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J Comp Neurol. Author manuscript; available in PMC 2017 September 01. Published in final edited form as: J Comp Neurol. 2016 September 1; 524(13): 2577–2603. doi:10.1002/cne.23978.
Individual Sympathetic Postganglionic Neurons Co-Innervate Myenteric Ganglia and Smooth Muscle Layers in the Gastrointestinal Tract of the Rat
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Gary C. Walter, Robert J. Phillips, Jennifer L. McAdams, and Terry L. Powley Department of Psychological Sciences, Purdue University, West Lafayette, Indiana 47907-2081, USA
Abstract
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A full description of the terminal architecture of sympathetic axons innervating the gastrointestinal (GI) tract has not been available. To label sympathetic fibers projecting to the gut muscle wall, dextran biotin was injected into the celiac and superior mesenteric ganglia (CSMG) of rats. Nine days post-injection, animals were euthanized, and stomachs and small intestines were processed as whole mounts (submucosa and mucosa removed) to examine CSMG efferent terminals. Myenteric neurons were counterstained with Cuprolinic Blue; catecholaminergic axons were stained immunohistochemically for tyrosine hydroxylase. Essentially all dextran-labeled axons (135 of 136 sampled) were tyrosine hydroxylase-positive. Complete postganglionic arbors (n=154) in the muscle wall were digitized and analyzed morphometrically. Individual sympathetic axons formed complex arbors of varicose neurites within myenteric ganglia/primary plexus and, concomitantly, long rectilinear arrays of neurites within circular muscle/secondary plexus or longitudinal muscle/ tertiary plexus. Very few CSMG neurons projected exclusively (i.e. ~100% of an arbor’s varicose branches) to myenteric plexus (~2%) or smooth muscle (~14%). With less stringent inclusion criteria (i.e. ≥ 85% of an axon’s varicose branches), larger minorities of neurons projected predominantly to either myenteric plexus (~13%) or smooth muscle (~27%). The majority (i.e., ~60%) of all individual CSMG postganglionics formed mixed, heterotypic arbors that co-
Corresponding Author: Terry L. Powley, Purdue University, Department of Psychological Sciences, 703 Third St, West Lafayette, IN 47907-2081, Phone: 765-494-6269, Fax: 765-496-1264, [email protected]. Conflict of Interest Statement The Authors declare no conflict of interest.
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Resources Cited RRID:RGD_61109 RRID:AB_2307337 RRID:AB_2336819 RRID:nig-0000-10294 RRID:rid_000081 RRID:AB_572268 RRID:AB_2313713 RRID:AB_2313574 RRID:AB10566286 RRID:AB_10562715 Role of Authors GCW participated in the design, data collection, statistical analyses, figure production and writing of the manuscript. RJP contributed to the design, development of the tracer protocol, statistical analyses, figure production and writing of the manuscript. JLM contributed to the development of the tracer protocol, surgery and ganglion injections, and development of the manuscript. TLP participated in the design, analyses of the data, and preparation of the manuscript.
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innervated extensively (> 15% of their varicose branches per target) both myenteric ganglia and smooth muscle. The fact that ~87% of all sympathetics projected either extensively or even predominantly to smooth muscle, while simultaneously contacting myenteric plexus, is consistent with the view that these neurons control GI muscle directly, if not exclusively.
Keywords Autonomic Nervous System; Celiac Ganglion; Enteric Nervous System; Muscularis Externa; Superior Mesenteric Ganglion
Introduction Author Manuscript
The sympathetic nervous system (SNS) supplies much of the extrinsic innervation of the gastrointestinal (GI) tract and profoundly influences motility and other GI functions (Furness and Costa, 1974; Furness, 2006a; Jobling, 2012). In spite of the key roles the SNS plays in gut physiology, much about the organization of the sympathetic postganglionic neurons innervating the smooth muscle wall of the GI tract remains unknown. In particular, the morphologies and features of the motor neuron axonal arbors, as these neurite terminals ramify and distribute to modulate function in the gut wall, have not been thoroughly analyzed.
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The current incomplete ideas about SNS postganglionic architecture are based on examinations with various protocols that have not evaluated the full distribution or complete morphology of individual postganglionic neurons. Thus the inferred patterns of innervation have been shaped—as well as potentially skewed—by the limitations of the techniques employed. The point is reinforced by the fact that the various patterns of sympathetic organization that have been adduced with different methodologies have been inconsistent.
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Classically, one school of thought inferred that sympathetic postganglionic fibers originating in the prevertebral ganglia and projecting to the GI tract innervate directly the smooth muscle fibers of the muscularis externa (Langley, 1903; Hill, 1927). This two-neuron view of the SNS outflow was based largely on the fact that, in other organ systems, autonomic preganglionic fibers originating in the CNS project to postganglionic neurons in prevertebral ganglia, and the axons of those postganglionic neurons then typically innervate smooth muscle effectors directly (Langley, 1903; Hill, 1927; Kuntz, 1934, 1953; Bennett, 1972). With the introduction of tissue protocols that induce catecholamine fluorescence in situ, however, evidence accumulated that the sympathetic postganglionic fibers innervate myenteric ganglion neurons (e.g., Falck et al., 1962; Capurso et al., 1968; Furness, 1970b; Furness and Costa, 1971; Furness and Malmfors, 1971), which were known to project to smooth muscle. In a series of influential investigations beginning with the experiments of Norberg and Sjoqvist, (Norberg, 1964; Norberg and Sjoqvist, 1966; Norberg, 1967), investigators observed that fluorescent catecholaminergic postganglionic fibers in the gut are densely packed in the myenteric plexus, conspicuously less densely packed in the circular muscle than in the myenteric plexus, and almost absent in the longitudinal muscle sheet (Norberg, 1964, 1967; Hollands and Vanov, 1965; Capurso et al., 1968). Such a view was also consistent with both earlier (e.g., Cajal, 1911; Muller, 1911; Carpenter, 1924) and more J Comp Neurol. Author manuscript; available in PMC 2017 September 01.
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recent (e.g., Llewellyn-Smith et al., 1984; Wiley and Owyang, 1987; Keast, 1994; Hayakawa et al., 2008) observations from other techniques that suggest sympathetic fibers contact myenteric neurons that perhaps in turn project to smooth muscle fibers.
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In spite of widespread illustration of three-neuron chains in reviews, textbooks, and schematics (e.g., Burnstock and Costa, 1973; Brading, 1999; Furness, 2006b; Janig, 2006), the organization has found limited direct support when other methodologies have been used to describe the SNS postganglionic distribution in the gut. Ultrastructural studies have routinely noted that sympathetic axons rarely contact or run in near-proximity to the somata or dendrites of myenteric neurons. Instead, EM assessments indicate that extrinsic sympathetic axons tend to course superficially in the ganglia of the plexuses, where the neurites’ varicosities appear to form appositions with other axons and, tellingly, with sites near the basal lamina and capsule of the plexuses that might allow transmitter diffusion directly to smooth muscle fibers (Gabella, 1970; Manber and Gershon, 1979; LlewellynSmith et al., 1981, 1984; Gordon-Weeks, 1982). Similarly, electrophysiological analyses have suggested limited, though certainly some, influences of catecholamines in the myenteric plexus. Some analyses indicated that catecholamines only weakly, if at all, affect the firing patterns or resting potentials of myenteric neurons (Holman et al., 1972; Nishi and North, 1973; Takayanagi et al., 1977). In contrast, however, other experiments have indicated that sympathetic projections to the intestine appear to presynatically inhibit cholinergic potentials in some myenteric neurons (Hirst and McKirdy, 1974), hyperpolarize some myenteric neurons (Galligan and North, 1991; Bian and Galligan, 2007), and potentially modulate reflex circuits within the myenteric plexus (Stebbing et al., 2001).
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Reinforcing the conclusion that direct sympathetic innervation of myenteric ganglion cells may be limited, it is also clear that essentially all extrinsic fibers projecting to the different tissues of the gut wall course initially through the myenteric plexus. Thus, even axons that merely traverse the plexus pathways to reach more distal target tissues, including even submucosal sites, might account for an appearance of a concentration of sympathetic fibers (and potential contacts) commonly observed within the plexus. Furthermore, immunohistochemistry of catecholamines and their synthetic enzymes, in contrast to the indirect fluorescence techniques, has regularly described substantial networks of catecholaminergic axons in the circular (though not longitudinal) muscle sheet throughout the rostral-caudal extent of the GI tract (Furness et al., 1990; Tassicker et al., 1999; Phillips et al., 2006, 2009, 2013; Tan et al., 2010).
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Most of the apparent discrepancies between the views provided by the different techniques seem to have been predicated on a working assumption that SNS postganglionic projections either innervate smooth muscle fibers directly (i.e., in a two-neuron-outflow pattern) or, instead, innervate the enteric neurons of the plexus and hence smooth muscle indirectly (i.e., in a three-neuron-outflow pattern). Evidence for one pattern has frequently been taken as evidence against the other, and vice versa. The organizational possibilities are not binary, however, and other architectures consistent with both two-neuron pattern and three-neuron pattern would be consistent with all of the previous observations. For example, separate populations of postganglionic fibers might project, respectively, to smooth muscle fibers and to enteric neurons. Or, yet again, individual fibers might course through the plexuses of the
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gut wall, supplying collaterals or varicosities en passage to myenteric neurons, and then continue on to also directly innervate the smooth muscle. Interestingly, we are unaware of any morphological experiment that has systematically evaluated such a co-innervation possibility for individual sympathetic fibers in the gut. Presumably, at least in part, it has been relatively impractical to assess this alternative for the sympathetic projections to the gut because the extensively intertwined postganglionic axons projecting to the gut are so densely packed and distributed through such distances and complex tissue elements, that the traditional methodologies (induced fluorescence, ultrastructural analysis, electrophysiology, immunohistochemistry) cannot readily isolate the full reach of individual autonomic axonal arbors in the gut wall.
