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Fall 2014 Morphometric characterization of individual sympathetic postganglionic axons innervating the muscle layers of the gastrointestinal tract of the rat: A complex effector model Gary C. Walter Purdue University
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Recommended Citation Walter, Gary C., "Morphometric characterization of individual sympathetic postganglionic axons innervating the muscle layers of the gastrointestinal tract of the rat: A complex effector model" (2014). Open Access Dissertations. 381. https://docs.lib.purdue.edu/open_access_dissertations/381
This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. 30 08 14 PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance
Gary Christopher Walter
MORPHOMETRIC CHARACTERIZATION OF INDIVIDUAL SYMPATHETIC POSTGANGLIONIC AXONS INNERVATING THE MUSCLE LAYERS OF THE GASTROINTESTINAL TRACT OF THE RAT: A COMPLEX EFFECTOR MODEL
Doctor of Philosophy
Terry L. Powley
Edward A. Fox
Jean-Christophe Rochet
John R. Burgess
To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification/Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.
Terry L. Powley Christine A. Hrycyna 10/01/2014
Department
MORPHOMETRIC CHARACTERIZATION OF INDIVIDUAL SYMPATHETIC
POSTGANGLIONIC AXONS INNERVATING THE MUSCLE LAYERS OF THE
GASTROINTESTINAL TRACT OF THE RAT: A COMPLEX EFFECTOR MODEL
A Dissertation
Submitted to the Faculty
of
Purdue University
by
Gary C. Walter
In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
December 2014
Purdue University
West Lafayette, Indiana ii
ACKNOWLEDGMENTS
I am grateful to J. McAdams for her assistance with tracer injections and tissue processing for microscopy. Also, L.A. Schier and J. McAdams for proof reading a draft of this dissertation. To Nikhilendra Singh for being an awesome friend, along with his girlfriend Mary Ann Origer and her family for being so nice to me and giving me a place to escape from the Lafayette area. A special acknowledgement to the women of my life who made some of the tough times in graduate school a little easier; my appreciation to Jean DiPirro and Lara Shonk. To all my friends. Special thanks to my parents and other family members for their love and support over the years.
iii
TABLE OF CONTENTS
Page
LIST OF TABLES ...... v
LIST OF FIGURES ...... vi
NOMENCLATURE ...... x
ABSTRACT ...... xi
INTRODUCTION ...... 1
MATERIALS AND METHODS ...... 7
Animals ...... 7 Tracer Injection Into the Sympathetic Ganglia ...... 7 Tissue Fixation and Dissection ...... 10 Staining ...... 10 Sympathetic Terminal Inventories and Analyses...... 11 Initial Screening ...... 11 Analyses of General Morphology ...... 12 Criteria for Myenteric Ganglia...... 13 Criteria for Sympathetic Postganglionic Innervation ...... 13 Criterion for Sympathetic Arbors Projecting Exclusively to Myenteric Neurons ...... 15 Photography ...... 17 Statistics ...... 18
RESULTS ...... 19
Screened and Digitized Endings ...... 19 Sympathetic Nerves Innervate Multiple Different Tissue Sites in the Muscle Wall of the GI Tract ...... 20 General Pattern of Innervation ...... 20 Single Tissue Type Innervated: Primary Plexus ...... 21
iv
Page
Single Tissue Type Innervated: Secondary Plexus/Circular Muscle ...... 23 Innervation of Longitudinal Muscle/Tertiary Plexus ...... 24 Multiple Tissue Types Innervated: Primary Plexus in Addition to the Secondary Plexus/Circular Muscle and/or the Tertiary Plexus ...... 25 Quantitative Analysis of Myenteric Ganglia Innervation ...... 26 Quantitative Analysis of the Relative Proportions of Individual Heterotypic Sympathetic Arbors Innervating Myenteric Plexus vs. Smooth Muscle...... 27 Quantitative Analysis of General Terminal Morphology ...... 30 Sympathetic Innervation of the GI Vasculature ...... 31 Bilateral Sympathetic Innervation of Stomach and Small Intestines . . . 32
DISCUSSION ...... 33
Sympathetic Morphology ...... 35 Primary Plexus and the Myenteric Ganglia ...... 37 Smooth Muscle ...... 39 Secondary plexus/circular muscle ...... 39 Longitudinal muscle/tertiary plexus ...... 41 Morphological and Multiple Target Mechanisms for Complex Integration ...... 42 Morphological Modulatory Mechanisms ...... 43 Ganglia and Muscle Modulatory Mechanisms ...... 44 Multiple Targets Integrate the Modulatory Mechanisms . . . . . 45 Blood Vessel Innervation ...... 48 Bilateral Innervation ...... 50 Limitations ...... 50 Perspectives ...... 54
LIST OF REFERENCES ...... 56
APPENDICES
Appendix A: Tables ...... 78 Appendix B: Figures ...... 81
VITA ...... 98
v
LIST OF TABLES
Appendix Table Page
1. Distribution of Traced Endings ...... 78
2. Heterotypic Endings ...... 79
3. Summary of General Morphometric Parameters ...... 80
vi
LIST OF FIGURES
Appendix Figure Page
1. A Neurolucida tracing of a single sympathetic arbor with collateral terminal branches and/or varicose fibers of passage located in the primary plexus, the secondary plexus/circular muscle, and the tertiary plexus. The parent axon (PA) can be seen extending from the right side of the tracing, running in the longitudinal direction. Figure 2 provides photomicrographs that correspond to the demarcated areas on the present tracing: A = ganglia; B = tertiary plexus; C = secondary/circular muscle; D = circular muscle. Scale bar = 500 μm ...... 81
2. Images were taken from the traced dextran-biotin labeled ending shown in Figure 1. The circular muscle is oriented from top-to-bottom in all panels. A: Innervation of a myenteric ganglion by branches of sympathetic axon. A relatively small diameter neurite can be seen branching from the larger diameter parent fiber at the top of the photomicrograph. As the neurite passes through the ganglion numerous collateral varicose branches (arrows) can be observed as they extend a short distance, diffusely meander through the neurons, or encircle a neuron. B: Varicose fiber segments terminating in the tertiary plexus. An interganglionic connective strand can be observed as it courses away from a myenteric ganglion (at the left). Along the trajectory a collateral branch forms into a relatively small internodal tertiary plexus array in which multiple bifurcations produce branch segments and terminal endings with clearly defined varicosities (arrows). C: Varicose fiber segments traversing within the secondary plexus and eventually projecting into the circular muscle coat. As the interconnective strand extends into the internodal region a relatively compact circularly oriented varicose array traverses through a 5μm thick plane. D: All-in-focus image of varicose fiber segments innervating the circular muscle. A single fiber, entering from the top of the image, branched at the level of the circular muscle into an array with a less strict smooth muscle alignment, as they passed over myenteric ganglia. The dextran-biotin labeled neurites were visualized using DAB (brown) and the myenteric neurons were counterstained with Cuprolinic Blue (blue). Scale bars in A, C, D = 50 μm; B = 20 μm...... 82 vii
Appendix Figure Page
3. A dextran-biotin labeled sympathetic axon that exclusively innervated the primary myenteric plexus. A: Multiple ganglia within the myenteric plexus were innervated. While varicose axons can be seen in and around the ganglia, portions of the axon can also be seen as merely interconnective strand elements (arrowhead) between the ganglia. B: A Neurolucida tracing of the entire axon from which panels A and C are taken. The neurite consisted of a long PA (entering at 12 o’clock) that repeatedly branched to innervate numerous ganglia and interconnective strands of the myenteric plexus. C: Varicose fiber branches can be seen around myenteric neurons and a transitory varicose fiber (arrowhead) of passage can be observed coursing through a ganglion. Arrows designate a small branching spur of the arbor running into the secondary plexus/circular muscle sheet. For purposes of categorizing the neuronal arbors in terms of their tissue targets, when such spurs constituted less than 15% of the cumulative length of the sympathetic arbor’s branches, they were not considered a substantial projection. Scale bars in A & C = 63 μm; B = 250 μm ...... 84
4. A dextran-biotin labeled sympathetic axon that innervates the primary plexus, the secondary plexus/circular muscle, and the tertiary plexus. A: The prominent feature of the traced axon, which is shown in its entirety, was the extensive innervation of the tertiary plexus between parallel myenteric ganglia. B: The smooth large diameter parent axon (entering bottom right; arrows) passes through a ganglion and gives off several thin varicose branches that come in close proximity to myenteric neurons before the parent axon takes a 90 degree turn into the tertiary plexus. Upon entering the tertiary plexus, the sympathetic arbor branches become highly varicose in appearance, divide extensively, and run predominately in the longitudinal direction (left to right) covering the area between two parallel strips of ganglia. C: Higher magnification of the rightmost ganglion in panel B. In the upper portion of the image, tertiary fibers are out of focus (arrowheads), and can be observed passing under a neuron, in the longitudinal direction. Tertiary plexus fiber segments can also be observed with varicosities and terminal endings (arrows) along the border of a myenteric ganglion. Scale bars in A = 250 μm; B = 31 μm; C = 10 μm ...... 86
viii
Appendix Figure Page
5. A dextran-biotin labeled sympathetic axon that innervates the primary plexus and the secondary plexus/circular muscle. A: Upon innervating a ganglion, an arbor segment branches extensively giving off numerous collateral varicose processes in the ganglion. While some areas of the ganglion have diffuse innervation other regions of the ganglion appear to receive a higher number of varicose branches. B: At 1000x magnification of one pole of the ganglion illustrates the close association between varicose processes (arrowheads) and the myenteric neurons that they encircle. C: Varicose innervation of the secondary plexus and circular muscle is shown in focus (arrowheads) with innervation of a myenteric ganglion by collaterals from the same parent axon shown out of focus (arrows). This displays fiber segments innervating distinct tissue types and the planar difference of their respective locations, 5μm apart, within the whole mount. D: A tracing of the entire ending highlights the morphology of the long rectilinear multi-branching arrays that run within the circular muscle and superficial to the circular muscle within the secondary plexus. E: An all-in-focus image of an axonal array innervating the secondary plexus and the circular muscle with collateral branches produced by the same sympathetic neuron innervating the myenteric ganglion which runs parallel to the circular muscle. Scale bars in A & C = 20 μm; B = 10 μm; D = 125 μm; E = 31 μm ...... 88
6. The number of myenteric ganglia innervated by terminal branches was greatest in the proximal gut and decreased in the distal gut (ANOVA; F(4, 122) = 5.363, P = 0.0005). B: The pattern was similar for varicose arbor length (ANOVA; F(4, 127) = 4.989, P = 0.0009). Asterisks indicate a significant difference (P < 0.05) compared to the stomach * or duodenum 0-3 cm **; Tukey’s HSD. Mean ± S.E.M...... 90
7. A: In the subset of axons that innervated both the primary plexus and secondary plexus/circular muscle, there was a greater proportion of the overall neurite length within the muscle compared to the ganglia and the interconnective strands (ANOVA; F(1.521, 18.25) = 27.89, P < 0.0001). B: In the subset of axons that innervated the primary plexus and the tertiary plexus, there was a greater proportion of the overall length within the tertiary plexus compared to the ganglia and the interconnective strands (ANOVA; F(1.733, 27.73) = 3.769, P = 0.0412). C: In the subset of axons that innervated all three sites, there was a similar proportion of the overall length within all sites (ANOVA; F(2.684, 163.7) = 2.745, P = 0.0509). Asterisks indicate a significant difference compared to the secondary/circular muscle * or compared to the tertiary plexus **; Tukey’s HSD. Mean ± S.E.M...... 91
ix
Appendix Figure Page
8. A: In the subset of axons, which innervated the primary plexus and secondary plexus/circular muscle, there was a trend for a greater proportion of the overall “terminal endings” to innervate the muscle compared to the ganglia and the interconnective strands (ANOVA; F(1.994, 23.93) = 1.901, P = 0.1714). B: In the subset of axons, which innervated the primary plexus and the tertiary plexus, there was a greater proportion of terminals branches within the tertiary plexus compared to the ganglia and the interconnective strands (ANOVA; F(1.327, 21.24) = 11.06, P = 0.0016). C: In the subset of axons, which innervated all three sites, there was a greater proportion of terminals branches within the ganglia and the tertiary plexus compared to the interconnective strands and the secondary plexus/circular muscle (ANOVA; F(2.629, 160.3) = 14.25, P < 0.0001). Asterisks indicate a significant difference compared to the ganglia * or compared to the tertiary plexus **; Tukey’s HSD. Mean ± S.E.M...... 93
9. Sympathetic innervation of the blood vessels was observed in some of the whole mounts. A: Sympathetic axonal branches silhouette the outer perimeter of a blood vessel before dividing and giving off several collateral branches that extend into the neighboring myenteric ganglia. B: A tracing of the entire ending documents how the parent axon (entering from the lower right) bifurcates and the resulting secondary fibers then run parallel in close association to the blood vessel with several collateral branches innervating the nearby myenteric ganglia. C: At higher magnification, the axonal branches in close apposition to the outer wall of a blood vessel are clearly varicose (arrows). D: The innervation of a myenteric ganglion is similarly varicose (arrows). Scale bars in A = 125 μm; B = 250 μm; C & D = 20 μm ...... 95
10. A tracing of a dextran-biotin labeled sympathetic axon that illustrates a terminal field extending within both the dorsal and ventral portions of the stomach through an interconnecting fiber that projects across the greater curvature ...... 97
x
NOMENCLATURE
Symbol Description
ABC Avidin-Biotin Complex
CNS Central Nervous System cm Centimeter
CSMG Celiac and Superior Mesenteric Ganglia
DAB 3, 3’-diaminobenzidine
DBH Dopamine Beta Hydroxylase dH2O Deionized Water
GI Gastrointestinal i.p. Intraperitoneal
NA Noradrenaline
NPY Neuropeptide Y
PBS Phosphate Buffered Saline
SEM Standard Error of the Mean
SOM Somatostatin s.c. Subcutaneous
TH Tyrosine Hydroxylase
xi
ABSTRACT
Walter, Gary C. Ph.D., Purdue University, December 2014. Morphometric Characterization of Individual Sympathetic Postganglionic Axons Innervating the Muscle Layers of the Gastrointestinal Tract of the Rat: A Complex Effector Model. Major Professor: Terry L. Powley.
