Clinical and Basic Neurogastroenterology and Motility

manner.

Section A

Foundations of neurogastroenterology and motility

Chapter 1

Nerves, smooth muscle cells and interstitial cells in the GI tract: Molecular and cellular interactions

Kenton M. Sanders    Department of Physiology and Cell Biology, School of Medicine, University of Nevada, Reno, Reno, NV, United States

Abstract

Multiple cell types generate and organize the movements of the gut muscles required to process nutrients and wastes in the GI tract. Central to motility are smooth muscle cells (SMCs) that generate the forces necessary to propel luminal contents or restrict movements. SMCs are not capable of organ level cooperation without complex regulatory mechanisms. Unrestrained, SMCs are a detriment to propulsive activity because of their intrinsic but cell-to-cell uncoordinated excitability properties. SMCs are coupled electrically to interstitial cells forming a regulatory unit called the SIP syncytium. SIP is an acronym that describes a syncytium of electrical network cells formed by the SMCs, ICC (Interstitial Cells of Cajal) and PDGFR2 + (Platelet-derived growth factor receptor alpha). Interstitial cells generate electrical rhythmicity and synchronize the contractions of SMCs into phasic contractions. Interstitial cells also receive and transduce neural inputs. Superimposed upon the regulation provided by the SIP syncytium, the enteric nervous system senses the volume and composition of luminal contents through intrinsic primary sensory nerves (IPANs) and generates motility patterns at the whole organ level. These effects are further mediated through 5-hydroxy tryptamine (Serotonin), acetyl choline, vasoactive intestinal peptide, nitric oxide and others. Neuropathies of the enteric nervous system are associated with serious motility disorders.

Keywords

Gastrointestinal motility; Smooth muscle cells; Interstitial cells of Cajal; PDGFRα+ cells; SIP syncytium; Enteric nervous system; IPAN; Motor neuron; Reflex

Key Points

●Smooth muscle cells (SMCs) are the power house that generates the forces responsible for gastrointestinal motility.

●SMCs are incapable of organizing tissue level or organ level motor patterns.

●The electrical network formed by the SMCs, interstitial cells of Cajal (ICC) and PDGFR+ interstitial cells, is known as the SIP syncytium.

●The SIP syncytium generates electrical rhythmicity, known as slow waves, and organizes SMC activity into the phasic contractions at the heart of peristalsis and segmentation.

●SIP cells receive and transduce inputs from the enteric motor neurons.

●Neurons of the enteric nervous system (ENS) sense the contents from the gut and organize appropriate motor responses in order to produce organ level motility patterns.

Acknowledgments

The author would like to thank the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) for many years of support from a Program Project Grant (P01DK41315), a MERIT Award (DK40569) and several R01 awards. The author is also very grateful for the many excellent collaborators he has been fortunate to work with over the past 40 years, most notably Professors Sean M. Ward and Sang Don Koh. The author is also grateful to Professor Jean-Pierre Timmermans for providing histological images of porcine enteric neurons and ganglia.

Introduction

Motility of the GI Tract

From the standpoint of motor functions, the gastrointestinal (GI) tract is a complicated series of hollow organs that are highly developed to move, store and process nutrients and waste for the most part in a manner that never rises to the level of conscious awareness. The esophagus moves food following a swallow by inducting a peristaltic contraction from the throat to the stomach and repeats this process with secondary peristaltic movements if the first sweep is not fully effective. Esophageal contents are pushed into the stomach after relaxation of the lower esophageal sphincter that forms a barrier at the junction of the esophagus and stomach.

The stomach is a truly complex organ that in spite of a common chamber is capable of storage of ingested food, reduction in the size of food particles and calibrated emptying of nutrient materials so as not to overwhelm the digestive and absorptive capacity of the small intestine. Relaxation of the proximal stomach begins before and proceeds after food reaches the stomach by receptive relaxation and gastric accommodation. Through these reflexes the volume of the proximal stomach increases to accommodate the mass of food ingested with a minimal increase in gastric pressure. Food is held in the proximal stomach and slowly released into the distal region where powerful antral peristaltic contractions force the mass of undigested food toward the pyloric sphincter. The pylorus closes as each peristaltic wave invades the terminal antrum such that only a minor amount of material is released into the duodenum with each cycle. Closure of the pyloric sphincter upon arrival of the peristaltic contraction creates a high pressure chamber in the terminal antrum and the contents of this chamber are rapidly repelled back toward the body of the stomach. This process causes trituration of the food particles, and together with the strongly acidic conditions and pepsin causes breakdown of larger pieces of food into tiny particles that are ready for digestion. When reduced in size, the solid particles of food are emptied slowly into the duodenum where further digestion and ultimate absorption of nutrients occurs.

Postprandial motility in the small intestine is best described as segmental contractions. In this pattern, segments of small bowel contract forcefully, as segments on either side of the contracting region relax. Then the contracted, segment relaxes and the formerly relaxed segments contract and so on. This alternating pattern is intermixed with very short sweeps of peristaltic movements that cause gradual aboral progression of the contents. The back and forth movements of segmentation are an effective means of stirring and delaying aboral progression of the contents to provide sufficient time for digestion and absorption of nutrients. Other intrinsic patterns of movements observed in the small intestine include the migrating myoelectric (motor) complex (MMC) that occurs during fasting and sweeps through the entire small intestine, long peristaltic responses to some laxative substances and pathogens, and the retroperistalsis that occurs in vomiting.

The colon also has a storage capacity and the capability for aboral and oral progression. A great volume of water and electrolytes are secreted into the GI tract from the mouth through the small intestine to lubricate food for swallowing and to serve as a vehicle for controlling pH and secretion of digestive enzymes. The proximal colon provides a means of recovering the water and electrolytes; so-called ‘the spin-dry cycle’ of the GI tract by Charles Code, one of the great pioneers of motility research. Liquid chyme reaching the colon from the small intestine is stirred and eventually slowly kneaded as it becomes a semi-solid in the proximal colon and then propelled to the rectum by the transverse and distal colon. Filling of the rectum initiates the urge to defecate that is conveyed to the conscious mind for a decision about a choice of a time and place. When the decision is made, intrinsic enteric inhibitory nerves release neurotransmitters such as nitric oxide and vasoactive intestinal peptide and relax the internal anal sphincter, somatic neurons regulating tone of the external sphincter cease firing allowing relaxation of the external anal sphincter, and a mass peristaltic movement through the transverse and distal colon expels the fecal mass.