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To circumvent some of the limitations associated with the conventional approaches, the present experiment used a different technique—namely labeling, in any one animal preparation, limited numbers of individual sympathetic postganglionic neuron axonal arbors in their entirety with the anterograde tracer dextran biotin (Walter et al., 2009). The isolated and complete arbors of individually identified axons were then traced, digitally reconstructed, and evaluated morphometrically in whole mounts of the muscularis externa to determine the distribution patterns of single sympathetic nervous system fibers as a means of evaluating and comparing them to the various putative patterns of innervation.
Materials and Methods Animals
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Virgin male rats (Fischer 344; n = 62; Harlan Laboratories, Indianapolis, IN; RRID:RGD_61109) were purchased at 2-months of age and weighed 175–200 g at the time of delivery. The animals were housed in an AAALAC-approved room kept at 20–23°C, on a 12:12 hour light:dark schedule, with access to pelleted chow (Laboratory diet no. 5001; PMI Feeds Inc., Brentwood, MO, USA) and filtered tap water available ad libitum. All Procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (8th ed., The National Academic Press, Washington, D.C.), and approved by the Purdue University Animal Care and Use Committee. Every effort was made to minimize suffering and the number of animals used. Tracer Injection in the Celiac and Superior Mesenteric Ganglia
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After a 2-week acclimation period in the colony, each animal was food-deprived overnight, anesthetized with Isoflurane (Isoflo®; Abbott Laboratories, North Chicago, IL, USA), and injected with Glycopyrrolate (0.2 mg/ml, s.c.; AmericanRegent Inc., Shirley, NY, USA) to reduce salivary, tracheobronchial, and gastric secretions. Each rat was then positioned on its back, and the abdominal cavity was opened by a midline laparotomy. The abdominal organs were draped with sterile saline moistened gauze and gently displaced inside the abdomen to visualize the sympathetic ganglia. To minimize disturbance of organs and surgery time, only the left celiac and superior mesenteric ganglia (CSMG), which are a main source of sympathetic innervation to the stomach and small intestine (Miolan and Niel, 1996; Quinson et al., 2001), were injected with tracer.
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More specifically, since the two ganglia are fused in the rat, the posterior pole of the CSMG (Hammer and Santer, 1981; Berthoud and Powley, 1993; Furness, 2014) was impaled using a Nanofil syringe with a beveled 35 gauge needle (NF35BV; World Precision Instruments, Sarasota, FL). The needle was advanced longitudinally, within and parallel to the long axis of the fused ganglia, until the tip rested at the rostral pole of the celiac ganglion part of the CSMG complex (Berthoud and Powley, 1996; Quinson et al., 2001). Dextran biotin, 6 µl of 10K MW lysine-fixable (7.5% concentration; Cat# D1956; Life Technologies, Grand Island, NY; RRID:AB_2307337), was then slowly injected as the needle was gradually retracted in a stepwise fashion within the ganglia. When the injection was completed and the infusion pressure within the CSMG had dissipated, the needle was slowly withdrawn from the ganglia. The displaced organs were then returned to their original position within the abdominal cavity, and the abdominal muscle was closed using interrupted sutures followed by closure of the skin with a single continuous suture.
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Prior to being returned to their home cages, rats recovered on a water circulating warming pad until their righting reflexes had completely returned. To minimize post-surgical discomfort and pain, Buprenorphine (0.01 mg/kg; s.c., Buprenex®, Reckitt Benckiser Pharmaceuticals Inc., Richmond, VA, USA) was administered prior to discontinuation of Isoflurane and then again once every 24 h over the following 72 h post-surgery. Also, to prevent post-surgical dehydration, rats were given an injection of warm sterile saline (6 ml; s.c.). Both body weight and intake of food and water were monitored throughout the duration of the recovery period to ensure that rats did not experience any post-surgical malaise.
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Control procedures were run to evaluate whether tracer leakage from the CSMG into the surrounding parenchyma or intraperitoneal space might be a source of neural labeling (Fox and Powley, 1989): Following laparotomy and exposure of the left CSMG, as described above, 6 µl of dextran biotin was dispensed over the exterior surface of the CSMG of control rats (n = 4). The complete absence of labeled terminal fields in the gut wall of these four rats indicated that labeled terminals in the ganglia-injected group were a result of tracer being incorporated into the neurons of the left CSMG and transported along their axons to their terminal arbors located in the gut wall and were not a byproduct of tracer leakage. Tissue Fixation and Dissection
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Nine days after tracer injection, rats were weighed, euthanized with a lethal dose of sodium pentobarbital (180 mg/kg, i.p.), and perfused through the left ventricle of the heart with 200 ml of 0.01M PBS at 40°C followed by 500 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 4°C. Prior to the perfusion, 0.5 ml of heparin was injected in the left ventricle to facilitate exsanguination. After fixation, the stomach and small intestine were removed and sampled according to the criteria of Hebel and Stromberg (1976). Specifically, the stomach was divided into dorsal and ventral halves by cutting along the greater and lesser curvatures. Whole mounts of the small intestine consisted of 3 cm long sections from the duodenum (the first 6 cm anal to the pyloric sphincter), mid-jejunum (middle 3 cm of the small intestine), and ileum (the first 6 cm oral to the ileocaecal junction). Intestinal whole mounts were opened by cutting along J Comp Neurol. Author manuscript; available in PMC 2017 September 01.
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the longitudinal axis of the mesenteric attachment. The specimens were rinsed under tap water and further fixed overnight in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 4°C. Intact muscularis externa whole mounts consisting of the circular and longitudinal muscle layers, with the myenteric plexus sandwiched in-between, were then isolated by removing the mucosa and submucosa from each specimen. Staining
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Free floating whole mounts were rinsed in PBS followed by a 30 min endogenous peroxidase block (methanol:3% H2O2; 4:1). Following additional PBS rinses, whole mounts were rinsed in dH2O and then stained in 0.5% Cuprolinic Blue (quinolinic phthalocyanine; 17052; Polysciences, Inc., Warrington, PA) in 0.05 M sodium acetate buffer containing 1.0 M MgCl2 (pH 4.9) for 2 h in a humidified slide warmer at 38°C. Cuprolinic Blue is a neuron-specific stain that is a pan-neuronal marker for myenteric neurons and is compatible with the dextran biotin protocol (Phillips et al., 2004; Walter et al., 2009). Whole mounts were then rinsed in dH2O, differentiated for 2 min in 0.05 M sodium acetate buffer containing 1.0 M MgCl2 (pH 4.9), and rinsed again in dH2O followed by 3 × 5 min PBS rinses. Whole mounts were then soaked for 3 d in PBS containing 0.5% Triton X-100 and 0.08% Sodium Azide to improve penetration through the muscle sheets. Next, free floating whole mounts were rinsed in PBS for 6 × 5 min followed by a 60 min soak in ABC (prepared according to the manufacturer’s directions; Cat# PK-6100; Vectastain Elite ABC kit; Vector Laboratories; RRID:AB_2336819). Whole mounts were then rinsed in PBS for 6 × 5 min, reacted in DAB solution for 3 min, followed by 6 × 5 min rinses in dH2O. The specimens were mounted on gelatin-coated slides, crushed for 90 min, air-dried overnight, dehydrated in an ascending series of alcohols, cleared in xylene, and coverslipped with Cytoseal (Richard-Allan Scientific, Kalamazoo, MI, USA).
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Fluorescent Immunohistochemistry To determine if tracer injection into the CSMG inadvertently labeled visceral afferent fibers of passage originating from the dorsal root ganglia (DRG), 12 rats were injected with dextran biotin into the CSMG as described above. Following fixation and dissection of the GI tract into regional whole mounts, a fluorescent double labeling protocol was employed to determine the relationship of dextran filled terminals with the sympathetic innervation of the gut visualized using antibodies to the noradrenaline- (as well as dopamine- and adrenaline-) synthesizing enzyme tyrosine hydroxylase (TH).
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Free floating whole mounts were incubated for 5 d in PBS containing 0.5% Triton X-100 and 0.08% sodium azide, followed by a 1 h soak in the same solution to which streptavidin ALEXA Fluor 594 (1:1000; Cat# S11227; Life Technologies; RRID:AB_2313574) had been added. Whole mounts were then rinsed in PBS followed by an overnight block in PBS containing 5% normal goat serum, 2% bovine serum albumin, 0.3% Triton X-100, and 0.08% sodium azide, and then incubated for an additional 24 h in a primary antibody diluted with the same blocking solution. Different whole mounts were exposed to either a mouse TH (1:500; Cat# 22941; ImmunoStar, Hudson, WI; RRID:AB_572268) or a rabbit TH (1:1000; Cat# P40101-0; Pel Freez Biologicals, Rogers, AR; RRID:AB_2313713) antibody. Whole mounts were then rinsed in PBS and 0.3% Triton X-100, and incubated for 2 h at
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room temperature in the appropriate secondary conjugated to ALEXA Fluor 488 (i.e., either goat anti-mouse, Cat# A11029, RRID:AB10566286 or goat anti-rabbit, Cat# A11034, RRID:AB_10562715; both purchased from Life Technologies) diluted 1:500 with the same solution used for the previous rinses. Finally, double-labeled whole mounts were rinsed in PBS, mounted on gelatin-coated slides, cover-slipped with Dako Fluorescent Mounting Medium (S3023; Dako North America, Inc., Carpinteria, CA), sealed with CoverGrip Coverslip Sealant (23005; Biotium, Inc., Hayward, CA), and stored at 2–8°C in the dark until examined for fluorescent signal.
