April 2003
Volume 44, Issue 4
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Cornea  |   April 2003
Sympathetic Neural Control of the Mouse Lacrimal Gland
Author Affiliations
  • Chuanqing Ding
    From the Vision Science Research Center, University of Alabama at Birmingham, Birmingham, Alabama; and the
  • Benjamin Walcott
    Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York.
  • Kent T. Keyser
    From the Vision Science Research Center, University of Alabama at Birmingham, Birmingham, Alabama; and the
Investigative Ophthalmology & Visual Science April 2003, Vol.44, 1513-1520. doi:10.1167/iovs.02-0406
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      Chuanqing Ding, Benjamin Walcott, Kent T. Keyser; Sympathetic Neural Control of the Mouse Lacrimal Gland. Invest. Ophthalmol. Vis. Sci. 2003;44(4):1513-1520. doi: 10.1167/iovs.02-0406.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To explore the sympathetic innervation pattern and the role of sympathetic nervous system control of protein secretion in the exorbital lacrimal glands of normal mice.

methods. Mouse lacrimal glands were processed for single- and double-label indirect immunofluorescence studies to show their innervation patterns. The sucrose-potassium phosphate-glyoxylic acid method was also used to visualize the adrenergic innervation. The effects of adrenergic and cholinergic agonists on protein secretion were evaluated.

results. The mouse lacrimal gland can be divided into two different areas based on the innervation density and distribution pattern. One area, approximately 10% to 30% of the gland, exhibited much higher innervation density, both parasympathetic and sympathetic, than the rest of the gland. The adrenergic agonists norepinephrine and phenylephrine induced increases in protein secretion that were of a magnitude similar to the increases induced by the cholinergic agonist carbachol at 10−6 to 10−4 M. Isoproterenol, the β-adrenergic agonist, also elicited protein secretion at 10−5 to 10−4 M.

conclusions. The data indicate that there is extensive sympathetic innervation of the mouse lacrimal gland and that sympathetic input can modulate protein secretion. The division of the lacrimal gland into two areas suggests that the mouse lacrimal gland is a mixed gland and that these two areas may play different roles in secreting tears of different compositions in various situations. These data appear to support the notion that differential secretion is accomplished by activating different populations of secretory cells that are differentially innervated.

