1Lehrstuhl für Neurobiologie, Fakultät für Biologie, Universität Bielefeld, D-33501 Bielefeld, Germany; and 2Centre for Visual Sciences, Research School for Biological Sciences, Australian National University, Canberra, ACT 2600, Australia
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ABSTRACT |
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Dürr, Volker and
Martin Egelhaaf.
In Vivo Calcium Accumulation in Presynaptic and Postsynaptic
Dendrites of Visual Interneurons.
J. Neurophysiol. 82: 3327-3338, 1999.
In this comparative in vivo study of
dendritic calcium accumulation, we describe the time course and spatial
integration properties of two classes of visual interneurons in the
lobula plate of the blowfly. Calcium accumulation was measured during
visual motion stimulation, ensuring synaptic activation of the neurons
within their natural spatial and temporal operating range. The compared cell classes, centrifugal horizontal (CH) and horizontal system (HS)
cells, are known to receive retinotopic input of similar direction selectivity, but to differ in morphology, biophysics, presence of dendrodendritic synapses, and computational task. 1) The time course of motion-induced calcium
accumulation was highly invariant with respect to stimulus parameters
such as pattern contrast and size. In HS cells, the rise of
[Ca2+]i can be described by a single
exponential with a time constant of 5-6 s. The initial rise of
[Ca2+]i in CH cells was much faster (
1 s). The decay time constant in both cell classes was
estimated to be at least 3.5 times longer than the corresponding rise
time constant. 2) The
voltage-[Ca2+]i relationship was best
described by an expansive nonlinearity in HS cells and an approximately
linear relationship in CH cells. 3) Both cell classes
displayed a size-dependent saturation nonlinearity of the calcium
accumulation. Although in CH cells calcium saturation was
indistinguishable from saturation of the membrane potential, saturation
of the two response parameters differed in HS cells. 4)
There was spatial overlap of the calcium signal in response to
nonoverlapping visual stimuli. Both the area and the amplitude of the
overlap profile was larger in CH cells than in HS cells. Thus calcium
accumulation in CH cells is spatially blurred to a greater extent than
in HS cells. 5) The described differences between the
two cell classes may reflect the following computational tasks of these
neurons: CH cells relay retinotopic information within the lobula plate
via dendritic synapses with pronounced spatial low-pass filtering. HS
cells are output neurons of the lobula plate, in which the slow, local
calcium accumulation may be suitable for local modulatory functions.
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INTRODUCTION |
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A central question about the computational
function of sensory interneurons is how the integrative properties of
their dendrites shape the overall tuning characteristics to certain
features of the stimulus (Borst and Egelhaaf 1994).
Because the dendrite represents the input zone of the neuron,
visualizing the spatiotemporal activity patterns, as was done here by
monitoring the intracellular concentration of ionic free calcium
[Ca2+]i, can be expected
to yield important clues about the computations performed by the
respective neuron.
The functional significance of
[Ca2+]i in invertebrate
neurons is manifold (Kostyuk 1992), comprising essential
mechanisms such as transmitter release (Katz 1969
),
synaptic plasticity (Denk et al. 1996
; Regehr and
Tank 1994
), triggering or modulation of biochemical and genetic
signaling pathways (Ghosh and Greenberg 1995
),
modulatory actions on ion channels in the outer cell membrane (Sah 1996
), and intracellular stores (Berridge
1997
; Hardie 1996
), as well as charge transfer,
leading to depolarization of the cell membrane (Skeer et al.
1996
).
Although calcium imaging has become a standard technique in
neurophysiology, in vivo studies of dendritic
[Ca2+]i dynamics have mainly been restricted
to insect sensory systems (Borst and Egelhaaf 1992;
Egelhaaf and Borst 1995
; Egelhaaf et al.
1993
; Ogawa et al. 1996
; Single and Borst
1998
; Sobel and Tank 1992
; but see
Svoboda et al. 1997
). The lobula plate of the fly
remains one of the few model systems, where dendritic calcium accumulation can be studied in vivo in individually identifiable neurons, the biophysical properties as well as the behavioral context
of which are known. In taking advantage of this detailed knowledge, the
present comparative study focuses on distinct differences in dendritic
calcium accumulation between two neuron classes, the centrifugal
horizontal (CH) and horizontal system (HS) cells, both of which
receive similar retinotopic input but drastically differ in their
functional properties. While CH cells are relay neurons within the
lobula plate, bearing presynaptic specializations across their entire
dendritic arborization (Gauck et al. 1997
), HS cells are
output elements of the lobula plate, possessing purely postsynaptic
dendrites (Hausen et al. 1980
). The comparison of calcium accumulation in CH and HS cells therefore promises new insights
into the general characteristics of information processing at dendritic
synapses such as the kinetics of calcium-mediated mechanisms and
the spatial scale of interactions within an array of dendritic synapses.
The lobula plate is a layered neuropile in the optic lobe, containing
the dendrites of a set of motion-sensitive, large-field visual
interneurons, the tangential cells. Among them, ~30 have been
identified and characterized in some detail (e.g., Douglass and
Strausfeld 1996; Eckert and Dvorak 1983
;
Egelhaaf 1985
; Hausen 1976
,
1981
, 1982a
,b
, 1984
). All
of these are directionally selective to visual motion and have been
discussed in the behavioral context of gaze stabilization, course
control and figure-ground discrimination (reviews: Egelhaaf and
Borst 1993
; Hausen and Egelhaaf 1989
).
