sinus. The reticular endings are in the mesenchymal sheath in close proximity to the glassy membrane (asterisk). The ringwulst (RW) is in the ring sinus.

nated after transection of the infraorbital nerve (Fig. 130. They were not observed terminating as morphologically distinct endings, but presumably form some type of FNE. Their source is primarily from the DVNs, although descending contributions from SVNs could not be ruled out.

Cavernoiis sinus. The anti-PGP 9.5 immunofluorescence revealed several sets of profiles within the cavernous sinus, most of which had not been observed previously with silver stains. All these profiles were eliminated by infraorbital nerve transection, indicating that they were indeed neuronal (Fig. 13C).

The most surprising set of endings was a dense reticular- like network of extensively branched neuronal profiles that arose from several large-caliber afferents (Figs. 3, 6). These reticular endings were located within the mesenchymal sheath in the upper part of the cavernous sinus, where the trabeculae completely span the lumen, and were in close proximity to the glassy membrane (Fig. 6). Previous light microscopic studies revealed only the proximal branches of these endings.

Beginning just below the level of the reticular endings, a loose network of medium and small-caliber profiles mean- dered haphazardly within the mesenchymal sheath all the way down to the dermal papilla (Fig. 7A,B). Many of these labeled profiles also ramified among the trabeculae. Little of this innervation had been observed before. The medium-

Fig. 7. A.B: Medium-power (~3001 photomicrographs of longitudinal sections showing haphazard medium- and fine-caliber profiles in the mesenchymal sheath at the level of the cavernous sinus. The broad arrowhead in A indicates a profile suspended in a trabecula within the lumen of the sinus. DP. dermal papilla. C: Higher power ( ~450) photomicrograph of a section cut longitudinally along the interface

between the mesenchymal sheath and the follicle at the level of the cavernous sinus. The circuniferentially oriented network of fine-caliber profiles is within the mesenchymal sheath close to the basement membrane. D: Fine-caliber profiles (arrowheads) in the dermal papilla, which arise from axons located deep to the F-SC (open arrow). ~ 3 0 0 ) .

Fig. 8. Innervation of the intervibrissal fur in sections perpendicular to the surface of the fur. x 150. A-C: Sections beginning near the neck of an F-SC (A) and moving progressively towards the center of the intervibrissal fur (i.e.. farther from a n F-SC). C, capsule: G. guard hair; V, vellus hair. Larger nerves (curved arrows) located nearer the F-SCs give rise to a deep plexus of smaller nerves (larger arrowheads) that supply a deeper stratum of innervation (between larger broad solid arrows) to the midshaft level of guard and vellus hairs. This midshaft innervation consists primarily of piloneural complexes (larger arrows), which are shown in detail in Figure 10. Still smaller bundles of iFYons

(smaller arrowheads) ascend to form a second, more superficial stratum of innervation (between smaller broad solid arrows) consisting of a plexus (small arrows) that supplies innervation to the epidermis (open arrows). Note that the innervation density especially in the epidermis diminishes further from the F-SCs. Near the neck of the F-SC the fur contains more widely spaced individual guard hairs each of which is innervated. Further from the F-SCs, there tend to be clusters of guard and vellus hairs. Note the small bundle of fine-caliber profiles (wavy arrow in A) within the capsule of the F-SC.


Figure 8


Fig. 9. Cluster endings in the epidermis of the intervibrissal skin in close proximity to guard hairs (GH) and vellus hairs (VH). Solid arrows indicate source profiles. A: Photomicrograph of cluster endings (open arrows) adjacent to the mouths of a guard hairs sectioned perpendicular to the skin surface. x300. B,C: High-power photomicrographs t ~ 6 0 0 ) of cluster endings in the epidermis as seen in sections perpendicular and parallel to the skin surface, respectively.

and small-caliber profiles appeared to be two distinct but intermingled populations, although the possibility could not be ruled out that the smaller profiles were branches from the larger. Neither type of profile terminated as a morphologically distinct type of ending.

