Central projections of primary afferent fibers from the rat trigeminal nerve labeled with isolectin B4-HRP

The central projections of unmyelinated primary afferent fibers from the rat trigeminal nerve were investigated using retrograde and transganglionic transport of isolectin B4-horseradish peroxidase (B4-HRP). After the injection of a small amount of the tracer into various areas of the facial dermis and oral mucosa, small neuronal somata were retrogradely labeled in the trigeminal ganglion. Clearly delineated transganglionic labeling was observed exclusively in lamina II of the medullary and upper cervical dorsal horn. Its distribution showed a somatotopic organization. Fibers from the oral mucosa terminated in the region 0-1.7 mm caudal to the obex, whereas those from the facial skin terminated in the region from 1.3 mm caudal to the obex through the second cervical segment. In each of these two terminal regions, the rostral areas in the periphery were represented more rostrally than the caudal areas, and the dorsal areas in the periphery were represented more ventrolaterally than the ventral areas.     

Quantitative and somatotopic mapping of neurones in the trigeminal mesencephalic nucleus and ganglion innervating teeth in monkey and baboon

Neurones of the trigeminal mesencephalic nucleus (Mes V) and ganglion innervating the periodontium of incisor, canine and molar teeth in 10 monkeys and 10 baboons were counted and mapped using the horseradish peroxidase (Hrp), retrograde axonal transport method. Periodontal afferent neurones of all these teeth were well represented in the Mes V, although the incisors had a significantly higher number of labelled neurones than the canines or molars. The primary cell bodies of the periodontal afferents were located mainly in the caudal part of the ipsilateral Mes V from the level of the inferior colliculus to the floor of the fourth ventricle in the pons. The caudal periodontal Mes V neurones may be favourably located to make collateral connections with the trigeminal motor nucleus for jaw reflexes. Incisors and canines had a large and predominantly ipsilateral representation of Hrp-labelled neurones in the ganglion. In contrast, molar representation in the ganglion was sparse and all labelled neurones supplied ipsilateral teeth. The maxillary and mandibular teeth had a somatotopic distribution within the respective maxillary (middle) and mandibular (posterolateral) compartments of the trigeminal ganglion. It is suggested that the anterior teeth with greater connections to the Mes V and the ganglion may impart greater sensory perception and be involved in jaw reflexes to ensure a good occlusal relation during mastication, while the afferent connections of the molars may initiate complex jaw reflexes during the occlusal phase of mastication.     

Functional connectivity in the rodent trigeminal pathway grown in vitro

In explant cocultures of the rat trigeminal pathway, embryonic trigeminal ganglion cells grow their axons into peripheral cutaneous and central nervous system targets (R.S. Erzurumlu, S. Jhaveri, Target influences on the morphology of trigeminal axons, Exp. Neurol, 135 (1995) 1-16; R.S. Erzurumlu, S. Jhaveri, H. Takahashi, R.D.G. McKay, Target-derived influences on axon growth modes in explant cocultures of trigeminal neurons, Proc. Natl. Acad. Sci. USA 90 (1993) 7235-7239). In heterochronic cocultures, composed of embryonic trigeminal ganglion, embryonic whisker pad and postnatal brainstem slice, trigeminal axons develop arbors and terminal boutons in the brainstem trigeminal nuclei. To determine whether these terminal arbors establish functional connections with the brainstem neurons, we examined the electrophysiological properties of brainstem neurons and their responsiveness to trigeminal ganglion stimulation. Intracellular recordings were done in vitro on cells of the trigeminal subnucleus interpolaris (SPI) in trigeminal pathway cocultures (E15 whisker pad, E15 trigeminal ganglion, and postnatal day (PND) 0-2 brainstem slice) or in the SPI of acutely prepared brainstem slices. Electrophysiological properties of SPI cells in both preparations were virtually identical. The voltage responses of SPI neurons to intracellular current injection were highly linear suggesting they lacked a number of voltage-dependent conductances. Depolarizing current injection produced trains of action potentials with a frequency that varied with stimulus intensity. In explant cocultures, electrical activation of the trigeminal ganglion evoked EPSPs, and EPSPs coupled with IPSPs in SPI cells. Bicuculline blockade of IPSP activity resulted in long lasting EPSPs whose duration increased with membrane depolarization. These results show that brainstem trigeminal neurons can retain their functional properties in culture and establish functional connections with primary sensory afferents.     