Many levels of control are required to produce the complex motility patterns necessary to achieve all of the motor functions of the GI tract. This chapter will discuss the types of cells responsible for generating contractile behaviors and regulating contractions to produce and organize the motor activity of normal GI motility.

Myogenic regulation—If movement occurs there must be a motor

Structural features of smooth muscle cells

The SMCs are the motor that generates the forces necessary to propel luminal contents or restrict movement. Like other muscles, GI smooth muscles rely upon the formation of cross-bridges between actin and myosin for the development of force [1]. The dominant myosin in GI muscles is Class II, composed of two heavy chains, making up the head and tail domains (encoded by Myh11), and four light chains (2 MLC20 and 2 MLC17) that incorporate into the heads and necks of the thick filaments. MLC20 (encoded by Mly9 or Mly12b) is known as the regulatory light chain, and MLC17 is called an essential light chain that is likely to serve a structural role. Upon activation of cells, MLC20 is phosphorylated at Ser¹⁹ by myosin light chain kinase (MLCK encoded by Mylk; Fig. 1). MLC20 phosphorylation facilitates binding of the myosin head to actin (cross-bridge formation) and activation of the ATPase constitutive to myosin. The activity of MLCK is regulated by Ca² +/calmodulin binding, such that enhanced cytoplasmic Ca² + leads to activation of MLCK, MLC20 phosphorylation and initiation of contraction. The dependence of muscle contraction upon cytoplasmic Ca² + is described by a intracellular Ca² + vs force relationship. The process of relaxation is dependent upon deactivation of cross-bridge formation, and this is accomplished by dephosphorylation of MLC20 by myosin light chain phosphatase (MLCP; Fig. 1) [2]. MLCP has three subunits, a catalytic subunit (PP1δ isoform), a regulatory subunit (MYPT1—Ppp1r16a), and a small subunit with unknown function (M20). The maximum contractile force and the duration of the response to a given stimulus depends upon the balance between activation of MLCK and MLCP.

Fig. 1 Major myogenic mechanisms in SMCs controlling contractions. Ca ² + required for excitation–contraction coupling enters cells through VDCC or NSCC (receptor-operated channels, ROC, or stretch-activated channels, SAC). Ca ² + entry through VDCC is increased ( circle with + sign ) when cells are depolarized. In some cases, for example stimulation by agonists, this occurs by openings of NSCC and influx of Na + or Ca ² + . VDCC are also negatively regulated by K + channels. Most inhibitory agonists suppress openings of VDCC by activation of K + channels. Ca ² + release from IP 3 receptor-operated Ca ² + channels in the sarcoplasmic reticulum (SR) membrane can supplement Ca ² + entry. Excitatory agonists in many cases lead to stimulation of IP 3 production by G protein-linked activation of PLCβ. When [Ca ² + ] i increases, it binds to calmodulin and these signaling molecules bind to and activate myosin light chain kinase (MLCK). MLCK phosphorylates MLC20 at S19 to activate cross-bridge formation. MLCP dephosphorylates MLC20 causing relaxation. MLCP is regulated by inhibitory factors. When MLCP is inhibited, the degree of MLC20 phosphorylation increases and contractile force increases. This process is known as Ca ² + sensitization because more contractile force is attained at a given level of [Ca ² + ] i (a leftward shift in the [Ca ² + ] i vs force relationship). MLCP is regulated by PKC which phosphorylates CPI-17 and inhibits the PP1c subunit of MLCP. MLCP is also regulated by Rho kinase (ROCK) which phosphorylates a regulatory subunit, MYPT. ROCK is activated by a G protein coupled pathway linked to several receptors. Abbreviations include: [Ca ² + ] i , cytoplasmic Ca ² + ; DAG , diacylglycerol; IP 3 , inositol 1,4,5-trisphosphate; MLC20 , 20kDA light chain of myosin; MLCK , myosin light chain kinase; MLCP , myosin light chain phosphatase; NSCC , nonselective cation channels; PLCβ , phospholipase Cβ; PKC , protein kinase C; VDCC , voltage-dependent Ca ² + channels.

GI smooth muscle express actin (encoded by Acta2) and this is commonly used as a standard for the smooth muscle phenotype. However, SMCs of the GI tract also express another isoform, gamma enteric actin (Actg2) that clearly has important motor functions in GI SMC contractility. Mutations in Actg2 have been found in patients with chronic intestinal pseudo-obstruction (CIPO) or megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) [3]. Other accessory proteins important in the contractile apparatus of GI SMCs include calponin, caldesmon and tropomyosin.

Both thin (actin and calponin) and thick filaments (myosin) are present in SMCs, however their organization differs from skeletal and cardiac muscles. Thin filaments are tethered to the plasma membrane at dense bodies (attached to plasma membrane and rich in α-actinin), and as cross bridges with myosin are formed, the thin filaments are pulled past the thick filaments to shorten the cells and develop force [1]. Other important structures in GI SMCs include the sarcoplasmic reticulum (SR), an intracellular store for Ca² +, and caveolae, which are invaginations of the plasma membrane containing signaling molecules and ion channels. Often caveolae are in close proximity to SR, creating a signaling structure called a plasma membrane-SR junction.

Excitation–contraction (E–C) coupling in GI smooth muscle cells

E–C coupling includes mechanisms activated by a large variety of natural stimuli in GI SMCs. SMC contractions are initiated by Ca² + entry into the cytoplasm. However, Ca² + entry can also be supplemented by Ca² + release from the SR and the eventual contractile response can be modulated by dynamic changes in the sensitivity of the contractile apparatus to Ca² + (Fig. 1) [4]. Bioactive agonists can induce depolarization and activation of Ca² + entry, cause release of Ca² + from stores or alter Ca² + sensitivity.

Electro-mechanical coupling in SMCs and ionic conductances

Because of the regulatory roles of Ca² + and calmodulin in activating MLCK, contractions of SMCs in GI muscles are typically initiated by a rise in cytoplasmic Ca² +. SMCs express a variety of ion channels in their plasma membranes that are activated by depolarization (i.e., voltage-dependent). Controlling the excitability of SMCs is a major task of the regulatory processes that serve to generate GI motility. Take for example an excitability state in which SMCs are spontaneously and tonically active. Such a condition would preclude the ability to organize contractile behaviors such as segmentation or peristalsis in which thousands of SMCs must contract and relax together or in a sequential manner. Setting the excitability of SMCs therefore is critical for developing and maintaining the appropriate level of readiness for higher levels of control to generate the patterns of contraction appropriate for motility.