In CH and HS cells, visual stimulation of the ipsilateral part of their
receptive field leads to graded depolarization of the membrane
potential during front-to-back horizontal motion, and graded
hyperpolarization during motion in the opposite direction. This
ipsilateral input is mediated to the lobula plate arborization by a
large array of retinotopically organized inhibitory and excitatory local elements. Previous optical imaging studies have demonstrated that
dendritic calcium accumulation in HS cells is fast and sustained, occurring within a few hundred milliseconds after onset of visual motion, with increasing [Ca2+]i over stimulus
periods up to 30 s (Egelhaaf and Borst 1995). The
same study also showed that [Ca2+]i in HS
cells was significantly raised in response to stimulus motion in the
preferred direction of the cells but remained virtually unchanged in
response to motion in the opposite direction (null direction). This
finding also indicated the presence of a voltage-dependent calcium
influx, because the activation of cholinergic, ligand-gated currents is
thought to be only weakly direction selective (Borst et al.
1995
; Single et al. 1997
). Voltage-dependent
calcium influx has also been confirmed by DC injection into the
dendrites of various tangential cells (Egelhaaf and Borst
1995
; Single and Borst 1997
,
1998
).
CH cells
The class of CH cells comprises two cells in each half of the
brain, one with an arborization within the dorsal part of the lobula
plate (DCH), the other with an arborization in the ventral part (VCH)
(Eckert and Dvorak 1983; Hausen 1976
).
Both CH cells are inhibitory, GABAergic neurons (Meyer et al.
1986
; Strausfeld et al. 1995
).
In a recent electron-microscopical study of the VCH cell, Gauck
et al. (1997) describe postsynaptic specializations in both the
main arborization in the lobula plate and a second, smaller arborization that is located in the protocerebrum. The only presynaptic specializations of VCH are synaptic connections between the dendrite of
VCH and other unidentified neurites within the lobula plate (Gauck et al. 1997
). Gauck et al. also describe one
finding of reciprocal synapses in this region. In spite of the lack of
detailed anatomic studies of DCH, light-microscopical and
electrophysiological characteristics of DCH suggest similar dendritic
synapses in both CH cells. In the context of dendritic calcium
accumulation, this is particularly interesting because the
two-dimensional distribution of
[Ca2+]i is likely to
reflect the distribution of presynaptic activity throughout the array
of dendritic chemical synapses. Because Gauck et al. have found pre-
and postsynaptic specializations within a range of 0.1 µm, the
presynaptic activity pattern is expected to be relayed into a very
similar postsynaptic activity pattern of reversed sign.
The dendritic inhibition mediated by VCH has been discussed in the
functional context of figure-ground discrimination, based on a laser
ablation study (Warzecha et al. 1993), optical imaging results (Egelhaaf et al. 1993
), and modeling approaches
(Borst and Egelhaaf 1993
).
HS cells
The class of HS cells comprises a set of three neurons in each
half of the brain (Hausen 1976, 1982a
,b
).
The dendritic arborizations of HS cells are exclusively located in the
lobula plate, and their receptive fields are juxtaposed in a
dorsoventral order relative to the visual equator (north: HSN;
equatorial: HSE; south: HSS). Their dendrites bear input
specializations on spinelike protrusions of their main branches as well
as on second and higher order branches (Hausen et al.
1980
). The graded depolarizations have superimposed on them
spikelike depolarizations, which disappear after treatment with
tetrodotoxin and therefore are thought to be caused by a voltage-activated Na current (Haag et al. 1997
)
rendering the dendrite a fast response element to oscillatory
stimulation (Haag and Borst 1996
).
HS cells make synaptic output connections only in the terminal region
located in the protocerebrum (Hausen et al. 1980). In contrast to CH cells, the transmitter of HS cells is unknown.
In a functional context, HS cells have been discussed as part of the
neural substrate mediating optomotor responses around the vertical axis
of the animal. This interpretation is supported by experiments with the
Drosophila mutant "optomotor blind," which lacks both HS
and VS cells (Heisenberg et al. 1978), by lesion experiments using laser ablation of individual cells (Geiger and Nässel 1981
, 1982
) and microsurgical
severing of certain fiber tracts (Hausen and Wehrhahn
1990
) and by the overall similarities of the electrical
response properties and the turning behavior of flying blowflies
(Hausen and Wehrhahn 1989
).
Although the identity of the presynaptic neurons of CH and HS cells is still unknown, their receptive fields, directional tuning properties, and region of arborization indicate that the ipsilateral input of CH and HS cells can be assumed to be very alike. Yet their output organization and membrane properties reveal fundamental differences. The present study will show that calcium accumulation in these cell classes differs considerably, manifesting the functional tasks of these neurons.
A preliminary account of this study has been published in abstract form
(Dürr and Egelhaaf 1997).
