Molecular Anatomy of the Gut-Brain Axis Revealed with Transgenic Technologies: Implications in Metabolic Research

REVIEW ARTICLE published: 31 July 2013 doi: 10.3389/fnins.2013.00134 Molecular anatomy of the gut-brain axis revealed with transgenic technologies: implications in metabolic research

Swalpa Udit and Laurent Gautron*

Division of Hypothalamic Research, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA

Edited by: Neurons residing in the gut-brain axis remain understudied despite their important role Kevin W. Williams, The University of in coordinating metabolic functions. This lack of knowledge is observed, in part, because Texas Southwestern Medical labeling gut-brain axis neurons and their connections using conventional neuroanatomical Center, USA methods is inherently challenging. This article summarizes genetic approaches that enable Reviewed by: Amanda J. Page, Hanson Institute, the labeling of distinct populations of gut-brain axis neurons in living laboratory rodents. Australia In particular, we review the respective strengths and limitations of currently available Julie A. Chowen, Hospital Infantil genetic and viral approaches that permit the marking of gut-brain axis neurons without Universitario Niño Jesús, Spain the need for antibodies or conventional neurotropic tracers. Finally, we discuss how these Gary Schwartz, Albert Einstein College of Medicine of Yeshiva methodological advances are progressively transforming the study of the healthy and University, USA diseased gut-brain axis in the context of its role in chronic metabolic diseases, including *Correspondence: diabetes and obesity. Laurent Gautron, Division of Hypothalamic Research, Keywords: vagus nerve, mouse models, autonomic nervous system, morphology, obesity Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Y6.220C - Dallas, TX 75390-9077,USA e-mail: laurent.gautron@ utsouthwestern.edu

“These nerves [to the bowels] are but small, because the parts serving viscera. Figure 1 provides a simplified overview of the anatomy of for nutrition, needed none but little nerves, for the performance of the mammalian gut-brain axis and its major components. While the third duty of the nerves, which is in the discerning and knowing the general organization of the gut-brain axis appears relatively ... of what is troublesome to them [ ]. We have this benefit by this simple compared to that of the CNS, the neurochemical, anatom- sense, that as soon as anything troubles and vellicates the bowels, we ical and functional relationships between different populations being admonished thereof may look for help in time” of gut-brain axis neurons can be highly complex (Anlauf et al., –Ambroise Paré, circa 1579. 2003; Travagli et al., 2003; Powley et al., 2005; Lomax et al., 2006; Bertrand, 2009; Brierley, 2010; Fox, 2013). Notably, the anatom- OVERVIEW OF THE ANATOMICAL STUDY OF THE GUT-BRAIN ical study of gut-brain axis has a remarkably long history. For AXIS instance, the vagus nerve was already known to Galen (circa A.D. The gut-brain axis comprises a network of autonomic neurons 130–200) (Ackerknecht, 1974). Furthermore, Renaissance physi- that connect the central nervous system (CNS)—specifically, the cianswereawareoftheimportanceofthenervesupplytothe caudal brainstem and spinal cord—to the esophagus, gastroin- gut in discerning, as put by Ambroise Paré (Paré, 1968), what testinal tract, liver, and pancreas (Loewy and Spyer, 1990; Janig, is troublesome to the bowels. However, the detailed anatomy of 1996; Powley, 2000; Gibbins et al., 2003; Furness, 2006). The the gut-brain axis remained inaccessible to biologists for a long axons of these neurons travel through the vagus, splanchnic, time because the nerves immediately cease to be distinguishable mesenteric and pelvic spinal nerves to innervate the abdominal as they penetrate into peripheral organs. Thus, it was not until the late nineteenth century that postganglionic neurons located Abbreviations: AP, area postrema; BAC, bacterial artificial chromosome; β-gal, in the gastrointestinal wall (also known as enteric neurons) were β –galactosidase; CNS, central nervous system; CGRP, calcitonin gene-related discovered by Auerbach and Meissner (Meissner, 1857; Auerbach, peptide; ChAT, choline acetyltransferase; CMV, cytomegalovirus; Cre, Cre- recombinase; DBH, dopamine β-hydroxylase; DMV, dorsal motor nucleus of the 1863). Enteric neurons, along with postganglionic neurons in vagus; DRG, dorsal root ganglion; eGFP, enhanced GFP; eGFPf, enhanced GFP far- the gallbladder and pancreas, are part of the gut-brain axis, as nesylated; GAD, glutamic acid decarboxylase; GHSR, ghrelin receptor; GFP, green they receive direct input from, and transmit information to, the fluorescent protein; IML, intermediolateral cell column; IRES, internal ribosome entry site; MC4R, melanocortin-4 receptor; mp, myenteric plexus; NTS, nucleus rest of the autonomic nervous system; however, enteric neurons of solitary tract; NG, nodose ganglion; PNS, peripheral nervous system; Phox2b, are also capable of operating independently of the gut-brain axis paired-like homeobox 2b; POMC, proopiomelanocortin; PRS, noradrenergic- (Gershon, 1981; Morris et al., 1985; Mawe et al., 1997; Powley, specific Cis element; preSVG, prevertebral sympathetic ganglion; rAAV, recombi- 2000). nant adeno-associated viruses; RSV, rous sarcoma virus; smp, submucosal plexus; TB, transcription blocker; TH, tyrosine hydroxylase; UTR, untranslated region; X, Using Golgi staining (DeFelipe, 2010), histologists in the vagus nerve. beginning of the twentieth century continuously refined our

