Distinct central representations for sensory fibers innervating either the conjunctiva or cornea of the rat

Abstract

The laminar sheet of epithelium (e.g., skin and mucous membrane) enclosing our bodies is represented in the dorsal horns of the medulla and spinal cord. The eyeball however indents this laminar sheet and is shrouded by different layers: the cornea/sclera, the conjunctiva, and hairy skin. This involution of the orb confounds defining the central representation of the cornea and its surrounding mucosa and skin. We used herein the transganglionic transport of a cocktail of HRP conjugated to cholera toxin and wheat germ agglutinin to determine the central representation of these epithelia in the dorsal horns of the rat. The HRP cocktail was injected either into the stroma of the cornea, the mucosa of the conjunctiva, or the supraorbital and infraorbital nerves. Injections of the cornea produced dense label in the interstitial islands in the ventral medullary dorsal horn (MDH), probably lamina I, and in neuropil in the ventromedial tip of the MDH, probably lamina II. There sometimes was variable, diffuse label in the C1 dorsal horn after corneal injections but more rostral parts of the trigeminal sensory complex were never labeled. Injections of the conjunctiva densely labeled laminae I-III in the C1 dorsal horn, while laminae IV-V were diffusely labeled. Sparser reaction product also was seen in lamina I in positions similar to the cornea projection. Label was seen ventrally in subnuclei interpolaris and oralis, as well as the principal trigeminal nucleus. Projections of the infraorbital nerve included all laminae in the trigeminocervical complex as well as large portions of the rostral subnuclei in the spinal trigeminal nucleus. The projections of the supraorbital nerve were similar, but were restricted to ventral parts of the trigeminal sensory complex. In other cases the cornea was injected either after cutting the supraorbital and infraorbital nerves or the conjunctiva was injected after enucleating the eyeball. Any reaction product from corneal injections was reduced dramatically in the C1 dorsal horn after transection of the infraorbital and supraorbital nerves. Injecting the conjunctiva after enucleating the eyeball densely labeled the C1 projection to the dorsal horn, a small patch in lamina I in the MDH, as well as the rostral trigeminal complex. We propose that the cornea has but a single representation in the trigeminocervical complex in its ventral part near the caudal end of the medulla. We also propose the palpebral conjunctiva mucosa is represented in the C1 dorsal horn, and speculate that the bulbar conjunctiva overlaps with that of the cornea in lamina I. We discuss these projections in relation to the circuitry for the supraorbital-evoked and corneal-evoked blink reflexes. The relationship of the cornea and conjunctiva is intimate, and investigators must be very careful when attempting to stimulate them in isolation.

Figure 1

Brightfield photomicrograph illustrating the distribution and heterogeneity of reaction product in the dorsal horn after an injection of an HRP-labeled cocktail of tracers into the infraorbital nerve. Note the differences in the size and density of reaction product in the different laminae. See text for details. Dashed line indicates separation between lamina II and lamina III. Abbreviation: sp5, spinal trigeminal tract

Figure 2

Darkfield photomicrographs of sections cut through the rostral trigeminal sensory complex showing the transganglionic transport of a mixture of HRP-labeled molecules after its application to periorbital nerves/receptive fields. Note that NO label was present in any part of the rostral trigeminal complex after corneal injections but double bursts of label were seen after injections of both superior and inferior parts of the conjunctiva (arrows). Also note that these partially overlap with projections from the supraorbital nerve and infraorbital nerve. Abbreviations: Subnucleus interpolaris, Sp5I; subnucleus oralis, Sp5O; Principal trigeminal nucleus, Pr5; icp, inferior cerebellar peduncle; sp5, spinal trigeminal tract; m5, motor trigeminal root.

Figure 3

Darkfield photomicrographs of sections cut through the trigeminocervical complex showing the transganglionic transport of a mixture of HRP-labeled molecules after its application to periorbital nerves/receptive fields. Note the cornea representation is shown almost exclusively in lamina I in the ventral MDH (D1; arrow) with only isolated fibers noted in the DH more caudally (B1; arrow). Both the infraorbital and supraorbital nerves were transected in this case. Injections into the conjunctival mucosa intensely labeled lamina I–III of the caudal C1 DH (B2, arrows), with only sparse label seen in lamina I where the corneal fibers terminate (D2, arrow). Note that the sparse label seen in lamina IV extended beyond that in lamina II and often ‘surrounded’ unlabeled neuropil (Figs A2–C2, arrows). The infraorbital and supraorbital nerves as well as the cornea remained intact in this case. All laminae were labeled somatotopically after injections of the supraorbital (A3–D3) and infraorbital (A4–D4) nerves. Note that the label from the conjunctiva (B2) and supraorbital nerve (B3, C3) ‘fill in’ areas in laminae II and III voided by the infraorbital nerve projections (B4, C4), but that projections to laminae I, IV, and V overlap.

Figure 4

Line drawings showing the distribution of reaction product in the trigeminocervical complex after the transganglionic transport of an HRP cocktail injected into the cornea, the conjunctiva, and the supraorbital and infraorbital nerves. Numbers above sections indicate distance in millimeters relative to obex. Arrows in the cornea series point to label in presumptive lamina II while those in the conjunctiva series point to the two bursts of label seen when the whole conjunctiva was injected.

Figure 5

Darkfield photomicrographs of sections cut through the trigeminal sensory complex showing the transganglionic transport of a mixture of HRP-labeled molecules (arrows) after its application to the conjunctiva in an enucleated rat. This suggests that label noted in the caudal C1 DH (A) and rostral trigeminal sensory complex (C, D) is from the conjunctiva and that there is potential overlap with corneal projections only in lamina I in the ventral MDH (B).