Friedrich-Miescher-Laboratory of the Max-Planck-Society, D-72076 Tubingen, Germany
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ABSTRACT |
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Oertner, Thomas G., Tilmann M. Brotz, and Alexander Borst. Mechanisms of Dendritic Calcium Signaling in Fly Neurons. J. Neurophysiol. 85: 439-447, 2001. We examined the mechanisms underlying dendritic calcium accumulation in lobula plate tangential cells of the fly visual system using an in vitro preparation of the fly brain. Local visual stimulation evokes a localized calcium signal in the dendrites of these cells in vivo. Here we show that a similar localized calcium accumulation can be elicited in vitro by focal iontophoretic application of the cholinergic agonist carbachol. The calcium signal had at least two sources: first, voltage-dependent calcium channels contributed to the carbachol-induced signal and were concentrated on the dendrite, the soma, and the terminal ramification of the axon. However, the dendritic calcium signal induced by carbachol stimulation was only weakly dependent on membrane depolarization. The most likely explanation for the second, voltage-independent part of the dendritic calcium signal is calcium entry through nicotinic acetylcholine receptors. We found no indication of second-messenger or calcium-mediated calcium release from intracellular stores. In summary, the characteristic spatiotemporal calcium signals in the dendrites of lobula plate tangential cells can be reproduced in vitro, and result from a combination of voltage- and ligand-gated calcium influx.
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INTRODUCTION |
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Calcium can serve both as a
charge carrier and a second messenger and therefore is able to link
electrical activity to biochemical processes in nerve cells. Its
functional role is best understood in presynaptic terminals where high
local calcium concentrations are needed for the release of synaptic
vesicles (Südhof 1995; Zucker
1996
). The somatic calcium concentration influences the transcription of numerous genes (Hardingham et al.
1997
). At the postsynaptic site, calcium is crucial for the
induction of long-term changes in synaptic transmission (Bear
and Malenka 1994
; Eilers et al. 1996
;
Neveu and Zucker 1996
; Tsumoto and Yasuda
1996
). Dendrites can be hyperpolarized by the activation of
Ca2+-dependent K+ channels,
effectively reducing the excitability of neurons (Sobel and Tank
1994
). Calcium may play many other roles depending on the type
of nerve cell where it occurs and exerts its action.
Calcium accumulation has been reported in vivo in fly lobula plate
tangential cells (Borst and Egelhaaf 1992). The
tangential cells represent a set of about 60 fairly large visual
interneurons each of which can be identified individually due to their
invariant anatomy and characteristic visual response properties (for
review, see Hausen 1984
). Located in the posterior part
of the third visual neuropile of the fly, the lobula plate, they
spatially pool the signals of thousands of local columnar elements
arranged in a retinotopic fashion (Haag et al. 1992
).
Tangential cells have large receptive fields and respond to visual
motion in a directionally selective way (Borst and Egelhaaf
1990
; Egelhaaf et al. 1989
). There are cells
tuned to horizontal motion, e.g., HS and CH cells (Eckert and Dvorak 1983
; Hausen 1982a
,b
)
and to vertical motion, e.g., VS cells (Hengstenberg
1982
; Hengstenberg et al. 1982
). The electrical
response of many tangential cells consists of a graded shift in
membrane potential, superimposed by irregular spikes in certain cell
types (Haag and Borst 1996
; Hengstenberg 1977
). Excitatory and inhibitory synaptic input to the
dendrites of VS and HS cells is mediated by nicotinic acetylcholine
receptors and by GABAA-like receptors,
respectively (Brotz and Borst 1996
).
The tangential cells are involved in the fly's visual course control
system. Because of their well-established functional role, they serve
as an attractive model for the analysis of single-cell computation
(Single et al. 1997). With the aid of realistic
compartmental models, the influence of anatomical and physiological
features of the cell on its information processing capabilities can be explored (Borst and Haag 1996
; Haag et al.
