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Functional alterations in gut contractility after connexin36 ablation and evidence for gap junctions forming electrical synapses between nitrergic enteric neurons
FEBS Letters
Abstract
Neurons in the enteric nervous system utilize numerous neurotransmitters to orchestrate rhythmic gut smooth muscle contractions. We examined whether electrical synapses formed by gap junctions containing connexin36 also contribute to communication between enteric neurons in mouse colon. Spontaneous contractility properties and responses to electrical field stimulation and cholinergic agonist were altered in gut from connexin36 knockout vs. wild-type mice. Immunofluorescence revealed punctate labelling of connexin36 that was localized at appositions between somata of enteric neurons immunopositive for the enzyme nitric oxide synthase. There is indication for a possible functional role of gap junctions between inhibitory nitrergic enteric neurons.
... Within the mouse small intestine, ICC networks, the circular muscle layer as well as the nitrergic nerves (Nagy et al., 2014) are coupled by gap junctions. Within the first 10 min after addition of carbenoxolone (10 µM) the clusters changed from increased gap distance to abolition of the clusters (Figure 7). ...
... Similarly, inhibition of nitric oxide synthesis abolished regular rhythmic contractions in the rat small intestine in vivo Ferens et al., 2005). In the small intestine under control conditions, nitrergic nerves may generate a more or less constant level of nitric oxide, possibly facilitated by their connectivity through gap junctions (Nagy et al., 2014). This control mechanism by nitric oxide appears similar to the orchestration of rhythmicity in small blood vessels which is dependent on a constant production of nitric oxide by endothelial cells (Matchkov, 2010). ...
... Since nitrergic nerves appear to be coupled by gap junctions (Nagy et al., 2014), which might facilitate continuous synchronized release of nitric oxide, we hypothesized that block of gap junction conductance would inhibit the minute rhythm facilitated by neurally derived nitric oxide. Since high concentrations of carbenoxolone were needed to interfere with the clusters and since this coincided with disruption of synchronization of contraction waves within the clusters without first affecting its rhythm, it is more likely that the main effect of carbenoxolone was due to inhibition of gap junction communication between ICC and smooth muscle cells. ...
Nitrergic nerves have been proposed to play a critical role in the orchestration of peristaltic activities throughout the gastrointestinal tract. In the present study, we investigated the role of nitric oxide, using spatiotemporal mapping, in peristaltic activity of the whole ex vivo mouse intestine. We identified a propulsive motor pattern in the form of propagating myogenic contractions, that are clustered by the enteric nervous system into a minute rhythm that is dependent on nitric oxide. The cluster formation was abolished by TTX, lidocaine and nitric oxide synthesis inhibition, whereas the myogenic contractions, occurring at the ICC-MP initiated slow wave frequency, remained undisturbed. Cluster formation, inhibited by block of nitric oxide synthesis, was fully restored in a highly regular rhythmic fashion by a constant level of nitric oxide generated by sodium nitroprusside; but the action of sodium nitroprusside was inhibited by lidocaine indicating that it was relying on neural activity, but not rhythmic nitrergic nerve activity. Hence, distention-induced activity of cholinergic nerves and/or a co-factor within nitrergic nerves such as ATP is also a requirement for the minute rhythm. Cluster formation was dependent on distention but was not evoked by a distention reflex. Block of gap junction conductance by carbenoxolone, dose dependently inhibited, and eventually abolished clusters and contraction waves, likely associated, not with inhibition of nitrergic innervation, but by abolishing ICC network synchronization. An intriguing feature of the clusters was the presence of bands of rhythmic inhibitions at 4–8 cycles/min; these inhibitory patches occurred in the presence of tetrodotoxin or lidocaine and hence were not dependent on nitrergic nerves. We propose that the minute rhythm is generated by nitric oxide-induced rhythmic depolarization of the musculature via ICC-DMP.
... Cx36, a neurotransmitter that is often found in gamma-aminobutyric acid neurons [63][64][65], was shown to localize at nitric oxide synthase (nNOS)containing neuronal cell bodies throughout the GI tract [66]. Abnormal patterns of contractility were detected in the gut after Cx36 knockdown: (1) enhanced amplitude of spontaneous smooth muscle activity in the colon, (2) inability to relax during electric-field stimulation, and (3) increased post-EFS contraction [66]. ...
... Cx36, a neurotransmitter that is often found in gamma-aminobutyric acid neurons [63][64][65], was shown to localize at nitric oxide synthase (nNOS)containing neuronal cell bodies throughout the GI tract [66]. Abnormal patterns of contractility were detected in the gut after Cx36 knockdown: (1) enhanced amplitude of spontaneous smooth muscle activity in the colon, (2) inability to relax during electric-field stimulation, and (3) increased post-EFS contraction [66]. Also, previous experiments have demonstrated that nitric oxide acts as a neurotransmitter in mouse colonic longitudinal muscle [66]. ...
... Abnormal patterns of contractility were detected in the gut after Cx36 knockdown: (1) enhanced amplitude of spontaneous smooth muscle activity in the colon, (2) inability to relax during electric-field stimulation, and (3) increased post-EFS contraction [66]. Also, previous experiments have demonstrated that nitric oxide acts as a neurotransmitter in mouse colonic longitudinal muscle [66]. Elevated colonic contractility in Cx36 knockdown mice associated with enhanced responsiveness to carbachol (cholinergic receptor agonist) was noted [66]. ...
Gap junctions are hexagonal arrays of protein molecules in the plasma membrane and were first described in Mauthner cell synapses of goldfish. They form pathways for coupling between cells, allowing passive, electrotonic spread of ions and also passage of larger molecules such as amino acids and nucleotides. They are expressed in both excitable and non-excitable tissues. Each gap junction is made of two connexons, which are hexameric proteins of the connexin subunit. In this review, the roles that connexins play in gastrointestinal motility, the mechanisms of altered connexin expression leading to inflammatory bowel disease, gastrointestinal infections, and gastrointestinal symptoms in autistic spectrum disorder are discussed in detail.
... In order to test the motility of the intestinal segments we measured contractility using an isolated organ bath (Glasapparatemeister, Topas GmbH, Germany) as performed by others (16). The tissue pieces were ligated at each end with a silk thread and suspended longitudinally between two platinum electrodes by attaching one end to the isometric force transducer (Transducer Typ SG4-45, SWEMA, Sweden) and the other end to the base of the 15 ml chamber filled with Krebs solution maintained at 37 • C and aerated with 95% O 2 /5%CO 2 . ...
... After several medium changes, samples were subjected to EFS using an electric stimulator (Föhr Medical Instruments, Seeheim/Ober Beerbach, Germany). The optimal EFS parameters were determined testing several values for voltage (5, 10, and 15 V) and frequencies (10,16, and 80 Hz) on test samples. We decided to stimulate the ENS from 2-months exposed mice using three different pulse durations (200, 400, and 800 µs) and reduced the number to two different pulse durations (200 and 400 µs) for the 4-months exposed mice. ...
Parkinson's disease (PD) is known to involve the peripheral nervous system (PNS) and the enteric nervous system (ENS). Functional changes in PNS and ENS appear early in the course of the disease and are responsible for some of the non-motor symptoms observed in PD patients like constipation, that can precede the appearance of motor symptoms by years. Here we analyzed the effect of the pesticide rotenone, a mitochondrial Complex I inhibitor, on the function and neuronal composition of the ENS by measuring intestinal contractility in a tissue bath and by analyzing related protein expression. Our results show that rotenone changes the normal physiological response of the intestine to carbachol, dopamine and electric field stimulation (EFS). Changes in the reaction to EFS seem to be related to the reduction in the cholinergic input but also related to the noradrenergic input, as suggested by the non-adrenergic non-cholinergic (NANC) reaction to the EFS in rotenone-exposed mice. The magnitude and direction of these alterations varies between intestinal regions and exposure times and is associated with an early up-regulation of dopaminergic, cholinergic and adrenergic receptors and an irregular reduction in the amount of enteric neurons in rotenone-exposed mice. The early appearance of these alterations, that start occurring before the substantia nigra is affected in this mouse model, suggests that these alterations could be also observed in patients before the onset of motor symptoms and makes them ideal potential candidates to be used as radiological markers for the detection of Parkinson's disease in its early stages.
... This stimulatory current stimulates all neurons within myenteric plexus, including excitatory and inhibitory elements, sympathetic and parasympathetic nerves, and cholinergic and non-cholinergic neurotransmitters (Kilbinger et al. 1982). One study showed that colonic muscle strips from normal mice produced a biphasic response to EFS, consisting of an inhibitory relaxation component and excitatory contractile component upon termination of EFS which were abolished in the presence of tetrodotoxin, demonstrating that EFS response in colon muscle was occurred through neuronal pathway (Nagy et al. 2014). Furthermore, inhibitory neurotransmitters are released during enteric nerve stimulation by EFS (Nagy et al. 2014). ...
... One study showed that colonic muscle strips from normal mice produced a biphasic response to EFS, consisting of an inhibitory relaxation component and excitatory contractile component upon termination of EFS which were abolished in the presence of tetrodotoxin, demonstrating that EFS response in colon muscle was occurred through neuronal pathway (Nagy et al. 2014). Furthermore, inhibitory neurotransmitters are released during enteric nerve stimulation by EFS (Nagy et al. 2014). In addition, nitric oxide contributes to the relaxation of EFS response in proximal and distal colon (Bayer et al. 2003;Ghia et al. 2005). ...
Amitriptyline, a well-known antidepressant, exerts inhibitory effect on electrically stimulated rat colon smooth muscle contraction. In this study, we investigated the signaling pathway of amitriptyline-induced inhibitory effect. Changes in isometric force of colon muscle were recorded on polygraph, and data were analyzed by measuring the inhibitory extent induced by amitriptyline. Firstly, muscles were contracted by stimulation with electric field stimulation (EFS), and then, amitriptyline was added cumulatively to determine its influence effect on EFS. Amitriptyline significantly inhibited EFS-induced contraction dose dependently. Then, the mechanism of inhibitory effect of amitriptyline was evaluated by pretreating with various antagonists such as L-NAME, methylene blue, atropine, 5-HT receptors blockers, guanethidine, prazosin, guanabenz, isoprenaline, Y27632 (Rho-kinase inhibitor), ML9 (myosin light chain kinase (MLCK) inhibitor), U73122 (PLC inhibitor), and chelerythrine (PKC inhibitor). Then, Ca2+ channel blocker (nifedipine) and K+channel blockers, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and glybenclamide, were used to determine the involvement of ion channels. L-NAME, guanabenz, 5HT4 receptor blocker, ML9, and Y27632 enhanced the effect of amitriptyline. Meanwhile, methylene blue, atropine, guanethidine, prazosin, methylsergide, ondansetron, U73122, and chelerythrine blocked its effect. It was also shown that nifedipine enhanced but TEA and glybenclamide blocked amitriptyline-induced inhibitory effect on EFS. Our results indicated that amitriptyline may exert inhibitory effect in response to EFS by inhibiting muscarinic receptors and then PLC-mediated PKC pathway leading to opening of ATP-sensitive potassium channel.