An ultrastructural study of the relationship between sensory trigeminal nerves and odontoblasts in rat dentin/pulp as demonstrated by the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP)

Because the ultrastructure of the trigeminal sensory nerves in dentin, especially in relation to odontoblasts, remains to be clarified, we investigated the relationship between the trigeminal sensory nerves and the odontoblast processes using the anterograde axonal transport technique by injecting wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the rat trigeminal ganglion. Light microscopically, the nerves labeled with WGA-HRP were mainly concentrated at the pulpal horn, forming a nerve plexus at the subodontoblastic region and penetrating the predentin/dentin about 50 to 70 microns. Ultrastructurally, HRP reaction products were observed intra-axonally in the myelinated (A delta) and unmyelinated (C) axons in the subodontoblastic region. Most nerves lost the Schwann sheath and were naked in the predentin/dentin. The labeled varicosities were close to the odontoblast processes in the dentinal tubules. No synaptic structures could be detected between the varicosities and the odontoblasts, but a gap about 20 nm wide was found between them. One type of varicosity was a rich mitochondria-containing varicosity, while the other was a rich vesicle-containing (large dense core vesicles and small clear vesicles) one. The reaction products were also found in the extracellular spaces surrounding the axons. Sometimes the reaction products were seen in the coated pits or the endocytotic vesicles of the odontoblast processes. The present study demonstrated that nerve endings (varicosities) derived from the trigeminal ganglion were present in the dentinal tubules, and that WGA-HRP extracellularly extruded from the sensory nerves in the odontoblastic layer or predentin/dentin. These findings thus suggest that sensory nerves may have some (e.g., trophic) effect on either odontoblasts or the environment around the sensory nerves in the dentin/pulp.     

Hydroxylamine treatment increases glutathione-protein and protein-protein binding in human erythrocytes

Functional development of the rat whisker somatosensory system was studied by using the (14C) 2-deoxyglucose (2DG) metabolic mapping technique. Restrained rat pups had their left mystacial vibrissae stroked for 30 minutes and their brains harvested, sectioned, and autoradiographed from the level of the lower medulla to the frontal cortex. Subjects were tested at postnatal days (PNDs) 0-9 and 21. At birth, all subjects exhibited a significant increase of 2DG uptake in the left spinal trigeminal nuclei, the principal trigeminal sensory nucleus, and a portion of the right ventral posteromedial thalamic nucleus. The primary somatosensory cortex exhibited significant 2DG uptake contralateral to stimulation by PND 6, followed by the secondary somatosensory cortex at PND 7. The pattern of 2DG uptake in the somatosensory cortices became more intense and well defined by PND 9. Given that the somatosensory system develops in an orderly fashion from the periphery to higher brain structures, the present results show that brain structures mediating whisker sensory input are not metabolically active until projections from lower somatosensory centers are established. Neurons become responsive to whisker stimulation in the subcortical structures at birth and in the somatosensory cortex a few days later. This cortical activity follows the organization of the upper tier of thalamocortical fibers into a "barrelfield." Moreover, there is a gradual enhancement in functional activity of the vibrissa neurons at different somatosensory nuclei as rats mature. The present study elucidates the time course of functional development in the rat somatosensory system.     

Early ontogeny of catecholaminergic cell lineage in brain and peripheral neurons monitored by tyrosine hydroxylase-lacZ transgene

As the first and rate limiting enzyme in the biosynthetic pathway for catecholamine (CA) neurotransmitters, tyrosine hydroxylase (TH) is a specific phenotypic marker for CA cells in the central and peripheral nervous systems of adult animals. During embryogenesis, TH expression appears permanently within cells destined to be CA-secreting during adult life, and transiently in several cell types that will not express TH in adulthood. In this study, we examined the early ontogeny of TH expression in transgenic mouse embryos by following the expression of a lacZ reporter, driven by the tissue-specific promoter of the rat TH gene. The lacZ reporter product, beta-galactosidase (beta-gal), visualized by X-gal staining, first became apparent in primordia of sensory ganglia serving the glossopharyngeal (IX) and vagal (X) cranial nerves at embryonic day (E)9.0. Between E9.5 and E10.5, beta-gal expression extended to the remaining cranial sensory ganglia serving the trigeminal (V) and facial (VII) nerves, dorsal root ganglia, ventrolateral neural tube and sympathetic ganglion primordia. During that same period, the first beta-gal expression in the embryonic brain also appeared within distinct regions, such as the ventral prosencephalon, the ventral and dorsolateral mesencephalon and the rostral and caudal rhombencephalon. The level of beta-gal expression in all these tissues decreased at E13.5, but a distinct adult pattern of beta-gal expression started to emerge in the substantia nigra and ventral tegmental area in the central nervous system and the adrenal medulla in the periphery. Our findings indicate that the proximal 9.0 kb of the 5' promoter region of the rat TH gene encodes sufficient information to direct development of the appropriate catecholaminergic lineage cells in the central and most peripheral nervous systems during embryogenesis.     

