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Vascular injection of the macromolecular tracer, horseradish peroxidase (HRP), was used to study the permeability of the odontoblast cell layer in developing and mature rat molar teeth, and to investigate the effect of cavity preparations on the permeability of this epithelioid cell layer in adult animals. HRP injected into the vascular system of normal animals 28 days of age and older was localized histochemically (from 5 to 90 min after injection) throughout the extracellular spaces of the maxillary dental pulps; however, the tracer did not penetrate beyond the tight junctions at the apical region of the odontoblast cell layer, and was absent from the predentin and dentin. In contrast, HRP injected into very young neonatal animals (e.g., day 3) resulted in free passage of HRP between odontoblasts and into the overlying predentin and dentin. When Class V cavities had been prepared in adult maxillary molars after HRP was injected into the blood stream, HRP reaction product penetrated the predentin and dentin immediately beneath the cavity preparation; however, adjacent, untraumatized areas of predentin and dentin in the operated teeth were devoid of reaction product. These results provide evidence that: (1) a physiological barrier develops between the distal segments of odontoblast cell bodies in normal rat molar teeth between days 15 and 28 of postnatal life, and this barrier prevents the passage of macro-molecules from the pulp into the predentin and dentin; and (2) this barrier is perturbed following routine restorative procedures in adult animals. [ABSTRACT FROM AUTHOR]

In vivo large-area confocal laser scanning microscopy (CLSM) of the human eye using EyeGuidance technology allows a large-scale morphometric assessment of the corneal subbasal nerve plexus (SNP). Here, the SNP of a patient suffering from diabetes and associated late complications was analyzed. The SNP contained multiple clusters of large hyperintense, stellate-shaped, cellular-like structures. Comparable structures were not observed in control corneas from healthy volunteers. Two hypotheses regarding the origin of these atypical structures are proposed. First, these structures might be keratocyte-derived myofibroblasts that entered the epithelium from the underlying stroma through breaks in Bowman's layer. Second, these structures could be proliferating Schwann cells that entered the epithelium in association with subbasal nerves. The nature and pathophysiological significance of these atypical cellular structures, and whether they are a direct consequence of the patient's diabetic neuropathy/or a non-specific secondary effect of associated inflammatory processes, are unknown.

Purpose: The subbasal nerve plexus (SNP) is the densest and most recognizable component of the mammalian corneal innervation; however, the anatomical configuration of the SNP in most animal models remains incompletely described. The purpose of the current study is to describe in detail the SNP architecture in eight different mammals, including several popular animal models used in cornea research., Methods: Corneal nerves in mouse, rat, guinea pig, rabbit, dog, macaque, domestic pig, and cow eyes were stained immunohistochemically with antiserum directed against neurotubulin. SNP architecture was documented by digital photomicrography and large-scale reconstructions, that is, corneal nerve maps, using a drawing tube attached to a light microscope., Results: Subbasal nerve fibers (SNFs) in mice, rats, guinea pigs, dogs, and macaques radiated centrally from the corneoscleral limbus toward the corneal apex in a whorl-like or spiraling pattern. SNFs in rabbit and bovine corneas swept horizontally across the ocular surface in a temporal-to-nasal direction and converged on the inferonasal limbus without forming a spiral. SNFs in the pig cornea radiated centrifugally in all directions, like a starburst, from a focal point located equidistant between the corneal apex and the superior pole., Conclusions: The results of the present study have demonstrated for the first time substantial interspecies differences in the architectural organization of the mammalian SNP. The physiological significance of these different patterns and the mechanisms that regulate SNP pattern formation in the mammalian cornea remain incompletely understood and warrant additional investigation.

This report characterizes the neurobiology of the ocular surface and highlights relevant mechanisms that may underpin contact lens-related discomfort. While there is limited evidence for the mechanisms involved in contact lens-related discomfort, neurobiological mechanisms in dry eye disease, the inflammatory pathway, the effect of hyperosmolarity on ocular surface nociceptors, and subsequent sensory processing of ocular pain and discomfort have been at least partly elucidated and are presented herein to provide insight in this new arena. The stimulus to the ocular surface from a contact lens is likely to be complex and multifactorial, including components of osmolarity, solution effects, desiccation, thermal effects, inflammation, friction, and mechanical stimulation. Sensory input will arise from stimulation of the lid margin, palpebral and bulbar conjunctiva, and the cornea.

Purpose: To delineate the clinical features and alterations in innervation and substance P (SP) content in spontaneous chronic corneal epithelial defects (SCCED) in dogs and to conduct a preliminary investigation evaluating the efficacy of topical SP, with or without insulin-like growth factor (IGF)-1, in the treatment of this disorder., Methods: Complete ophthalmic examinations, including Cochet-Bonnet aesthesiometry, were performed in 45 canine patients that had spontaneous corneal epithelial defects of at least 3 weeks' duration and with no identifiable cause. Eighteen patients had superficial keratectomies performed, and the corneal nerves were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, SP, vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH). Relative fiber densities were assessed qualitatively and quantitatively. Corneal epithelial cell and tear SP contents were determined in affected and normal dogs by an enzyme immunoassay. A preliminary open-label treatment trial of topical SP, with and without IGF-1, was conducted in 21 dogs., Results: The duration of the erosion before admittance into the study was a mean of 9.22 weeks (range, 3-52). The average patient was middle aged (mean, 9.25 +/- 1.85 years [SD]); no sex predisposition of the disease was identified. Boxers, golden retrievers, and keeshonds were overrepresented when compared with the normal hospital population. Corneal sensation was normal. Marked alterations in corneal innervation were identified in affected dogs with abnormal increased SP and calcitonin gene-related peptide (CGRP)-immunoreactive nerve plexuses identified surrounding the periphery of the epithelial defect. The SP content of epithelial cells surrounding the defect increased, whereas the tear SP content remained unchanged. Of the canine patients treated with SP, with or without IGF-1, 70% to 75% had complete healing of the defect., Conclusions: This idiopathic spontaneous corneal disease in dogs shares clinical features with chronic epithelial defects in humans. The presence of marked alterations in peptidergic innervation and positive response to topical therapy with SP suggest that SP plays a critical role in corneal wound-healing processes.