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molecular markers that could be used to selectively label different types of autonomic nerve endings in a systematic manner are still lacking. In addition, the staining obtained with antibodies might not always be distributed evenly in a neuron, and the expression levels of many molecular markers can fluctuate due to experimental conditions. Second, studies employing anterograde tracer (brain-to-gut) injections have been essential to our current understanding of the basic morphology and anatomical distribution of gut-brain axis neurons (Berthoud et al., 1991; Fox et al., 2000; Wang and Powley, 2000; Walter et al., 2009; Zagorodnyuk et al., 2010). Needless to say, those previous studies have served as invaluable references for scientists interested in the gut-brain axis and have revealed many different types of specialized neuronal endings with distinct shapes, functions and tissue distributions in the gut. Details of the variety of receptors found in the gut are available in several publications (Fox et al., 2000; Berthoud et al., 2004; Powley et al., 2011). On a more practical level, however, tracer experiments inherently produce variable results and some degree of tissue damage at the site of injection. Furthermore, tracer injections are laborious and are not always compatible with long-term physiological experiments given the relatively short FIGURE 1 | Simplified organization of the gut-brain axis. The main life of conventional neural tracers, and, in the case of peripheral anatomical and functional subdivisions of a mammalian gut-brain axis are represented with neurons belonging to the enteric and extrinsic autonomic ganglia, tracer injections remain technically challenging. systems in different colors. Importantly, the enteric nervous system is Due to the aforementioned limitations, there are still gaps in entirely contained within the gastrointestinal wall. For purposes of our knowledge of the impact of various physiological and patho- simplification, all the nerve branches supplying the gut, pancreatic physiological conditions on gut innervation. A better understand- post-ganglionic neurons and the sacral parasympathetic system are not depicted in this figure. ing of the anatomy and plasticity of the gut-brain axis will help to advance our understanding of autonomic neural circuits and numerous chronic diseases that affect the gut-brain axis, including but not limited to inflammatory bowel diseases, metabolic understanding of the intricate networks of neurons of the syndrome, visceral pain, and eating disorders (Mayer and Collins, gut-brain axis. Despite its indubitable value, Golgi staining 2002; Powley et al., 2005; Faris et al., 2008; Blackshaw et al., 2010; remained capricious. As a result, neuroanatomists have developed Cluny et al., 2012; Larauche et al., 2012; Raybould, 2012). The more reliable ways of interrogating the organization of the gut- primary goal of this article is to review the principles, advantages, brain axis in recent decades. First, a large number of anatomical and limitations of various approaches that permit the genetic studies using the injection of retrograde tracers or neurotropic labeling of specific populations of neurons within the gut-brain viruses into visceral tissues have revealed the connections between axis in temporally and spatially controlled manners. We will focus autonomic nerves and their gastrointestinal targets (Sharkey on components of the vagus and spinal nerves that innervate et al., 1984; Sterner et al., 1985; Altschuler et al., 1989; Rinaman the gut; however, the methods described in this article can also et al., 1999; Rinaman and Schwartz, 2004). Although useful, be applied to other components of the peripheral nervous sys- these newer approaches did not provide a means of labeling tem (PNS). Further information on the mapping of peripheral autonomic peripheral endings. In addition, retrograde tracing nociceptive, somatosensory and somatomotor pathways using techniques (gut-to-brain) using neurotropic viruses or tracers reporter mice can be found elsewhere (Tucker et al., 2001; Nguyen to verify connections between autonomic neurons and periph- et al., 2002; Basbaum and Braz, 2010; Li et al., 2011; Whitney et al., eral tissues can potentially produce false-positive results (Fox and 2011). Although this article focuses on the anatomy of the adult Powley, 1986; Berthoud et al., 2006). Moreover, the interpreta- gut-brain axis (rather than its physiology), the usefulness of trans- tion of data obtained with neurotropic rabies virus (which travels genic tools in studying the metabolic functions of the gut-brain in a multisynaptic manner) is complicated by the fact that the axis will be briefly discussed in the last section. vagus nerve innervates sympathetic and pelvic ganglia (Berthoud and Powley, 1993), which makes it difficult to ascertain the neu- LABELING GUT-BRAIN AXIS NEURONS WITH TRANSGENIC ral routes taken by these viruses. Alternatively, immunolabeling TECHNOLOGIES can be useful in identifying select neuropeptides, neurotrans- SURROGATE REPORTERS mitters, enzymes and receptors found in vagal and/or spinal Principle, advantages, and limitations visceral endings (Furness and Costa, 1980; Green and Dockray, Several mouse models that express fluorescent reporter proteins 1987; Patterson et al., 2002; Chiocchetti et al., 2003; Wang and in subsets of gut-brain axis neurons have been described in the Neuhuber, 2003; Lindsay et al., 2006; Phillips and Powley, 2007; literature (Table 1). These mice generally possess a transgene Mitsui, 2009; Bellier and Kimura, 2011). Unfortunately, specific incorporating the regulatory elements of a gene endogenously

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Table 1 | Non-exhaustive list of the genetic models employed to introduce reporter expression in the rodent gut-brain axis.

Mouse line Construct Reporter(s) Labeled gut-brain Reference(s) or virus name axis neurons

CONSTITUTIVE EXPRESSION OF REPORTER CGRPα-GFP Knockin of eGFPf in Calca locus eGFPf CGRPα DRG neurons McCoy et al., 2012 ChATBAC-eGFP BACwith78and36kbat5and 3 ends of eGFP Cholinergic neurons Tallini et al., 2006 Chat andeGFPinexon2 ChAT-6417-LacZ LacZ driven by 5 fragments of the mouse β-gal Naciff et al., 1999 ChAT gene ChAT-tauGFP BAC with entire Chat gene and tauGFP in tauGFP Grybko et al., 2011 exon3 DBH-lacZ LacZ driven by 5 fragments of the human β-gal Noradrenergic neurons Mercer et al., 1991 DBH gene GAD-EGFP eGFP driven by 1.2 kb of regulatory eGFP Gabaergic NTS neurons Oliva et al., 2000; Bailey elements upstream of mouse Gad1 et al., 2008; Gao et al., 2009 GHSR-eGFP BAC containing eGFP driven by GHSR eGFP Subgroup of Furness et al., 2011, 2012 promoter preganglionic sympathetic neurons MC4R-GFP BAC containing Tau-sGFP inserted in the tau-sGFP Subgroup of NG, DMV Kishi et al., 2003; ATG site of Mc4r and DRG neurons Gautron et al., 2010a, 2012 Peripherin-eGFP BAC with eGFP driven by the entire human eGFP Peripherin-containing McLenachan et al., 2008 hPRPH-1 gene neurons Phox2b-H2BCFP BAC with Histone2B-fused with cerulean cerulean Autonomic neural Corpening et al., 2008 inserted into the ﬁrst exon of Phox2b locus crest-derived cells POMC-EGFP EGFP inserted in exon 2 of murine Pomc eGFP NTS POMC neurons Fan et al., 2004; with 13 kb of 5 and 2 kb of 3 regulatory Appleyard et al., 2005; sequences Padilla et al., 2012 CRE-DEPENDENT EXPRESSION OF REPORTER ChAT-IRES-Cre IRES-Cre inserted in ChAT exon 15 GFP, tdTomato Cholinergic neurons Rossi et al., 2011; Gautron et al., 2013a ChAT-Cre BAC with Cre inserted into the ﬁrst exon of eGFP Cholinergic neurons Gong et al., 2003 ChAT locus

