THEME
Cutting-Edge Technology
III. Imaging and the gastrointestinal tract: mapping the human enteric nervous system

Michael Schemann1, Klaus Michel1, Saskia Peters1, Stephan C. Bischoff2, and Michel Neunlist1

1 Department of Physiology, School of Veterinary Medicine, D-30173 Hannover; and 2 Department of Gastroenterology, Hepatology, and Endocrinology, Medical School of Hannover, D-30623 Hannover, Germany


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Monitoring membrane potentials by multisite optical recording techniques using voltage-sensitive dyes is ideal for direct analysis of network signaling. We applied this technology to monitor fast and slow excitability changes in the enteric nervous system and in hundreds of neurons simultaneously at cellular and subcellular resolution. This imaging technique presents a powerful tool to study activity patterns in enteric pathways and to assess differential activation of nerves in the gut to a number of stimuli that modulate neuronal activity directly or through synaptic mechanisms. The optical mapping made it possible to record from tissues such as human enteric nerves, which were, until now, inaccessible by other techniques.

multisite optical recording; voltage-sensitive dyes; enteric nervous system


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WHY USE IMAGING TECHNIQUES? Interaction between cells and multiple layers of functionally different cells is crucial for normal function of the gut. Recordings from individual cells or subcellular structures reveal important information on regulatory mechanisms and signaling cascades but fail to advance our understanding of how this activity pattern is integrated within complex systems. One of the greatest challenges that gut physiologists face is to explain how activity in a network of cells is regulated and how this affects behavior of the gut. This requires integration of vast data acquired at the cellular and subcellular level. This is tedious and often not feasible. To understand how networks function, one has to directly record cell interactions with a temporal and spatial resolution, which does justice to the hundreds of cells being active in concert.

The gut has to fulfill functions as diverse as the transport of luminal contents, the transport across the mucosa, and defense against noxious stimuli. The activity of smooth muscle, blood vessels, secretory, absorptive, and immune cells has to be controlled. All control functions are intimately linked to the gut's own nervous system, the enteric nervous system (ENS). Located within the wall of the gastrointestinal tract, the ENS has many functional and structural similarities to the brain. The ENS consists anatomically and functionally mainly of two ganglionated plexuses. The ENS is not readily accessible because it is, in the case of the myenteric plexus, sandwiched between muscle layers or, in the case of the submucosal plexus, embedded in layers of the submucosa. A number of experimental approaches has advanced our knowledge and appreciation of the ENS. Immunohistochemistry has revealed species-, region-, and target-specific neurochemical coding of enteric cell populations (see Ref. 9). Tracing studies have helped to identify functionally distinct populations in the ENS (see Ref. 3). Intracellular recordings have characterized the neurophysiological and neuropharmacological properties of enteric nerve cells (see Refs. 2 and 20). These studies have led to concepts in which specialized enteric pathways consist of intrinsic primary afferent neurons, interneurons, and motoneurons. One of the best-studied functions mediated by such a pathway is the so-called peristaltic reflex. This was functionally identified a century ago (1). Chemo- or mechanosensors in the gut activate a cascade of events that leads to a descending inhibition and an ascending excitation. Projections of secretomotor pathways have also been identified (see Ref. 9). Activity within and along the enteric neuronal pathways, such as signals among cell networks, remains largely unknown. This would require one to map signal spread within the ENS by monitoring fast voltage changes. This is because the membrane potential controls major neuronal processes such as receptor potentials, the generation of action potentials and their spread along processes, pre- and postsynaptic potentials, neurotransmitter release, and soma excitability.