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METHODS |
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Animal preparation
Experiments were carried out on 1- to 3-day-old female blowflies of the genus Calliphora (C. erythrocephala in Bielefeld, Germany and C. stygia in Canberra, Australia). C. erythrocephala was taken from laboratory stock, bred at the University of Bielefeld. C. stygia pupae were supplied by the CSIRO Division of Entomology in Canberra. Generally, electrophysiological responses and calcium signals in the lobula plate neurons of both species were indistinguishable. Also, the overall morphology of the neurons was the same in both species, although C. stygia neurons were larger than C. erythrocephala neurons. Minor anatomic differences such as the slightly more distal location of the soma in VS cells of C. stygia did not contradict pooling of data from both species. Still, separate treatment of the experimental results obtained from the two species was necessary where exact matching of the stimulus parameters was required, e.g., in measurements of contrast dependency of calcium accumulation. Unless explicitly stated for particular figures and data sets, all results presented were acquired on C. erythrocephala. The animals were anesthetized with carbon dioxide, and the thorax was waxed to a glass support. Then the legs were removed and the wings and abdomen were immobilized with wax. The head was pitched downward, and the genae were waxed to the thorax. Subsequently, a hole was cut into the back of the head, such that the brain was accessible and the right lobula plate was visible from above. Neck muscles were severed, and antennae, haustellum, gut, fat body and heart were removed. The abdomen was filled with Ringer solution (concentrations in mM: 128.3 NaCl, 5.4 KCl, 1.9 CaCl2, 4.8 NaHCO3, 3.3 Na2HPO4, 3.4 KH2PO4, 13.9 glucose, pH 7.0; all chemicals from Merck), and all wounds were sealed with wax. Finally the main tracheae running across the optic lobes were plugged.
Stimulus procedure
The fly was mounted under an epifluorescence microscope (Zeiss
Universal III-RS) with its head facing downward into an opaque sphere
( 36 mm). Two stripe patterns (
66°) could be projected onto
this sphere from below. The spatial wavelength of the stimulus pattern
amounted to ~33° (with local distortions due to the projection), and the contrast was 0.68 (except for experiments on contrast dependency) at a mean light intensity of 230 cd/m2. The spectrum of the stimulus light was
that of a Hg-arc-lamp (Osram HBO 100 W). Note that these measures only
give a lower estimate of the physiologically relevant light intensity
to a fly, because Hg-arc lamps emit blue and ultraviolet (UV) light at
high intensities, which excite Dipteran photoreceptors considerably (Hardie 1982
), while this spectral range is largely
neglected in the usual photometric measures as obtained from a
luminance meter (Minolta). Both stripe patterns could be shifted along
the horizontal and vertical axis as seen from the fly, and each of them
could be occluded separately (single-field stimulus). Furthermore, they
could be rotated independently by means of dove prisms, such that the
orientation of the pattern could be adjusted to the preferred direction
of the recorded visual interneuron. The temporal frequency of the
moving pattern was always set to 2 Hz, corresponding to a pattern
velocity of ~66°/s. The location of the two visual stimulus fields
was adjusted individually for each electrophysiological recording, such
that motion in either field elicited significant change in membrane
potential. The stimulus fields covered the ipsilateral frontal region
of the receptive field of the recorded neuron. Stimulation of the
contralateral eye was prevented by shifting the stimuli such that the
characteristic electrical response components for contralateral
stimulation disappeared [excitatory postsynaptic potentials (EPSPs)
and inhibitory postsynaptic potentials (IPSPs) in CH cells, EPSPs in
HSE and HSN]. Precision of stimulus positioning was estimated to
~5°, which was insufficient to measure size tuning curves or
delineation of receptive fields. However, all experiments involved
measurement of responses to a reference stimulus (simultaneous motion
in both stimulus fields) and the relative response differences between
this reference condition and the test condition (motion in single
stimulus field, or reduced pattern contrast). Thus the experimental
design did not rely on exact positioning of the stimulus pattern.
Electrophysiology
The cells were recorded from intracellularly, using sharp glass
electrodes (GC100F-10, Clark) pulled on a Brown-Flaming puller (Sutter
Instruments P80-PC or P97). Their resistance when filled with 1 M KCl
was 30-40 M. The indifferent electrode was a wide-tip glass
pipette, also used for supplying the preparation with Ringer solution.
To allow focusing during an experiment, both electrode micromanipulors
(Narashige), the experimental animal and the stimulus projection sphere
were mounted on a common moveable platform. Neurons were penetrated in
the axon by capacitance overcompensation. Voltage was amplified 10-fold
(Axoclamp-2A, Axon Instruments), low-pass filtered at 2 kHz,
AD-converted at an amplitude resolution of 0.244 mV per count (DT2801A,
Data Translation) and stored on a computer (IBM-AT). Data were sampled
at 1 kHz and stored as single traces and/or as averaged traces of 3-10
recordings. Computer programs for stimulus control,
electrophysiological data acquisition, and data analysis were written
in Turbo Pascal (Borland; some TP units were kindly supplied by R. Feiler, MPI für biologische Kybernetik in Tübingen,
Germany). Electrical recordings were often carried out during weak
tonic hyperpolarization with current injections ranging from 0.4 to 1.5 nA, i.e., during iontophoresis of the fluorescent dye. Comparison of
stimulus-induced depolarizations during current injections in the
mentioned range and without injection did not yield significant
correlation between injected current and the amplitude or the time
course of the electrical responses of the neuron.