Nav1.8-Cre Cre knocked into Nav 1. 8 locus β-gal, tdTomato Nav1.8-expressing Stirling et al., 2005; afferents Gautron et al., 2011; Shields et al., 2012

Nav1.8-Cre (or SNS-Cre) BAC of Nav1. 8 locus containing Cre β-gal Agarwal et al., 2004 Phox2bcre BAC containing Cre inserted in Phox2b YFP All autonomic neurons D’Autreaux et al., 2011 exon 2 but sympathetic preganglionic Phox2b-Cre BAC containing Cre inserted in Phox2b EGFP DMV and NTS Gong et al., 2003 exon 1 Phox2b-Cre Cre driven by Phox2b with >75 kb at 3 and GFP, β-gal, DMV, NG, subset of Rossi et al., 2011; Scott 5 ends tdTomato NTS and enteric et al., 2011; Gautron neurons et al., 2013b POMC-Cre BAC containing Pomc locus with Cre eGFP, NTS POMC neurons Balthasar et al., 2004; insertedinexon2 tdTomato Padilla et al., 2012 TH-Cre IRES-Cre knocked in 3UTR of TH gene YFP, β-gal Catecholaminergic Lindeberg et al., 2004; neurons Obermayr et al., 2013 VIRALLY DELIVERED REPORTER AAV (serotypes) CMV-driven eGFP eGFP Majority of NG neurons Kollarik et al., 2010 AdRSVlacZ RSV-driven LacZ β-gal Majority of cultured NG Meyrelles et al., 1997 neurons AdCMVlacZ CMV-driven LacZ AdRSVgfp RSV-driven GFP GFP

(Continued)

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Table 1 | Continued

Mouse line Construct Reporter(s) Labeled gut-brain Reference(s) or virus name axis neurons

PRSx8-AlstR-eGFP-LV eGFP driven by Phox2b-activated promoter eGFP DMV neurons at site of Mastitskaya et al., 2012 injection PRSx8-ChIEFtdTomato-AV tdTomato driven by Phox2b-activated tdTomato promoter rAAV8-GFP CMV-driven GFP GFP Subgroup of DRG Storek et al., 2008; neurons innervating the Vulchanova et al., 2010; colon Schuster et al., 2013

For abbreviations and references, see the text. expressed in gut-brain axis neurons and a green fluorescent pro- Review of available tools tein (GFP) gene or one of its variants. Different approaches for The choline acetyltransferase (ChAT) gene encodes the enzyme inserting a transgene into the genome exist, but most of the necessary for the synthesis of acetylcholine, the principal neu- mice discussed in this article were generated either using bacterial rotransmitter of preganglionic parasympathetic and sympathetic artificial chromosome (BAC transgene) or knock-in (inserted in neurons and a large proportion of enteric neurons (Arvidsson the endogenous allele) approaches. Both approaches have advan- et al., 1997). A transgenic mouse with lacZ expression that is tages and issues, which are described in more specialized articles restricted to cholinergic neurons was generated more than a (Gong et al., 2003; Dhaliwal and Lagace, 2011; Heffner et al., decade ago (Naciff et al., 1999). This study also established the 2012; Murray et al., 2012). Nonetheless, it is important to remem- regulatory sequence in the ChAT locusthatisnecessarytodrive ber that the insertion of a transgene into the genome with the specific lacZ expression in cholinergic neurons. Dorsal motor BAC approach is random. Due to this random insertion, trans- nucleus of the vagus (DMV) neurons have been stained in this gene expression does not always perfectly recapitulate that of the mouse, but other gut-brain axis neurons have not been examined. endogenous gene. In contrast, the knock-in approach produces More recently, ChATBAC-eGFP and ChATBAC-tau-GFP animals reporter expression that is under the control of endogenous reg- were generated using a BAC strategy (Tallini et al., 2006; Grybko ulatory sequences, which results faithful expression patterns. In et al., 2011). In both lines, the expression of either enhanced all cases, ectopic transgene expression can occur, and the distri- GFP (eGFP) or tau-GFP is under the control of the ChAT pro- bution of the reporter must be systematically compared to that of moter. Tau is a microtubule-binding protein and, consequently, the endogenous gene of interest. tau-GFP fusion protein is more effectively transported to termi- GFP is a widely used fluorescent reporter that is well- nal processes than GFP alone. As anticipated, the fluorescence transported and allows for the labeling of terminal endings in in these two lines has been reported in all cholinergic neurons both the periphery and CNS. In addition, constitutive reporters including, DMV and enteric neurons. A study by Grybko and closely reflect the endogenous expression of one particular gene colleagues demonstrated bright native GFP fluorescence that per- of interest at any given time and can allow for the labeling of fectly colocalized with ChAT immunoreactivity. Although these cells that are otherwise difficult to detect using conventional animals have not been characterized in depth (e.g., with ectopic anatomical methods (Padilla et al., 2012). GFP reporters, how- expression), they appear to be useful for the study of cholinergic ever, are not without caveats. For instance, GFP is not always gut-brain axis neurons. bright enough to be observed without immunostaining (Liu et al., Melanocortin-4 receptor (MC4R) is an important molecular 2003). Fortunately, antisera against GFP that can be used to stain player in the regulation of feeding, metabolic, and autonomic GFP-containing structures are widely available. The marking of functions (Cone, 2005). Not surprisingly, MC4R is expressed cells with GFP reporters is not permanent, and, consequently, in gut-brain axis neurons including subsets of both motor and these reporters can be useful in studies that aim to examine sensory vagal and spinal neurons (Kishi et al., 2003; Gautron dynamically regulated proteins (via the resulting up- or down- et al., 2010a, 2012). MC4R-GFP mice that express tau-sapphire- regulation of GFP). However, this feature can also be a drawback GFP fusion protein under the control of the MC4R regulatory if the primary goal of the study is fate mapping or the permanent sequences have been generated (Liu et al., 2003)(Figure 2A). labeling of a group of neurons across physiological circumstances. These mice express GFP in subgroups of nodose ganglion (NG) Finally, GFP has been reported to be potentially toxic to brain and DMV neurons, as well as in preganglionic sympathetic neu- cells at high expression levels (Howard et al., 2008). Although rons (Liu et al., 2003; Gautron et al., 2010a), and the GFP is it does not have the versatility of fluorescent reporters, another present only in MC4R-expressing neurons. Interestingly, GFP is reporter protein commonly employed in molecular biology is the transported to the terminal endings of vagal neurons within the β-galactosidase enzyme, which is encoded by the lacZ gene. Cells gastrointestinal tract and, hence, reveal the peripheral targets expressing lacZ can be labeled in straightforward manner with a of MC4R in the hepatic artery, myenteric plexus and intestinal blue dye. mucosa (Gautron et al., 2010a).