A full description of the terminal morphology of sympathetic postganglionic axons innervating the musculature of the gastrointestinal (GI) tract has not been available.
Furthermore, common assumptions about the morphology and distribution of catecholaminergic terminal fields have been strongly shaped by the limitations of the techniques employed to distinguish the fibers and complicated by inconsistent findings generated with various methodologies. Thus, the present experiment used modern neural tracer techniques to provide high-resolution labeling of sympathetic fibers projecting to the smooth muscle wall of the GI tract. Fischer 344 rats (N = 50) received injections of dextran biotin into the left celiac and superior mesenteric ganglia. Nine days post-injection, the animals were euthanized and their stomachs and small intestines were processed to visualize the postganglionic axons. Myenteric neurons were counterstained with Cuprolinic Blue. Individual sympathetic arbors (n =
154) in the gut wall were inventoried, digitized and analyzed morphometrically. After entering the GI tract wall, sympathetic axons branched extensively to form complex terminal arrays within the myenteric ganglia and adjacent tertiary plexus. Sympathetic xii arbors also formed long rectilinear multiply branched arrays of neurites within the circular muscle and its associated secondary plexus. A small subset (13%) of individual sympathetic axons terminated solely in the myenteric ganglia, while another minority subset (27%) exclusively innervated the secondary plexus and circular muscle. In contrast, the largest group of endings (i.e., 60%) formed heterotypic arbors that innervated the myenteric ganglia and either the tertiary plexus and/or the secondary plexus/circular muscle. Though past experiments relying on fluorescence histochemistry have emphasized sympathetic axons projecting to the myenteric plexus, and thus apparently controlling smooth muscle function indirectly through these enteric ganglion projections, the fact that some 87% of sympathetic axons project extensively, or even exclusively, to the smooth muscle sheets is consistent with the view that sympathetic postganglionic axons are organized to control smooth muscle directly.
1
INTRODUCTION
The sympathetic nervous system coordinates autonomic programs that influence gastrointestinal (GI) motility, secretion, and blood vessel diameter (Furness,
2007; Furness and Costa, 1974; Jobling, 2011). Analyses of the neural circuits controlling these functions have been limited, to some degree, by insufficient structural information about the sympathetic postganglionic projection patterns, especially those located within the smooth muscle wall of the gut. While prior observations have produced a partial description of the pathways, the information has remained incomplete, and the available observations have been heavily influenced by the inherent limitations of the indirect techniques used to visualize catecholaminergic postganglionic axons.
Classically, autonomic efferent outflows are depicted as two-neuron pathways that consist of more proximal preganglionic neurons projecting to postganglionic neurons that terminate directly on effectors (see Figure 1 in Hill, 1927; Figure 3 in
Langley, 1903). In the specific case of the sympathetic innervation of the gut wall, this motor outflow would consist of preganglionic neurons in the intermediolateral column of the spinal cord projecting to postganglionic neurons that are located in the prevertebral celiac and superior mesenteric ganglia (CSMG) and that project directly to smooth muscle effectors in the gut wall. 2
Such a two-neuron model of the sympathetic outflow was largely abandoned for the GI tract, however, with the introduction of histofluorescence protocols to visualize sympathetic catecholaminergic fibers (Baker and Santer, 1989; Capurso et al.,
1968; Falck et al., 1962, 1982; Furness, 1970a; Furness and Costa, 1971, 1975; Furness and Malmfors, 1971; Furness et al., 1977; Gabella and Costa, 1967, 1969; Gillespie and Maxwell, 1971; Hollands and Vanov, 1965; Llewellyn-Smith et al., 1984;
Norberg, 1964; Read and Burnstock, 1968, 1969; Scheuermann and Stach, 1983, 1984;
Silva et al., 1971). These histofluorescence observations of the sympathetic innervation of the alimentary canal revealed a dense pattern of fibers within the myenteric plexus, with comparatively sparser catecholaminergic innervation of the circular muscle, and with essentially no fibers in the longitudinal muscle (Capurso et al., 1968; Hollands and Vanov, 1965; Norberg, 1964, 1967). The resulting assumption that sympathetic postganglionic axons project--not to smooth muscle effectors, but-- predominately to intercalated myenteric neurons led to the influential and widely adopted view that GI muscle activity is indirectly regulated by the sympathetic nervous system through transmitter release within myenteric ganglia (Furness, 2007; Furness and Costa, 1974; Norberg, 1964, 1967).
While there has been wide acceptance of this three-neuron or indirect- innervation-of-muscle model of sympathetic circuitry (see Figure 38 in Norberg,
1967), the findings from a small number of studies that used techniques other than histofluorescence fail to fully support the conclusion that sympathetic fibers primarily innervate myenteric ganglia. Instead, the studies raise the possibility, consistent with the two-neuron model, that postganglionic catecholamine release reaches smooth 3 muscle. In ultrastructural investigations, for example, though sympathetic axons course through the myenteric plexus, the fibers appear to be in close apposition to other axons as well as to be more superficially located around the basal lamina and capsule of the myenteric ganglia. Furthermore, these catecholaminergic fibers rarely make direct synaptic contacts on myenteric neurons. Thus, ultrastructural observations suggest the possibility of a somewhat more direct pathway to smooth muscle, one in which released transmitter diffuses from the myenteric plexus (Gabella, 1970; Gordon-
Weeks, 1982; Llewellyn-Smith, 1981; Manber and Gershon, 1979). In keeping with such a view, electrophysiological evaluations suggested that the membrane potential and electrical activity of myenteric neurons are negligibly affected by noradrenaline or sympathetic stimulation (Nishi and North, 1973; Takayanagi et al., 1977); whereas noradrenaline has been shown to have a direct effect on the circular muscle (Gershon,
1967).
A further discrepancy is apparent in pharmacological investigations that indicate that sympathetic activity reduced acetylcholine outflow from the plexus, which was initially deemed to be the result of stimulating myenteric somata (Jansson and
Lisander, 1969; Jansson and Martinson, 1966), but may in fact be a result of sympathetic presynaptic modulation of cholinergic parasympathetic terminals coursing with sympathetic fibers rather than an influence on the myenteric neurons (Knoll and
Vizi, 1971; Paton and Vizi, 1969; Vizi and Knoll, 1971). Finally, immunohistochemical visualization of tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) (Hokfelt et al., 1973; 1977a; 1977b; Lindh et al., 1986; Lundberg et al., 1982; 1983; Udenfriend, 1966) in sympathetic postganglionic axons within the 4 gut wall has routinely demonstrated heavy direct innervation of the circular muscle
(Furness et al., 1990; Olsson et al., 2004; Phillips et al., 2006; 2009; Tan et al., 2010;
Tassicker et al., 1999).
Some, though not all, of the apparent discrepancies between two- versus three- neuron models might be reconciled with an inclusive view that one subset of sympathetic postganglionic neurons project to myenteric neurons, whereas a second pool of the postganglionics projects to smooth muscle. Indeed, in the same study,
Gabella and Costa (1969) found evidence for both types of projection. Given such observations, these two types of circuits are typically discussed and schematically illustrated as involving different prevertebral neurons. Commonly, though, the three- neuron pathways in which enteric ganglia act as distal second-stage postganglionic neurons prevail in models, and the alternative more direct muscle innervation is treated as of little consequence (Furness, 2007; Furness et al., 1990; Lomax et al., 2010). Still further, of course, the two- and three- neuron alternatives may not always be mutually exclusive, as both myenteric ganglia and smooth muscle sites could receive collateral axon projections from the same sympathetic neuron (Scheuermann and Stach, 1984).
Such alternate organizational possibilities have not been systematically evaluated, in part due to the difficulty that previous techniques have had in isolating the profile of any single sympathetic axon. In fact, although attempts have been made to qualitatively identify an individual axon’s trajectory within the myenteric plexus by performing a partial sympathectomy followed by histofluorescence (c.f., Furness and
Costa, 1974), the fibers were eventually lost among other axons (Llewellyn-Smith et al., 1981). 5
Along with the difficulty in isolating the full extent and pattern of an individual sympathetic axon, the indirect labeling techniques rarely discriminate between dopaminergic and noradrenergic fibers, and/or lack certainty as to the origin of labeled fibers. In fact, evidence has been presented for the existence of TH-positive myenteric neurons (Anlauf et al., 2003; Li et al., 2004), vagal efferent (Armstrong et al., 1990;
Gwyn et al., 1985; Tayo and Williams, 1988; Tsukamoto et al., 2005) and afferent neuronal projections (Katz et al., 1983; Kummer et al., 1993), and possibly fibers from dorsal root neurons (Brumovsky et al., 2006). Further complicating histochemical surveys, it is also the case that a small fraction of cell bodies within the celiac and superior mesenteric ganglia are TH-negative (Elfvin et al., 1993).
To circumvent some of the technical limitations, the present study used an alternative approach to either histofluorescence or immunohistochemistry by employing the anterograde tracer dextran biotin to directly assess the topography of individual postganglionic axons within the muscularis externa of the stomach and small intestine. Dextran biotin has proven to be a successful anterograde tracer for other extrinsic sources of innervation to the enteric nervous system (ENS) and can provide a level of control over the number of neurons that are labeled (e.g. vagal afferent and efferent; Powley and Phillips, 2005; Phillips et al., 2008; Walter et al., 2009). In the present study, the tracer was injected into the left CSMG to attain the desired enteric labeling (Quinson et al., 2001; Trudrung et al., 1994). Following the injection into the prevertebral ganglia, individual axonal arbors were traced in their entirety, digitally reconstructed, and morphometrically analyzed to evaluate the distribution of individual 6 neurites within the wall of the gut and to determine how terminal projection patterns relate to models of postganglionic innervation.
7
MATERIALS AND METHODS
Animals
Virgin male Fischer 344 (F344; n = 50) rats were purchased from Harlan
Laboratories (Indianapolis, IN) at 2-months of age and weighed 175-200 g at the time of delivery. Rats 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 Into the Sympathetic Ganglia
Dextran biotin injected into peripheral ganglia (Nodose: Fox et al., 2000,
Phillips and Powley, 2000, Powley and Phillips, 2005; Dorsal Root: Kyloh and
Spencer, 2014) yields excellent labeling of gut terminal fields equivalent to Golgi staining, and the tracer was used in the present study to produce the high definition structural detail of the sympathetic innervation needed to generate complete characterizations of the axon topography. Overnight food-deprived rats were 8 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. For tracer injections, each rat was then positioned on its back, and the abdominal cavity was opened by a midline laparotomy. The abdominal organs were draped with 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.
Using a Leica MZ16 microscope, the distinct U formed by the celiac and mesenteric arteries branching from the descending aorta was located. The two arteries provided an easily identifiable anatomical landmark for the left CSMG, as the anterior and posterior poles of the jointly encapsulated ganglia drape, respectively, across each vessel (Hamer and Santer, 1981; Berthoud and Powley, 1993). Once the left CSMG was visualized, the posterior pole of the CSMG was impaled using a 35 gauge Nanofil syringe with a beveled needle (NF35BV; World Precision Instruments, Sarasota, FL), and the needle was advanced longitudinally, within and parallel to, the ganglia, until the tip rested at the anterior pole of the CSMG. Dextran biotin, 6 μl of 10K MW lysine-fixable (7.5% concentration; D1956; Invitrogen, Carlsbad, CA), was then slowly injected as the needle was gradually retracted within the ganglia in a stepwise fashion.
