One difficulty in these experiments is that a typical change. in light intensity recorded during a single voltage oscillation

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1 Proc. Natl. Acad. Sci. USA Vol. 86, pp , March 1989 Neurobiology Spatially and temporally resolved calcium concentration changes in oscillating neurons of crab stomatogastric ganglion (arseiazo l/bursting/optical recording/cancer irroratus/cancer borealis) W. N. ROSS*t AND K. GRAUBARDt* *Department of Physiology, New York Medical College, Valhalla, NY 10595; tdepartment of Zoology, University of Washington, Seattle, WA 98195; and tmarine Biological Laboratory, Woods Hole, MA Communicated by Theodore H. Bullock, November 14, 1988 ABSTRACT Calcium concentration changes during oscillations of the membrane potential of crab (Cancer irroratus or Cancer borealis) stomatogastric neurons were monitored at many positions by using the calcium indicator dye arsenazo m and a photodiode array. Data analysis algorithms using signal averaging techniques were developed to improve the time resolution of the measured calcium changes. As previously reported, calcium oscillations were detected from all regions of the neuropil but not from the soma or axon. In some cells step increases in intracellular neuropil calcium were correlated with each of the action potentials in the burst (on the peak of the voltage oscillation). In other cells we observed calcium oscillations phase-locked to the membrane potential with no spikerelated component. A few cells had both spike-evoked and graded potential components- to the calcium oscillations. In those cells, the spatial distribution of the spike-correlated calcium influx differed from that of the voltage-oscillationcorrelated calcium influx, suggesting that different neurites might interact with their postsynaptic targets with different mixtures of graded and spike-correlated transmitter release. The stomatogastric ganglion of decapod crustaceans is one of the best understood nervous systems. All of its neurons have been identified and the synaptic connections among them have been characterized (reviewed in ref. 1). Several circuit models have been developed to summarize the current understanding of this system (reviewed in ref. 2). In these models each neuron is usually conceived of as a single entity, interacting with pre- and postsynaptic cells at a single point. Yet it is clear that the synaptic interactions are spatially distributed along the fine neurites (3-5). This suggests that it is important to examine not just which cells are interacting but also where in the neuropil they contact each other and the nature of the interactions at those locations. For example, it has been shown that many stomatogastric neurons interact with their target cells by using a mixture of spike-evoked and graded synaptic potentials (reviewed in ref. 6). Thus it would be useful to know if these two components are found at all neuropil locations and with what relative strength. We have begun to use optical techniques to examine the regional properties of stomatogastric neurons. In our previous paper (7) we reported the use of the calcium indicator arsenazo III and a photodiode array to detect calcium changes at many locations in these cells. We found that depolarizing stimuli to the cell body elicited calcium changes over all of the neuron except the axon, suggesting that calcium channels were distributed over all the nonaxonal surface membrane of the cells. Pyloric neurons normally undergo membrane-potential oscillations which can include a burst of spikes during the depolarized phase. We found that such voltage oscillations were correlated with oscillations in calcium concentration that were restricted to the neuropil region of the neuron. One difficulty in these experiments is that a typical change in light intensity recorded during a single voltage oscillation is 5 X 10-5 of the average intensity. Even with careful attention to noise reduction, the oscillations in calcium concentration can rarely be detected without averaging. Since the membrane potential oscillation is never exactly repeated, the simple averaging procedure we used in our earlier experiments gave averaged records which lost the time resolution inherent in the raw data. For the experiments reported here we implemented data taking and analysis procedures that maintained this time resolution. Using these techniques, we now report the following results: for many bursting neurons, two components of the neuropil calcium oscillation can be detected, one correlated with spike activity and a slower component related to the underlying voltage oscillation. Often the spike-correlated changes are relatively larger in those neuropil regions expected to be electrotonically close to the axon. Neuropil regions that appear to be electrotonically far from the axon sometimes show little or no spike-correlated jump in intracellular calcium concentration, but instead show a smooth rise in calcium that often begins before the first spike in the burst. As a result, some neurons show regional variations in the time course of calcium concentration changes. Finally, in some neurons which have membrane potential oscillations that are below threshold for spikes, it is still possible to demonstrate voltage-correlated oscillations in neuropil calcium concentration. Some of these results have been reported in abstract form (8). MATERIALS AND METHODS The stomatogastric ganglion