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GABAergic interneurons located in the nucleus of solitary tract (NTS) play a critical role in vago-vagal reflexes (Bailey et al., 2008). GAD-EGFP mice express eGFP under the control of the glutamic acid decarboxylase-67 gene (aka Gad1)(Oliva et al., 2000). The vast majority of GFP-positive cells are immunoreactive for GABAergic markers. Many brain sites known to contain GABAergic neurons do not display GFP fluorescence, which sug- gests GFP underexpression. Brainstem slices from the GAD-EGFP mouse have been employed to facilitate the patch-clamping of interneurons in the dorsovagal complex to conduct detailed mea- surements of their electrophysiological properties in response to vagal afferent stimulation (Bailey et al., 2008; Gao et al., 2009). Paired-like homeobox 2b (Phox2b) is a transcription factor that is critically involved in the early differentiation of autonomic and viscerosensory pathways (Brunet and Pattyn, 2002). The neural precursors of many gut-brain axis neurons express Phox2b during development including virtually all parasympathetic, enteric and sympathetic ganglia neurons (Tiveron et al., 1996). One mouse line expressing a fused Histone2B-cerulean protein under the control of Phox2b was generated using a BAC strategy to mark enteric progenitors (Corpening et al., 2008). The reporter strictly localizes to the nucleus, rendering the identification of adjacent neurons straightforward. Expression of this transgene has been demonstrated in Phox2b cells in the CNS, sympathetic chain and enteric system, which agrees with the known distribution of Phox2b. However, it is not clear whether FIGURE 2 | Representative examples of transgenic approaches that vagal neurons express the transgene. Double-labeling with an enable the marking of restricted gut-brain axis neurons in the mouse. antibody against Phox2b further validated the genuine distribu- (A) Vagal sensory neurons in the NG of MC4R-GFP mouse stained with an tion of the transgene in Phox2b cells in the developing and adult anti-GFP antibody and diaminobenzidine. Notably, both the cell bodies and gut. Thus, far, this model has primarily been used to monitor the axons of vagal sensory neurons are clearly labeled. (B,D) Fluorescently labeled terminal fibers in the dorsovagal-complex and duodenum mucosa of migration of enteric progenitors in the developing murine gut aNav1.8-Cre-tdTomato mouse. (C) Whole-mount of the ileal myenteric and to identify adult neural crest derived cells (Corpening et al., plexus of a Phox2b-Cre-tdTomato mouse revealing vagal extrinsic 2008). innervation and isolated innervation of enteric neurons. (E) Whole-mount of Proopiomelanocortin (POMC) is a precursor of several pitu- the gastric myenteric plexus of a ChAT-Cre-tdTomato mouse containing fluorescent preganglionic fibers intermingled with cholinergic enteric itary and hypothalamic peptides that is important in neuroen- neurons that reveals the myenteric plexus. The above images are all docrine and metabolic functions (Liu et al., 1992; Cowley et al., unpublished and were generated in our laboratory using mouse lines 2001). The NTS contains a small population of POMC-expressing described in the article. neurons (Padilla et al., 2012). Unlike other POMC cells, NTS POMC neurons produce little POMC transcript, rendering their detection challenging with conventional neuroanatomical tools. A mouse that expresses eGFP under the ghrelin receptor Fortunately, a mouse expressing eGFP under the control of the (GHSR) promoter was recently characterized (Furness et al., regulatory sequence of the murine Pomc gene has been generated 2011). Simply expressed, ghrelin is a hormone secreted by the and widely used to visualize NTS POMC neurons (Cowley et al., stomach epithelium that promotes hunger and modulates auto- 2001; Fan et al., 2004; Appleyard et al., 2005; Padilla et al., 2012). nomic functions (Nogueiras et al., 2008). Using double-labeling Specifically, electrophysiological studies using POMC-EGFP ani- and retrograde tracer experiments, a subgroup of preganglionic mals have identified NTS POMC neurons as part of a vagovagal sympathetic neurons, including neurons connected to the gut circuitthatisimplicatedinsatiation(Appleyard et al., 2005). (as evidenced by retrograde tracing experiments), has been Other GFP reporter lines that are potentially interesting for identified to contain eGFP (Furness et al., 2012). The projec- the study of gut-brain axis neurons include the peripherin-eGFP tions of these neurons terminating in sympathetic ganglia are (McLenachan et al., 2008)andαCGRP-farnesylated-GFP mice nicely labeled by eGFP immunohistochemistry. These data sug- (McCoy et al., 2012). Peripherin is an intermediate filament that gest that GHSR-eGFP mice are a valid model for identifying is not unique to the gut-brain axis but is widely distributed in GHSR-expressing gut-brain axis neurons. As GHSR mRNA has the PNS (Troy et al., 1990). The peripherin-eGFP mouse shows been reported to be present in NG and DMV neurons (Date fluorescence in many peripheral sensory neurons and enteric et al., 2002; Zigman et al., 2006), further work to determine neurons and their peripheral projections (McLenachan et al., whether vagal neurons are labeled in this reporter model is 2008). It remains unclear whether GFP expression recapitulates warranted. endogenous peripherin gene expression. Calcitonin gene-related