When the 6 μl injection had been completed and the infusion pressure within the
CSMG had dissipated, the needle was removed from the ganglia. The displaced organs 9 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.
Prior to being returned to their home cages, rats recovered on a water circulating warming pad until the complete return of their righting reflex. 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 every 12 h over the following 72 h post- surgery. Also, to prevent post-surgical dehydration, rats were given an injection of warm saline (3 ml; s.c.). Body weight along with food and water intake were monitored throughout the duration of the post-surgery recovery period to ensure that rats did not experience any post-surgical malaise.
Control procedures were run to rule out tracer leakage from the left CSMG into the surrounding parenchyma as a potential 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 confirmed 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 the terminals located in the gut wall and were not a byproduct of tracer leakage. 10
Tissue Fixation and Dissection
Nine days post-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, 0.1 ml of heparin was injected in the left ventricle of the heart to facilitate exsanguination.
Following 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, while 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 the longitudinal axis of the mesenteric attachment. Whole mounts were rinsed under tap water and further fixed overnight in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 4°C. The intact smooth muscle 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
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 11
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 panneuronal marker for myenteric neurons (Phillips et al., 2004) and is compatible with our dextran biotin protocol (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 x 5 min PBS rinses. Whole mounts were then soaked for 3 d in PBS containing 0.5% Triton X-100 and 0.8% Sodium Azide to improve penetration through the muscle sheets. Free floating whole mounts were then rinsed in PBS for 6 x 5 min followed by a 60 min soak in ABC (prepared according to the manufacturer’s directions; PK-6100; Vectastain Elite ABC kit; Vector
Laboratories). Whole mounts were then rinsed in PBS for 6 x 5 min, reacted in DAB solution for 3 min, followed by 6 x 5 min rinses in dH2O. Whole mounts 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).
Sympathetic Terminal Inventories and Analyses
Initial Screening
Whole mounts were systematically scanned at 400x magnification under brightfield illumination using a Leica DMRE microscope. Once an arbor was identified it was further inspected to determine if it met the following four criteria: (a) well labeled, (b) completeness of the axon, (c) minimal tissue artifact such as folds and tears, and (d) free of multiple overlapping, unrelated axons that might obscure the analyses of a given terminal field. Individual sympathetic terminal arbors that 12 adequately satisfied the four criteria for full analyses were digitized, reconstructed, and evaluated quantitatively using a Neurolucida (MicrobrightField Inc., Williston, VT,
USA) workstation employing a Zeiss Axio Imager Z2 microscope (Carl Zeiss
Microimaging, Gottingen, Germany) equipped with DIC optics and both a 40x dry and a 63x water immersion long working distance objective.
Analyses of General Morphology
The following standardized morphometric measures (Powley et al., 2012,
2013a, 2013b, 2014), captured with the Neurolucida software, were analyzed for all digitized neurites: total arbor length, parent axon length (using Strahler Analysis), total number of terminal branches, highest branch order, and terminal field size (using
Convex Hull Analysis).
Enteric circuitry and smooth muscle tissues were distinguished according to conventional criteria (cf. Furness, 2007; Scheuermann and Stach, 1984). Briefly, the primary myenteric plexus was considered to be both the myenteric ganglia and 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, 13 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 (Furness, 2006; Wilson, et al.,
1987), whereas the tertiary plexus carries axons that innervate the longitudinal muscle sheet (Llewellyn-Smith et al., 1993). These conventional inferences were used in categorizing the tissue targets of the labeled CSMG neurons.
Criteria for Myenteric Ganglia
According to 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 distance of three or more average neuron diameters, then they were considered two separate ganglia (Berthoud et al., 1997). Also, if a group of neurons was isolated from another group by a string of successive single-file neurons, which were three average neuronal diameters in length, then the two groupings were considered to be separate ganglia (Berthoud et al., 1997).
Criteria for Sympathetic Postganglionic Innervation
Two different indices were used to independently estimate the extent of innervation of tissue targets by sympathetic axons: 1) Consistent with the common assumption (Levitan and Kaczmarek, 2002), the highest order branches, or terminal tips, of sympathetic motor fiber arbors were assumed to be sites of transmitter release.
2) Similarly, consistent with conventional observations (Falck and Owman, 1966;
Furness et al., 2014), segments of sympathetic axons with substantial varicosities were assumed to release transmitter from the varicosities. 14
Assessments of proportion or extent of innervation patterns of tissues were developed from the above criteria of postganglionic innervation. In the case of the myenteric 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 connective neighboring a ganglion. The second measure involved counting the number of ganglia with varicose fibers passing through or in close proximity to a ganglion. 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 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 (Furness,
1970a; Gillespie and Maxwell, 1971; Read and Burnstock, 1969).
Both criteria of postganglionic innervation were similarly used to evaluate the more direct projections to the smooth muscle sheets. 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. 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 15 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.
Criterion for Sympathetic Arbors Projecting Exclusively to Myenteric Neurons
Given that the three-neuron model, in which sympathetic postganglionic arbors are assumed to project essentially exclusively to myenteric plexus neurons and hence only indirectly to the smooth muscle, has recently been the dominant model of the organization of sympathetic control of the GI tract (e.g., Furness, 2007), we scrutinized the criterion—or criteria—for assessing morphometrically this predicted pattern.
The initial tissue survey of sympathetic projections for strong staining quality and whole mount integrity suggested that there was a subset of axonal arbors that approximated the conventional idea of CSMG neurons innervating the myenteric primary plexus, which contains the myenteric neuronal somata and their dendritic processes, but the preliminary survey also suggested that the majority of even this subset of CSMG neuronal arbors still issued some percentage of its collaterals and branches to the muscle sheets and their associated networks of higher order connectives. In the initial screenings, this limited percentage of non-primary-plexus collaterals of the subset of CSMG neurons seemed to amount typically to 15% of the cumulative length of all of a postganglionic arbor’s branches (see Figure 3 below). To avoid setting too stringent a criterion for myenteric-plexus-projecting sympathetic arbors, one that would effectively exclude almost all the sympathetic projections from the subset of CSMG arbors most closely approximating the three-neuron-model prediction, we adopted the cutoff criterion of 15%. With this criterion arbors with 16
15% of their total length outside the primary plexus were considered to project nearly exclusively to myenteric ganglion cells, whereas arbors with >15% of their total neurite segment length outside the primary plexus were considered more complex or “mixed” or specifically “heterotypic” projections fields.
Three other considerations reinforced the use of the 15% cutoff point that was adopted for the basic quantitative and categorical comparisons. First, after all of the sympathetic arbors had been digitized and characterized morphometrically, the distribution of the percentage of the cumulative neurite length of each of the 154 neurons that projected to sites in the muscle wall other than the primary plexus was examined. The scatter plot of the percentages revealed a trimodal distribution, with the first peak occurring with ~10% to ~15% of each arbor’s total neurite length distributed outside of the primary plexus (and, conversely ~85% to ~90% within the primary plexus). The scatter plot also suggested a nadir between the first and second peaks in the distribution that occurred at around 25%.
Second, though we routinely performed our calculations using the 15% criterion for a cutoff, we also checked calculations and groupings of arbors using break point criteria of both 10% and 20%. Though shifting the criterion cutoffs did somewhat affect the numbers of cases in the different subsets, of course, the shifts did not change any of the basic patterns observed or conclusions reached with the 15% cutoff.
Third, and finally, we adopted the 15% cutoff rather than a still higher percentage in part because it seemed incautious to assume that as much as, say, 20% or more of the total neurite length of an arbor could be summarily discounted. 17
It should also be noted, though, that even the cutoff criterion of 15% that we used would, arguably, have still discounted significant and quite functional elements of the arbors that projected to non-primary-plexus targets. Nonetheless, using a more stringent cutoff criterion [i.e. <15% (and particularly <10%) of an arbor’s neurite length outside the primary myenteric plexus] to define sympathetic postganglionic arbors that “exclusively” innervated the primary plexus dramatically diminished the size of that subset of sympathetic neurons which conformed to the conventional three- neuron model of the sympathetic control of the gut.
Photography
Single high power images were acquired with a Spot Flex camera controlled by
Spot Software (V4.7 Advanced Plus; Diagnostic Instruments, Sterling Heights, MI) 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
(630x; 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). 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 in Photoshop CS5 (Adobe Systems, San Jose, CA) to generate an all-in-focus image. Photoshop CS5 was also used in final figure production to: (a) apply text and scale bars; (b) adjust brightness, contrast, color, hue, and sharpness; and (c) organize the final layouts of the figures.
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Statistics
Statistical analyses and graph generation were accomplished using GraphPad
Prism (V. 5.0; GraphPad Software, San Diego, CA, USA). 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
Screened and Digitized Endings
The high-definition permanent dextran biotin labeling of sympathetic axons in whole mounts that consisted of both longitudinal and circular muscle with the myenteric plexus sandwiched in-between made it practical to reconstruct digitally and analyze morphologically individual axons that originated from the left CSMG and terminated in the muscle wall of the gut.
Our initial survey identified a total of 254 sympathetic axons that satisfied the histological quality criteria for analysis. Of the full inventory, 160 axons were randomly selected for digitization and analysis using the Neurolucida tracing software.
Of the random sample, final quantitative analysis was performed on 154 axons, as we determined during the tracing process that six cases needed to be dropped because of either incomplete Cuprolinic Blue staining of neurons (N = 2) or innervation of blood vessels by the neurites (N = 4). The digitized sympathetic terminals analyzed were representative of the different gut regions sampled; see Table 1 for a detailed regional breakdown of the 154 analyzed neurites.
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Table 1 goes about here.
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Sympathetic Nerves Innervate Multiple Different Tissue Sites in the Muscle Wall
of the GI Tract
General Pattern of Innervation
Sympathetic axons were prominently and extensively distributed throughout the smooth muscle wall in all four regions of the GI tract sampled. Along the length of the stomach and small intestine (i.e. duodenum, jejunum and ileum), the basic architecture and organization of individual sympathetic terminal fields consisted of single parent axons entering the gut wall and then branching repeatedly to form extensive terminal arbor arrays extending over large innervation fields. More particularly, three patterns of sympathetic efferent arborization—differing in terms of the tissues they contacted— were observed. These patterns address the different models of sympathetic innervation that have been previously inferred from observations with fluorescence histochemistry, electron microscopy and other earlier protocols (see Introduction). In brief:
A small minority (13%, or 20 of the 154 arbors) innervated essentially exclusively the primary myenteric plexus and thus conformed, presumably, to the three-neuron or indirect-innervation of muscle model of sympathetic circuitry that has tended to monopolize recent discussions.
A separate minority (27%, or 42 of the 154 arbors) directly—and nearly exclusively—innervated smooth muscle (i.e., circular muscle and/or secondary plexus), consistent with the two-neuron or direct postganglionic innervation of smooth muscle model of the sympathetic outflow.
The majority (60%, or 92 of the 154 cases) of the sympathetic terminal arbors, however, as discussed more fully below, were more complex and extensive, ending 21 heterotypically in different target tissues at different sites. Specifically, this majority of sympathetic fibers directly innervated both the primary myenteric plexus, providing substantial numbers of collaterals or contacts directly to myenteric neurons, and, in parallel, smooth muscle, by issuing significant numbers of collaterals or contacts directly to a muscle sheet. See Figures 1 & 2; also Table 1.
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Figures 1 & 2 go about here.
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While the majority of the traced cases comprised this broad group of heterotypic or “mixed” ending arbors (i.e., a single parent axon with terminations in multiple sites of different tissue types), these mixed arbors could be further broken down into three subgroups. Notably, the common feature between the three groups was that the heterotypic endings always included innervation of the primary plexus.
The three mixed ending subgroups consisted of endings that innervated (a) the primary plexus and the secondary plexus/circular muscle, (b) the primary plexus and the tertiary plexus, and (c) the primary plexus, the secondary plexus/circular muscle, and the tertiary plexus; see Table 2. The different types of sympathetic terminal fields are discussed more fully below.
Single Tissue Type Innervated: Primary Plexus
Though even a cursory and qualitative assessment of the random sample of endings indicated that a relatively small percentage of the sympathetic arbors innervated, almost exclusively, the primary myenteric plexus, the estimate of that percentage was heavily affected by our “length of varicose arbors” criterion for 22 innervation. Most, if not all, of the 154 arbors evaluated had a few or more short branches or axonal arrays that extended away from the tissue site receiving the preponderance of the apparent innervation. Of those sympathetic arbors projecting predominately to the primary plexus, some of these short branches and/or arrays extended to the plexus-smooth muscle boundary. In Figure 3, for example, a sympathetic axon appears to predominately innervate only the primary plexus, with minor short collateral branches to the smooth muscle (designated with arrows in Figure
3C). In the case of the axon illustrated in Figure 3, such collateral branches accounted for only 8.5% of the total cumulative length of the fiber’s arbor. Whether these minor branches (which technically could force a reclassification of an arbor from being considered as innervating a single type of target tissue, i.e. the plexus, to being considered as projecting heterotypically to innervate both plexus and muscle) were discounted, or not, materially affected estimates of the number of fibers that projected exclusively to the primary plexus.