and connecting nerves and ganglia from crabs (Cancer irroratus or Cancer borealis) were dissected and desheathed by standard techniques (9, 10). The preparation was mounted on the stage of a compound microscope and individual cells were impaled with microelectrodes and filled with arsenazo III by ionophoresis. Some of the cells were identified by suction electrode recordings from nerves. Changes in intracellular calcium concentration were measured as changes in absorbance at 660 nm and were recorded at all locations by using the apparatus described previously (7). Soma membrane potential was recorded simultaneously with a microelectrode (usually with the same electrode used for dye injection, or occasionally, with a KCI-filled pipette). Cells were later filled with Lucifer yellow and reconstructed from photographs taken in situ before fixation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact Abbreviations: LP neuron, lateral pyloric neuron; VD neuron, ventriculator dilator neuron; IC neuron, inferior cardiac neuron; PD neuron, pyloric dilator neuron.

2 1680 Neurobiology: Ross and Graubard 6rP) 64 gm 1 sec soma voltage FIG. 1. Spatial distribution of calcium concentration oscillations in an LP neuron. Each trace shows the time course of the calcium change detected by an element of the array. At the magnification used for this study each array element recorded from a 64-Am x 64-,m square of tissue. One hundred and twenty-five 1-sec segments were averaged by using the procedure described in Materials and Methods. The calcium scale for the traces is arbitrary, but it is the same for all traces. The shape of the neuron was determined from a through-focus series of photographs of the fluorescence from the Lucifer-yellow-filled cell. There are calcium oscillations in all neuropil locations, but not over the soma or axon. The soma voltage trace shown in all figures is the average of the soma voltages for the same times as the optical signal averages. In individual sweeps, the typical soma peak-to-trough voltage level was 16 mv with 7-mV spikes for a total voltage change of 23 mv. The trough voltage was approximately -56 mv. Computer programs were written for data acquisition and analysis. For data acquisition, a series of long time segments of optical and electrical data was recorded and stored on a hard disc. Optical records were low-pass filtered with a time Proc. Natl. Acad. Sci. USA 86 (1989) constant of 10 msec and were typically sampled every 8 msec. [The optical records shown in Fig. 1 were additionally filtered with a digital algorithm (11) to a high-frequency cut-off of 50 Hz.] Electrical records were sampled at a 10 times higher rate than the optical records. Each segment of 1024 optical data points (8 sec) contained 6-20 oscillations ( Hz). For analysis, the soma-voltage data segments were read back and displayed one at a time. A cursor was used to mark the time of the first action potential in a burst or (if there were no action potentials) the time of the peak of the slow oscillation. A 250-point interval in the optical records, centered at the marked time, was then selected. To improve the signalto-noise ratio of the optical records, the data for all of the intervals were added together. The process of selection ensured that the time of the first action potential or oscillation peak was always the same. However, the second or later action potentials were not so precisely aligned. Therefore, in the averaged electrical records the first action potential appears larger and sharper than the others; this is a consequence of averaging data with jitter in the interspike intervals. RESULTS Fig. 1 shows a montage of calcium changes detected during natural oscillations of the somatically recorded membrane potential of a lateral pyloric (LP) neuron. Calcium oscillations occur at all locations over the neuropil but are not observed over the soma or the axon. This is the same result described in our previous paper (7) except the new averaging procedure has greatly improved the signal-to-noise ratio of the optical records. Fig. 2 shows a comparison between the averaged membrane potential and the calcium changes detected simultaneously from two neuropil locations of a ventricular dilator (VD) or inferior cardiac (IC) neuron. Each action potential in the burst matches a step change in calcium entry. In fact, for this neuron, it appears that all of the calcium entry can be accounted for by these steps; there is no obvious increase during the slowly rising membrane potential which preceded the burst (although there is a slowing in the rate of fall of a calcium which could be due to either a reduced removal or to an increased influx). This was true for all neuropil regions of this neuron. FIG. 2. Comparison between the time course of the calcium concentration change at two neuropil locations and the somatically recorded membrane potential. (A) One hundred traces of 592 msec each were averaged with the time alignment determined by the peak of the first action potential. The calcium traces have been scaled to the same peak-to-peak amplitude. The rise in calcium occurs in steps (arrows) which correlate with the action potentials. In individual sweeps, the soma voltage oscillation (including spikes) was about 25 mv. (B) Reconstruction of the VD or IC neuron with the locations of the two neuropil calcium traces shown in A indicated by stippling. Each stippled pixel element is 64,.m x 64 um in all figures.