Multiple roles of neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1 adhesion molecules during sensory innervation of the otic epithelium in vitro

To explore the role of cell adhesion molecules in the innervation of the inner ear, antibody perturbation was used on histotypic co-cultures of the ganglionic and epithelial anlagen derived from the otocyst. When unperturbed, these tissues survived and differentiated in this culture system with outgrowth of fasciculated neuronal fibers which expressed neural cell adhesion molecule and L1. The fibers exhibited target choice and penetration, then branching and spreading within the otic epithelium as individual axons. Treatment of the co-cultures, or of the ganglionic anlagen alone, with anti-neural cell adhesion molecule or anti-L1 Fab fragments produced a defasciculation of fibers but did not affect neurite outgrowth. In the co-cultures this defasciculation was accompanied by a small increase in the number of fibers found in inappropriate tissues. However, the antibodies did not prevent fiber entry to the otic epithelium. In contrast, removal of polysialic acid from neural cell adhesion molecule with endoneuraminadase-N, while producing a similar fiber defasciculation, also increased the incidence of fibers entering the epithelium. Nevertheless, once within the target tissue, the individual fibers responded to either Fab or to desialylation by spreading out more rapidly, branching, and growing farther into the epithelium. The findings suggest that fasciculation is not essential for specific sensory fibers to seek out and penetrate the appropriate target, although it may improve their tracking efficiency. Polysialic acid on neural cell adhesion molecule appears to limit initial penetration of the target epithelium. Polysialic acid as well as neural cell adhesion molecule and L1 function are involved in fiber-target interactions that influence the arborization of sensory axons within the otic epithelium.     

Genocide in Bosnia: the case of Dr. Radovan Karadzic

In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread between neurons in the central nervous system (CNS) by distinctly different routes. The steps of viral infection and spread in a single neuron type and nearby glial cells in vivo have been determined for three different strains of HSV (F, H129, and McIntyre-B). The corneas of mice were inoculated with equivalent titers of the strains. Two to 5 days later, the animals were killed. The spread of viral proteins within trigeminal cells was examined using immuno- and electron microscopy and Western blots with anti-HSV polyclonal antiserum. McIntyre-B virus infection resulted in fewer labeled ganglion cells, possibly as a result of reduced viral production in the corneal epithelium or trigeminal ganglion cells. Although the McIntyre-B strain was at least as, if not more efficient, at retrograde transport than the other strains, the amount of McIntyre-B virus that was transported in the trigeminal roots in an anterograde direction was significantly less than the other strains. Uptake by ganglionic satellite cells was qualitatively similar for the three strains, but maturation and release of virus from satellite cells to other neurons were reduced in the McIntyre-B strain. These characteristics may account for the preferential retrograde transneuronal spread of McIntyre-B strain.     

Morphological characteristics of primary sensory and post-synaptic sympathetic neurones supplying the temporomandibular joint in the cat

The cells of origin of peripheral nerves that supply the temporomandibular joint were investigated by examining the centripetal transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Following WGA-HRP injection into the temporomandibular joint capsule of the cat, a large number of labelled neurones were observed in the trigeminal and superior cervical ganglia ipsilateral to the injection site, while no labelled neurones were detected in the cervical dorsal-root ganglia. Only one labelled neurone was seen in the stellate ganglion. Labelled neurones were primarily located in the posterolateral and dorsal regions of the trigeminal ganglion, but their distribution in the superior cervical ganglion was not localized to specific regions. The labelled neurones in the trigeminal ganglion were significantly larger than those in the superior cervical ganglion but the sizes of smaller neurones overlapped, suggesting that trigeminal ganglion neurones send both myelinated and unmyelinated fibres to the temporomandibular joint. The innervation of the temporomandibular joint by somatosensory and sympathetic fibres suggests that sympathetic nerves could be responsible for allodynia or neuropathic pain caused by temporomandibular disorders.     