1997
). Using this strategy, the functional role of fast
Na+ channels for the amplification of
high-frequency synaptic input could be demonstrated (Haag and
Borst 1996
). Because of their position on the posterior surface
of the brain and their flat, two-dimensional geometry, lobula plate
tangential cells are easily accessible to imaging techniques.
Stimulating the fly visually with gratings moving in the cell's
preferred direction resulted in elevated calcium concentrations in the
dendrite, the soma, and the terminal region of the axon (Borst
and Egelhaaf 1992
). Interestingly, the dendritic calcium
signals were restricted to the stimulated branches of the dendritic
arbor in a retinotopic fashion. To investigate the functional role of
calcium in the dendrites of these cells, the mechanisms of calcium
accumulation need to be clarified first. Here, we examine the sources
of calcium accumulation in VS and CH cells in an in vitro preparation
of the fly brain (Brotz et al. 1995
) where the natural
geometry and connectivity of the tangential cells are retained and
extracellular ion and drug concentrations can be easily controlled.
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METHODS |
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Preparation and set-up
The in vitro preparation of the blowfly brain has been
described previously (Brotz et al. 1995). Briefly,
female blowflies (Calliphora erythrocephala), 1-3 days old,
were cold anesthetized and decapitated. The brain was removed under
ice-cold saline by cutting the first optic chiasm between the lamina
and the medulla and glued with its posterior surface onto a brass
washer. The neurolemma of the anterior side was gently peeled off to
ease the penetration with microelectrodes. The preparation was then transferred to the recording chamber where it was perfused with oxygenated fly saline (95% O2-5%
CO2, Messer Griesheim GmbH, Germany) at a rate of
8 ml/min. The recording chamber was mounted on an inverted microscope
(Axiovert 35 M with Plan-Neofluar 10×/0.30 Objective, Zeiss,
Oberkochen, Germany). All experiments were performed at room
temperature (23-25°C).
Solutions
As standard perfusion saline, we used a modified hemolymph-like
solution after Stewart et al. (1994), composed of (in
mM) 70 NaCl, 5 KCl, 1.5 CaCl2, 5 MgCl2, 10 NaHCO3, 5 trehalose, 115 sucrose, and 5 HEPES, adjusted to pH 7.2 with 1 M HCl.
In low-Ca2+ saline, CaCl2
was replaced by MgCl2. In
low-Na+ saline, NaCl was replaced by
N-methyl-D-glucamine
(NMDG+). To investigate the contribution from
intracellular calcium stores to the calcium signals, ryanodine (10 µM, Alomone Labs), thapsigargin (1 µM, Alomone Labs), or caffeine
(10 mM) was added to the perfusion solution. Chemicals were from Sigma
unless otherwise noted.
Recording of membrane potential
Electrodes were pulled on a Brown-Flaming micropipette puller
(P-97) using thin-walled glass capillaries with an outer diameter of 1 mm (GB100TF-10, Vitrex Modulohm, Herlev, Denmark). When filled with 500 mM KCl and 12 mM fura-2 (Molecular Probes, Eugene, OR), electrodes had
resistances of 80-110 M. Fura-2 was iontophoretically injected by
applying negative current pulses of 1nA (1-Hz square wave, 50% duty
cycle) for 3-5 min. The membrane potential was recorded using an
Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). The
headstage with the electrode holder was mounted on a stepmotor-driven
micromanipulator (Luigs and Neumann, Ratingen, Germany). The output
signal of the amplifier was digitized via a 12 bit A/D converter
(CIO-DAS16F, Computerboards, Mansfield, MA) at a sampling rate of 10 Hz
and fed into an Intel 486 based PC. The programs for data acquisition
and evaluation were written in Turbo-Pascal (Borland).
