Spatial Distribution and Characteristics of Voltage-Gated Calcium Signals Within Visual Interneurons

Juergen Haag and Alexander Borst

Friedrich-Miescher-Laboratory of the Max-Planck-Society, D-72076 Tuebingen, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Haag, Juergen and Alexander Borst. Spatial Distribution and Characteristics of Voltage-Gated Calcium Signals Within Visual Interneurons. J. Neurophysiol. 83: 1039-1051, 2000. Most of our knowledge about insect calcium currents is derived from studies on cultured or dissociated somata. So far, only little data on calcium currents are available for neurons including their dendritic and presynaptic structures. Here we combined the switched-electrode voltage-clamp technique with optical recording using calcium-sensitive dyes in identified fly visual interneurons in vivo to characterize the voltage dependence and dynamics of calcium currents quantitatively and in a spatially resolved way. For all three cell types considered, i.e., centrifugal horizontal (CH), horizontal system (HS), and vertical system (VS) cells, the activation curve is rather flat and covers a voltage range from -60 to -20 mV in dendritic as well as presynaptic areas of the cells. The calcium increase is fastest for CH cells with a time constant of ~70 ms. In HS and VS cells, the time constant amounts to 400-700 ms. The calcium dynamics as determined in different regions of the cells are similar except for a small segment between the axon and the dendrite in HS and VS cells, where the calcium increase is significantly faster. In summary, the results show the existence of a low-voltage-activated calcium current with little or no inactivation in dendritic as well as presynaptic regions of fly lobula plate tangential cells.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Calcium currents have been studied in vertebrate neurons in detail. Numerous studies revealed a great diversity of underlying subtypes which are nowadays classified into at least six different types called L, N, P, Q, R, and T type (for review, see: Olivera et al. 1994; Randall 1998; Tsien and Tsien 1990). Each subtype is characterized by its biophysical properties like the voltage range and kinetics of activation and inactivation, its selective permeability for other bivalent cations, and its pharmacological profile. According to this classification, the low-voltage-activated (LVA) family has only one member, the T-type calcium channel, characterized by its low activation threshold and transient response property, whereas the remaining five types, i.e., L-, N-, P-, Q-, and R-type calcium currents all belong to the high-voltage-activated (HVA) family.

In contrast, calcium currents in insect neurons are less well understood. In the studies on insect calcium currents published so far (Bickmeyer et al. 1994a,b; Grolleau and Lapied 1996; Grünewald and Levine 1998; Hayashi and Levine 1992; Laurent et al. 1993; Leung and Byerly 1991; Leung et al. 1989; Mills and Pitman 1997; Pearson et al. 1993; Schäfer et al. 1994; Wicher and Penzlin 1997), it became obvious that the classification established for vertebrate calcium currents does not hold for insects: many calcium currents revealed a high activation threshold but were found to be resistant to dihydropyridines, which typically block HVA currents in vertebrates. Moreover certain fractions of spider toxins like agatoxin from Agelenopsis aperta (Bindokas and Adams 1989; Bindokas et al. 1991) or atracotoxin from Hadronyche versuta (Fletcher et al. 1997) were found to differentiate between vertebrate and invertebrate calcium channels. However, most studies on insect calcium currents were performed on cultured or acutely dissociated somata and not on whole neurons with all their ramifications in situ. Thus today, only little data are available on calcium currents in other areas of insect neurons beside the soma (but see Laurent et al. 1993), where activation and inactivation characteristics might well differ from the somatic calcium currents described so far. Using calcium-sensitive dyes, we therefore set out to study calcium currents in a group of neurons in the fly, which, located directly underneath the rear surface of the brain, are easily accessible for optical recording in vivo in all their various ramifications (Borst and Egelhaaf 1992; Single and Borst 1998).

These cells, called lobula plate tangential cells (LPTCs), represent a set of ~60 fairly large neurons per brain hemisphere each of which can be identified individually due to its invariant anatomy and characteristic visual response properties (Hausen 1982, 1984; Hengstenberg et al. 1982). They are located in the posterior part of the third visual neuropile of the fly called the lobula plate (LP). With their large dendrites they spatially pool the signals of thousands of local, columnar elements arranged in a retinotopic fashion (Borst and Egelhaaf 1992; Haag et al. 1992). They thus have large receptive fields and respond to visual motion in a directionally selective way (Borst and Egelhaaf 1989, 1990). Among them, cells are found responding preferentially to vertical motion like the vertical system (VS) cells as well as cells that are best activated by horizontal motion like the horizontal system (HS) and centrifugal horizontal (CH) cells. There exist two CH cells per brain hemisphere [a dorsal (DCH) and a ventral (VCH) cell], three HS cells [the northern (HSN), the equatorial (HSE), and the southern (HSS) cells], and 11 VS cells (VS1-VS11) together covering almost completely the visual space surrounding the animal. The different members of each family occupy different regions within the lobula plate and, because of the retinotopic organization, have different but often overlapping receptive fields. CH cells exhibit a different synaptic organization than HS and VS cells, whereas HS and VS cells are purely postsynaptic in the lobula plate (Hausen et al. 1980), the branches of CH cells in the lobula plate are pre- and postsynaptic (Gauck et al. 1997). The protocerebral (PC) branches of VS and HS cells are output regions of cells. HSN and HSE cells receive an additional contralateral input on these PC branches by a spiking motion sensitive neuron. The PC branches of CH cells are purely postsynaptic. During preferred direction (PD) motion, the cells depolarize in a graded way. This depolarization, sometimes superimposed by action potentials of small and irregular amplitude (Haag and Borst 1996; Hengstenberg 1977), is accompanied by an increase in the intracellular calcium level. During antipreferred or null direction (ND) motion, the cells hyperpolarize and the calcium concentration decreases (Single and Borst 1998). Simultaneous recordings of the electrical response and the calcium signal under different stimulus conditions revealed that the calcium accumulation was proportional to changes in membrane potential (Single 1998; Single and Borst 1998). Furthermore injection of depolarizing current into the axon of various VS, CH, and HS cells elicited a strong calcium influx (Egelhaaf and Borst 1995; Single 1998). In CH cells, voltage-clamp experiments revealed a voltage-dependent calcium current that was blocked by cobalt ions (Haag et al. 1997). In HS and VS cells, however, no calcium current could be detected electrophysiologically (Haag 1994).