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The degree of co-localization between the injected tracer and the TH antigen was determined using an Olympus DSU (Disk Scanning Unit) spinning disk confocal microscope attached to a BX61 motorized microscope (Olympus, Center Valley, PA). The confocal microscope was controlled using SlideBook digital microscopy software (V5.0; Intelligent Imaging Innovations, Denver, Co). Double-labeled sections were imaged using highly selective filter sets specific for the visualization of ALEXA Fluor 594 (XF102-2; Omega Optical) and ALEXA Fluor 488 (QMAX-Green; Omega Optical, Brattleboro, VT). A 40× water objective lens (NA = 1.15) was used. Extended-focus images or z-series consisting on average of 21 optical sections at an optimized z-increment of 0.38 µm were created. In some cases, z-stacks were compressed into one focal plan (i.e., a maximum value projection). To test for co-localization, a whole mount was systematically scanned for ALEXA Fluor 594. When a well labeled terminal neurite was identified, the upper and lower limits of the terminal were determined, with the experimenter blind to the ALEXA Fluor 488 wavelength, and a z-stack was captured using both filter sets. Each focal plane with the two channels merged was then examined individually for co-localization throughout the entire z-stack.
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Antibody Characterization
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Table 1 summarizes information about the primary antibodies used in this study to identify the sympathetic motor/tyrosine hydroxylase-positive innervation of the smooth muscle layers of the gut wall. The mouse monoclonal TH antibody (ImmunoStar Cat# 22941, RRID:AB_572268) was raised against TH purified from rat PC12 cells, and the antibody is described as having wide species crossreactivity (manufacturer’s datasheet). The immunogen used to produce the rabbit polyclonal TH antibody (Pel-Freez Biologicals Cat# P40101, RRID:AB_2313713) was SDS-denatured TH from rat pheochromocytoma, and shown by the manufacturer using western blot analysis to be specific for the ≈60 kD TH protein in rat caudate lysate. Immunostaining in the gut wall for both antibodies was consistent with the pattern of sympathetic innervation reported previously in GI whole mounts (Tan et al., 2010; Phillips et al., 2013), which is eliminated following celiac and superior mesenteric ganglionectomy surgery (Phillips et al., 2013). No positive reactions were observed in any whole mounts in which TH antibodies were omitted from the protocol. Whole Mount Evaluation and Analysis Criteria for myenteric ganglia—In keeping with the criteria of Bar-Shai and coworkers (2004), two or more myenteric neurons clustered together were considered a ganglion. If a cluster of cell bodies was separated from another cluster of cell bodies by a
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distance of three or more average neuron diameters, then they were considered two separate ganglia (cf. Berthoud et al., 1997). Also, if a group of neurons was isolated from another group by a string of successive single-file neurons, that was as much as, or more than, three average neuronal diameters in length, then the two groupings were considered to be separate ganglia (cf. Berthoud et al., 1997).
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Criteria for myenteric plexus connectives—Enteric circuitry and smooth muscle tissues were distinguished according to commonly used criteria (cf. Scheuermann and Stach, 1984; Furness, 2006a). Briefly, the primary myenteric plexus was considered to be formed of both the myenteric ganglia and the major connectives interconnecting those ganglia (also referred to as interganglionic connectives or interconnective strands). The secondary plexus was defined as those higher order connectives that (a) issue from the myenteric ganglia or interganglionic connectives, (b) lie in a network deep to the primary plexus and in contact with the circular muscle, and (c) consist of fascicles running predominately parallel to the circular muscle fibers. The tertiary plexus was defined as the network of higher order ENS connectives that (a) issue from the myenteric ganglia, interganglionic connectives, or secondary plexus, (b) lie in a network situated at the level of, or just superficial to, the primary plexus and abutting the deeper face of the longitudinal muscle sheet wall, and (c) consist of fascicles running predominately parallel to longitudinal muscle fibers. The secondary plexus carries axons of extrinsic (and intrinsic) efferent (and afferent) neurons that innervate the circular muscle sheet (Wilson, et al., 1987; Furness, 2006b), whereas the tertiary plexus carries axons that innervate the longitudinal muscle sheet (Llewellyn-Smith et al., 1993). These conventional distinctions and inferences were used in categorizing the tissue targets of the labeled CSMG axonal arbors.
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Sympathetic Terminal Arbor Inventories and Morphometry Initial screening—The whole mounts were first systematically scanned at 400× magnification with brightfield illumination. Once an arbor was identified, it was further inspected to determine whether it satisfied the following four criteria: (a) well labeled, (b) completeness of the axon, (c) minimal tissue artifacts such as folds and tears, and (d) free of multiple independent axons interdigitating or overlapping in a manner that might confuse the evaluation of a given terminal field.
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Morphometry—A total of 254 sympathetic axons that satisfied the histological criteria for morphometry were located in the initial screening for staining quality and specimen integrity. Of that inventory, 160 axons were randomly selected for digitization, and individual sympathetic terminal arbors were then subsequently traced, digitally reconstructed, and evaluated quantitatively using a Neurolucida (MicrobrightField Inc., Williston, VT, USA; RRID:nig-0000-10294) workstation employing a Zeiss Axio Imager Z2 microscope (Carl Zeiss Microimaging, Gottingen, Germany) equipped with DIC optics and both a 40× dry and a 63× water immersion long working distance objective. For each of the digitized sympathetic arbors, the following standardized morphometric measures captured with the Neurolucida software were analyzed for all neurites: total arbor length, parent axon length (using the Strahler Analysis algorithm), varicose neurite length, J Comp Neurol. Author manuscript; available in PMC 2017 September 01.
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total number of terminal branches, highest branch order, and two-dimensional terminal field size (using the Convex Hull algorithm [cf. Powley et al., 2012, 2013a, 2013b, 2014]). Criteria for Sympathetic Postganglionic Innervation or Contacts Two different indices were used to independently estimate the extent of innervation of, or contact with, tissue targets by sympathetic axons: 1) Consistent with conventional observations (e.g., Falck and Owman, 1966; Furness et al., 2014), beaded segments of sympathetic axons exhibiting substantial numbers of varicosities were assumed to release transmitter to the immediately adjacent tissues from those varicosities. 2) Similarly, consistent with the common assumption (e.g., Levitan and Kaczmarek, 2002), the highest order branches, or distal terminal tips, of sympathetic motor fiber arbors were assumed to be sites of transmitter release.
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Assessments of the proportions or extent of innervation patterns of different tissues in the gut wall were developed from the above criteria of postganglionic innervation. In the case of the primary plexus (myenteric ganglia and their primary interconnective strands), both of the measures were recorded. The first measure consisted of counting the number of ganglia that had either a terminal branch within a ganglion or the end of the terminal branch in a primary connective neighboring a ganglion. The second measure involved counting the number of ganglia with varicose fibers passing through or in close proximity. In the case of the myenteric plexus, both measures, that is both a terminal branch and a beaded varicose axon, needed to satisfy a neurite-to-ganglion proximity rule were defined as having a distance from a ganglion of less than or equal to half the average neuron diameter of Cuprolinic blue labeled myenteric neurons. This distance was chosen based on the expected distance of neurotransmitter diffusion (Read and Burnstock, 1969; Furness, 1970b; Gillespie and Maxwell, 1971). The total length of each arbor’s varicose segments located in conjunction with each of the types of tissue (i.e., myenteric ganglia and primary plexus; circular muscle sheet and secondary connectives; longitudinal muscle sheet and tertiary connectives) was also determined and then expressed as a percentage of the total length of all of that arbor’s segments.
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Both criteria of postganglionic innervation were similarly used to evaluate the more direct projections to the smooth muscle sheets: 1) Labeled terminal axonal arbor branches and varicose segments of the arbor within the circular muscle or in the secondary plexus in immediate apposition to the muscle were assumed to directly innervate muscle fibers. 2) Similarly, labeled terminal axonal branches and varicose arbor segments in the longitudinal muscle or in the tertiary plexus in immediate apposition with the muscle sheet were assumed to directly innervate longitudinal muscle fibers. For these estimates of muscle sheet innervation, both the number of terminal branches and the length of varicose arbor segments in association with the muscle sheets were determined and used as separate measurements of degree of innervation to confirm and cross-validate the sympathetic terminal field innervation of muscle in the gut wall. As in the case of the primary myenteric plexus, the proximity rule (i.e. within half an average neuron’s diameter of the putative target tissue) was used.
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Final Postganglionic Samples
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In the final analysis, after the 160 sympathetic arbors had been traced and digitized, the reconstructions of 154 of the fibers were ultimately used for quantitative comparisons of myenteric ganglion and smooth muscle innervation. These 154 axonal terminal arbors yielded representative samples for each of the five regions prepared as whole mounts (stomach: n = 22; duodenum 0–3 cm: n = 33; duodenum 3–6 cm: n = 29; jejunum: n = 20; ileum: n = 50).