Optical imaging
Changes in intracellular ionic calcium concentration
[Ca2+]i were measured as
relative fluorescence changes of an intracellular calcium-sensitive
dye. Typically, electrode tips were filled with a Fura-2 solution (20 mM Fura-2 pentapotassium salt, Molecular Probes; 33.3 mM KCl, 1.7 mM
KOH, Merck; 29.2 mM HEPES, Sigma; pH 7.3). In some experiments with
C. stygia a Calcium Green solution was used instead
(Calcium-Green-1 hexapotassium salt, 8.7 mM, Molecular Probes; 140 mM
K-acetate, Merck; 15 mM HEPES, Sigma; pH 7.3). In most cases the shaft
end of the electrode was filled with Polyvinylpyrrolidon (Fluka)
dissolved in 1 M KCl (Merck) to minimize dilution of the dye. After
injection, the dye was left to diffuse throughout the cytoplasm for
several minutes. An Hg-arc-lamp (HBO 100W, Osram; with stabilized power
supply VXHC 75/100 W, Zeiss) was used for fluorescence excitation. The epifluorescence filter set for optical recording with Fura-2 consisted of a 380-nm band-pass excitation filter (bandwidth 10 nm), a 410-nm dichroic mirror and a 500- to 530-nm band-pass barrier filter. For
experiments with Calcium Green the filter combination was a 472-nm
excitation filter (bandwidth 17 nm), a 510-nm dichroic mirror and a
529- to 560-nm barrier filter. Image magnification was 20-fold (UD20
long-distance lens, Zeiss; numerical aperture 0.56). The focal depth
was determined to be ~8-10 µm, enough for the entire dendritic
arborizations of single CH or HS cells to be in focus. Fluorescence
images were acquired with a Photometrics S200 camera system (16-bit
Peltier-cooled charge-coupled device) driven by a Macintosh IIfx
computer equipped with a NU200 controller board (Photometrics).
Temporal resolution of the camera system was 0.64 Hz (128 × 128 pixel images and 200-ms exposure). In this configuration the pixel
diameter was 4.5 µm. Image acquisition and processing was done by
means of IPlab (Signal Analytics). During each measurement a series of
20-42 images was taken. The first exposure was used to trigger both
visual stimulation and electrophysiological recording. The second image
was used as the "base image" with reference to which the relative
fluorescence change F/F in all subsequent
images of the series was calculated. To achieve higher sampling rates,
single wavelength measurements were taken (excitation at
= 380 nm). Relative fluorescence change
F/F is
approximately proportional to
[Ca2+]i up to 30%
(Lev-Ram et al. 1992
). For tangential cells of the fly,
Borst and Egelhaaf (1992)
reported a resting
[Ca2+]i in a VS1-cell to
be 50 nM. Based on this concentration, they calculated
F/F amplitudes of 10 and 30% to correspond to
[Ca2+]i increments of 34 and 135 nM, respectively. Despite the fact that Fura-2 at the used
excitation wavelength exhibits fluorescence decrements in association
with calcium,
F/F amplitudes will be inverted
throughout this paper, to describe increments of
[Ca2+]i with positive
F/F values. For quantitative analyses of
calcium accumulation, calculation of
F/F was
limited to an image region, subsequently called "analyzed region."
The relative fluorescence change
F/F was
calculated for each frame of a series of images relative to a base
frame, taken before stimulus presentation (for equation, see
Vranesic and Knöpfel 1991
). Background
fluorescence was determined in an image region away from the dyed
neuron. This image region remained the same for all measurements of a
given cell and was chosen such that the mean background fluorescence was ~2% below the lowest fluorescence value in the analyzed region. The background fluorescence was determined separately in each frame to
eliminate time-varying effects such as tissue bleaching and slight
stimulus-dependent intensity changes of scattered light. For further
aspects of
F/F calculation that are more
specific to this preparation, see Dürr (1998)
.
A crucial aspect of F/F calculation is the
choice of the analyzed region. Earlier optical-imaging studies on
tangential cells (Borst and Egelhaaf 1992
;
Egelhaaf and Borst 1995
; Egelhaaf et al.
1993
) have used masks that covered only the main dendritic branches. The main argument for this restriction of the analyzed region
was the exclusive visibility of the main branches, and the robust
F/F calculation for regions with high
fluorescence compared with the ambient background. To allow consistent
placement of the analyzed region in all cells, the masks used in this
study covered the entire dendrite, spanning the area included by the two marginal main branches and the most distal visible fine branches. The rationale underlying this choice of the analyzed region was based
on the facts that 1) the most sensitive region within the receptive fields of HS cells is located in the frontal visual field,
corresponding to the most distal zone of their dendritic arborization
in the lobula plate (Hausen 1982b
). Here, a given stimulus causes the largest depolarization; 2)
reconstructions of the dendrites of both cell classes have shown that
their fine distal arborizations virtually cover the entire area of the
lobula plate between their main dendritic branches (CH: Gauck et
al. 1997
; HS: Hausen 1982a
; Hausen et al.