The adequate production of tears of the appropriate composition is essential for the maintenance of clarity in the cornea. Most of the fluid- and tear-specific proteins that supply the mammalian eye are from the major lacrimal glands. In rodents, a major lacrimal gland is the exorbital lacrimal gland found near the ear on the lateral side of the skull. This lacrimal gland is innervated by the autonomic nervous system, with parasympathetic input predominating, 1 2 3 although some sympathetic innervation has been reported in some species. 1 2 3 4 5 6 7 The innervation is reported to be evenly distributed, 8 9 10 although the innervation in rat has been reported to be most abundant in the hilus. 11  
Although it is generally accepted that the parasympathetic innervation of the lacrimal gland is responsible for inducing most tear secretion, there is still some controversy about the functional relevance of the sympathetic innervation. There is evidence that the sympathetic nervous system plays a direct and significant role in initiating and modulating tear secretion in some species, instead of modulating only blood flow to the gland. 12 13 The sympathetic innervation varies greatly among species, and there are some discrepancies in the literature. For example, there are reports that every acinar cell is in proximity with an adrenergic fiber in cats, 4 dogs, 5 7 humans, and monkeys. 6 However, other data from studies in humans 14 and monkeys 15 are inconsistent with these observations. In rat 5 10 and mouse, 5 9 16 only sparse adrenergic nerve fibers have been reported, mostly in association with blood vessels, with only a few being found among acini. In guinea pig, rich adrenergic innervation in lacrimal glands has been reported, 10 whereas others have described a virtual absence in the gland, except along the blood vessels. 5 Although in our previous report we showed that only sparse adrenergic nerves were present in the mouse lacrimal gland, 16 we occasionally found that some areas of the gland demonstrated much more labeling. To resolve these inconsistencies in our data and to further our understanding of the innervation of the mouse lacrimal gland, a useful model system in the study of lacrimal gland function, we used histochemical and immunohistochemical techniques to examine the sympathetic innervation pattern. We also determined whether various α- and β-adrenergic agonists induced glandular protein secretion. We found that some lobes of the mouse lacrimal gland displayed a high density of tyrosine hydroxylase (TH)–immunoreactive fibers, a marker for sympathetic fibers, 9 among the acini, whereas in other lobes the sympathetic innervation to the acini was sparse. Antibodies to the vesicular acetylcholine transporter (VAChT) and to synaptophysin also showed that the cholinergic and total innervations were not distributed evenly among the lobes of the gland. Both cholinergic and adrenergic agonists induced protein secretion by gland fragments, suggesting that both have a role in the regulation of gland function. 
Materials and Methods
Chemicals
Carbamylcholine chloride (carbachol), isoproterenol bitartrate, norepinephrine bitartrate, l-phenylephrine hydrochloride, phentolamine hydrochloride, and dl-propranolol hydrochloride, were from Sigma (St. Louis, MO). All other reagents were of the highest purity available. 
Animals
C57 female mice (∼18 g body weight), aged from 2 to 12 months, were purchased from commercial vendors (Taconic Farms, Germantown, NY, or Charles River, Wilmington, MA). All animals were kept in a 12-hour light–dark cycle and maintained in an accredited animal facility with freely available food and water. They were managed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Lacrimal glands were obtained by killing the animals with an overdose of halothane approximately at midday and rapidly removing and trimming the glands from surrounding membranes and fatty tissues under a dissecting microscope. 
Tissue Preparation for Immunohistochemistry
For immunohistochemistry, lacrimal glands were fixed in freshly prepared 4% buffered paraformaldehyde. After 3 to 4 hours of fixation at 4°C, the tissue was placed in 0.1 M phosphate buffer containing 30% sucrose at pH 7.4 for at least 12 hours at 4°C. The glands were then placed in optimal cutting temperature (OCT) embedding medium (Sakura Finetek USA, Torrance, CA), serially sectioned at 10 μm on a cryostat (Leica, Deerfield, IL), and collected on slides (SuperFrost Plus; VWR Scientific, West Chester, PA). The sections were dried and stored at −20°C until used. 
Immunohistochemistry
The antibodies used were sheep (dilution 1:800) and rabbit (dilution 1:400) anti-TH polyclonal antibodies (Chemicon International, Temecula, CA); rabbit anti-synaptophysin polyclonal antibody (Dako, Carpinteria, CA) at a dilution of 1:200; and goat anti-VAChT polyclonal antibody (Chemicon) at dilution of 1:2000. The VAChT antibody has been well characterized and is reported to colocalize with classic cholinergic markers, such as choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), in both the central and peripheral nervous systems and in the lacrimal gland. 17 18  
Sections were incubated in primary antibody diluted with 0.1 M sodium phosphate buffer (PBS) overnight or for 48 hours for TH, at 4°C. For the control, primary antibodies were omitted. Secondary antibodies used were fluorescein isothiocyanate (FITC)–conjugated donkey anti-rabbit, sheep or goat IgG, and Texas red- or rhodamine red-X–conjugated donkey anti-goat, sheep or rabbit IgG (Jackson ImmunoResearch, West Grove, PA), all at a dilution of 1:200. The secondary antibodies were applied for an hour at room temperature. The slides were then washed with three changes of PBS and one change of 4 mM sodium carbonate (pH 10.0), coverslipped, and examined with a conventional fluorescence microscope (Eclipse E800M; Nikon, Melville, NY). The images were captured with a digital camera (SpotCam; Diagnostic Instruments, Sterling Heights, MI) and analyzed on a computer (PhotoShop; Adobe Systems, San Diego, CA). At least 21 sections were examined per gland to examine all lobes within the gland. 
Sucrose-Potassium Phosphate-Glyoxylic Acid Method
The sucrose-potassium phosphate-glyoxylic acid (SPG) technique, as developed by De la Torre, 19 is a very sensitive and powerful method for visualizing adrenergic nerves. 7 10 Lacrimal glands were quickly removed from the animals and frozen in OCT medium on chucks in the cryostat at −20°C. Sections (14-μm-thick) were cut, picked up on room-temperature slides, and immediately dipped three times for 1 second in the glyoxylic acid solution. The sections were dried with a blow dryer set at cool. When dry, the sections were covered with 1 to 2 drops of mineral oil (USP grade) and placed on an aluminum tray in a 95°C oven for 1 to 3 minutes. The slides were drained of excess oil, covered with 2 drops of fresh oil, and coverslipped. The sections were examined with a conventional fluorescence microscope. 
Protein Secretion
Lacrimal glands were weighed before being cut into fragments of 1 to 2 mm with a scalpel blade. The fragments were washed in 5 mL of saline solution (116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.81 mM MgCl2, 1.01 mM NaH2PO4, 26.2 mM NaHCO3, and 5.6 mM dextrose [pH 7.4]), maintained at 37°C and vigorously bubbled with 95% O2 and 5% CO2 in a beaker for 10 minutes. The solution was changed three times and discarded. The gland fragments were then incubated in 1 mL saline for 10 minutes, and the saline was removed and replaced with fresh medium. This cycle was repeated two or three times, and the saline was collected after each of the 10-minute incubations. The proteins in these samples represent the basal secretion from the glands. In the last exchange, the medium that was added contained one of the drugs at a specific concentration. After another 10-minute incubation, the medium was removed and saved, and the proteins in these solutions represented the stimulated secretion in response to various drugs. One gland was used in each experiment. 