... The existence of such axoaxonic coupling would be expected to yield situations where AISs intersect at points where they harbour Cx36-puncta. In a variety of other systems, Cx36-puncta can readily be visualized at points of contact between appropriately labelled gap-junctionally coupled neuronal elements [6,40]. However, in the many images examined here, we found only a few instances of Cx36-puncta that may be localized at AIS intersections. ...
Electrical synapses formed by gap junctions occur at a variety of neuronal subcellular sites in the mammalian central nervous system (CNS), including at somatic, dendritic and axon terminal compartments. Numerous electrophysiological studies using mice and rats, as well as computer modelling approaches, have predicted the additional occurrence of electrical synapses between axons near their emergence from neuronal somata. Here, we used immunofluorescence methods to search for localization of the neuronal gap junction-forming protein connexin36 (Cx36) along axon initial segments (AISs) labelled for the AIS marker ankyrinG. Immunofluorescent Cx36-puncta were found to be associated with AISs in several CNS regions of mice, including the spinal cord, inferior olive and cerebral cortex. Localization of Cx36-puncta at AISs was confirmed by confocal single scan and 3D imaging, immunofluorescence intensity profiling and high resolution structured illumination microscopy (SIM). AISs measuring up to 30 µm in length displayed typically a single Cx36-punctum and the incidence of these long AISs displaying Cx36-puncta ranged from 3% to 7% in the inferior olive and in various layers of the cerebral cortex. In the inferior olive, the gap junction associated protein zonula occludens-1 (ZO-1) was found to be co-localized with Cx36-puncta on AISs, indicating that these puncta have some of the molecular constituents of gap junctions. Our results add to the neuronal subcellular locations at which Cx36 is deployed, and raise possibilities for its involvement in novel functions in the AIS compartment.
... In order to test the motility of the intestinal segments we measured contractility using an isolated organ bath (Glasapparatemeister, Topas GmbH, Germany) as performed by others [15]. The tissue pieces were ligated at each end with a silk thread and suspended longitudinally between two platinum electrodes by attaching one end to the isometric force transducer (Transducer Typ SG4-45, SWEMA, Sweden) and the other end to the base of the 15 ml chamber filled with Krebs solution maintained at 37°C and aerated with 95% O 2 /5%CO 2 . ...
Parkinson′s disease (PD) is known to involve the peripheral nervous system (PNS) and the enteric nervous system (ENS). Functional changes in PNS and ENS appear early in the course of the disease and are responsible for some of the non-motor symptoms observed in PD patients like constipation, that can precede the appearance of motor symptoms by years. We have shown that environmental toxins can trigger the disease by acting on the ENS and on the autonomic nervous system. Oral exposure to the pesticide rotenone, a mitochondrial Complex I inhibitor, leads to decreased stool depositions in mice. Here we analyzed the effect of rotenone on the function and structure of the ENS by measuring intestinal contractility in a tissue bath and by analyzing related protein expression. Our results show that rotenone changes the normal physiological response of the intestine to carbachol, dopamine and electric field stimulation. The magnitude and direction of these alterations varies between intestinal regions and exposure times and is associated with an early up-regulation of dopaminergic, cholinergic and adrenergic receptors and an irregular reduction in the amount of enteric neurons in rotenone-exposed mice. The early appearance of these alterations makes them ideal candidates to be used as biomarkers for the detection of Parkinson′s disease in its early stages.
... All subtypes of ICC form electrically coupled networks, similarly, the smooth muscle cells in both the longitudinal and circular muscle layer from coupled networks as shown by numerous electrical recordings and calcium imaging (40). The nerves in the enteric nervous system are coupled through synaptic contacts and gap junctions (41). There is no reason to assume that the advances in brain research through the study of its network properties will not be applied to the enteric nervous system. ...
Gastrointestinal smooth muscle research has evolved from studies on muscle strips to spatiotemporal mapping of whole organ motor and electrical activities. Decades of research on single muscle cells and small sections of isolated musculature from animal models has given us the groundwork for interpretation of human in vivo studies. Human gut motility studies have dramatically improved by high-resolution manometry and high-resolution electrophysiology. The details that emerge from spatiotemporal mapping of high-resolution data are now of such quality that hypotheses can be generated as to the physiology (in healthy subjects) and pathophysiology (in patients) of gastrointestinal (dys) motility. Such interpretation demands understanding of the musculature as a super-network of excitable cells (neurons, smooth muscle cells, other accessory cells) and oscillatory cells (the pacemaker interstitial cells of Cajal), for which mathematical modeling becomes essential. The developing deeper understanding of gastrointestinal motility will bring us soon to a level of precision in diagnosis of dysfunction that is far beyond what is currently available.
... Of note, a tyrosine residue at the second to last position can be found in at least 3 other connexins, which are preferentially expressed in the nervous system and probably NO sensitive: Cx33, 45,46 Cx35, 47,48 and Cx36. 49,50 Unfortunately, in none of the studies it was analyzed whether NO affects the phosphorylation of the tyrosine residues similarly as described here. ...
Objective:
Because of its strategic position between endothelial and smooth muscle cells in microvessels, Cx37 (Connexin 37) plays an important role in myoendothelial gap junctional intercellular communication. We have shown before that NO inhibits gap junctional intercellular communication through gap junctions containing Cx37. However, the underlying mechanism is not yet identified.
Approach and results:
Using channel-forming Cx37 mutants exhibiting partial deletions or amino acid exchanges in their C-terminal loops, we now show that the phosphorylation state of a tyrosine residue at position 332 (Y332) in the C terminus of Cx37 controls the gap junction-dependent spread of calcium signals. Mass spectra revealed that NO protects Cx37 from dephosphorylation at Y332 by inhibition of the tyrosine phosphatase SHP-2. Functionally, the inhibition of gap junctional intercellular communication by NO decreased the spread of the calcium signal (induced by mechanical stimulation of individual endothelial cells) from endothelial to smooth muscle cells in intact vessels, while, at the same time, augmenting the calcium signal spreading within the endothelium. Consequently, preincubation of small resistance arteries with exogenous NO enhanced the endothelium-dependent dilator response to acetylcholine in spite of a pharmacological blockade of NO-dependent cGMP formation by the sGC inhibitor 1H-1,2,4Oxadiazolo[4,3-a]quinoxalin-1-one.
Conclusions:
Our results identify a novel mechanism by which NO can increase the efficacy of calcium, rising vasoactive agonists in the microvascular endothelium.
... Spinal cord motor systems, where electrical synapses between interneurons provide synchronization of subthreshold potentials and rhythmic firing that is in phase with ventral root motor output, suggesting a contribution of these synapses to operation of the locomotor central pattern generator [221]; 11. Spinal cord sensory systems, where the wellknown phenomenon of primary afferent presynaptic inhibition was found to be impaired in Cx36 null mice [146], indicating that electrical synapses play an essential role somewhere in the circuitry governing this inhibition; and 12. Gastrointestinal system, where enteric neurons of the myenteric plexus were found to be linked by Cx36-containing gap junctions, and where Cx36 null mice displayed physiological deficits in regulation of gut smooth-muscle contractility [222]. ...
Gap junctions provide the basis for electrical synapses between neurons. Early studies in well-defined circuits in lower vertebrates laid the foundation for understanding various properties conferred by electrical synaptic transmission. Knowledge surrounding electrical synapses in mammalian systems unfolded first with evidence indicating the presence of gap junctions between neurons in various brain regions, but with little appreciation of their functional roles. Beginning at about the turn of this century, new approaches were applied to scrutinize electrical synapses, revealing the prevalence of neuronal gap junctions, the connexin protein composition of many of those junctions, and the myriad diverse neural systems in which they occur in the mammalian CNS. Subsequent progress indicated that electrical synapses constitute key elements in synaptic circuitry, govern the collective activity of ensembles of electrically coupled neurons, and in part orchestrate the synchronized neuronal network activity and rhythmic oscillations that underlie fundamental integrative processes.
... For example, the heart has a four-chambered configuration, which along with rhythmic contraction governed by the intrinsic electrical system, exhibits a strong and regular pumping behavior to achieve blood circulation through the body [23]. In addition, similar electrical signals transmitted from the brain can induce the movement of the musculoskeletal system, such as the peristaltic moves of esophagus or gut that consist of sequential, alternating waves of contraction and relaxation of alimentary wall smooth muscles, which act to propel food along [24][25][26]. The movements can also be directed by other biochemical cues such as nitric oxide that leads to vasodilation and caffeine that causes vasoconstriction. ...
Four-dimensional (4D) bioprinting, encompassing a wide range of disciplines including bioengineering, materials science, chemistry, and computer sciences, is emerging as the next-generation biofabrication technology. By utilizing stimuli-responsive materials and advanced three-dimensional (3D) bioprinting strategies, 4D bioprinting aims to create dynamic 3D patterned biological structures that can transform their shapes or behavior under various stimuli. In this review, we highlight the potential use of various stimuli-responsive materials for 4D printing and their extension into biofabrication. We first discuss the state of the art and limitations associated with current 3D printing modalities and their transition into the inclusion of the additional time dimension. We then suggest the potential use of different stimuli-responsive biomaterials as the bioink that may achieve 4D bioprinting where transformation of fabricated biological constructs can be realized. We finally conclude with future perspectives.
... 26 Gap junctions and connexons serve multiple functions, which are directly correlated to the specific needs of the cell. For example, connexins are widely expressed in the vascular bed, [27][28][29][30][31][32][33] heart, 34-36 lungs, [37][38][39][40] pancreas, [41][42][43][44] liver, [45][46][47] bowel, [48][49][50][51][52][53] bone, 54-58 skin, 59-61 placenta, 62 and central and peripheral nervous systems. [63][64][65][66] In these systems, gap junctions and connexons have been found to mediate signal propagation, the response to mechanical stimuli, coordination of electric activity, cell polarization, control over growth and differentiation, hormone secretion, and many other processes. ...