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The remaining six of the 160 randomly chosen motor arbors were evaluated separately but not included in quantitative analyses of terminal patterns: two of the six arbors were judged problematic for complete quantitative assessment because the specimens traversed regions with incomplete Cuprolinic Blue staining. The remaining four of the six cases projected to the vasculature rather than the smooth muscle sheets; these vascular efferents were evaluated and are described separately. Photography
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Single high power images were acquired with a Spot Flex camera controlled by Spot Software (V4.7 Advanced Plus; Diagnostic Instruments, Sterling Heights, MI; www.spotimaging.com) mounted on a Leica DMRE. Multi-field composite (or mosaic) images containing the entire area of innervation by a sympathetic axon were generated at high magnification (630×; water immersion objective) with a Leica DFC310 FX digital CD color camera mounted on a Leica DM5500, running Surveyor with Turboscan software (V. 6.0.5.3; Objective Imaging, Cambridge, UK; www.objectiveimaging.com). To capture the varying depth of a neurite within a smooth muscle whole mount, each image in a mosaic typically consisted of multiple focal z-planes that were merged using either Photoshop CS5 (Adobe Systems, San Jose, CA; www.adobe.com) or Helicon Focus Pro X64 software (V5.3.7; HeliconSoft Ltd, Kharkov, Ukraine; www.heliconsoft.com) to generate an all-infocus image. Photoshop CS5 was also used in final figure production to: 1) apply text and scale bars; 2) adjust brightness, contrast, color, hue, and sharpness; and 3) organize the final layouts of the figures. Statistics Statistical analyses and graph generation were accomplished using GraphPad Prism (V. 5.0; GraphPad Software, San Diego, CA, USA; RRID:rid_000081). A one-way ANOVA with Tukey’s post hoc test (corrected for multiple comparisons) was performed to determine significant differences between groups. Data is presented as means ± SEM. Statistical significance was considered p < 0.05.
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Results Co-localization of Dextran Biotin Labeled Terminals with Tyrosine Hydroxylase Co-localization analysis was done on 136 dextran labeled terminals located in the muscle wall of jejunal whole mounts. One hundred dextran labeled terminals were evaluated for colocalization with the polyclonal rabbit TH antibody, and an additional 36 dextran labeled endings were evaluated for co-localization with the monoclonal mouse TH antibody. The
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distribution of arbors sampled consisted of 54 in the myenteric ganglia/1° plexus, 43 in the longitudinal muscle/3° plexus, and 39 in the circular muscle/2° plexus; Figure 1A–I. In all, only one dextran labeled terminal (located in the circular muscle/2° plexus) was found to be TH-negative. Qualitative evaluation of randomly chosen dextran labeled arbors located in the smooth muscle wall of the stomach, duodenum, and ileum were found to be similarly extensively co-localized with TH. Omission of the primary resulted in complete loss of TH-positive fibers; Figure 1J–L. Examination of a) dextran-negative/TH-positive arbors in the double labeled material and b) dextran labeled terminals in the primary deletion control material confirmed that the emission spectra of the two fluorophores combined with the filter sets used during the acquisition of the images sufficiently avoided spectral bleed-through artifact and were suitable for co-localization analysis; Figure 1C,F,I, and K.
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General Architecture of Sympathetic Postganglionic Terminal Arbors Dextran-biotin labeling of sympathetic axons in whole mounts that preserved the myenteric plexus in situ between the longitudinal and circular muscle sheets provided high-definition delineation of complete motor terminal arbors terminating in the muscle wall of the gut and made it practical to routinely digitize and analyze morphologically individual CMSG axons. Figure 2 illustrates a Neurolucida tracing of a typical CSMG axonal arbor, and Figure 3 contains photomicrographs of different representative sites within the tracing in Figure 2, as designated by the lettering.
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In each of the four gut regions sampled (i.e. stomach, duodenum, jejunum and ileum), the basic architecture of individual sympathetic terminal fields throughout the smooth muscle wall consisted of a parent axon entering the gut wall within a bundle or fascicle of fibers, coursing some distance through the myenteric plexus network, and then branching repeatedly to form an extensive terminal array of varicose neurites extending over a large innervation field, making presumptive contacts in multiple different tissues.
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The initial screening to identify traceable CSMG arbors produced observations indicating both a need to distinguish different groupings of axonal terminal fields, particularly with reference to the three-neuron and two-neuron models of the sympathetic outflow, and a need to evaluate the disparate ending patterns quantitatively. In brief, one small minority of postganglionic arbors appeared to project strongly to myenteric neurons and the primary plexus, in a configuration consistent with a three-neuron-outflow model, and a second small minority of postganglionic arbors appeared to predominately innervate one or both smooth muscle sheets directly, in a pattern consistent with a two-neuron-outflow model. In contrast, however, a large majority of CSMG postganglionics, as illustrated in Figures 2 and 3, distributed a substantial percentage of their neurites, in parallel, to both the myenteric ganglia/1° plexus and the smooth muscle. Criteria Distinguishing Subpopulations of Sympathetic Postganglionics The fine architecture of sympathetic postganglionics in the muscularis externa of the gut wall has not previously been well characterized, so a working criterion to distinguish between the identified subpopulations was generated. First, the 154 arbors in the random J Comp Neurol. Author manuscript; available in PMC 2017 September 01.
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sample of postganglionic neurons that innervated the smooth muscle wall were traced and digitized. Since all branches of these arbors projected to only two types of tissue, the myenteric plexus and/or smooth muscle, the proportions or total lengths of each arbor’s varicose neurite branches in myenteric ganglia/1° plexus and in smooth muscle, respectively, were then calculated and used to plot innervation profiles.
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More specifically, for innervation profiles, the composite or combined length of all varicose branch segments and terminal branches was obtained separately for each of the individual 154 arbors. Then, for each arbor, as analyzed below, the separate summed lengths of those varicose neurites in apposition to each of the basic types of target tissues innervated (myenteric ganglia/1° plexus; circular muscle/2° plexus; or longitudinal muscle/3° plexus) were separately expressed as percentages of the respective arbor’s full cumulative length of varicose branches. Further, for the purposes both of generating criteria that distinguished subpopulations and of making general comparisons between the two-neuron and the threeneuron models, the projections to the longitudinal and circular muscle layers were then also combined and collectively considered as innervation of “smooth muscle.”
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Once the data for the muscle sheets had been combined, the frequency distributions of the numbers of arbors within the sample of 154 cases with different percentages of neurites projected to each of the two broad tissue types (myenteric ganglia/1° plexus; smooth muscle) of the gut wall were examined. Figure 4 illustrates the distribution of all 154 arbors with the different percentages (on the lower x-axis) of their varicose segments projecting specifically to smooth muscle and/or their respective interconnectives. Reciprocally, since all varicose neurites not terminating in muscle terminated in the primary plexus, Figure 4 also illustrates, at the left end of the abscissa (on the upper x-axis), the subpopulation of arbors that project specifically to myenteric ganglia/1° plexus.
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From this neurite distribution in Figure 4, two factors emerged and were used to establish the operationally defined criteria for grouping the 154 arbors examined in the series of specific quantitative comparisons reported below. The most salient factor for identifying subpopulations was that the distribution of arbors was polymodal, including at least three identifiable peaks. The second factor shaping the provisional groupings of the CSMG postganglionic arbors was a set of pragmatic observations about cutoffs that would distinguish the distinct subpopulations that appeared to form clusters in the polymodal distribution of arbor segments illustrated in Figure 4. A strict construction of the commonly used three-neuron and two-neuron ideas of the sympathetic outflow generally might extrapolate to the prediction that an individual arbor would project exclusively to, respectively, the primary myenteric plexus or the smooth muscle, but as Figure 4 indicates, few arbors projected 100% of their varicose neurites to smooth muscle and even fewer arbors projected 0% of their varicose neurites to smooth muscle (and hence 100% to myenteric ganglia/1° plexus). The typical architecture of the postganglionics is illustrated by the axonal terminal arbor in Figure 5 which exemplifies the fact that, even in the case of most of those sympathetic arbors constituting the left-most peak in Figure 4 and projecting predominately to the myenteric ganglia/1° plexus, some of their short branches and/or arrays also typically
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extended to smooth muscle. Like most of the other arbors in the left-most cluster in Figure 4, the axon in Figure 5 innervated predominately the myenteric ganglia and associated primary plexus, but, nonetheless, the arbor distributed short collateral branches (example designated with arrows in Figure 5C) to the smooth muscle. Specifically, in the case in Figure 5, these collateral varicose branches to the muscle sheet accounted for only 8.5% of the total cumulative length of the fiber’s arbor. Examination of the left-most and right-most peaks in Figure 4 (corresponding, respectively, to arbors that terminate nearly exclusively in the myenteric ganglia/1° plexus or the smooth muscle) makes it clear that, a restrictive or “exclusive” decision rule requiring that 100% of an arbor’s segments need project to the relevant target tissue, in order for that individual postganglionic to be classified as projecting to a particular target, does not include all of the arbors comprising the two clusters close to the limits of the distribution in Figure 4.
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A strategy of defining a less restrictive and more practical cutoff, one that better reflected the clustering patterns in the distribution of Figure 4, was to find the nadir immediately to the right of the left-most peak and the nadir just to the left of the right-most peak and then to use the average of the two nadirs to generate a cutoff criterion. Given that the average of the two nadirs rounded to essentially 15%, we used that value for the cutoff of the left-most peak and the complementary value of 85% as the cutoff of the right-most peak. Arbors with 85% or more of their varicose segments projecting to a particular type of tissue were considered to predominantly innervate that tissue. In contrast, individual postganglionic arbors that comprised the subpopulation in the broad middle peak or cluster in Figure 4, which distributed roughly equivalent proportions of their neurites to the two tissues and did not distribute as much as 85% of their varicose branches to either the myenteric ganglia/1° plexus or the smooth muscle/associated interconnectives, were considered mixed or heterotypic neurons with substantial inputs to both types of tissues and a predominant input to neither type of tissue. Thus, based on these observed clustering patterns and considerations, we adopted a standard terminology of designating arbors that supplied 85% or more (but less than 100%) of their beaded branch segments to one tissue or the other as predominately innervating that tissue, and more equally distributed arbors that did not issue as much as 85% of their varicose branches to either single tissue type as providing mixed or heterotypic innervation of the two tissues.1 Arbors that projected 100% of their varicose neurites to either one tissue or the other were considered as exclusively innervating that tissue.