To set resting membrane potentials, SMCs express K+ channels, which, as in other excitable cells, maintain inside negative membrane potentials that vary from about − 40 mV to − 80 mV depending upon the region of the gut and the species (Fig. 1) [5]. K+ channels contributing to the negative resting membrane potentials of SMCs include nucleotide regulated channels (e.g., KATP), inward rectifiers (KIR), Ca² +-activated K+ channels, two-pore K+ channels and delayed rectifier K+ channels. The relative contributions of each of these conductances (often described as the open probability of the channels) in setting the level of SMC excitability depends upon the numbers of ion channels available in the plasma membrane, the current–voltage characteristics and the regulatory milieu at any given moment in time (e.g., cytoplasmic Ca² + levels, activity of cyclic nucleotide-dependent phosphorylation mechanisms, etc.).

A second important class of ion channels in GI SMCs is voltage-dependent Ca² + channels (VDCC; Fig. 1; [6]), predominantly due to CaV1.2 channels (aka L-type Ca² + channels), but in some cases also due to CaV3 family channels (aka T-type Ca² + channels) [5]. These channels activate when SMCs are depolarized. Depolarization of SMCs can occur through direct effects of bioactive regulatory substances (e.g., neurotransmitters, hormones, paracrine substances, or inflammatory mediators) on SMCs or through the influences of other types of cells that are electrically coupled to SMCs (see SIP syncytium—input from middle management section). Depolarization from actions of bioactive regulatory substances frequently occurs through activation of non-selective cation channels (NSCC) that might allow Na+ or Ca² + entry into the cells [7]. Depolarization of SMCs can lead to the development of Ca² + action potentials that are due to the opening of VDCC. In phasic regions of the GI tract the open probabilities of the Ca² + channels tend to activate at membrane potentials somewhat more positive than the resting potential. Opening of VDCC allows influx of Ca² + into SMCs, and as discussed above, this is the switch that activates contraction. CaV1.2 channels are blocked by dihydropyridines, and drugs of this class, sometimes used clinically to keep coronary arteries relaxed, can have side effects of weakening contractions of GI muscles.

In phasic regions of the GI tract membrane potential oscillates between negative potentials to less negative potentials on an ongoing basis. These events are called electrical slow waves (Fig. 2) [7]. During the negative periods of time, Ca² + entry is minimal because the open probability for VDCC is extremely low. During the depolarized periods at the peaks of the slow waves, the open probability for VDCC increases, Ca² + entry occurs and phasic contractions are evoked [8]. In some regions of the gut slow wave depolarization evokes development of smooth muscle action potentials, due to a rapid increase in VDCC. These events couple to a large increase in cytoplasmic Ca² + and forceful contractions. In tonic regions of the gut membrane potentials are relatively more positive and lie in the range of − 40 to − 50 mV, which is within the range of activation for VDCC. Ca² + leaks into these cells constantly and produces tonic contraction. Cytoplasmic Ca² + must be returned to low levels to accomplish relaxation, and this occurs through active membrane transport or through ion exchange transporters, that either extrude the Ca² + to the extracellular space or reduce cytoplasmic Ca² + by uptake into intracellular storage organelles, such as the SR. Mechanisms are also available for restoration of Na+ and K+ gradients provided by the Na+ K+ ATPase.

Fig. 2 Electrical slow waves recorded from an intracellular impalement of canine antral circular muscle. Slow waves occur spontaneously in antral muscles, but at a lower frequency than shown in this example. These slow waves were paced to approximate the normal rate at which they occur in the intact stomach. Slow waves are composed of a rapid upstroke depolarization, a partial repolarization, a plateau phase that is sustained for several seconds and then repolarization back to the resting potential (which is the most negative potential during the cycle). The open probability of VDCC is increased by the SW depolarization; open probability of VDCC is very low between slow waves. Slow waves are generated in ICC and conduct to SMCs (see Fig. 3). Ca² + entry into SMCs during each slow wave cycle causes development of a phasic contraction. Slow waves propagate from the corpus (site of dominant pacemaker) to the pylorus, providing the mechanism for gastric peristalsis.

Pharmaco-mechanical coupling in SMCs

Some bioactive regulatory substances and exogenous drugs have significant motor effects that occur either in harmony with electro-mechanical coupling or in the absence of major electrophysiological events. These drugs, initiate responses through G protein-coupled receptors or the activation of guanylyl cyclase to either increase or decrease the sensitivity of the contractile apparatus to Ca² +[9]. Shifting the Ca² +/force relationship in a leftward direction leads to greater force development at lower Ca² + concentrations. This type of response leads to enhanced contractions and is known as increased Ca² + sensitivity [10]. Ca² + sensitization is accomplished by parallel pathways in SMCs, mediated by protein kinase C or activation of Rho-associated kinase that phosphorylates the MYPT1 regulatory subunit of MLCP, reducing the activity of the phosphatase (Fig. 1). As discussed in the section on regulation of contractions, reducing the activity of MLCP causes increased myosin phosphorylation and increased contractile force.

SIP syncytium—Input from middle management

In the older literature regulation of motility by mechanisms intrinsic to SMCs was referred to as ‘myogenic’ [11]. Now it is known that SMCs are electrically coupled to at least two additional types of cells, interstitial cells of Cajal (ICC; labeled with antibodies to c-Kit) and cells labeled with antibodies to platelet-derived growth factor receptor α (aka PDGFRα+ cells). The electrical network formed by SMCs, ICC and PDGFRα+ cells is known as the SIP syncytium (Fig. 3) [12]. ICC and PDGFRα+ cells express ionic channels and mechanisms that contribute significantly to the regulation of SMC excitability. Thus, the term ‘myogenic regulation’ has been expanded dramatically to include the regulatory actions of SIP cells.