Another loose network of fine-caliber, but more circumferentially oriented, profiles was observed for the first time in close proximity to the glassy membrane around the follicle (Fig. 7C). We could not determine whether these axons arose from the more haphazard centrifugally located profiles described above or whether they were a separate set of innervation.

The dermal papilla contained a few fine-caliber profiles that appeared to arise from a few labeled axons located at the base of the F-SC (Fig. 7D). This innervation penetrated the dermal papilla in the company of a few small blood vessels. These were the only profiles that were completely eliminated by removal of the superior cervical ganglia. Thus they appeared to be sympathetic.

Intervibrissal fur innervation The intervibrissal

fur is composed of an epidermis, dermis, numerous follicles of guard and vellus hairs, and the papillary muscle slings for the protraction of the vibrissal F-SCs. The distinction between guard and vellus hairs within the intervibrissal skin is based on size and complexity of innervation, although there is a continuum between two extremes (see Rice and Munger, '86). Guard hairs are less numerous, are usually larger, and have more complex innervation. Vellus hairs are far more numerous, are usually smaller, and may

Dermal papilla.

Structiire of the intervibrissal fur.

have little if any innervation. The skin in close proximity to the vibrissae contains mostly individually spaced guard hairs (Fig. 8A). Farther from the vibrissae, the hairs are organized into clusters containing one or two prominent guard hairs and several smaller guard and vellus hairs (Fig. 8B,C). The PGP 9.5 immunofluorescence revealed numerous axonal profiles throughout the intervibrissal fur with extensive termination around the intervibrissal hair follicles, in the epidermis between the hairs, and along the surface of vascular profiles in the dermis (Figs. 8-1 1).

Deep within the dermis, most of the labeled profiles were multiaxonal fascicles from the infraorbital nerve or facial nerve (Figs. 2, 11A). The facial nerve fascicles supplied exclusively the papillary muscles of the F-SCs (Fig. 12) and their label was preserved after transection of the infraorbital nerve (Fig. 2D). PGP 9.5-LIR was expressed in the motor endplates as well as in the axons (Fig. 12). The smallest fascicles deep in the dermis were usually in close proximity to vascular profiles (Fig. 11A). The walls of each vascular profile throughout the dermis were laced with labeled single and bundled small-caliber axonal profiles (Fig. 11A). Otherwise, there were no obvious, morphologically distinct axonal endings within the deep dermis or in affiliation with the papillary muscles.

An extensive plexus of tortuous branching and intermingled individual profiles and small fascicles was labeled in the superficial part of the intervibrissal dermis (Fig. 8). This was especially dense nearer the necks of the vibrissal F-SCs, where the source nerves for the fur innervation were located (Fig. 8A). These source nerves arose in common with SVNs from small infraorbital nerve branches

Dermis irtneruation.


Fig. 10. Photomicrographs of guard hair iGH) and vellus hair iVH) innervation as seen in sections perpzndicular to the skin surface. Piloneural complexes in A 1 x300X) and B 1 x450) consist of a palisade of longitudinally oriented lanceolate endings (large arrows) surrounded by fine caliber circumferential endings (small arrows). Ascending axons

in A provide Merkel endings (open arrows) at the mouth of the guard hair. The Merkel cells are not labeled. C: Fine-caliber circular endings iarrowheads) around the neck of a guard hair follicle. ~ 4 5 0 . D: Sparse, fine-caliber innervation (arrow) commonly associated with vellus hairs. ~ 4 5 0 . Sparse innervation of a vellus hair (VH) is also seen in A.

that ascend in close proximity to the F-SCs (Dorfl, '85; Munger and Rice, ' 86) .

The plexus in the superficial part of the dermis provided small bundles of axons that innervated guard and vellus hairs at about midshaft level throughout the intervibrissal fur (Figs. 8, 10A,B). This plexus in turn gave rise to smaller ascending branches that ascended to form a subepidermal plexus immediately subjacent and supplying innervation to the epidermis (Figs. 8,9).