Purpose: To determine the architectural pattern and neuropeptide content of canine corneal innervation., Methods: Corneal nerve fibers in normal dog eyes were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), and tyrosine hydroxylase (TH). Relative innervation densities and distribution patterns for each fiber population were assessed qualitatively by serial line-drawing reconstructions and quantitatively by computer-assisted analyses., Results: More than 99% of all corneal PGP-9.5-immunoreactive (IR) nerves contained both CGRP and SP, approximately 30% contained TH, and none contained VIP. Distribution patterns of corneal PGP-9.5-, CGRP-, SP-, and TH-IR nerves were indistinguishable, except that TH-IR fibers were absent from the corneal epithelium. Morphologically, canine corneal innervation consisted of a rich anterior stromal plexus, divided on the basis of morphologic criteria into anterior and posterior levels, and a rich epithelial innervation, characterized by large numbers of horizontally oriented, basal epithelial "leash" formations. Leash axons in all quadrants of the corneal epithelium oriented preferentially toward a common locus in the perilimbal cornea., Conclusions: The results of this study demonstrate for the first time the detailed architectural features, distinctive basal epithelial leash orientations, and peptidergic content of canine corneal innervation. The normal innervation pattern described in this study will provide other investigators with essential baseline data for assessing corneal nerve alterations in canine patients with spontaneous chronic corneal epithelial defects (SCCED) and other ocular diseases or injuries.

Purpose: To determine the ability of norepinephrine to modulate proliferation, adhesion, and migration of SV-40 transformed human corneal epithelial cells., Methods: Assays were performed using SV-40 transformed human corneal epithelial cells. For proliferation assays, cells were plated in 96-well plates coated with fibronectin and collagen (FNC). A dose-response curve was generated for norepinephrine in concentrations of 100 nM-100 microM. The cell number in each well was evaluated using the fluorochrome Calcein AM (an intracellular esterase cleavage substrate), and fluorescence was determined using an automated fluorescent plate reader. For cell adhesion, 25 x 10(-3) cells were plated onto FNC-coated 96-well plates, incubated in 10 nM-100 microM norepinephrine for 90 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-microm pore size and FNC-coated filters. Cells (250 x 10(3)) were added to the upper chamber, incubated for 18 h in the presence of factors, after which time the cells that had migrated through the filter were quantitated. The toxicity of norepinephrine was evaluated using a standard Live/Dead assay employing the combined fluorochromes of ethidium homodimer (to indicate dead cells) and Calcein AM (to indicate viable cells). Varying concentrations of norepinephrine were added, and the cells incubated for 3 h and the fluorometric assay performed., Results: Norepinephrine stimulated corneal epithelial cell proliferation and migration over a wide range of concentrations. It did not modulate cell adhesion and demonstrated cell toxicity only at the highest (supraphysiologic) concentration tested., Conclusions: Norepinephrine is normally found in the cornea and may be important in the maintenance of normal corneal homeostasis and in wound-healing processes.

Corneal nerves regulate corneal epithelial integrity, proliferation, and wound healing. The mechanisms by which the nerves mediate their effects remain poorly understood; however, the release of biologically active neuropeptides has been hypothesized. The purpose of the current investigation was to determine the relative densities, distribution patterns, and origins of rat corneal nerves containing each of eight different neuropeptides, calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), neuropeptide Y (NPY), methionine-enkephalin (M-ENK), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), and cholecystokinin (CCK). In the first set of experiments, immunohistochemical demonstrations of the above neuropeptides were performed on free-floating corneal sections cut tangential to the corneal surface. The results showed that six of the peptides, CGRP, SP, GAL, NPY, M-ENK, and VIP were present in rat corneal nerves. The innervation patterns of corneal nerves containing each of these six peptides were then documented by mapping all fibers in serial sections from select corneal quadrants onto a series of line drawings by using a drawing tube. In the second set of experiments, the origins of the corneal peptidergic nerve fibers were determined by selective ocular denervations. Unilateral combined sensory and sympathetic ocular denervations or unilateral sympathetic ocular denervations were performed in adult rats by transecting the ophthalmomaxillary nerve and/or removing the superior cervical ganglion. After 5-7 days, each of the ipsilateral corneas was sectioned and processed immunohistochemically for the presence of one of the six peptides found in experiment one, and the fibers that survived the ocular denervations were plotted onto line drawings. Ocular denervations revealed that corneal peptidergic nerves have sensory (CGRP, SP, and GAL), sympathetic (NPY), and parasympathetic (GAL, NPY, M-ENK, and VIP) origins. The results of this investigation have shown that the peptidergic innervation of the rat cornea is more extensive and complex than previously reported. This is the first investigation to show the presence of GAL in the rat cornea, and the first to demonstrate the presence of NPY-, VIP-, and M-ENK-IR nerve fibers in the cornea of any species., (Copyright 1998 Academic Press Limited.)

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., (Copyright 1998 Academic Press Limited.)

Purpose: The authors determined the effect of topically applied substance P (SP) on the rate of corneal epithelial wound closure in the rabbit., Methods: Uniform circular lesions, 6.5 mm in diameter, were made bilaterally in the corneal epithelium of 24 rabbits using N-heptanol. Substance P was applied repeatedly to one eye, and the SP1-7 fragment was applied to the contralateral (control) eye until wound closure was obtained. Three concentrations of peptide solution (5 x 10(-5) M, 5 x 10(-4) M, and 5 x 10(-3) M) were tested in separate groups of eight animals each. An additional eight animals received topical applications (5 x 10(-7) M) of the neurokinin-1 (NK1)-specific SP receptor antagonist CP-99,994-01 or its ineffective enantiomer CP-100,263-01. The mean rates of wound healing for each group of experimental and control eyes were determined by linear regression and analyzed by analysis of variance., Results: There were no statistically significant differences in the mean rates of wound closure (range, 0.083 to 0.106 mm/hour) between experimental- and control-treated corneas for any of the four groups tested., Conclusions: The topical application of SP or its NK1 receptor antagonist has no significant effect on the rate of corneal epithelial wound closure in the rabbit.