Connexins are membrane-spanning proteins that allow for the formation of cell-to-cell channels and cell-to-extracellular space hemichannels. Many connexin subtypes are expressed in kidney cells. Some mutations in connexin genes have been linked to various human pathologies, including cardiovascular, neurodegenerative, lung, and skin diseases, but the exact role of connexins in kidney disease remains unclear. Some hypotheses about a connection between genetic mutations, endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) in kidney pathology have been explored. The potential relationship of kidney disease to abnormal production of connexin proteins, mutations in their genes together with ER stress, or the UPR is still a matter of debate. In this scenario, it is tantalizing to speculate about a possible role of connexins in the setting of kidney pathologies that are thought to be caused by a deregulated podocyte protein expression, the so-called podocytopathies. In this article, we give examples of the roles of connexins in kidney (patho)physiology and propose avenues for further research concerning connexins, ER stress, and UPR in podocytopathies that may ultimately help refine drug treatment.
... Slide-mounted sections could be routinely stored at −35 °C for several months before use. Considerations regarding the differential sensitivity of Cx36 vs. EGFP to tissue fixation and consequent difficulties in immunohistochemical detection of these proteins in combination using double labelling procedures with current commercially available antibodies have been previously discussed ( Nagy et al., 2013Nagy et al., , 2014). Thus, for immunolabelling of EGFP in EGFP-Cx36 mice, animals were transcardially perfused as above, except with fixative containing 4% formaldehyde, where labelling for Cx36 was substantially diminished and often abolished. ...
Electrical synapses formed by gap junctions composed of connexin36 (Cx36) are widely distributed in the mammalian central nervous system (CNS). Here, we used immunofluorescence methods to document the expression of Cx36 in the cochlear nucleus and in various structures of the auditory pathway of rat and mouse. Labelling of Cx36 visualized exclusively as Cx36-puncta was densely distributed primarily on the somata and initial dendrites of neuronal populations in the ventral cochlear nucleus, and was abundant in superficial layers of the dorsal cochlear nucleus. Other auditory centers displaying Cx36-puncta included the medial nucleus of the trapezoid body (MNTB), regions surrounding the lateral superior olivary nucleus, the dorsal nucleus of the medial lemniscus, the nucleus sagulum, all subnuclei of the inferior colliculus, and the auditory cerebral cortex. In EGFP-Cx36 transgenic mice, EGFP reporter was detected in neurons located in each of auditory centers that harboured Cx36-puncta. In the ventral cochlear nuclei and the MNTB, many neuronal somata were heavily innervated by nerve terminals containing vesicular glutamate transporter-1 (vglut1) and Cx36 was frequently localized at these terminals. Cochlear ablation caused a near total depletion of vglut1-positive terminals in the ventral cochlear nuclei, with a commensurate loss of labelling for Cx36 around most neuronal somata, but preserved Cx36-puncta at somatic neuronal appositions. The results suggest that electrical synapses formed by Cx36-containing gap junctions occur in most of the widely distributed centers of the auditory system. Further, it appears that morphologically mixed chemical/electrical synapses formed by nerve terminals are abundant in the ventral cochlear nucleus, including those at endbulbs of Held formed by cochlear primary afferent fibers, and those at calyx of Held synapses on MNTB neurons.
Copyright © 2015. Published by Elsevier Ltd.
... It is colocalized with nitric oxide synthase in the myenteric plexus of mouse colon, suggesting a role for gap junctions in inhibitory nitrergic enteric neuronal activity. This is further substantiated by the notion that Cx36 knockout mice exhibit modified spontaneous contractility properties and altered responses to electrical field stimulation and cholinergic agonists in the gut [112]. Besides conveying electrical signals, Cx43 represents an important component of the protective innate immune response of the intestinal epithelium. ...
Connexin proteins are abundantly present in the digestive system. They primarily form gap junctions, which control the intercellular exchange of critical homeostasis regulators. By doing so, gap junctions drive a plethora of gastrointestinal and hepatic functional features, including gastric and gut motility, gastric acid secretion, intestinal innate immune defense, xenobiotic biotransformation, glycogenolysis, bile secretion, ammonia detoxification and plasma protein synthesis. In the last decade, it has become clear that connexin hemichannels, which are the structural precursors of gap junctions, also provide a pathway for cellular communication, namely between the cytosol and the extracellular environment. Although merely pathological functions have been described, some physiological roles have been attributed to connexin hemichannels, in particular in the modulation of colonic motility. This equally holds true for cellular channels composed of pannexins, connexin-like proteins recently identified in the intestine and the liver, which have become acknowledged key players in inflammatory processes and that have been proposed to control colonic motility, secretion and blood flow.
Cell-to-cell communication through gap junction channels is very important to coordinate the functions of cells in all multicellular biological tissues. It allows the direct exchange of ions and small molecules (including second messengers, such as Ca2+, IP3, cyclic nucleotides, and oligonucleotides). In this study, a total of 48 members of the gap junction (GJ) protein family were identified from Sebastes schlegelii. In S. schlegelii, GJ proteins were classified into two types, connexin, and pannexin, and then connexins were divided into five subfamilies. The naming of 48 genes was verified through phylogenetic analysis and syntenic analysis. The connexin proteins contained four transmembrane fragments and two extracellular loops, the lengths of the intracellular loop and C-terminal was quite different, and the C-terminal region was highly variable after post-translational modification. PPI analysis showed that GJs interacted with tight junctions, adhesive junctions, and cell adhesions to form a complex network and participated in cell-cell junction organization, ATP binding, ion channel, voltage-gated conduction, wnt signaling pathway, Fc-γ receptor signaling pathway, and DNA replication. In addition, the S. schlegelii GJ protein was highly expressed in intestinal tissues and remarkably regulated after Edwardsiella tarda and Streptococcus iniae infection. The expression of GJs in intestinal cells of S. schlegelii was significantly regulated by LPS and poly (I:C), which was consistent with the results of intestinal tissue stimulation by pathogens. In conclusion, this study can provide valuable information for further research on the function of S. schlegelii GJ proteins.
The rapid development of 3D printing has led to considerable progress in the field of biomedical engineering. Notably, 4D printing provides a potential strategy to achieve a time-dependent physical change within tissue scaffolds or replicate the dynamic biological behaviors of native tissues for smart tissue regeneration and the fabrication of medical devices. The fabricated stimulus-responsive structures can offer dynamic, reprogrammable deformation or actuation to mimic complex physical, biochemical, and mechanical processes of native tissues. Although there has been notable progress made in the development of the 4D printing approach for various biomedical applications, its more broad-scale adoption for clinical use and tissue engineering purposes has been complicated by a notable limitation of printable smart materials and the simplistic nature of achievable responses possible with current sources of stimulation. In this review, we highlight the recent progress made in the field of 4D printing by discussing the various printing mechanisms that have been achieved with great emphasis on smart ink mechanisms of 4D actuation, construct structural design, and printing technologies. We then summarize recent 4D printing studies which focus on the applications of tissue/organ regeneration and medical devices. Finally, the current challenges and future perspectives of 4D printing are also discussed.
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The peculiar organization of cells of the gastrointestinal tract, in part, is due to the integrity of the anatomical architecture of the linkages or junctions between the neighboring cells. In this chapter, the structural and functional characteristics of these junctions are discussed.
Background:
Despite having an optimal pull-through operation, some children with Hirschsprung's disease (HSCR) continue to experience persistent bowel symptoms. Coordinated colonic electrical activity depends on intercellular communication between the enteric nerves, interstitial cells of Cajal (ICCs), smooth muscle cells, and fibroblast-like (platelet-derived growth factor receptor-alpha-positive) cells. Gap junctions are low-resistance channels composed of connexin (Cx) proteins which couple cells electrically and chemically. We aimed to evaluate the expression patterns of Cx26, Cx36, and Cx43 in the colon of children with HSCR and in healthy control colon.
Materials and methods:
Entire resected colonic specimens were collected from children undergoing pull-through surgery for HSCR (n = 10). Colonic controls were collected at colostomy closure in children with imperforate anus (n = 8). Distribution of Cx26, Cx36, and Cx43 was assessed using double-labeled immunofluorescence and confocal microscopy. Protein expression was quantified using Western blot analysis.
Results:
Sparse punctate Cx36 expression was seen in the myenteric plexus in nerve trunks and some platelet-derived growth factor receptor-alpha-positive cell and ICC fibers. Cx26 was similarly distributed, although it was not coexpressed in ICCs. Cx43 was only coexpressed with ICCs in the myenteric plexus. Expression of Cx26 and Cx43 was markedly reduced in the aganglionic colon in HSCR compared to controls, while Cx26 expression was also moderately reduced in the ganglionic bowel in HSCR.
Conclusions:
Reduced expression of Cx26 and Cx43 is implicated in the pathophysiology of colonic dysmotility in the aganglionic bowel as well as, in the case of Cx26, the ganglionic bowel in HSCR.
The present study was performed on whole-mount preparations to investigate the chemical neuroanatomy of the equine myenteric plexus throughout its distribution in the intestinal wall. The objective was to quantify neurons of the myenteric plexus, especially the predominant cholinergic and nitrergic subpopulations. Furthermore, we investigated the distribution of vasoactive intestinal polypeptide and the calcium-binding protein calretinin. Samples from different defined areas of the small intestine and the flexura pelvina were taken from 15 adult horses. After fixation and preparation of the tissue, immunofluorescence labeling was performed on free floating whole-mounts. Additionally, samples used for neuropeptide staining were incubated with colchicine to reveal the neuropeptide distribution within the neuronal soma. The evaluation was routinely accomplished using confocal laser-scanning microscopy. For quantitative and qualitative analysis, the pan-neuronal marker anti-HuC/D was applied in combination with the detection of the marker enzymes for cholinergic neurons and nitrergic nerve cells. Quantitative data revealed that the cholinergic subpopulation is larger than the nitrergic one in several different locations of the small intestine. On the contrary, the nitrergic neurons outnumber the cholinergic neurons in the flexura pelvina of the large colon. Furthermore, ganglia are more numerous in the small intestine compared with the large colon, but ganglion sizes are bigger in the large colon. However, comparison of the entire population of neurons in the different locations of the gut showed no difference. The present study adds further data on the chemoarchitecture of the myenteric plexus which might facilitate the understanding of several gastrointestinal disorders in the horse. ava i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s ev i e r. c o m / l o c a t e / b r a i n r e s
A great deal is now known about the protein components of tight junctions and adherens junctions, as well as how these are assembled. Less is known about the molecular framework of gap junctions, but these also have membrane specializations and are subject to regulation of their assembly and turnover. Thus, it is reasonable to consider that these three types of junctions may share macromolecular commonalities. Indeed, the tight junction scaffolding protein zonula occluden-1 (ZO-1) is also present at adherens and gap junctions, including neuronal gap junctions. On the basis of these earlier observations, we more recently found that two additional proteins, AF6 and MUPP1, known to be associated with ZO-1 at tight and adherens junctions, are also components of neuronal gap junctions in rodent brain and directly interact with connexin36 (Cx36) that forms these junctions. Here, we show by immunofluorescence labeling that the cytoskeletal-associated protein cingulin, commonly found at tight junctions, is also localized at neuronal gap junctions throughout the central nervous system. In consideration of known functions related to ZO-1, AF6, MUPP1, and cingulin, our results provide a context in which to examine functional relationships between these proteins at Cx36-containing electrical synapses in brain--specifically, how they may contribute to regulation of transmission at these synapses, and how they may govern gap junction channel assembly and/or disassembly.