3 Neurobiology: Ross and Graubard Proc. Natl. Acad. Sci. USA 86 (1989) 1681 B FIG 3. Comparison between the time course of the neuropil calcium concentration change and the membrane potential in another VD or IC neuron. (A) Two hundred and eighty-six traces of 744 msec each were averaged with the time aligned on the peak of the first spike in the burst. The calcium traces have been scaled to the same peak-to-peak amplitude. In individual sweeps, the soma peak-to-trough voltage was 15 mv with a 8-mV spike for a total voltage excursion of 23 mv. The trough voltage was approximately -67 mv and the bath temperature was approximately 18'C. The calcium increase begins before the action potential and there is little additional influx correlated with the times of the spikes. (B) The reconstructed cell with the three pixels shown in A marked by stippling. Calcium entry in the neuropil was not always exclusively associated with the occurrence of action potentials. Fig. 3 shows a similar comparison from another VD or IC neuron. In this case the rise in calcium concentration clearly precedes the first action potential. This signal implies that there is also a calcium increase associated with the slow membrane potential change. This conclusion was made even clearer in experiments where there were no action potentials on the peaks of the slow oscillations. We examined long records of membrane potential oscillations for intervals where the peak of the oscillation failed to reach spike threshold. Only these intervals were averaged. Even in these cases, slow calcium oscillations were phase locked to the membrane potential oscillation (Fig. 4). These signals were found over the entire neuropil but not in the soma or axon. For most cells the time course of the calcium oscillations was similar at all neuropil locations. However, other studies led us to believe that calcium entry might vary in different A voltage regions. Spikes in stomatogastric neurons are initiated near the region where the axons leave the ganglion and are only passively spread through the neuropil and to the soma (12, 13). Also, the conductances responsible for the plateau are probably nonsomatic (14). If calcium entry is through a voltage-gated calcium channel, then the variation in potential in different regions might lead to different calcium signals in those regions. If there is more than one type of calcium channel in these cells (7, 12), this could lead to further spatial variation. Therefore, we examined our records for variations in time course in different regions. Fig. 5 shows two clear examples of spatial heterogeneity. For each cell two neuropil locations are compared in detail. The locations closer to the place where the axon exits the ganglion have clear spike-related signals. The locations at the far end of the neuropil appear to have mostly a graded signal which begins to rise before the spike. Locations in between had signals that resembled a mixture of these two components. & BAB axon v I I I f ff' I' II II\ IIA 300 mse; C I FIG. 4. Oscillations in calcium concentration occur in the absence of spikes. (A) One hundred and ninety traces of 536-msec duration were averaged with the time aligned on the peak of the soma voltage oscillation. The calcium traces have been scaled to the same peak-to-peak amplitude. Calcium oscillations from three neuropil locations (solid lines) show similar waveforms and phase relationships compared to the soma voltage (broken line). In individual sweeps, the soma peak-to-trough voltage excursion was approximately 10 mv. (B) The reconstructed neuron (probably a pyloric cell) with the locations of the three pixel elements whose output is shown in A marked by stippling. Each pixel is 64 gum x 64 lim.