In the present report, we studied the voltage-dependent calcium accumulation by voltage-clamp experiments while measuring simultaneously the change in calcium concentration by optical recording using fluorescent calcium indicators. This allowed us to characterize the voltage dependence and dynamics of intracellular calcium signals in dendritic, axonal, and presynaptic regions of insect neurons.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation and set-up

Female blowflies (Calliphora erythrocephala) were anesthetized briefly with CO2 and mounted ventral side up with wax on a small preparation platform. The head capsule was opened from behind; the trachea and airsacs that normally cover the lobula plate were removed. To eliminate movements of the brain caused by peristaltic contractions of the esophagus, the proboscis of the animal was cut away and the gut was pulled out. This allowed stable intracellular recordings of <= 45 min. The fly then was mounted on a heavy recording table with the stimulus monitors in front of the animal. The fly brain was viewed from behind through a microscope (Axiotech vario 100HD, Zeiss).

Stimulation

Stimuli were generated on Tektronix 608 monitors by an image synthesizer (Picasso, Innisfree) and consisted of a one-dimensional grating of 14° spatial wavelength and 87% contrast displayed at a frame rate of 200 Hz. The mean luminosity of the screen was 11.2 cd/m2. The intensity of the pattern was square-wave modulated along its horizontal axis. The angular width of the stimulus fields was 40° in the horizontal and 28° in the vertical direction as seen by the fly. To identify the cells by their visual response properties, cells first were stimulated by the pattern moving at 28°/s back and forth before the voltage-clamp protocol was applied.

Electrical recording

For intracellular recordings of the cells, electrodes were pulled on a Brown-Flaming micropipette puller (P-97) using thin-wall glass capillaries with an outer diameter of 1 mm (Clark, GC100TF-10). The tip of the electrode was filled with 8.8 mM Calcium Green (Ca-Green -1 hexapotassium salt, Molecular Probes). The shaft of the electrode was filled with a solution containing 2 M KAc + 0.5 M KCl. They had resistances of ~15 MOmega . A SEL10-amplifier (npi-electronics) was used throughout the experiments and was operated in the discontinuous mode with a switching frequency of 20 kHz. To study the intrinsic active membrane properties, we used the following protocol: starting from an initial value of -50 mV, cells were held at -70 mV for 300 ms, then clamped to -80 mV for 400 ms to remove inactivation, and then stepped to various potentials for 600 ms to measure the activation characteristic. Then the voltage returned to the usual holding potential of -50 mV between all runs. The current following the step from -70 to -80 mV was used for leak subtraction. Because the different cells of a family (e.g., HSN, HSE, and HSN) had response properties that differed only with respect to their receptive field locations, data from these cell types were pooled and are collectively referred to as "HS cells" in the following. All recordings were made from the axons of the cells. For data analysis, the output signal of the amplifier was fed to a PS/2 PC via an 12-bit A/D converter (Metra Byte µCDAS-16G, Keithley Instruments) at a sampling rate of 1 kHz and stored to hard disk.

Optical recording

We used the microscope with the FITC filter set 9 from Zeiss (BP, 450-490 nm; beamsplitter, 510 nm; LP, 520 nm) for the Calcium-Green dyes and (BP, 380 nm; beamsplitter, 425 nm; BP, 500-520 nm) for fura-2, an UD ×20/0.57 objective and a CCD camera (PXL, Photometrics, equipped with a EEV-chip, 1,024 × 512 pixel) connected to a Power-Mac (Apple). Images were taken at 10 Hz at 128 × 128 pixel resolution and were evaluated using IPLab (Scanalytics) and IDL (Research Systems) software. The first frame of each image series was taken as the reference frame, which was subtracted from each following image. This resulted in a series of difference images (Delta F) that subsequently were divided by the reference frame (Delta F/F). To investigate the time course of the calcium signals with a high temporal resolution, we used a photodiode array of five times five photodiodes as an alternative to the CCD camera. The array (MD25-5T, Centronic, Croydon) was mounted to the camera port of the microscope. It was connected to an initial amplification stage housing 25 current-voltage transducers with low-noise resistors of 108 Omega . The signals were then fed to a 5 × 5 matrix from which individual diodes could be selected and connected to a four-channel sample-and-hold (S&H) amplifier. The area of illumination was defined by an iris diaphragm and typically had a diameter of ~40 µm. The final photodiode output signals then were fed, together with the electrophysiological signals, to the computer at a sampling rate of 1 kHz and stored to hard disk. Original photodiode signals were transformed into measures of relative change in fluorescence by switching to the DC mode of the diode amplifier and determining the absolute fluorescence before and after each sweep. The difference signal as obtained from the amplifier in the S&H mode was filtered by a 10 point (i.e., 10 ms) adjacent average function and then divided by the DC signal.