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peptide (CGRP)-α and -β are neuropeptides produced in enteric, nociceptive and viscerosensory neurons (Mulderry et al., 1988). The CGRPα-GFP mouse shows GFP immunoreactivity in many dorsal root ganglion (DRG) neurons and nerve bundles in the intestines but not in enteric neurons (McCoy et al., 2012). While all CGRP-immunoreactive DRG neurons display GFP, approx- imately 30% of GFP neurons are not CGRP immunoreactive, which raises the question of transgene ectopic expression and/or subthreshold expression of CGRP. The dopamine β-hydroxylase (DBH) gene is responsible for the synthesis of noradrenaline in the autonomic nervous system (Elfvin et al., 1993). The human β-hydroxylase gene has previously been employed to drive LacZ expression in the mouse to identify noradrenergic neurons (Mercer et al., 1991). Several lines have been produced, including one that expresses β-gal fused with a nuclear translocation signal. As anticipated, transgene expression has been reported in sympathetic ganglia, cranial parasympathetic ganglia, the enteric nervous system, and in a small portion of neurons in the peripheral sensory ganglia (Mercer et al., 1991). However, the presence of transgene expression in FIGURE 3 | Principle of an experiment using the Cre-LoxP system to cholinergic ganglia may be due to ectopic expression. label specific gut-brain axis neurons in the mouse. Finally, while the development of the enteric nervous system is a field of study that is too vast to be reviewed in the current article, it is noteworthy that a number of transgenic reporters reporter protein. A few examples the utilization of the aforemen- have been created to study the fate mapping and differentiation tioned strategy of labeling gut-brain axis neurons are described of enteric neurons during early development (Hanna et al., 2002; below (see also Table 1). Nonetheless, caution must be taken Young et al., 2004; Deal et al., 2006; Corpening et al., 2008, 2011; when interpreting the distribution of any given Cre-expressing Mundell et al., 2012). To avoid oversimplifying this complex area cell population. Indeed, Cre expression can occur transiently in of research, we deliberately avoid reviewing these models. developing neurons that do not express the transgene of interest in adulthood, which results in the labeling of cells other Cre/LoxP TECHNOLOGY than the intended target neurons (Heffner et al., 2012; Padilla Principle, advantages, and limitations et al., 2012). Thus, off-target and inconsistent Cre activity must Cre/LoxP technology deserves specific attention, as this approach be considered as potential drawbacks when using the Cre-LoxP profoundly changed how neuroanatomy is conceived and per- approach. formed (Dymecki and Kim, 2007; Livet et al., 2007; Madisen et al., 2010; Weissman et al., 2011). Figure 3 describes the principles Review of available tools of the Cre/LoxP technology applied to the labeling of gut-brain Nav1.8 is a tetrodotoxin-resistant sodium voltage-gated chan- axis neurons. Briefly, by transgenically directing the expression nel that is enriched in C-fiber peripheral afferents, including of Cre-recombinase (Cre) to discrete populations of neurons, a significant proportion of spinal nociceptors, and subsets of including gut-brain axis neurons (using BAC or gene targeting), low-threshold mechanoreceptors (Djouhri et al., 2003; Fukuoka itispossibletoinduceandmodulatetheexpressionofatarget et al., 2008; Shields et al., 2012). It has been clearly estab- fluorescent reporter in a Cre-responsive manner. Recently, differ- lished that Nav1.8 is essential to the electrogenesis of noci- ent laboratories have taken advantage of available mouse Cre lines ceptors and, as a corollary, certain pain phenotypes including to specifically manipulate gene expression in autonomic neurons. visceral pain (Laird et al., 2002; Zimmermann et al., 2007). To label neurons, select Cre mice can be systematically crossed More surprisingly, expression of Nav1.8 has been demonstrated with reporter mice that possess a loxP-flanked stop cassette that in a majority of vagal sensory neurons (Stirling et al., 2005; prevents the expression of LacZ or a fluorescent reporter protein. Gautron et al., 2012). Transgenic mice expressing Cre under the In Cre-expressing neurons, however, the transcriptional termina- control of the Nav1.8 promoter have been generated and char- tion sequence is excised allowing reporter production (Madisen acterized (Stirling et al., 2005). In these mice, the translational et al., 2010). As an example, mice that express tdTomato in a start-site of Nav1.8 was substituted with that of Cre recom- Cre-dependent manner are often utilized because tdTomato is binase. Mice carrying one Nav1.8-cre allele show normal pain one of the brightest red fluorescent proteins available (Shaner behavior, and their DRG neurons exhibit normal electrophys- et al., 2004). Thus, native fluorescence can be observed not only iological properties (Stirling et al., 2005). However, mice with in the cell bodies but also the central relays and peripheral two Cre alleles are equivalent to Nav1.8 knock-out mice and, endings, which reveals the full extent of Cre-expressing neuron thus, tetrodotoxin-sensitive currents are absent in these mice. connectivity. One major advantage of this approach is that it Embryonic and adult Cre recombinase expression reported by allows for the permanent labeling of non-replicating cells with a β-galactosidase activity is specific to small diameter neurons in