4 1682 Neurobiology: Ross and Graubard -2./ "\.-1.V 200 msec FIG. 5. Spatial variation in the time course of calcium concentration oscillations in different neuropil regions of two neurons. Comparison of two calcium signals with the somatically recorded membrane potential. (A and B) Same neuron and data set as in Fig. 3. The location closer to the axon (solid line in A, left pixel marked by arrow in B) has a strong spike-related component and rises later than the signal at the second location (broken line in A, right pixel in B). (C and D) Similar behavior in a pyloric dilator (PD) neuron. One hundred and ninety-six time segments of 975 msec were averaged. In individual sweeps, the soma trough-to-peak voltage was typically 7 mv with a 13-mV spike for a total voltage excursion of 20 mv. The trough voltage was approximately -48 mv. There is one arbitrary calcium scale for the two calcium traces in A and a second arbitrary scale for the two traces in C. The pixels in B and D are each 64,um x 64 Am. DISCUSSION The spatial distribution of calcium oscillations observed in this series of experiments was the same as previously reported-i.e., oscillations were observed in all neuropil locations but not in the soma or axon. However, the improved averaging procedure gave signals whose time course could be better correlated with the components of the oscillating membrane potential. This information allows us to draw stronger conclusions from the spatial distribution. In many cells it appeared that all of the increase in intracellular calcium during the oscillation occurred in steps, each one of which correlated with the attenuated action potential recorded in the soma. If all of the calcium increase is spike-related, we would expect that the strongest signals might be observed from the axon which has a fully regenerative action potential. The fact that no signals were observed there, even in cells with large spike-correlated jumps in calcium in neuropil regions, strongly suggests that the density Proc. Natl. Acad. Sci. USA 86 (1989) of calcium channels in the axon membrane is much lower than in the neuropil processes. A second conclusion concerns the reason why calcium oscillations were not observed in the soma. One possibility is that the amplitude of the membrane potential oscillation is much higher in the neuropil than in the soma and therefore the calcium channels in the soma are not activated. This might be possible for cells in which all the calcium increase appears to be spike correlated, since action potentials propagate passively into the soma and have only a 4- to 15-mV amplitude there. However, calcium oscillations were observed in the neuropil and not the soma even under conditions where there were no action potentials. For a slowly changing membrane potential the space constant should be much longer than for a passively decrementing action potential, and we would expect only a small potential difference between soma and neuropil if neuropil processes are all depolarized together (reduced load conditions). We previously argued that the lack of observed calcium oscillations in the soma was not likely to be due to a low membrane area relative to the neuropil or to a lack of calcium-permeable channels in the soma. This conclusion was made because (i) strong calcium signals of rapid onset were detected from the soma when the cell was intracellularly stimulated with an electrode in the cell body (7); and (it) these signals disappeared when calcium was removed or when cadmium was added to the bath, showing that the signals were dependent on extracellular calcium and thus were probably caused by voltage-dependent calcium influx and not by calcium release from internal stores (15). The remaining possibility is that the processes that admit calcium into the soma and neuropil are different or have different properties. For example, calcium entry into the soma might be through a voltage-correlated calcium exchanger or pump, while neuropil calcium entry might occur through voltage-gated calcium channels. Or, all calcium entry might be through voltage-gated ion channels, but the channels in the soma might differ from those in the neuropil. For instance, if the channels in the soma had a higher threshold