Experimental procedure

The dye was injected into the cell with a hyperpolarizing current of -3 nA for 3-5 min until the cell was clearly visible under illumination. The experiments with the photodiode array were repeated five times for each pulse injection or each activation step and then averaged. To study the influence of the dye-filled electrode on the measured currents, the electrode was withdrawn and the filled cell was penetrated again using an electrode filled with 2 M KCl (Figs. 2 and 5).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We first examined whether all the different cell families of tangential cells possess voltage-activated calcium currents. Cells were filled with the calcium indicator Calcium Green-1, depolarized by stepping the potential from -80 to -20 mV via current injection into the axon and imaged by means of a CCD camera before and after depolarization. Raw fluorescence images of the different cells as seen within such experiments are shown in Fig. 1, left. The resulting differences in fluorescence, i.e., the difference between the fluorescence images during and before depolarization, expressed in relation to the control image, (Delta F/F), are shown in Fig. 1, right. After injection of depolarizing current, all three cell types revealed a strong increase in fluorescence of <= 10% indicating a rise of cytosolic calcium. The signal was strongest in the lobula plate arborizations of the cells and rather weak in the axon. This experiment thus demonstrates the existence of voltage-activated calcium influx in all three cell types.



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Fig. 1. Raw fluorescence images (left) of centrifugal (CH), horizontal system (HS), and vertical system (VS) cells (from top to bottom) and color-coded images (right) showing the relative change of fluorescence (in %) 400 ms after a voltage step from -80 to -20 mV was applied by current injection into their axon. Cells were loaded with Calcium Green-1.

To relate the optical signals with the currents recorded during voltage-clamp experiments, we optically recorded the change of fluorescence in LP branches of a CH cell and voltage-clamped the cell to various potentials. The cell was penetrated with an electrode containing Calcium Green-1 and was filled with the dye for 4 min. After the cell was clearly visible, the electrode was withdrawn, and the cell was penetrated again with an electrode containing 2 M KCl solution. The different current traces (Fig. 2A) show the leakage subtracted currents for a potential step from -80 mV to the different potential values as indicated in the figure. A transient inward current is activated at steps more positive than -60 mV. It is followed by an outward current with a long time constant. The measured currents look similar to those recorded with the dye-filled electrode (data not shown). Furthermore the currents recorded from the dye-filled cell did not differ from currents recorded from an unfilled cell (Haag et al. 1997). Earlier experiments on CH cells, including the application of potassium (TEA) and calcium channel blocker (cobalt) had revealed that the transient inward current is due to calcium influx, whereas the outward current is carried by potassium (Haag et al. 1997). Under TEA, the isolated Ca-current exhibited two components: a fast transient component and a steady-state component with a smaller amplitude. Without the blocking of potassium channels, this steady-state component is masked by the outward current. The change of fluorescence recorded from the dendrite of the same CH cell depended on the command voltage in a linear way for step potentials more positive than -60 mV (Fig. 2B). This also can be seen in Fig. 2C where the steady-state change in fluorescence (right y axis) is shown as a function of membrane potential together with the peak amplitude of the initial inward current of this experiment and the steady-state amplitude of the inward current from another experiment with TEA (left y axis) (data from Haag et al. 1997). Both measures depend on the membrane potential in a similar way. Thus optical recording of calcium-dependent fluorescent changes allow inferences to be made on calcium currents with the benefit that the calcium signals can be studied in a spatially resolved way.



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Fig. 2. Recorded currents as a function of time for different step potentials of a dorsal CH (DCH) cell (A) and the simultaneously change in fluorescence measured in the lobula plate (LP) branches (B). C: steady-state change in fluorescence (right y axis) shown as a function of step potential together with the peak amplitude of the initial inward current shown in B and the steady-state amplitude of the inward current from another experiment with TEA (left y axis) (data from Haag et al. 1997).

To examine the activation characteristics of the voltage-dependent calcium accumulation, we next performed a series of voltage-clamp experiments with simultaneous optical recording of the fluorescent changes in different regions of CH, HS, and VS cells. The voltage-clamp protocol was the following: from a holding potential of -50 mV, the membrane potential was stepped to -70 mV for 300 ms. From there the potential was clamped to -80 mV for 400 ms. From -80 mV the potential was stepped to more positive values ranging from -60 to -20 mV. The optical signal was recorded by means of photodiodes, one placed over the protocerebral branches of the cells, the other covering a small spot of their lobula plate arborizations (Fig. 3, top). In Fig. 3A, the steady-state fluorescence changes in PC branches of CH cells for different step potentials is shown together with the respective data from HS and VS cells. Figure 3B shows the steady-state fluorescence changes measured in the LP branches of CH, HS, and VS cells. In general, the activation characteristics of the fluorescence signals in LP and PC branches were similar. Also, in both areas the time course of the fluorescence change was independent of the step potential (data not shown), and no marked differences were seen between the different cell-families. Starting at around -60 mV, the signal increased in a rather linear way with increasing step potential up to the maximum potential tested, i.e., -20 mV. However, in VS cells, the current as recorded from PC branches seems to differ slightly from HS and CH cells: here, the fluorescence signal at -30 mV reaches almost the same value as for -20 mV. Beside this, there was no indication of different types of voltage-gated calcium entry with different activation characteristics. In summary, the signals from all three cell types, from PC as well as LP branches, depend on the membrane potential in an almost identical manner. This dependence is rather linear and ranges from -60 mV to at least -20 mV: higher membrane potentials could not be reached due to outward rectification (Haag et al. 1997). Blocking of potassium currents to prevent outward rectification and thus to extend the voltage range was experimentally difficult because the concomitant depolarization of all other neurons in the circuit caused a large synaptic bombardment and made the recording unstable.