Purpose: To determine the effect of ocular sympathetic nerves on corneal epithelial proliferation in the rat., Methods: Osmotic pumps filled with bromodeoxyuridine (BrdU) were implanted subcutaneously in adult rats to label corneal epithelial cells entering the S-phase of the cell cycle during a 24-hour period. Corneas in some animals were wounded with n-heptanol. Several days or weeks before pump implantation, animals were subjected to either unilateral superior cervical ganglionectomy (SCGectomy), unilateral transection of the cervical sympathetic trunk (sympathetic decentralization), bilateral SCGectomy plus unilateral topical norepinephrine administration, or unilateral SCGectomy plus systemic capsaicin administration. Differences in BrdU-labeling indices between experimental and control eyes in each group were determined from cell counts on paraffin sections., Results: Superior cervical ganglionectomy and sympathetic decentralization significantly decreased epithelial proliferation in nonwounded and wounded corneas. Topical applications of norepinephrine to eyes that had been deprived of their sympathetic innervation completely reversed the antiproliferative effect of ocular sympathectomy. Systemic administration of the neurotoxin capsaicin, in conjunction with unilateral SCGectomy, did not alter the proliferative rate; comparison was made to animals that received unilateral SCGectomy only., Conclusions: Ocular sympathetic nerves stimulate rat corneal epithelial proliferation under normal physiological conditions and during corneal wound healing. The promotion of DNA synthesis by these nerves occurs independently of functional interactions with capsaicin-sensitive, ocular sensory nerves and appears to be related, at least in part, to the release of norepinephrine.

The aims of this study were: (1) to demonstrate how reproducible variations in incomplete anesthesia of the inferior alveolar nerve can be used as a guide to locate the etiologic sites of referred trigeminal pain emanating from the mandible; (2) to describe the salient histopathologic features of a lowgrade, nonsuppurative osteomyelitis seen in this patient population. Forty-six patients with idiopathic facial pain were subjected to a specific protocol of local anesthetic injections to sequentially block branches of the mandibular nerve to determine the effects on his/her pain. If this significantly reduced or altered the pain on three separate appointments, then exploratory surgery was conducted near identified zones of unanesthetized gingiva. Blocking (92%), bridging (4%), and divergence (4%) were observed patterns of anesthetic resistance of the mucogingival tissues used to categorize the incomplete anesthesia. A 100% correlation was found between the identified zones of unanesthetized gingiva and the discovery of intramedullary pathology. Medullary fibrosis with ischemic and degenerative changes in the cancellous bone were common findings, along with chronic inflammatory cell infiltrates and clusters of lymphocytes. It is concluded that Ratner's method of diagnostic anesthesia be implemented when searching for occult pain producing pathology of the jaws.

Purpose: To determine the effect of diabetes mellitus on the levels of substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP) in the rat cornea and iris., Methods: Corneas and irides from control and diabetic rats were processed for neuropeptide radioimmunoassay 3 months after induction of diabetes with streptozotocin. Corneas and irides also were processed for SP and CGRP immunohistochemistry and were evaluated qualitatively., Results: The radioimmunoassay data revealed no significant differences in either the content or concentration of SP, CGRP, and VIP between control and diabetic corneas. In contrast, iridial levels of CGRP and SP were significantly increased by 38% and 256%, respectively, in the diabetic animals. Iridial VIP levels were unchanged in the diabetic versus control groups. Immunohistochemical demonstrations of corneal and iridial SP- and CGRP-immunoreactive fiber plexuses were indistinguishable on the basis of purely qualitative criteria., Conclusions: The results of this study have demonstrated a target- and peptide-specific effect of short-term diabetes on SP and CGRP expression in ocular nerves of the anterior eye segment. The absence of demonstrable changes in corneal neuropeptide levels argue against the theory that corneal abnormalities seen in clinical diabetes are caused, in part, by deficits in synthesis or axonal transport of "trophic" peptides in corneal sensory nerves. In contrast, elevated iridial SP and CGRP levels may be responsible for reported clinical deficits in pupillary diameter regulation.

The sympathetic innervation of the mammalian cornea is thought to play an important role in the regulation of epithelial ion transport, mitogenesis, and wound healing following corneal injuries. Anatomically, the three-dimensional organization and relative density of corneal sympathetic innervation in many species remains inadequately described. In the present study, the sympathetic innervation of five different mammals (guinea pig, rat, mouse, hamster, and human) was studied in corneas sectioned parallel to the main axis of fiber orientation by labeling the fibers immunohistochemically with antiserum against tyrosine hydroxylase and an avidin-biotin-diaminobenzidine technique. The results showed that each species displayed a distinctive pattern and density of tyrosine hydroxylase immunoreactive (TH-IR) corneal innervation that was unique to that species. The overall level of TH-IR innervation was highest in the guinea pig, moderate in the human, hamster, and rat, and lowest in the mouse. In all species examined, TH-IR nerves were most numerous in the corneoscleral limbus where they either formed intimate associations with blood vessels or coursed through the connective tissue matrix apparently unrelated to vascular elements. Other TH-IR nerves entered the cornea proper in radially directed stromal nerve bundles to give rise to subepithelial plexuses of varying complexity. Occasional intraepithelial penetrations were observed in the guinea pig, human, and rat. Removal of the superior cervical ganglion resulted in the total loss of TH-IR staining from guinea pig and hamster corneas and in the substantial but incomplete loss of TH-IR staining from rat and mouse corneas, thus demonstrating their predominantly sympathetic origin. Combined sympathetic and sensory ocular denervation in the rat eliminated almost all corneal and limbal TH-IR immunostaining, thus suggesting a minor TH-IR sensory component in this species. In agreement with this conclusion, small numbers of TH-IR sensory neurons and an abundance of TH-IR fibers were observed in the trigeminal ganglia of the rat and guinea pig. Removal of the rat main ciliary ganglion resulted in the loss of additional TH-IR fibers from the chamber angle and iris, thereby confirming a partial parasympathetic contribution to the rat iridial TH-IR innervation. Following unilateral removal of the superior cervical ganglion in rats and guinea pigs, the contralateral cornea contained increased numbers of TH-IR nerves, suggesting an upregulation of tyrosine hydroxylase (TH) expression in some contralateral axons.(ABSTRACT TRUNCATED AT 400 WORDS)