Optical recording and image processing
Tangential cells stained with fura-2 were illuminated by a
monochromator (DeltaRAM, P. T. I., South Brunswick, NJ) via
quartz fiber optics and imaged with a Peltier-cooled CCD-camera (CH250, Photometrics, Tucson, AZ). Images were acquired by an Apple Macintosh Quadra 900 and further processed with custom-written software in IDL
(Research Systems, Boulder, CO). For ratiometric measurements (Grynkiewicz et al. 1985), 100 images were acquired at 1 Hz (binning factor, 2; exposure time, 500 ms), alternating between 340- and 380-nm excitation. To correct for spatial variation in the
illumination, a thin film of fluorescein-solution under a coverslip was
imaged at 340- and 380-nm excitation. The resulting correction images were low-pass filtered and normalized to their highest values, resulting in pixel values between 0.7 and 1. The raw images of the cell
at 340- and 380-nm illumination were divided pixel by pixel by the
corresponding correction images to achieve a uniform background
intensity. For evaluation, two different procedures were used: for a
qualitative, image-based interpretation of the spatiotemporal changes,
pairs of 340/380-nm images were divided pixel by pixel without
background subtraction. Discarding the first image, the second 340/380
image of a sequence served as reference image and was subtracted on a
pixel-by-pixel basis from all the following ratio images. When viewed
as a movie, the resulting stack of images gave an impression of the
spatiotemporal changes in the 340/380-nm ratio. All false color images
shown are based on this evaluation procedure, providing a qualitative
representation of intracellular calcium changes. For quantitative
analysis of [Ca2+]i, the
background was subtracted in the raw images: The average pixel value of
an unstained region of the tissue was determined for each image and
subtracted from every pixel of that image. When the ratio 340/380 was
calculated for a small region within the cell (10 pixels,
200
µm2), care was taken not to include pixels
outside the cell as these pixels are especially prone to noise. The
resulting ratios were converted to absolute
[Ca2+] using a linear approximation of the
calibration curve (fura-2 calibration kit, Molecular Probes) between a
ratio of 1 (=100 nM Ca2+) and 2 (=400 nM
Ca2+) and plotted as a function of time.
Pharmacological stimulation
Cells were stimulated by iontophoretic application of the
nonspecific cholinergic agonist carbachol. A Neurophore BH2
micro-iontophoresis system (Medical Systems, Greenvale, NJ) was
connected to a low-resistance glass microelectrode (10-20 M) filled
with 0.1 M carbachol. By means of a micromanipulator (Narishige,
Japan), the electrode was inserted about 20 µm into the tissue of the
lobula plate at the ventrolateral margin of the neuropil. A retaining
current of
10 nA was applied to prevent drug leakage. If not
otherwise stated, the carbachol ejection current was adjusted between
20 and 100 nA to depolarize the cell to 3-6 mV from the resting potential.
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RESULTS |
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General characteristics of electrical and calcium signal on carbachol stimulation
In the intact fly, local visual stimulation leads to a
depolarization and a localized calcium signal in the dendrite of
tangential cells (Borst and Egelhaaf 1992). Here, we
show that a similar localized dendritic calcium signal could be
elicited in vitro by focal application of the cholinergic agonist
carbachol. The general layout of the experiments is shown in Fig.
1A. A single tangential cell
was filled with the calcium indicator fura-2 from a sharp intracellular
electrode placed in the axon (Fig. 1B). This electrode was
also used to monitor the membrane potential of the cell during the
whole experiment. For stimulation, carbachol was released by
iontophoresis from an extracellular microelectrode placed in the lobula
plate. Images were taken at a rate of 1 Hz, switching the excitation
wavelength between 340 and 380 nm. We observed a large change in the
fluorescence ratio 8 s after the onset of a 1-s carbachol pulse
(Fig. 1C). Warm colors encode large increases in the 340/380
fluorescence ratio. The calcium signal was strongest in the dendrite
close to the stimulation electrode (Fig. 1C, filled
triangle). In addition, a weaker calcium signal was detectable in the
terminal region of the axon (Fig. 1C, open triangle). There
were no changes in fluorescence in the upper main branch of the
dendrite or in the axon. The soma of the cell was not in the focal
plane in this example. Figure 1D shows the response to a
weaker ejection current (25 nA) at the same scaling. The amplitude of
the signal is smaller and the spatial extension of the dendritic
calcium accumulation is more restricted.