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Figure 3 goes about here.
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When the “length of varicose arbors” criterion was stringently applied, even to short axonal spurs with minimal muscle contact, an analysis of the raw data determined that only three axons or 2% of the 154 arbors had terminal branches and varicose fibers of passage exclusively limited to the primary plexus. In these three cases, innervation of the primary plexus consisted of 59% of the axon length contained within the ganglia 23 while the remaining 41% of the axon length innervated the interconnective strands of the plexus.
Because such an extremely restrictive decision rule almost certainly distorted the survey and biased the analysis against recognizing arbors that, practically speaking, innervated a single tissue site, we relaxed the arbor length criterion to exclude the minor collateral branches and re-analyzed the findings. Such collateral branches or spurs were considered minor projections and ignored for purposes of the analysis of the target tissues of any postganglionic fiber, if less than 15% of the fiber’s total length occurred in the secondary tissue site. Even after lowering our criteria for arbors innervating a single site to exclude those minor collateral branches reaching tissue sites other than the primary plexus, the number of sympathetic axons classified as exclusively innervating the primary plexus still remained relatively low (N = 20); see
Table 1.
Single Tissue Type Innervated: Secondary Plexus/Circular Muscle
Compared to the primary plexus, analysis of the raw data determined that a greater number (N = 22) of traced neurites innervated exclusively the secondary plexus/circular muscle. These cases were typically morphologically simple and usually consisted of a parent axon that extended a short distance from the mesenteric border towards the anti-mesentery and terminated as arrays that projected within the secondary plexus/circular muscle coat. The number of cases that only innervated the secondary plexus/circular muscle almost doubled (N = 42) after lowering our criteria for innervation to include cases with minor collateral branches (i.e., branches with less 24 than 15% of their overall length contained within a site) innervating sites other than the secondary plexus/circular muscle; see Table 1.
Innervation of Longitudinal Muscle/Tertiary Plexus
While direct innervation of the longitudinal muscle by dextran labeled fiber segments was extremely rare (N = 2; 1% of the 154 arbors), there was considerable innervation of the tertiary plexus. In the present study the decision was made to classify the sympathetic innervation of the tertiary plexus as targeting the longitudinal muscle. We based this decision on the current assumption that sympathetic fibers innervating the tertiary plexus play an effector role in longitudinal muscle function
(Furness, 1970a; Gillespie and Maxwell, 1971; Read and Burnstock, 1969).
Specifically, because of the tertiary plexus’s close proximity to longitudinal muscle fibers, and the presence of varicosities along the lengths of the terminal branches within the tertiary plexus, neurotransmitters released from these neurites are thought to directly influence the longitudinal muscle (Llewellyn-Smith et al., 1993).
Innervation associated with the tertiary plexus by dextran labeled fiber segments consisted of branches that tended to have a winding nature as they projected in a predominately longitudinal manner; Figure 2B and 4A. While some axons making up the tertiary plexus were observed within the same focal plane as the myenteric plexus, other fibers were just-superficial to the ganglia and traversed primarily in the longitudinal axis; Figure 4B. Bifurcating arbors within the tertiary plexus exhibited varicosities along their length and frequently terminated either within the interganglionic space or adjacent to the myenteric ganglia; Figure 4C.
25
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Figure 4 goes about here.
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Relevant to the present classification scheme, the tertiary plexus was never the singular target of any of the analyzed sympathetic axons. Rather, if a neurite was associated with the tertiary plexus then it was always the case that the same neurite also innervated one or more other sites. Thus, traced axons associated with the tertiary plexus were always analyzed as part of one or more of the heterotypical or mixed ending subgroups discussed below; see Table 2.
Multiple Tissue Types Innervated: Primary Plexus in Addition to the Secondary
Plexus/Circular Muscle and/or the Tertiary Plexus
Analysis of the raw data determined that the majority of the traced terminal arbors (N = 129) were heterotypic, innervating multiple different types of sites within the gut wall. As discussed above, a more conservative criterion for innervation was therefore again adopted so as to not overestimate the role of minor collaterals in smooth muscle function. Specifically, minor collateral branches (i.e., branches with less than 15% of their overall length contained within a site) were not included when axons were classified based on site(s) of innervation. Even using this more conservative criterion for innervation, it was still the case that the largest group of dextran labeled arbors were mixed endings that innervated different types of sites (N =
92); see Table 2. As mentioned above, these complex mixed endings always innervated the primary plexus and one or more additional sites. In fact, the largest group of mixed endings consisted of neurites that innervated the primary plexus, 26 tertiary plexus, and secondary plexus/circular muscle (N = 62); Figures 2 & Figure 4, followed by neurites that innervated the primary plexus and the tertiary plexus (N =
17), and lastly, neurites that innervated the primary plexus and the secondary plexus/circular muscle (N = 13); Figure 5.
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Table 2 goes about here.
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Figure 5 goes about here.
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Quantitative Analysis of Myenteric Ganglia Innervation
We and others have shown that there are regional differences in the myenteric plexus located within the smooth muscle wall of the gut (e.g., Karaosmanoglu et al.,
1996; Phillips and Powley, 2001), so we sought to determine if these regional differences were also reflected in differences of the sympathetic innervation of the myenteric ganglia. Specifically, we sought to determine if the number of ganglia innervated by individual sympathetic neuronal arbors located in the proximal gut wall
(i.e., stomach and proximal duodenum) differed in the number of ganglia innervated by individual arbors in the distal gut (i.e., distal duodenum, mid-jejunum, and ileum).
Our two dependent measures used to determine ganglia innervation by sympathetic arbors (i.e. terminal branches and varicosities) differed in the number of ganglia estimated to be innervated per axon. Specifically, a greater number of ganglia were found to be innervated by the “varicose neurites” criterion compared to the 27
“terminal branches” criterion; however both measures revealed the same proximal-gut- versus-distal-gut pattern of innervation, with a greater number of ganglia in the proximal gut innervated compared to the distal gut. Analysis of ganglia innervation by terminal branches revealed a significant regional difference [F(4, 122) = 5.363, P =
0.0005] with a greater number of ganglia innervated per axon in the stomach compared to the distal gut (vs duodenum 3-6 cm, P = 0.0195; vs mid-jejunum, P = 0.0468; vs ileum, P = 0.0096), while the proximal duodenum innervation was comparable to the stomach (P > 0.05) but had a greater number of innervated ganglia per axon compared to the distal duodenum (P = 0.0399) and ileum (P = 0.0179); Figure 6A. Analysis of ganglia innervated by varicose axons revealed a similar regional pattern [F(4, 127) =
4.989, P = 0.0009], with a greater number of ganglia innervated per sympathetic arbor in the stomach compared to the distal intestines (vs duodenum 3-6 cm, P = 0.0214; vs mid-jejunum, P = 0.0725; vs ileum, P = 0.0149), while the proximal duodenum was comparably innervated to the stomach (P > 0.05) but had a greater number of ganglia innervated compared to the distal intestines (vs duodenum 3-6 cm, P = 0.0400; vs mid- jejunum, P = 0.1390; vs ileum, P = 0.0250); Figure 6B.
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Figure 6 goes about here.
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Quantitative Analysis of the Relative Proportions of Individual Heterotypic
Sympathetic Arbors Innervating Myenteric Plexus vs. Smooth Muscle
With collateral innervation to the primary myenteric plexus (i.e., ganglia and interconnective strands), secondary plexus/circular muscle, and tertiary plexus, 28 analyses were done to provide an indication of the extent of sympathetic projections to each area. The first evaluation extracted the extent of sympathetic innervation within the various plexuses and muscle (see methods) by identifying the total length devoted to each area and subsequently the proportion of the overall length. Statistical comparisons of the proportional lengths innervating each target were performed using the traced axons that comprised the mixed endings described in Table 2. For the 13 cases in which a neurite innervated both the primary plexus and the secondary plexus/circular muscle, a significant [F(1.521, 18.25) = 27.89, P < 0.0001] percentage of the total arbor length innervated the secondary plexus/circular muscle (60%) compared to the ganglia (16%; P = 0.0002) or the interconnective strands (24%; P =
0.0006); Figure 7A. Analysis of the 17 neurites that innervated both the primary plexus and the tertiary plexus revealed an overall significant difference [F(1.733,
27.73) = 3.769, P = 0.0412], with a greater percentage of the total length innervating the tertiary plexus (45%) compared to the ganglia (29%; P = 0.1856) and the interconnective strands (26%; P = 0.0483); Figure 7B. Conversely, a more uniform pattern of innervation was determined for the 62 heterotypic cases that innervated the primary plexus, secondary plexus/circular muscle, and the tertiary plexus [F(2.684,
163.7) = 2.745, P = 0.0509] with very similar percentages of overall length innervating each site: ganglia (23%), interconnective strands (29%), secondary plexus/circular muscle (27%), and tertiary plexus (21%); Figure 7C.
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Figure 7 goes about here.
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In addition to the proportion of total arbor length associated with each of the different target tissues, the proportion of terminal branch innervation was similarly assessed for each site. Although terminal branches are not a direct indication of synaptic modulation due to vesicle release from the varicosities along the axon length, the terminal branches may have some relation to plexus- or site-directed innervation.
This analysis was performed on the subclasses of mixed endings; Table 2. The total number of terminal branches within the defined plexuses and circular muscle were summed and a proportion of the overall terminations was calculated. Analysis of the sympathetic arbors that had terminal branches in both the primary plexus and the secondary plexus/circular muscle revealed a trend for the secondary plexus/circular muscle (46%) to have a greater proportion of terminal branches compared to the ganglia (29%) or the interconnective strands (24%); Figure 8A. Analysis of the neurites that had terminal branches in both the primary plexus and the tertiary plexus revealed an overall significant difference [F(1.327, 21.24) = 11.06, P = 0.0016], with the largest percentage of terminal branches located in the tertiary plexus (52%) followed by the ganglia (33%; vs. tertiary plexus, P = 0.1974) and then the interconnective strands (14%; vs. tertiary plexus, P < 0.0001; vs ganglia, P = 0.0272);
Figure 8B. Lastly, analysis of the terminal branches for the subset of arbors with terminal branches in all three tissue sites revealed a difference [F(2.629, 160.3) =
14.25, P < 0.0001]. There was a similar proportion of terminal branches within the ganglia (35%) and tertiary plexus (30%), which were both significantly greater compared to the interconnective strands (17%; vs. ganglia, P < 0.0001; vs. tertiary 30 plexus, P = 0.0026) and the secondary plexus/circular muscle (18%; vs ganglia, P <
0.0001; vs. tertiary plexus, P = 0.0200); Figure 8C.
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Figure 8 goes about here.
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Quantitative Analysis of General Terminal Morphology
Employment of the MicroBrightField algorithms for analyses of general morphometric parameters (see Table 3) revealed that the averages for total arbor length, parent axon length (determined using Strahler analysis of branch ordering), total number of terminal branches, and highest branch order (centrifugal branch order) were typically highest in the stomach and proximal duodenum compared to the distal intestine. This is suggestive of a larger, more complex arrangement of sympathetic terminal fields in the upper GI tract. It may be premature, however, to conclude that such a difference is functionally relevant (Mawe et al., 1989), because it could just as likely reflect the sheer size of the organ and thus the length of an axon that is necessary to cover the overall area of innervation. To potentially address this concern, terminal field size was determined using a Convex Hull Analysis. To avoid inflating the size of the terminal field due to the extended distance that some parent fibers project before the occurrence of an initial bifurcation, convex hull analysis only included the length of the neurite after the initial bifurcation. Both ANOVA and post hoc analyses of the terminal arbors determined that the stomach had a greater area covered compared to the intestinal regions (all P values < 0.0001) while the different intestinal regions all had similar terminal field sizes (all P values > 0.05). 31
The fact that sympathetic arbors in the stomach were significantly larger for every measure is likely a result of the stomach’s greater surface area. In contrast, while the arbors in the duodenum 0-3 cm had a greater total arbor length, parent axon length, higher branching order, and total number of terminal branches compared to more distal intestinal regions, they had a similar terminal field size, possibly indicating that terminal complexity in the duodenum was not a byproduct of organ area but rather indicative of having a more densely packed terminal field (Gillespie and Maxwell,
1971; Hollands and Vanov, 1965).