Imaging techniques that detect activity at many sites have become very potent tools in gut physiology. At an organ level, observation of gut movements together with measurements of muscle activity at closely spaced recording sites have identified motor patterns and their role for transport of luminal content (6, 14). At a cellular level, visualization of cytochrome oxidase activity (8) or of markers for synaptic release (7), activity-dependent expression of immediate early genes (see Ref. 16), or monitoring of intracellular calcium levels in multilayer preparations (17, 19) has been used to observe changes in ENS activity. These approaches have limitations in that they record either only slow events, are not suitable to record dynamic changes, or do not provide direct information on cellular excitability. We believe these are crucial issues in the imaging of nerve activity. Multisite recordings should be used in combination with voltage-sensitive dyes (VSD) to directly monitor fast potential changes in neurons. This requires that multisite optical recording techniques (MSORT) have the spatial and temporal resolution to reveal the action potential discharges of hundreds of individual cells simultaneously (Fig. 1; see Refs. 10 and 21). We have validated that changes in fluorescence of VSDs correspond to signals recorded with intracellular electrodes (10). Thus imaging of fast and slow synaptic and nonsynaptic responses is possible with MSORT (Figs. 1 and 2).


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Fig. 1.   Imaging in the enteric nervous system (ENS). A: principles of multisite optical recording technique (MSORT). 1-(3-sulfonatopropyl)-4-[beta [2-(di-n-octylamino)-6-hapthyl]vinyl]pyridinium betaine (Di-8-ANEPPS)-stained tissue is excited with a xenon arc lamp (lamp house model 770 with 1600 series power supply, Opti-Quip, Highland Mills, NY). The fluorescence changes are detected either with a 468-photodiodes array or a cooled charge-coupled device (CCD) camera made of 70 × 70 pixels (RedshirtImaging, Fairfield, CT). Optical signals are processed with a computer; frame rate is 1.6 kHz with the photodiode-system and 2.7 kHz with the CCD-system, enabling the detection of action potentials. With a ×40 objective (UAPO/340, 1.35 n.A., Olympus, Hamburg, Germany), we detected fractional changes (Delta F/F = change in fluorescence divided by the resting light level) in the range of 0.05-4%. With the ×40 objective, the photodiode system has a spatial resolution of ~280 µm2 per diode, whereas the one of the CCD system is ~24 µm2 per pixel. Both systems allow resolution at a cellular range; the CCD system, even at a subcellular range. B: DI-8-ANEPPS-labeled myenteric ganglion of the guinea pig ileum. The dark outlines of stained cell bodies are visible (inverted image). C: the signals of the photodiodes have been projected into the image, allowing spatial identification of optical signals. Each of the 464 traces represents the condensed signals of individual photodiodes during the recording period. The signals are responses evoked by single-pulse stimulation (0.5 ms, arrow) of an interganglionic fiber tract and represent single sweeps (the color-highlighted traces are shown at an expanded scale in E). For each trace, time course is from left to right and upward deflections correspond to signal amplitude (see scale bars in the bottom right corner). The image also contains a spatial grid (see scale bar in the bottom left corner), and the orientation is that top is oral and bottom is anal. Although no signals are recorded outside the ganglion, deflections can be seen in the traces of those photodiodes that detect activity in the ganglion. The most pronounced deflections representing compound action potentials are from the nerve strand that projects from top to bottom and crosses the ganglion. D: results on immunohistochemical localization of calbindin (in blue)- or choline acetyltransferase (ChAT) immunoreactivity (in green) are demonstrated; mixed colors indicate colocalization. Again, the same traces have been projected into the image. E: the expanded responses of individual sites identified by color highlights in C. Illustrated from left to right (E) are a nerve action potential recorded from an interganglionic fiber tract, a neuron with no response, a neuron with an action potential and a fast excitatory postsynaptic potential (EPSP; see hump after action potential), another neuron showing an action potential and fast EPSP, a neuron discharging an action potential and 1 spontaneous action potential just before the stimulus-evoked action potential. Note that the cell with no response is calbindin positive, the neurons with the fast EPSP and the spontaneously active neuron are ChAT positive but calbindin negative. A/D, analog to digital; Delta F/F, change in fluorescence divided by the resting light level.