The expression pattern of the orphan nuclear receptor RORbeta in the developing and adult rat nervous system suggests a role in the processing of sensory information and in circadian rhythm

RORbeta is an orphan nuclear receptor related to retinoid and thyroid hormone receptors and is exclusively expressed in the central nervous system (CNS). Here we present an in situ hybridization analysis of the distribution of RORbeta mRNA in the developing and adult rat CNS. The receptor localizes to areas involved in the processing of sensory information. In the cerebral cortex, RORbeta mRNA was exclusively detected in non-pyramidal neurons of layer IV and, less so, layer V. The highest expression was found in primary sensory cortices. In the thalamus highest RORbeta expression was found in the sensory relay nuclei projecting to the respective cortical areas. In contrast, sensory projection neurons in the periphery, for example retinal ganglion cells and neurons of the sensory ganglia showed only little RORbeta expression. RORbeta is also expressed in areas involved in the generation and maintenance of circadian rhythms - the suprachiasmatic nucleus, the pineal gland and the retina. In the latter two tissues, RORbeta mRNA abundance oscillates with circadian rhythmicity peaking during the hours of darkness. RORbeta mRNA could not be detected in striatum, hippocampus, cerebellum, the motor nuclei of the cranial nerves or the ventral part of the spinal cord. During development, RORbeta is expressed in many areas as early as embryonic day (E) 15, anticipating the distribution pattern in the adult. Our data suggest that RORbeta regulates genes whose products play essential roles in the context of sensory input integration as well as in the context of circadian timing system.     

Trigeminal denervation and operant behavior in the rat.

39 male Wistar rats underwent selective sectioning of trigeminal sensory and motor nerves in 2 experiments to examine the contribution of the sensorimotor system to the control of instrumental responses reinforced with food or water. It was hypothesized that trigeminal denervation would disrupt motivational systems mediating Ss' thirst and hunger. Results show that unilateral sectioning of either Section V sensory or V motor nerves had no significant effect on leverpressing. Bilateral sectioning significantly reduced leverpressing, and the deficit was greatest in Ss with trigeminal motor nerve sectioning. It is concluded that trigeminal orosensory or oromotor denervation disrupts performance on a food- or water-reinforced task in which execution does not require a trigeminally mediated response. Findings are discussed in terms of motivational and reinforcement accounts of instrumental learning. (32 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Organization of primary afferent axons in the trigeminal sensory root and tract of the rat

A combination of immunocytochemical and electron microscopic methods were employed to assess the organization of the trigeminal (V) spinal tract in adult rats. Immunostaining was employed at the light microscopic level to selectively label large myelinated (by using antibodies against neurofilament protein) and small unmyelinated (by using antibodies against calcitonin gene-related peptide) primary afferents. In addition, the plant lectin Bandeiraea simplicifolia-I was employed to histochemically label small unmyelinated primary afferents. Results from these experiments indicated that larger myelinated axons were distributed throughout the cross-sectional extent of the V spinal tract (TrV), whereas smaller fibers were most numerous just below the pial surface. These results were confirmed with quantitative electron microscopy which demonstrated that the central portion of the V sensory root and TrV were composed primarily of larger myelinated fibers, whereas the periphery of the root and the portion of TrV just below the pial surface contained a higher percentage of smaller myelinated and unmyelinated axons. When considered together with results regarding the birthdates of neurochemically defined classes of V ganglion cells (White et al. [1994] J. Comp. Neurol. 350:397-411), these results suggest that TrV is laid down in a chronotopic fashion with the first axons forming its deeper portion and later arriving axons being added more superficially.     