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Table 3 goes about here.
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Sympathetic Innervation of the GI Vasculature
Individual axons originating from the left CSMG were observed to issue branches in small bundles silhouetting the outer perimeter of blood vessels within the muscle wall of the stomach (N = 4; Fig 9). The blood vessels were distinguished by the pattern of staining outlining the vascular lumen and were made even more evident by varicose sympathetic arbors and terminal branches lining the outer limits; Figure 9.
Although it has typically been thought that arteries are supplied by a distinct set of sympathetic fibers, the present experiment observed that a single axon along a blood vessel also formed collateral branches that were associated with neighboring myenteric neurons; Figure 9. Also, the evidence suggested that the vascular innervation, provided by a solitary axon, could project to the secondary plexus/circular muscle as well as localized regions of several distinct arterial branches. 32
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Figure 9 goes about here.
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Bilateral Sympathetic Innervation of Stomach and Small Intestines
The experimental capability, provided by the dextran-biotin, of distinguishing single sympathetic postganglionic axons throughout their trajectories produced cases in which a single axon traversed across both the ventral and the dorsal sides of the stomach, providing bilateral innervation of the organ; Figure 10. Similar instances were observed in the intestines as neurites were observed extending from the mesenteric attachment and then continuing beyond the point of the anti-mesentery into the other hemisphere of the small intestine. Additionally, sympathetic nerves taking a perivascular route from the ganglia to the border of the GI walls, upon entering the
ENS, appeared to form collateral branches that extended bilaterally within parallel pathways.
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Figure 10 goes about here.
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33
DISCUSSION
Given the still-incomplete understanding of sympathetic postganglionic axonal organizational patterns in the GI tract, the present study was designed to provide a more comprehensive analysis of the morphology and topography of sympathetic axon terminals located in the smooth muscle wall of the GI tract. To assess this motor neuron architecture, we combined a high-definition anterograde tracer protocol, using dextran biotin, with quantitative microscopy and digital reconstruction of terminal fields that were then evaluated morphologically and stereologically. Such techniques are widely used in the central nervous system (CNS) for analysis of neuronal circuitry
(Donohue and Ascoli, 2011; Lemmens et al., 2010; Lu, 2011; Senft, 2011), but have not been applied to the study of the sympathetic innervation of the ENS. Similarly, there is a limited amount of research using modern anterograde tracers to investigate the sympathetic nervous system.
A few studies have employed anterograde labeling to identify the sympathetic postganglionic innervation of the GI tract, but these were done in vitro by applying biotinamide to a severed mesenteric nerve trunk, a procedure which has the potential to label indiscriminately any cut fiber in the trunk, followed by indirect verification of the sympathetic fibers based on TH-expression (Olsson et al., 2004; Tan et al., 2010;
Tassicker et al., 1999). Additionally, the in vitro experiments used whole mounts in 34 which the circular muscle was removed, so it was not possible to study the sympathetic innervation of the secondary plexus/circular muscle (Olsson et al., 2004; Tan et al.,
2010; Tassicker et al., 1999). In light of the evidence that the circular muscle is heavily innervated by postganglionic sympathetic fibers (present results; also see
Furness et al., 1990; Olsson et al., 2004; Phillips et al., 2006; 2009; Tan et al., 2010;
Tassicker et al., 1999), exclusion of the circular muscle sheet presumably resulted in the biotinamide surveys missing significant aspects of the topographical organization and distribution of sympathetic terminals. Given the demonstrated utility of combining anterograde tracing and axon reconstruction within the CNS (Lemmens et al., 2010), the present investigation overcame prior limitations by direct labeling of sympathetic neurons by tracer injections within the CSMG and the use of intact whole mounts of the complete smooth muscle wall of the gut. In combination, the protocols provided unequivocal labeling of intact sympathetic terminal fields located in the gut wall and made practical detailed qualitative and quantitative analyses of digitally reconstructed terminal arbors.
Several of the morphometric parameters and findings reported here have not been described in prior investigations of postganglionic axons. The present study found, in the rodent GI tract, that there is extensive sympathetic innervation of the primary myenteric plexus, secondary plexus/circular muscle, and tertiary plexus.
These postganglionic projections fell into three broad categories. One subset of CSMG sympathetic neurons, a minority (13%), projected almost exclusively to the myenteric ganglia and their primary interconnective strands, in a pattern consistent with the currently prevailing model of a three-neuron outflow in which myenteric neurons are 35 intercalated between the postganglionic neurons and the target tissue effectors they control. A second subset of CSMG neurons, again a minority (27%), projected essentially exclusively directly to smooth muscle, in keeping with the classical view that autonomic motor outflows are comprised of two-neuron projections in which the postganglionic neurons project directly onto smooth muscle effectors.
In contrast to the first two types of sympathetic postganglionic neuronal projections found in CSMG, the largest subset actually consisted of a sympathetic motor neuron pool (60%) that issued extensive and complex axonal arbors, which projected heterotypically to both the primary myenteric plexus and one or more smooth muscle sheet. This substantial pool of postganglionics with mixed or complex terminal arbors has an architecture in which single motor neurons would appear capable of coordinating or directly crosslinking myenteric plexus and smooth muscle operations locally, within a given neuron’s terminal innervation field.
Sympathetic Morphology
The sympathetic prevertebral ganglia have multiple sources of synaptic input
(Gibbins, 1995; Miolan and Niel, 1996) and, as a result, are considered to share features with networks within the CNS that operate as integrative centers. The apparent integration within the prevertebral ganglia is classically considered to occur among subcategories of neuronal somata that are distinguished on the basis of neurochemical makeup (Gibbins, 1992; Lindh et al., 1986), and to some extent on topography (Lindh et al., 1986; Quinson et al., 2001), morphology (Purves, 1988), and electrophysiological signature (Boyd et al., 1996; Jobling and Gibbins, 1999; Keast et al., 1993). Practically, though, it has not been feasible to associate the prevertebral 36 ganglia subcategories with distinct motor neuron terminal specializations within the muscularis externa of the GI tract. Currently, the characteristics of noradrenergic innervation in the myenteric plexus and smooth muscle layers are ordinarily represented schematically and in models as distinct neuronal projections to either primary, secondary, or tertiary plexus, with little indication that the fibers project extensively to smooth muscle and that there are identifying features of an individual axon beyond the simplified organization (Furness, 2007; Scheuermann and Stach,
1984).
A number of the early descriptions of the adrenergic sympathetic projections in the ENS came from the application of histofluorescence techniques to cryostat sectioned tissue, which suggested minimal innervation of the muscle layers and a large number of varicose fibers within the plane of the myenteric plexus (Norberg, 1964;
Hollands and Vanov, 1965; Jacobowitz, 1965; Capurso et al., 1968). In the years that followed, an important technical advancement came from the use of stretched tissue whole mounts, which allowed the complete branching pattern of axons to be observed in the myenteric plexus and muscle layers (Furness and Malmfors, 1971; Gabella and
Costa, 1967; Read and Burnstock, 1969; Silva et al., 1971). These early histofluorescence studies suggested that the largest density of innervation is at the level of the myenteric plexus, preferentially projecting to the ganglia, and the number of fluorescent fibers in the longitudinal muscle appeared to be nonexistent compared to the labeling in the circular muscle (Furness and Malmfors, 1971; Gabella and Costa,
1967; Jacobowitz, 1965; Norberg, 1964; Read and Burnstock, 1968, 1969; Silva et al.,
1971). 37
Primary Plexus and the Myenteric Ganglia
The present study found that 13% of the labeled CSMG neuronal arbors projected exclusively to the primary plexus (N = 20/154; see Table 1). Consistent with previous reports, a single axon could be seen innervating a variable number of ganglia along its trajectory (Scheuermann and Stach, 1984). As sympathetic axons coursed from one ganglion to another through the interconnective strands of the primary plexus, some neurite segments contained varicosities and ended as terminal branches (Furness,
1970a; Mawe et al., 1989). In both ganglia and their connectives, the varicose neurites not conspicuously in apposition with myenteric neurons apparently made contacts either on myenteric ganglion cell dendrites or on axons coursing within the interganglionic interconnectives (Furness, 1970a; Mawe et al., 1989) and associated branches. Also, varicosities that are suggestive of terminal contacts were present within the ganglia (Gabella and Costa, 1969; Olsson et al., 2004) and several en passage varicosities on the same axon could be observed in relation to a single neuron
(Gabella, 1971, 1972). Although the typical organization of sympathetic axons around a myenteric neuron has been referred to as basket-like networks (Read and Burnstock,
1968), these were rarely seen in the present observations (see also Llewellyn-Smith et al., 1981). Further, consistent with our observation for the CSMG terminal arbors,
Tassicker and coworkers (1999) estimated that basket-like calices of varicosities occur for only 1% of myenteric perikarya.
In contrast to the idea of basket-like or other extensive clustering of the CSMG axonal varicosities in close apposition to individual myenteric neurons, many of the dextran-labeled arbors we evaluated made only limited varicose contacts in close 38 proximity to myenteric perikarya (see Figures 3 & 5). Additionally, as has been observed in ultrastructural (Gordon-Weeks, 1982; Llewellyn-Smith et al., 1984) and immunohistochemical (Tassicker et al., 1999) surveys, labeled CSMG fibers often appeared to course around or through myenteric ganglia in relatively superficial paths just inside the ganglionic capsule. Some of these superficial CSMG fibers appeared to course through the ganglia without elaborating varicosities; others contained only limited numbers of varicosities in the proximity of myenteric neurons. In some instances, a ganglion only received a single short branch or varicose terminal spur that was juxtaposed to one peripherally located neuron.
This limited and dispersed pattern of innervation was also apparent in the analyses of heterotypic axons with mixed target tissues where only a modest percentage of the overall or total arbor length was comprised of varicose terminal branches (29- 35%) and varicose neurite segments (16- 29%; see Figure 7) in close apposition to the myenteric neurons, a pattern which is in contrast to the view that sympathetic axons preferentially innervate the ganglia (Furness, 2007; Furness and
Costa, 1974; Norberg, 1964). Along with the present finding of a low percentage of fibers exclusively innervating myenteric ganglia, previous ultrastructural identification of direct synapses between adrenergic axons and myenteric neurons (Taxi, 1969) suggests that the prototypical pre- and postsynaptic connections are rarely found, in part because many of the axons course along the outer perimeter of the ganglia
(Gordon-Weeks, 1982; Llewellyn-Smith et al., 1984).
39
Smooth Muscle
Secondary plexus/circular muscle. The sympathetic innervation of circular muscle is an area that has typically received relatively little attention due both to earlier investigations revealing a paucity of catecholamine innervation within the smooth muscle layers and to the proposal that sympathetic innervation modulates myenteric neurons rather than directly influencing the muscle (Norberg, 1964, 1967). In fact, many studies ignored the circular muscle by removing it from their whole mount preparations, thus limiting their observations to only the longitudinal muscle and myenteric plexus (e.g. Baker and Santer, 1989; Furness and Costa, 1971; 1975; Furness and Malmfors, 1971; Olsson et al., 2004; Tan et al., 2010; Tassicker et al., 1999).
Even though there has been a relative lack of attention in previous research, some evidence for sympathetic neurites within the circular muscle, in a range of species, has still been observed (Baker and Santer 1989; Capurso et al., 1968; Furness et al. 1990;
Gabella and Costa, 1969; Tan et al. 2010).
As previously observed in both light microscopy (Furness, 1970a; Furness and
Malmfors, 1971; Gabella and Costa, 1969; Mann and Bell, 1993; Mawe et al., 1989;
Olsson et al., 2004; Phillips et al., 2006; 2009; Read and Burnstock, 1969; Silva et al.,
1971; Tan et al., 2010), and electron microscopy (Gabella, 1970; Gabella and Costa,
1969; Wong, 1977), varicose intramuscular nerve fibers are present amid the bundles of smooth muscle cells in the circular muscle. The main feature of axons in this muscle layer is parallel branches that run along the circular muscle, parallel to the muscle fibers, with varying densities of collateral fibers across samples (Furness,
1970a). Also, some of the fibers that extend in the circular muscle have branches that 40 varied slightly from this parallel array. However, another pattern emerged that has no previous mention in the literature; this pattern involved a more irregular expansion and meandering of the branched fibers, rather than a relatively strict alignment with the circular muscle. Besides neurites within the plane of the smooth muscle, there were also collateral branches that had a similar appearance and orientation as the parallel intramuscular arrays, but were superficial to the circular muscle, and have been considered the secondary plexus (Furness, 2007; Llewellyn-Smith et al., 1993;
Scheuermann and Stach, 1984). While some of the secondary plexus fibers extended into the depth of the circular muscle (Llewellyn-Smith et al., 1993), others ended below the surface of the smooth muscle. At times this connection between the secondary plexus and circular muscle produced distinct features of relatively short bundles of axonal branches that extended between the two planes.