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Footnote 1: Given that the choice of what to use as cutoff criteria was arbitrary, we also explored the consequences of using cutoffs of
J Comp Neurol. Author manuscript; available in PMC 2017 September 01. Published in final edited form as: J Comp Neurol. 2016 September 1; 524(13): 2577–2603. doi:10.1002/cne.23978.
Individual Sympathetic Postganglionic Neurons Co-Innervate Myenteric Ganglia and Smooth Muscle Layers in the Gastrointestinal Tract of the Rat
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Gary C. Walter, Robert J. Phillips, Jennifer L. McAdams, and Terry L. Powley Department of Psychological Sciences, Purdue University, West Lafayette, Indiana 47907-2081, USA
Abstract
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A full description of the terminal architecture of sympathetic axons innervating the gastrointestinal (GI) tract has not been available. To label sympathetic fibers projecting to the gut muscle wall, dextran biotin was injected into the celiac and superior mesenteric ganglia (CSMG) of rats. Nine days post-injection, animals were euthanized, and stomachs and small intestines were processed as whole mounts (submucosa and mucosa removed) to examine CSMG efferent terminals. Myenteric neurons were counterstained with Cuprolinic Blue; catecholaminergic axons were stained immunohistochemically for tyrosine hydroxylase. Essentially all dextran-labeled axons (135 of 136 sampled) were tyrosine hydroxylase-positive. Complete postganglionic arbors (n=154) in the muscle wall were digitized and analyzed morphometrically. Individual sympathetic axons formed complex arbors of varicose neurites within myenteric ganglia/primary plexus and, concomitantly, long rectilinear arrays of neurites within circular muscle/secondary plexus or longitudinal muscle/ tertiary plexus. Very few CSMG neurons projected exclusively (i.e. ~100% of an arbor’s varicose branches) to myenteric plexus (~2%) or smooth muscle (~14%). With less stringent inclusion criteria (i.e. ≥ 85% of an axon’s varicose branches), larger minorities of neurons projected predominantly to either myenteric plexus (~13%) or smooth muscle (~27%). The majority (i.e., ~60%) of all individual CSMG postganglionics formed mixed, heterotypic arbors that co-
Corresponding Author: Terry L. Powley, Purdue University, Department of Psychological Sciences, 703 Third St, West Lafayette, IN 47907-2081, Phone: 765-494-6269, Fax: 765-496-1264, [email protected]. Conflict of Interest Statement The Authors declare no conflict of interest.
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Resources Cited RRID:RGD_61109 RRID:AB_2307337 RRID:AB_2336819 RRID:nig-0000-10294 RRID:rid_000081 RRID:AB_572268 RRID:AB_2313713 RRID:AB_2313574 RRID:AB10566286 RRID:AB_10562715 Role of Authors GCW participated in the design, data collection, statistical analyses, figure production and writing of the manuscript. RJP contributed to the design, development of the tracer protocol, statistical analyses, figure production and writing of the manuscript. JLM contributed to the development of the tracer protocol, surgery and ganglion injections, and development of the manuscript. TLP participated in the design, analyses of the data, and preparation of the manuscript.
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innervated extensively (> 15% of their varicose branches per target) both myenteric ganglia and smooth muscle. The fact that ~87% of all sympathetics projected either extensively or even predominantly to smooth muscle, while simultaneously contacting myenteric plexus, is consistent with the view that these neurons control GI muscle directly, if not exclusively.
Keywords Autonomic Nervous System; Celiac Ganglion; Enteric Nervous System; Muscularis Externa; Superior Mesenteric Ganglion
Introduction Author Manuscript
The sympathetic nervous system (SNS) supplies much of the extrinsic innervation of the gastrointestinal (GI) tract and profoundly influences motility and other GI functions (Furness and Costa, 1974; Furness, 2006a; Jobling, 2012). In spite of the key roles the SNS plays in gut physiology, much about the organization of the sympathetic postganglionic neurons innervating the smooth muscle wall of the GI tract remains unknown. In particular, the morphologies and features of the motor neuron axonal arbors, as these neurite terminals ramify and distribute to modulate function in the gut wall, have not been thoroughly analyzed.
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The current incomplete ideas about SNS postganglionic architecture are based on examinations with various protocols that have not evaluated the full distribution or complete morphology of individual postganglionic neurons. Thus the inferred patterns of innervation have been shaped—as well as potentially skewed—by the limitations of the techniques employed. The point is reinforced by the fact that the various patterns of sympathetic organization that have been adduced with different methodologies have been inconsistent.
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Classically, one school of thought inferred that sympathetic postganglionic fibers originating in the prevertebral ganglia and projecting to the GI tract innervate directly the smooth muscle fibers of the muscularis externa (Langley, 1903; Hill, 1927). This two-neuron view of the SNS outflow was based largely on the fact that, in other organ systems, autonomic preganglionic fibers originating in the CNS project to postganglionic neurons in prevertebral ganglia, and the axons of those postganglionic neurons then typically innervate smooth muscle effectors directly (Langley, 1903; Hill, 1927; Kuntz, 1934, 1953; Bennett, 1972). With the introduction of tissue protocols that induce catecholamine fluorescence in situ, however, evidence accumulated that the sympathetic postganglionic fibers innervate myenteric ganglion neurons (e.g., Falck et al., 1962; Capurso et al., 1968; Furness, 1970b; Furness and Costa, 1971; Furness and Malmfors, 1971), which were known to project to smooth muscle. In a series of influential investigations beginning with the experiments of Norberg and Sjoqvist, (Norberg, 1964; Norberg and Sjoqvist, 1966; Norberg, 1967), investigators observed that fluorescent catecholaminergic postganglionic fibers in the gut are densely packed in the myenteric plexus, conspicuously less densely packed in the circular muscle than in the myenteric plexus, and almost absent in the longitudinal muscle sheet (Norberg, 1964, 1967; Hollands and Vanov, 1965; Capurso et al., 1968). Such a view was also consistent with both earlier (e.g., Cajal, 1911; Muller, 1911; Carpenter, 1924) and more J Comp Neurol. Author manuscript; available in PMC 2017 September 01.
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recent (e.g., Llewellyn-Smith et al., 1984; Wiley and Owyang, 1987; Keast, 1994; Hayakawa et al., 2008) observations from other techniques that suggest sympathetic fibers contact myenteric neurons that perhaps in turn project to smooth muscle fibers.
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In spite of widespread illustration of three-neuron chains in reviews, textbooks, and schematics (e.g., Burnstock and Costa, 1973; Brading, 1999; Furness, 2006b; Janig, 2006), the organization has found limited direct support when other methodologies have been used to describe the SNS postganglionic distribution in the gut. Ultrastructural studies have routinely noted that sympathetic axons rarely contact or run in near-proximity to the somata or dendrites of myenteric neurons. Instead, EM assessments indicate that extrinsic sympathetic axons tend to course superficially in the ganglia of the plexuses, where the neurites’ varicosities appear to form appositions with other axons and, tellingly, with sites near the basal lamina and capsule of the plexuses that might allow transmitter diffusion directly to smooth muscle fibers (Gabella, 1970; Manber and Gershon, 1979; LlewellynSmith et al., 1981, 1984; Gordon-Weeks, 1982). Similarly, electrophysiological analyses have suggested limited, though certainly some, influences of catecholamines in the myenteric plexus. Some analyses indicated that catecholamines only weakly, if at all, affect the firing patterns or resting potentials of myenteric neurons (Holman et al., 1972; Nishi and North, 1973; Takayanagi et al., 1977). In contrast, however, other experiments have indicated that sympathetic projections to the intestine appear to presynatically inhibit cholinergic potentials in some myenteric neurons (Hirst and McKirdy, 1974), hyperpolarize some myenteric neurons (Galligan and North, 1991; Bian and Galligan, 2007), and potentially modulate reflex circuits within the myenteric plexus (Stebbing et al., 2001).
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Reinforcing the conclusion that direct sympathetic innervation of myenteric ganglion cells may be limited, it is also clear that essentially all extrinsic fibers projecting to the different tissues of the gut wall course initially through the myenteric plexus. Thus, even axons that merely traverse the plexus pathways to reach more distal target tissues, including even submucosal sites, might account for an appearance of a concentration of sympathetic fibers (and potential contacts) commonly observed within the plexus. Furthermore, immunohistochemistry of catecholamines and their synthetic enzymes, in contrast to the indirect fluorescence techniques, has regularly described substantial networks of catecholaminergic axons in the circular (though not longitudinal) muscle sheet throughout the rostral-caudal extent of the GI tract (Furness et al., 1990; Tassicker et al., 1999; Phillips et al., 2006, 2009, 2013; Tan et al., 2010).
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Most of the apparent discrepancies between the views provided by the different techniques seem to have been predicated on a working assumption that SNS postganglionic projections either innervate smooth muscle fibers directly (i.e., in a two-neuron-outflow pattern) or, instead, innervate the enteric neurons of the plexus and hence smooth muscle indirectly (i.e., in a three-neuron-outflow pattern). Evidence for one pattern has frequently been taken as evidence against the other, and vice versa. The organizational possibilities are not binary, however, and other architectures consistent with both two-neuron pattern and three-neuron pattern would be consistent with all of the previous observations. For example, separate populations of postganglionic fibers might project, respectively, to smooth muscle fibers and to enteric neurons. Or, yet again, individual fibers might course through the plexuses of the
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gut wall, supplying collaterals or varicosities en passage to myenteric neurons, and then continue on to also directly innervate the smooth muscle. Interestingly, we are unaware of any morphological experiment that has systematically evaluated such a co-innervation possibility for individual sympathetic fibers in the gut. Presumably, at least in part, it has been relatively impractical to assess this alternative for the sympathetic projections to the gut because the extensively intertwined postganglionic axons projecting to the gut are so densely packed and distributed through such distances and complex tissue elements, that the traditional methodologies (induced fluorescence, ultrastructural analysis, electrophysiology, immunohistochemistry) cannot readily isolate the full reach of individual autonomic axonal arbors in the gut wall.