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The time course of the dendritic calcium signal after background subtraction is shown in Fig. 2A. [Ca2+]i rose about 50 nM above the resting value after the weak stimulation (grey squares) and about 160 nM after the stronger stimulation (filled squares). The open squares correspond to the color coded images shown in Fig. 1, C and D. The peak [Ca2+]i was reached 6-8 s after the onset of stimulation. Removal of free calcium was slow, with time constants of about 25-30 s. The onset of the voltage response as measured in the axon of these nonspiking cells was correlated with the calcium signal, but the rise time and decay were faster (Fig. 2B). After strong depolarizations, the time constant of repolarization was shortened and a slight afterhyperpolarization was often seen (black line). The amplitude of both depolarization and calcium signal were dependent on the carbachol dose. The dose-response curves of two different VS cells are given in Fig. 3, A and B. Here, the ejection time was used to change the carbachol dose. The voltage response always saturated at lower doses than the local dendritic calcium signal. The fact that individual cells saturated at different carbachol doses is most likely due to the variable position of the stimulation electrode relative to the dendrite in each experiment. The sigmoid curve fitted to the calcium responses was shifted about 0.3 log units to the right relative to the log dose-response curve of the voltage response, corresponding to a doubling of the carbachol dose necessary to reach the half-maximal response.
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Calcium enters the tangential cells from outside
In principle, the observed elevation in [Ca2+]i on cholinergic stimulation could arise from the influx of Ca2+ through channels in the plasma membrane, by calcium release from internal stores, or by a combination of both processes. To dissect these alternatives, we stimulated tangential cells after 10 min of perfusion with low-calcium saline. Calcium chelators were not added to prevent emptying of potential intracellular stores. Given the fact that the cells under scrutiny were still embedded in the tissue, we do not assume a complete washout of all extracellular Ca2+ ions under these conditions. Control values of the calcium response and depolarization were recorded in standard saline before and after low-calcium perfusion.
Figure 4 gives an example of one such
experiment, together with average values. The placement of the
electrodes is indicated on the photo of the dye-filled cell in Fig.
4A. After 10 min of perfusion with low-calcium saline, the
calcium signal on carbachol stimulation was completely abolished in
this experiment (Fig. 4F). Response amplitudes across
experiments were normalized to the respective control responses. In
low-calcium saline, the calcium signal was on average reduced to 21%
of the control experiments (Fig. 4D, n = 3).
This reduction was completely reversible after switching back to
standard saline. From this we conclude that the observed calcium signal
is caused primarily by an influx of Ca2+ from the
extracellular space. There are two more features in the responses which
deserve closer inspection. 1) Resting values of
[Ca2+]i were lower in
low-calcium saline, suggesting a permanent influx of
Ca2+ ions under control conditions. Experiments
in vivo corroborate a constant calcium influx in the absence of
stimulation (Haag and Borst 2000). 2)
Examining the electrical response of the cell, we found that the
depolarization in low-Ca2+ saline was on average
13% stronger than under control conditions (Fig. 4D). The
electrical response was more strongly enhanced in low-calcium
experiments with virtually abolished calcium responses than in
low-calcium experiments with residual calcium responses. The most
likely explanation for the weaker depolarization in the control
experiment is a K+ current activated by elevated
[Ca2+]i. This current
would limit the depolarization after calcium influx and could also
account for the afterhyperpolarization seen after strong
depolarizations (Fig. 2).
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The absence of a Ca2+ signal in low-calcium
saline is in accordance with a purely nicotinic pharmacology of
the acetylcholine receptors (AChRs). Inositol 1,4,5-triphosphate
(IP3)-mediated Ca2+ release from
internal stores triggered by muscarinic AChRs would be independent of
the extracellular Ca2+ concentration. This is in
line with a previous pharmacological study in which muscarinic
antagonists were unable to block carbachol-induced depolarization and
muscarinic agonists did not lead to a depolarization of tangential
cells (Brotz and Borst 1996). The possibility of an
additional contribution by calcium-induced calcium release (CICR) from
internal calcium stores cannot be excluded by low-calcium experiments
but has to be addressed by use of specific pharmacological tools (see
following text).