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Fig. 3. Steady-state fluorescence changes, normalized to the maximum value reached in each experiment for highest command voltage, in protocerebral (PC) branches (A) and LP branches (B) for different step potentials in voltage clamp for CH, HS, and VS cells. Areas depicted are shown for a ventral CH (VCH) cell on top. In both areas and all 3 cell types the fluorescence signal is raised for membrane potentials more positive than -60 mV. Data represent the average of 7 CH, 5 HS, and 4 VS cells.

From the experiments shown in Fig. 3, there was no indication for different type of voltage-activated calcium entry. However, within different subregions within, e.g., the dendrite, calcium entry could occur with different activation characteristics. This should result in an inhomogeneous distribution of fluorescence increase for different step potentials. Using a CCD camera at 10-Hz frame rate, we voltage-clamped a CH cell to different potentials and looked whether or not calcium signals were spatially homogeneous for each potential (Fig. 4). The false color images (Delta F/F, top) show that the calcium signal was distributed homogeneously for each voltage step. Only the amplitude increased for more positive potentials. To evaluate these images quantitatively, we marked three areas in the cell (A, B, and C, shown in the inset together with a binary image of the cell) and plotted the resulting relative fluorescence changes as a function of membrane potential for each area independently. As can be seen, within each area, fluorescence increases equally with increasing depolarization. Furthermore the fluorescence changes in the different LP branches of the cell did not reveal any differences in their time course (data not shown). Similar results were obtained in three other experiments (data not shown). We conclude that the command voltage reached the different LP branches equally well and that calcium entry occurred in a spatially homogeneous way within the LP branches of CH cells.



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Fig. 4. Voltage-clamp experiment of a DCH cell. Cell was clamped to different membrane potentials. False color images shown on top (same color scale as in Fig. 1, from -1.25 to +5%) represent the relative change in fluorescence 200 ms after the step to the indicated membrane potential. Graphs show the dependence of relative fluorescence change 200 ms after the step to the indicated membrane potential within the 3 areas marked on the schematic image in the inset. Within all 3 areas, fluorescence increases in a linear way with increasing command voltage.

The data in Fig. 3 indicated a slight increase of fluorescence when the potential was stepped from -80 to -50 mV. Because the resting potential in LPTCs is typically around -50 mV, this would suggest a permanent calcium influx at resting potential of the cells. To test this question explicitly, we applied hyperpolarizing voltage steps from -50 to -80 mV and measured the resulting change in fluorescence. Figure 5 shows the result from an experiment on a VCH. Similar data were obtained on HS cells (data not shown). Clearly, the negative potential step was accompanied with a decrease in fluorescence. When the membrane potential was returned to -50 mV, the fluorescence increased again to the value before the negative potential step. This demonstrates that there exists a significant permanent calcium entry at the resting potential of these cells. To investigate to what extent this resting calcium influx is artificially produced by leak currents accompanying the penetration of the cells by sharp microelectrodes, we shifted the membrane potential of a CH cell through its synaptic input rather than through current injection. To do so, we displayed excitatory (preferred direction, front-to-back) and inhibitory (null direction, back-to-front) motion stimuli in front of the fly's eye on the ipsilateral side. Starting at a resting potential of -48 mV, back-to-front motion led to hyperpolarization by ~4 mV. Front-to-back motion depolarized the cell by 6 mV (Fig. 5B). Simultaneously with the electrical recording, i.e., while the electrode still was inserted in the cell, this synaptically induced potential shift in both directions was accompanied by a decreased as well as increased fluorescence, respectively (Fig. 5C). Compared with the voltage-step from -50 to -80 mV, the relative change of fluorescence was, of course, of significantly smaller amplitude (compare Fig. 5, C with A). Importantly though, after the electrode was withdrawn and the same stimuli were applied as before, almost identical changes of fluorescence were observed. Again fluorescence decreased by the same amount as before when the pattern was moving back to front and increased by ~3% when the pattern moved in the opposite direction. This experiment thus suggests that the permanent calcium influx observed in these cells at resting potential is not an artifact: Modulation of the membrane potential by natural synaptic input indeed modulates the calcium concentration in the cells, leading to decreased or increased calcium levels depending on the prevailing direction of motion in front of the fly's eye. It also shows that the cells are not significantly depolarized by the penetration of sharp microelectrodes: Rather their true resting membrane potential seems to be around -50 mV.



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Fig. 5. A: hyperpolarizing voltage steps from -50 to -80 mV (left axis) applied to a CH cell and the resulting change in fluorescence (right axis). Negative potential step is accompanied with a decrease in fluorescence. B: membrane potential of a CH cell as a function of time in response to null and preferred direction motion displayed in front of the ipsilateral eye. C: fluorescence change in response to the motion stimuli recorded simultaneously with the intracellular recording (pre, right y axis) and shortly after the electrode was withdrawn (post, left y axis). For easier distinction between the fluorescence signals before and after the electrode was withdrawn, pre and post traces are displayed with a vertical offset. Fluorescence decreases during null direction motion both times.