Although research using histofluorescence to visualize sympathetic nerves has suggested a relatively sparse innervation of the circular muscle compared to the innervation of the ganglia, the present investigation found a greater number of arbors that essentially exclusively innervated the secondary plexus/circular muscle (N =
42/154; see Table 1) compared to the subset that projected to the primary plexus alone.
Also, in the major subset of heterotypic arbors with multiple target tissues, calculations suggest that the circular muscle receives an extensive percentage of sympathetic fiber length (27- 60%) compared to the ganglia (16- 29%); see Figure 7. Further, while these observations may not directly equate to density, it is the case that the direct physiological responses on the smooth muscle layers may not require a high density of noradrenergic innervation due to the fact that smooth muscle cells can pass the change 41 in potential between adjoining membranes to neighboring cells (Furness, 1970b) through interconnected gap junctions (Baluk and Gabella, 1987; Brock and Cunnane,
1992; Gabella, 1973), electrical coupling (Gabella, 1973), and/or fibroblasts (Baluk and Gabella, 1987).
Longitudinal muscle/tertiary plexus. As opposed to those studies that thoroughly investigated the circular muscle, some early ultrastructural research revealed a complete lack of sympathetic nerve fibers within the longitudinal muscle sheet (Gabella, 1973; Baluk and Gabella, 1987), while other evidence has shown the existence of the rare axon penetrating the first muscle cell layer or residing within shallow grooves in the sheet (Llewellyn-Smith et al., 1993). In line with this observation, we observed longitudinal muscle innervation (see also Furness, 1970a;
Furness and Malmfors, 1971; Gabella and Costa, 1969; Mann and Bell, 1993; Mawe et al., 1989; Olsson et al., 2004; Read and Burnstock, 1969; Silva et al., 1971; Tan et al.,
2010), consisting of one or two short collateral terminal fiber segments running longitudinally. Despite the paucity of direct longitudinal muscle innervation there were instances, analogous to the relationship between the secondary plexus and circular muscle fibers, in which fibers coursed in a longitudinal array superficial to the myenteric plexus. Although this pattern of innervation may be suggestive of a longitudinal effector the functional relevance is unknown. On the other hand, early research considered the nervous tissue in the tertiary plexus as neuroeffectors of the longitudinal muscle (Furness, 1970a; Gillespie and Maxwell, 1971; Read and
Burnstock, 1969). Ultrastructural observations indicated that this plexus of fibers was indeed selectively juxtaposed with longitudinal muscle cells (Llewellyn-Smith et al., 42
1993). The meandering tertiary plexus axons in the present dextran-labeled series were observed as either short wavy fibers with several varicose branches protruding into the internodal area, a wandering expansion across the zone between two adjacent ganglia, or a protracted extension across several internodal regions.
Morphological and Multiple Target Mechanisms for Complex Integration
The present study found that the largest group of sympathetic axons (60%; N =
92/154; see Table 1) consisted of mixed endings that branched extensively to innervate multiple sites in both the myenteric plexus and the smooth muscle wall of the gut. It should be noted, too, that the estimate of 60% is a conservative one, achieved when the stringency of the criteria used to classify an arbor as innervating solely the myenteric plexus or exclusively smooth muscle targets was relaxed. If more stringent criteria were applied to the 154 sympathetic arbors assessed, however, over 84% of all terminal arbors would have been classified as heterotypic or mixed-tissue projecting axons.
Consistent with earlier research that has been mostly discounted or overlooked, we observed fibers exiting the myenteric ganglia and extending into the circular muscle
(Capurso et al., 1968; Silva et al., 1971), longitudinal muscle (Capurso et al., 1968), the tertiary plexus (Furness, 1970a), and secondary plexus. Along with the primary plexus fibers that branched and continued to project to either one of the muscle sheets, we also observed single fibers that projected through the primary plexus and continued to both secondary (presumptive circular muscle) and tertiary (provisional longitudinal muscle) plexuses, which confirmed a previously postulated pattern (Scheuermann and Stach,
1984). Also, various terminal field patterns and sizes within the different plexuses and circular muscle were observed. This distributed arrangement of sympathetic fibers is 43 suggestive of a complex mixed-target-site model for modulating GI function, which appears to simultaneously incorporate both the classical view (Hill, 1927; Langley,
1903) and the three-neuron chain in autonomic circuitry (Norberg, 1967). By the efferent axonal outflow of single sympathetic neurons projecting both to the myenteric plexus and to one or both muscle sheets, individual sympathetic fibers might provide a coordinating input to the different target tissue types.
Morphological Modulatory Mechanisms
The presently observed co-connectivity of individual, morphologically unique axons projecting both in the different plexuses and the muscle sheets, could be an example in which structurally different collateral branches from the same axon produce unique functional consequences due to the release of disparate transmitters (Burnstock,
1976; Kandel and Gardener, 1972). The sympathetic nervous system employs neurochemical coding (Costa et al., 1984; Gibbins, 1991, 1992), and prevertebral neurons synthesize multiple transmitters and/or neuromodulators (Burnstock, 1976;
Hokfelt et al., 1977a; Kandel and Gardner, 1972). This has allowed researchers to distinguish separate neurochemical classes of neurons that produce either noradrenergic/somatostatin (NA/SOM), noradrenergic/neuropeptide Y (NA/NPY), or solely noradrenergic (NA/-) signaling mechanisms (Lindh et al., 1986; Lundberg et al.,
1982; Macrae et al., 1986), which apparently target distinct regions within the ENS: the submucosa; the intestinal blood vessels; the myenteric plexus and muscle, respectively
(Costa and Furness, 1984). However, these subcategories also co-express other transmitters, and some of the alternative substances show immunoreactivity in a fraction of CSMG neurons that are non-noradrenergic (Elfvin et al., 1993). These 44 mixes of neurotransmitters or neuromodulators could suggest both distinct functional properties and the possible presence of transmitters that have not been thoroughly considered for sympathetic junctions within the ENS.
Besides neurochemical coding, the various innervation patterns that were observed in the present work could be designed to process the incoming signal in a frequency dependent manner (Grossman et al., 1973). Such distinct morphology could create different strengths of spatially and temporally organized nerve impulses that modulate the smooth muscle patterns involved in motility. Additionally, it is the case that vesicle release from varicosities relies on the frequency of a nerve impulse, as the presence of an action potential does not always correspond to the discharge of a vesicle from all varicosities (Brock and Cunnane, 1987). In fact there is a low probability of vesicle release from any given varicosity (Brock and Cunnane, 1992). Varicosities could filter the incoming signal, just as different diameter axons or branch points along the axon modify the conduction of the nerve impulse (Waxman, 1972).
Ganglia and Muscle Modulatory Mechanisms
The present comparisons of the proportions of axonal segment lengths and terminal branches within myenteric ganglia to those within the tertiary plexus and the secondary plexus/circular muscle, showed that the myenteric neurons receive a small proportion of varicose fiber length (see Figure 7) and “terminal endings”, which suggests that, quantitatively, the predominate target for sympathetic innervation is the smooth muscle. This organizational analysis suggests a substantial role for direct sympathetic modulation of smooth muscle inhibition. Two types of inhibitory mechanisms have been previously suggested, whereby the myenteric ganglia and 45 muscle input either reinforce one another or operate under distinct circumstances (Silva et al., 1971). Whether the mechanism at any given period is direct, indirect, or synergistic could depend on how abruptly an inhibitory response must occur, or it could fluctuate depending on the existing level of tension in the smooth muscle at the time of sympathetic transmission. The complexity of the direct and indirect mechanism are in line with the Gabella and Costa finding (1969), which suggested the existence of both the classical or two-neuron view of preganglionic neuron to postganglionic neuron to muscle, and the three-neuron chain, involving an intercalated
“postganglionic” myenteric neuron. The present investigation illustrates the possibility that the sympathetic nervous system may have separate pools of neurons consistent with each of these candidate views of connectivity, while also showing that the majority of neurons were a complex mixture of both.
Multiple Targets Integrate the Modulatory Mechanisms
This heterotypic or multiple-target model involves a larger amount of direct smooth muscle modulation than previously considered. Not only is it possible that NA diffuses from within the myenteric plexus to the surrounding muscle layers (Furness,
1970a; Gillespie and Maxwell, 1971; Read and Burnstock, 1969), causing relaxation of smooth muscle (Garry and Gillespie, 1955; Gillespie and MacKenna, 1961), but the present findings substantiate earlier pharmacological manipulations demonstrating that noradrenaline has a direct effect on the circular muscle (Gershon, 1967; Venkova and
Krier, 1995), where immunoreactive Į2a, ȕ1, and ȕ2 adrenoceptors have been identified
(Nasser et al., 2006). Also, the large amount of tertiary plexus innervation that was observed could be associated with the physiological mechanism of longitudinal 46 relaxation through modulation of ȕ-adrenoceptors (Gillespie and Khoyi, 1977;
Kosterlitz et al., 1970; Kostka et al., 1989; Vizi, 1974), as well as both ȕ- and Į- adrenoceptors in the stomach (Bailey, 1971).
Previous physiological findings that sympathetic fibers modulate acetylcholine release from within the myenteric plexus were unclear as to whether the noradrenergic influence was on myenteric somata or vagal parasympathetic axons (Jansson and
Lisander, 1969; Jansson and Martinson, 1966). Minimal ganglionic modulation is evident by the vague alteration in myenteric neuronal activity due to noradrenaline or sympathetic stimulation (Nishi and North, 1973; Takayanagi et al., 1977), as well as the rarity of axosomatic synaptic specializations observed in electron microscopic investigation (Gordon-Weeks, 1982; Llewellyn-Smith et al., 1984). Those adrenergic synapses that do occur appear to lack selective contact for any of the Dogiel types of neurons (Scheuermann and Stach, 1983). Specificity has also been difficult to analyze given the observed expression pattern of Į2a- and ȕ1-adrenoceptors on calbindin-, calretinin-, and nitrergic-positive myenteric neurons, along with the fact that not every cell of a particular phenotype expressed an adrenergic receptor (Nasser et al., 2006).
On the other hand, sympathetic fibers may cause a presynaptic inhibition of vagal terminals (Knoll and Vizi, 1971; Paton and Vizi, 1969; Vizi and Knoll, 1971) by diminishing the number of varicosities that are stimulated by an action potential
(Kadlec et al., 1986), which is thought to occur through presynaptic Į-adrenoceptors
(Kosterlitz et al., 1970; Surprenant and North, 1988; Vizi, 1974). However, myenteric neurons and vagal parasympathetic axons may not be the only recipients of sympathetic influence within the myenteric plexus. In fact, it has been shown that 47 catecholamine fluorescent varicose terminals also surround glial cells (Gabella, 1971;
Gulbransen et al., 2010), which express the Į2a adrenoceptor (Nasser et al., 2006).
It is also the case that sympathetic varicose branches or neurite segments within the primary plexus may not be limited to the ganglia but may also occur in interconnective strands. Noradrenergic nerves have been observed making synapse- like contacts with other neurites (Manber and Gershon, 1979; Llewellyn-Smith et al.,
1984). This structural layout suggests the possibility of axo-axonal presynaptic inhibition via the Į2 receptor on acetylcholine, as well as non-cholinergic output (Tack and Wood, 1992), like serotoninergic (Gershon and Sherman, 1987) and catecholamines themselves by autoinhibition of adrenergic varicosities (Brock and
Cunnane, 1992).
So, although it is parsimonious to assume separate neuronal classes are responsible for simple direct and indirect projections modifying the activity of the muscularis externa, given the data, it would appear as though the sympathetic neurite modulation occurs at various levels, through interactions with parasympathetic varicosities, myenteric neurons and processes, glial cells, fibroblast cells, direct neuromuscular junctions, diffusion, and autoinhibition. Adding to the apparent complexity of the sympathetic circuitry is the presently observed heterotypic or mixed- target axons. Such a highly intricate system suggests that the postganglionic sympathetic terminal input to the GI tract affects—and could perhaps integrate the operation of— multiple targets. Such operations would have parallels to the complex processing that occurs within the prevertebral ganglia.
48
Blood Vessel Innervation
Over half of all sympathetic neurons appear to have a role in regulating vasomotor activity (Gibbins, 2011). It has been shown that sympathetic vascular neurons have some of the smallest dendritic trees and soma (Gibbins et al., 2003), which is related to the small overlapping terminal fields on blood vessels (Gibbins et al., 1998). Although the present investigation showed that the terminal area around a given GI vessel was small, different degrees of vascular innervation were observed, from one small vascular region receiving sympathetic contact to several distant arterial branches having connections from a single axon. There could be different populations of sympathetic vasoconstrictor neurons, based on cellular morphology and neurochemical content, which innervate specific regions of the vascular bed in the wall of the GI tract, like that observed in other tissues (e.g. cutaneous; Gibbins et al., 1996).