Corneal sensory and sympathetic nerves exert opposing actions on corneal mitogenesis and wound healing. The mechanisms by which these nerves exert their actions are unknown; however, the release of axonally transported neuropeptides has been postulated. In the present study, we investigated changes in innervation densities of calcitonin gene-related peptide (CGRP-) and tyrosine hydroxylase (TH-)immunoreactive (IR) nerves of the rat cornea following neonatal capsaicin administration, and the relationships between these changes and the development of neuroparalytic keratitis. Newborn rats were injected with capsaicin on each of the first 3 days of life. Forty-eight hours after the last injection, corneal CGRP immunostaining had totally disappeared from the cornea, whereas TH immunostaining was relatively unaffected. Over the next several weeks, a dramatic reinnervation of the cornea took place. By 6-8 weeks both the CGRP- and TH-IR corneal innervation density in the capsaicin-treated animals exceeded that of age-matched control or normal animals; that is, the corneas had become "hyper-reinnervated." The pattern of innervation that returned was grossly abnormal and was characterized by the presence of a bizarre subepithelial plexus of fine stromal sprouts; an abundance of myelinated axons; and complex, atypical, epithelial leash morphologies. Retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) from the central cornea in control and capsaicin-treated adult animals labeled an average of 143 and 47 trigeminal ganglion cells, respectively (with mean diameters of 25.7 +/- 0.49 microns and 34.3 +/- 0.72 microns), suggesting a 67% decrease in corneal afferent neurons in the capsaicin-treated animals. Transection of the ophthalmomaxillary nerve in adult capsaicin-treated animals completely eliminated corneal CGRP-IR staining, and extirpation of the superior cervical ganglion resulted in the loss of 70-80% of corneal TH-IR nerves, thus demonstrating the sensory and predominantly sympathetic origins, respectively, of these fiber populations. Chronic keratitis and neovascularization developed in the capsaicin-treated animals by approximately 3 weeks of age, achieved a maximum intensity between 4 and 6 weeks, and showed some gradual improvement thereafter. However, the keratitis never completely disappeared, even after 13 months. In conclusion, these data show that corneal sensory (CGRP-IR) and sympathetic (TH-IR) nerve fibers undergo extensive sprouting following partial corneal sensory denervation with the neurotoxin capsaicin. However, the resultant "hyper-reinnervation" is morphologically abnormal and, for reasons unknown, functionally incapable of preventing or totally reversing the keratitis.

The development of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerves was studied in neonatal and adult rat corneas stained immunohistochemically according to an avidin biotin peroxidase procedure. At birth, rat corneas already contained dense plexuses of CGRP-LI nerve fibers. Most of the nerves entered the cornea in 12-15 prominent stromal nerve bundles located at regular intervals around the circumference of the cornea. Fibers in these bundles entered the epithelium approximately midway between the limbus and the center of the cornea and supplied extensive central and pericentral areas of the tissue. In addition, smaller numbers of axons entered the cornea individually and in small fascicles located in between the larger bundles and supplied mainly peripheral territory. In the epithelium, the CGRP-LI nerves formed a complex, highly anastomotic meshwork that ramified uniformly throughout central and peripheral areas of the tissues. Fibers in the plexus gave origin to numerous short, stout terminal axons that extended into the adjacent epithelium in all directions with no preferred orientation. During the first week of neonatal life, several changes in CGRP-LI innervation occurred: 1) the innervation density of the central and pericentral cornea increased relative to the peripheral cornea; 2) intraepithelial axons became progressively longer, increased in branching complexity, and oriented preferentially towards the center of the cornea; and 3) a dense innervation of the corneoscleral limbus and, in particular, the branches of the marginal artery, developed. Midway through the second week of life, immature versions of corneal epithelial "leashes," the dominant feature of the adult corneal innervation, were first observed. Over the next 10 days, the leash formations in the central and pericentral cornea gradually became more complex and gave rise to greater numbers of terminal axons, compared to developing leashes in the peripheral cornea. The mature pattern of corneal CGRP-LI innervation was reached on day 21 and remained constant (except for compensatory growth-related elongation of axons) for at least the first 6 months of life. Transection of the ophthalmomaxillary nerve or neonatal administration of the sensory neurotoxin capsaicin resulted in the total loss of CGRP-LI staining from the cornea. In contrast, removal of the superior cervical ganglion had no effect on corneal CGRP-LI staining. The extraordinary density and complexity of the CGRP-LI innervation of the rat cornea demonstrated at all stages of development in this study suggests that these nerves may play important roles in corneal sensory, reflex, and trophic functions.

The sensory innervation of the rat kidney and ureter was investigated in wholemount preparations and sectioned materials by labeling the afferent nerve fibers with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) transported anterogradely from dorsal root ganglia. Labeled fibers were seen in large numbers in the ureter and in the lining of the renal pelvis, where they were located in the adventitia, smooth muscle, subepithelial connective tissue, and epithelium. Most of the fibers in the ureter and ureteropelvic junctional zone traveled parallel to the long axis of the organ. In contrast, fibers in the widest part of the funnel-shape renal pelvis were oriented predominantly in a circumferential fashion. Many of the pelvic afferents were extremely fine and appeared to terminate as free nerve endings. Modest networks of labeled axons were also observed around branches of the renal artery; the greatest innervation was supplied to the distal portions of the interlobar arteries and to the arcuate arteries. Only single axons were observed around the interlobular arteries, and very few fibers were seen around afferent arterioles or near glomeruli. In contrast to the arteries, branches of the renal vein were relatively sparsely innervated. Occasional labeled fibers entered the renal cortex and formed intimate associations with renal tubules; however, the vast majority of renal tubular elements were not contacted by labeled sensory fibers. Labeled fibers were never observed in the renal medulla or in the papilla. The present study represents the first time that the sensory innervation of the kidney and ureter has been investigated by using a highly specific anterograde nerve tracing technique. The pattern of innervation demonstrated here reveals an anatomical configuration of ureteral and renal pelvic sensory nerves consistent with a role in detection of ureteral and pelvic pressure and chemical changes and a renal vascular sensory innervation that may monitor changes in renal arterial and venous pressure and chemical content. Still other renal afferent nerve endings may signal renal pain.