Electrical synapses are known to form networks of extensively coupled neurons in various regions of the mammalian brain. The mesencephalic trigeminal (MesV) nucleus, formed by the somata of primary afferents originating in jaw-closing muscles, constitutes one of the first examples supporting the presence of electrical synapses in the mammalian CNS; however, the properties, functional organization, and developmental emergence of electrical coupling within this structure remain unknown. By combining electrophysiological, tracer coupling, and immunochemical analysis in brain slices of rat and mouse, we found that coupling is mostly restricted to pairs or small clusters of MesV neurons. Electrical transmission is supported by connexin36 (Cx36)-containing gap junctions at somato-somatic contacts where only a small proportion of channels appear to be open (∼0.1%). In marked contrast with most brain structures, coupling among MesV neurons increases with age, such that it is absent during early development and appears at postnatal day 8. Interestingly, the development of coupling parallels the development of intrinsic membrane properties responsible for repetitive firing in these neurons. We found that, acting together, sodium and potassium conductances enhance the transfer of signals with high-frequency content via electrical synapses, leading to strong spiking synchronization of the coupled neurons. Together, our data indicate that coupling in the MesV nucleus is restricted to mostly pairs of somata between which electrical transmission is supported by a surprisingly small fraction of the channels estimated to be present, and that coupling synergically interacts with specific membrane conductances to promote synchronization of these neurons.
Electrical synapses between interneurons contribute to synchronized firing and network oscillations in the brain. However, little is known about how such networks respond to excitatory synaptic input. To investigate this, we studied electrically coupled Golgi cells (GoC) in the cerebellar input layer. We show with immunohistochemistry, electron microscopy, and electrophysiology that Connexin-36 is necessary for functional gap junctions (GJs) between GoC dendrites. In the absence of coincident synaptic input, GoCs synchronize their firing. In contrast, sparse, coincident mossy fiber input triggered a mixture of excitation and inhibition of GoC firing and spike desynchronization. Inhibition is caused by propagation of the spike afterhyperpolarization through GJs. This triggers network desynchronization because heterogeneous coupling to surrounding cells causes spike-phase dispersion. Detailed network models predict that desynchronization is robust, local, and dependent on synaptic input properties. Our results show that GJ coupling can be inhibitory and either promote network synchronization or trigger rapid network desynchronization depending on the synaptic input.
Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozymes I (in neuronal and epithelial cells), II (in cytokine-induced cells), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity. Across species, amino acid sequences for each isoform are well conserved (> 90% for isoforms I and III, > 80% for isoform II). All isoforms use L-arginine and molecular oxygen as substrates and require the cofactors NADPH, 6(R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide. They all bind calmodulin and contain heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. Expression of isoform II of NO synthase can be induced with lipopolysaccharide and cytokines in a multitude of different cells. Based on sequencing data there is no evidence for more than one inducible isozyme at this time. NO synthase II is not regulated by Ca2+; it produces large amounts of NO that has cytostatic effects on parasitic target cells by inhibiting iron-containing enzymes and causing DNA fragmentation. Induced NO synthase II is involved in the pathophysiology of autoimmune diseases and septic shock. Isoform III of NO synthase has been found mostly in endothelial cells. It is constitutively expressed, but expression can be enhanced, eg, by shear stress. Its activity is regulated by Ca2+ and calmodulin. NO from endothelial cells keeps blood vessels dilated, prevents the adhesion of platelets and white cells, and probably inhibits vascular smooth muscle proliferation.
Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43
(Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos
to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result
of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart.
This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among
connexins in other parts of the developing fetus.
The enteric nervous system is a collection of neurons in the gastrointestinal tract1 that constitutes the “brain of the gut” and can function independently of the central nervous system.2 This system controls the motility,3,4 exocrine and endocrine secretions,5 and microcirculation6 of the gastrointestinal tract; it is also involved in regulating immune and inflammatory processes.7 In the past decade, major advances in the understanding of the enteric nervous system have led to a greater appreciation of its importance in clinical medicine. In this review we highlight some of these advances. Development of the Enteric Nervous System The enteric nervous system . . .
Gap junctions are the morphological substrate of one class of electrical synapse. The history of the debate on electrical vs. chemical transmission is instructive. One lesson is that Occam's razor sometimes cuts too deep; the nervous system does its operations in a number of different ways and a unitarian approach can lead one astray. Electrical synapses can do many things that chemical synapses can do, and do them just as slowly. More intriguing are the modulatory actions that chemical synapses can have on electrical synapses. Voltage dependence provides an important window on structure function relations of the connexins, even where the dependence may have no physiological role. The new molecular approaches will greatly advance our knowledge of where gap junctions occur and permit experimental manipulation with high specificity.
Mediators of nonadrenergic, noncholinergic (NANC) relaxation in longitudinal muscle of several regions of ICR mouse intestine were studied. An inhibitor of synthesis of nitric oxide, N(G)-nitro-L-arginine (L-NOARG) at 10 microM significantly inhibited NANC relaxations induced by electrical field stimulation (EFS) in the jejunum, ileum, and the proximal and distal colon. Especially in the ileum extent of the inhibition was more than 80%. An antagonist of vasoactive intestinal peptide (VIP) receptors, VIP(10-28) at 3 microM partially inhibited the EFS induced relaxations in the jejunum and proximal colon, but very slightly in the distal colon and had no effect in the ileum. An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptor, PACAP(6-38) at 3 microM partially inhibited the EFS-induced relaxations in the proximal and distal colon, but not in the jejunum and ileum. Totals of the percentages of relaxant components mediated by nitric oxide, VIP and PACAP in every region are roughly equal to a hundred percent. In another series of experiments, EFS-induced relaxations were almost completely inhibited by the treatment of the segments with L-NOARG and VIP(10-28) in the jejunum, with L-NOARG, VIP(10-28) and PACAP(6-38) in the proximal colon, and with L NOARG and PACAP(6-38) in the distal colon. The present results suggest that nitric oxide solely mediates the relaxation of longitudinal muscle of the ileum of ICR mice, whereas nitric oxide and VIP co-mediate it in the jejunum, nitric oxide, VIP and PACAP in the proximal colon, and nitric oxide and PACAP in the distal colon.
Physiological and ultrastructural evidence indicates that gap junctions link many classes of neurons in mammalian central nervous system (CNS), allowing direct electrical and metabolic communication. Among at least six gap junction-forming connexin proteins in adult rat brain, connexin- (Cx) 32, Cx36, and Cx43 have been reported to occur in neurons. However, no connexin has been documented at ultrastructurally defined neuronal gap junctions. To address this question directly, freeze-fracture replica immunogold labeling (FRIL) and immunofluorescence (IF) were used to visualize the subcellular and regional localization of Cx36 in rat brain and spinal cord. Three antibodies were generated against different sequences in Cx36. By Western blotting, these antibodies detected protein at 36 and 66 kDa, corresponding to Cx36 monomer and dimer forms, respectively. After double-labeling for Cx36 and Cx43 by FRIL, neuronal gap junctions in inferior olive, spinal cord, and retina were consistently immunogold-labeled for Cx36, but none were labeled for Cx43. Conversely, Cx43 but not Cx36 was detected in astrocyte and ependymocyte gap junctions. In >250 Cx32/Cx43 single- and double-labeled replicas from 10 CNS regions, no neuronal gap junctions were labeled for either Cx32 or Cx43. Instead, Cx32 and Cx43 were restricted to glial gap junctions. By IF, Cx36 labeling was widely distributed in neuropil, including along dendritic processes and within neuronal somata. On the basis of FRIL identification of Cx36 in neuronal gap junctions and IF imaging of Cx36 throughout rat brain and spinal cord, neuronal gap junctions containing Cx36 appear to occur in sufficient density to provide widespread electrical and metabolic coupling in adult CNS.
The transmembrane connexin proteins of gap junctions link extracellularly to form channels for cell-to-cell exchange of ions and small molecules. Two primary hypotheses of gap junction coupling in the CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in both neurons and glia, and (2) intercellular junctional coupling is restricted to specific coupling partners, with different connexins expressed in each cell type. There is consensus that gap junctions link neurons to neurons and astrocytes to oligodendrocytes, ependymocytes, and other astrocytes. However, unresolved are the existence and degree to which gap junctions occur between oligodendrocytes, between oligodendrocytes and neurons, and between astrocytes and neurons. Using light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling of adult rat CNS, we investigated whether four of the best-characterized CNS connexins are each present in one or more cell types, whether oligodendrocytes also share gap junctions with other oligodendrocytes or with neurons, and whether astrocytes share gap junctions with neurons. Connexin32 (Cx32) was found only in gap junctions of oligodendrocyte plasma membranes, Cx30 and Cx43 were found only in astrocyte membranes, and Cx36 was only in neurons. Oligodendrocytes shared intercellular gap junctions only with astrocytes, with each oligodendrocyte isolated from other oligodendrocytes except via astrocyte intermediaries. Finally, neurons shared gap junctions only with other neurons and not with glial cells. Thus, the different cell types of the CNS express different connexins, which define separate pathways for neuronal versus glial gap junctional communication.
Minute-to-minute behavior of the alimentary tract reflects the integrated functioning of the gut's musculature, secretory glands and blood–lymphatic vasculature. Activity of the three effector systems to generate functionally effective patterns of behavior, which are adaptive for differing digestive states, is organized and coordinated by the enteric nervous system (i.e., the brain-in-the-gut). The heuristic model for the enteric nervous system (ENS) is the same as for all integrative nervous systems, whether in vertebrate or invertebrate animals. Like other integrative nervous systems, such as the spinal cord and brain stem, the ENS functions with sensory neurons, interneurons and motor neurons. That the gut does not work without the ENS can be made as an absolute statement. This is apparent in its absence in terminal regions of the large intestine in Hirschsprung's disease in humans and animals where it is reflected by dysfunctional motility, failure of defecation and proximal fecal compaction ...