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Fig. 2.   MSORT allows one to detect a variety of electrical signals. All responses illustrated here are from guinea pig myenteric ganglion cells. A: a single-pulse stimulation of a nerve strand evoked an action potential (AP) followed by a fast EPSP. B: the response of a nerve strand to a 10-µA single-pulse stimulation is shown. C: the spike amplitude increased with 20-µA stimulus strength, indicating compound AP. D: a 500-ms microejection of 0.5 µM acetylcholine evoked a depolarization and spike discharge. E: the response of a ganglion cell to pulse-train stimulation of a nerve strand (50 Hz for 1 s). During the stimulus, each pulse evoked an AP. This neuron started to discharge APs after the pulse train, indicating that a slow EPSP has been generated. Electrical stimulation of nerve strands is indicated by arrows.


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VSDs have been recently used to monitor the activity of nerves in the ENS (10, 11). The use of MSORT and VSD signals was made possible in the ENS by adapting techniques initially used in invertebrates, central nervous system, and cardiac muscle (4, 5, 13, 21). In the ENS, the membrane-bound styryl dye 1-(3-sulfonatopropyl)-4-[beta [2-(di-n-octylamino)-6-hapthyl]vinyl]pyridinium betaine (Di-8-ANEPPS) has been used to monitor fast changes in membrane potentials (10, 11). The molecular mechanism of VSD is very likely electrochromism, i.e., the dye absorption and emission spectra change with the membrane potential. Depending on the excitation and emission spectra, membrane depolarization can be measured either as an increase or a decrease in fractional change in fluorescence (Fig. 1). In the ENS, VSD respond linearly to changes in membrane potential in the physiological range (10). MSORT, in combination with VSD, has numerous advantages over conventional recording techniques: 1) it allows simultaneous recordings of excitability in a large neuronal population; 2) it is a noninvasive method, in contrast to microelectrode recordings, which may damage the cell membrane and thereby alter ion concentration and homeostasis; 3) it enables recordings from cells that are otherwise inaccessible either because they are too small or hidden between tissue layers. Nevertheless, when using VSD, one has to overcome certain obstacles: 1) specific labeling of cells with VSD, 2) reported phototoxicity of VSD, 3) bleaching of VSD occurs, and 4) a decent signal-to-noise ratio is crucial for the interpretation of optically recorded signals. We have continuously improved our imaging technique to overcome these limitations. The loading procedure is one crucial step. Currently, we use local microejection techniques to apply the VSD through a micropipette directly onto the ganglia (10). This improved the specificity of labeling, the signal-to-noise ratio, and it reduced phototoxicity. Still it remains to be established whether all single-cell bodies in a given ganglion are sufficiently labeled by VSD to detect signals. At present, using short recording periods of up to 3 s, we can record as often as 30 times from the same ganglion or cell without any sign of bleaching or phototoxicity and with a signal-to-noise ratio that often exceeds 5:1. This renders the method useful to record small-amplitude signals, such as subthreshold fast excitatory postsynaptic potentials (EPSPs) (10), and for neuropharmacological studies, which require multiple recording periods to assess the effects of agonists or antagonists with recovery periods interspersed.

Originally, MSORT was used to record activity from dissociated guinea pig submucosal plexus (11). We have developed protocols to use MSORT in freshly dissected preparations from guinea pig, mouse, and human (10, 12). The myenteric or submucosal plexus is kept at physiological temperature, is carbogen-gassed perfusate, and is not treated with antioxidants. An important step here was the ability to selectively label individual ganglia with VSD, thereby improving the signal-to-noise ratio.