Internal calcium stores could not be activated
Influx of calcium ions could, in principle, trigger
calcium-induced calcium release from intracellular stores by activation of ryanodine receptors. To trigger the release of calcium from putative
intracelluar stores, we perfused the preparation with saline containing
10 µM ryanodine for 5 min, then stimulated presumptive ryanodine
receptors by bath application of caffeine (10 mM, 2 min). During the
caffeine application,
[Ca2+]i was monitored.
For these experiments, VS and HS cells were used. We found no change in
[Ca2+]i using this
paradigm (n = 5) or by applying caffeine alone
(n = 4). In a second set of experiments, we tested
the effects of thapsigargin, a potent blocker of sarco/endoplasmic
reticulum Ca2+-ATPase (SERCA) (Jackson et
al. 1988). Bath application of 1 µM thapsigargin for 10 min
did not trigger calcium release in unstimulated cells
(n = 8) nor did it reduce carbachol-induced calcium
responses (Fig. 5). We therefore conclude
that intracellular stores do not significantly contribute to the evoked
calcium transients in these cells.
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Calcium influx through voltage-dependent calcium channels (VDCCs)
The experiments in low-calcium saline described in the preceding text indicate a contribution of extracellular Ca2+ to the calcium signal in tangential cells. However, since stimulation of nAChRs by carbachol led to a depolarization of the cells, influx through nAChRs and through VDCCs could not be discriminated in these experiments. To examine voltage-dependent calcium influx in isolation, we manipulated the cell's membrane potential by current injection instead of carbachol iontophoresis. In CH cells, depolarizing current injections of 10 and 15 nA into the axon caused a strong increase in [Ca2+]i in the dendritic arborizations in the lobula plate and in the protocerebrum (Fig. 6C). The calcium influx into the axon and primary dendritic branches was much smaller and reached its peak several seconds after the termination of the current injection (Fig. 6D, blue curve). This delayed signal in the main dendritic branches and the axon was most probably caused by calcium diffusion from smaller branches of the dendrite. If the voltage-gated calcium influx in these experiments was due to the activation of VDCCs, they are not distributed homogeneously across the membrane, but concentrated on the dendritic branches of higher order. In VS1 cells, depolarizing current injections also caused a strong dendritic calcium influx (n = 2), whereas in VS2 and VS3 cells the dendritic calcium signal was weak or absent (n = 5, data not shown).
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To exclude the possibility of calcium influx through the sodium-calcium
exchanger operating in reverse mode (Hoyt et al. 1998), we analyzed the influence of the extracellular sodium concentration on
the calcium signal induced by depolarizing current injections. After
wash-in of low-Na+ saline, resting
[Ca2+]i rose about 30-50
nM above the resting level in standard saline. This effect was reversed
after washing with standard saline. For better comparison of the time
courses, the calcium transients in Fig. 7
are shown relative to resting
[Ca2+]i before the
stimulation. In low-Na+ saline, the peak calcium
influx during the depolarizations was unchanged, but the time constant
of calcium removal was prolonged. This effect was reversible after
perfusion in standard saline. From this we conclude that the calcium
influx during depolarizations was caused mainly by the activation of
VDCCs and not by the sodium-calcium exchanger operating in reverse
mode. The slow return to resting calcium levels hints to the importance
of the sodium-calcium exchanger for the fast removal of calcium ions
from the cytoplasm.