1980
); 3) even though the fine distal arborizations
could not be resolved optically, the fluorescent dye diffused into
those branches, causing a detectable increase in fluorescence in the
regions between the visible dendritic branches and significant
fluorescence changes were consistently recorded from these zones. Hence
data on calcium accumulation in this paper represent spatial averages
of calcium accumulation in the entire dendrite. Exceptions are the
analysis of time course differences in proximal and distal branches
(Fig. 3, B and C), where the original mask was
divided in two parts, and the analysis of spatial overlap (Figs. 7 and
8), where the distal margin of the original mask was used to align a
dorsoventral transect.
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RESULTS |
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Time course of calcium accumulation
For illustration of the correlated changes in membrane potential and [Ca2+]i, two representative simultaneous recordings are shown in Fig. 1. After motion onset there was a steep rise in depolarization. Due to the switched epifluorescence illumination the depolarization is superimposed by brief, large transients after each shutter opening. In HS cells, the graded depolarization showed a transient early phase, reaching a steady-state phase after a few seconds of stimulus motion. The transient onset was less pronounced in CH cells. In Fig. 1 it is followed by a sustained shallow increase of depolarization. Calcium accumulation typically resembled a low-pass filtered version of the electrical response, the rise time constant being much longer in HS cells than in CH cells. After stop of motion, depolarization decreased with comparable slope to the rising phase, overshooting into a transient afterhyperpolarization. The decay of the calcium response, on the other hand, was much slower than its rise. An after-response of reversed sign was never observed for the calcium response. In CH cells, the slope of the decreasing calcium response changes from an initial steep phase to a delayed slow decline.
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An interesting feature of F/F time courses is
their striking similarity even under very different stimulus
conditions. An example is given in Fig.
2, where an HSS cell was excited by four stimuli of different size and location within the receptive field of
the cell, but also of different pattern contrast. The invariance of the
F/F time courses became evident after
normalizing each
F/F trace with respect to its
maximum amplitude (ranging from 7.5 to 34.3%
F/F). Although each stimulus condition was of
different strength, the kinetics describing the accumulation appeared
to be identical. This observation even held true when the calcium accumulation took place in different parts of the dendritic
arborization, as revealed by the time courses in response to two
nonoverlapping stimuli (Size 2 and Size 3 in Fig. 2).
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For quantitative comparison of calcium accumulation in the two
cell classes, we mathematically described the
F/F time courses by the exponential function
F/F(t) = S[1
exp(
t/
)] (Eq. 1). When time
constant
and saturation level S were varied to
numerically fit the average
F/F traces
obtained from HS cells and CH cells of C. erythrocephala,
the resulting time constant was much longer in HS cells than in CH
cells at very similar saturation levels (
= 5.3 s and
= 1 s, respectively, Fig.
3A). While the HS cell response is well described by a single exponential, there is some discrepancy in case of the CH cell response, particularly after the
initial 2 s of the response. Because the fast initial rise of the
calcium signal in CH cells was followed by a slower sustained increase,
the time course in CH cells may have been better described by two time
constants. The decay time constants of the signal after stop of
stimulus motion were estimated to be in the range of 10 s for CH
cells and 20 s in HS cells. Fits were not calculated because time
constants were longer than the measured time period. Similar to the
signal increase, the course of the declining signal in CH cells
suggests that it is not well described by a single time constant.
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Because the branching pattern of the two cell classes differs
considerably, it remained to be shown that the observed difference in
kinetics of calcium accumulation was not due to the larger proportion
of fine distal branches covered by the analyzed region in CH cells.
Because the fine distal branches were consistently found to show the
largest changes in
[Ca2+]i, the differences
found between the cell classes could well be due to differences in
kinetics between distal and proximal parts of the dendritic
arborization. To test the hypothesis that distal HS dendrites behave
like CH dendrites, F/F traces were calculated
in two subregions of the original analyzed region, one covering only
the thick main dendritic branches, the other covering the remaining
area (Fig. 3, B and C). During stimulus motion,
the mean response amplitudes in the fine dendrites were larger than the
corresponding amplitudes in the main dendrites (CH: fine 12.0%, main
7.9%; HS: fine 15.5%, main 12.6%). The corresponding pairs of single
recordings significantly differed in CH cells but not in HS cells. In
both cell classes the time constants were significantly shorter in
distal than in main dendrites (mean traces in Fig. 3, B and
C, CH: fine 0.88 s, main 1.33 s HS: fine 4.77 s, main 6.71, see figure legend). Comparison between the two cell classes yielded significantly longer time constants and larger amplitudes in HS cells than in CH cells, both in fine and main dendritic branches. Thus the observed difference in calcium
accumulation between the two cell classes cannot be attributed to
branching pattern. Interestingly, the decay time course in main and
fine branches differ only for a few seconds after stop of pattern
motion, superimposing after 5-10 s.
Dependency of calcium accumulation on membrane potential
To reveal the dependency of calcium accumulation on membrane
potential, it was necessary to measure the cell responses at various
stimulus strengths. Egelhaaf and Borst (1995) have used pattern velocity for this purpose. However, changes in pattern velocity
strongly affect the dynamic response components of tangential cells.