Epidermis innervation. The epidermis contained a rich distribution of labeled fine-caliber profiles arising from the

subepidermal plexus (Figs. 8, 9). Some individual fine- caliber profiles, which are presumably FNEs, extend through the epidermis and nearly reach the stratum corneum. These presumptive individual FNEs were more frequent and tortuous than those in the epidermis of the F-SC rete ridge collar. They were more frequent towards the F-SC than towards the middle of a patch of intervibrissal fur. At frequent intervals, dense clusters of extremely fine profiles were contributed to the epidermis by medium-caliber source profiles that appear to contain more than one axon (Fig. 9). These clusters were often in close proximity but were not


Fig. 11. Photomicrographs of the network of innervation around blood vessels in the dermis (arrows) before (A) and 2 weeks after iB) infraorbital nerve transection. x 350. Note the almost complete elimination of the vascular innervation. Labeled axons indicated by the broad arrow in A are from the intact infraorbital nerve. Other labeled axons in A and all the labeled profiles in B are intact axons from the facial nerve intermingled with papillary muscle fibers.

consistently affiliated with the mouth of guard or vellus hairs. None of these intraepidermal endings was noted in previous studies of mystacial pad innervation.

Four distinctly different types of labeled ending profiles could be seen in direct affiliation with guard hairs (Fig. 10). All four were rarely seen on a single hair, which was typically a relatively larger-caliber guard hair. Two of the endings formed a combination referred to as a pilo-Ruffini or piloneural complex (Munger and Ide, '88; Biemesderfer et al., '78; Rice and Munger, '86). These were typically located about midway along the length of the follicle, below the attachment of occasional small sebaceous glands (Figs. 10A,B). In a complete piloneural complex, medium- to large-caliber axonal profiles formed short, longitudinally oriented, lanceolate terminals arranged as a palisade around the follicle. Slightly finer caliber axonal profiles provided thin endings oriented circumferentially around the lanceolate palisade.

Some guard hairs had only the lanceolate palisade component of the piloneural complex. In general, the lanceolate palisade became less complete as the caliber of the hairs decreased. On rare occasions, a few of these lanceolate endings were affiliated with vellus hairs. Typically, vellus hairs were innervated by only a few fine-caliber profiles that had more of a circumferential orientation (Figs. 10A,D).

Merkel endings were a third type of ending associated with guard hairs (Fig. 10A). These appeared as a few labeled

Inneroation of guard and vellus hair follicles.

discs along the stratum basalis of a thickened epidermal pad usually located at the posterior edge of the mouth of most larger guard hairs. The Merkel endings were provided by a few medium- to large-caliber axonal profiles that ascended directly from the vicinity of the piloneural complexes. In contrast to the Merkel cells in the external root sheath of F-SCs, the Merkel cells affiliated with the guard hairs were like those in the rete ridge collars and did not express PGP

Finally, PGP 9.5-LIR was expressed in a sparse, circumferentially oriented network of fine profiles that encircled the neck of occasional large guard hairs (Fig. 1OC). These profiles, which had not been reported previously, had a distinctly finer caliber and more superficial location than the circumferential components of the piloneural complexes affiliated with the same follicles. They appear to arise from the subepidermal plexus.

9.5-LIR.

Sympathetic innervation All but the facial nerve innervation of the papillary

muscles was eliminated by transection of the infraorbital nerve as it exits the infraorbital foramen (Figs. 13,14). This included the innervation of the vasculature (Fig. 11B). Thus the sensory and presumably autonomic innervation of the mystacial pad distributes entirely via the infraorbital nerve. However, removal of one superior cervical ganglion had no appreciable impact on most of the labeled innerva-


Fig. 12. A,B: Photomicrographs of axons and motor endplates (broad arrows) affiliated with papillary muscles. X400. Fine processes in A (arrow) are from the grazed edge of a blood vessel.

tion of the ipsilateral mystacial pad 2 weeks later. Only the innervation to the F-SC dermal papillae was completely eliminated. This suggests that most of the fine-caliber innervation within the mystacial pad, including the dense innervation of the vasculature, arises from the trigeminal ganglion, with possibly a parasympathetic contingent from the pterygopalatine ganglion.