MSORT allowed us to record directly the activity of hundreds of nerves simultaneously with a high temporal and spatial resolution (Fig. 1). Activity may occur spontaneously, be induced by exogenous substances, or be evoked by electrical stimulation of fiber tracts, resulting in activation of synaptic release of transmitters or generation of process potentials (Fig. 2). Fast as well as slow EPSPs have been successfully recorded (Fig. 2). Inhibitory synaptic potentials remain difficult to identify, because the underlying hyperpolarization can only be resolved with the direct-current-coupled charge-coupled device (CCD) system. The potential to record fast activity makes it possible to record action potentials and fast synaptic events such as fast EPSPs (Fig. 2). In addition, we recorded action potentials that likely represent compound action potentials from nerve fibers (Fig. 2). This has been revealed by studies using omega -conotoxin GVIA (conotoxin; 0.5 µM) to inhibit synaptic release or TTX (1 µM) to block conduction of action potentials (Fig. 3). Optically recorded signals that were evoked by electrical stimulation of fiber tracts consisted of two components. One is totally blocked by conotoxin as well as by TTX and hence is a fast EPSP. Blockade by hexamethonium (200 µM) further revealed the nicotinic cholinergic nature of the fast EPSP. The other component is an action potential, which occurred with shorter delay than the fast EPSP and which corresponded to a conduction velocity of 0.3-0.5 m/s. This response is not affected by conotoxin or hexamethonium but is totally abolished by TTX and can be recorded from nerve strands as well as from ganglia. This likely represents compound action potentials of nerve processes running with interganglionic fiber tracts or crossing ganglia. Alternatively, it may represent antidromic activation of the cell soma (see below).


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Fig. 3.   Optically recorded fast EPSPs and nonsynaptic responses in a guinea pig myenteric ganglion. A-C: control responses to a single pulse stimulus of interganglionic fiber tracts (arrows). D-F: responses after 15-min application of 0.5 µM omega -conotoxin GVIA. Signal in A is a nerve AP recorded from interganglionic fiber tracts; signals in B and C are from 2 different ganglion cells. Conotoxin blocked the fast EPSPs but not the nerve AP. The AP in B had a conotoxin-sensitive component, indicating that the fast EPSP triggered an AP.

Records of simultaneous fast events at multiple sites of the preparation allow one to create spatiotemporal maps that show excitability spread (Fig. 4). This is one of the prerequisites to map sequential activation within a cellular network and to understand how various substances affect signaling in particular pathways.


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Fig. 4.   Spread of activity evoked by single-pulse stimulation of interganglionic nerve strands in a human submucosal ganglion (A) and a guinea pig myenteric ganglion (B, C). The images are transformations of the optically recorded signals into color codes (see color bar). Dark purple color represents low level, i.e., resting membrane potential, and red the highest, i.e., peak of an AP. Color-coded spatiotemporal maps of fluorescence intensity reveal the spread of electrical activity and illustrate how a signal invades a ganglion in time and space. The frames run sequentially from right to left and from top left to bottom right. Note that the magnification is different for A vs. B and C (compare scale bars indicating the size of the ganglia). First frame in A illustrates DI-8-ANEPPS-labeled human submucosal ganglion. The white outlines of the cells are visible; 9 individual cells are numbered. Next frame to the right shows superimposed the color-coded signals detected by the CCD system just as the initial stimulus response invaded the ganglion (0 ms). At 4 ms, the initial AP exits the ganglion, and activity can still be seen at cell 9. After this initial response, APs (red color) occur at different times in different cells. At 10 ms, cells 4 and 6 show activity that appears to propagate to cell 8 at 14 ms. At 24 ms, cell 9 fires an AP. APs appear to leave the ganglion along the nerve strand at the bottom left. At 47 ms, cell 8 is active again, and at 90 ms, cell 1 is active. In B and C, the outline of a guinea pig myenteric ganglion is visible. Optical recordings were made with the photodiode system. This ganglion contains ~40 nerve cell bodies. The 2 panels illustrate stimulus-dependent signal spread along the ganglion (compare B and C). Nerve strands projecting from above (B) or projecting from the right side (C) have been stimulated. First frame in each panel was acquired before the stimulus (0 ms). Note that the area at which the stimulated fiber tract enters the ganglion is activated first (compare images at 3 ms in B and C). This initial response (red color) is an AP and lasts only for a few milliseconds. However, activation persists for a long time, which is due to fast EPSPs in some neurons. We refer to Fig. 3, B and C, in which the original stimulus-evoked APs and fast EPSPs of 2 of the neurons in this ganglion are illustrated. The color code has been adjusted so that the peak of the AP as well as the peak of the fast EPSP correspond to red color. As with the initial AP, regional variation in fast EPSP discharge occurs, although convergence of inputs is also obvious. For example, compare patterns between B and C at 24, 36, or 42 ms.


