1 Hebrew University-Hadassah Medical School, Jerusalem 91240, Israel; and 2 Department of Physiology, New York Medical College, Valhalla, New York 10595
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ABSTRACT |
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The spatial distribution and changes in intracellular calcium concentration ([Ca2+]i) in myenteric neurons were measured using fura 2 in the longitudinal muscle-myenteric plexus preparation from the guinea pig duodenum. These measurements were made simultaneously with intracellular voltage recordings. The generation of action potentials in the cell bodies of both S- and AH-type neurons increased [Ca2+]i in the processes and cell bodies. There was no measurable delay between the [Ca2+]i changes in the somata and the processes, indicating that these changes were caused by the spread of electrical signals and not by diffusion. The rate of Ca2+ removal was faster in the processes than in the somata, apparently due to the large surface-to-volume ratio in the former. In AH neurons, the [Ca2+]i transient was shorter than the duration of the after-spike hyperpolarization. It is concluded that the two main types of myenteric neurons possess voltage-gated Ca2+ channels in both somata and processes.
fura 2; enteric nervous system; electrophysiology; guinea pig
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INTRODUCTION |
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THE IMPORTANCE OF THE concentration of intracellular calcium ions ([Ca2+]i) in regulating the electrical behavior of myenteric neurons has been recognized since the pioneering electrophysiological work of Nishi and North (17) and Hirst and co-workers (7, 10). This is particularly evident for AH neurons, in which action potentials are carried to a large extent by Ca2+ (10). The prolonged hyperpolarization [afterhyperpolarization (AHP)] that follows a spike in AH neurons was found to be Ca2+ dependent (18, 25, 26). This was further supported by single-electrode voltage-clamp studies in which voltage-dependent Ca2+ currents and Ca2+-activated K+ currents were measured (8, 9). It had been assumed that in S cells no Ca2+ enters the cells during a spike, because spikes were completely blocked by tetrodotoxin (10). In a patch-clamp study of cultured myenteric neurons, two types of Ca2+ currents were identified, but no distinction was made between cell types (1). Tatsumi et al. (23) used fura 2 to measure [Ca2+]i after action potentials and also as a result of a depolarization induced by high extracellular K+. Tatsumi et al. (23) concluded that AH cells possess voltage-dependent Ca2+ channels and also showed a partial correlation between AHP and increased [Ca2+]i. Tatsumi et al. (23) did not report results on S-type neurons. Trouslard et al. (24) measured [Ca2+]i with indo 1 in cultured myenteric neurons and also recorded membrane currents using the patch-clamp technique. They concluded that in S neurons a depolarization, induced electrically or chemically, caused an increase in [Ca2+]i. In these two studies (23, 24) both the spatial and temporal resolutions were low and therefore Ca2+ signals were measured from the soma only; also, the precise time course of the Ca2+ transients was not recorded. Thus the distribution of Ca2+ channels in the cell processes is still unknown. In the present work, we addressed these questions by making intracellular electrical recordings from myenteric neurons and simultaneously measuring [Ca2+]i with a fast cooled charge-coupled device (CCD) camera (14). This enabled [Ca2+]i measurements with high resolution in both the spatial and temporal domains.
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MATERIALS AND METHODS |
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Male guinea pigs weighing 300-400 g were used. The animals were stunned and bled. The duodenum was removed and placed in cold Krebs solution containing (in mM) 120.9 NaCl, 5.9 KCl, 14.4 NaHCO3, 2.5 MgSO4, 1.2 NaHPO4, 2.5 CaCl2, and 11.5 glucose. Longitudinal muscle-myenteric plexus preparations were obtained by removing the mucosa, submucosa, and circular muscle. The preparation was pinned on the bottom of a Sylgard covered chamber. The chamber was superfused with Krebs solution bubbled with 95% O2-5% CO2, and the temperature was 30°C. To prevent muscle movements, the solution contained nicardipine (1 µM). In several experiments, nicardipine was omitted and the results were the same in both cases. Intracellular recordings were made with sharp microelectrodes pulled from thick-wall 1.5-mm glass. The tips of the electrodes were filled with 2 mM fura 2 (Molecular Probes) dissolved in 0.2 M potassium acetate, and the shanks were filled with 4 M potassium acetate.
Fura 2 fluorescence was measured with a filter cube containing 380 ± 5 nm excitation filter, 410-nm dichroic mirror, and 495-nm long-pass emission filter. High-speed optical recordings were made with a cooled slow-scan CCD camera (Photometrics, Tucson, AZ) operated in the frame transfer mode (14). Frame intervals were 25-30 ms. Membrane potentials and [Ca2+]i transients were monitored simultaneously. In some experiments, both biocytin (3.5%) and fura 2 were injected into the neurons. After these experiments, the tissue was fixed in 4% paraformaldehyde overnight. It was then incubated in avidin-horseradish peroxidase (Sigma; Ref. 11) for 2 h and reacted for 40 min in a solution containing 0.2% diaminobenzidine, 0.01% H2O2 in 0.1 M tris(hydroxymethyl)aminomethane buffer (pH 7.2). The tissue was then dehydrated in alcohol, cleared in xylene, and mounted in Entellan (Merck).