In all experiments reported so far, the relative change of fluorescence was evaluated disregarding the fact that the background contributes to the fluorescence in the images to a different amount depending on the diameter of the cellular process: the smaller the diameter of the branch considered, the larger the contribution of background fluorescence and the more the real relative fluorescence change becomes underestimated. Evaluating the images this way, therefore attenuates the real relative fluorescence changes systematically in thin branches as compared with thick ones. To examine to what extent this background fluorescence affects our measurements, we evaluated the relative change of fluorescence in four different areas of one CH, HS, and VS cell after voltage steps from -80 to -20 mV, where the areas called 1-4 were shifted increasingly toward the dendritic tips of the cells (Fig. 6, left). In these areas, we first calculated the relative fluorescence change in the usual way, i.e., without subtracting the background cells (Fig. 6, middle). In CH cells, there is a trend toward an increased fluorescence when moving outward from area 1 to 4 and maximum values were at about 10%. In HS and VS cells, largest fluorescence changes occurred in the thick diameter branches and decreased continuously toward small diameter branches. This situation changed when Delta F/F signals were evaluated after background subtraction. We defined four background areas adjacent to each one of areas 1 to 4 where we determined the average fluorescence and subtracted this value from the average fluorescence within areas 1 to 4 before calculating the ratio of Delta F and F. Now, the Delta F/F values increased systematically toward smaller processes in all three cell types and reached maximum values of ~50% in small dendritic branches (Fig. 6, right). Such an increase of peak calcium concentration for smaller diameter processes is indeed to be expected for constant current density per membrane area due to the larger surface-to-volume ratio in smaller branches.



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Fig. 6. Relative fluorescence changes in a CH, an HS, and a VS cell (from top to bottom) in response to a voltage step from -80 to -20 mV. To the left, schematic images of the cells are shown as derived from their raw fluorescence images in the respective experiment. On these cells, 4 areas are marked within which the relative fluorescence changes were evaluated before (middle) and after (right) background subtraction ("BGS"). Background was determined for each area separately within an adjacent region. Whereas without background subtraction, the signals tend to decrease for more distal locations, they consistently increase after background subtraction in all 3 cell types. Note different y-axis scales in middle and right columns.

Having considered the spatial distribution of steady-state signals and their voltage dependence so far, we investigated the dynamics of the calcium signal induced by voltage steps in the following series of experiments. The voltage protocol was the same as in the experiments before and is shown in Fig. 7, bottom, where a voltage step from -80 to -20 mV was applied to the axon of the cells. The resulting time course of fluorescence changes was determined from a large area covering most of the LP branches of the cells and was normalized with respect to the maximum value reached during depolarization. The results obtained from several such experiments on CH, HS, and VS cells were averaged and are shown in Fig. 7, top. In CH cells, the preceding steps from -50 to -70 and -80 mV led to a decrease in fluorescence. In response to the step from -80 to -20 mV, fluorescence increased fastest in CH cells with a time constant of ~70 ms. Compared with that, the rise in fluorescence was much slower in HS and VS cells. Here, the average time constant for the rise amounted to ~400-500 ms. The decay of the fluorescence signal had somewhat larger time constants in all three cell types. Again, CH cells had by far shorter decay time constants than HS and VS cells.



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Fig. 7. Mean fluorescence changes measured in the LP branches of CH, HS, and VS cells in response to the voltage protocol shown in the bottom graph. Note that fluorescence signals were not stable at the beginning of the voltage step to -20 mV. For better comparison, the signals for the different cells are normalized with respect to the maximal signal. Signal in CH cells is significantly faster than in HS and VS cells, both in its rising as well as in its falling phase.

To look for any different dynamics of the calcium signal within different regions of the cells, we evaluated the time course of fluorescence signals of the experiment shown in Fig. 6 within the same areas as marked there (Fig. 8, left). As can be seen in Fig. 8, right, the time course of the fluorescence signal in CH cells was almost identical in all small-diameter parts of the cell. However, within the central neurite (left-most area), the signal was significantly slower. In contrast, the signals in all dendritic areas in HS and VS cells were as slow as already documented in Fig. 7. The time constant of the rise in fluorescence again amounted to ~500 ms in dendritic areas. However, within a small axonal area (marked by a red circle) right next to where the dendrite branches off, the signal was significantly faster and reached time constants within the range of values determined for CH cells otherwise. The signals of HS and VS cells further toward the axon terminal were again as slow as the dendritic ones (data not shown). Thus CH cells seem to have a faster calcium dynamics than HS and VS cells. This, however, is only true for the lobula plate branches of the cells: the axonal signal in CH cells is in general slower. In HS and VS cells, there exists one area where the calcium signal is faster by almost an order of magnitude compared with the rest of the cell.



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Fig. 8. Time course of fluorescence changes in a CH, an HS, and a VS cell (from top to bottom) in response to the voltage protocol shown in the bottom graph. The same set of experiments was used as for the data in Fig. 6. To the left, schematic images of the cells are shown as derived from their raw fluorescence images in the respective experiment. On these cells, four areas are marked within which the relative fluorescence changes were evaluated. For better comparison the signals for the different cells are normalized with respect to the maximal signal. CH cell signals are significantly than faster than HS and VS cells in all dendritic areas. In the axon (red lines), the situation is reversed.