The peptidergic and serotoninergic innervation of the rat dura mater was investigated by reacting dural wholemounts immunohistochemically with antibodies to calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and serotonin (5-HT). CGRP and SP innervations of the dura were robust and the patterns of distribution of these neuropeptides were essentially the same. The majority of the fibers were perivascular and distributed to branches of the anterior and middle meningeal arteries and to the superior sagittal and transverse sinuses. Other CGRP/SP fibers appeared to end "free" within the dural connective tissue. NPY-immunoreactive fibers were extremely numerous and also distributed heavily to the branches of the meningeal arteries, the venous sinuses, and to the dural connective tissue. The pattern of NPY innervation resembled in many ways that of CGRP/SP; however, NPY innervation of the sinuses was greater and NPY perivascular fibers supplying the meningeal arteries formed more intimate contacts with the walls of the vessels. The pattern of VIP innervation was, in general, similar to that observed for the three previous neuropeptides; however, the overall density was considerably less. Small to moderate numbers of serotoninergic nerve fibers were observed in some, but not all, of the duras processed for 5-HT. The latter fibers were almost exclusively perivascular in distribution. Dural mast cells were prominently stained in the 5-HT preparations because of their serotonin content. Mast cells were also labeled in a nonspecific fashion in some of the tissues reacted immunohistochemically for neuropeptides; some of them were located in close apposition to passing nerve fibers. This study represents, to our knowledge, the first comprehensive work on the peptidergic and serotoninergic innervation of the mammalian dura mater. The results should increase our understanding of the roles that these fibers play in normal dural physiology and of their potential interactions in the pathogenesis of vascular headache.

The central projections of rat trigeminal primary afferent neurons to various "non-trigeminal" areas of the central nervous system were examined by labeling the fibers with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) transported anterogradely from the trigeminal ganglion. This technique produced a clear and comprehensive picture of trigeminal primary afferent connectivity that was in many ways superior to that which may be obtained by using degeneration, autoradiography, cobalt labeling, or HRP transganglionic transport techniques. Strong terminal labeling was observed in all four rostrocaudal subdivisions of the trigeminal brainstem nuclear complex, as well as in the dorsal horn of the cervical spinal cord bilaterally, numerous brainstem nuclei, and in the cerebellum. Labeling in the ipsilateral dorsal horn of the cervical spinal cord was very dense at C1, moderately dense at C2 and C3, and sparse at C4-C7. Numerous fibers crossed the midline in the medulla and upper cervical spinal cord and terminated in the contralateral pars caudalis and dorsal horn of the spinal cord from C1-C5. The latter axons terminated most heavily in the mandibular and ophthalmic regions of the contralateral side. Extremely dense terminal labeling was observed in the ipsilateral paratrigeminal nucleus and the nucleus of the solitary tract, moderate labeling was seen in the supratrigeminal nucleus and in the dorsal reticular formation, and small numbers of fibers were observed in the cuneate, trigeminal motor, lateral and superior vestibular nuclei, and in the cerebellum. The latter fibers entered the cerebellum in the superior cerebellar peduncle and projected to the posterior and anterior lobes as well as to the interposed and lateral deep cerebellar nuclei. Most projections in this study originated from fibers in the dorsal part of the spinal tract of V, suggesting a predominantly mandibular origin for these fibers. Projections from the ophthalmic and maxillary divisions, in contrast, were directed mainly to the cervical spinal cord bilaterally, to contralateral pars caudalis, and to certain areas of the reticular formation. In conclusion, this study has demonstrated that somatosensory information from the head and face may be transmitted directly to widespread and functionally heterogeneous areas of the rat central nervous system, including the spinal cord dorsal horn, numerous brainstem nuclei, and the cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

The purpose of the present investigation was to determine if the horseradish peroxidase (HRP) technique could be used as a method for labeling sensory nerve fibers (specifically, tooth pulp afferents) for detailed ultrastructural analyses. HRP injected into the trigeminal ganglion of adult rats was taken up by ganglion cell bodies and transported anterogradely to their peripheral endings in the dental tissues. Following perfusion-fixation, the teeth were decalcified in EDTA, sectioned, reacted for HRP activity according to the tetramethylbenzidine (TMB) technique, and processed for electron microscopy. The HRP-TMB reaction product was clearly visible within most of the axons in the dental pulp, appearing as conspicuous, rectangular shaped aggregates of fine rods or needles.

The purpose of the present investigation is to introduce the enzyme horseradish peroxidase (HRP) for the study of the morphology and peripheral distribution of sensory nerve endings. HRP was injected into the trigeminal ganglion or trigeminal brainstem nuclear complex (TBNC) in separate adult rats. HRP injected into the trigeminal ganglion was taken up by the neuronal perikarya and transported anterogradely in massive amounts to sensory nerve endings in the cornea, vibrissal hair follicles, tooth pulps, and periodontal ligaments. HRP injected into the TBNC was taken up by trigeminal primary afferent fibers that terminated there and transported transganglionically, i.e., past or through the trigeminal ganglion, to peripheral sensory endings. The results of the present study demonstrate for the first time that: (1) anterograde HRP transport is a highly successful method of labeling with an intracellular marker trigeminal sensory endings in a variety of oro-facial tissues, and (2) trigeminal primary sensory neurons possess intra-axonal transport mechanisms by which HRP, and possibly other substances, taken up in the central nervous system may be transported to the periphery.