Pools of motoneurons in the lumbar spinal cord innervate the sexually dimorphic perineal musculature, and are themselves sexually dimorphic, showing differences in number and size between male and female rodents. In two of these pools, the dorsomedial nucleus (DMN) and the dorsolateral nucleus (DLN), dimorphic motoneurons are intermixed with non-dimorphic neurons innervating anal and external urethral sphincter muscles. As motoneurons in these nuclei are reportedly linked by gap junctions, we examined immunofluorescence labeling for the gap junction-forming protein connexin36 (Cx36) in male and female mice and rats. Fluorescent Cx36-labeled puncta occurred in distinctly greater amounts in the DMN and DLN of male rodents than in other spinal cord regions. These puncta were localized to motoneuron somata, proximal dendrites, and neuronal appositions, and were distributed either as isolated or large patches of puncta. In both rats and mice, Cx36-labeled puncta were associated with nearly all (> 94%) DMN and DLN motoneurons. The density of Cx36-labeled puncta increased dramatically from postnatal days 9 to 15, unlike the developmental decreases in these puncta observed in other central nervous system regions. In females, Cx36 labeling of puncta in the DLN was similar to that in males, but was sparse in the DMN. In enhanced green fluorescent protein (EGFP)-Cx36 transgenic mice, motoneurons in the DMN and DLN were intensely labeled for the EGFP reporter in males, but less so in females. The results indicate the presence of Cx36-containing gap junctions in the sexually dimorphic DMN and DLN of both male and female rodents, suggesting coupling of not only sexually dimorphic but also non-dimorphic motoneurons in these nuclei.
Axon terminals forming mixed chemical/electrical synapses in the lateral vestibular nucleus of rat were described over forty years ago. Because gap junctions formed by connexins are the morphological correlate of electrical synapses, and with demonstrations of widespread expression of the gap junction protein connexin36 (Cx36) in neurons, we investigated the distribution and cellular localization of electrical synapses in the adult and developing rodent vestibular nuclear complex, using immunofluorescence detection of Cx36 as a marker for these synapses. In addition, we examined Cx36 localization in relation to that of the nerve terminal marker vesicular glutamate transporter-1 (vglut-1). An abundance of immunolabelling for Cx36 in the form of Cx36-puncta was found in each of the four major vestibular nuclei of adult rat and mouse. Immunolabelling was associated with somata and initial dendrites of medium and large neurons, and was absent in vestibular nuclei of Cx36 knockout mice. Cx36-puncta were seen either dispersed or aggregated into clusters on the surface of neurons, and were never found to occur intracellularly. Nearly all Cx36-puncta were localized to large nerve terminals immunolabelled for vglut-1. These terminals and their associated Cx36-puncta were substantially depleted after labyrinthectomy. Developmentally, labelling for Cx36 was already present in the vestibular nuclei at postnatal day 5, where it was only partially co-localized with vglut-1, and did not become fully associated with vglut-1-positive terminals until postnatal day 20 to 25. The results show that vglut-1-positive primary afferent nerve terminals form mixed synapses throughout the vestibular nuclear complex, that the gap junction component of these synapses contain Cx36, that multiple Cx36-containing gap junctions are associated with individual vglut-1 terminals and that the development of these mixed synapses is protracted over several postnatal weeks.
The hypothesis that Chromogranin A (CgA)-derived peptides are involved in mechanisms modulating altered colonic motility was tested. Rat distal colonic strips were studied using an organ bath technique. Acetic acid (AA)-induced effects were characterized on spontaneous mechanical activities (SMA) in the presence of CgA4–16 or CgA47–66. In preparations with mucosa, AA induced a transient hyperactivity followed by a decrease in tone. The first phase is sensitive to tetrodotoxin (TTX) and capsaicin. The second phase was sensitive to BAYK8644 but insensitive to l-nitro-arginine-methyl-ester (l-Name)/apamin together. CgA4–16 or CgA47–66 alone produced no change on SMA. The administration of CgA4–16 prior to AA increased the duration of the excitatory component and reduced tone inhibition. CgA47–66 prior to AA only decreased duration of the excitatory phase. In preparations without mucosa, AA decreased tone. This effect was sensitive to BAYK8644 and CgA4–16. Trypsin decreased basal tone. This effect was suppressed by TTX, BAYK8644 or l-Name/apamin and were reduced by CgA4–16. AA-induced effects on rat colonic motility in vitro may be mediated through activation of primary afferents and an action at L-Type calcium channels. CgA-derived peptides are shown to decrease AA-induced effects on motility.
Although connexin36 (Cx36) has been studied in several tissues, it is notable that no data are available on Cx36 expression in the carotid body and the intestine. The present study was undertaken to evaluate using immunohistochemistry, PCR and Western blotting procedures, whether Cx36 was expressed in the mouse carotid body and in the intestine at ileum and colon level. In the carotid body, Cx36 was detected as diffuse punctate immunostaining and as protein by Western blotting and mRNA by RT-PCR. Cx36 punctate immunostaining was also evident in the intestine with localization restricted to the myenteric plexus of both the ileum and the colon, and this detection was also confirmed by Western blotting and RT-PCR. All the data obtained were validated using Cx36 knockout mice. Taken together the present data on localization of Cx36 gap-junctions in two tissues of neural crest-derived neuroendocrine organs may provide an anatomical basis for future functional investigations.
Electrical synaptic transmission via gap junctions has become an accepted feature of neuronal communication in the mammalian brain, and occurs often between dendrites of interneurons in major brain structures, including the hippocampus. Electrical and dye-coupling has also been reported to occur between pyramidal cells in the hippocampus, but ultrastructurally-identified gap junctions between these cells have so far eluded detection. Gap junctions can be formed by nerve terminals, where they contribute the electrical component of mixed chemical/electrical synaptic transmission, but mixed synapses have only rarely been described in mammalian CNS. Here, we used immunofluorescence localization of the major gap junction forming protein connexin36 to examine its possible association with hippocampal pyramidal cells. In addition to labeling associated with gap junctions between dendrites of parvalbumin-positive interneurons, a high density of fine, punctate immunolabeling for Cx36, non-overlapping with parvalbumin, was found in subregions of the stratum lucidum in the ventral hippocampus of rat brain. A high percentage of Cx36-positive puncta in the stratum lucidum was localized to mossy fiber terminals, as indicated by co-localization of Cx36-puncta with the mossy terminal marker vesicular glutamate transporter-1, as well as with other proteins that are highly concentrated in, and diagnostic markers of, these terminals. These results suggest that mossy fiber terminals abundantly form mixed chemical/electrical synapses with pyramidal cells, where they may serve as intermediaries for the reported electrical and dye-coupling between ensembles of these principal cells. This article is part of a Special Issue entitled Electrical Synapses.
Cellular prion protein (PrP(C) ) is expressed in the enteric nervous system (ENS), however, its physiological role has not been identified. Studies suggest that PrP(C) can function as a metal-binding protein, as absence of the protein has been linked to altered copper metabolism and atypical synaptic activity. Because copper is known to modulate smooth muscle relaxation, we tested the hypothesis that PrP(C) deficiency would alter intestinal contractility.
We examined electrically evoked ileal contractility in Prnp(-/-) or wild type littermate mice and the effects of copper or copper chelation. PrP(C) expression was studied in whole mount ileal preparations of mice and guinea pigs by immunohistochemistry. Key
Relative to wild type mice, ileal tissues of Prnp(-/-) mice exhibited reduced electrical field stimulation (EFS)-evoked contractility. Furthermore, EFS-induced relaxation, as a percentage of that induced by a nitric oxide donor, was enhanced. Addition of a copper donor to the organ bath increased, whereas the addition of a copper chelator inhibited, nitric oxide donor-induced ileal relaxation in Prnp(-/-) mice. PrP(C) was expressed on nerve fibers or terminals, and some cell bodies in the myenteric and submucosal plexuses of wild type mice. PrP(C) colocalized with a neuron-specific ectonucleotidase, nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), but to only a limited extent with GFAP, a marker of enteric glia. Guinea pigs expressed PrP(C) in nerve fibers or terminals and enteric glia in the myenteric and submucosal plexuses.
Our findings suggest that PrP(C) , which is abundant in the ENS, has a role in the regulation of ileal contractility.
The immunohistochemical localization of connexin (Cx) 43 and Cx 45 in the musculature of the rat small intestine was studied at the ultrastructural level, with special reference to the interstitial cells of Cajal in the deep muscular plexus region (ICC-DMP). Cx 43 was localized at gap junctions formed between every group of cells, i.e., smooth muscle cell~smooth muscle cell, smooth muscle cell~ICC-DMP and ICC-DMP~ICC-DMP. In contrast, Cx 45 immunoreactivity was only detected at gap junctions between ICC-DMP~ICC-DMP. Since different types of Cx molecules have different properties for electrical and chemical coupling of cells, it is suggested that the homotypic network of ICC-DMP connected with Cx 45 gap junctions may function as an independent compartment segregated from the whole cellular network including the smooth muscle cells connected with Cx 43 gap junctions. It is further speculated that the ICC-DMP of the rat small intestine communicate with each other and with smooth muscle cells via the passage of messenger molecules through Cx 43, but they may use an additional mechanism, as yet unknown, for communications restricted to other ICC-DMP.
Key points
Reflexes evoked by sensory information from muscles and skin play an important role in controlling muscle activity during movement.
The strength of these reflexes is regulated in part by presynaptic inhibition, a process controlling the release of chemical transmitters from sensory fibres terminating on spinal neurones.
Our study is the first to show that electrical synapses among spinal neurones in young animals are essential for normal operation of processes that presynaptically regulate synaptic transmission between large diameter sensory fibres and spinal cord neurones.
Transgenic mice lacking connexin36, a protein that mediates electrical communication via gap junctions between neurones, suffer a severe impairment of presynaptic inhibition and a similar impairment can be produced in normal mice with drugs that disrupt gap junction function.
The wide distribution of connexin36 in the spinal cord suggests that neuronal gap junctions also play a role in other physiological processes.