Fig. 3 Cellular components of the SIP syncytium. SMCs, ICC and PDGFRα + cells are electrically coupled via gap junctions in SMCs, forming a syncytium of cells. This structure provides regulatory control of SMCs and is called the SIP syncytium. Two types of ICC are depicted in the figure. ICC-MY are cells found in the myenteric space between the circular and longitudinal (not show) muscle layers. These cells are branched and form a network. SWs are generated in these cells and propagate cell-to-cell actively within the ICC-MY network, causing propagation of slow waves and coordination of SMCs. SWs conduct to SMCs and are not regenerated by SMCs because these cells lack ionic mechanisms necessary for active propagation of SWs. Depolarization caused by SWs activates voltage-dependent conductances in SMCs, including the VDCC that permits Ca ² + entry and initiates excitation–contraction coupling (see Fig. 1). ICC-IM and PDGFRα+ cells are found wrapped around the processes of enteric motor neurons and these cells are closely associated with varicosities from which neurotransmitters are released. Neurotransmitters regulate Ca² + release in ICC and PDGFRα+ cells. Ca² + transients couple to Ca² + dependent ion channels in these cells. Excitatory neural inputs (ACh and SubP) increase Ca² + transients in ICC and activate Ano1 channels, causing net depolarization of the SIP syncytium. Inhibitory neural input (NO) suppresses Ca² + transients in ICC, causing net hyperpolarization in the SIP syncytium. Inhibitory neural inputs (purines) in PDGFRα+ cells increase Ca² + transients in PDGFRα+ cells, and Ca² + release is coupled to activation of SK channels in these cells. Activation of SK channels causes hyperpolarization of the SIP syncytium.

Structure and function of ICC

ICC are cells of mesenchymal origin that develop during the embryonic period and form networks at the borders of the circular and longitudinal muscle layers [13]. ICC are called ICC-MY when found in the region of the myenteric plexus between the circular and longitudinal muscle layers, ICC-SM at the submucosal border of the circular muscle (ICC-SM) or ICC-SS at the serosal surface of the longitudinal muscle layer [12]. Other types of ICC lie between or within muscle bundles and are known as intramuscular ICC (ICC-IM) or septal ICC (ICC-SEP when lying in the spaces between muscle bundles). Cells that develop into networks are multi-processed and electrically coupled to each other through gap junctions. Network types of ICC (ICC-MY, ICC-SM and ICC-SS) and intramuscular types of ICC (ICC-IM and ICC-SEP) are electrically coupled to SMCs. From an ultrastructural perspective ICC have a granular cytoplasm, an abundance of mitochondria and endoplasmic reticulum, gap junctions with SMCs and many display caveolae.

Pacemaker ICC and mechanism of electrical slow wave

ICC-MY and ICC-SM generate the pacemaker activity that organizes the contractile behaviors of GI SMCs into phasic contractions [12]. Pacemaker activity leads to the development of rhythmic electrical activity known as slow waves. Slow waves originate from negative membrane potentials and are composed of two phases: an upstroke depolarization that repolarizes partially and then development of a plateau phase that can last from one to many seconds depending upon the organ and the species (Fig. 2). Slow waves propagate actively through the networks of ICC and conduct passively into the syncytium of SMCs. SMCs do not have the ion channel apparatus to regenerate or actively propagate slow waves, so electrical coordination between regions of SMCs must occur through the integrity of the ICC networks. Slow waves recorded from ICC have fast upstroke depolarizations and large amplitudes. Slow waves can also be recorded from SMCs due to the electrical coupling with ICC.

Depolarizations of SMCs due to slow waves cause membrane potential to exceed the threshold for activation of CaV1.2 channels and cause either the development of Ca² + action potentials or continuous Ca² + entry during the plateau phase of the slow waves [7]. Basal cytoplasmic Ca² + levels recover between slow waves, so slow waves organize contractile activity into phasic contractions.

Slow waves are generated by two major ionic conductances in ICC [12]. The first conductance is ANO1, a Ca² +-activated Cl− channel. Due to the distribution of Cl− ions across the plasma membranes of ICC, opening of Cl− channels causes inward current and depolarization. ANO1 channels are activated by periodic release of Ca² + from Ca² + stores. Depolarization caused by activation of ANO1 causes activation of CaV3 channels, and this conductance is required for the active propagation of slow waves. Entry of Ca² + through CaV3 channels causes Ca² + release, a process known as Ca² +-induced Ca² + release. Cell-to-cell depolarization, induction of Ca² + release and activation of ANO1 Cl− channels regenerates slow waves throughout the network of ICC. In some cases, slow waves propagate through the ICC network over long distances (as in the stomach), organizing sequential activation of SMCs from the corpus to the pylorus [14]. This type of slow wave propagation causes peristaltic contractions. In other cases, slow waves propagate for a more limited distance due to collisions with slow waves originating at sites more proximal or distal. This limits activation of SMCs into segments, leading to a segmental type of contractile pattern (as in the small intestine).

Neural regulation via ICC

Intramuscular ICC (ICC-IM) also express ANO1 channels and display spontaneous Ca² + release events [12]. These events occur in a stochastic manner, however, and do not develop into slow waves, as in ICC-MY. Release of Ca² + in ICC-IM initiates activation of ANO1 channels, and these currents, activated in thousands of ICC-IM muscle bundles, can cause depolarization of membrane potential, a net excitatory influence on SMCs. Activation of Ca² + release in ICC-IM can enhance the excitatory influence, and turning off Ca² + release produces an inhibitory influence. Intramuscular ICC are found in very close proximity to the varicose processes of enteric motor neurons [15]. Intercellular junctions of only about 20 nm exist between these cells, but junctions of this type are sometimes seen between varicosities and SMCs, as well. Current information suggests that ICC-IM are innervated by motor neurons, and these junctions with enteric motor neurons represent synapse-like structures [16]. In the tiny volumes of these junctions, neurotransmitter concentration can be very high, making them ideal places for neuro-ICC transmission. As discussed in more detail below, excitatory and inhibitory motor neurons exist in the GI tract. Excitatory motor neurons release acetylcholine (ACh) or neurokinins (e.g., substance P) and inhibitory motor neurons release nitric oxide (NO), purines, and neuropeptides, such as vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase-activating polypeptide (PACAP). Imaging Ca² + release events in ICC has suggested that ICC are directly innervated by excitatory neurons and responsive to ACh and Substance P [17]. Similarly ICC-IM are also innervated by nitrergic neurons and Ca² + transients are inhibited by NO [18]. Loss of ICC in mutant animals with defective c-Kit signaling has suggested that an important part of cholinergic and nitrergic neural responses are due to transduction of these neurotransmitters by ICC.