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To circumvent some of the limitations associated with the conventional approaches, the present experiment used a different technique—namely labeling, in any one animal preparation, limited numbers of individual sympathetic postganglionic neuron axonal arbors in their entirety with the anterograde tracer dextran biotin (Walter et al., 2009). The isolated and complete arbors of individually identified axons were then traced, digitally reconstructed, and evaluated morphometrically in whole mounts of the muscularis externa to determine the distribution patterns of single sympathetic nervous system fibers as a means of evaluating and comparing them to the various putative patterns of innervation.
Materials and Methods Animals
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Virgin male rats (Fischer 344; n = 62; Harlan Laboratories, Indianapolis, IN; RRID:RGD_61109) were purchased at 2-months of age and weighed 175–200 g at the time of delivery. The animals were housed in an AAALAC-approved room kept at 20–23°C, on a 12:12 hour light:dark schedule, with access to pelleted chow (Laboratory diet no. 5001; PMI Feeds Inc., Brentwood, MO, USA) and filtered tap water available ad libitum. All Procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (8th ed., The National Academic Press, Washington, D.C.), and approved by the Purdue University Animal Care and Use Committee. Every effort was made to minimize suffering and the number of animals used. Tracer Injection in the Celiac and Superior Mesenteric Ganglia
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After a 2-week acclimation period in the colony, each animal was food-deprived overnight, anesthetized with Isoflurane (Isoflo®; Abbott Laboratories, North Chicago, IL, USA), and injected with Glycopyrrolate (0.2 mg/ml, s.c.; AmericanRegent Inc., Shirley, NY, USA) to reduce salivary, tracheobronchial, and gastric secretions. Each rat was then positioned on its back, and the abdominal cavity was opened by a midline laparotomy. The abdominal organs were draped with sterile saline moistened gauze and gently displaced inside the abdomen to visualize the sympathetic ganglia. To minimize disturbance of organs and surgery time, only the left celiac and superior mesenteric ganglia (CSMG), which are a main source of sympathetic innervation to the stomach and small intestine (Miolan and Niel, 1996; Quinson et al., 2001), were injected with tracer.
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More specifically, since the two ganglia are fused in the rat, the posterior pole of the CSMG (Hammer and Santer, 1981; Berthoud and Powley, 1993; Furness, 2014) was impaled using a Nanofil syringe with a beveled 35 gauge needle (NF35BV; World Precision Instruments, Sarasota, FL). The needle was advanced longitudinally, within and parallel to the long axis of the fused ganglia, until the tip rested at the rostral pole of the celiac ganglion part of the CSMG complex (Berthoud and Powley, 1996; Quinson et al., 2001). Dextran biotin, 6 µl of 10K MW lysine-fixable (7.5% concentration; Cat# D1956; Life Technologies, Grand Island, NY; RRID:AB_2307337), was then slowly injected as the needle was gradually retracted in a stepwise fashion within the ganglia. When the injection was completed and the infusion pressure within the CSMG had dissipated, the needle was slowly withdrawn from the ganglia. The displaced organs were then returned to their original position within the abdominal cavity, and the abdominal muscle was closed using interrupted sutures followed by closure of the skin with a single continuous suture.
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Prior to being returned to their home cages, rats recovered on a water circulating warming pad until their righting reflexes had completely returned. To minimize post-surgical discomfort and pain, Buprenorphine (0.01 mg/kg; s.c., Buprenex®, Reckitt Benckiser Pharmaceuticals Inc., Richmond, VA, USA) was administered prior to discontinuation of Isoflurane and then again once every 24 h over the following 72 h post-surgery. Also, to prevent post-surgical dehydration, rats were given an injection of warm sterile saline (6 ml; s.c.). Both body weight and intake of food and water were monitored throughout the duration of the recovery period to ensure that rats did not experience any post-surgical malaise.
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Control procedures were run to evaluate whether tracer leakage from the CSMG into the surrounding parenchyma or intraperitoneal space might be a source of neural labeling (Fox and Powley, 1989): Following laparotomy and exposure of the left CSMG, as described above, 6 µl of dextran biotin was dispensed over the exterior surface of the CSMG of control rats (n = 4). The complete absence of labeled terminal fields in the gut wall of these four rats indicated that labeled terminals in the ganglia-injected group were a result of tracer being incorporated into the neurons of the left CSMG and transported along their axons to their terminal arbors located in the gut wall and were not a byproduct of tracer leakage. Tissue Fixation and Dissection
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Nine days after tracer injection, rats were weighed, euthanized with a lethal dose of sodium pentobarbital (180 mg/kg, i.p.), and perfused through the left ventricle of the heart with 200 ml of 0.01M PBS at 40°C followed by 500 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 4°C. Prior to the perfusion, 0.5 ml of heparin was injected in the left ventricle to facilitate exsanguination. After fixation, the stomach and small intestine were removed and sampled according to the criteria of Hebel and Stromberg (1976). Specifically, the stomach was divided into dorsal and ventral halves by cutting along the greater and lesser curvatures. Whole mounts of the small intestine consisted of 3 cm long sections from the duodenum (the first 6 cm anal to the pyloric sphincter), mid-jejunum (middle 3 cm of the small intestine), and ileum (the first 6 cm oral to the ileocaecal junction). Intestinal whole mounts were opened by cutting along J Comp Neurol. Author manuscript; available in PMC 2017 September 01.
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the longitudinal axis of the mesenteric attachment. The specimens were rinsed under tap water and further fixed overnight in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 4°C. Intact muscularis externa whole mounts consisting of the circular and longitudinal muscle layers, with the myenteric plexus sandwiched in-between, were then isolated by removing the mucosa and submucosa from each specimen. Staining
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Free floating whole mounts were rinsed in PBS followed by a 30 min endogenous peroxidase block (methanol:3% H2O2; 4:1). Following additional PBS rinses, whole mounts were rinsed in dH2O and then stained in 0.5% Cuprolinic Blue (quinolinic phthalocyanine; 17052; Polysciences, Inc., Warrington, PA) in 0.05 M sodium acetate buffer containing 1.0 M MgCl2 (pH 4.9) for 2 h in a humidified slide warmer at 38°C. Cuprolinic Blue is a neuron-specific stain that is a pan-neuronal marker for myenteric neurons and is compatible with the dextran biotin protocol (Phillips et al., 2004; Walter et al., 2009). Whole mounts were then rinsed in dH2O, differentiated for 2 min in 0.05 M sodium acetate buffer containing 1.0 M MgCl2 (pH 4.9), and rinsed again in dH2O followed by 3 × 5 min PBS rinses. Whole mounts were then soaked for 3 d in PBS containing 0.5% Triton X-100 and 0.08% Sodium Azide to improve penetration through the muscle sheets. Next, free floating whole mounts were rinsed in PBS for 6 × 5 min followed by a 60 min soak in ABC (prepared according to the manufacturer’s directions; Cat# PK-6100; Vectastain Elite ABC kit; Vector Laboratories; RRID:AB_2336819). Whole mounts were then rinsed in PBS for 6 × 5 min, reacted in DAB solution for 3 min, followed by 6 × 5 min rinses in dH2O. The specimens were mounted on gelatin-coated slides, crushed for 90 min, air-dried overnight, dehydrated in an ascending series of alcohols, cleared in xylene, and coverslipped with Cytoseal (Richard-Allan Scientific, Kalamazoo, MI, USA).
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Fluorescent Immunohistochemistry To determine if tracer injection into the CSMG inadvertently labeled visceral afferent fibers of passage originating from the dorsal root ganglia (DRG), 12 rats were injected with dextran biotin into the CSMG as described above. Following fixation and dissection of the GI tract into regional whole mounts, a fluorescent double labeling protocol was employed to determine the relationship of dextran filled terminals with the sympathetic innervation of the gut visualized using antibodies to the noradrenaline- (as well as dopamine- and adrenaline-) synthesizing enzyme tyrosine hydroxylase (TH).
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Free floating whole mounts were incubated for 5 d in PBS containing 0.5% Triton X-100 and 0.08% sodium azide, followed by a 1 h soak in the same solution to which streptavidin ALEXA Fluor 594 (1:1000; Cat# S11227; Life Technologies; RRID:AB_2313574) had been added. Whole mounts were then rinsed in PBS followed by an overnight block in PBS containing 5% normal goat serum, 2% bovine serum albumin, 0.3% Triton X-100, and 0.08% sodium azide, and then incubated for an additional 24 h in a primary antibody diluted with the same blocking solution. Different whole mounts were exposed to either a mouse TH (1:500; Cat# 22941; ImmunoStar, Hudson, WI; RRID:AB_572268) or a rabbit TH (1:1000; Cat# P40101-0; Pel Freez Biologicals, Rogers, AR; RRID:AB_2313713) antibody. Whole mounts were then rinsed in PBS and 0.3% Triton X-100, and incubated for 2 h at
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room temperature in the appropriate secondary conjugated to ALEXA Fluor 488 (i.e., either goat anti-mouse, Cat# A11029, RRID:AB10566286 or goat anti-rabbit, Cat# A11034, RRID:AB_10562715; both purchased from Life Technologies) diluted 1:500 with the same solution used for the previous rinses. Finally, double-labeled whole mounts were rinsed in PBS, mounted on gelatin-coated slides, cover-slipped with Dako Fluorescent Mounting Medium (S3023; Dako North America, Inc., Carpinteria, CA), sealed with CoverGrip Coverslip Sealant (23005; Biotium, Inc., Hayward, CA), and stored at 2–8°C in the dark until examined for fluorescent signal.