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the DRG and trigeminal ganglion and many neurons in the AP, NTS, DMV, NG, and the enteric nervous system. Phox2b- NG. Other than a few neurons in the superior cervical ganglion, Cre mice created by the Gensat project display Cre activity in no Cre activity has been observed in peripheral tissues or the the NTS and DMV (Gong et al., 2003). The extent to which CNS. We, and others, crossed Nav1.8-Cre mice with tdTomato this mouse exhibits Cre in peripheral ganglia is unknown. The reporter animals (Gautron et al., 2011; Shields et al., 2012). Phox2b-Cre line generated by the Elmquist group was crossed In the offspring, all Nav1.8-expressing neurons, including many with a GFP or lacZ reporter mouse and proven to express Cre vagal and spinal afferents and their connections within the gut in all NG, DMV, and a subgroup of NTS neurons (Rossi et al., and CNS, are fluorescently marked (Gautron et al., 2011). As 2011; Scott et al., 2011). The latter mouse did not display Cre with other examples, we were able to visualize vagal tension activity in sympathetic neurons and displayed Cre activity in only and mucosal endings in the myenteric plexuses of Nav1.8-Cre- a small subset of enteric neurons, suggesting that, presumably tdTomato mice (Figures 2B,D). Importantly, the numbers and due to positional effects, the expression of Cre underestimates distribution patterns of tdTomato-positive cells in the DRG and that of the endogenous Phox2b gene. Nonetheless, this mouse NG are directly comparable to those of Nav1.8 mRNA, suggest- has proven useful for selectively tagging the vagus nerve. In ing that Cre activity occurs only in Nav1.8-expressing neurons. Phox2b-Cre-tdTomato mice, distinguishing vagal motor vs. sen- Another mouse expressing Cre under the Nav1.8 promoter also sory endings remains difficult in locations in which they are exists (Agarwal et al., 2004). Because this model was generated intermingled (i.e., the myenteric plexus); however, the anatomical with a BAC, it has the advantage of not disrupting the endoge- integrity of the entire vagus nerve can easily be gauged (Gautron nous Nav1.8 gene in contrast to the knock-in approach described et al., 2013b)(Figure 2E). In the latter study, we also showed previously. that Roux-en-Y gastric bypass surgery results in significant vagal ChAT-Cre mice have recently been generated by targeting the denervation of the stomachs of Phox2b-Cre-tdTomato mice. This ChAT gene with IRES-Cre (Rossi et al., 2011). Double-labeling finding is in agreement with observations that suggest signif- experiments have shown that these mice faithfully express Cre icant morphological and electrophysiological changes in vagal in cholinergic neurons both in the CNS and PNS. When crossed neurons following gastrointestinal surgical intervention in the rat with a tdTomato reporter line, the offspring produce tdTomato (Phillips and Powley, 2005; Guijarro et al., 2007; Browning et al., in all cholinergic neurons, which allows for distinct labeling of 2013b). the cell bodies and projections of all preganglionic sympathetic Postganglionic sympathetic neurons express tyrosine hydrox- and parasympathetic neurons and cholinergic enteric neurons ylase (TH), the rate-limiting enzyme in catecholamine biosyn- (Gautron et al., 2013a)(Figure 2C). Importantly, Cre activity thesis (Elfvin et al., 1993). Several mice expressing Cre under appears to be limited to ChAT-immunoreactive cells. Compared the control of the TH promoter have been generated in the past to the ChAT-GFP lines described previously, ChAT-Cre mice (Gelman et al., 2003; Lindeberg et al., 2004; Savitt et al., 2005). offer the advantage of allowing the reporter to be invariably Using a TH-Cre knock-in, catecholaminergic gut-brain axis neu- expressed in ChAT cells regardless of ChAT expression levels. This rons, including sympathetic postganglionic neurons and a small is important because neuronal ChAT mRNA levels may be dif- population of enteric neurons transiently expressing TH during ferentially regulated under different physiological circumstances development, can be marked (Lindeberg et al., 2004; Obermayr (Gibbs, 1996; Castell et al., 2002). Several mouse lines express- et al., 2013). The patterns of Cre activity have been validated ing Cre under the control of ChAT have also been created in the using TH staining, Cre mRNA in situ hybridization and reporter context of the Gensat project (Gong et al., 2003). These mice distributions (β-gal and YFP). appear to show restricted Cre activity in cholinergic neurons Remarkably, numerous reagents expressing Cre under the con- including DMV neurons. Little information is available about the trol of transcription factors implicated in the development of distribution of Cre in other gut-brain axis neurons. neural–crest derived tissues (other than Phox2b) are available A mouse expressing Cre under the control of Pomc regulatory (Druckenbrod and Epstein, 2005; Stine et al., 2009; Mundell et al., elements has been generated with a BAC (Balthasar et al., 2004). 2012). While these models are indispensable for tracking enteric POMC-Cre mice crossed with different inducible reporters have progenitors migrating in the developing gut, as mentioned before, been used in several laboratories to identify POMC hypothala- it is beyond the scope of this article to review the transgenic tools mic and/or NTS neurons (Balthasar et al., 2004; Huo et al., 2006; used in developmental biology. Lastly, new Cre lines are continu- Zheng et al., 2010). A recent study revealed significant discrep- ously being generated and are readily available to investigators. A ancies between cells with POMC-Cre activity and POMC-EGFP few examples include neuropeptide Y-, Transient receptor poten- transgene expression (Padilla et al., 2012). Specifically, the pop- tial vanilloid receptor 1-, TH-, advilin- and peripherin-Cre mice, ulations of NTS cells labeled by the coexpression of both the which have all been demonstrated to induce Cre expression in POMC-EGFP and POMC-Cre overlap minimally, which raises populations of peripheral afferents and/or brain neurons (Zhou again the issue of possible ectopic and off-target expression. et al., 2002; Gelman et al., 2003; Braz and Basbaum, 2009; Mishra Several Phox2b-Cre mice have recently been generated by dif- et al., 2011; Zurborg et al., 2011). While the genes employed ferent laboratories (D’Autreaux et al., 2011; Rossi et al., 2011). to drive Cre expression in the latter transgenics are known to Phox2b-Cre mice from the group of Dr. Brunet were crossed be transcriptionally active in gut-brain axis neurons, insufficient with a YFP reporter mouse to label Phox2b-Cre-expressing neu- information is available to assess their usefulness in targeting the rons (D’Autreaux et al., 2011). As a result, Phox2bCreYFP embryos gut-brain axis. Therefore, we will not include these animals in our show YFP staining in the progenitors of many neurons of the review.