Mechanosensitive responses of ICC

The walls of the GI tract are continuously changing due to loading of organs with food, storage and passage of food, digestion products, and fecal matter and contractile activities. ICC also possess mechanosensitive mechanisms that can affect the frequency of slow wave activity or the responses to enteric neurotransmitters [19]. The range of mechanosensitive mechanisms is poorly understood at the present time, but in the stomach, the chronotropic effects of stretching antral muscles were related to the generation of prostaglandins. Effects may also be stimulated by mechanosensitive nerves organized into reflexes or potentiation or suppression of neural responses.

Structure and function of PDGFRα+ cells

PDGFRα+ cells were originally identified by electron microscopy and referred to as fibroblast-like cells based on ultrastructural features, such as an abundance of rough endoplasmic reticulum [20]. These cells are electrically coupled to SMCs via gap junctions and recognized as more than just fibroblast-like after it was discovered that they express the growth factor receptor PDGFRα [20, 21]. Having this receptor as a positive biomarker for these cells allowed co-labeling of the cells with antibodies against other proteins that were known to be involved in enteric neurotransmission. It was found that PDGFRα+ cells have a high expression of P1Y1 receptors and small-conductance Ca² +-activated K+ (SK3) channels [22]. Isolated PDGFRα+ cells respond to P1Y1 agonists with the activation of SK3 channels.

PDGFRα+ cells in intact muscles generate localized Ca² + transients, as described above in ICC-IM [23]. However, the electrophysiological response of PDGFRα+ cells is opposite to that of the ICC-IM due to the expression of outward current channels (SK3) instead of inward current channels (ANO1). Therefore, PDGFRα+ cells have a net hyperpolarizing or inhibitory effect on the excitability of SMCs. Stimulation of purinergic neurons in GI muscles elicits activation of Ca² + release in PDGFRα+ cells and this occurs in a temporal sequence equivalent to the generation of inhibitory junction potentials in intact muscles. P2Y1 receptors mediate purinergic inhibitory signals in GI muscles, as these responses were lost in transgenic mice with genetic deactivation of P2ry1[24].

SIP syncytium as the basis for myogenic regulation of motility

A major function of the integrated output of the SIP syncytium is regulating the excitability of SMCs, the ultimate determinant of motor output in each organ and region of the GI tract. As discussed above Ca² + release events in ICC generate a net depolarizing or excitatory effect in the SIP syncytium and Ca² + release events PDGFRα+ cells generate a net hyperpolarizing or inhibitory effect [12]. Areas like the colon where there is significant suppression of SMC contraction on an ongoing basis are characterized by the phenomenon of ‘tonic inhibition’. This suppression comes from tonic inhibitory neural input to ICC in the form of suppressing Ca² + release in ICC.

A second feature provided by the integration of behaviors in the SIP syncytium is electrical and contractile pattern generation. All phasic regions of the GI tract have readily distinguishable contractile patterns that form the basis for regional motility [12]. In the stomach electrical slow waves at a relatively slow frequency lead to phasic contractions that develop into a ring and propagate from the proximal corpus to the pyloric sphincter. The rate of propagation and the bandwidth of contracting cells constitute the pattern of motor activity that accomplishes trituration of solids and normal gastric emptying. In the small intestine the contractile pattern changes into a dominantly segmental motif with short peristaltic contractions interspersed. This pattern is optimum for stirring and mixing of chyme to facilitate neutralization of acid, secretion and mixing of digestive enzymes, digestion of macromolecules, solubilization of fats, transport of digestion products across unstirred layers and absorption of nutrients. Here again the patterning of contractions and the restriction of slow wave propagation into segmental rings is accomplished by ICC. Colonic contractions and facilitation of propagating into colonic migrating motor contractions (CMMC) also depend upon the pattering and generation of contractions by ICC.

Defects in the SIP syncytium leading to motility dysfunction

Loss or defects in the SIP syncytium can lead to severely altered motility patterns [18]. SMCs are not necessarily deprived of their contractile functions, as neural inputs, acting directly upon SMCs may convey contractile activity. However, loss or defects in ICC can cause loss of the normal patterns of motor activity. Diabetes can cause loss or at least damage to ICC networks resulting in delayed gastric emptying. Also this may lead to either a disturbance in the pacemaker function of ICC, but more likely to produce breaks in the normal proximal to distal flow of slow wave propagation from the proximal corpus to the pylorus. This might permit the emergence of ectopic pacemaker sites and disturbances in the normal pattern of peristaltic contractions. Lesions in ICC have also been noted in small intestinal pseudo-obstructions and in slow transit constipation. Similar loss of normal slow wave propagation may underlie these motor defects. The susceptibility of ICC to defects in a number of additional motility disorders makes them an important target for future research including how can damage to ICC be avoided, and once defects develop how can ICC networks or its physiological functions be restored.

Neurogenic regulation—Contributions from the executive suite

Another level of regulation of motor functions in the GI tract is provided by the enteric nervous system (ENS). The ENS has multiple functions, including control of motor patterns, regulation of acid secretion in the stomach, regulation of epithelial transport and regulation of mucosal blood flow. Fig. 4 shows anatomical information and images of neurons and enteric glial cells in enteric nervous system. In this section, we will concentrate on the circuitry and reflexes involved in modulating the motor patterns established by the myogenic mechanisms discussed above.