DISCUSSION Labeling specificity

Antihuman PGP 9.5 immunofluorescence revealed consid- erably more neuronal-like profiles in the mystacial pad in addition to those observed previously with reduced silver techniques or conventional electron microscopy (Tello, '23; Andres, '66; Patrizzi and Munger, '66; Renehan and Munger, '86; Rice et al., '86a). The newly observed profiles were in the vibrissal F-SCs and in the intervibrissal skin, where they were especially numerous in close proximity to the F-SCs. Previously Unseen innervation, especially fine caliber, has also been revealed routinely with PGP 9.5 irnmunocytochemistry in a variety of other tissues (Heppel- man et d., '88; Lauweryns and Van Ranst, '88; Wilson et al,, '88; Edvinsson et al., '89; Karanth et al., '90; ~ ~ ~ i ~ ~ i et

jg0; Wang et al., jg0; Griinblad et al., '91; Rydh et al,, '91 1. Transection of the infraorbital nerve eliminated PGP

Fig. 13. photomicrographs illustrating the loss of labeled endings in the rete ridge (A), ICB (B) , and ring sinus (Ci 2 weeks after infraorbital nerve transection. x300. Note persisting labeling along the normal paths of medium- to large-caliber axons !open arrows) and in Merkel cells at the level of the ring sinus. In A, cells with dendritic-like processes (arrows) also remain labeled in the rete ridge and ICB.


Fig. 14. Cells with dendritic-like processes (arrows) in the epidermis and guard hair follicles that have persisting labeling after infraorbital nerve transections. A. ~ 3 0 0 . B: x 600.

9.5-LIR in nearly all of the neuronal-like profiles, support- ing the interpretation that they were indeed neuronal. Although the possibility exists that some of the eliminated profiles may be nonneuronal structures that express PGP 9.5-LIR in the presence of innervation, similar appearing neuronal-like profiles were usually labeled in a subsequent anterograde transport study in which horseradish peroxi- dase (HRP), wheat germ agglutinin HRP, or cholera-toxin B fragment HRP was injected into the trigeminal ganglion (Fundin et al., '93).

It was clear, however, that PGP 9.5-LIR was not exclusively neuronal. First, moderate levels of PGP 9.5-LIR were present in the outer perimeter of myelin sheaths in normal nerve fascicles. Residual, highly vacuolated PGP 9.5-LIR persisted in denervated nerve fascicles 2 weeks after nerve transection. This suggests that PGP 9.5-LIR may be expressed in Schwann cells normally and is perhaps enhanced after denervation. Second, moderate levels of PGP 9.5-LIR were observed in Merkel cells, but only those in the outer root sheath of F-SCs, even after nerve transection. Merkel cells labeled with PGP 9.5 had been seen previously in human epithelium (Ramieri et al., '90; Wang et al., '90). However, the Merkel cells in the rete ridge of the F-SCs and in the intervibrissal skin were not labeled. This differential expression of PGP 9.5-LIR in the mystacial pad of the rat may be related to differences in the origin of these populations of Merkel cells (Nurse and Farraway, '88). Finally, PGP 9.5-LIR was also expressed in cells, usually seen in the epidermis, that had fine dendritic-like processes. These cells resemble Langerhans's cells and/or dendritic cells, which appear to be related to the thymic based T-cell population (Havran and Allison, '90; Shiohara et al., '90; Tigelaar et al., '90). Similar cells have also been observed in human epithelium (Wanget al., '90). These cells seemed to be especially evident in the denervated mystacial pad. Whether the nonneuronal labeling was due to the presence of PGP 9.5-like antigen or to a cross reaction with another compound with a PGP 9.5-like epitope remains to be determined.