Although isoproterenol is subject to inactivation by oxidation in oxygenated medium, our relatively short incubation period (10 minutes), did not cause a significant change in concentration. 12 Also, although isoproterenol becomes brownish pink on oxidation, the color did not become visually perceptible until the concentration exceeded 10−4 M. However, to ensure that the discoloration did not decrease accuracy, control samples with corresponding isoproterenol concentrations were included in each experiment and corrections were made at various concentrations. 
Protein Assay
The samples were analyzed for total protein with a Coomassie protein assay kit (Pierce, Rockford, IL). Bovine serum albumin (BSA) was used as a standard protein and standards were run with each assay. Protein concentrations were determined from the standard curves measured with each assay. The assays were performed on a microplate reader (model EL 808; Bio-Tek Instruments, Winooski, VT) at 595 nm. Both samples and standards were read in duplicate on 96-well flat-bottomed microplates (Costar; Corning Inc., Corning, NY). Total protein concentration was determined with the software provided by the manufacturer (KC4; Bio-Tek). Proteins secreted in response to various agonists (stimulated secretion) were the difference between total and basal secretions. The readings were then converted to micrograms per milliliter per gram tissue per minute. 
Quantification of VAChT Immunoreactivity
Under low-power magnification, 10 randomly selected rectangular areas (30 × 40 μm) in both low- and high-density innervation areas were chosen. Then, with a 40× objective, the punctate VAChT labeling was counted, and the results in each area were averaged. The averages represent the amount of VAChT immunoreactivity in either low- or high-density innervation areas. 
Statistical Analysis
Data were expressed as the mean ± SEM, when appropriate. Student’s paired or unpaired t-tests were performed on a computer (SigmaPlot 5.0 software; SPSS Inc., Chicago, IL). P < 0.05 was considered to be significant. 
Results
Sympathetic Innervation in the Gland
Examination of the lacrimal gland stained with antisera to TH (host in either rabbit or sheep) showed that some lobes of the gland had relatively sparse TH immunoreactivity (low innervation density areas), whereas other lobes had extensive TH immunoreactivity (high innervation density areas). In all lobes, TH immunoreactivity was associated with the blood vessels (Fig. 1A) . In the lobes with low-density innervation, in addition to the TH immunoreactivity associated with the blood vessels, a few positive fibers were seen among the acini. These fibers were varicose and were close to some acini (Fig. 1B) . However, most of the acini did not have TH-immunoreactive fibers associated with them. In some cases, there were regions in the low-density innervation areas that did have a higher density of TH immunoreactivity but they were not common (Fig. 1C) . In the lobes with high-density innervation TH immunoreactivity, there was an extensive network of positively staining fibers. The immunoreactivity was so intense that the fibers appeared smooth with few obvious varicosities (Fig. 1D) . In these lobes, virtually every acinar cell had a TH-immunoreactive fiber close to it. In serial sections of the whole glands, we found that the areas of high-density innervation were usually situated in the rostral–ventral side of the gland, often beneath areas of low-density innervation in the gland. The size of high-density innervation areas varied from 10% to 30% of the whole gland. 
The SPG method revealed a pattern of fibers similar to that observed with the TH antibodies. In all parts of the gland, fluorescent fibers were associated with the blood vessels that are found within the lobes (Fig. 2A) . In some lobes, there were occasional varicose fibers among the acini (Fig. 2B) , whereas in other lobes there was a very dense meshwork of fibers among the acini (Fig. 2C)
Parasympathetic Innervation in the Gland
VAChT immunoreactivity was found in all lobes of the gland. Varicose fibers were found among acini, presumably representing synaptic vesicles containing acetylcholine (ACh). These VAChT-positive varicosities were close to the basolateral membrane of the acini and formed a loose network around them (Fig. 3A) . Some larger nonvaricose nerve fibers were also observed to be VAChT immunoreactive. 
In the lobes of the gland with high-density TH immunoreactivity, more VAChT-positive fibers were also observed (Fig. 3A) . Because VAChT immunoreactivity was always punctate with obvious varicosities, we were able to quantify VAChT immunoreactivity in the low-density and high-density innervation areas. We estimated that the number of VAChT immunoreactivity in the high-density innervation areas was approximately 3.7 times of the low-density areas (Fig. 4)
Total Innervation in the Gland
In the low-density innervation areas, the synaptophysin immunoreactivity appeared to be evenly distributed among most of the acini, although some acini exhibited less synaptophysin immunoreactivity. The synaptophysin immunoreactivity was characteristically punctate, and outlined the acini. Some synaptophysin-immunoreactive nerve fibers were also observed. However, in the high-density innervation areas, synaptophysin-immunoreactive features were present in higher density than in low-density innervation areas. Most synaptophysin-immunoreactive nerve fibers were thick and had smooth surfaces, displaying morphologic properties similar to those observed with the TH antibody, and the punctate synaptophysin immunoreactivity appeared to merge, forming a dense plexus of nerve fibers (Figs. 3B 3D)
Double Labeling
Double labeling showed that in the high-density innervation areas, most of the synaptophysin-immunoreactive fibers also exhibited TH immunoreactivity (Figs. 3C 3D) . Some VAChT immunoreactive nerves also showed TH immunoreactivity (Figs. 3E 3F) , suggesting that nerves in this area are mixed, containing both parasympathetic and sympathetic nerve fibers. However, it appeared that the sympathetic innervation predominated in the high-density innervation areas. 
In the low-density innervation areas, most of the synaptophysin immunoreactivity colocalized with VAChT immunoreactivity (Figs. 3A 3B) , suggesting that most of the innervation of these lobes was of parasympathetic origin, with the exception of a few nerve fibers that were TH immunoreactive in the interstitial areas and blood vessels. 
Protein Secretory Responses to Autonomic Agonists at 10−5 M
The immunohistochemical studies raised the possibility that the sympathetic system may play a major role in the regulation of lacrimal gland function in the mouse. To test this possibility, we exposed lacrimal gland fragments to adrenergic agonists and measured the amount of protein secreted in response to each drug. Norepinephrine (an α- and β-adrenergic receptor agonist), phenylephrine (an α1-adrenergic agonist), and isoproterenol (a β-adrenergic agonist) all induced protein secretion by the gland fragments (Fig. 5) . For comparison, we also used carbachol, which is a muscarinic cholinergic agonist. 
Norepinephrine, carbachol, and phenylephrine induced the most secretion, whereas isoproterenol induced the least. Norepinephrine and phenylephrine elicited almost the same magnitude of protein secretion as that elicited by carbachol (P > 0.05, no significant difference between any two of them). Isoproterenol appeared to be a relatively weaker stimulant, as the increase in protein secretion was significantly different from the increases induced by the other three agonists (P < 0.01). 
Discussion