Fig. 4 Structure of the enteric nervous system. (A) Schematic of the small intestine and anatomical organization of the myenteric and submucosal plexus and longitudinal, circular and muscularis mucosae layers of muscle. The deep muscular plexus is a region dense in varicose projections of motor neurons, but no cell bodies. (B) Porcine myenteric ganglia labeled with NADPH diaphorase, a histological stain for neural nitric oxide synthase (nNOS). Note the distribution of nNOS + neurons in most ganglia. (C) Porcine myenteric ganglion labeled by silver impregnation. Dogiel type I (S-type) neurons are encircled by yellow; Dogiel type II (AH-type) are encircled by red; and Dogiel type III are encircled by green in this image. Images from pig are shown because of the morphological diversity found in these animals and in human ganglia (e.g., type III neurons). Most studies have been performed on laboratory rodents that do not display the same level of morphological diversity. Thus, the full chemical coding and functions of type III and other neurons in higher mammals and humans are not fully characterized. (D) Porcine submucous ganglia displayed with immunostaining for galanin. (E) Higher resolution of porcine submucous ganglion with galanin immunolabeling. (F–H) Triple labeling of a human colonic myenteric plexus ganglion with antibodies for Hu (F), choline acetyltransferase (ChAT) (G) and nNOS (H). Note Hu labels most neurons, but specific populations of neurons are labeled by ChAT ( arrows ) and nNOS ( open arrows ) antibodies. Occasional cells were positive for both ChAT and nNOS ( large arrow heads ), but some neurons displayed expression for neither antigen ( small arrow heads ). Scale bar calibration is the same in panels (F – H ) . (I–J) Double labeling of a human colonic myenteric ganglion with antibodies for Hu (I) and S100 (J). Hu labels myenteric neurons ( arrows in I) and S100 labels enteric glia ( arrows in J) that wrap around the neurons and provide structure to the ganglion. Scale bar calibration is the same in panels (I–J). (Images in (B), (D) and (E) were provided by Professor Jean-Pierre Timmermans, and image in (C) was provided by Professor Timmermans and Professor emeritus Werner Stach. (I-J) From Murphy EM, Defontgalland D, Costa M, Brookes SJ, Wattchow DA. Quantification of subclasses of human colonic myenteric neurons by immunoreactivity to Hu, choline acetyltransferase and nitric oxide synthase. Neurogastroenterol Motil 2007;19(2):126–134.)

Structural organization of the ENS

Cell bodies of the ENS are concentrated in two major networks of ganglia, the myenteric plexus and the submucous plexus (Fig. 4A–E) [25]. A smaller plexus of neurons lies along the submucosal surface of the circular muscle layer in the colon and is known as Henle's plexus. Enteric neurons are encased in a mesh of glial cells (Fig. 4I and J). Enteric glia are not excitable cells, but they respond to extrinsic and intrinsic nerve stimulation and to a variety of neurotransmitter substances via Ca² + signaling mechanisms [26, 27]. Glial cells are electrically coupled to each other by gap junctions and thought to modulate GI motility, possibly through interactions with enteric neurons or with other cellular neighbors [28]. Motor regulation comes mainly from neurons of the myenteric plexus, and thus the discussion here is focused upon the organization and functions of neurons with cell bodies in the myenteric plexus.

Development of the ENS

Neurons and glia of the ENS develop as a result of colonization of the gut by vagal and sacral neural crest cells (NC cells). To a major extent colonization occurs in a rostrocaudal manner beginning in the esophagus and progressing toward the colon. Migrating NC cells are developmentally immature and express chemical markers, such as Sox10, RET, Phox2b and p75 [29]. Mesodermal tissues prolong the precursor status and promote cell proliferation by the expression of factors, such as glial derived neurotrophic factor (GDNF), endothelin-3 (ET-3) and BMP2/4, thus sustaining the precursor cell population that is capable of further migration and development [30]. Through RET expression precursor NC cells are attracted and continue their migration in response to local expression of its ligand, GDNF, that displays both mitogenic and chemotactic properties [31]. Cells from the sacral neural crest enter the gut at the posterior end and migrate to meet the vagal neural crest front through a caudorostral progression [32].

Cells behind the migrating front of NC cells develop neural phenotypes and organize into ganglia. Neurons develop prior to the development of glia and may manifest various phenotypes before the mature phenotype is established. Fully differentiated neurons don't migrate, so if migratory influences and proliferative processes are compromised, as occurs in various mutations of the critical factors responsible for these functions, migrating NC cells can fail to colonize the entire colon, leading to an aganglionic segment of the colon, a condition known as Hirschsprung's disease (HD) [33]. Patients with HD have a tightly constricted segment of the colon and a functional obstruction due to the absence of intrinsic neurons that typically impart tonic inhibition on the SIP syncytium. This obstruction blocks passage of fecal material and leads to enlargement of the colon proximal to the obstruction. HD is a classic example of a polygenic disease, and a great number of mutations and combinations of mutations in key genes have been identified as risk factors for the development of this disorder.

Reflex activation of ENS

The ENS in the small and large intestines, and more sparsely in the stomach, contains sensory neurons that are typically called intrinsic primary afferent (sensory) neurons (IPANs). With large cell bodies and Dogiel type II morphology (Fig. 4C) these cells have multiple projections to the mucosa for sensing and transducing information about luminal volume and contents. Terminals of IPANs innervate the lamina propria in the mucosa and come into close contact with enterochromaffin (EC) cells that respond to a variety of stimuli, mechanical, chemical and nutrients, by releasing 5-hydroxytryptamine (5-HT). This compound activates 5HT3 receptors on IPAN terminals, an ionotropic receptor that upon activation causes depolarization of the nerve terminal and evokes action potentials. These events travel antidromically back to the cell bodies in the myenteric plexus to initiate GI reflexes (Fig. 5).

Fig. 5 Arrangement of enteric neurons within myenteric ganglia in intestinal muscles. Intrinsic primary sensory neurons (IPANs) have processes that innervate the lamina propria of the mucosa (not shown) and receive information about luminal contents. IPANs are heavily interconnected forming a sensory network which tends to amplify and distribute sensory input. IPANs synapse with ascending (AIN) and descending (DIN) interneurons to further distribute the sensory information and with longitudinal muscle motor neurons (LMMN) and circular muscle motor neurons to elicit reflex stimulation or inhibition of muscle layers. Activation of IPANs, distribution of information via interneurons and motor responses elicited by motor neurons constitutes the peristaltic reflex. Polarization of this reflex results in activation of ascending interneurons and excitatory motor above a site of mucosal stimulation and descending interneurons and inhibitory motor neurons below a site of mucosal stimulation. (Image is constructed from information provided in Furness JB. The enteric nervous system and neurogastroenterology. Nat Rev Gastroenterol Hepatol 2012;9(5):286–294.)