Newly revealed innervation Most of the newly revealed innervation consisted of

small-caliber fibers. These included 1) frequent clusters of intraepidermal endings in the intervibrissal skin; 2 ) thin, tortuous, or radially oriented individual endings throughout the epidermis, including the F-SC rete ridge; 3) a few

small bundles of fine-caliber axons in the neck of the sinus capsule; 4) a loose network of fine-caliber fibers distributed circumferentially and centrifugally to the lanceolate endings in the upper portion of mesenchymal sheath at the level of the F-SC ring sinus; 5) a loose network of fine- caliber and medium-caliber axons distributed haphazardly among the trabeculae and mesenchymal sheath of the cavernous sinus; 6) another network of fine-caliber axons distributed circumferentially within the mesenchymal sheath of the cavernous sinus but in close proximity to the glassy membrane; 7) a few endings in all the vibrissal dermal papillae; 8 ) small networks of fine-caliber fibers distributed circumferentially around the neck of some guard hairs; and 9) extensive innervation of the mystacial pad vasculature.

Although this fine-caliber innervation was rarely or never stained by reduced silver techniques, other fine- caliber profiles were revealed equally well by both PGP 9.5 immunofluorescence and reduced-silver techniques in locations such as the ICB in F-SCs and the piloneural complexes of guard hairs (Rice et al., '86a; Rice and Munger, '86). In the ICB the vast majority of profiles are C fibers and their terminals (Mosconi et al., '93). Thus some populations of C-fibers are apparently argyrophilic, whereas others are not.

PGP 9.5-LIR was intensely expressed in all Merkel and lanceolate endings, which were provided by medium-caliber (A6) or large-caliber (AP) afferents and were previously observed by reduced-silver techniques and/or EM (Tello, '23; Andres, '66; Patrizzi and Munger, '66; Renehan and Munger, '86; Rice et al., '86a). However, PGP 9.5 immunofluorescence also revealed some endings of medium- to large-caliber afferents that were apparently not argyrophilic. Merkel endings in the intervibrissal fur were prevalent with PGR 9.5 immunofluorescence, but were rarely stained with reduced-silver techniques (Rice and Munger, '86). Also, reduced-silver preparations only revealed the large-caliber source axons and proximal branches that provide the dense network of reticular-like endings which was labeled by anti-PGP 9.5 in the mesenchymal sheath of the F-SC cavernous sinus (Renehan and Munger, '86; Rice et al., '86a). These axons were previously interpreted as a source of corpuscular endings, Ruffini endings, or free nerve endings Andres ('66) apparently observed the reticular endings at the EM level and, based on their ultrastructure, referred to them as branched lanceolate endings.


Yohro ('77) may have also seen them in the shrew and interpreted them as circumferential lanceolate endings. However, neither ultrastructural study gave a full apprecia- tion of their dense concentration and extensive, irregular branching.

Although corpuscular andlor Ruffini endings were reported in the cavernous sinuses of other species (Andres, '661, they were not observed with EM, reduced-silver techniques, or PGP immunofluorescence in the mystacial F-SCs in the rat. Corpuscular endings were observed in reduced-silver and anti-PGP immunofluorescence preparations of forelimb carpal F-SCs of the rat (Fundin and Rice, '91; and unpublished observations). Thus some endings such as these may be species or location specific.

Overview of mystacial pad innervation The results of this study and other studies now indicate

that the innervation of the mystacial pad is even more complex than was previously demonstrated. The F-SCs are innervated by an astounding array of endings provided by several SVNs converging at the neck and by a single DVN entering the cavernous sinus. The SVNs provide nearly all of the innervation to the rete ridge collar and ICB. In addition to the newly discovered fine-caliber profiles in the rete ridge collar, the SVN innervation includes Merkel-neurite complexes in the rete ridge collar as well as a dense circumferentially oriented array of lanceolate endings and bundled C-fiber terminals in the ICB (Mosconi et al., '93). The DVNs supply most if not all of the various networks of fine-caliber innervation at the level of the ring and cavernous sinuses mentioned above. Some of the fine-caliber axons at the level of the ring sinus mesenchymal sheath continue into the lower portion of the ICB. The DVNs also supply the dense lattice of reticular endings in the mesenchymal sheath of the cavernous sinus as well as the palisade of lanceolate endings in the mesenchymal sheath of the ring sinus and the extensive array of Merkel-neurite complexes around the circumference of the follicle external root sheath at the level of the ring sinus. Finally, all of the dermal papillae are innervated by a few fine-caliber axons, presumably of sympathetic origin.