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RESULTS |
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Neurons were classified as S or AH according to their responses to depolarizing current pulses 200 ms in duration. Cells responding with long trains of action potentials were classified as S cells, whereas neurons that responded with only 1-2 action potentials and displayed a prolonged (>4 s) hyperpolarization after a single spike were classified as AH cells (7). Only cells that were well filled with the dye, had a stable resting potential, and fired action potentials were used for further study.
We recorded electrical activity from 19 neurons; 7 were S-type cells,
10 were AH-type cells, and 2 cells could not be clearly classified.
Spikes evoked by a depolarizing current pulse caused a rise in
[Ca2+]i
in the somata of all the neurons; most of them also showed a change in
[Ca2+]i
over their processes (Fig. 1). When the
cell was stimulated by a train of brief depolarizing pulses, a distinct
[Ca2+]i
increase was associated with every spike (Fig.
1B,
trace
1). There was no measurable delay
(resolution limited by the frame rate) between the spike and the onset
of
[Ca2+]i
increases. Measurements in areas in which no dendrites were seen did
not show any change in
[Ca2+]i
(Fig. 1B,
trace
5), indicating that fluorescence
changes in the dendrites were not due to light scatter from the soma.
The fluorescence changes (F) measured in the dendrites were always lower than those in the soma, but the fractional change (
F/F) was
nearly the same (Fig. 1C) or higher.
This is probably due to the higher surface-to-volume ratio in the
dendrites, but does not indicate that
Ca2+ current density is the same
in the different compartments.
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Figure 2 shows measurements from an S-type neuron. The morphological classification of this cell as a Dogiel type I cell was verified by the injection of biocytin, which revealed (Fig. 2E) the typical structure of this cell type, with a single long process and several short, broad dendrites (3). During a 1-s depolarizing pulse, the cell fired repetitively and as a result a rise in [Ca2+]i was detected both in the soma (Fig. 2A, trace 1) and in the dendrites (Fig. 2A, trace 2). The decay of the fluorescence transient started immediately after the last action potential, indicating that the dendritic signal was not a result of Ca2+ diffusion from the soma. Figure 2B shows the same traces normalized to the same peak amplitude to facilitate a comparison of the recovery time courses in the two compartments. Clearly [Ca2+]i was removed faster in the dendrites than in the soma, with half-recovery times (t1/2) of 1,250 and 3,000 ms, respectively.
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The recovery rate of [Ca2+]i to resting level depends on the different mechanisms for Ca2+ removal available in a cell, such as buffers or membrane pumps. Introducing a Ca2+ indicator, which in itself is a Ca2+ buffer, into a cell interferes with the removal process (16). Because fura 2 is a relatively strong Ca2+ buffer, the higher its concentration in the cell, the slower the decay time is of the fluorescence transient. This relationship is demonstrated in Fig. 2C in which fluorescence transients from the soma of the same cell at three different times during the experiment are compared. The bar graph in Fig. 2C shows the relative resting fluorescence (proportional to the fura 2 concentration) measured just before stimulation at these three times. As the fura 2 concentration increased, the Ca2+ removal rate became slower. Because we used sharp electrodes, we do not know the exact dye concentration in the cells. To visualize the transients in the dendrites, we used high fura 2 concentrations in the electrodes, which apparently affected the kinetics. Values for t1/2 for both S- and AH-type neurons ranged from 1 to 15 s.
An example of the Ca2+ transients recorded in an AH neuron is shown in Fig. 3. The cell was stimulated to fire five spikes by injecting brief depolarizing current pulses. The spikes were followed by a very prolonged (>15 s) AHP. [Ca2+]i transients were recorded in both the soma and processes of this cell, again with a faster recovery time in the dendrite (Fig. 3A). Note that each spike was associated with a nearly equal step increase in [Ca2+]i and that the AHP reached maximum more than 2 s after [Ca2+]i reached its peak. Whereas the [Ca2+]i transient decayed to half maximum in about 10 s, the AHP remained almost constant for this duration, indicating that the Ca2+ removal process is faster than the AHP decay. Similar observations were made in all AH cells studied. Figure 3B shows, on a normalized scale, the time course of the [Ca2+]i change in the soma compared with that in the nucleus. Note that the fluorescence signal in the nucleus continued to increase after the fifth spike.