Because the dynamic properties of the electrical responses differ
between CH and HS cells, stimulus strength was altered with variable
pattern contrast instead. Within the chosen range, variation of pattern
contrast produced large changes in the calcium response of both cell
classes, whereas the effect on membrane potential was pronounced in CH
cells but not in HS cells. Figure 4 shows
a representative example of this finding. Both the electrical and
calcium responses of an HSS cell are displayed for three pattern contrasts. Increase of the pattern contrast corresponded to a 64%
increment of steady-state membrane depolarization and a 479% increase
in the mean calcium signal. The gray-level coding of the images
illustrates our common finding, that
[Ca2+]i increase was
largest in the distal region of the dendrite, where it received input
from the frontal region in visual space.
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More systematic investigation of the voltage-[Ca2+]i relationship revealed an almost linear dependency in CH cells but an expansive nonlinearity in HS cells (Fig. 5). After normalization with respect to the responses to the stimulus with highest contrast, the data of each cell class showed significant positive correlation between calcium signal and membrane depolarization. The qualitative observation, that HS cell data, on average, lay below the diagonal lines in Fig. 5 was statistically confirmed. In the case of HS cells, the data significantly deviated from a linear relationship, whereas the data of CH cells could not be discriminated from the linear relationship. Additionally to this statistical test, a parabolic curve fit was determined to describe the voltage-[Ca2+]i relationship in each cell class. Parabolas were used for two reasons: First, the sought function needed to intersect the origin and, to account for normalization, the point (1/1). Second, the numerical procedure could be reduced to fit a single parameter (the exponent), which could be interpreted as a measure of nonlinearity. The exponent of the best-fitting parabola was 2.8 for HS cells and 1.7 for CH cells. Similar results were obtained when individual measurements were considered rather than the means of each cell.
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Because the contrast dependency of the calcium accumulation was virtually the same in both cell classes (data not shown), the differing behavior of HS cells must be due to differences in the contrast dependency of electrical responses. At low pattern contrast, HS cells generally responded with larger membrane depolarization than CH cells, although the modulation range of the electrical responses was about the same in both cell classes.
Size-dependent saturation of the calcium response
When the moving pattern was presented in either one of the two
stimulus fields, stimulus motion in the dorsal part of the receptive
field caused a pronounced increase of F/F in
the dorsal branches of the dendrite, whereas ventral stimulation
resulted in a pronounced increase of
F/F in
ventral branches. Such retinotopic calcium accumulation was found in
all neurons examined. Qualitatively, the local
F/F amplitudes in response to motion in both
stimulus fields correlated well with the corresponding
F/F amplitudes in response to motion in two
single-field responses. To quantify how well the responses to the
single-field stimuli superimpose, we investigated whether there was
size-dependent saturation of the calcium response similar to, and
possibly related to the size-dependent saturation characteristic of the
membrane potential (Hausen 1982b
, 1984
).
In a second step, the spatial spread of the calcium signal was measured.
A directly comparable measure of size-dependent saturation of both
response parameters was defined as the difference between the
normalized linear prediction and unity: SAT = (R1 + R2)/Ref 1 (Eq. 2), where the two responses to motion in either of
the single stimulus fields, R1 and R2, are normalized by the reference response to simultaneous motion in both stimulus fields, Ref. The sum
R1 + R2 can be interpreted as the expected response to motion in both
stimulus fields in case of linear superposition. The use of a linear
prediction was sensible, because the single stimulus fields did not
overlap and the responses were recorded independently of each other.
Both response parameters displayed significant saturation in CH and HS cells, as denoted by positive values (Fig. 6). In other words, the increments of both response parameters became smaller the larger the size of the stimulus. Saturation of the calcium and electrical responses could not be statistically discriminated in CH cells, but they marginally differed in HS cells, corroborating the previous finding of a nonlinear voltage-[Ca2+]i relationship in HS cells, when stimulus contrast was varied rather than size. Comparison of the same response parameters between CH and HS cells revealed a significant difference in the saturation of the membrane potential but not in the saturation of the calcium signal.
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Overlapping calcium signals in response to nonoverlapping stimuli
In spite of the general validity of the observation that calcium
accumulation is a retinotopic process, it was evident that there was
considerable spatial overlap of the calcium responses to nonoverlapping
stimuli. The area and amplitude of spatially overlapping calcium
signals in response to nonoverlapping stimuli were determined from the
same experiments as those performed to investigate the size dependency.
Given the retinotopic columnar structure of the optic lobes
(Strausfeld 1976), excitation of two spatially disjunct
areas of the retina will lead to excitation of different populations of
input elements to the lobula plate. Because the exact border of the
dendritic region that actually received synaptic input cannot be
determined, the spatial spread of the signal was quantified by relating
the two single-field responses (Fig.
7).To do so, a
topographic map of the overlapping signal was calculated from two
corresponding
F/F images. Each pixel contained
the smaller one of the two
F/F amplitudes that were recorded at this location in response to either stimulus field
(Fig. 7D). Two parameters were quantified to describe the overlapping signal: area and amplitude profile.
|
The area of overlap was quantified for a given threshold
F/F amplitude. To make the threshold
comparable between different cells, it was expressed as a fraction of
the maximum amplitude recorded in response to simultaneous motion in
both stimulus fields. Because the choice of a particular threshold was
arbitrary, this procedure was repeated for a range of four thresholds.