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Calcium influx through nicotinic AChRs
To demonstrate Ca2+ influx through nicotinic AChRs (nAChRs), we tried to prevent the cell from depolarizing on carbachol stimulation. Because of the high impedance of the recording electrode and the large size of the cells, conditions were unfavorable for reliable voltage-clamp experiments. By replacing extracellular Na+ with NMDG+, reducing [Na+]o from 80 to 10 mM, we could prevent the fast depolarization on carbachol stimulation (Fig. 8C). Under these conditions, the rise in dendritic [Ca2+]i was very similar to the control experiment during the first 20 s after carbachol stimulation (Fig. 8F). In the next 60 s, the calcium levels returned to baseline in the control experiments but continued to rise in the low-Na+ saline. On average, the depolarization was reduced to about 26% of the control responses, but the dendritic calcium signal still reached 87% of the control amplitude (Fig. 8D, n = 3). Measurements were taken at the time of peak depolarization in the control experiments (15 s after stimulus onset). From this we conclude that under control conditions, the fast depolarization was carried mainly by Na+ ions, under control conditions, Ca2+ entered the cell mainly through ligand-gated channels, and removal of Ca2+ is dependent on the function of the Na+/Ca2+ exchanger. These experiments suggest a small contribution of VDCCs to the bulk calcium signal, which seems to consist mainly of voltage-independent calcium influx. This is in accordance with experiments in which the effect of simultaneous hyperpolarizing current injection on the carbachol-induced calcium transients was examined (data not shown). In these experiments, hyperpolarization of VS and HS cells could not prevent dendritic calcium influx after carbachol stimulation.
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DISCUSSION |
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Focal application of the cholinergic agonist carbachol in vitro led to a localized dendritic calcium signal in the dendrites of tangential cells. In principle, this calcium signal could be caused by calcium influx through ligand- or voltage-gated channels or by calcium release from internal calcium stores. Reducing the extracellular calcium concentration strongly attenuated the carbachol-induced calcium signal (Fig. 4), demonstrating an influx of calcium ions from the extracellular space. CICR could, in principle, amplify this initial calcium influx. However, stimulation of putative ryanodine receptors did not change the intracellular calcium concentration. Blocking calcium pumps on the endoplasmic reticulum with thapsigargin did not evoke calcium transients nor did it reduce the amplitude of carbachol-induced calcium transients (Fig. 5). From this we conclude that CICR does not contribute significantly to the carbachol-evoked calcium transients in these cells.
We also considered the possibility of
IP3-mediated calcium release from intracellular
calcium stores, triggered by the stimulation of muscarinic receptors.
In cockroach motoneurons, for example, the stimulation of muscarinic
receptors is known to activate calcium release (David and Pitman
1996). However, second-messenger-mediated calcium release from
internal stores is not dependent on calcium influx from the
extracellular space and should be strongly influenced by blocking
calcium pumps of the endoplasmic reticulum. Exactly the opposite was
found in the experiments reported here, making a muscarinic mechanism
in tangential cells highly unlikely. This corroborates a previous
electrophysiological study of fly tangential cells, where the
pharmacology of AChRs has been characterized as being nicotinic
(Brotz and Borst 1996
).
The dendritic voltage-gated calcium influx demonstrated here in vitro
(Fig. 6) has been characterized by voltage-clamp experiments in vivo
(Haag and Borst 2000). It is activated over a broad
range of membrane potentials between
60 and
20 mV in a fairly
linear fashion. This unusual characteristic has led to speculations
whether the voltage-gated influx is due to the activation of
voltage-gated calcium channels or caused by the sodium-calcium
exchanger operating in reverse mode. Due to the stochiometry of the
exchanger (3 sodium ions are extruded for 1 calcium ion to enter),
calcium influx through the exchanger would be accompanied by a net
outward current (Blaustein and Lederer 1999
). This,
however, is inconsistent with the electrophysiological characterization
of calcium currents in CH cells: the cobalt-sensitive current remaining
after blocking voltage-gated K+ and
Na+ channels is an inward current (Haag et
al. 1997
). A second argument against calcium influx through the
exchanger comes from the experiments reported here, where replacing
extracellular sodium ions by NMDG+ did not reduce
the amplitude of the calcium signals (Fig. 7). We conclude that the
depolarization-induced calcium influx is indeed due to the activation
of low-threshold voltage-gated calcium channels. At the resting
potential of about
50 mV, this current will be partially activated,
leading to a balance between permanent calcium influx and
energy-consuming removal by calcium pumps and sodium-calcium
exchangers. Although sodium-calcium exchangers do not consume ATP
themselves, they are driven by the sodium gradient established by
ATPases. It comes as no surprise that these graded-response neurons
employ different mechanisms of calcium homoeostasis than the more
extensively studied spiking neurons: a persistent calcium current would
provide a mechanism for encoding continuous voltage changes in both
depolarizing and hyperpolarizing directions.