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VIRALLY MEDIATED GENE DELIVERY the guinea pig NG (Kollarik et al., 2010). Stable expression of Principles, advantages, and limitations eGFP was successfully obtained in 50–80% of vagal sensory neu- Stereotaxic injections of viral vectors of varying packaging capac- rons depending on the serotype. The same study indicated that ity, capsid serotype, and cellular tropism are commonly used to the injection of an AAV into vagally innervated tissue is suffi- deliver transgenes that encode fluorescent proteins into the CNS cient to transfect vagal neurons innervating that tissue (i.e., the (Klein et al., 2002; Tenenbaum et al., 2004; Luo et al., 2008; esophagus) and that the eGFP-filled terminal endings of trans- Betley and Sternson, 2011). As illustrated by the examples below, fected vagal sensory neurons can be observed in the periphery viral vectors appear adequate to transfer GFP expression in gut- including the trachea and esophagus. In the case of vagal sensory brain axis neurons; however, few studies have attempted to do neurons, it is also known that a vast majority of primary-cultured this (Table 1). Generally speaking, viral vectors confer long-term dissociated NG neurons can be successfully transfected with ade- gene expression and are deliverable to laboratory rodents, non- noviruses driving either LacZ or GFP expression (Meyrelles et al., human primates and to humans for therapeutic purposes (Kay 1997). et al., 2000; Christine et al., 2009; Bu et al., 2012). While immune responses to viral particles and toxicity are a concern with certain AREAS OF IMPROVEMENT viral vectors (Sawada et al., 2010), recombinant adeno-associated Although the rapidly evolving transgenic technologies described viruses (rAAV) produce little (if any) inflammation (Chamberlin in this article are not meant to replace more conventional and Saper, 1998; Lowenstein and Castro, 2002), and virally deliv- approaches, they offer numerous advantages over classical tracing ered transgenes do not integrate into the host genome (Schnepp and immunohistochemical techniques. In the future, we foresee et al., 2003). To further minimize neuronal injury, using a glass that the approaches described in this article will change many micropipette coupled to an iontophoretic or air-pressure set-up areas of research that require the visualization of visceral affer- for injection is preferable (Chamberlin et al., 1998; Krenzer et al., ents, vagal and enteric neurons, especially for scientists who are 2011). Interestingly, transgenes transferred by viral vectors can not familiar with neuroanatomical techniques. However, trans- be Cre-dependent, which allows specific gene expression that is genic technologies have numerous caveats. First, the paucity of limited to molecularly defined neurons at the site of injection well-characterized mouse lines that permit targeting gut-brain (Lazarus et al., 2007; Gautron et al., 2010b; Harris et al., 2012). neurons greatly limits our ability to manipulate the full range of However, this strategy is inherently limited by the fact that inject- the different types of gut-brain axis neurons described in the liter- ing the NG, DRG, or enteric system is technically challenging. ature. For example, the development of Cre lines that are adequate Finally, the number of transfected neurons at the site of injec- to differentiate vagal stretch (intramuscular arrays) vs. tension tion is always inherently variable, and it is not completely clear receptors (intraganglionic laminar endings) from mucosal end- whether all gut-brain axis neurons are equally sensitive to viral ings would be useful in clarifying the physiological role of each transfection. of the aforementioned vagal endings. It is particularly important considering that subpopulations of vagal afferents may have com- Review of available tools pletely different roles in appetite regulation. Moreover, the ability Mastitskaya and colleagues recently demonstrated the feasibil- to differentiate the non-neuronal cells implicated in the normal ity of delivering a few different transgenes encoding engineered functioning of the gut-brain axis, such as glial cells and intersti- receptors (i.e., allatostatin and channel rhodopsin receptors) into tial cells of Cajal, would be useful (McDougal et al., 2011; Powley the rat DMV using a lentivirus and an adenovirus (Mastitskaya and Phillips, 2011; Gulbransen and Sharkey, 2012). We expect et al., 2012). Of note, both vectors incorporated an artificial that this problem will become smaller in the future as the number Phox2 promoter to ensure the preferential expression of the of strains generated by large-scale Cre-driver projects continues transgenes in the DMV. Each transgene was also coupled with to grow and become available in public repositories (Gong et al., the expression of a specific fluorescent reporter, either eGFP or 2003; Murray et al., 2012). The lack of temporal specificity of the tdTomato. As a result, DMV neurons in brainstem slices could models described in this article is another problem. For exam- be visualized by their respective eGFP and tdTomato fluores- ple, it would be useful to have the ability to restrict Cre to a cence and then patched for electrophysiological recordings. This desired life stage. Among other strategies, this could be achieved study is a good illustration of the usefulness of viral vectors in using mice that express a Cre-estrogen receptor-fused protein. In manipulating gene expression in DMV neurons. There are few these animals, Cre-induced recombination occurs only following studies that have attempted virally mediated gene delivery in the exogenous administration of tamoxifen (Badea et al., 2003), neurons residing in the peripheral ganglia. Intrathecal delivery which restricts Cre activity to a desired temporal stage and facil- of rAAVs carrying a GFP transgene has been performed in rats itates cell lineage studies. Likewise, the tetracycline system is a and mice and resulted in the labeling of DRG neurons (Storek popular technique that is employed to control gene transcription et al., 2008; Vulchanova et al., 2010). Despite the variable transfec- in a reversible and temporally restricted manner (Schonig et al., tion efficiency of this approach, it was recently reported that this 2013). More inducible Cre drivers relevant to the gut-brain axis approach can be used to label subsets of DRG neurons that supply will become available in the future. As mentioned before, ectopic the mouse colon (Schuster et al., 2013). Specifically, GFP-positive and/or inconsistent transgene expressions are issues that need to spinal sensory terminals have been observed in the colonic myen- be addressed when working with transgenic technologies. Lastly, teric plexus and mucosa of injected mice. Thus, far, only one it must be noted that the physiology of the gut-brain axis has been study has directly administered AAVs of different serotypes into extensively studied in guinea pigs and rats instead of mice, and