The sympathetic innervation varies greatly among species, and there are some discrepancies between reported observations. In rat 5 10 and mouse, 5 9 16 only sparse adrenergic nerve fibers have been observed, mostly in association with blood vessels, with only a few found among acini. In guinea pig, some researchers reported rich adrenergic innervation in the lacrimal gland, 10 whereas others described a virtual absence in the gland except along the blood vessels. 5 Our observations of TH immunoreactivity in the low-density innervation areas appeared to be in agreement with previous reports in the mouse, 5 9 16 whereas the observation of TH immunoreactivity in the high-density innervation areas is similar to the sympathetic innervation observed in guinea pig extraorbital lacrimal gland. 10 However, the division of the mouse lacrimal gland into two areas, based on the differences of innervation pattern and density, is a novel finding. By using serial sections from the whole gland, we estimated that the high-density innervation areas represent approximately 10% to 30% of the gland and are situated on the rostral side of the gland, mostly near the ventral side. The low-density innervation areas clearly showed blood vessels with associated staining, which suggests that these areas are not simply inaccessible to the antisera. Further, by using a totally different technique, the SPG method which has been shown to be very sensitive in visualizing adrenergic nerves, 7 10 we found similar differences in the adrenergic innervation density between different lobes. 
Increasing evidence in some species 12 13 indicates that the sympathetic nervous system plays a role in influencing tear secretion, not only by modulating blood flow to the gland and its distribution within it, but also by direct effects on the secretory acini. In the present study, the observation of TH-immunoreactive nerve fibers with numerous varicosities in close association with acini in some lobes of the mouse gland support this notion. Axonal varicosities have been shown to represent synaptic junctions. This observation contrasts with published data from mouse lacrimal gland, which showed extensive adrenergic innervation of blood vessels, but only sparse nerve fibers associated with the secretory acini. 5 9 16  
The results of the protein secretion experiments reported herein, with norepinephrine used as the agonist for both α- and β-adrenergic receptors and phenylephrine and isoproterenol for α1- and β-adrenergic receptors, 12 20 respectively, suggest the presence of both α1- and β-adrenergic receptors in the mouse lacrimal gland and their association with the protein secretion process. The robust responses elicited by norepinephrine and phenylephrine suggest that the sympathetic innervation of the mouse lacrimal gland has more functional significance than previously thought. Although there are TH-immunoreactive fibers associated with the blood vessels within the lacrimal gland, the protein secretion data described earlier were obtained from in vitro lacrimal gland fragments and could not be caused by the modulation of blood flow in the gland. The significant response of the gland fragments to adrenergic agonists suggests there is extensive direct adrenergic control of acinar cells. 
In the mouse lacrimal gland, there was α1-adrenergic–mediated protein secretion. Phenylephrine has been shown to increase intracellular Ca2+ concentrations 21 and to induce significant peroxidase secretion (an index of protein secretion) in the mouse. 22 Intracellular recordings of mouse acinar cells indicate that epinephrine elicits hyperpolarization and a marked reduction of membrane resistance. 23 24 In rat lacrimal gland, phenylephrine stimulates protein secretion from acini in a time- and concentration-dependent manner, with the maximum reached at 10−4 M. 25 α-Adrenergic agonists, such as phenylephrine and norepinephrine, can induce K+ release and secretion of both peroxidase and newly synthesized protein from rat acinar cells. 13 25 26 27  
The selective α1 adrenergic agonist, phenylephrine, appears to have intrinsic activity similar to that of norepinephrine, a mixed α- and β-receptor agonist, in eliciting protein secretion. In rat, 85% of the adrenergic regulation of protein secretion was achieved through activation of the α1-adrenergic receptor, and the remaining 15% was assumed to be due to the activation of the β receptor. 20 Our data are in agreement with those obtained in rats. At 10−5 M, protein secretion induced by phenylephrine is approximately 89% of that induced by norepinephrine, suggesting that the adrenergic stimulation in the mouse lacrimal gland is also mainly achieved through α1-receptor activation. 
Protein secretion data suggest that a β-adrenergic pathway is present in the mouse lacrimal gland as well. Although the isoproterenol-induced protein secretion was much smaller than that induced by other agonists, it doubled the secretion rate. These results appear to contradict a report that the sympathetic influence in mouse lacrimal gland is mediated only by α1-adrenoceptors. 28 Intracellular recording of mouse lacrimal gland acinar cells, for example, revealed that isoproterenol has no detectable effects on membrane potential and resistance. 23 24 However, in the lacrimal gland of rat, isoproterenol stimulated both fluid and protein (peroxidase) secretion, although the response was slight and no K+ release was observed. 13 29 30 In vivo experiments on cannulated rabbit lacrimal gland also demonstrated that there are norepinephrine-responsive β-adrenergic receptors present, and that isoproterenol was more effective than norepinephrine and epinephrine in inducing lacrimal flow. 31 Reports from in vitro studies using rabbit lacrimal gland fragments also show that isoproterenol can induce protein secretion. 12  
Although there were no significant differences between protein secretion rates induced by norepinephrine, phenylephrine, and carbachol, the significant increase in protein secretion induced by norepinephrine and phenylephrine appears to be in contrast to the prevailing view that the parasympathetic system is responsible for most of the protein secretion in the mouse lacrimal gland. Previous studies reported that there was only sparse sympathetic innervation of the mouse lacrimal gland 5 9 16 and that there is much more parasympathetic innervation present in the gland. These reports suggested that the sympathetic innervation observed may not be sufficient to induce robust protein secretion in response to adrenergic agonists. 
The sympathetic nerves within the lacrimal gland primarily originate from the superior cervical ganglion (SCG), which is the uppermost ganglion of the sympathetic trunk. In the lacrimal gland of rats, 32 retrograde-tracing experiments showed both ipsi- and contralateral sympathetic contributions from SCG neurons, although only a few labeled somata were observed in the contralateral SCG. Although no comparable data concerning the sympathetic source in mouse lacrimal gland are available, it seems likely that the mouse lacrimal gland is similar to that of rat, with both ipsi- and contralateral SCG contributions. We speculate that there may be some relationship between the origin of the sympathetic input to the lacrimal gland and the density of innervation—that is, perhaps the sympathetic innervation in the high-density innervation areas comes from the ipsilateral SCG, whereas that in the low-density innervation areas originates contralaterally, or vice versa. Retrograde labeling studies, with dye injections into either the high- or low-density innervation areas of the lacrimal gland, and subsequent evaluation of the labeling in the ipsi- and contralateral SCG could test this idea. 
In addition to TH immunoreactivity, regional differences were also observed in both synaptophysin and VAChT immunoreactivity. Much more synaptophysin and VAChT immunoreactivity was observed in the high-density innervation areas. Synaptophysin has been well documented as a marker of total innervation, 8 and VAChT has been extensively documented as a marker for parasympathetic nerves. 17 18 Our data suggest that the mouse lacrimal gland is divided into two areas, one with much higher innervation density (both parasympathetic and sympathetic) and the other with lower density. After counting the number of VAChT-immunoreactive puncta, we estimated that the parasympathetic innervation in the high-density innervation areas was approximately 3.7 times that of the low-density innervation area. This is a novel observation that contrasts with data from studies of the autonomic supply to lacrimal glands in mouse, 5 9 rat, 11 human, and monkey, 6 which showed relatively even innervation density. 