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MSORT proved to be a valuable tool to record from nerves that are inaccessible by conventional intra- or extracellular electrophysiological techniques. We were able to reliably and reproducibly record from submucosal as well as myenteric neurons even of humans (Figs. 4 and 5). To demonstrate why MSORT is currently our preferred method to record from the human ENS, we report here our results in surgical specimen removed during cancer surgery (30 preparations from 30 patients). Recordings were made from macroscopically normal tissue dissected so as to expose the submucous plexus or the myenteric plexus (n = 3 preparations). Tissue sample and procedures were approved by the local ethics committee of the Medical School of Hannover (Hannover, Germany).


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Fig. 5.   Optically recorded signals from human submucosal (A, B) and myenteric (C-H) ganglia. In A, the first single-pulse stimulation of a nerve strand (arrow) evoked a fast EPSP-triggered AP; the second pulse induced a subthreshold fast EPSP. B: a ganglion cell showed spontaneous APs. C-E: responses to single-pulse stimulation in a myenteric ganglion cell (C, D) and from a nerve strand (E); note that the neuron in D discharged 2 APs in response to the stimulus. Responses in F and G are abolished by 200 µM hexamethonium, indicating the cholinergic nature of the fast EPSP. However, the AP response of the nerve strand is not changed.

Single-shock electrical stimulation of interganglionic fiber tracts usually activated 55 ± 32% of the neurons per ganglion in the submucosal plexus (based on 256 neurons, 24 ganglia, 11 preparations). Two types of responses were recorded. One was an early action potential occurring at a short delay after the stimulus pulse. The second response was a synaptically evoked fast EPSP. Frequently, several fast EPSPs summated and reached threshold for spike discharge (Fig. 5). Of 247 neurons, 68 showed a fast EPSP only, whereas 169 showed signals consisting of an additional early response (action potential) immediately before the fast EPSP. The fast EPSPs had durations of 42 ± 12 ms (22 signals, 10 ganglia, 10 preparations) and were hexamethonium-sensitive (247/247 neurons), whereas the early response remained unchanged (169/169 neurons; Fig. 5). The early response was, however, totally blocked by TTX in all 75 neurons tested and was thus of neural origin. The conduction velocity of these action potentials was 0.34 ± 0.11 m/s, suggestive of C fiber activation. These action potentials had a half-maximal duration of 2.4 ± 0.4 ms, and some of them might represent antidromic activation of cell bodies because these signals were encountered at the same location as synaptic signals. More likely, they represent compound action potentials of nerve fibers that project within interganglionic fiber tracts and cross through the ganglia because most signals increased with the current strength of the stimulation pulse and because they can also be detected along nerve bundles. In 10 of 179 submucosal neurons, we found evidence for a hexamethonium-insensitive component of the fast EPSP, but the identity of the transmitter mediating such noncholinergic EPSPs remains open. The noncholinergic fast EPSPs had durations of 31 ± 5 ms. The strong dominance of cholinergic fast EPSPs is supported by the Ach-evoked excitation. Local microejection of 0.5 mM Ach by brief pressure pulses (5-200 ms) directly onto the ganglion studied activated 91 neurons (20 ganglia, 14 preparations). Of 18 neurons, the Ach response was totally abolished by hexamethonium in 6 and partially blocked in 12 neurons. The remaining Ach response was blocked by the muscarinic blocker atropin (11/11 neurons).