In the intervibrissal skin, the dermal plexus provides at least two morphologically distinct intraepidermal endings: individual and clustered. At least four other morphologically distinct endings are affiliated with guard hairs: Merkel- neurite complexes at the mouth of follicles, occasional circumferential networks of fine-caliber processes around the neck, and lanceolate and circumferential endings in the piloneural complexes about midway along the length of the follicles. Finally, vellus hairs are frequently innervated by single or partial combinations of endings like those affiliated with guard hairs.

Several types of afferents have been characterized electrophysiologically in the mystacial pad or hairy skin (Brown and Iggo, '67; Zucker and Welker, '69; Burgess and Perl, '73; Gottschaldt et al., '73; Pubols et al., '73; Dykes, '75; Gibson and Welker, '83a,b; Hayashi, '85; Jacquin et al., '86a,b; Renehan et al., '86; Lichtenstein et al., '90). How- ever, none of these has yet been directly correlated with a particular morphology. Some studies have simply characterized vibrissal afferents as rapidly or slowly adapting, whereas others have attempted more detailed subcategories based on thresholds, directional selectivity, or discharge patterns. On the other hand, Gibson and Welker ('83a,b) have described the vibrissal afferent population as a continuum

ranging from slowly to rapidly adapting extremes having a broad range of thresholds and directional characteristics. The wide variety of ending types and distributions as revealed by PGP immunofluorescence and other techniques seems consistent with Gibson and Welker's interpretation, although it remains to be seen how the various endings fit within such a continuum, or if some endings have yet to be functionally characterized.

Large-caliber DVN afferents to F-SCs The Merkel, lanceolate, and reticular endings are all

likely low-threshold mechanoreceptors based on their highly specialized morphology and large-caliber afferents. Of these, only the response characteristics of Merkel endings have been definitively identified as slowly adapting in response to pressure, but this was determined in glaborous skin (Munger and Ide, '88). Lanceolate endings are purportedly rapidly adapting (Dykes, '83), but this has not been directly established.

Based on the location of the endings and their purported response characteristics, the Merkel endings at the level of the ring sinus in F-SCs have been hypothesized to be slowly adapting endings with highly selective directional sensitivity to vibrissal deflections (Rice et al., '86; Lichtenstein et al., '90). In contrast, the lanceolate endings at the level of the ring sinus have been hypothesized to function as rapidly adapting endings that may detect more transient characteristics of deflection but with relatively poor directional sensitivity (Rice et al., '86; Lichtenstein et al., '90). At the time when these hypotheses were proposed, the dense network of reticular endings at the upper end of the cavernous sinus was unknown, although endings with a lanceolate-like ultrastructure had been identified in this location (Andres, '66). Based on their ultrastructure, reticular and lanceolate endings may be responsive to similar types of mechanical distortion. However, the location and widespread distribution of the reticular endings suggest that they should have a function different from that of the lanceolate endings. At the level of the cavernous sinus, where the reticular endings are located, trabeculae span between the mesenchymal sheath and the sinus capsule (Rice, '93), and the shaft of the vibrissa is less keratinized and thus more pliable than at the level of the ring sinus (Wrobel, '65; Yohro, '77; Rice, unpublished observation). Considering this structural arrangement along with their widespread distribution, the reticular endings may be more easily subjected to mechanical distortions but have less directional specificity than the lanceolate endings.

Fine-caliber networks in F-SC sinuses The networks of fine-caliber and medium-caliber axons at

various sites within the mesenchymal sheath and among the trabeculae may have a variety of components and functions. Some could be related to a recently elucidated capillary network within the mesenchymal sheath (Rice, '93). Nilsson ('69, '72) hypothesized that the regulation of blood flow in the F-SC may alter the sensitivity of the sensory endings. Consistent with this hypothesis, Gottschaldt et al. ('73) observed fluctuations in responsive- ness that seemed to correlate with changes in blood pressure. Thus a t least some of the fine-caliber innervation may be autonomic.