In a final series of experiments, we also studied the influence of the affinity of the dye used as calcium indicator on the time course of the fluorescence signal. Three dyes with different affinities were used: the two high-affinity dyes, fura-2 and Calcium Green-1 (Kd = 145 nM and Kd = 190 nM, Molecular Probes), and the low-affinity dye, Calcium Green-5N (Kd = 14 µM, Molecular Probes). Calcium accumulation was induced by injection of depolarizing current pulses of 1-s duration and 10-nA amplitude. Fluorescence changes were measured with photodiodes placed over the PC branches of the cells. The dye was injected iontophoretically with an ongoing hyperpolarizing current of -3-nA amplitude and measurements were taken ~5 min after filling when the cells were clearly discernable from background. Figure 9 shows the results obtained from different CH and HS cells filled with Calcium Green-5N (Fig. 9A), Calcium Green-1 (Fig. 9B) and fura-2 (Fig. 9C). For easier comparison, the fluorescence changes were normalized with respect to their maximum value reached during depolarization. The results can be summarized as follows: 1) both Calcium Green-5N and Calcium Green-1 resulted in similar time courses. However, one of the high-affinity dyes, i.e., fura-2, led to a significant increase of the time constants both in CH and HS cells. 2) However, for all dyes tested, the signal was always faster in CH than in HS cells, both for the increase in response to depolarization as well as the decrease in fluorescence after depolarization ceased. The difference in time course found between the cells thus is likely not be artificially produced by different buffering effects of the indicator dye but rather due to real differences in the calcium dynamics in the different cell types.



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Fig. 9. Influence of the dye affinity on the time course of the fluorescence signals in CH and HS cells. Kd values of the dyes decrease from A to C. Change in fluorescence was induced by injection of a 10-nA depolarizing current pulse into the axon of the 2 cells. For better comparison, the signals are normalized with respect to the maximal value at the end of the current pulse. Signals depend on the affinity of the dye: especially for 1 of the high-affinity dyes, fura-2, the signals are slowed down significantly. For all dyes used, however, the signal in CH cells is faster than the signal measured in HS cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the experiments reported in the preceding text, we filled insect neurons with a fluorescent calcium indicator, applied voltage steps to their axon through a microelectrode, and optically measured the concomitant fluorescence changes in vivo in various regions of the cells. Ideally we would like to interpret our results in terms of activation characteristics of calcium currents and compare them with previous reports on the same cells and with measurements of calcium currents in other insect neurons. Before doing so, however, we will critically discuss the methods to reveal the limitations of possible conclusions that can be drawn.

Space-clamp

A critical difference between voltage-clamp experiments in spatially extended neurons and isolated somata is the fact that in somata a perfect space clamp is guaranteed, whereas in spacious structures it is not a priori clear to what extent an applied voltage at the recording site reaches distal branches of the cell. This problem thus has to be considered when activation ranges for optical measurements in various regions of the cell are indicated quantitatively in terms of voltages applied to the axon. In our case, however, the neurons under study are considered as electrically compact from a series of modeling studies (Borst and Haag 1996; Borst and Single 1999; Haag et al. 1997, 1999). The estimates on membrane parameters of LPTCs were derived from two sorts of experiments: in one case, membrane parameters of precisely reconstructed LPTC models were fitted to current- and voltage-clamp data obtained from axonal measurements (Borst and Haag 1996; Haag et al. 1997); in the other case the responses of the cells to visual input of various size was taken as control (for HS and VS cells) or as additional target data (for CH cells) for the parameter fit (Haag et al. 1999). Although the first sort of experiments allows us to draw precise conclusions only for axonal membrane properties and estimates for dendritic membrane properties that are based mainly on the assumption of spatial homogeneity, the latter case involves synaptic stimulation and measures electrical cross-talk between the postsynaptic sites. Here the cells exhibited a pronounced response saturation reaching only ~50% of the response expected for linear summation (Haag et al. 1999), indicating an electrically compact dendrite for all neurons tested in the present report. In conclusion from the modeling studies on LPTCs, for the voltage range used here, a minimum of ~70% should reach the dendritic tips in CH, HS, and VS cells. Thus it is likely that for remote branches of the cells the real voltage range tested amounted to only -57 to -30 mV instead of -60 to -20 mV as indicated on the x axis. This is about the deviation to be expected at maximum. The statements remains valid that within this range the fluorescence signal increases about linearly with increasing membrane voltage.