Evidence has shown that, as they enter a particular area within the GI walls, the sympathetic nerves will follow a given mesenteric arterial branch with both the axons and the branching artery coexisting within the same confined segment of the ENS
(Furness and Costa, 1974). Once inside the ENS, the conventional view has been that fibers projecting to the myenteric plexus arise from a separate population of neurons than those supplying the vasculature (Costa and Furness, 1973; Llewellyn-Smith et al.,
1984), although any connectivity between the two regions has been difficult to distinguish (Furness, 1970a). While the present work observed sympathetic axons around the borders of blood vessels alone, there were also cases that provided evidence for collaterals to the myenteric ganglia and muscle layers. Despite the proposal that the
NA/NPY subclass of neurons have the distinct function of influencing vascular 49 responses, it may be the case that NPY neurons also modulate GI smooth muscle activity (Lundberg et al., 1983; Mawe et al., 1989), which could be due to the collateral branches observed in the current study. It has also been suggested that NPY may not be the only transmitter associated with vasomotor activity (Gibbins and Morris, 1990).
Thus, there may be a subpopulation of neurons that contain an alternate chemical substance, which have processes contacting both the vasculature and surrounding enteric structures.
It was unclear, using the present protocols, if the dextran-labeled fibers contacted a specific phenotype of myenteric neuron. As this is the first observation of axons with collateral branches to both the blood vessels and neighboring plexuses, their functional connectivity is unknown. The short branches that projected away from the vasculature to nearby regions may provide input that synchronizes local smooth muscle activity with the vasoresponse, in order to provide some accommodation that prevents damage, or these branches could produce responses that improve local oxygen utilization.
The blood vessel innervation was not the main focus of the present experiment.
The relatively low number of fibers projecting to the GI vasculature may have been due to the injection protocol, along with the number and location of vascular neurons within the CSMG. Segregation of neuronal chemical phenotypes has been observed in the CSMG, and the largest number of NPY-positive neurons is located in the posterior- superior part of the complex (Lindh et al., 1986).
50
Bilateral Innervation
Various instances of a single axon projecting bilaterally across both ventral and dorsal halves of the stomach or both hemispheres of the small intestine were observed.
In line with previous reports, an occasional axon was observed to extend from the mesenteric attachment beyond the point of the anti-mesentery into the other hemisphere of the small intestine (Tan et al., 2010; Tassicker et al., 1999). Also, as the main nerve bundle projected along its perivascular route at the border of the GI tissue, there appeared to be collateral branches extending bilaterally into the ENS with parallel pathways.
Limitations
Although the current injection technique is well suited for studying the CSMG innervation of the GI tract, several possible limitations of the protocol should be pointed out. First, while it has been suggested that the left CSMG has a greater number of neurons projecting to the GI tract in comparison to the right (Miolan and Niel,
1996), there could be morphological differences between the left and right CSMG. So, due to the unilateral injections of the dextran biotin, the present characterization of sympathetic axons might not have captured every morphological variant that celiac/superior mesenteric neurons produce.
Second, although dextran tracers are incorporated at the cell body (Reiner et al.,
2000; Raju and Smith, 2006), and do not cross the cell membrane, it is possible that the tracer could enter fibers of passage if they have been damaged (Wouterlood and
Jorritsma-Byham, 1993). If the surgical procedure happened to damage any fibers that pass through the CSMG, then it is possible that such fibers could have incorporated 51 dextran biotin and transported it into the GI tract. One such route would be intestinofugal neurons in the myenteric plexus that project to the sympathetic prevertebral ganglia (Furness et al., 2000; Szurszewski et al., 2002). However, the majority of intestinofugal neurons reside in the colon (Szurszewski et al., 2002), and when the tissue was thoroughly scanned there was no evidence of myenteric neuronal labeling in the small intestine or stomach.
Another possibility is that spinal afferent fibers coursing through the CSMG could have been inadvertently damaged and labeled by the tracer injections (Gibbins,
1995; Lee et al., 1987; Ma and Szurszewski, 1996). While different functional classes of afferent neurons have been identified, the exact location and morphology within the
GI tract have only been recently described for one category, referred to as intramuscular nerve ending (Kyloh and Spencer, 2014). These intramuscular nerve endings have some similarities to the appearance of parallel postganglionic fibers described here and previously defined as noradrenergic circular muscle fibers. The complex morphology of the CSMG terminal arbors we observed within the layers of the muscularis externa suggests that sympathetic postganglionic nerves were identified in the present study. However, it cannot be completely ruled out that there could be a small percentage of the currently identified smooth muscle innervation that arises from dorsal root neurites and their endings. Most ambiguous in this regard would probably be six cases (4% of the pool of 154 axons) that did not project to the myenteric plexus and that exclusively innervated the muscle. This group of six were somewhat morphologically similar to what would be expected for a terminal originating from the dorsal root ganglia (Kyloh and Spencer, 2014), although they did differ from recently 52 described visceral afferent terminal morphology (Kyloh and Spencer, 2014), due to the postganglionic axons having narrow bundles of collateral branches and more extensive circumferential length.
Besides the limitations described above, the overall length, terminal field size, and ganglia counts of some sympathetic projections may have been underestimated in the present study. This occurred in some cases, which had: 1) the visibility of a portion of the axon and/or ganglia obstructed by artifact; 2) the tracing discontinued due to a broken arbor; 3) a diminished staining intensity on some branches; 4) a slightly misaligned cut along the mesenteric attachment and dorsal-ventral halves of the stomach, during tissue preparation, causing a single axon to be severed into shortened neurites. The first-pass survey to identify potential specimens attempted to reduce these complications by having a set of criteria for an axon to be inventoried for tracing, but some axons with minimal infractions were allowed. With the present sample size these limitations may have been overcome by the overall average of those pathways that were represented by a more complete and unobstructed axon.
While the main qualitative features were identified and morphometric analyses were then performed within the smooth muscle layers of laminar whole mounts, the submucosa and mucosa were dissected from the smooth muscle and discarded. It is possible that the axons observed in the present investigation potentially could have elaborated branches that continued toward regions within the submucosa and mucosa
(Gabella, 2012). This would mean some of the branches that were considered to produce a terminal ending may in fact be a fiber passing through the circular muscle. 53
Thus, the total number of terminal branches would have been slightly inflated in the present investigation.
Still another limitation of the present analysis is that the survey preserved only the smooth muscle wall of the gut and adopted the working assumptions that all labeled axons innervated that wall and that the fibers controlled smooth muscle. Such assumptions do not take into account the fact that, as just mentioned, some branches of some of the arbors could have continued on into the submucosa and mucosa (both excluded in the whole mount preparation). Furthermore, the assumptions of the experiment do not incorporate the possibility that some fibers that innervated the primary myenteric plexus or the secondary and tertiary plexuses might be organized, perhaps indirectly, to innervate immune, vascular, and/or secretory effector tissues.
Nevertheless though, any ambiguities of the particular effector tissues innervated notwithstanding, the present experimental observations would suggest that a preponderance of the CSMG sympathetic neuronal arbors directly, without an obligatory myenteric plexus relay, innervate the smooth muscle sheets and (possibly other) tissues in the gut wall.
Another possible limitation when considering the pattern of innervation was that the morphometric analyses did not take into account the changes in smooth muscle thickness and the axon’s movements between the different planes. The regions of GI tissue with a thicker muscle layer could have longer axons, depending on the amount of wandering between the different planes along the course of an arbor’s trajectory. Also, if a thicker smooth muscle receives greater innervation, then this could potentially have 54 an effect on the level of direct versus indirect sympathetic modulation and the number of branches in the different plexuses.
Perspectives
The present investigation shows the utility of neuronal reconstruction to investigate the sympathetic postganglionic projections in the healthy alimentary canal.
On the other hand, as in the CNS, the technique applied here also has potential usefulness for the study of aging and neuropathology. With more specific details on sympathetic terminal patterns, future research on aging and disease could identify aberrant anatomy that could be specific to a particular target site or category of sympathetic postganglionic neuron, thus illuminating the mechanism(s) of the associated functional deficit(s).
Synuclienopathies are related to a number of nervous system complications and disease progressions (Wakabayashi et al., 2010). The protein alpha-synuclein has been observed in its normal state throughout the GI tract, in relation to some intrinsic sources, as well as extrinsic efferent pathways (Phillips et al., 2008). On the other hand, pathological alpha-synuclein has been observed as aggregates within dystrophic axons of various phenotypes including TH (Phillips et al., 2009). Tyrosine hydroxylase-positive fibers have previously been observed to deteriorate in the aging
GI tract (Baker and Santer, 1988), and to form easily identifiable swollen axons that could be seen around myenteric neurons and in the smooth muscle (Phillips et al.,
2006). Since abnormal alpha-synuclein is apparent in multiple sources further research involving the present techniques may elucidate a sympathetic axonal category or target 55 site susceptible to alpha-synuclein aggregation and possible disease development in aging.
Diabetes is complicated by gastrointestinal symptoms of diarrhea, periods of constipation, and reduced peristaltic time, various dysfunctions all of which point to alterations in the functioning of smooth muscle (Feldman and Schiller, 1983). There is a potential link between the present findings that sympathetic postganglionic axons project within the smooth muscle and previous findings that sympathetic activity appears to be compromised in diabetic patients. In fact, despite the sparing of sympathetic prevertebral neurons, the sympathetic arbors appear to be abnormal
(Schmidt et al., 2004; 2008). Also, neuropathology has been shown in the wall of the
GI tract (Lomax et al., 2010), with a possible reduction in TH immunoreactivity (He et al., 2001). Tyrosine hydroxylase-positive fibers have been observed adjacent to interstitial cells of Cajal (ICC) at the junction between the myenteric plexus and circular muscle (Cobine et al., 2010). The ICC are thought to directly activate both the circular and longitudinal muscle fibers (Sanders et al., 2010), and have been found to be affected in diabetes (He et al., 2001; Wang et al., 2009). By incorporating the present methodology, future work could potentially identify sympathetic patterns that are affected in diabetes, whether they are associated with ICC in the circular muscle layer, or another target.
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Appendix A: Tables
Table 1 Distribution of Traced Endings ______Site of Innervation* Region Sample Size Primary Plexus Muscle Heterotypic ______Stomach 22 2 5 15 Duodenum 0-3 cm 33 4 10 19 Duodenum 3-6 cm 29 0 8 21 Mid Jejunum 20 2 3 15 Ileum 50 12 16 22 ______6 154 20 42 92 % 13 27 60 ______
Note. The total number of tracings in the stomach and small intestinal tissues are provided in the column labeled sample size. These overall sample sizes were then broken down in the columns designating each of the observed sites of innervation. Three patterns of innervation were observed forming collateral branches within the primary plexus, the smooth muscle, or a mix of both tissue sites. The total number of tracings for each site of innervation were acquired by summing the number of traced axons in each of the gastrointestinal regions. Percentages are based on the total number of tracings (N = 154). *Minor collateral branches with less than 15% of their total length contained within a site were not considered.
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Table 2 Heterotypic Endings ______Sites of Innervation Region Sample Size P/CM P/T P/CM/T ______
Stomach 15 2 0 13
Duodenum 0-3 cm 19 1 2 16
Duodenum 3-6 cm 21 3 8 10
Mid Jejunum 15 2 5 8
Ileum 22 5 2 15 ______
6 92 13 17 62
% 14 19 67 ______
Note. The total number of tracings in the stomach and small intestinal tissues are provided in the column labeled sample size, which were taken from the column labeled “Heterotypic,” in
Table 1. These overall sample sizes were then broken down in the columns designating each of the observed sites of innervation for the heterotypic or mixed category of axons. Three patterns of innervation were observed forming collateral branches within the primary plexus and secondary plexus/circular muscle, the primary plexus and tertiary plexus, or all three targeted tissues. The total number of tracings for each site of innervation were acquired by summing the number of traced axons in each of the gastrointestinal regions. Percentages are based on the total number of arbors that produced innervation to the primary plexus and smooth muscle tissues (N = 92). Primary Plexus (P); Secondary Plexus/Circular Muscle (CM);
Tertiary Plexus (T).
