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First published online July 25, 2005
Journal of Experimental Biology 208, 2981-3001 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01732

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Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system

A. L. Obaid¹, M. E. Nelson¹, J. Lindstrom¹ and B. M. Salzberg^1,2,*

¹ Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6074, USA
² Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6074, USA

View larger version (73K): [in a new window]   Fig. 1. mAb 313, which selectively recognizes 3-nAChRs, shows a pattern of immunoreactivity very similar to that exhibited by mAb 210 in permeabilized tissue. (A) Submucous ganglion stained with mAb 210 (17 nmol l–1) and FITC-goat-anti-rat (1:1000). Scale bar, 25 µm. (B) Submucous ganglion stained with mAb 313 (35 nmol l–1) and FITC-goat-anti-rat (1:1000). Scale bar, 20 µm. In both preparations, the tissue was permeabilized with 0.5% Triton X-100.

View larger version (124K): [in a new window]   Fig. 2. The pattern of immunofluorescence obtained using mAb 306 mimics the distribution of BgT-binding in submucosal ganglia, and the block of BgT-binding by nicotine confirms that BgT reacts specifically with enteric 7-nAChRs. (A) Submucosal ganglion stained with mAb 306 (17 nmol l–1) and FITC-conjugated goat-anti-mouse. Fixed tissue permeabilized with Triton X-100 (0.5%). (B) Negative control for A, obtained from the same preparation in the absence of mAb 306. (C) Binding of Alexa 594-conjugated BgT (250 nmol l–1) to a live segment of submucosal plexus. (D) The binding of Alexa 594-conjugated BgT was blocked by a 16 h co-incubation with 1 mmol l–1 nicotine. Scale bar, 25 µm.

View larger version (73K): [in a new window]   Fig. 3. 7-nAChRs are also present in myenteric plexus, but their distribution is not as ubiquitous as in the submucous plexus. (A,C) Red (Alexa 594-conjugated mAb 210, 7 nmol l–1) and green (17 nmol l–1 mAb 306/FITC-conjugated goat-anti-mouse 1:1000) channels of a confocal micrograph taken from a myenteric ganglion double-stained for 3/5- and 7-containing nAChRs. (B) Combined immunofluorescence from A and C. In A–C, the arrow points to a cell that expresses 3/5- but not 7-nAChRs. The upward arrow head points to a cell that expresses 7- but not 3/5-nAChRs. The downward arrow head points to a cell that expresses both types of nAChRs. Scale bar, 50 µm.

View larger version (49K): [in a new window]   Fig. 4. The pattern of ß2-immunoreactivity in the submucous plexus is very similar to the pattern of immunoreactivity for 3/5-nAChRs, suggesting that these subunits are structural partners in the same heteromeric nAChRs. (A,C) Green (Alexa 488-conjugated mAb 210, 7 nmol l–1) and red (33 nmol l–1 mAb 295/TR-conjugated goat-anti-rat 1:1000) channels from a confocal micrograph taken from a submucous ganglion double-stained for 3/5- and ß2-nAChRs. (B) Combined immunofluorescence from A and C. The upward arrow points to one of the multiple neurones that express 3/5- as well as ß2-nAChRs. The horizontal arrow head points to a cell that expresses predominantly ß2-nAChRs. The vertical arrow head points to a cell that expresses mostly 3/5-nAChRs. Scale bar, 50 µm.

View larger version (40K): [in a new window]   Fig. 5. High-speed camera recordings from submucous ganglia reveal the differential sensitivities of individual neurones to specific nAChR-antagonists. (A) High-resolution image of a submucous ganglion stained with the naphthylstyryl-pyridinium dye di-4-ANEPPDHQ (inverted grey scale), showing the individual neurones identified by numbers. The stimulation electrode, indicated schematically, was on an adjacent ganglion to the upper right, out of the field of view. (B) Pixel map of the NeuroCCD-SM camera depicting, in red, the pixels within the area of interest. (C) Experimental protocol. (D) High-speed optical recordings from the ganglion in A. Data are presented in two ways: signals spatially averaged over the whole ganglion [row of bars labelled `Ganglion', whose heights represent the amplitude of the voltage change averaged over the area of interest (red pixels in B)] and signals spatially averaged over individual neurones (`Cells' numbered 1–11). The colours match the conditions illustrated in C and reflect the steady-state responses obtained during successive drug applications (see Fig. 6B,C). Since no absolute membrane potential calibration is possible in this type of experiment, the vertical axis in this and all subsequent optical recordings represents changes in fluorescence intensity in arbitrary units (F). Illumination was reduced 21-fold by inserting neutral density filters in the light path; its duration was limited to 1.8 s per trial. Magnification, 100x. All traces were band-pass filtered between 6.6 and 200 Hz.

View larger version (18K): [in a new window]   Fig. 6. The serial effects of -CTx MII, -CTx AuIB and Mec are additive and not the result of gradual deterioration of the preparation. (A) Actual optical traces, spatially averaged over the entire ganglion from the experiment in Fig. 5, are shown instead of bars to highlight the progression of the drug effects. Here, all the trials recorded during the experiment are shown. Note the obvious clustering of responses according to colour. (B) Inhibitory effects on evoked synaptic responses (spatially averaged over the whole ganglion) of sequential application of nAChR-antagonists. (C) Examples of differential sensitivity of individual neurones to these antagonists. As in Fig. 5, all traces were band-pass filtered between 6.6 and 200 Hz.