Cellular localization of dopamine D2 receptor messenger RNA in the rat trigeminal ganglion

The actions of dopamine are mediated by specific, high-affinity, G protein-coupled receptors. Multiple subtypes of dopamine receptors have been characterized, including the D2 subtype (D2R). Cells within the dorsal root and petrosal ganglia of the rat express D2R messenger RNA (mRNA) consistent with D2R expression by primary sensory neurons. We hypothesized that neurons of the trigeminal ganglion express D2R mRNA. Total cellular RNA from rat trigeminal ganglia was analyzed on Northern blots under high stringency conditions. Hybridization of trigeminal ganglion RNA resulted in a signal which comigrated with striatal, pituitary, and hypothalamic D2R mRNA. To determine the distribution of D2R expressing cells in the trigeminal ganglion, cryostat sections were analyzed by in situ hybridization followed by emulsion autoradiography. We identified a population of clustered cells labeled with dense grain concentrations over their cytoplasms. These findings demonstrate the expression of D2 dopamine receptor mRNA in discrete subpopulations of neurons in the rat trigeminal ganglion. Our observations suggest that drugs active at dopamine receptors of the D2 subtype are potential modulators of sensory activity of neurons whose cell bodies reside in the trigeminal ganglion. D2 dopamine receptors may thus have a role in clinical pain syndromes involving the head and neck.     

Electrophysiologic study of propagation of excitation in the cat pterygopalatine ganglion

Action potentials were studied in nerves of the cat pterygopalatine ganglion evoked by stimuli applied to other nerves of the ganglion (in situ). It is established that most fibres passing through the ganglion are continuous sympathetic postganglionic fibres (not less than 3 groups). Most parasympathetic preganglionic fibres are synaptical on the ganglionic neurons and are represented by a group of fibres with the same threshold of excitation. Intracellular recording from single neurons of the pterygopalatine ganglion showed that stimulation of the Vidian nerve caused orthodromic spikes with short latency in one group of neurons and spikes with long latency in other neurons (2.5-6 ms and 10-40 ms respectively). Only fast fibres appear to terminate on most neurons of the ganglion, and only slow fibres do on some other neurons. Tonic activity was not observed when was performed from intact nerves of the pterygopalatine ganglion. The interacellular recording from single neurons of the ganglion showed that frequency of spike potentials either is low (1-3 per second) in some neurons or the potentials are absent in general in other neurons.     

Growth-associated protein GAP-43 and nerve cell adhesion molecule in sensory nerves of cornea

We used dual-wavelength fluorescence microscopy and monoclonal antibodies to growth-associated protein (GAP-43) and nerve cell adhesion molecule (N-CAM) to identify these proteins in nerve fibers of normal rat and rabbit corneas. Overlapping immunoreactivity of GAP-43 and N-CAM was evident along nerve fibers of rabbit corneal sections, suggesting that GAP-43 is constitutively expressed in these sensory nerves. The immune reaction of monoclonal antibody to GAP-43 and [125I]protein A was used to quantitate relative amounts of GAP-43 in the normal cornea and in a cornea subjected to a de-epithelializing wound. Collectively these findings imply that GAP-43 is axoplasmically transported from cells in the trigeminal (or superior cervical) ganglion to the cornea. Moreover, these data indicate that GAP-43 appears to be involved in the remodeling of corneal nerves that is necessary for normal innervation.     

Evolution of nerve development in frogs. I. The development of the peripheral nervous system in Discoglossus pictus (Discoglossidae)

The gross anatomical development of the peripheral nervous system (PNS) during embryogenesis and metamorphosis in the frog Discoglossus pictus is described based on whole-mount immunostaining for nerves and muscles. In the head, neurite outgrowth starts with the mandibular ramus of the trigeminal nerve at the tailbud stage. Cranial muscles are innervated as soon as they differentiate, beginning at mid-embryonic stages. During late embryonic stages, the course of the trigeminal and facial nerves becomes greatly distorted and changes again drastically during metamorphosis accompanying the reorganization of the jaw muscles. Two occipital somites and nerves develop transitorily but degenerate at late embryonic stages. The hypoglossal nerve develops by fusion of the first and second spinal nerves and receives a transitory contribution of the third and fourth spinal nerve at embryonic stages. In the trunk, several classes of Rohon-Beard neurites could be identified at embryonic stages, one of which forms intersegmental sensory nerves that prefigure the course of the sensory rami of spinal nerves at later stages. We give detailed schedules of PNS and cranial muscle development which, in comparison with data on other frog species described in a companion paper, will serve as a basis to evaluate heterochronic shift during evolution of PNS development in frogs.     