In addition to these stimulus-evoked signals, we were able to detect spontaneous activity in ~18% of the neurons in the human colonic submucous plexus (88/500; 51 ganglia, 23 preparations; Fig. 5). On the basis of the analysis of interspike intervals in 64 spontaneously active neurons, a prominent peak was found at 134 ms, which corresponds to a 7.5-Hz spike-discharge rate. Hexamethonium blocked spontaneous activity in only 12 of 34 neurons.

In some experiments, interganglionic fiber tracts on the oral and the anal side of the ganglia were stimulated. This revealed that most of the neurons (128/256) responded to stimulation on either side of the ganglion, whereas a smaller group of neurons did not respond at all (69/256). A subset of neurons responded only to stimulation on the oral or the anal side of the ganglion, and it was found that more neurons responded to oral (37/256) than to anal stimulation (23/256; P = 0.014, z-test). This analysis revealed that spread of activity is, in part, pathway dependent, although a substantial degree of convergence occurs.

After the experiments, we performed triple immunohistochemistry to label neurons for vasoactive intestinal polypeptide (VIP), choline acetyltransferase (ChAT), and substance P. For 365 neurons whose signals we had recorded, it was possible to determine the neurochemical code. The proportion of neurons with a particular neurochemical code was in good agreement with a previous study (15), indicating that there was no experimental bias in that VSD may preferentially label a certain neuronal population. A potential problem is that the quality of the immunohistochemistry decreased with longer recording periods.

On the basis of studies in which we randomly stimulated single nerve fibers entering a ganglion, we found that 67% of 175 neurons exhibiting a fast EPSP were VIP positive and 33% were ChAT positive but VIP negative. Of 119 neurons with no fast EPSP, 56% were VIP positive and 44% were ChAT positive but VIP negative. Thus neurons responding with fast EPSPs were significantly more often VIP positive (chi 2-test; P = 0.042). Of those neurons with spontaneous activity, many were VIP positive (78% of 46 neurons), and this was significantly more than in the population of nonspontaneously active neurons (60% of 310 neurons).


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Imaging techniques using fast optical recordings are powerful tools to analyze signal spread in the gut. Multisite recording approaches provide valuable tools to study how cells interact and to identify specific pathways. A particular advantage is that these imaging techniques can detect cell activity in tissues difficult to access from small laboratory animals, such as the mouse, to larger mammals, widely, humans. After the basic neuropharmacological, synaptic, and electrical properties of human enteric neurons is established, it should be rewarding to look for changes in tissue from patients suffering from functional, inflammatory, or structural bowel disease. Equally important should be to use MSORT to record nerve activity in response to a variety of physiological stimuli that activate mechano- or chemoreceptors.

Mathematical and computer modeling of the enteric nervous system have been tackled (18). It is essential that these models are based on experimental evidence and that their predictions are tested in live tissue with appropriate recording techniques. MSORT is ideal to provide such data.

In addition to the continuous technological progress for MSORT and improved fluorescent probes, novel approaches using animals genetically modified to express encodable activity-sensitive fluorescent indicators is of particular interest. Also, MSORT is not restricted to the use of voltage-sensitive dyes but, in principle, may be used with all fluorescent dyes. Other fluorescent dyes may prove of value to study additional events that involve multiple sites.


    ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 280 to M. Schemann).


    FOOTNOTES

Present Address of M. Neunlist: INSERM U 539 Hopital Hôtel Dieu, 3ème Nord Place Alexis Ricordeau, 44035 Nantes, France.

Address for reprint requests and other correspondence: M. Schemann, Dept. of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15/102, D-30173 Hannover, Germany (E-mail: Michael.Schemann{at}tiho-hannover.de).

First published February 27, 2002;10.1152/ajpgi.00043.2002


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