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)
2 6 6 6 6 6 m
P * * * * 6.5 x 10 6.5 2.0 x 10 2.0 x 10 0.9 x 10 1.2 x 10 0.8 r r r r r
2.3 2.0 † † * * 3.3 3.3 3.2 3.2 3.1 2.6 1.6 r r r r r
19.83 18.77 er of Highest Field Terminal † † * * 71.3 71.3 59.1 59.1 38.3 14.5 12.2 r r r r r
67.33 77.88 m) Branches Terminal Order Branch Size ( 296.6 296.6 7.6 36.38 180.6 2.9 27.13 † † P
* * * * 1454 783.3 783.3 375.4 250.2 206.3 r r r r r
1787 ( m) Length 4658 2484 1472 † P * * * * 17890 6682 3013 2342 1955 r r r r r
Significant difference comparedSignificant difference stomach to the Significant difference compared toduodenum 0-3 cm Duodenum 36887 Duodenum 19580 Mid- 10263 Ileum 12786 ______± Means SEM ______Stomach 77118 7456 312.9 36.11 30.0 Table 3 Summary General of Morphometric Parameters ______Total Arbor Parent Axon Total Numb * † Region Length ( Region Length 0-3 cm cm 0-3 Jejunum
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Appendix B: Figures
Figure 1. A Neurolucida tracing of a single sympathetic arbor with collateral terminal branches and/or varicose fibers of passage located in the primary plexus, the secondary plexus/circular muscle, and the tertiary plexus. The parent axon (PA) can be seen extending from the right side of the tracing, running in the longitudinal direction.
Figure 2 provides photomicrographs that corresspond to the demarcated areas on the present tracing: A = ganglia; B = tertiary plexus; C = secondary/circular muscle; D = circular muscle. Scale bar = 500 μm. 82
Figure 2. Images were taken from the traced dextran-biotin labeled ending shown in
Figure 1. The circular muscle is oriented from top-to-bottom in all panels. A:
Innervation of a myenteric ganglion by branches of sympathetic axon. A relatively small diameter neurite can be seen branching from the larger diameter parent fiber at the top of the photomicrograph. As the neurite passes through the ganglion numerous collateral varicose branches (arrows) can be observed as they extend a short distance, diffusely meander through the neurons, or encircle a neuron. B: Varicose fiber segments terminating in the tertiary plexus. An interganglionic connective strand can be observed as it courses away from a myenteric ganglion (at the left). Along the trajectory a collateral branch forms into a relatively small internodal tertiary plexus array in which multiple bifurcations produce branch segments and terminal endings with clearly defined varicosities (arrows). C: Varicose fiber segments traversing within the secondary plexus and eventually projecting into the circular muscle coat.
As the interconnective strand extends into the internodal region a relatively compact circularly oriented varicose array traverses through a 5μm thick plane. D: All-in- focus image of varicose fiber segments innervating the circular muscle. A single fiber, entering from the top of the image, branched at the level of the circular muscle into an array with a less strict smooth muscle alignment, as they passed over myenteric ganglia. The dextran-biotin labeled neurites were visualized using DAB (brown) and the myenteric neurons were counterstained with Cuprolinic Blue (blue). Scale bars in
A, C, D = 50 μm; B = 20 μm. 83
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Figure 3. A dextran-biotin labeled sympathetic axon that exclusively innervated the primary myenteric plexus. A: Multiple ganglia within the myenteric plexus were innervated. While varicose axons can be seen in and around the ganglia, portions of the axon can also be seen as merely interconnective strand elements (arrowhead) between the ganglia. B: A Neurolucida tracing of the entire axon from which panels
A and C are taken. The neurite consisted of a long PA (entering at 12 o’clock) that repeatedly branched to innervate numerous ganglia and interconnective strands of the myenteric plexus. C: Varicose fiber branches can be seen around myenteric neurons andatransitory varicose fiber (arrowhead) of passage can be observed coursing through a ganglion. Arrows designate a small branching spur of the arbor running into the secondary plexus/circular muscle sheet. For purposes of categorizing the neuronal arbors in terms of their tissue targets, when such spurs constituted less than 15% of the cumulative length of the sympathetic arbor’s branches, they were not considered a substantial projection. Scale bars in A & C = 63 μm; B = 250 μm. 85
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Figure 4. A dextran-biotin labeled sympathetic axon that innervates the primary plexus, the secondary plexus/circular muscle, and the tertiary plexus. A: The prominent feature of the traced axon, which is shown in its entirety, was the extensive innervation of the tertiary plexus between parallel myenteric ganglia. B: The smooth large diameter parent axon (entering bottom right; arrows) passes through a ganglion and gives off several thin varicose branches that come in close proximity to myenteric neurons before the parent axon takes a 90 degree turn into the tertiary plexus. Upon entering the tertiary plexus, the sympathetic arbor branches become highly varicose in appearance, divide extensively, and run predominately in the longitudinal direction
(left to right) covering the area between two parallel strips of ganglia. C: Higher magnification of the rightmost ganglion in panel B. In the upper portion of the image, tertiary fibers are out of focus (arrowheads), and can be observed passing under a neuron, in the longitudinal direction. Tertiary plexus fiber segments can also be observed with varicosities and terminal endings (arrows) along the border of a myenteric ganglion. Scale bars in A = 250 μm; B = 31 μm; C = 10 μm. 87
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Figure 5. A dextran-biotin labeled sympathetic axon that innervates the primary plexus and the secondary plexus/circular muscle. A: Upon innervating a ganglion, an arbor segment branches extensively giving off numerous collateral varicose processes in the ganglion. While some areas of the ganglion have diffuse innervation other regions of the ganglion appear to receive a higher number of varicose branches. B: At
1000x magnification of one pole of the ganglion illustrates the close association between varicose processes (arrowheads) and the myenteric neurons that they encircle.
C: Varicose innervation of the secondary plexus and circular muscle is shown in focus
(arrowheads) with innervation of a myenteric ganglion by collaterals from the same parent axon shown out of focus (arrows). This displays fiber segments innervating distinct tissue types and the planar difference of their respective locations, 5μm apart, within the whole mount. D: A tracing of the entire ending highlights the morphology of the long rectilinear multi-branching arrays that run within the circular muscle and superficial to the circular muscle within the secondary plexus. E: An all-in-focus image of an axonal array innervating the secondary plexus and the circular muscle with collateral branches produced by the same sympathetic neuron innervating the myenteric ganglion which runs parallel to the circular muscle. Scale bars in A & C =
20 μm; B = 10 μm; D = 125 μm; E = 31 μm. 89
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Figure 6. A: The number of myenteric ganglia innervated by terminal branches was greatest in the proximal gut and decreased in the distal gut (ANOVA; F(4, 122) =
5.363, P = 0.0005). B: The pattern was similar for varicose arbor length (ANOVA;
F(4, 127) = 4.989, P = 0.0009). Asterisks indicate a significant difference (P < 0.05) compared to the stomach * or duodenum 0-3 cm **; Tukey’s HSD. Mean ± S.E.M
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Figure 7. A: In the subset of axons that innervated both the primary plexus and secondary plexus/circular muscle, there was a greater proportion of the overall neurite length within the muscle compared to the ganglia and the interconnective strands
(ANOVA; F(1.521, 18.25) = 27.89, P < 0.0001). B: In the subset of axons that innervated the primary plexus and the tertiary plexus, there was a greater proportion of the overall length within the tertiary plexus compared to the ganglia and the interconnective strands (ANOVA;F(1.733, 27.73) = 3.769, P = 0.0412). C: In the subset of axons that innervated all three sites, there was a similar proportion of the overall length within all sites (ANOVA; F(2.684, 163.7) = 2.745, P = 0.0509).
Asterisks indicate a significant difference compared to the secondary/circular muscle * or compared to the tertiary plexus **; Tukey’s HSD. Mean ± S.E.M. 92
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Figure 8. A: In the subset of axons, which innervated the primary plexus and secondary plexus/circular muscle, there was a trend for a greater proportion of the overall “terminal endings” to innervate the muscle compared to the ganglia and the interconnective strands (ANOVA; F(1.994, 23.93) = 1.901, P = 0.1714). B: In the subset of axons, which innervated the primary plexus and the tertiary plexus, there was a greater proportion of terminals branches within the tertiary plexus compared to the ganglia and the interconnective strands (ANOVA; F(1.327, 21.24) = 11.06, P =
0.0016). C: In the subset of axons, which innervated all three sites, there was a greater proportion of terminals branches within the ganglia and the tertiary plexus compared to the interconnective strands and the secondary plexus/circular muscle
(ANOVA; F(2.629, 160.3) = 14.25, P < 0.0001). Asterisks indicate a significant difference compared to the ganglia * or compared to the tertiary plexus **; Tukey’s
HSD. Mean ± S.E.M. 94
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Figure 9. Sympathetic innervation of the blood vessels was observed in some of the whole mounts. A: Sympathetic axonal branches silhouette the outer perimeter of a blood vessel before dividing and giving off several collateral branches that extend into the neighboring myenteric ganglia. B: A tracing of the entire ending documents how the parent axon (entering from the lower right) bifurcates and the resulting secondary fibers then run parallel in close association to the blood vessel with several collateral branches innervating the nearby myenteric ganglia. C: At higher magnification, the axonal branches in close apposition to the outer wall of a blood vessel are clearly varicose (arrows). D: The innervation of a myenteric ganglion is similarly varicose
(arrows). Scale bars in A = 125 μm; B = 250 μm; C & D = 20 μm. 96
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Figure 10. A tracing of a dextran-biotin labeled sympathetic axon that illustrates a terminal field extending within both the dorsal and ventral portions of the stomach through an interconnecting fiber that projects across the greater curvature.
VITA 98
VITA
Gary C. Walter
Education: Purdue University – West Lafayette, IN (2006–2014) Ph.D. in Integrative Neuroscience- Purdue University Life Sciences (PULSe) - anticipated Advisor: Terry L. Powley Ph.D. Dissertation: Morphological characterization of individual sympathetic postganglionic axons innervating the muscle layers of the gastrointestinal tract of the rat: A complex effector model. Buffalo State College – Buffalo, NY (2002–2006) B.S. in Mathematics and Psychology Advisor: Jean M. DiPirro Ph.D.
Professional Experience: Experimental Procedure, Instrumentation, and Analysis Graduate Research Assistant, Purdue University (West Lafayette, IN) (2006–2014) o Altered amino acid sequences in a calcium channel inserted into bacteria. (lab rotation) o Isolated a protein fraction from rat brains for the study of DJ-1. (lab rotation) o Executed a method for visualizing the vagal efferent innervation of the myenteric plexus. (See publication below: Walter et al., 2009) o Performed the methods to identify alpha synuclein localization within intrinsic and extrinsic elements of the enteric nervous system in juvenile and aged rats. (See publication below: Phillips et al., 2008; 2009) o Examined the sympathetic prevertebral axon morphology within the muscle layers of the gastrointestinal tract via brightfield microscopy and digitally reconstructed axons. (Dissertation) o Familiarized with sectioning tissue on a vibratome and a cryostat.
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Academic and Undergraduate Work Graduate Teaching Assistant, Purdue University (West Lafayette, IN) (2005–2014) o Graded essays reports, quizzes, and exams, and provided office hours for Motivation, Drugs & Behavior, Ethology, and Learning & Behavior; additional duty of lecturing for Introduction to Statistics for Psychology and Introduction to Behavioral Neuroscience; held recitation for Elementary Psychology.
Buffalo State College (Buffalo, NY) (2004–2006) o Researched the effect of a predator scent on neuropeptide and lipid neurotransmitter levels in the catecholaminergic system that mediates fear and anxiety. o Studied the influence of an argument strength and humor in an advertisement on persuasiveness. o Examined the results of social pressure and participant sex on pain threshold.
Publications: o Powley TL, Walter GC, Phillips RJ. Vagal afferent and efferent endings interact in the myenteric plexus. (Manuscript in process) o Phillips RJ, Walter GC, Powley TL. Age-related changes in vagal afferents innervating the gastrointestinal tract. Auton Neurosci. 2010 Feb 16;153(1-2):90-8. o Phillips RJ, Walter GC, Ringer BE, Higgs KM, Powley TL. Alpha-synuclein immunopositive aggregates in the myenteric plexus of the aging Fischer 344 rat. Exp Neurol. 2009 Nov;220(1):109-19. o Walter GC, Phillips RJ, Baronowsky EA, Powley TL. Versatile, high-resolution anterograde labeling of vagal efferent projections with dextran amines. J Neurosci Methods. 2009 Mar 30;178(1):1-9. o Phillips RJ, Walter GC, Wilder SL, Baronowsky EA, Powley TL. Alpha-synuclein- immunopositive myenteric neurons and vagal preganglionic terminals: autonomic pathway implicated in Parkinson's disease? Neuroscience. 2008 May 15;153(3):733- 50.
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Awards: o Purdue University Graduate School Summer Research Grant (Summer 2013) o NIMH Summer Fellowship Program in Applied Life Span Developmental Psychology (Summer 2005) o Research Foundation at Buffalo State College Summer Research Grant (Summer 2004)
Professional Affiliation: o Ronald E. McNair Post Baccalaureate Scholar Program (5/04- 5/06)