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The degree of co-localization between the injected tracer and the TH antigen was determined using an Olympus DSU (Disk Scanning Unit) spinning disk confocal microscope attached to a BX61 motorized microscope (Olympus, Center Valley, PA). The confocal microscope was controlled using SlideBook digital microscopy software (V5.0; Intelligent Imaging Innovations, Denver, Co). Double-labeled sections were imaged using highly selective filter sets specific for the visualization of ALEXA Fluor 594 (XF102-2; Omega Optical) and ALEXA Fluor 488 (QMAX-Green; Omega Optical, Brattleboro, VT). A 40× water objective lens (NA = 1.15) was used. Extended-focus images or z-series consisting on average of 21 optical sections at an optimized z-increment of 0.38 µm were created. In some cases, z-stacks were compressed into one focal plan (i.e., a maximum value projection). To test for co-localization, a whole mount was systematically scanned for ALEXA Fluor 594. When a well labeled terminal neurite was identified, the upper and lower limits of the terminal were determined, with the experimenter blind to the ALEXA Fluor 488 wavelength, and a z-stack was captured using both filter sets. Each focal plane with the two channels merged was then examined individually for co-localization throughout the entire z-stack.
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Antibody Characterization
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Table 1 summarizes information about the primary antibodies used in this study to identify the sympathetic motor/tyrosine hydroxylase-positive innervation of the smooth muscle layers of the gut wall. The mouse monoclonal TH antibody (ImmunoStar Cat# 22941, RRID:AB_572268) was raised against TH purified from rat PC12 cells, and the antibody is described as having wide species crossreactivity (manufacturer’s datasheet). The immunogen used to produce the rabbit polyclonal TH antibody (Pel-Freez Biologicals Cat# P40101, RRID:AB_2313713) was SDS-denatured TH from rat pheochromocytoma, and shown by the manufacturer using western blot analysis to be specific for the ≈60 kD TH protein in rat caudate lysate. Immunostaining in the gut wall for both antibodies was consistent with the pattern of sympathetic innervation reported previously in GI whole mounts (Tan et al., 2010; Phillips et al., 2013), which is eliminated following celiac and superior mesenteric ganglionectomy surgery (Phillips et al., 2013). No positive reactions were observed in any whole mounts in which TH antibodies were omitted from the protocol. Whole Mount Evaluation and Analysis Criteria for myenteric ganglia—In keeping with the criteria of Bar-Shai and coworkers (2004), two or more myenteric neurons clustered together were considered a ganglion. If a cluster of cell bodies was separated from another cluster of cell bodies by a
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distance of three or more average neuron diameters, then they were considered two separate ganglia (cf. Berthoud et al., 1997). Also, if a group of neurons was isolated from another group by a string of successive single-file neurons, that was as much as, or more than, three average neuronal diameters in length, then the two groupings were considered to be separate ganglia (cf. Berthoud et al., 1997).
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Criteria for myenteric plexus connectives—Enteric circuitry and smooth muscle tissues were distinguished according to commonly used criteria (cf. Scheuermann and Stach, 1984; Furness, 2006a). Briefly, the primary myenteric plexus was considered to be formed of both the myenteric ganglia and the major connectives interconnecting those ganglia (also referred to as interganglionic connectives or interconnective strands). The secondary plexus was defined as those higher order connectives that (a) issue from the myenteric ganglia or interganglionic connectives, (b) lie in a network deep to the primary plexus and in contact with the circular muscle, and (c) consist of fascicles running predominately parallel to the circular muscle fibers. The tertiary plexus was defined as the network of higher order ENS connectives that (a) issue from the myenteric ganglia, interganglionic connectives, or secondary plexus, (b) lie in a network situated at the level of, or just superficial to, the primary plexus and abutting the deeper face of the longitudinal muscle sheet wall, and (c) consist of fascicles running predominately parallel to longitudinal muscle fibers. The secondary plexus carries axons of extrinsic (and intrinsic) efferent (and afferent) neurons that innervate the circular muscle sheet (Wilson, et al., 1987; Furness, 2006b), whereas the tertiary plexus carries axons that innervate the longitudinal muscle sheet (Llewellyn-Smith et al., 1993). These conventional distinctions and inferences were used in categorizing the tissue targets of the labeled CSMG axonal arbors.
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Sympathetic Terminal Arbor Inventories and Morphometry Initial screening—The whole mounts were first systematically scanned at 400× magnification with brightfield illumination. Once an arbor was identified, it was further inspected to determine whether it satisfied the following four criteria: (a) well labeled, (b) completeness of the axon, (c) minimal tissue artifacts such as folds and tears, and (d) free of multiple independent axons interdigitating or overlapping in a manner that might confuse the evaluation of a given terminal field.
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Morphometry—A total of 254 sympathetic axons that satisfied the histological criteria for morphometry were located in the initial screening for staining quality and specimen integrity. Of that inventory, 160 axons were randomly selected for digitization, and individual sympathetic terminal arbors were then subsequently traced, digitally reconstructed, and evaluated quantitatively using a Neurolucida (MicrobrightField Inc., Williston, VT, USA; RRID:nig-0000-10294) workstation employing a Zeiss Axio Imager Z2 microscope (Carl Zeiss Microimaging, Gottingen, Germany) equipped with DIC optics and both a 40× dry and a 63× water immersion long working distance objective. For each of the digitized sympathetic arbors, the following standardized morphometric measures captured with the Neurolucida software were analyzed for all neurites: total arbor length, parent axon length (using the Strahler Analysis algorithm), varicose neurite length, J Comp Neurol. Author manuscript; available in PMC 2017 September 01.
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total number of terminal branches, highest branch order, and two-dimensional terminal field size (using the Convex Hull algorithm [cf. Powley et al., 2012, 2013a, 2013b, 2014]). Criteria for Sympathetic Postganglionic Innervation or Contacts Two different indices were used to independently estimate the extent of innervation of, or contact with, tissue targets by sympathetic axons: 1) Consistent with conventional observations (e.g., Falck and Owman, 1966; Furness et al., 2014), beaded segments of sympathetic axons exhibiting substantial numbers of varicosities were assumed to release transmitter to the immediately adjacent tissues from those varicosities. 2) Similarly, consistent with the common assumption (e.g., Levitan and Kaczmarek, 2002), the highest order branches, or distal terminal tips, of sympathetic motor fiber arbors were assumed to be sites of transmitter release.
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Assessments of the proportions or extent of innervation patterns of different tissues in the gut wall were developed from the above criteria of postganglionic innervation. In the case of the primary plexus (myenteric ganglia and their primary interconnective strands), both of the measures were recorded. The first measure consisted of counting the number of ganglia that had either a terminal branch within a ganglion or the end of the terminal branch in a primary connective neighboring a ganglion. The second measure involved counting the number of ganglia with varicose fibers passing through or in close proximity. In the case of the myenteric plexus, both measures, that is both a terminal branch and a beaded varicose axon, needed to satisfy a neurite-to-ganglion proximity rule were defined as having a distance from a ganglion of less than or equal to half the average neuron diameter of Cuprolinic blue labeled myenteric neurons. This distance was chosen based on the expected distance of neurotransmitter diffusion (Read and Burnstock, 1969; Furness, 1970b; Gillespie and Maxwell, 1971). The total length of each arbor’s varicose segments located in conjunction with each of the types of tissue (i.e., myenteric ganglia and primary plexus; circular muscle sheet and secondary connectives; longitudinal muscle sheet and tertiary connectives) was also determined and then expressed as a percentage of the total length of all of that arbor’s segments.
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Both criteria of postganglionic innervation were similarly used to evaluate the more direct projections to the smooth muscle sheets: 1) Labeled terminal axonal arbor branches and varicose segments of the arbor within the circular muscle or in the secondary plexus in immediate apposition to the muscle were assumed to directly innervate muscle fibers. 2) Similarly, labeled terminal axonal branches and varicose arbor segments in the longitudinal muscle or in the tertiary plexus in immediate apposition with the muscle sheet were assumed to directly innervate longitudinal muscle fibers. For these estimates of muscle sheet innervation, both the number of terminal branches and the length of varicose arbor segments in association with the muscle sheets were determined and used as separate measurements of degree of innervation to confirm and cross-validate the sympathetic terminal field innervation of muscle in the gut wall. As in the case of the primary myenteric plexus, the proximity rule (i.e. within half an average neuron’s diameter of the putative target tissue) was used.
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Final Postganglionic Samples
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In the final analysis, after the 160 sympathetic arbors had been traced and digitized, the reconstructions of 154 of the fibers were ultimately used for quantitative comparisons of myenteric ganglion and smooth muscle innervation. These 154 axonal terminal arbors yielded representative samples for each of the five regions prepared as whole mounts (stomach: n = 22; duodenum 0–3 cm: n = 33; duodenum 3–6 cm: n = 29; jejunum: n = 20; ileum: n = 50).