In the experiments in low-sodium saline, resting
[Ca2+]i was elevated and
the return of [Ca2+]i to
resting values after short depolarizing current injections was
prolonged (Fig. 7). If the cell was stimulated with carbachol under
these conditions, it was literally flooded with calcium (Fig.
8F). Even after switching back to standard saline,
[Ca2+]i remained elevated
for several minutes. These conditions were clearly deleterious for the
cells as the responses to control stimulations in standard saline were
strongly reduced after extended periods of elevated
[Ca2+]i (data not shown).
The prolonged depolarization seen after carbachol stimulation in
low-Na+ saline might be partly caused by
presynaptic cells: since the calcium metabolism is affected in all
cells in the tissue, a carbachol pulse might lead to extensive
transmitter release due to elevated calcium levels in presynaptic
terminals. If only the cell under scrutiny was depolarized by a short
current injection in low-sodium saline,
[Ca2+]i slowly returned
to baseline after 1-2 min (Fig. 7). These observations, together with
the elevated resting calcium levels seen in
low-Na+ saline, are in accordance with the
sodium-calcium exchanger mainly mediating
[Ca2+]i recovery after
carbachol stimulation. However, since removal of extracellular sodium
ions affects all exchange mechanisms that rely on the sodium gradient,
more complicated interactions are possible (Storozhevykh et al.
1998). In the tangential cells, the transport capacity of
ATP-dependent calcium pumps alone seems to be insufficient to restore
resting calcium levels while the cell is depolarized.
In vertebrate neurons, calcium influx through nAChRs can contribute
significantly to changes in postsynaptic calcium concentration (Mulle et al. 1992; Vernino et al. 1994
).
For insect CNS neurons, there are only few studies of the calcium
fluxes through nAChRs. A study on cultured Kenyon cells of the bee
reports a calcium-to-sodium permeability ratio of 6.4 (Goldberg
et al. 1999
). In cultured neurons from embryonic cockroach
brains, calcium influx through nAChRs was below detection threshold
(van Eyseren et al. 1998
). In experiments on isolated
somata from locust thoracic ganglia, the influx associated with the
opening of nAChRs contributed about 25% to the total calcium signal
after carbachol stimulation (Oertner et al. 1999
).
However, these results cannot be transferred to lobula plate tangential
cells because the relative density of nAChRs and VDCCs might strongly
vary in different kinds of neurons. In the dendrites of fly tangential
cells, the experiments in low-Na+ saline reported
here suggest a large contribution of ligand-gated calcium influx.
Qualitatively the permeability of dendritic nAChRs for calcium is
supported by a series of calcium measurements in tangential cells in
vivo. Lack of congruence between membrane potential and calcium signal
was first described in HS cells during visual motion stimulation at
high temporal frequencies (Egelhaaf and Borst 1995
).
Under these conditions, the membrane potential as measured in the axon
was unchanged from rest after an initial transient, but the dendritic
calcium signal was large and continued to rise after the membrane
potential had returned to rest. This suggests a voltage-independent
component of the visually induced calcium signal. In experiments on VS
cells, a rise in [Ca2+]i
was observed in the fine terminal branches of the dendrite during
visual stimulation against the preferred direction in spite of a
general hyperpolarization of the cell (Borst and Single
2000
). Since both excitatory and inhibitory inputs become
activated during visual stimulation against the preferred direction
(Single et al. 1997
), the observed rise in
[Ca2+]i can be readily
explained by calcium influx through nAChRs.