The regional differences of innervation suggest that the mouse lacrimal gland is a mixed gland. In the high-density innervation areas, in which dense nerve fibers were observed around blood vessels and acini, the density and distribution patterns of synaptophysin and TH immunoreactivity were similar to the sympathetic innervation in guinea pig extra- and intraorbital glands. 10 In the low-density innervation areas, however, the density of TH immunoreactivity was similar to the sympathetic innervation observed in lacrimal glands of rat 10 and mouse. 5 9 This is reminiscent of the innervation pattern in avian harderian gland, the main lacrimal gland in birds, which has two anatomically distinct areas, the cortex and the medulla. The cortex consists of secretory tubules made of columnar epithelium, whereas the medulla contains only a few. Both cholinergic and adrenergic nerves are observed in the harderian gland, but only sparse fibers are observed around the cortical secretory acini in pigeon 33 and chicken, 34 35 in contrast to the dense innervation observed in the medulla. 
Anatomically, the mouse exorbital lacrimal gland consists of several lobes, and each lobe has a duct that runs to the surface of the eye. There are six to seven ducts that come together to form the main duct. Therefore, it is conceivable that the lobes have different innervation patterns and functions. A lobe itself consists of many lobules, which are further composed of acini, the functional units of tear secretion. Because tear flow is highly regulated, division of the mouse lacrimal gland into two distinct areas according to the differences of innervation density and distribution pattern suggests that the two areas may have different functions. 
In addition to classic neurotransmitters, such as ACh and norepinephrine, various neuropeptides 14 15 36 37 38 and other unconventional transmitters such as nitric oxide 16 have been found in the mammalian lacrimal gland, including that of the mouse. The available data suggest that both classic neurotransmitters and at least two neuropeptides are present in the lacrimal glands of most species. 35 36  
The complexity of the innervation pattern of the lacrimal gland, in conjunction with the multiple neurotransmitters and modulators present, may influence the protein, ion, and water composition of tears. 12 35 Based on the results obtained from the rabbit, Bromberg 12 suggested that both parasympathetic and sympathetic systems work together to effect secretion of tears of the appropriate composition. For example, the parasympathetic system may regulate the flow rate and electrolyte content, and the sympathetic system may regulate protein secretion. Differential secretion could be accomplished by stimulating different autonomic nervous pathways in acinar and ductal cells, by activating different populations of secretory cells, or by the activation of different intracellular signal systems. There is precedence for this idea. 
Based on data from muscarinic ACh receptor immunohistochemical observations in the rat lacrimal gland, it has been reported that even though the immunoreactivity was associated with each acinus, the labeling appeared to be unevenly distributed both within an acinus and between them. 35 The two separate areas within the mouse lacrimal gland, with distinctive innervation densities and distribution patterns, and the regional differences of innervation even in the low-density innervation areas, suggest that the secretory cells in these two areas may be differentially stimulated. Thus, tears of varying composition, resulting from differential sympathetic and parasympathetic stimulation, could be produced, depending on the needs of a given situation. 
To our knowledge, no studies have reported the existence of different innervation density and distribution patterns in the lacrimal glands of mouse and other mammals, including our own observations in mouse. 16 There are several possible explanations for this. (1) It may be that only portions of the lacrimal gland, primarily the central area, were sectioned. Alternatively, when sectioning the gland, investigators may have retained sections from the larger central portion of the gland and discarded smaller section through the peripheral portions of the gland. Therefore, sections through the high-density innervation area, which is usually in the peripheral gland, may have been discarded. (2) Only some sections were examined. As the high-density innervation area encompasses only 10% to 30% of the whole gland, the number of sections that include these areas represent only a small portion of all sections. These two factors explain in part why we, and perhaps others, did not report the differential innervation pattern in previous papers. 16 Indeed, that the dense sympathetic innervation was restricted to one portion of the gland became clear to us only after we examined glands that had been serially sectioned. (3) The dense immunoreactivity in the high-density region of the gland may have been interpreted as artifactual or nonspecific staining. Such dense sympathetic innervation is inconsistent with many previous reports and the prevailing consensus concerning the autonomic innervation of the mouse lacrimal gland. 
It is unlikely that we observed a pathologic condition, because the animals used, aged between 2 to 12 months, were healthy at the time of experiment. Tissues obtained from many animals yielded similar results, and it is very unlikely that all these animals had the same pathologic condition. 
It should be pointed out that because our observations were made in the course of a pilot study of possible changes in the gland due to Sjögren’s syndrome, we used only female mice, so the data reported herein may not be applicable to male mice. Another caveat is that the protein secretion measurements were made using fragments of the lacrimal gland, and for this reason likely included nerve fiber fragments, ductal cells, myoepithelial cells, mast cells, and plasma cells. However, as acinar cells represent at least 80% of the rat lacrimal gland mass, 13 39 40 we believe the data presented in the current report are representative of the acinar cells. 
Sjögren’s syndrome is a chronic autoimmune disease that affects lacrimal and salivary glands and is sometimes associated with other connective tissue disease. The syndrome is characterized by lymphocyte infiltration in these tissues and subsequent functional impairment. In Sjögren’s syndrome, tear secretion is decreased, even that resulting from reflex stimulation, 41 suggesting that the neural control pathway may be compromised. The differential innervation density and distribution pattern in the mouse lacrimal gland described in the current study suggests that the high- and low-density innervation areas may have functional differences, and it is reasonable to speculate that the progression and effects of the disease differ in the different parts of the gland. These novel findings extend our overall understanding of lacrimal gland control and function and may have implications for the cause and progression of Sjögren’s syndrome. 
In summary, these data indicate that there is extensive sympathetic innervation in the mouse lacrimal gland and that it appears to be directly associated with the protein secretory process. Immunohistochemistry and SPG observations suggest the mouse lacrimal gland can be divided into two areas based on the density and distribution pattern of the innervation. One area, comprising approximately 10% to 30% of the gland, has higher innervation density, both parasympathetic and sympathetic. The rest of the gland is less densely innervated. These two areas may play different roles in secreting tears that are of different compositions in various situations. These data support the notion that differential secretion is accomplished by activating different populations of secretory cells, which are differentially innervated. 
 