for activation (the reverse of the model in ref. 16, reviewed in ref. 17), then our observations could be explained. In a related species, Panulirus interruptus, the threshold for transmitter release in PD and LP neurons is about -60 mv, which is near the trough voltage in the soma for PD and about midway between peak and trough for the soma voltage in LP neurons (18). Thus, if Panulirus and Cancer neurons are similar, then somatic and synaptic calcium entry must occur through different channel types. The relationship of the time course of the calcium concentration oscillation to the time course of the membrane potential has implications for synaptic transmission in these cells, since the level of intracellular calcium just under the presynaptic membrane is presumably a direct regulator of transmitter release. In some cells calcium entry appeared to be entirely spike evoked. In others there was no clear jump in calcium at the time of an action potential; instead there was a smoothly graded, slow oscillation. However, in most neurons there was a clear mixture of the two components. This observation is consistent with the fact that both spikeevoked and graded synaptic transmission have been described for these cells in P. interruptus (reviewed in ref. 6). Our measurements are a way of estimating the relative importance of each component. To make these measurements more meaningful, the actual calcium concentrations at the peak and trough of the oscillation should be determined as well as the relationship between calcium concentrations and the amount of transmitter release. One step in this direction is the use of fura-2 instead of arsenazo III as the dye to track calcium concentrations (8). For this indicator the ratio of fluorescence at two different excitation wavelengths

5 Neurobiology: Ross and Graubard can be calibrated in terms offree calcium concentration in the cytoplasm (19). It is interesting that the time course of calcium oscillation differed in different regions of the neuropil. This implies that the relative importance of spike-evoked and graded calcium entry (and by implication synaptic transmission) varies in different regions. Since the inputs and outputs of these cells are distributed over the neurites of stomatogastric neurons, we might expect different messages to be sent from processes in different regions. A complete model of the stomatogastric ganglion must take this spatial heterogeneity into account. We thank N. Lasser-Ross for most of the computer programming, Galen Eaholtz for technical assistance, and W. H. Calvin for helpful discussions. This research was supported by U.S. Public Health Service Grants NS16295, NS15697, NS25505, National Science Foundation Grant BNS , and the Irma T. Hirschl Foundation. 1. Selverston, A. I. & Moulins, M., eds. (1987) The Crustacean Stomatogastric System (Springer, New York). 2. Hartline, D. K. (1987) in The Crustacean Stomatogastric System, eds. Selverston, A. I. & Moulins, M. (Springer, New York), pp King, D. G. (1976) J. Neurocytol. 5, King, D. G. (1976) J. Neurocytol. 5, Hall, D. H., Marder, E. & Bennett, M. V. L. (1985) Soc. Neurosci. Abstr. 11, 506. Proc. Natl. Acad. Sci. USA 86 (1989) Russell, D. F. & Graubard, K. (1987) in The Crustacean Stomatogastric System, eds. Selverston, A. I. & Moulins, M. (Springer, New York), pp Graubard, K. & Ross, W. N. (1985) Proc. Nati. Acad. Sci. USA 82, Graubard, K., Eaholtz, G., Ross, W. N. & Connor, J.A. (1987) Soc. Neurosci. Abstr. 13, Maynard, D. M. & Selverston, A. I. (1975) J. Comp. Physiol. 100, Hooper, S. L., O'Neil, M. B., Wagner, R., Ewer, J., Golowasch, J. & Marder, E. (1986) J. Comp. Physiol. A 159, Hamming, R. W. (1977) Digital Filters (Prentice-Hall, Englewood Cliffs, NJ), pp Miller, J. P. (1975) Soc. Neurosci. Abstr. 1, Raper, J. A. (1979) Dissertation (Univ. of California, San Diego). 14. Gola, M. & Selverston, A. I. (1981) J. Comp. Physiol. 145, Graubard, K. & Ross, W. N. (1986) Soc. Neurosci. Abstr. 12, Llinds, R. & Yarom, Y. (1981) J. Physiol. (London) 315, Llinds, R. R. (1983) in Brain Slices, ed. Dingledine, R. (Plenum, New York), pp Graubard, K., Raper, J. A. & Hartline, D. K. (1983) J. Neurophysiol. 50, Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol. Chem. 260,