Friedrich-Miescher-Laboratory of the Max-Planck-Society, D-72076 Tuebingen, Germany
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 M
. 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 (F) that subsequently were divided by
the reference frame (
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
. 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).
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RESULTS |
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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, (
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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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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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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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
(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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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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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
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
F and F. Now,
the
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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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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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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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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DISCUSSION |
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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 F/F
images because only when background fluorescence is subtracted reliably do
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,
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
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
F/F signals in small
neural processes quantitatively are bound to fail in an in vivo
situation. Our estimates of cell-born
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
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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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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