Obviously, the area of overlap was smaller, the higher the threshold
(Fig. 8A). The area of overlap
largely scaled with the size of the dendritic tree. Thus in spite of
the much larger arborizations in CH cells than in HS cells, the
relative area remained comparable, although the mean values of CH cells
were consistently larger than those of HS cells, irrespective of the
chosen threshold. At a threshold of 50% of the maximum response, the
area of overlap was close to zero in both cell classes.
|
Quantification of the amplitude profile across the dendrite was carried
out for six VCH cells and six HS cells (Fig. 8B). In these
cells, the local F/F amplitudes were
determined along a vertical transect across the distal part of the
dendrite. This was done by calculation of the mean
F/F amplitudes in horizontal pixel lines,
equivalent to a 90-µm-wide stripe across the distal lobula plate,
corresponding to the input zone of the frontal visual field
(Strausfeld 1976
). An example of a single overlap
profile is shown in Fig. 7D. Because the exact midline
between the excited input regions on the dendrite could not be
determined, comparison of individual profiles required them to be
centered with respect to their maximum. The average overlap profiles
are given in Fig. 8B. Because, on average, HS cells
exhibited stronger calcium accumulation than CH cells, normalization
resulted in further separation of the overlap profiles in VCH and HS
cell. The amplitude of overlap in VCH cells reached the mean response
level in this region and was about twice as large as in HS cells.
Similar results were obtained for an earlier time window (1st 4.6 s, data not shown), indicating that the spatial extent of the overlap
does not greatly vary in time.
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DISCUSSION |
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Visual motion stimuli induced calcium accumulation in combined pre- and postsynaptic CH cell dendrites and purely postsynaptic HS cell dendrites. Dendritic calcium accumulation followed an invariant time course in both cell classes with time constants being shorter in CH cells than in HS cells. In both cell classes, calcium accumulation was stronger in fine distal branches than in thick proximal branches. The relationship of calcium accumulation and concurrent membrane potential differed considerably between types of dendrite. Both cell classes exhibited size-dependent saturation of the calcium response. The spatial spread of the calcium signal was investigated by evaluating the spatial overlap of the calcium responses to spatially nonoverlapping stimuli. Both the amplitude and the area of overlap of the calcium signal were larger in CH than in HS cells, indicating a larger extent of blurring of the retinotopic input pattern across the CH dendrite compared with the HS dendrite.
Time course of calcium accumulation and decay
The major entry mode of calcium ions into the cytoplasm can be
considered a voltage-dependent calcium influx via the cell membrane.
Voltage-dependent calcium accumulation has been reported for several
kinds of tangential cells in vivo (Egelhaaf and Borst 1995; Single and Borst 1997
,
1998
) and in vitro (Oertner and Borst 1997
). To date, a cobalt-blockable calcium current has only
been shown for CH cells (Haag et al. 1997
). Because, in
principle, voltage-dependent calcium accumulation could also arise from
electrogenic Na/Ca exchangers in the cell membrane (Blaustein
1988
; Kostyuk 1992
), experimental evidence for
the existence of voltage-dependent calcium channels is still required
for HS cells. Calcium entry via cholinergic ligand-gated ion channels
may contribute to some extent (Oertner and Borst 1997
)
but must be expected to be negligible compared with voltage-dependent
calcium influx, because null direction motion does not induce large
changes in HS cells (Egelhaaf and Borst 1995
), although
cholinergic ion channels are thought to be active also during null
direction motion (Borst et al. 1995
; Single et
al. 1997
). Recently claimed calcium decrease during null
direction motion in all tangential cells of the lobula plate (Single and Borst 1998
) was found in our own experiments
only in case of CH cells but not in HS cells (Dürr, Kurtz, and
Egelhaaf, unpublished observations). The latter difference may cause
the faster decline of
[Ca2+]i in CH cells than
in HS cells during poststimulus afterhyperpolarization.
Both the invariance of the time course and the dependency of the
maximum [Ca2+]i on
stimulus parameters are in accordance with an analytic model of
residual intracellular calcium dynamics by Tank et al.
(1995). Although their model predicts the rise and decay
kinetics of [Ca2+]i to be
strongly dependent on the concentration of intracellular calcium
buffers, the plateau concentration is expected to remain unaffected by
these buffers. Rather, the maximum
[Ca2+]i is determined by
the equilibrium of calcium influx and extrusion across the cell
membrane. Applying this model to the dendrites under investigation, the
similar
F/F amplitudes observed in CH and HS
cells suggest a similar balance of calcium entry and extrusion in each
of both cell classes. Moreover, the considerable differences between
the kinetics of the calcium response in CH and HS cells may be due to
different native buffer systems. In principle, a systematic difference
in the Fura-2 concentration in both cell classes, e.g., due to a larger
cytoplasm volume in CH cells, could mimic the same effect. However,
because fluorescence in branches of comparable diameter in both cell
classes did not vary systematically, a biasing effect due to dye
concentration should be negligible. The single time constant describing
the calcium accumulation in HS cells (Fig. 3A) suggests that
a single cellular mechanism is dominating the dynamics of the calcium signal.