Figure 1.
 
Sympathetic innervation in the mouse lacrimal gland. (A) TH-immunoreactive nerve fibers were observed to be associated with blood vessels (arrows). (B) Nerve fibers that were TH immunoreactive were observed to have a large number of varicosities and were in close contact with acini in the low-density innervation areas. (C) In some regions within the low-density innervation areas, TH-immunoreactive fibers were observed to be in close association with acini and in relatively higher density than other regions. These regions were mostly found in the periphery of the gland. (D) TH immunoreactivity visualized with FITC in the high-density innervation. Thick nerve bundles and fibers that exhibit TH immunoreactivity appeared smooth and formed a dense plexus around the acini. Scale bar, 40 μm.
Figure 1.
 
Sympathetic innervation in the mouse lacrimal gland. (A) TH-immunoreactive nerve fibers were observed to be associated with blood vessels (arrows). (B) Nerve fibers that were TH immunoreactive were observed to have a large number of varicosities and were in close contact with acini in the low-density innervation areas. (C) In some regions within the low-density innervation areas, TH-immunoreactive fibers were observed to be in close association with acini and in relatively higher density than other regions. These regions were mostly found in the periphery of the gland. (D) TH immunoreactivity visualized with FITC in the high-density innervation. Thick nerve bundles and fibers that exhibit TH immunoreactivity appeared smooth and formed a dense plexus around the acini. Scale bar, 40 μm.
Figure 2.
 