Abstract Electrical synapses formed by gap junctions containing connexin36 (Cx36) promote synchronous activity of interneurones in many regions of mammalian brain; however, there is limited information on the role of electrical synapses in spinal neuronal networks. Here we show that Cx36 is widely distributed in the spinal cord and is involved in mechanisms that govern presynaptic inhibition of primary afferent terminals. Electrophysiological recordings were made in spinal cord preparations from 8‐ to 11‐day‐old wild‐type and Cx36 knockout mice. Several features associated with presynaptic inhibition evoked by conditioning stimulation of low threshold hindlimb afferents were substantially compromised in Cx36 knockout mice. Dorsal root potentials (DRPs) evoked by low intensity stimulation of sensory afferents were reduced in amplitude by 79% and in duration by 67% in Cx36 knockouts. DRPs were similarly affected in wild‐types by bath application of gap junction blockers. Consistent with presynaptic inhibition of group Ia muscle spindle afferent terminals on motoneurones described in adult cats, conditioning stimulation of an adjacent dorsal root evoked a long duration inhibition of monosynaptic reflexes recorded from the ventral root in wild‐type mice, and this inhibition was antagonized by bicuculline. The same conditioning stimulation failed to inhibit monosynaptic reflexes in Cx36 knockout mice. Immunofluorescence labelling for Cx36 was found throughout the dorsal and ventral horns of the spinal cord of juvenile mice and persisted in mature animals. In deep dorsal horn laminae, where interneurones involved in presynaptic inhibition of large diameter muscle afferents are located, cells were extensively dye‐coupled following intracellular neurobiotin injection. Coupled cells displayed Cx36‐positive puncta along their processes. Our results indicate that gap junctions formed by Cx36 in spinal cord are required for maintenance of presynaptic inhibition, including the regulation of transmission from Ia muscle spindle afferents. In addition to a role in presynaptic inhibition in juvenile animals, the persistence of Cx36 expression among spinal neuronal populations in the adult mouse suggests that the contribution of electrical synapses to integrative processes in fully mature spinal cord may be as diverse as that found in other areas of the CNS.
Nitric oxide was identified as a biological intercellular messenger just over 20 years ago, and its presence and potential importance in the nervous system was immediately noted. With the cloning of NO synthase and the physiological NO receptor soluble guanylyl cyclase, a variety of histochemical methods quickly led to a rather complete picture of where NO is produced and acts in the nervous system. However, the details regarding the subcellular localization of NO synthase and the identity of its molecular binding partners require further clarification. Although the hypothesis that calcium influx via activation of NMDA receptors is a key trigger for NO production has proven very popular and led to suggested roles for NO in synaptic plasticity, there is little direct evidence to support this notion. Instead, studies from the peripheral nervous system indicate a key role for voltage-sensitive calcium channels in regulating NO synthase activity. A similar mechanism may also be important in central neurons, and it remains an important task to identify the precise sources of calcium regulating NO production in specific NO neurons. Also, although cGMP production appears to mediate the physiological signaling by NO, the specific roles of cGMP-dependent ion channels, protein kinases and phosphodiesterases in mediating NO action remain to be determined.
Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) occur in most major structures in the mammalian central nervous system. These synapses link ensembles of neurons and influence their network properties. Little is known about the macromolecular constituents of neuronal gap junctions or how transmission through electrical synapses is regulated at the level of channel conductance or gap junction assembly/disassembly. Such knowledge is a prerequisite to understanding the roles of gap junctions in neuronal circuitry. Gap junctions share similarities with tight and adhesion junctions in that all three reside at close plasma membrane appositions, and therefore may associate with similar structural and regulatory proteins. Previously, we reported that the tight junction-associated protein zonula occludens-1 (ZO-1) interacts with Cx36 and is localized at gap junctions. Here, we demonstrate that two proteins known to be associated with tight and adherens junctions, namely AF6 and MUPP1, are components of neuronal gap junctions in rodent brain. By immunofluorescence, AF6 and MUPP1 were co-localized with Cx36 in many brain areas. Co-immunoprecipitation and pull-down approaches revealed an association of Cx36 with AF6 and MUPP1, which required the C-terminus PDZ domain interaction motif of Cx36 for interaction with the single PDZ domain of AF6 and with the 10th PDZ domain of MUPP1. As AF6 is a target of the cAMP/Epac/Rap1 signalling pathway and MUPP1 is a scaffolding protein that interacts with CaMKII, the present results suggest that AF6 may be a target for cAMP/Epac/Rap1 signalling at electrical synapses, and that MUPP1 may contribute to anchoring CaMKII at these synapses.
The aim of this study was to investigate whether the pig colon descendens might be a good model for the responses mediated via the different locations of human colonic 5-HT(4) receptors. The intrinsic excitatory and inhibitory motor neurotransmission in pig colon descendens was therefore first characterized. In circular smooth muscle strips, electrical field stimulation (EFS) at basal tone induced only in the combined presence of the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) and the SK channel blocker apamin voltage-dependent on-contractions. These on-contractions were largely reduced by the neuronal conductance blocker tetrodotoxin (TTX) and by the muscarinic receptor antagonist atropine, illustrating activation of cholinergic neurons. The 5-HT(4) receptor agonist prucalopride facilitated submaximal EFS-evoked cholinergic contractions and this effect was prevented by the 5-HT(4) receptor antagonist GR113808, supporting the presence of facilitating 5-HT(4) receptors on the cholinergic nerve endings innervating circular muscle in pig colon descendens. Relaxations were induced by EFS in strips pre-contracted with substance P in the presence of atropine. The responses at lower stimulation voltages were abolished by TTX. L-NAME or apamin alone did not influence or only moderately reduced the relaxations, but L-NAME plus apamin abolished the relaxations at lower stimulation voltages, suggesting that NO and ATP act as inhibitory neurotransmitters in a redundant way. Prucalopride did not influence the EFS-induced relaxations at lower stimulation voltage, nor did it per se relax contracted circular muscle strips. No evidence for relaxing 5-HT(4) receptors, either on inhibitory neurons or on the muscle cells was thus obtained in pig colon descendens circular muscle.
In 1958, Akira Watanabe made three seminal proposals about neuronal communication. First, he demonstrated that electrical synapses interconnect neurons in the cardiac ganglion of Japanese lobsters, and he deduced that ionic current could pass directly from cell to cell in either direction. Second, he proposed that electrical synapses have low-pass filtering characteristics; that is, slow fluctuations of membrane voltage pass more effectively between cells than do fast events such as action potentials. Third, he found that membrane potential oscillations were coordinated among neurons and concluded that “synchronization is due to the presence of the electrical connection between the cells” (Watanabe, 1958).
The present study was performed on whole-mount preparations to investigate the chemical neuroanatomy of the equine myenteric plexus throughout its distribution in the intestinal wall. The objective was to quantify neurons of the myenteric plexus, especially the predominant cholinergic and nitrergic subpopulations. Furthermore, we investigated the distribution of vasoactive intestinal polypeptide and the calcium-binding protein calretinin. Samples from different defined areas of the small intestine and the flexura pelvina were taken from 15 adult horses. After fixation and preparation of the tissue, immunofluorescence labeling was performed on free floating whole-mounts. Additionally, samples used for neuropeptide staining were incubated with colchicine to reveal the neuropeptide distribution within the neuronal soma. The evaluation was routinely accomplished using confocal laser-scanning microscopy. For quantitative and qualitative analysis, the pan-neuronal marker anti-HuC/D was applied in combination with the detection of the marker enzymes for cholinergic neurons and nitrergic nerve cells. Quantitative data revealed that the cholinergic subpopulation is larger than the nitrergic one in several different locations of the small intestine. On the contrary, the nitrergic neurons outnumber the cholinergic neurons in the flexura pelvina of the large colon. Furthermore, ganglia are more numerous in the small intestine compared with the large colon, but ganglion sizes are bigger in the large colon. However, comparison of the entire population of neurons in the different locations of the gut showed no difference. The present study adds further data on the chemoarchitecture of the myenteric plexus which might facilitate the understanding of several gastrointestinal disorders in the horse.
Laminar preparations of fixed segments of the guinea-pig intestine were examined for nitric oxide synthase activity using reduced nicotinamide adenine dinucleotide phosphate and nitroblue tetrazolium salt as substrates. Under conditions specific for detecting nitric oxide synthase-related diaphorase activity, a subpopulation of neural elements in the myenteric plexus, deep muscular plexus and submucosa were intensely stained. Intensely stained nerve fibres were distributed throughout the meshworks of the myenteric plexus and its innervation of the circular muscle, and in the submucosa within Henle's plexus. Intensely stained nerve cells and their processes were evident in most myenteric ganglia but were rare in ganglia of Henle's plexus. Stained ganglion cells comprised types I, II and VI of the morphologically defined enteric nerve cells. Stained neural elements were increasingly prevalent within successively more caudal segments of the intestine. In addition to neuronal staining, arterioles of the submucosal vascular network displayed distinct, punctate patches of staining distributed over their surface. Perivascular nerve fibre staining was absent. These results show nitric oxide synthase activity to be present within neurons and fibres of the major enteric nerve layers and within submucosal blood vessels throughout the guinea-pig small and large intestine.
The ultrastructure of the region shown to be essential for pacemaking activity of the circular muscle of the canine colon was studied. This region, at the inner border of the circular muscle, consists of a network of several layers of interstitial cells of Cajal type III. These are interconnected to one another and to the adjacent circular muscle cells by numerous gap junctions. Elsewhere in circular muscle, gap junctions are rare and small. In addition, interstitial cells are in close (often less than 20 nm) contact with nerve varicosities containing large granular vesicles or sometimes small granular vesicles. The morphology of interstitial cells resembles that of others of type III. It is suggested that this arrangement of interstitial cells, circular smooth muscles, and nerves allows for a tightly coupled network of membrane oscillators to be subject to neural modulation.
We examined in vitro changes in contractility of jejunal longitudinal muscle strips in rats infected with the nematode parasite Trichinella spiralis. Length-passive tension relationships were unchanged. However, muscle from infected rats on days 5 and 6 postinfection (PI) generated maximal active tension induced by carbachol at significantly less stretch (39.9 +/- 1.0 and 34.3 +/- 6.3%, respectively) than control tissues (66.0 +/- 2.3%). In infected rats on day 5 PI, the maximum tension generated by carbachol (1.6 +/- 0.4 g/mm2) and by 5-hydroxytryptamine (5-HTP) (2.6 +/- 0.1 g/mm2) was significantly greater than in control tissue (0.5 +/- 0.2 g/mm2). On removal of calcium from the medium, responses of muscle from control and infected rats were reduced in a proportionate manner. The increased responsiveness to carbachol and 5-HTP was maximal by day 5 PI and was associated with a decrease in the ED50 value for 5-HTP but not for carbachol. All changes were reversed by 23 days PI. These results indicate that T. spiralis infection in the rat is associated with alterations in jejunal longitudinal smooth muscle function.
The distribution of gap junctions (nexuses) in various parts of the small and large intestines of the guinea-pig was studied using the freeze-fracture technique and in thin sections. The percentage area of smooth muscle cell surface occupied by gap junctions varies from 0.50% in the circular muscle of the duodenum to zero in the longitudinal muscle of the ileum. In the circular muscle of the jejunum and ileum the area occupied by nexuses is 0.22% (or about 11 μm2 per cell). The sizes of junctions range from less than 0.01 μm2 to 0.20 μm2, with two-thirds of them being smaller than 0.05 μm2. In the colon, gap junctions are rare, very small and confined to the circular muscle layer. Even the smallest aggregates of intramembrane particles correspond to areas of close apposition between the membranes of adjacent cells; it is therefore justified to interpret them as being gap junctions. Some gap junctions are formed between a smooth muscle cell and an interstitial cell. Gap junctions are not found in the longitudinal muscle of the small intestine; this is in sharp contrast to the abundance of gap junctions in the adjacent circular layer.