The methods of transganglionic transport of horseradish peroxidase (HRP) and horseradish peroxidase--wheat germ agglutinin (HRP-WGA) were used to determine the location within the trigeminal ganglion of the primary afferent neurons that innervate the rat central cornea, and the brainstem and spinal cord termination sites of these cells. In each of 18 animals, solutions of HRP or HRP-WGA were applied to the scarified corneal surface and allowed to infiltrate into the corneal epithelium and stroma for 15 minutes. Postmortem examination of the corneal whole mounts from the experimental animals, and of corneas and neural tissues from several control animals, showed that the HRP/HRP-WGA remained confined to the central cornea with no spread into adjacent intra- or extraorbital tissues. HRP-labeled corneal afferent somata were located in the dorsal part of the ophthalmic region of the ipsilateral trigeminal ganglion. The central fibers of the corneal afferent neurons projected very heavily to interstitial nuclei of Cajal in the spinal tract of V at the level of caudal pars interpolaris and rostral pars caudalis, lightly to the pars caudalis/C1 transition zone, and sparsely to the dorsal horn of spinal cord segments C1-C3. The trigeminal main sensory nucleus, pars oralis, the rostral three-fourths of pars interpolaris, and an extensive midregion of pars caudalis were totally devoid of reaction product. Terminal fields in caudal pars caudalis and in the spinal cord dorsal horn were concentrated largely in the outer half of lamina II, with lesser accumulations in lamina I, the deeper half of lamina II, and in lamina III. The present study demonstrates for the first time by means of an anatomical tracing procedure the brainstem termination sites of corneal afferent neurons in the rat. The patchy, discontinuous nature of the corneal afferent projection to the caudal trigeminal brainstem nuclear complex (TBNC), and the total lack of corneal projections to rostral subdivisions of the TBNC, provide an exception to the general rule of trigeminal organization in which most areas of the head and face are represented as continuous columns throughout the rostrocaudal extent of the ipsilateral TBNC.

The central projections of five peripheral branches of the trigeminal nerve were investigated by the method of transganglionic transport of horseradish peroxidase (HRP). In separate animals, the corneal, supraorbital, infraorbital, mental, or inferior alveolar branches were transected and soaked in concentrated solutions of HRP. Forty-eight to 72 hours after surgery, the brain-stem, upper cervical spinal cord, and trigeminal ganglia were perfusion-fixed and processed according to the tetramethylbenzidine technique. The results show that trigeminal primary afferent neurons which innervate the cornea project mainly to the levels of caudal pars interpolaris and caudal pars caudalis. In contrast, trigeminal primary afferent neurons whose peripheral processes course through the supraorbital, infraorbital, or mental nerves project most heavily to the trigeminal main sensory nucleus, pars interpolaris, and the rostrocaudal middle three-fifths of pars caudalis. Trigeminal primary afferent neurons which give origin to the inferior alveolar nerve project heavily and in approximately equal numbers of all rostrocaudal levels of the trigeminal brainstem nuclear complex (TBNC). A small number of fibers from each of the latter four cell populations project directly to the contralateral C1-C2 dorsal horn. A small number of fibers from each cell population studied end in the reticular formation immediately adjacent to the spinal nucleus of V. It is concluded that the cornea and facial skin regions of the cat are represented nonuniformly along the rostrocaudal length of the TBNC.

Horseradish peroxidase-wheat germ agglutinin injected into the superior cervical ganglion is taken up by the neuronal perikarya and transported anterogradely in large quantities into peripheral fibers and axonal terminals in a variety of orofacial and cerebral tissues, including the iris, cornea, pineal gland, facial skin and the adventia of cerebral and facial blood vessels. Subsequent histochemical processing according to the tetramethylbenzidine procedure produces a unique, highly visible intraaxonal marker that makes the identification and 3-dimensional tracing of the sympathetic nerve fibers clear and unambiguous at both the light and electron microscopic levels.

The method of transganglionic transport of horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) was used to determine the location within the monkey trigeminal ganglion of the primary afferent neurons that innervate the cornea, and the brainstem and spinal cord termination sites of these cells. In each of four animals. Gelfoam pledgets were saturated with 2% HRP-WGA in saline and applied to the scratched surface of the central cornea for 30 minutes. Postmortem examination of the corneal whole mounts revealed that the tracer solution remained confined to approximately the central one-fourth of the cornea with no spread into the peripheral cornea or limbus. Seventy-two to 96 hours after tracer application, 126-242 labeled cell bodies were observed in the medial region of the ipsilateral trigeminal ganglion. The majority of neurons were concentrated in an area of the ganglion that lay directly caudal to the entering fibers of the ophthalmic nerve, but smaller numbers of cells lay somewhat more laterally, near the region where the ophthalmic and maxillary nerves come together. A very small number of neurons in one animal innervated the cornea by sending their fibers into the maxillary nerve. HRP-WGA-labeled terminal fields were present to some extent in all four major rostrocaudal subdivisions of the ipsilateral trigeminal brainstem nuclear complex (TBNC), but the size of the terminal fields and the intensity of labeling differed markedly from one level of the TBNC to the next. Labeled fibers projected heavily to the transitional zone between caudal pars interpolaris and rostral pars caudalis (i.e., the "periobex" region of the TBNC) and moderately to the trigeminal main sensory nucleus, pars oralis, and caudal pars caudalis at the level of the pyramidal decussation. Remaining areas of the TBNC, including rostral pars interpolaris and the midlevel of pars caudalis, received few, if any, corneal afferent projections. Occasional labeled fibers were observed in the dorsal horn of C1 and in the rostral half of C2. It is hoped that data generated in the current investigation of nonhuman primates will contribute to a better understanding of the neural substrates that subserve corneal sensation and the blink reflex in humans.

Horseradish peroxidase-wheat germ agglutinin (HRP-WGA) applied to proximal stumps of tooth pulp primary afferent neurons in rats was taken up and transported transneuronally to neurons in the ipsilateral trigeminal brainstem nuclear complex. The results of this study suggest that HRP-WGA transport may be a novel means of labeling both primary and higher order neurons that transmit tooth pulp sensory information in the rat and may be used to investigate the fine structure and synaptic contacts of central nervous system neurons that receive tooth pulp afferent input.