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The remaining six of the 160 randomly chosen motor arbors were evaluated separately but not included in quantitative analyses of terminal patterns: two of the six arbors were judged problematic for complete quantitative assessment because the specimens traversed regions with incomplete Cuprolinic Blue staining. The remaining four of the six cases projected to the vasculature rather than the smooth muscle sheets; these vascular efferents were evaluated and are described separately. Photography
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Single high power images were acquired with a Spot Flex camera controlled by Spot Software (V4.7 Advanced Plus; Diagnostic Instruments, Sterling Heights, MI; www.spotimaging.com) mounted on a Leica DMRE. Multi-field composite (or mosaic) images containing the entire area of innervation by a sympathetic axon were generated at high magnification (630×; water immersion objective) with a Leica DFC310 FX digital CD color camera mounted on a Leica DM5500, running Surveyor with Turboscan software (V. 6.0.5.3; Objective Imaging, Cambridge, UK; www.objectiveimaging.com). To capture the varying depth of a neurite within a smooth muscle whole mount, each image in a mosaic typically consisted of multiple focal z-planes that were merged using either Photoshop CS5 (Adobe Systems, San Jose, CA; www.adobe.com) or Helicon Focus Pro X64 software (V5.3.7; HeliconSoft Ltd, Kharkov, Ukraine; www.heliconsoft.com) to generate an all-infocus image. Photoshop CS5 was also used in final figure production to: 1) apply text and scale bars; 2) adjust brightness, contrast, color, hue, and sharpness; and 3) organize the final layouts of the figures. Statistics Statistical analyses and graph generation were accomplished using GraphPad Prism (V. 5.0; GraphPad Software, San Diego, CA, USA; RRID:rid_000081). A one-way ANOVA with Tukey’s post hoc test (corrected for multiple comparisons) was performed to determine significant differences between groups. Data is presented as means ± SEM. Statistical significance was considered p < 0.05.
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Results Co-localization of Dextran Biotin Labeled Terminals with Tyrosine Hydroxylase Co-localization analysis was done on 136 dextran labeled terminals located in the muscle wall of jejunal whole mounts. One hundred dextran labeled terminals were evaluated for colocalization with the polyclonal rabbit TH antibody, and an additional 36 dextran labeled endings were evaluated for co-localization with the monoclonal mouse TH antibody. The
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distribution of arbors sampled consisted of 54 in the myenteric ganglia/1° plexus, 43 in the longitudinal muscle/3° plexus, and 39 in the circular muscle/2° plexus; Figure 1A–I. In all, only one dextran labeled terminal (located in the circular muscle/2° plexus) was found to be TH-negative. Qualitative evaluation of randomly chosen dextran labeled arbors located in the smooth muscle wall of the stomach, duodenum, and ileum were found to be similarly extensively co-localized with TH. Omission of the primary resulted in complete loss of TH-positive fibers; Figure 1J–L. Examination of a) dextran-negative/TH-positive arbors in the double labeled material and b) dextran labeled terminals in the primary deletion control material confirmed that the emission spectra of the two fluorophores combined with the filter sets used during the acquisition of the images sufficiently avoided spectral bleed-through artifact and were suitable for co-localization analysis; Figure 1C,F,I, and K.
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General Architecture of Sympathetic Postganglionic Terminal Arbors Dextran-biotin labeling of sympathetic axons in whole mounts that preserved the myenteric plexus in situ between the longitudinal and circular muscle sheets provided high-definition delineation of complete motor terminal arbors terminating in the muscle wall of the gut and made it practical to routinely digitize and analyze morphologically individual CMSG axons. Figure 2 illustrates a Neurolucida tracing of a typical CSMG axonal arbor, and Figure 3 contains photomicrographs of different representative sites within the tracing in Figure 2, as designated by the lettering.
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In each of the four gut regions sampled (i.e. stomach, duodenum, jejunum and ileum), the basic architecture of individual sympathetic terminal fields throughout the smooth muscle wall consisted of a parent axon entering the gut wall within a bundle or fascicle of fibers, coursing some distance through the myenteric plexus network, and then branching repeatedly to form an extensive terminal array of varicose neurites extending over a large innervation field, making presumptive contacts in multiple different tissues.
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The initial screening to identify traceable CSMG arbors produced observations indicating both a need to distinguish different groupings of axonal terminal fields, particularly with reference to the three-neuron and two-neuron models of the sympathetic outflow, and a need to evaluate the disparate ending patterns quantitatively. In brief, one small minority of postganglionic arbors appeared to project strongly to myenteric neurons and the primary plexus, in a configuration consistent with a three-neuron-outflow model, and a second small minority of postganglionic arbors appeared to predominately innervate one or both smooth muscle sheets directly, in a pattern consistent with a two-neuron-outflow model. In contrast, however, a large majority of CSMG postganglionics, as illustrated in Figures 2 and 3, distributed a substantial percentage of their neurites, in parallel, to both the myenteric ganglia/1° plexus and the smooth muscle. Criteria Distinguishing Subpopulations of Sympathetic Postganglionics The fine architecture of sympathetic postganglionics in the muscularis externa of the gut wall has not previously been well characterized, so a working criterion to distinguish between the identified subpopulations was generated. First, the 154 arbors in the random J Comp Neurol. Author manuscript; available in PMC 2017 September 01.
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sample of postganglionic neurons that innervated the smooth muscle wall were traced and digitized. Since all branches of these arbors projected to only two types of tissue, the myenteric plexus and/or smooth muscle, the proportions or total lengths of each arbor’s varicose neurite branches in myenteric ganglia/1° plexus and in smooth muscle, respectively, were then calculated and used to plot innervation profiles.
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More specifically, for innervation profiles, the composite or combined length of all varicose branch segments and terminal branches was obtained separately for each of the individual 154 arbors. Then, for each arbor, as analyzed below, the separate summed lengths of those varicose neurites in apposition to each of the basic types of target tissues innervated (myenteric ganglia/1° plexus; circular muscle/2° plexus; or longitudinal muscle/3° plexus) were separately expressed as percentages of the respective arbor’s full cumulative length of varicose branches. Further, for the purposes both of generating criteria that distinguished subpopulations and of making general comparisons between the two-neuron and the threeneuron models, the projections to the longitudinal and circular muscle layers were then also combined and collectively considered as innervation of “smooth muscle.”
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Once the data for the muscle sheets had been combined, the frequency distributions of the numbers of arbors within the sample of 154 cases with different percentages of neurites projected to each of the two broad tissue types (myenteric ganglia/1° plexus; smooth muscle) of the gut wall were examined. Figure 4 illustrates the distribution of all 154 arbors with the different percentages (on the lower x-axis) of their varicose segments projecting specifically to smooth muscle and/or their respective interconnectives. Reciprocally, since all varicose neurites not terminating in muscle terminated in the primary plexus, Figure 4 also illustrates, at the left end of the abscissa (on the upper x-axis), the subpopulation of arbors that project specifically to myenteric ganglia/1° plexus.
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From this neurite distribution in Figure 4, two factors emerged and were used to establish the operationally defined criteria for grouping the 154 arbors examined in the series of specific quantitative comparisons reported below. The most salient factor for identifying subpopulations was that the distribution of arbors was polymodal, including at least three identifiable peaks. The second factor shaping the provisional groupings of the CSMG postganglionic arbors was a set of pragmatic observations about cutoffs that would distinguish the distinct subpopulations that appeared to form clusters in the polymodal distribution of arbor segments illustrated in Figure 4. A strict construction of the commonly used three-neuron and two-neuron ideas of the sympathetic outflow generally might extrapolate to the prediction that an individual arbor would project exclusively to, respectively, the primary myenteric plexus or the smooth muscle, but as Figure 4 indicates, few arbors projected 100% of their varicose neurites to smooth muscle and even fewer arbors projected 0% of their varicose neurites to smooth muscle (and hence 100% to myenteric ganglia/1° plexus). The typical architecture of the postganglionics is illustrated by the axonal terminal arbor in Figure 5 which exemplifies the fact that, even in the case of most of those sympathetic arbors constituting the left-most peak in Figure 4 and projecting predominately to the myenteric ganglia/1° plexus, some of their short branches and/or arrays also typically
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extended to smooth muscle. Like most of the other arbors in the left-most cluster in Figure 4, the axon in Figure 5 innervated predominately the myenteric ganglia and associated primary plexus, but, nonetheless, the arbor distributed short collateral branches (example designated with arrows in Figure 5C) to the smooth muscle. Specifically, in the case in Figure 5, these collateral varicose branches to the muscle sheet accounted for only 8.5% of the total cumulative length of the fiber’s arbor. Examination of the left-most and right-most peaks in Figure 4 (corresponding, respectively, to arbors that terminate nearly exclusively in the myenteric ganglia/1° plexus or the smooth muscle) makes it clear that, a restrictive or “exclusive” decision rule requiring that 100% of an arbor’s segments need project to the relevant target tissue, in order for that individual postganglionic to be classified as projecting to a particular target, does not include all of the arbors comprising the two clusters close to the limits of the distribution in Figure 4.
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A strategy of defining a less restrictive and more practical cutoff, one that better reflected the clustering patterns in the distribution of Figure 4, was to find the nadir immediately to the right of the left-most peak and the nadir just to the left of the right-most peak and then to use the average of the two nadirs to generate a cutoff criterion. Given that the average of the two nadirs rounded to essentially 15%, we used that value for the cutoff of the left-most peak and the complementary value of 85% as the cutoff of the right-most peak. Arbors with 85% or more of their varicose segments projecting to a particular type of tissue were considered to predominantly innervate that tissue. In contrast, individual postganglionic arbors that comprised the subpopulation in the broad middle peak or cluster in Figure 4, which distributed roughly equivalent proportions of their neurites to the two tissues and did not distribute as much as 85% of their varicose branches to either the myenteric ganglia/1° plexus or the smooth muscle/associated interconnectives, were considered mixed or heterotypic neurons with substantial inputs to both types of tissues and a predominant input to neither type of tissue. Thus, based on these observed clustering patterns and considerations, we adopted a standard terminology of designating arbors that supplied 85% or more (but less than 100%) of their beaded branch segments to one tissue or the other as predominately innervating that tissue, and more equally distributed arbors that did not issue as much as 85% of their varicose branches to either single tissue type as providing mixed or heterotypic innervation of the two tissues.1 Arbors that projected 100% of their varicose neurites to either one tissue or the other were considered as exclusively innervating that tissue.
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Footnote 1: Given that the choice of what to use as cutoff criteria was arbitrary, we also explored the consequences of using cutoffs of