In the small intestine of cats and rabbits, gap junctions are abundant in the circular muscle layer, whereas they are very small in size and very few in number in the longitudinal muscle layer.
The distribution of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining and its colocalisation with nitric oxide synthase immunoreactivity was examined in neurons of the quail gastrointestinal tract. Immunoreactivity and enzyme activity were found in identical populations of neurons. Nitric oxide synthase activity was present in 30-40% of myenteric nerve cells in each region. Numerous reactive cells were also in the submucous plexus and many positive nerve fibres innervated the muscle. In the small intestine, some of the fibres innervating the circular muscle formed a deep muscular plexus and in the large intestine there was a submuscular plexus. Nerve fibres formed baskets around nerve cells of myenteric and submucous ganglia. Intramural arterioles were innervated, most prominently in the oesophagus and stomach. Very few fibres innervated the mucosa, except for some oesophageal glands and the core of villi in the cloaca. It is concluded that nitric oxide is probably involved in transmission to muscle of the avian intestine, as it is in mammals, and that it is probably also produced at neuroneuronal and neurovascular junctions.
The distribution and abundance of nitric oxide synthase (NOS)-containing neurons and their terminals in the gastrointestinal tract of the guinea-pig were examined in detail using NADPH diaphorase histochemistry and NOS immunohistochemistry. NOS-containing cell bodies were found in the myenteric plexus throughout the gastrointestinal tract and in the submucous plexus of the stomach, colon and rectum. NOS-containing neurons comprised between 12% (in the duodenum) and 54% (in the esophagus) of total myenteric neurons. In the ileum, NOS neurons represented 19% of total myenteric neurons. Most of the NOS neurons throughout the gastrointestinal tract possessed lamellar dendrites and a single axon. NOS-containing terminals were abundant in the circular muscle, including that of the sphincters, but were rare in the longitudinal muscle, except for the taeniae of the caecum. The muscularis mucosae of the esophagus, stomach, colon and rectum received a medium to dense innervation by NOS terminals. Within myenteric ganglia, NOS-containing terminals were extremely sparse in the esophagus, stomach and duodenum, common in the ileum and distal colon and extremely dense in the proximal colon and rectum. The submucous plexus in the ileum and large intestine contained a sparse plexus of NOS-containing terminals. NOS terminals were not observed in the mucosa of any region. We conclude that throughout the gastrointestinal tract of the guinea-pig, NOS neurons are inhibitory motor neurons to the circular muscle; in the ileum and large intestine, NOS neurons may also function as interneurons.
Nitric oxide (NO) is considered an important inhibitory neurotransmitter in the gut. Nitric oxide synthase (NOS)-containing neurons were visualized by immunocytochemistry using antibodies against neuronal NOS in the oesophagus, the gastrointestinal tract and the coeliac ganglion of rat. NOS-containing nerve cell bodies were numerous in the myenteric but fewer in the submucous ganglia all along the gut. Synthesis of NOS in enteric nerve cell bodies was confirmed by in situ hybridization, demonstrating the presence of NOS mRNA. Varicose nerve fibers formed extensive networks in the circular smooth muscle and the myenteric ganglia. The pyloric sphincter contained abundant NOS-containing nerve fibers. NOS-containing nerve terminals were frequently found around the Brunner glands in the duodenum; scattered nerve terminals occurred in the gastric and colonic mucosa and around blood vessels in the submucosa all along the gut. The neuronal cell bodies in the coeliac ganglion were non-immunoreactive but frequently surrounded by baskets of NOS-immunoreactive nerve fibers. Double staining for NOS and neuropeptides in oesophagus, stomach and small and large intestine revealed that a small subpopulation of the NOS-containing nerve cell bodies stored in addition vasoactive intestinal peptide (VIP), and in oesophagus, stomach and small intestine also neuropeptide Y (NPY). However, NOS-containing nerve terminals, particularly those in the circular muscle of the gut, frequently contained VIP throughout the gut; in the oesophagus, stomach and the small intestine they contained also NPY.(ABSTRACT TRUNCATED AT 250 WORDS)
The localization of nitric oxide synthase, the enzyme responsible for producing the short-acting messenger nitric oxide, has been determined in the digestive tract of the rat using histochemistry for reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity, a specific marker for neuronal nitric oxide synthase. Positively stained neurons were found throughout the entire digestive tract from the esophagus to the rectum. Positive neuronal somata were very common in the myenteric ganglia. Dense positive fibers were distributed in internodal strands, the secondary plexus, the tertiary plexus, and were particularly abundant in the deep muscular plexus, while very few were observed in the submucosal ganglia. The density of these positive structures was higher in the small and large intestine than in the esophagus and stomach. The pattern of distribution suggested that some of these positive cells innervate gut muscles. Double-staining revealed that in these enteric neurons, nitric oxide synthase does not co-localize with acetylcholinesterase. Instead, vasoactive intestinal polypeptide almost always coexists with nitric oxide synthase in the myenteric plexus. Thus, nitric oxide and vasoactive intestinal polypeptide may be co-transmitters in a population of non-adrenergic, non-cholinergic neurons in the enteric nervous system.
Acetylcholine‐induced contractions of the rat isolated anococcygeus muscle were blocked by atropine (0.1 μ m ), slightly enhanced by hexamethonium (0.1 m m ) and tetrodotoxin (1 μ m ), but little affected by prazosin (0.1 μ m ).
In the presence of the α 2 ‐adrenoceptor agonist, UK14304, which raised the tone of the muscle, acetylcholine had a biphasic effect consisting of an initial relaxation followed by a contraction.
Atropine (0.1 μ m ) enhanced the relaxant component and abolished the contractile component of the response, whereas tetrodotoxin, ω‐conotoxin GVIA or hexamethonium abolished or greatly reduced the relaxant component.
The nitric oxide synthesis inhibitor N G ‐nitro‐ l ‐arginine methyl ester ( l ‐NAME, 100 μ m ) increased acetylcholine‐induced contractions in the absence of UK14304 and markedly reduced the relaxant component to acetylcholine in the presence of UK14304. The effects of l ‐NAME were annulled by l ‐arginine (300 μ m ).
The results suggest that acetylcholine acts concurrently on muscarinic receptors of the smooth muscle to cause contraction and nicotinic receptors of nitrergic nerves to cause relaxation. The observed response is the resultant of these two opposing effects and depends also on the prevailing tone.
Electron-microscopic studies have revealed a heterogeneous distribution of gap junctions in the muscularis externa of mammalian intestines. This heterogeneity is observed at four different levels: among species; between small and large intestines; between longitudinal and circular muscle layers; and between subdivisions of the circular muscle layer. We correlated results obtained with two immunomethods, using an antibody to the known gap-junctional protein (connexin43) with ultrastructural findings, and further evaluated the respective sensitivity of these two approaches. For comparative reasons we also included the vascular smooth muscle of coronary arteries into our study. Two versions of the immunotechnique (peroxidase-antiperoxidase and fluorescence methods) were applied to frozen sections of murine, canine, and human small and large intestines, as well as to pig coronary artery. In the small intestine of all three species a very strong reactivity marked the outer main division of the circular muscle layer, while the longitudinal muscle layer as well as the inner thin division of the circular muscle layer were negative. In murine and human colon both muscle layers were negative, while in canine colon the border layer between the circular muscle and the submucosa reacted strongly, and scattered activity was found in the portion of the circular muscle layer (one tenth of its thickness) closest to the submucosa. The remainder of the circular muscle layer and the entire longitudinal muscle layer were negative in the canine colon. In the coronary artery we could not confirm the positive, specific labeling reported by other investigators (l.c.).(ABSTRACT TRUNCATED AT 250 WORDS)
In the present study we demonstrate that gap junction coupling between developing layer II/III pyramidal cells in rat sensorimotor cortex is strongly modified by the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system. Dye coupling was revealed by intracellular injection of the gap junction-permeable tracer neurobiotin into single neurones. Following incubation of slices with sodium nitroprusside, a source of NO, the size of dye-coupled cell clusters was significantly reduced. In many cases, 2-3 cells remained strongly dye-coupled. These effects were blocked by intracellular injection of the guanylyl cyclase inhibitor cystamine and mimicked by both application of the membrane-permeant cGMP analogue 8-Br-cGMP and intracellular injection of cGMP. cGMP injection also induced a 60% increase in neuronal input resistance. These results indicate that NO modulates gap junction coupling in the developing neocortex via stimulation of guanylyl cyclase.
Nitric oxide is a ubiquitous cellular messenger that plays a role in a variety of biological mechanisms. Within the central nervous system, it is formed in glia and neuronal cells and can diffuse away from its site of origin to modulate membrane conductances, neurotransmitter release, behavior, or to control the blood flow within its region of action. Nitric oxide has been shown to modulate gap junction conductance in the retina and cortex, an action it shares with a number of neurotransmitters. In this study, we found that the activation of cortical afferents increased dye coupling between rat neostriatal neurons recorded in vitro. This effect was mimicked by a nitric oxide donor and prevented by a nitric oxide synthase inhibitor, suggesting that activation of corticostriatal fibers may open putative gap junctions in the striatum via release of nitric oxide. This is the first report showing that synaptic modulation of gap junctions can be mediated by nitric oxide, and may provide a mechanism to explain cortical modulation of subcortical pattern formation within this highly integrative structure.
The neurochemical composition of nerve fibres and cell bodies in the myenteric plexus of the proventriculus, stomach and small and large intestines of the golden hamster was investigated by using immunohistochemical and histochemical techniques. In addition, the procedures for localising nitric-oxide-utilising neurones by histochemical (NADPH-diaphorase) and immunohistochemical (nitric oxide synthase) methods were compared. The co-localisation of vasoactive intestinal polypeptide and nitric oxide synthase in the myenteric plexus of all regions of the gut was also assessed. The results demonstrated the presence of nerve fibres and nerve cell bodies immunoreactive to protein gene product, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, tyrosine hydroxylase, 5-hydroxytryptamine and nitric oxide synthase in all regions of the gastrointestinal tract examined. The pattern of distribution of immunoreactive nerve fibres and nerve cell bodies containing the above markers was found to vary in different regions of the gut. Myenteric neurones and nerve fibres containing immunoreactivity to nitric oxide synthase and NADPH-diaphorase reactivity, however, were shown to have an identical distribution throughout the gut. In contrast to some studies on the guinea-pig and rat, the co-existence of vasoactive intestinal polypeptide and nitric oxide synthase was seen in only a small population of myenteric neurones.