Relative change of fluorescence: real versus measured values

Another problem that arises when calcium measurements are made using fluorescent dyes in vivo consists in the uncertainty of the background contribution to the local fluorescence signal. This is an important point for the interpretation of Delta F/F images because only when background fluorescence is subtracted reliably do Delta F/F signals relate to indicator-bound calcium independent of the size of the cellular process (Grynkiewicz et al. 1985). Otherwise the extent to which real, i.e., cell-born, Delta F/F signals become underestimated may increase when smaller process are studied because of an artificially increased denominator in the expression. The in vivo situation, however, poses the problem of a spatially inhomogeneous background, and any pixel-based background subtraction becomes highly unreliable where small dendritic branches optically melt with the background (see also the discussion in Borst and Egelhaaf 1992). If background fluorescence is underestimated, the denominator of Delta F/F expression still may be too large leading to an underestimate of local relative fluorescence change. If background fluorescence is overestimated, the local signal may explode or even invert its sign. Thus with conventional optics, any attempts to measure Delta F/F signals in small neural processes quantitatively are bound to fail in an in vivo situation. Our estimates of cell-born Delta F/F signals therefore were not done on a pixel-by-pixel basis but rather after spatially integrating the values within larger areas within the processes and adjacent to them (Fig. 6). We tried to carefully select the control areas as close to the test area as possible but clearly staying away from any cellular processes. The resulting estimates suggest an increasing signal toward smaller processes with maximum values of >50% of relative change of fluorescence. Because of an increasing surface-to-volume ratio, this is to be expected for a spatial voltage profile that is rather homogeneous when calcium current density per membrane area is not systematically decreased in smaller branches of the neuron. In summary, an immediate interpretation of Delta F/F images without background subtraction like the ones shown in Fig. 1, right, in terms of relative changes of calcium changes is not possible. Nevertheless conclusions about the dependence of any local signal on membrane voltage, with or without background subtraction, are not affected by these limitations because as long as the same area is considered, the introduced error is a constant factor that affects all measurements at the same location in the same way.

Time course of the fluorescence signals

We found a significant difference in the time course of the fluorescence signals between CH cells on the one and HS and VS cells on the other hand. The time course of the voltage-activated fluorescence signal as obtained from the calcium indicator dye in principle is determined by a variety of different sources: the time course of a voltage-activated calcium inward current, an additional calcium-induced calcium release (CICR) from internal stores (McPherson et al. 1991; Simpson et al. 1995), the kinetics and affinity of internal buffers, ATP-dependent calcium pumps and Na-Ca exchanger (Hoyt et al. 1998; Reeves 1998; Yashar et al. 1998), and the kinetics, affinity, and free concentration of the calcium indicator dye (Regehr and Atluri 1995). Because in HS and VS cells no calcium current could be detected electrophysiologically (Haag 1994), indirect reasoning has to be applied to sort out the different possibilities mentioned in the preceding text that could lead to the observed differences in the dynamics of fluorescence signals between the different cell types. Compared with most previous reports on calcium measurements in fly tangential cells (Borst and Egelhaaf 1992; Duerr 1998; Egelhaaf and Borst 1995), the calcium signals presented here and in Single and Borst (1998) are in general significantly faster by almost an order of magnitude. For example, Duerr (1998) reports a positive slope time constant of 5-6 s for HS cells, whereas our measurements are in the range of 400-700 ms. Given our data on the influence of dye affinity (Fig. 9), the easiest way to explain these discrepancies is that most experiments in the earlier reports were done using fura-2, probably at higher concentrations. Nevertheless although an influence of the indicator dye on the time course was demonstrated (Fig. 9), the difference between the various cell types are unlikely to be caused by the dye because the difference was found for all dyes tested and equal injection times were used for all cell types. Nevertheless free dye concentration still could be affected differently in the different cell types by differences in binding to internal proteins. Furthermore in vitro experiments on fly LPTCs with application of ryanodine and caffeine revealed no indication of calcium-releasable calcium stores (Oertner and Borst 1999). This leaves basically two explanations of the differences in the time course between the cell types: HS and VS cells have a slower calcium entry than CH cells or the cells have different buffer kinetics. We found that not only the increase of fluorescence was faster in CH cells than in HS and VS cells but also the decay. If the calcium entry had different activation time constants, the decay on deactivation is not expected to differ in such a way as was observed in the experiments. Thus the internal calcium removal mechanisms like buffers, pumps, and exchangers are likely to be different in the cell types and therefore lead to a faster signal in CH cells than in HS and VS cells. There is one observation, however, that at first sight seems not to be reconcilable with the arguments presented in the preceding text, and that is the existence of a small subdendritic region in HS and VS cells where the time course is significantly faster than in the rest of the cells. Close inspection of the raw fluorescence images revealed that at exactly this location where the calcium signal is fast, the dye seems to stain the cell significantly less than in the neighboring areas (data not shown). Taking into account the low magnification and small numerical aperture of the objectives used here leading to a pronounced integration of photons along the z axis of the specimen, a likely explanation of the faint staining is that the dye fills the tubular process only in a peripheral, submembrane shell. In this case, higher calcium concentrations are expected to be reached much earlier than when the whole volume of the tube is being measured. Of course, this explanation deserves further investigation. So far, however, it is the most parsimonious explanation compatible with all experimental observations available at the moment. Interestingly, the situation in CH cells seems opposite: there, the fluorescence changes were found to be rather slow in the axon as compared with the PC or LP branches. Because the change in fluorescence signal detectable in the CH axon is weak compared with the rest of cell, the slow time course could hint to a rather low density of calcium channels where most of the calcium that appears in the axon is produced by lateral diffusion from the protocerebral and lobula plate branches. Whatever the exact mechanism may be underlying the differences in the time course of the calcium signals in CH cells on the one and in HS and VS cells on the other hand, it is interesting to note that they go along with the different synaptic organizations of these groups of LPTCs thus possibly reflecting differences in the function that calcium exerts in the different cells types.