Adrenergic innervation in the lacrimal gland as revealed by SPG. (A) Adrenergic fibers were in close contact with blood vessels. (B) In the low-density innervation areas, sparse nerve fibers were observed in association with acini, along with those around blood vessels (not shown in this figure). (B) A dense meshwork of adrenergic fibers was apparent among acini in the high-density innervation areas. Scale bar, 30 μm.
Figure 2.
 
Adrenergic innervation in the lacrimal gland as revealed by SPG. (A) Adrenergic fibers were in close contact with blood vessels. (B) In the low-density innervation areas, sparse nerve fibers were observed in association with acini, along with those around blood vessels (not shown in this figure). (B) A dense meshwork of adrenergic fibers was apparent among acini in the high-density innervation areas. Scale bar, 30 μm.
Figure 3.
 
Double labeling of the innervation in both low- and high-density areas in the lacrimal gland. (A) VAChT immunoreactivity was observed in both low- and high-density innervation. (B) Synaptophysin immunoreactivity (SIR visualized with FITC) colocalized with all VAChT immunoreactivity in both low- and high-density innervation. (C) Dense TH-immunoreactive nerve fibers were observed in the high-density innervation, mostly in thick bundles of fibers with a smooth appearance. No TH immunoreactivity was visualized in low-density innervation in this section. (D) Dense SIR (with FITC) observed in the high-density innervation areas colocalized with TH immunoreactivity, whereas SIR in low-density innervation did not. Note that even in the low-density innervation areas, there were regional differences in the SIR density. (E) In high-density innervation, some VAChT immunoreactivity (visualized with Rhodamine Red) appeared to colocalize or be in close association with TH immunoreactivity (arrows) which was visualized with FITC (F), and one nerve bundle appeared to include both VAChT immunoreactivity and TH immunoreactivity fibers, suggesting these nerves contain both parasympathetic and sympathetic fibers. Scale bar: (AD) 100 μm; (E, F) 40 μm.
Figure 3.
 
Double labeling of the innervation in both low- and high-density areas in the lacrimal gland. (A) VAChT immunoreactivity was observed in both low- and high-density innervation. (B) Synaptophysin immunoreactivity (SIR visualized with FITC) colocalized with all VAChT immunoreactivity in both low- and high-density innervation. (C) Dense TH-immunoreactive nerve fibers were observed in the high-density innervation, mostly in thick bundles of fibers with a smooth appearance. No TH immunoreactivity was visualized in low-density innervation in this section. (D) Dense SIR (with FITC) observed in the high-density innervation areas colocalized with TH immunoreactivity, whereas SIR in low-density innervation did not. Note that even in the low-density innervation areas, there were regional differences in the SIR density. (E) In high-density innervation, some VAChT immunoreactivity (visualized with Rhodamine Red) appeared to colocalize or be in close association with TH immunoreactivity (arrows) which was visualized with FITC (F), and one nerve bundle appeared to include both VAChT immunoreactivity and TH immunoreactivity fibers, suggesting these nerves contain both parasympathetic and sympathetic fibers. Scale bar: (AD) 100 μm; (E, F) 40 μm.
Figure 4.
 
The regional difference of VAChT immunoreactivity in the mouse lacrimal gland. Data are the mean ± SEM of results in number of sampling areas shown in parentheses. Each sampling area was 30 × 40 μm, or 1200 μm2. *Significant difference from area of low-density innervation.
Figure 4.
 
The regional difference of VAChT immunoreactivity in the mouse lacrimal gland. Data are the mean ± SEM of results in number of sampling areas shown in parentheses. Each sampling area was 30 × 40 μm, or 1200 μm2. *Significant difference from area of low-density innervation.
Figure 5.
 
Protein secretory responses of the mouse lacrimal gland to agonists at 10 μM. Ordinate is total protein secretion rate. Data are the mean ± SEM of results in the numbers of experiments shown in parentheses. There are no significant differences between any two responses evoked by carbachol, norepinephrine, and phenylephrine. The secretion induced by isoproterenol, however, was significantly lower than those induced by carbachol, norepinephrine, and phenylephrine. *Significant difference from the corresponding basal secretion rate of each agonist.
Figure 5.
 
Protein secretory responses of the mouse lacrimal gland to agonists at 10 μM. Ordinate is total protein secretion rate. Data are the mean ± SEM of results in the numbers of experiments shown in parentheses. There are no significant differences between any two responses evoked by carbachol, norepinephrine, and phenylephrine. The secretion induced by isoproterenol, however, was significantly lower than those induced by carbachol, norepinephrine, and phenylephrine. *Significant difference from the corresponding basal secretion rate of each agonist.
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Figure 1.
 
Sympathetic innervation in the mouse lacrimal gland. (A) TH-immunoreactive nerve fibers were observed to be associated with blood vessels (arrows). (B) Nerve fibers that were TH immunoreactive were observed to have a large number of varicosities and were in close contact with acini in the low-density innervation areas. (C) In some regions within the low-density innervation areas, TH-immunoreactive fibers were observed to be in close association with acini and in relatively higher density than other regions. These regions were mostly found in the periphery of the gland. (D) TH immunoreactivity visualized with FITC in the high-density innervation. Thick nerve bundles and fibers that exhibit TH immunoreactivity appeared smooth and formed a dense plexus around the acini. Scale bar, 40 μm.
Figure 1.
 