IPANs have abundant connectivity with each other, forming a reinforcing sensory network (Fig. 5). IPANs also make synaptic connections with ascending and descending interneurons, and with motor neurons that project to the muscle layers [34]. The neural network within the ENS is capable of rapid local amplification of sensory information and determination of responses through direct linkage to motor neurons and distributed linkage to motor neurons through ascending and descending interneurons (Fig. 5). This organization is capable of mediating the stereotypical reflexes important for processing nutrients and wastes in the GI tract. Processes of monopolar neurons with Dogiel Type 1 morphology (Fig. 4C) within the muscle layers also display mechanosensitivity and contribute to reflexes [35].

IPANs are also often called AH neurons because of their electrophysiological characteristics. These cells generate action potentials due to both tetrodotoxin-resistant Na+ and Ca² + channels and intermediate-conductance Ca² +-activated K+ (SK4) channels that are activated during action potentials and cause a slow ‘after-hyperpolarization’ (thus the name AH) following action potentials. The after-hyperpolarization is slow to recover, most likely due to the time required to sequester the Ca² + that enters cells during action potentials, and activation of SK4 channel leaves the neurons refractory and unable to regenerate the action potentials converging on the cell bodies from sensory terminals. Thus, AH neurons have a restricted response to depolarizing stimuli and can only fire a single or a few action potentials before the extended refractory period blocks development of further excitability.

Interneurons run in ascending or descending chains through myenteric ganglia (Fig. 5). These cells are involved in both local motility reflexes and the oral to anal movement of the migrating myoelectric complex [36]. Interneurons provide connectivity between sensory neurons (IPANs) and motor neurons and integrate and distribute sensory information to evoke appropriate motor responses (Fig. 5).

Motor neurons projecting to the muscle layers are either excitatory or inhibitory in nature. Excitatory neurons express the neurotransmitter, acetylcholine (ACh), but these neurons also express the neurokinin, substance P. Excitatory neurons are often labeled with antibodies to either the transporter that sequesters ACh into secretory vesicles (VAChT), choline acetyl transferase (ChAT; Fig. 4F and G) or by antibodies to substance P. Inhibitory neurons release multiple transmitters, including nitric oxide (NO), a purine substance, VIP or PACAP. These neurons are typically labeled for evaluation by antibodies to nitric oxide synthase (NOS1; also known as neural NOS; Fig. 4F and H) or by antibodies to VIP. The identity of the purine neurotransmitter was thought for many years to be adenosine triphosphate (ATP), but more recent evidence suggests that nicotinamide adenine dinucleotide (NAD+) or its metabolite adenosine diphosphate ribose (ADPR) are more likely candidates. Since these molecules are present in all cells, precise labels for the purine neurotransmitter are not yet available.

Motor neuron distribution is polarized. From any given ganglion the major projection of excitatory motor neurons is typically in the oral direction, and projection of inhibitory neurons is typically in the distal direction. This organization means that stimulation of IPANs at a given point in the bowel will produce a contractile response oral to the stimulus and an inhibitory response in the distal direction. This reflex is known as the peristaltic reflex, and it has also become known as the ‘law of the intestine’ [37].

As described above, 5-HT (aka serotonin) is an important mediator that conveys sensory information from EC cells to afferent neurons, contributes to integration within the enteric nervous system, has neuroprotective and trophic influences on neurons and ICC, and imparts a proinflammatory influence in mucosal tissues of the gut. Synthesis, uptake and receptors for 5-HT have been pursued extensively for therapeutic treatments of motility disorders. The gut is the largest source of 5-HT in the body [38]. Most of this mediator is made by mucosal cells by the action of tryptophan hydroxylase 1 (encoded by TPH1), however 5-HT is also synthesized in enteric neurons through the action of TPH2, expressed mainly in myenteric descending interneurons. Stimulation of the neural network between myenteric ganglia causes fast and slow excitatory synaptic potentials, most of which are mediated by cholinergic nicotinic receptors, but some are shown to be mediated by 5-HT3 receptors [39]. Mice lacking Tph1 have aberrant colonic motility patterns [40] Mice lacking Tph2 displayed slower GI transit and reduced myenteric neuronal density, suggesting a role for 5-HT in neuronal development and/or survival [41]. After release of 5-HT, deactivation of these sensory signals is accomplished by uptake into mucosal cells for recycling or degradation. Uptake of 5-HT is accomplished by serotonin-selective reuptake transporter (SERT encoded by SLC6A4). Several serotonin receptors, representing 5 of the known families of these receptors are present in the GI tract (5-HT1, 5-HT2, 5-HT3, 5-HT4, and 5-HT7). It is logical to assume that antagonists for 5-HT3 receptors might be useful for reducing sensory input, and possibly could be effective in reducing motility or pain. For example, antagonists for 5-HT3 reduce emesis and are used to reduce nausea in response to chemotherapy [42]. Agonists for 5-HT4 receptors were found to have prokinetic effects and used for constipation and to accelerate gastric emptying [43]. Varicosities containing 5-HT are closely associated with ICC, and 5-HT has been found to promote the survival of these cells and expansion of neurons in culture conditions. Proinflammatory effects in mucosal tissues are also attributed to 5-HT, and levels of this mediator are increased in inflammatory disorders in humans.

Neural reflexes regulate the tone of sphincters such that the lower esophageal sphincter (LES) relaxes to permit passage of food from the esophagus to the stomach after a swallow, the pyloric sphincter closes shortly after slow waves propagate into the terminal antrum to create high pressures, facilitating trituration of solids and regulation of gastric emptying, the ileocecal sphincter opens to facilitate ileal emptying, and the internal anal sphincter relaxes to facilitate defecation. Reciprocal innervation maintains tone in the LES, at other times to restrict reflux of highly acidic gastric contents into the esophagus, and likewise the internal anal sphincter tone, maintains fecal continence between bowel movements.

Ongoing inhibitory neural input is particularly important in the distal GI tract. SMCs in this region tend to exhibit spontaneous activity that would preclude organization of constructive motor patterns. Therefore, the intrinsic excitability of SMCs must be suppressed, a function provided by tonic low level activation of inhibitory neurons and release of NO. Inhibition of NO synthesis leads to significant activation of contractile activity that summates to tone in the colon. This is the major functional defect in Hirschsprung's disease. Here, loss of intrinsic neurons, particularly loss of inhibitory motor neurons, results in loss of tonic inhibition causing tonic contraction of the aganglionic segment and pseudo-obstruction.