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DISCUSSION |
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The results show that depolarizations in both AH and S-type myenteric neurons caused increases in [Ca2+]i. The fast and nearly equal step increases in [Ca2+]i associated with each spike strongly suggest that these changes are due to opening of voltage-gated Ca2+ channels, leading to Ca2+ influx. Previous studies (except Ref. 23) measuring [Ca2+]i in myenteric neurons were done on cultured neurons, whereas we recorded from neurons in a freshly dissected tissue in which the ganglia remained intact. Gelperin et al. (4) found in cultured myenteric neurons that bradykinin induced [Ca2+]i elevations from both intracellular and extracellular sources. The intracellular sources gave rise to a fast, early [Ca2+]i transient, whereas the plateau phase was associated with entry of extracellular Ca2+. These results are in apparent contrast to ours and may be explained by the particular action of bradykinin on myenteric neurons. In agreement with our results, Tatsumi et al. (23) showed that [Ca2+]i transients were absent in a Ca2+-free and high-magnesium solution.
Our results are consistent with previous studies on myenteric neurons (2, 23, 24), which indicated the presence of voltage-gated Ca2+ channels in these cells. However, these investigations (2, 23, 24) provided information only on [Ca2+]i changes in the cell bodies. The high spatial resolution of our experiments enabled us to show for the first time that the processes of both cell types have voltage-gated Ca2+ channels. It could be argued that the measured changes were due to diffusion of Ca2+ from the soma. However, the rise of [Ca2+]i in the processes was virtually simultaneous with that recorded in the soma, ruling out this interpretation (Figs. 1 and 3). It was found (6) that all the processes of AH neurons conduct action potentials. We have no direct evidence that the somatic depolarization propagated actively into the dendrites. As most of the dendritic [Ca2+]i changes were measured within ~100 mm from the soma, this could have resulted from passive propagation of the action potentials. However, the simultaneous change of [Ca2+]i in the soma and the dendrites does indicate the existence of voltage-activated Ca2+ channels in the membrane of both compartments in both S- and AH-type neurons. Such a distribution of Ca2+ channels has been found previously for central neurons such as cerebellar Purkinje cells (21) and pyramidal neurons in the hippocampus (20).
The magnitude and time course of [Ca2+]i changes in the processes are of great functional importance as they are related to transmitter release and excitability changes. It is therefore noteworthy that [Ca2+]i recovered much faster in the dendrites than in the somata. Similar findings were made in central neurons, in which recovery times as fast as 50 ms were measured (5). The fast removal may be explained by the large surface-to-volume ratio in the dendrites compared with the somata. If we assume an equal activity of Ca2+ pumps per membrane area in the two regions, this larger ratio will enable a faster removal of Ca2+ in the dendrites.
Another heterogeneity found in the myenteric neurons was the difference in time course of [Ca2+]i changes in the nucleus and the soma cytoplasm. In the cytoplasm [Ca2+]i started to decline at the end of the electrical activity, whereas in the nucleus its removal was delayed, indicating a slow Ca2+ diffusion into the nucleus. Similar kinetics of the nuclear Ca2+ transient were found in amphibian sympathetic neurons (19).
Our direct [Ca2+]i measurements in AH neurons verified previous studies on these cells, suggesting that AHP depends on Ca2+ influx. We found that the AHP was much more prolonged than the changes in [Ca2+]i (Fig. 3). It has been established that a Ca2+-activated K+ conductance underlies this AHP (9, 26), but no simple explanation is available for the difference in the time courses. A similar discrepancy between AHP duration and [Ca2+]i change was observed in vagal motoneurons (15). The fact that the AHP in the myenteric neurons lasts longer than the change in [Ca2+]i may indicate that Ca2+ is needed for its activation but not for its maintenance, implicating an additional controlling factor. Further studies are obviously needed to clarify the mechanisms of AHP.
In view of the large [Ca2+]i transients measured in S cells, it is interesting to ask why a prolonged AHP is absent in these cells. One possible explanation is that these cells have very few Ca2+-activated K+ channels. Alternatively, such channels may exist in these neurons, but their activation requires higher levels of [Ca2+]i than in AH cells. Such an interpretation is consistent with observations that long spike trains were followed by prolonged AHP in S neurons (7). Kunze et al. (13) suggested that large-conductance (BK) K+ channels mediate the prolonged AHP and that these channels are not activated in S neurons.
In conclusion, we measured increases in [Ca2+]i in both AH- and S-type myenteric neurons in the guinea pig duodenum, which appeared to be due to the activation of voltage-gated Ca2+ channels. Such channels are apparently present on the somata, axons, and dendrites of these cells.
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ACKNOWLEDGEMENTS |
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We thank Drs. W. N. Ross and Y. Yarom for support.
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FOOTNOTES |
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This work was supported by the United States-Israel Binational Science Foundation (BSF 91-00135 and 95-00568) and National Institute of Neurological Disorders and Stroke Grant NS-16295.
Address for reprint requests: M. Hanani, Laboratory of Experimental Surgery, Hadassah Univ. Hospital, Mount Scopus, il-91240 Jerusalem, Israel.
Received 18 March 1997; accepted in final form 22 September 1997.
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