Sympathetic innervation in the mouse lacrimal gland. (A) TH-immunoreactive nerve fibers were observed to be associated with blood vessels (arrows). (B) Nerve fibers that were TH immunoreactive were observed to have a large number of varicosities and were in close contact with acini in the low-density innervation areas. (C) In some regions within the low-density innervation areas, TH-immunoreactive fibers were observed to be in close association with acini and in relatively higher density than other regions. These regions were mostly found in the periphery of the gland. (D) TH immunoreactivity visualized with FITC in the high-density innervation. Thick nerve bundles and fibers that exhibit TH immunoreactivity appeared smooth and formed a dense plexus around the acini. Scale bar, 40 μm.
Figure 2.
 
Adrenergic innervation in the lacrimal gland as revealed by SPG. (A) Adrenergic fibers were in close contact with blood vessels. (B) In the low-density innervation areas, sparse nerve fibers were observed in association with acini, along with those around blood vessels (not shown in this figure). (B) A dense meshwork of adrenergic fibers was apparent among acini in the high-density innervation areas. Scale bar, 30 μm.
Figure 2.
 
Adrenergic innervation in the lacrimal gland as revealed by SPG. (A) Adrenergic fibers were in close contact with blood vessels. (B) In the low-density innervation areas, sparse nerve fibers were observed in association with acini, along with those around blood vessels (not shown in this figure). (B) A dense meshwork of adrenergic fibers was apparent among acini in the high-density innervation areas. Scale bar, 30 μm.
Figure 3.
 
Double labeling of the innervation in both low- and high-density areas in the lacrimal gland. (A) VAChT immunoreactivity was observed in both low- and high-density innervation. (B) Synaptophysin immunoreactivity (SIR visualized with FITC) colocalized with all VAChT immunoreactivity in both low- and high-density innervation. (C) Dense TH-immunoreactive nerve fibers were observed in the high-density innervation, mostly in thick bundles of fibers with a smooth appearance. No TH immunoreactivity was visualized in low-density innervation in this section. (D) Dense SIR (with FITC) observed in the high-density innervation areas colocalized with TH immunoreactivity, whereas SIR in low-density innervation did not. Note that even in the low-density innervation areas, there were regional differences in the SIR density. (E) In high-density innervation, some VAChT immunoreactivity (visualized with Rhodamine Red) appeared to colocalize or be in close association with TH immunoreactivity (arrows) which was visualized with FITC (F), and one nerve bundle appeared to include both VAChT immunoreactivity and TH immunoreactivity fibers, suggesting these nerves contain both parasympathetic and sympathetic fibers. Scale bar: (AD) 100 μm; (E, F) 40 μm.
Figure 3.
 
Double labeling of the innervation in both low- and high-density areas in the lacrimal gland. (A) VAChT immunoreactivity was observed in both low- and high-density innervation. (B) Synaptophysin immunoreactivity (SIR visualized with FITC) colocalized with all VAChT immunoreactivity in both low- and high-density innervation. (C) Dense TH-immunoreactive nerve fibers were observed in the high-density innervation, mostly in thick bundles of fibers with a smooth appearance. No TH immunoreactivity was visualized in low-density innervation in this section. (D) Dense SIR (with FITC) observed in the high-density innervation areas colocalized with TH immunoreactivity, whereas SIR in low-density innervation did not. Note that even in the low-density innervation areas, there were regional differences in the SIR density. (E) In high-density innervation, some VAChT immunoreactivity (visualized with Rhodamine Red) appeared to colocalize or be in close association with TH immunoreactivity (arrows) which was visualized with FITC (F), and one nerve bundle appeared to include both VAChT immunoreactivity and TH immunoreactivity fibers, suggesting these nerves contain both parasympathetic and sympathetic fibers. Scale bar: (AD) 100 μm; (E, F) 40 μm.
Figure 4.
 
The regional difference of VAChT immunoreactivity in the mouse lacrimal gland. Data are the mean ± SEM of results in number of sampling areas shown in parentheses. Each sampling area was 30 × 40 μm, or 1200 μm2. *Significant difference from area of low-density innervation.
Figure 4.
 
The regional difference of VAChT immunoreactivity in the mouse lacrimal gland. Data are the mean ± SEM of results in number of sampling areas shown in parentheses. Each sampling area was 30 × 40 μm, or 1200 μm2. *Significant difference from area of low-density innervation.
Figure 5.
 
Protein secretory responses of the mouse lacrimal gland to agonists at 10 μM. Ordinate is total protein secretion rate. Data are the mean ± SEM of results in the numbers of experiments shown in parentheses. There are no significant differences between any two responses evoked by carbachol, norepinephrine, and phenylephrine. The secretion induced by isoproterenol, however, was significantly lower than those induced by carbachol, norepinephrine, and phenylephrine. *Significant difference from the corresponding basal secretion rate of each agonist.
Figure 5.
 
Protein secretory responses of the mouse lacrimal gland to agonists at 10 μM. Ordinate is total protein secretion rate. Data are the mean ± SEM of results in the numbers of experiments shown in parentheses. There are no significant differences between any two responses evoked by carbachol, norepinephrine, and phenylephrine. The secretion induced by isoproterenol, however, was significantly lower than those induced by carbachol, norepinephrine, and phenylephrine. *Significant difference from the corresponding basal secretion rate of each agonist.
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