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these species have not been traditionally targeted for transgenics. several laboratories are currently examining the contributions of However, transgenic rats, including rat Cre-lines, are becoming the different branches of the vagus nerve to the beneficial effects more widely available (Witten et al., 2011; Schonig et al., 2012). of bariatric surgeries (Bueter et al., 2010; Breen et al., 2012; Shin et al., 2012). PERSPECTIVES IN METABOLIC RESEARCH While the above observations taken together link the gut-brain Many investigators acknowledge that identifying the mechanisms axis to diabetes and obesity, we do not yet possess a clear picture and pathways underlying the central integration of visceral infor- of the contributions of the visceral nerves to the pathophysiolo- mation is a true challenge that could provide a better understand- gies of chronic metabolic diseases because vagotomy studies have ing of diseases including obesity (Powley et al., 2005; Berthoud, generated confounding results and remain inherently limited in 2008; Yi and Tschop, 2012). The lack of adequate methods to terms of manipulating specific population of vagal afferents. As interrogate neural pathways linking the gut and the brain has the vagus nerve is a mixed nerve, surgical techniques, in addition largely contributed to the slow progress in this area—especially to being technically challenging, also result in full or partial loss of compared to other areas of sensory biology and somatomotor sys- efferent (motor) function. Pharmacological techniques, including tems. To aid the investigation of these pathways, we and others the administration of capsaicin to destroy small-diameter sen- have begun using and developing new transgenic tools to manip- sory neurons, have been used. However, this method also kills ulate gene expression in the gut-brain axis. In the last section, some neurons in the CNS without killing all vagal sensory neu- we will briefly review what is known about the implications of rons (Ritter and Dinh, 1992; Czaja et al., 2008; Browning et al., gut-brain axis neurons in diabetes and obesity and will make 2013a). Additionally, by 60-days post-capsaicin treatment, neu- the case that transgenic models can be instrumental in decipher- ronal nuclei in the NG of rats are not different from controls ing the role played by the gut-brain axis in chronic metabolic (Czaja et al., 2008), which limits the applicability of this approach diseases. for studying long-term regulation by the sensory vagus nerve. Although hepatic glucose flux is primarily under the direct Furthermore, regeneration/plasticity of some sensory terminals control of insulin (Cardin et al., 2002), numerous studies have after vagotomy has been reported (Phillips et al., 2000). Newly found that stimulating vagal efferents alters peripheral glu- developed genetic tools have emerged to circumvent many of the cose flux and insulin secretion (Shimazu and Fujimoto, 1971; aforementioned problems. First, transgenic reagents can be par- Shimazu, 1971; Rohner-Jeanrenaud et al., 1983; Berthoud et al., ticularly useful in studies seeking to knock-out or “reactivate” 1990; Rozman and Bunc, 2004; Peitl et al., 2005). Moreover, intact genes in autonomic pathways relevant to the regulation of periph- vagal fibers and capsaicin-sensitive afferents are required for the eral glucose flux and insulin secretion. For example, a few recent normal regulation of hepatic and pancreatic functions (Obici studies have employed Cre-LoxP technology to selectively reac- et al., 2001; Pocai et al., 2005; Razavi et al., 2006; Uno et al., 2006; tivate MC4R expression in preganglionic vagal and sympathetic Gram et al., 2007) and the anti-diabetic effects of gastric bypass neurons and demonstrated the key role of this receptor in ame- (Troy et al., 2008). Together, these observations strongly support liorating diabetes in MC4R null mice (Rossi et al., 2011; Zechner the idea that gut-brain axis neurons significantly contribute to et al., 2012). Other studies have focused on examining deficits regulating glucose homeostasis. Furthermore, diabetes is a lead- in neurotrophic factor innervation of the gastrointestinal tracts ing cause of injury to the PNS (Westerman et al., 1989; Drel of knockout mice and the physiological consequences of these et al., 2006), and these injuries may profoundly alter the func- deficits on feeding (Rossi et al., 2003; Fox, 2013). Second, new tioning of gut-brain axis as suggested by the many gastrointestinal neuromodulation techniques have recently been developed that and autonomic symptoms encountered by people with diabetes. allow the genetic targeting of specific neurons with engineered Manipulating the neurons that supply the viscera has also been transmembrane proteins that can exert varied effects on neuronal proposed as a potentially relevant weight-loss strategy on the activity (Aponte et al., 2011; Krashes et al., 2011). Specifically, basis of the known regulatory effect of vagal afferents on satia- optogenetic techniques involve light-sensitive proteins known as tion (Powley et al., 2005). Subdiaphragmatic vagotomy disrupts opsins that alter neuronal activity in response to a blue light. food consumption in rodents (Phillips and Powley, 1998; Powley Pharmacogenetic techniques involve G-coupled proteins recep- et al., 2005) and induces weight-loss in humans (Kral, 1978), tors that only respond to ligands with no other biological activity. but the results of this procedure are difficult to interpret because While these approaches vary in their kinetics and invasiveness, vagotomy also impairs gastrointestinal motility. In lean animals, they both allow the reversible and “remote” control of depolar- most studies agree that the selective surgical, genetic, or capsaicin- ization or hyperpolarization of targeted neurons within intact induced deafferentation of the vagus nerve results in altered meal neural circuits. To the best of our knowledge, only one study has patterns (Chavez et al., 1997; Schwartz et al., 1999; Fox et al., employed these new strategies to modulate the activity of DMV 2001; Chi et al., 2004) without causing frank obesity or long-term neurons (Mastitskaya et al., 2012). While this study aimed to hyperphagia due to compensatory changes in feeding behavior. interrogate the cardiovascular functions of DMV neurons rather In obese animals, however, capsaicin-induced deafferentation of than gut-related functions, it did provide a proof-of-concept the truncal vagus nerve partially prevents diet-induced obesity experiment demonstrating that opto- and pharmacogenetics have (Stearns et al., 2012). Lastly, device–assisted stimulations of the the potential to deeply transform the study of the gut-brain axis. vagus nerve and stomach wall have entered preclinical trials for Finally, Cre-LoxP can be used to ablate selective neurons in the the treatment of obesity (Aronne and Waitman, 2004; Bodenlos CNS or PNS using an inducible diphtheria toxin system (Luquet et al., 2007; Camilleri et al., 2008; Val-Laillet et al., 2010), and et al., 2005; Abrahamsen et al., 2008). In the future, these methods

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couldbeappliedtothegut-brainaxistoperform“molecular to, patch clamp (Bailey et al., 2008; Gao et al., 2009)andflow vagotomies” of specific population of vagal neurons. cytometry sorting (Buehler et al., 2012) of fluorescently tagged The above considerations are meant only to illustrate how neurons. Obviously, research on gut-brain axis neurons is not transgenic tools can be useful to further our understanding and to limited to metabolic diseases but is pertinent to numerous ques- predict the beneficial and deleterious consequences of gut-brain tions relevant to visceral pain, gut flora homeostasis, whole-body axis activity modulation by surgical, pharmacological, or device- inflammation and eating disorders, among other examples (Faris assisted means. As the number of transgenic reagents available et al., 2008; Andersson and Tracey, 2011; Sharkey and Mawe, to gut-brain axis scientists continues to grow and become more 2012). In summary, the rapidly evolving techniques described sophisticated, we predictable that these new tools will play a in this article have become indispensable and empowered both critical role in the advancement of our knowledge of the devel- anatomists and physiologists with unique tools to understand opment, morphological plasticity, molecular phenotyping, and better the gut-brain axis in the context of intact animals. connectivity of the healthy and diseased gut-brain axis. In addition to performing neuroanatomy, one can easily imagine many ACKNOWLEDGMENTS applications for these transgenic tools, including, but not limited I thank Dr. Chen Liu (UTSW) for discussions.

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