Calcium currents in fly visual interneurons and in other invertebrates

The experiments shown in the preceding text demonstrate that all three cell types investigated exhibit a voltage-dependent calcium influx. The calcium influx starts being activated at potentials more positive than -60 mV. At resting potential (around -50 mV), it is therefore already activated and can be shut down by hyperpolarization. This could be demonstrated either artificially by current injection or via their natural synaptic input by presenting null direction motion (Fig. 5). Furthermore the calcium current depends on the membrane potential in an almost linear way. This corroborates earlier findings that came to the same conclusion using visual stimuli of different strength where axonal membrane potential was measured together with the spatiotemporal calcium distribution (Single 1998; Single and Borst 1998). Although the voltage range tested here certainly covers the usual operating range of these neurons, it is not sufficient for a detailed comparison with the calcium currents described in other studies (see following text). The calcium current is present in almost all parts of the cell with similar activation characteristics: It is found in the lobula plate arborizations of the cells as well as in their axons and protocerebral branches. Given the relative uncertainty about the local voltage applied in the distal branches, it is safe to conclude that at least in the voltage range up to about -30 mV, a fairly linear relationship holds between calcium current and membrane potential in all regions of the cells. Another uncertainty pertains to the exact time course of the currents in the various parts of the cell: As was shown in Fig. 2 for CH cells, the transient response component is not resolved by our optical recordings, which only monitor the persistent part of the current. Thus it may well be that the calcium current has different transient characteristics in different parts of the cell that escaped our notice. However, in all parts of the cells, a non-inactivating calcium current undoubtedly exists with the voltage dependence indicated in the preceding text. Because the cells have a resting potential around -50 mV and the recorded calcium currents are found in pre- as well as postsynaptic areas, this finding offers the possibility to modulate a permanent transmitter release around a spontaneous level and thus to transmit information about preferred direction motion as well as about motion in the opposite direction to their postsynaptic target cells. Whether the calcium signals found in purely dendritic structures lead to adaptation of visual sensitivity via calcium-dependent potassium currents, analogous to what has been reported for auditory neurons in the cricket (Sobel and Tank 1994), remains to be seen.

It is not a priori clear whether such a voltage-dependent calcium entry is through calcium channels or brought by the action of a sodium-calcium exchanger: both mechanism will produce an increase of cytosolic calcium on increasing membrane potential. For the sodium-calcium exchanger, however, such an increased calcium influx will be accompanied by an increased net outward current (3 sodium ions are extruded for 1 calcium ion to enter) (De Schutter and Smolen 1998; DiFrancesco and Noble 1985), whereas calcium channels carry a net inward current. In CH cells, the calcium inward current also can be seen in the current traces during voltage-clamp experiments. Experiments with TEA to block K outward currents demonstrated that the inward current consists of a fast transient and a non-inactivating component (Haag et al. 1997), which both could be blocked by cobalt. Therefore voltage-dependent calcium accumulation in CH cells can be unambiguously attributed to voltage-activated calcium channels. Because in HS and VS cells no such measurements are available (see preceding text), it cannot be definitely decided which mechanism is responsible for the voltage-dependent calcium accumulation described here. However, from the similarity of the activation characteristics to CH cells, it seems likely that, as in CH cells, the voltage-dependent calcium entry in HS and VS cells is carried by voltage-activated calcium channels, too.

Voltage-gated calcium currents of insect neurons have been described previously in a number of studies (Bickmeyer et al. 1994a,b; Grolleau and Lapied 1996; Grünewald and Levine 1998; Hayashi and Levine 1992; Laurent et al. 1993; Leung and Byerly 1991; Leung et al. 1989; Mills and Pitman 1997; Pearson et al. 1993; Schäfer et al. 1994; Wicher and Penzlin 1997). Their key response characteristics like activation/inactivation behavior as well as their pharmacological profiles are summarized in Table 1. Beside the references that entered the Table 1, calcium currents also were found in a number of other studies on insect neurons (Christensen et al. 1988; Hayashi and Hildebrand 1990; Nightingale and Pitman 1988). As can be seen from this table, many of the insect calcium current listed are high-voltage activated. As an emerging general characteristic of insect HVA calcium currents, in contrast to vertebrate HVAs, they were found, as far as tested, to be insensitive to dihydropyridines like nifedipine and nidendripine. Moreover, BAY K8644 did not act as an analog in these studies. A comparison of the data presented here with the studies cited in the preceding text is critical because the voltage range within which calcium currents in fly LPTCs were tested is rather limited and so far no blocking agents were applied to characterize the calcium current in fly LPTCs pharmacologically or to dissect the current into possible components. Given these limitations, the fly calcium current described in this report seems closest to the M-LVA current described by Wicher and Penzlin (1997) and the maintained LVA current described by Mills and Pitman (1997) and Grolleau and Lapied (1996) in cockroach neurons. Interestingly, it is also reminiscent on a calcium current that has been described in interneurons of the leech (Angstadt and Calabrese 1991; Lu et al. 1997). Because many insect neurons are operating and transmitting signals across synapses in a graded rather than in a spiking mode, it is to be expected that more of such non-inactivating LVA calcium currents, similar to the ones described here for fly LPTCs, will be found in neurons of other insect species too once these are recorded from in the neuropile.


                              
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Table 1. Overview of voltage-activated calcium currents in insect neurons


    ACKNOWLEDGMENTS

We are grateful to M. Herre and the workshop of the Max-Planck-Institute for Biological Cybernetics, Tuebingen, for the design and construction of the electronics for the photodiode array and to T. Oertner and S. Single for critically reading the manuscript.


    FOOTNOTES

Present address and address for reprint requests: A. Borst, 201 Wellman Hall, ESPM-Division of Insect Biology, University of California, Berkeley, CA 94720-3112.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 June 1999; accepted in final form 5 October 1999.


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