However, much if not most of this innervation is apparently not sympathetic, since removal of the superior cervical ganglion caused no noticeable elimination except in the


dermal papillae. All this fine-caliber innervation was eliminated by infraorbital nerve transection, suggesting that most may be trigeniinal in origin or parasympathetic distributing through the infraorbital nerve. Since the vibrissal follicle is presumably a site of substantial cellular proliferation, some of this innervation may subserve effer- ent functions, such as maintenance or proliferation of extracellular matrix components or fibroblasts (Kruger, '87, '88; Silverman and Kruger, '87, '88, '89; Kruger et al., '89a). Other fine-caliber innervation may have a nociceptive function typically attributed to C fibers. With the availabil- ity of anti-PGP 9.5 as a general label, double-labeling studies using lectin binding (Rice, '93), other antibodies, and axonal transport (Fundin et al., '93) are in progress to further characterize this innervation.

SVN innervation Recent physiological and axon transport studies have

demonstrated that SVN and DVN axons are separate populations (Waite and Jacquin, '92) that have different constellations of central terminations (Arvidsson, '82; Arvidsson and Rice, '91). This suggests that these two populations subserve different functions in spite of the fact that both contain Merkel and lanceolate endings as well as fine-caliber afferents (albeit to different targets), and both contain afferents with slowly and rapidly adapting characteristics (Waite and Jacquin, '92). In the absence of any obvious sensory innervation of the papillary muscles (Semba and Egger, '86), which was confirmed by PGP 9.5 immunofluorescence, the endings in the ICB have been hypothesized to function as proprioceptors for whisking (Rice et a]., '86). This hypothesis is based on the location of the ICB, which is near the attachment of the F-SC to the epidermis, and the lack of ICB innervation in species that do not whisk (Rice et al., '86a,b). A detailed ultrastructural study of the ICB revealed a combination of 1 ) en passant, circumferentially oriented lanceolate endings arising from relatively few thinly myelinated AS afferents and 2) numerous highly varicose processes arising from several dozen unmyelinated C fibers (Mosconi et al., '93). No Ruffini endings were present, as had been previously believed (Renehan et al., '86). Based on their numbers and close affiliation with collagen sheaves, Mosconi et al. ('931 hypothesized that both sets of endings were mechanoreceptors, although possible nociceptive or effector roles attributed to C fibers were also discussed. This innervation is also being further characterized in double-labeling studies in combination with anti-PGP 9.5 immunocytochemistry.

Intraepidermal endings in the intervibrissal skin

The thin-caliber individual endings are diffusely distributed throughout the epidermis and are more concentrated in the intervibrissal fur than in the rete ridge of the F-SCs. Similar endings have been observed with PGP 9.5 in the epidermis of human skin as well as with antisubstance P and anticalcitonin gene-related peptide (CGRP) in the epidermis of the rat (Vaalasti et al., '88; Dalsgaard et al., '89; Silverman and Kruger, '89; Wang et al., '90). These apparently free nerve endings are likely candidates for C-fiber nociceptors and/or high-threshold mechanoreceptors characterized electrophysiologically in the rat mysta- cia1 pad and other hairy skin in rats and other species (Kumazawa and Perl, '78; Shea and Perl, '85; Sugiura et al.,

'86; Kruger, '8'7; Kruger et al., '89a). However, the immunofluorescence technique could not always distinguish between bundles of C fibers or single larger afferents in the underlying dermis. Therefore, we cannot rule out that some of the individual intraepithelial endings might arise from A6 as well as C fibers.