The ability to use the tetramethylbenzidine (TMB) method for studying neuronal connections at the electron microscopic level is often difficult because the conditions of osmification and dehydration used in processing the tissue may result in significant loss and/or decreased electron density of the reaction product. In the present study, we report that stabilization of TMB reaction product with 5% ammonium molybdate (AM) prior to osmificating the tissue results in the formation of TMB-AM crystals that are many times more electron dense and resistant to ethanol extraction than non-stabilized TMB crystals. The nature of the chemical interaction that underlies the stabilization of TMB by AM is uncertain, but it may involve the formation of an insoluble salt between molybdic ions and the TMB polymer. The use of this simple procedure increases the sensitivity of the TMB procedure at the electron microscopic level and may be used to label neuronal pathways in the peripheral and central nervous systems with equal success.

The sympathetic innervation of the rat cornea was studied by using the method of intraaxonal transport of horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA). In the first set of experiments, the relative number of superior cervical ganglion neurons that innervate the rat central cornea was estimated by the method of retrograde HRP-WGA transport. Following tracer application to the scarificed central corneal surface, 49-198 labeled neurons were observed in the ophthalmic region of the ipsilateral trigeminal ganglion and zero to four cells in the rostral pole of the ipsilateral superior cervical ganglion. In the second set of experiments, the three-dimensional distribution and termination sites of the corneal sympathetic nerve fibers were investigated by the technique of HRP-WGA anterograde transport from the superior cervical ganglion. HRP-WGA-labeled axons in corneal whole mounts were identified by the presence within their axoplasm of linear arrays of HRP-TMB reaction product, and their distribution was plotted faithfully onto line drawings made with a drawing tube attachment. Large numbers of HRP-labeled fibers were found in all animals within the corneoscleral limbus where the majority were associated with blood vessels. Fewer fibers (zero to 14 per animal) entered the cornea proper. The latter fibers entered the peripheral cornea in the deep to middle layers of the stroma and ascended into progressively more superficial layers as they coursed centrally. The majority of fibers branched infrequently in the peripheral cornea and increased in branching complexity near the central cornea. HRP-labeled axonal varicosities suggestive of terminal and preterminal expansions were located preferentially in the subepithelial layer of the corneal stroma and in the basal epithelium. Approximately 75% of the axonal varicosities were located in the central half of the cornea. The results of the current investigation reveal that the rat cornea is innervated sparsely by sympathetic nerve fibers derived from the superior cervical ganglion. These data provide additional support to current theories that corneal sympathetic nerve fibers may influence select aspects of corneal physiology, including ion transport and hydration, mitogenesis and wound healing, and sensitivity.

The origins of the renal efferent and afferent nerves in 5 cynomolgus monkeys (Macaca fascicularis) were studied by using the retrograde transport of horseradish peroxidase (HRP) and horseradish peroxidase-wheat germ agglutinin (HRP-WGA). The cut ends of the right renal nerves were soaked for 30-45 min in solutions consisting of 15% HRP and 1% HRP-WGA. Three or four days later the animals were killed and the tissues examined for the presence of retrogradely labeled neurons, HRP-filled cells were observed, with rare exceptions, only in ganglia ipsilateral to the side of tracer application. Renal efferent neurons (4648-14565 cells per animal) were found in relatively equal numbers in prevertebral and paravertebral (sympathetic chain) ganglia. Labeled prevertebral cells were distributed among the renal (52%), aorticorenal (32%) and superior mesenteric (16%) ganglia, whereas labeled paravertebral neurons were mainly located in chain ganglia T11-L3, with 94% of these located in L1-3. Labeled renal sensory neurons (31-543 per animal) constituted less than 5% of all labeled cells and were found in ipsilateral dorsal root ganglia T10-L3, with (80%) in T12 and L1. The labeled sensory neurons ranged from 18-64 microns in diameter (X = 32.4 microns). With the exception of a single cell in one animal, no labeled neurons were observed in the nodose ganglia. Many parallels were observed between the organization of the renal plexuses of macaques and humans, suggesting the utility of the non-human primate as an experimental model for functional studies of renal innervation in humans.

Transganglionic transport of horseradish peroxidase (HRP) or horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) was used to map in detail the central projections of trigeminal primary afferent neurons that innervate the dental pulp organ of the rat. In each of ten animals, 0.5-2.0 microliters of enzyme solution was injected into the pulp chamber of the first maxillary molar tooth. Postmortem examination of the decalcified teeth in all cases showed that the HRP/HRP-WGA remained confined to the pulp chamber and pulp roots, with no spread of enzyme into periapical tissues. HRP-labeled tooth pulp afferent fibers projected to all four rostrocaudal subdivisions of the ipsilateral trigeminal brainstem nuclear complex (TBNC) and to the upper cervical spinal cord. The labeled terminal fields formed a column that stretched relatively uninterrupted from just caudal to the rostromedial tip of the trigeminal principal sensory nucleus to at least the C2 segment of the spinal cord. The density of the afferent projection varied markedly from one rostrocaudal level of the TBNC to the next but was heaviest in an area encompassing the caudal one-half of the principal sensory nucleus and the rostral two-thirds of pars oralis. Fibers projected only lightly to pars caudalis, where they terminated preferentially in laminae I, IIa, and the junctional zone between laminae IV and V. HRP-labeled terminals in C1 and C2 were located almost exclusively in laminae I. In the dorsoventral axis, the terminal fields in the TBNC were located in a surprisingly dorsal part of the complex, well within what has been shown by others to be largely an area of termination for mandibular division fibers. Most fibers ended in medial parts of the TBNC, with the exception of two modestly labeled terminal fields located in the lateral aspects of rostral pars oralis and rostral pars caudalis. No labeled fibers terminated in the contralateral TBNC or contralateral cervical spinal cord.