While the ENS has powerful influences over the motor activity of the GI tract, its control is superimposed upon the myogenic regulation provided by the SIP syncytium. Basic patterning of contractions is a product of the SIP syncytium (slow wave activity and phasic contractions). Input from motor neuron can amplify the amplitude, in some cases affect the frequency of contractions or suppress contractile activity. Neural input also has control over the contractile tone in sphincters. However, neural activity cannot recreate the underlying intrinsic patterns of phasic contractions that underlie GI motility. Defects in myogenic regulation without defined neuropathies are likely to contribute to gastroparesis and slow transit constipation where loss of ICC has been documented.

Connectivity of the ENS with the central nervous system

While the neurons of the ENS are capable of directing normal motility patterns through the GI tract, extrinsic neurons are capable of additional regulatory inputs. Extrinsic regulation is provided by the sympathetic and parasympathetic neurons of the autonomic nervous system. A brief overview of the connections of autonomic neurons with the neurons and muscles of the GI tract and some basic principles are discussed.

Stimulation of sympathetic noradrenergic neurons inhibits gastric contractile activity during periods of vagal neuron activity, but only minor inhibitory responses are noted from activation of noradrenergic neurons in the presence of atropine [44]. Noradrenergic nerve fibers innervating myenteric ganglia were found to produce presynaptic inhibition of cholinergic neurotransmission [45]. These and other studies suggested that norepinephrine causes prejunctional inhibition of ACh release by inhibiting the firing of motor neurons. This inhibitory effect is a major mechanism of sympathetic actions in the GI tract and occurs through binding of norepinephrine (NE) to α2 adrenergic receptors on motor neurons. Direct actions of NE on the SIP syncytium also occur and are mediated either by α or β adrenergic receptors, but the cells mediating these effects have not been clearly identified. Sympathetic input to sphincter muscles leads to contraction in most animals and sphincters studied. These effects are mediated by α adrenergic receptors, however β receptors may also be present and inhibitory effects due to these receptors can in some cases be unmasked by blockade of α receptors. Sympathetic neurons also participate in intestino-intestinal reflexes in which stimuli in the gut, such as distension, evoke reflex responses via synaptic contacts with efferent sympathetic, post-ganglionic neurons in the pre-vertebral ganglia. The afferent arm of this reflex is provided by intestinofugal neurons [46]. IPANs may directly or indirectly activate intestinofugal neurons to initiate these reflexes. In general, input from sympathetic neurons restricts oral to anal movements of luminal contents by inhibiting the muscular propulsion in organs and constriction of sphincters.

Important regulation occurs via parasympathetic input, particularly from the vagus in the proximal GI tract. A large percentage of gastric enteric neurons are innervated by vagal fibers and many vagal afferent nerve endings are present in muscle layers and in region of the myenteric plexus [47, 48]. Relaxation of the proximal stomach begins before food reaches the stomach (receptive relaxation) and proceeds as the stomach fills with food (accommodation) [49]. Both of these reflexes are blocked if vagal nerves are cut. Gastric accommodation is mediated through vagal activation of enteric nitrergic neurons [50]. Distension of the distal stomach leads to enhanced antral contractions, a response that is mediated primarily through a vagal reflex [51].

Conclusions

This chapter has described the major cellular components that are responsible for GI motility. Much has been learned about the basic mechanisms of SMC contraction and its dependence upon voltage-dependent Ca² + entry to initiate contraction. More investigation is needed on Ca² + sensitization mechanisms, as this pathway, particularly its regulation by cGMP-dependent mechanisms is not sufficiently understood. An important concept is that SMCs have intrinsic excitability and contractile capabilities, but at the tissue level this activity is uncoordinated. When left unrestrained, the smooth muscle component of the tissue is overactive and unable to produce useful movements. The contributions of interstitial cells and the importance of the SIP syncytium is a novel aspect in GI motility research. These cells provide pacemaker activity (ICC) that organizes SMC activity into phasic contractions, which are the basis for peristaltic contractions and segmentation. Interstitial cells are also important for transduction of neural inputs, and they mediate cholinergic and nitrergic (ICC) and purinergic (PDGFRα+ cells) regulation. Lesions in interstitial cells have been reported in many GI motility disorders. More investigation is needed to understand the factors responsible for damage to interstitial cells and how these cells or their responses might be recovered. The ENS is extremely important in generating organ-level motility behaviors, but it should be recognized that regulation by the ENS is superimposed upon the behaviors of the SIP syncytium. Thus, if the excitability of SMCs is not set correctly by changes in the SIP syncytium (e.g., loss of the excitatory influences of ICC or the inhibitory influences of PDGFRα+ cells) normal functions of the ENS and activation of reflexes may be incapable of producing normal motility. Because of the important role of interstitial cells in mediating neural responses, damage to these cells may appear to be a problem related to the ENS. Basic investigations and clinical assessments need to delineate between problems due to ENS failure vs. defects in the cells that transduce neural inputs. Several drugs have powerful influences over the functions of the ENS and influences on GI motility, particularly drugs specific for 5-HT receptors. However, to date it has been difficult to use these drugs effectively in the treatment of GI motility disorders. Thus, more research to better understand the pharmacology and physiology of the ENS is needed. Several chemical regulatory mechanisms were left undiscussed in this chapter due to space, including hormonal mechanisms, regulation by paracrine substances, effects of inflammatory mediators and effects of recreational drugs such as opiates and cannabinoids.

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Further reading

[52] Murphy E.M., Defontgalland D., Costa M., Brookes S.J., Wattchow D.A. Quantification of subclasses of human colonic myenteric neurons by immunoreactivity to Hu, choline acetyltransferase and nitric oxide synthase. Neurogastroenterol Motil. 2007;19(2):126–134.

Chapter 2

Gut and brain interactions

Anthony C. Johnsona; Tijs Louwiesb; Tian Yuanb; Albert Orockb; Beverley Greenwood-Van Meervelda,b,c    a Oklahoma City VA Health Care System, Oklahoma City, OK, United States

b Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States

c Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States

Abstract

This chapter will discuss key mechanisms that influence the bidirectional communication between the gastrointestinal (GI) system and the central nervous system (CNS). The interaction between the GI tract and the CNS is complex. At the level of the GI tract, intrinsic enteric nervous system signaling regulates GI functions through interactions with the immune system and microbiota. Extrinsic signals modify gut functions while also sending sensory information to the