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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ABSTRACT |
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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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INTRODUCTION |
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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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SPATIOTEMPORAL IMAGING WITH MSORT AND VSD |
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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-[[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
-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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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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MAPPING THE HUMAN ENS |
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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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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
(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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SUMMARY, CONCLUSION, AND PERSPECTIVES |
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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.
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ACKNOWLEDGEMENTS |
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This work was supported by the Deutsche Forschungsgemeinschaft (SFB 280 to M. Schemann).
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FOOTNOTES |
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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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