Cellular networks of pacemaker activity in intestinal movements are still a matter of debate. Because gap-junctional intercellular communication in the intestinal wall may provide important clues for understanding regulatory mechanisms of intestinal movements, we have attempted to clarify the distribution patterns of three types of gap junction proteins. Using antibodies for connexin40, connexin43, connexin45, smooth muscle actin, and vimentin, immunocytochemical observations were made with the confocal laser scanning microscope on cryosections of fresh-frozen small intestine and colon of the dog and rat. Connexin 45 was localized along the deep muscular plexus of the small intestine in both dog and rat. Double labeling studies revealed that connexin45 overlapped with vimentin -, but not actin-positive areas, indicating the fibroblast-like nature of the cells, rather than their being smooth muscle-like. Connexin43 immunoreactivity appeared along the smooth muscle cell surface in the outer circular layer of the small intestine of both animals. Connexin 40 immunoreactivity was not observed in the muscle layer other than in the wall of large blood vessels. It is suggested that connexin45-expressing cells along the deep muscular plexus of dog and rat small intestine are likely to act as a constituent of a pacemaker system, which may include a conductive system, by forming a cellular network operating via specific types of gap junctions.
Nitrergic (NO) neurons play crucial inhibitory roles in the control of gut motility. Variations in the density of these neurons within the gastrointestinal tract (GI) may provide useful functional information, but, most surveys available have employed limited and/or highly localized samples. It remains unclear to what extent (a) NO neurons are concentrated disproportionately in particular GI regions, or (b) variations in NO cell number merely reflect changes in overall myenteric neuron density. This experiment surveyed the distributions of neuronal nitric oxide synthase-positive (NOS+) and other myenteric neurons in the GI tract, using immunohistochemical and Cuprolinic blue counterstaining techniques. Adjustable sampling grids superimposed on wholemounts were used to investigate the topographic patterns in the stomach (90 sampling sites; 45 per side) and proximal duodenum (63 loci). We present four major findings: First, variations were detected in the number of NOS+ neurons in specific regions of the stomach (e.g., corpus > antrum approximately equal to forestomach) and along both longitudinal (oral > anal) and circumferential (mesenteric > antimesenteric) axes in the duodenum. Second, the variations in NOS+ neuronal counts within each organ covaried with the total number of myenteric neurons at different locations (stomach, r=0.77; duodenum, r=0.59), suggesting that local myenteric plexus density is a factor determining NOS+ cell concentrations. Third, in contrast to such a principle of covariation within each organ, NOS+ neurons constituted a consistently smaller proportion of gastric (20%) than of duodenal (28%) myenteric plexus neurons, suggesting that a second principle controls the characteristic percentages of the myenteric plexus that express NOS in different organs. Fourth, the regional samples were used to extrapolate the overall number of NOS+ and total myenteric cells in the rat stomach (43,000; 217,000) and first 3.5 cm of the small intestine (29,000; 103,000). These results, taken together, also suggest that the surveying protocol used is capable of detecting subtle differences in cellular distributions, thus providing a practical strategy for investigating patterns of chemical phenotypes within the GI tract.
To identify the enzymatic source of nitric oxide (NO) in the lower esophageal sphincter (LES), studies were performed in wild-type and genetically engineered endothelial nitric oxide synthase [eNOS(-)] and neuronal NOS [nNOS(-)] mice. Under nonadrenergic noncholinergic (NANC) conditions, LES ring preparations developed spontaneous tone in all animals. In the wild-type mice, electrical field stimulation produced frequency-dependent intrastimulus relaxation and a poststimulus rebound contraction. NOS inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM) abolished intrastimulus relaxation and rebound contraction. In nNOS(-) mice, both the intrastimulus relaxation and rebound contraction were absent. However, in eNOS(-) mice there was no significant difference in either the relaxation or rebound contraction from the wild-type animal. Both nNOS(-) and eNOS(-) tissues showed concentration-dependent relaxation to NO donor diethylenetriamine-NO and there was no difference in the sensitivity to the NO donor in nNOS(-), eNOS(-), or wild-type animals. These results indicate that in mouse LES, nNOS rather than eNOS is the enzymatic source of the NO that mediates NANC relaxation and rebound contraction.
This manuscript reviews gap junctions' roles in control of intestinal motility. Gap junctions (GJs) of small intestine (SmIn) are found between circular muscle (CM) cells, between interstitial cells of Cajal (ICC) of deep muscular plexus (DMP) and between them and adjacent outer circular muscle (OCM). GJs between longitudinal muscle (LM) cells or between cells of inner circular muscle (ICM) have not been reported. Occasional GJs have been reported between ICC of the myenteric plexus (MyP) and rarely between these ICC and adjacent LM or CM cells, or between ICC within CM and smooth muscle cells. In the colon (Co) of several species a special network of ICC lines the inner border of CM, the submuscular plexus (SP). GJs are found between ICCs and between them and CM cells. The ICC of MyP of Co are associated with LM and CM; occasional GJs exist between ICC and each muscle layer. Small GJs are missed by electron microscopy or light microscopic Immunocytochemistry. Therefore, GJ coupling may exist without demonstrated GJs. The consequences for the pacemaking functions of ICC networks of varied densities of GJ between ICC and between ICC of MyP or DMP or of SP and CM are considered. Connexins (Cxs) that compose intestinal GJs may affect coupling, but are incompletely known. Understanding of the role of GJs in coordinating intestinal motility requires knowing: (1) what passes through gap junctions to couple ICC to smooth muscle cells; (2) what Cx with what conductances and what modulatory controls connect ICC and smooth muscle cells; (3) whether smooth muscles can generate slow waves independent of ICC networks; and (4) what happens to motility, slow waves, and IJPs when GJs are selectively uncoupled.
This paper, written for the symposium in honour of more than 40 years' contribution to autonomic research by Professor Geoffrey Burnstock, highlights the progress made in understanding the organisation of the enteric nervous system over this time. Forty years ago, the prevailing view was that the neurons within the gut wall were post-ganglionic neurons of parasympathetic pathways. This view was replaced as evidence accrued that the neurons are part of the enteric nervous system and are involved in reflex and integrative activities that can occur even in the absence of neuronal influence from extrinsic sources. Work in Burnstock's laboratory led to the discovery of intrinsic inhibitory neurons with then novel pharmacology of transmission, and precipitated investigation of neuron types in the enteric nervous system. All the types of neurons in the enteric nervous system of the small intestine of the guinea-pig have now been identified in terms of their morphologies, projections, primary neurotransmitters and physiological identification. In this region there are 14 functionally defined neuron types, each with a characteristic combination of morphological, neurochemical and biophysical properties. The nerve circuits underlying effects on motility, blood flow and secretion that are mediated through the enteric nervous system are constructed from these neurons. The circuits for simple motility reflexes are now known, and progress has been made in analysing those involved in local control of blood flow and transmucosal fluid movement in the small intestine.
In the canine gastrointestinal tract, the roles that gap junctions play in pacemaking and neurotransmission are unclear. Using antibodies to connexin (Cx)43, Cx45, and Cx40, we determined the distribution of these connexins. Cx43 was present in all locations where structural gap junctions occur. Cx40 was also widely distributed in the circular muscle of the lower esophageal sphincter (LES), stomach, and ileum. Cx45 was sparsely distributed in circular muscle of the LES. In the interstitial cells of Cajal (ICC) networks of myenteric plexus, in the deep muscular and submuscular plexuses, sparse Cx45 and Cx40 immunoreactivity was present. In colon, immunoreactivity was found only in the myenteric and submuscular plexus and nearby circular muscle cells. No immunoreactivity was found in sites lacking structural gap junctions (longitudinal muscle, inner circular muscle of the intestine, and most circular muscle of the colon). Studies of colocalization of connexins suggested that in the ICC networks, some colocalization of Cx43 with Cx40 and/or Cx45 occurred. Thus gap junctions in canine intestine may be heterotypic or heteromeric and have different conductance properties in different regions based on different connexin compositions.
Inhibitory interneurons often generate synchronous activity as an emergent property of their interconnections. To determine the role of electrical synapses in such activity, we constructed mice expressing histochemical reporters in place of the gap junction protein Cx36. Localization of the reporter with somatostatin and parvalbumin suggested that Cx36 was expressed largely by interneurons. Electrical synapses were common among cortical interneurons in controls but were nearly absent in knockouts. A metabotropic glutamate receptor agonist excited LTS interneurons, generating rhythmic inhibitory potentials in surrounding neurons of both wild-type and knockout animals. However, the synchrony of these rhythms was weaker and more spatially restricted in the knockout. We conclude that electrical synapses containing Cx36 are critical for the generation of widespread, synchronous inhibitory activity.
Interstitial cells of Cajal (ICC) pace gastrointestinal phasic activity and transmit nerve activity. Gap junctions may couple these cells to smooth muscle, but no functional evidence exists. The objective of this study was to use uncouplers of gap junctions, 18 alpha-glycyrrhetenic acid and its water-soluble analogue carbenoxolone, to evaluate if gap junctions function in pacing and neurotransmission. After inhibition of nerve function with tetrodotoxin (TTX) and N(G)-nitro-L-arginine (L-NOARG), ionomycin- or carbachol-initiated regular phasic activities of circular muscle strips from canine colon and ileum. In some cases, the primary ICC network responsible for pacing was removed. The effects of inhibitors of gap junction conductance (10(-5)-10(-4) mol L(-1)) on frequencies and amplitudes of contraction were compared to appropriate time controls. Lower oesophageal sphincter (LOS) relaxations to nerve stimulation were studied before and after inhibition of gap junction functions. No major changes in LOS relaxations or frequencies of colonic or ileal contractions occurred, but amplitudes of contractions decreased from these agents. Similar results were obtained when the myenteric plexus-ICC network of ileum was removed. Regular phasic activity was not obtained after removal of the colon submuscular plexus ICC. These findings suggest that mechanisms other than gap junctions couple gut pacemaking activity and nerve transmission.
Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to approximately 1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.
Certain neurons in the mammalian brain have long been known to be joined by gap junctions, which are the most common type of electrical synapse. More recently, cloning of neuron-specific connexins, increased capability of visualizing cells within brain tissue, labeling of cell types by transgenic methods, and generation of connexin knockouts have spurred a rapid increase in our knowledge of the role of gap junctions in neural activity. This article reviews the many subtleties of transmission mediated by gap junctions and the mechanisms whereby these junctions contribute to synchronous firing.