The origin, density, and distribution of sympathetic nerve fibers in the supratentorial dura mater of the rat were examined in detail in the current study by using wheat germ agglutinin horseradish peroxidase (WGA-HRP) retrograde tracing procedures, glyoxylic acid-induced fluorescence, and dopamine beta-hydroxylase (DBH) immunocytochemical staining of dural whole mount preparations. Application of WGA-HRP to the superior sagittal sinus and adjacent areas of the supratentorial dura mater labeled numerous neurons in each of the left and right superior cervical ganglia. Glyoxylic acid and DBH immunocytochemical staining of fixed dural whole mount preparations revealed prominent plexuses of sympathetic nerves about the middle meningeal artery and its branches, about the superior sagittal and transverse sinuses, and "free" within the dura mater, i.e., apparently unassociated with any vasculature. Significantly, in all of these areas, the density of sympathetic innervation revealed in this study was considerably greater than that previously demonstrated by other workers. An impressive population of mast cells also was observed within the dura mater of the glyoxylic acid-treated preparations. The majority of these cells were perivascular; however, a significant number were also present within the dura unrelated to the vasculature, and occasional cells were seen in close apposition to fluorescent sympathetic nerve fibers. Taken together, the identification of a robust sympathetic plexus and prominent mast cell population associated with a dura mater that also receives significant sensory projections from the trigeminal system raises interest regarding the functional interactions of these elements. These observations warrant further consideration regarding their role in the pathogenesis of vascular headache and head pain.

The somatotopic organization of the cat trigeminal ganglion has been investigated in the present study by using the horseradish peroxidase (HRP) technique. In separate animals, the corneal, supraorbital, infraorbital, inferior alveolar, or mental branches of the trigeminal nerve have been transected and then soaked in concentrated solutions of HRP. Retrogradely labeled corneal and supraorbital neurons have been found, with extensive overlap between the two cell populations, in the anteromedial region of the trigeminal ganglion. Inferior alveolar and mental neurons have been found to possess similar distributions within the posterolateral part of the trigeminal ganglion. Infraorbital cells have been localized in a central position. The cell bodies of any given nerve are found in at least minimal numbers in all dorsoventral levels of the trigeminal ganglion. However, cell bodies of origin of the supraorbital nerve and the lateral branch of the infraorbital nerve, innervating more posterior or lateral areas of the head and face, are found in greater numbers dorsally. Conversely, cell bodies of origin of the medial branch of the infraorbital nerve, the inferior alveolar nerve, and the mental nerve, supplying more rostral or intraoral areas of the orofacial region, are present in greater numbers ventrally. In contrast, corneal neurons are distributed uniformly in the dorsoventral axis. The ophthalmic and maxillary regions of the trigeminal ganglion appear to be well segregated, whereas the maxillary and mandibular regions exhibit a somewhat greater degree of overlap. Cell bodies of corneal afferent neurons range from 20 to 50 micrometer in diameter, whereas those of supraorbital, infraorbital, inferior alveolar and mental neurons measure from 20 to 85 micrometer. It is concluded from the findings of the present work that much of the cat trigeminal ganglion is organized somatotopically in not only the mediolateral axis but also in the dorsoventral axis.

The method of retrograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) was used to study the locations, numbers and somata diameters of corneal afferent and efferent neurons in four different mammalian species. A 2% solution of HRP-WGA was applied to the central cornea of the rat, rabbit, cat and monkey and the animals were perfusion-fixed 48-96 hr later. HRP-WGA-labeled sensory neurons were distributed relatively uniformly throughout the ophthalmic region of the ipsilateral trigeminal ganglion of the rat, cat and monkey. In marked contrast, labeled cells in the rabbit trigeminal ganglion were clustered in a sharply defined longitudinal column located in the midregion of the ophthalmic area. Occasional cells in some cats and monkeys were observed near, and possibly within, the maxillary region of the ganglion. There was no evidence for a dorsoventral somatotopy of corneal afferent neurons in any species. The majority of the labeled afferent somata were small or medium in size, although some larger diameter neurons were also observed. Modest numbers of labeled neurons were observed in the ipsilateral superior cervical ganglion (SCG) of the rabbit and cat; however, only occasional labeled neurons were observed in the SCG of the rat, and none were seen in the monkey. The labeled SCG cells, when present, were concentrated in the rostral half of the ganglion, although many cells in the cat SCG were also found further caudally. No labeled neurons were found in the middle cervical or stellate ganglia of any animal. The results of this study have revealed the existence of subtle interspecies differences in the organization of the mammalian corneal afferent and efferent innervations.

Damage to septohippocampal neurons in the adult rat results in sprouting of sympathetic axons into the denervated hippocampal formation. However, the distribution of sympathohippocampal fibers has only been assessed with light microscopic techniques, and it is not known if the sprouted fibers leave the blood vessels, along which they migrate into the hippocampal formation, to enter the hippocampal neuropil and, if they do, whether they form synaptic contacts with central neurons. Using the tetramethylbenzidine technique to visualize anterogradely transported wheat germ agglutinin-horseradish peroxidase conjugate, we identified sprouted sympathetic fibers in the hippocampal formation at both the light and electron microscopic level in albino rats receiving medial septal lesions. The majority of labeled fibers were observed within the regions immediately above and below the granule cell layer. Although most of the labeled sprouts were observed in association with intraparenchymal blood vessels, where they were usually apposed to the basal lamina, approximately a third of the labeled profiles were present within the neuropil with no obvious vascular relationships. Most of the profiles were identified as unmyelinated axons or vesicle-filled varicosities. Many of the latter structures contained small dense-cored vesicles, but in our sample none of the labeled profiles were observed to form membrane specializations with adjacent structures, and many were partly surrounded by presumed astrocytic processes. These results document the invasion of the CNS by sprouting axons of peripheral origin indicating that axonal elongation from uninjured neurons can occur within the mature mammalian CNS under certain circumstances. In addition, the presence of significant numbers of sympathetic fibers within the hippocampal neuropil indicates that they may be in a strategic position to influence hippocampal function.