NADPH-diaphorase localization in the CNS and peripheral tissues of the predatory sea-slug Pleurobranchaea californica

The distribution of putative nitric oxide synthase (NOS)-containing cells in the opisthobranch mollusc Pleurobranchaea californica was studied histochemically via NADPH-diaphorase (NADPH-d) reduction of Nitro Blue Tetrazolium (NTB). Whole mounts and cryostat sections were prepared from the central nervous system and peripheral organs, including the buccal muscles, esophagus, salivary glands, foot, mantle, and gills. NADPH-d-positive neurons were localized predominantly to the buccal and pedal ganglia as well as to distinct areas of the cerebropleural and visceral ganglia. A variety of identified neurons were positive for NADPH-diaphorase in various central ganglia, including the metacerebral cells of the cerebropleural ganglion, putative locomotor neurons of the pedal ganglia, and buccal motoneurons. Specific staining was observed only in somata of central neurons, whereas neuropil areas remained unstained. However, NADPH-d-reactive axons were dense in buccal ganglion nerves, whereas peripheral nerves and connectives of other ganglia had few or no NADPH-d positive terminals. In the periphery, NADPH-d activity was detected only in a few neurons of the rhinophore and tentacle ganglia. NADPH-d staining was marked in the salivary glands and gills, but there was no or very little staining in the esophagus, buccal mass, and foot. Histochemical stain production required the presence of both beta-NADPH and NBT; alpha-NADPH could not substitute for beta-NADPH. The inhibitor of NOS, 2,6-dichlorophenol-indophenol, at 10(-3) M, totally abolished NADPH-d-positive staining. The apparent high activity of central NADPH-d contrasts with much lower activity in the ganglia of the related gastropod Tritonia. These data suggest a role for nitric oxide as a signal molecule in the central nervous system of Pleurobranchaea.     

Fc gamma RIIIB gene duplication: evidence for presence and expression of three distinct Fc gamma RIIIB genes in NA(1+,2+)SH(+) individuals

To investigate when the neurotrophic cytokines ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin-M (OSM), interleukin-6 (IL-6) and cardiotrophin-1 (CT-1) act on developing sensory neurones and whether they co-operate with neurotrophins in regulating neuronal survival, we studied the in vitro trophic effects of these factors on two well-characterized populations of cranial sensory neurones at closely staged intervals throughout embryonic development. The cutaneous sensory neurones of the trigeminal ganglion, which show an early, transient survival response to BDNF and NT3 before becoming NGF-dependent, were supported by CNTF, LIF, OSM and CT-1 during the late fetal period, several days after the neurones become NGF-dependent. At this stage of development, these cytokines promoted the survival of a subset of NGF-responsive neurones. The enteroceptive neurones of the nodose ganglion, which retain dependence on BDNF throughout fetal development, were supported throughout their development by CNTF, LIF, OSM and CT-1, and displayed an additional survival response to IL-6 in the late fetal period. These findings indicate that populations of sensory neurones display different developmental patterns of cytokine responsiveness and show that embryonic trigeminal neurones pass through several phases of differing neurotrophic factor survival requirements.     

A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition

The mammalian cornea receives a dense sensory innervation and a modest sympathetic innervation. The purpose of the current study was to determine if the rat cornea is also innervated by parasympathetic nerves. In the first set of experiments, unilateral combined sympathetic and sensory ocular denervations were performed in adult rats by surgical removal of the superior cervical ganglion and intracranial transection of the trigeminal ophthalmomaxillary nerve. Completeness of the denervation procedure was verified postmortem by a variety of macroscopic and immunohistochemical methods. Five to twelve days later, the corneas were serially sectioned tangential to the ocular surface and processed immunohistochemically with antibodies against the pan-neuronal markers, protein gene product 9.5 (PGP-9.5) and peripherin. In every animal a small, but constant, population of corneal and limbal immunoreactive fibers were unaffected by the surgical denervations and were concluded to derive from parasympathetic ganglia. In the second set of experiments, the origins of the rat corneal innervation were determined by applying the neuroanatomical tracer, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to the central cornea. Two to four days later, the trigeminal, superior cervical, ciliary, accessory ciliary and pterygopalatine ganglia were sectioned and analysed for the presence of HRP-labeled neurons. Examination of the corneal application site and associated ocular tissues revealed no evidence of tracer spread into neighbouring structures. Small numbers (0-6 per animal) of HRP-labeled neurons were observed in the ipsilateral ciliary and accessory ciliary ganglia of most animals. The results of these carefully controlled studies provide strong anatomical evidence of a modest parasympathetic innervation of the rat cornea.