View larger version (33K): [in a new window]   Fig. 7. The serial effects of -CTx AuIB and -CTx MII are also additive, suggesting that 3ß2- and 3ß4-nAChRs can exist as independent entities in submucosal neurones. (A) High-resolution image of a submucosal ganglion (inverted grey scale), showing the individual neurones identified by numbers. The position of the stimulation electrode is shown schematically. (B) Pixel map of the NeuroCCD-SM camera depicting, in red, the area of interest. (C) Experimental protocol. (D) Spatially averaged optical outputs from the red pixels in B (represented by bars whose heights indicate the amplitude of the voltage change under each experimental condition in the row labelled `Ganglion') and from selected individual neurones (`Cells'). Magnification, 100x. `Ganglion' signals were high-pass filtered at 6.6 Hz. `Cell' traces were low-pass filtered at 200 Hz.

View larger version (39K): [in a new window]   Fig. 8. 7-nAChRs contribute substantially to the evoked response in submucosal neurones. (A) High-resolution image of a submucosal ganglion (inverted grey scale), showing the individual neurones identified by numbers. The position of the stimulation electrode is shown schematically. (B) Pixel map of the NeuroCCD-SM camera depicting, in red, the area of interest. (C) Experimental protocol. (D) Spatially averaged optical outputs from the red pixels in B (represented by bars whose heights indicate the amplitude of the voltage change under each experimental condition in the row labelled `Ganglion') and from the pixels that outline individual neurones (`Cells'). Magnification, 100x. `Ganglion' signals were high-pass filtered at 6.6 Hz. `Cell' traces were unfiltered.

View larger version (46K): [in a new window]   Fig. 9. The effects of -CTx AuIB and -CTx MII on submucosal ganglia can be reproduced on myenteric ganglia, confirming that 3ß2- and 3ß4-containing nAChRs are expressed in both enteric plexuses. (A) High-resolution image of a myenteric ganglion (inverted grey scale). The position of the stimulation electrode is shown schematically. (B) Pixel map of the NeuroCCD-SM camera depicting, in dark grey, the area of interest. Notice that the highlighted area is considerably bigger than the ganglion itself. (C) Experimental protocol. (D) Spatially averaged optical outputs from the dark-grey pixels in B, represented by bars whose heights indicate the amplitude of the voltage change under each experimental condition. Magnification, 40x. All signals were high-pass filtered at 9.8 Hz.

View larger version (37K): [in a new window]   Fig. 10. nAChR antagonists can also enhance synaptically evoked responses in the myenteric plexus. (A) High-resolution image of a myenteric ganglion (inverted grey scale), showing three individual neurones identified by numbers. (B) Pixel map of the NeuroCCD-SM camera depicting, in dark grey, the area of interest. Notice that, as in Fig. 8, the highlighted area is considerably bigger than the ganglion itself. (C) Low-magnification image of the preparation (20x), illustrating the location of the stimulating electrode (Es) with respect to the recording area. (D) Experimental protocol. (E) Spatially averaged optical outputs from the dark-grey pixels in B (represented by bars whose heights indicate the amplitude of the voltage change under each experimental condition in the row labelled `Ganglion') and from the pixels that outline neurones 1, 2 and 3 (`Cells'). Magnification, 100x. `Ganglion' signals were high-pass filtered at 6.6 Hz. `Cell' traces were unfiltered.

View larger version (102K): [in a new window]   Fig. 11. Myenteric neurones exhibit differential pharmacological responses to a given nAChR antagonist depending upon the location of the stimulation electrode, oral or aboral with respect to the ganglion. (A) Low-magnification image of the preparation (20x), illustrating the location of the stimulating electrodes (E1 and E2) with respect to the recording area. (B) High-resolution image of the myenteric ganglion of interest (inverted grey scale), showing four individual neurones identified by numbers. (C) Serial images of the selected myenteric ganglion taken at 20 min intervals throughout the experiment. Notice the changes in ganglion shape (and therefore in registration with respect to the camera) from one frame to another. The arrows have been inserted as fixed references to draw attention to the magnitude of the ganglionic movements. Magnification, 100x. (D) Stimulation protocol. (E) Experimental protocol. (F) Optical responses evoked by each of the stimulation electrodes (E1 and E2) according to the protocol described in D. Data are presented in two ways: signals spatially averaged over the whole ganglion (row of bars labelled `Ganglion', whose heights represent the amplitude of the voltage change evoked by each electrode, averaged over the area of interest) and signals spatially averaged over individual neurones (`Cells' numbered 1–4). (G) High-speed camera movies showing the spatial pattern of responses to stimulation by electrodes E1 and E2, before and after 25 min exposure to 100 nmol l–1 -CTx MII. Frame acquisition 1 kHz. Unfiltered.

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