Consistent with the distribution of these endings and their likely C-fiber origin, projections to outer lamina I1 of trigeminal nucleus caudalis from afferents in the intervibrissal fur were more dense than those from SVNs innervating the mouth and neck of the F-SCs (Arvidsson and Rice, '91). Outer lamina I1 has been shown to be a major site of termination for C-fiber nociceptors (Kumazawa and Perl, '73; Light and Perl, '79; Sugiura et al., '86; Kruger et al., '89b).

The clustered epidermal endings appear to be morphologically similar to the punctate, thinly myelinated mechanical nociceptors described previously in cat hairy skin (Kruger et al., '81), whose full penetration into the epidermis was not observed. Unmyelinated and thinly myelinated axons have also been shown to terminate as highly branched pencillate endings distributed just beneath the epidermis (Cauna, '73). The PGP 9.5 immunofluorescence indicates that the clustered epidermal endings in the rat mystacial pad are elaborately branched and extend through the epidermis nearly to the level of the stratum corneum. They appear to arise from A6 afferents, although it is possible that their source is a bundle of C fibers.

Because of their close proximity to the intervibrissal hairs, it is also possible that the clustered endings may function as a hair related afferent such as the A6 low- threshold D (down) hair afferents (Burgess and Perl, '73). In relation to the pencillate endings, Cauna ('73) also raised the possibility of a multimodal ending, although this has never been demonstrated physiologically. Consistent with previous observations of A6 central terminations, transganglionic transport of HRP from intervibrissal fur afferents produced label in lamina I and deep lamina I1 of nucleus caudalis. Lamina I is a principal site of A6 nociceptor and thermoreceptor terminations, whereas deep lamina I1 is principal site of D hair afferent projections (Brown, '81).

Endings affiliated with intervibrissal hairs The PGP 9 5 immunofluorescence indicates that Merkel

endings are more regularly affiliated with the mouths of guard hairs than previously observed (Rice and Munger, '86). Based on their position, they would seem to be a good candidate for the directionally sensitive, slowly adapting G2 unit (Burgess and Perl, '731, since they tend to be located to one side of the guard hairs and have an afferent in the medium to larger-caliber range. In contrast, the endings of the piloneural complex are embedded more deeply along the guard hair follicle, where they may require more rigorous hair deflections for stimulation as has been observed for electrophysiologically defined G1 and intermediate hair units (Burgess and Perl, '73).

Vascular innervation As in other tissues, the PGP 9.5 immunofluorescence

revealed extensive innervation of the vasculature (Edvins- son et al., '89; Wanget al., '90). This presumably indicates a high degree of vascular control within the mystacial pad. Studies using antibodies to a variety of neuropeptides have demonstrated at least two presumptive autonomic compo-


nents and one sensory component to vascular innervation (Lindh et al., '89). The total elimination of vascular innervation labeling after infraorbital nerve transection indicated that even the autonomic component gains access to the mystacial pad along with the infraorbital nerve branches. Surprisingly, most of the innervation remained unilateral to removal of the superior cervical ganglion on one side. This suggests that the sensory or parasympathetic components are predominant or perhaps sprout subsequent to the loss of the sympathetic axons.

CONCLUSIONS PGP 9.5 immunofluorescence revealed a far more dense

and greater variety of innervation in the mystacial pad than was previously detected by reduced silver techniques and electron microscopy. The new discoveries include primarily sets of fine-caliber innervation as well as a few endings of large-caliber afferents. Even though the trigeminal system in the rat has become one of the major models for studying the somatosensory system, none of the sensory endings has yet been definitively linked to a particular function. Fur- ther physiological, morphological, and biochemical studies are needed to obtain this information.

ACKNOWLEDGMENTS This project was supported by the Swedish Medical

Research Council, Project 5420,6540, and 8333 (to F.L.R.); by grants from IngaBritt och Arne Lundbergs Forskningss- tiftelse and Torsten och Ragnar Soderbergs Stiftelser; and by funds from the Medical and Dental Faculties of the Karolinska Institute. Excellent assistance was given by A. Dahlberg and C. Lilliehook.

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The innervation of the mystacial pad of the rat as revealed by PGP 9.5 immunofluorescence

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