Low-Na+ saline reduced the carbachol-induced
dendritic calcium signals to about 87% of the control responses (Fig.
8D). The electrical responses were more strongly reduced to
about 26% of the controls. Assuming that the voltage-gated calcium
influx is proportional to the depolarization, it follows that under
control conditions only about 18% of the total calcium signal was
dependent on membrane depolarization. This is in contrast to the
above-mentioned in vivo experiments, which concluded that on visual
stimulation, the majority of the dendritic calcium signal is due to
influx through VDCCs (Borst and Single 2000;
Single and Borst 1998
). First, injection of a
hyperpolarizing current during visual stimulation strongly reduced the
pattern-induced calcium modulations in the dendrite (Single and
Borst 1998
). Second, model calculations based on the relative
amount of dendritic calcium elicited during preferred versus
nonpreferred motion stimulation concluded that about 60% of the total
calcium accumulation during preferred direction motion is due to influx
through VDCCs (Borst and Single 2000
). How can this
discrepancy be explained? Unlike acetylcholine, carbachol is not
degraded by cholinesterases in the tissue. Application of carbachol
will therefore activate AChRs more extensively and with a prolonged
time course compared with synaptic stimulation in vivo. Considering the
sensitivity of VDCCs to changes in the intracellular calcium and ATP
concentration (Hao et al. 1999
), it is also possible
that during the preparation procedure a large fraction of VDCCs had
been lost or inactivated. This would shift the balance toward the more
robust ligand-gated calcium entry. The larger depolarizing currents
needed to elicit a calcium influx in vitro support this explanation.
Therefore the particular relation of voltage- to ligand-gated calcium
influx found in the experiments reported here cannot be transferred
uncritically to the situation in vivo.
The functional role of dendritic calcium in the tangential cells
remains to be investigated. The afterhyperpolarization seen after
strong depolarization (Fig. 2) and the enhanced electrical response in
low-calcium experiments with virtually abolished calcium responses
(Fig. 4) suggest a calcium-activated K+ current
in the dendrites of VS cells. This could also account for the fact that
strong carbachol stimulations saturated the voltage response while the
calcium response still increased (Fig. 3). A positive correlation
between the calcium influx and the size of the afterhyperpolarization
has also been reported in HS cells in experiments with visual
stimulation in vivo (Kurtz et al. 1999). A
calcium-mediated inhibitory mechanism could provide local adaptation
within a single cell due to the local restriction of the calcium
signals to the stimulated regions of the dendrite. Because of the slow
time constant of calcium removal, the internal calcium concentration
provides a temporal average of the local excitatory input. Up to now,
local adaptation to visual motion stimulation (Maddess and
Laughlin 1985
) has always been attributed to neuronal circuits
presynaptic to the tangential cells. The possibility of
calcium-dependent local adaptation within the dendrite of single
tangential cells is intriguing and will be addressed in subsequent studies.
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ACKNOWLEDGMENTS |
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We are grateful to K. Götz from the Max-Planck-Institute of Biological Cybernetics in Tübingen for generous loan of part of the equipment, to T. Martin for excellent technical assistance, and to J. Haag and K. Zito for critically reading the manuscript.
Present addresses: T. M. Brotz, National Cancer Institute, Experimental Immunology Branch, 10 Center Dr., Bethesda, MD 20892-1360; A. Borst, Department of Environmental Science, Policy, and Management, Division of Insect Biology, University of California, 201 Wellman Hall, Berkeley, CA 94720-3112.
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FOOTNOTES |
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Present address and address for reprint requests: T. G. Oertner, Cold Spring Harbor Laboratory, PO Box 100, 1 Bungtown Rd., Cold Spring Harbor, NY 11724 (E-mail: oertner{at}cshl.org).
Received 27 June 2000; accepted in final form 15 September 2000.
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REFERENCES |
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