Although the simple model by Tank et al. (1995)
plausibly describes accumulation of residual calcium in tangetial
cells, other mechanisms, not included in this model, possibly
contribute their share to the time course of neuronal
[Ca2+]i. For instance, in
spite of limited intracellular diffusion of calcium ions in neurons
(Gabso et al. 1997
) mobile buffers can greatly enhance
intracellular diffusability (Zhou and Neher 1993
). Furthermore, sequestration of calcium ions in
mitochondria as well as in the endoplasmatic reticulum is known to be
effective in neurons on a short time scale (Markram et al.
1995
; Pivovarova et al. 1997
;
Pozzo-Miller et al. 1997
).
Voltage dependency and spatial overlap
Variation of stimulus strength by means of pattern contrast
revealed an expansive nonlinearity of the
voltage-[Ca2+]i
relationship in HS cells (Fig. 4). This suggests the presence of a
voltage threshold for calcium entry into the cell. Because in CH cells
this relationship did not significantly deviate from linearity, a
possible voltage threshold of calcium entry in CH cells is expected to
be lower than in HS cells. Different thresholds of calcium entry in CH
and HS cells are likely to reflect different kinds of voltage-gated
calcium channels in the dendrites of CH and HS cells. In spite of the
qualitative finding of local calcium accumulation in CH and HS cells
(Borst and Egelhaaf 1992; Egelhaaf and Borst
1995
; Egelhaaf et al. 1993
; Single and
Borst 1998
) and the availability of detailed compartmental
models of the intrinsic properties of these cells (Borst and
Haag 1996
; Haag et al. 1997
), very little is
known about the link between membrane potential and spatial spread of
[Ca2+]i.
Both cell classes reveal size-dependent saturation of both calcium
accumulation and membrane depolarization (Fig. 6). While the
size-dependent saturation of the calcium signal in CH cells paralleled
the saturation observed for the electrical response, such matching did
not exist in HS cells. This corroborates our measurements of contrast
dependency. Size-dependent saturation of the membrane depolarization
has been reported earlier for HS cells (Hausen 1982b)
and CH cells (Egelhaaf et al. 1994
). The latter results
also suggested considerable spatial spread of motion-induced depolarization in the dendritic arborization of CH cells, quite contrary to what would be expected from their passive membrane properties (Borst and Haag 1996
).
Whereas we found a saturation nonlinearity in size-dependent calcium
accumulation in the CH cells, a size-dependent expansive nonlinearity
was postulated in a previous study (Egelhaaf et al. 1993). While the conclusions drawn in the present study are
based on quantitative analysis of calcium accumulation in a
sufficiently large sample of CH cells and could be tested
statistically, this was not the case in the earlier study, where the
conclusions drawn with respect to calcium accumulation were solely
derived from qualitative evidence. As a consequence, the current
hypothesis of small-field tuning in certain tangential cells
(Borst and Egelhaaf 1993
) will have to be refined.
Because the spatial divergence of retinotopic columnar processing has
never been examined on a comparably small range, it cannot be ruled out
that the observed overlap is due to presynaptic divergence of the
input, rather than due to postsynaptic expansion of the depolarization.
Yet, because several lines of evidence (see Comparison of purely
postsynaptic dendrites with pre- and postsynaptic dendrites)
indicate similar retinotopic input to CH and HS cells, divergence of
the retinotopic input, if present, should result in similar overlap of
the calcium signal in both cell classes. According to Hausen
(1981), divergence of the excitation pattern to CH cells could
be caused by nonretinotopic, recurrent excitation of the CH cell via
the two heterolateral H1 cells and the contralateral CH cells. Although
this scenario requires a succession of activity changes across four
synaptic interfaces, the effect of which seems debatable, it needs to
be tested in further experiments using microsurgical lesions of the
heterolateral pathways (e.g., Hausen and Wehrhahn
1990
). More pronounced diffusion in the spread of calcium in
VCH than in HS cells is unlikely for the following reasons.
1) The dense branching pattern of CH cells does not
support fast diffusion due to short distances. 2)
Significantly enhanced diffusion in CH cells would require raised
concentration of mobile calcium buffers (see Time course of
calcium accumulation and decay). This, however, is in conflict
with the finding of the short time constant, which should rather be
prolonged by high buffer concentrations (Tank et al.
1995
).
Voltage-dependent calcium influx must be dependent on the spatial
spread of the postsynaptic depolarization, but also on the activation
properties of the voltage-dependent process underlying the influx.
Compartmental-model simulations of the active properties in HS cells
suggest that "HS cells become significantly less compact [than
expected from the passive membrane properties] during depolarization due to the increased potassium conductance" (Haag et al.
1997, p. 363). Both the high-threshold activation of calcium
entry that was suggested above and the reduced compactness during
depolarization are active membrane properties favoring highly local
dendritic calcium accumulation in HS cells. On the other hand, the
absence of either of these two active mechanisms in CH cells is in
agreement with spatial blurring of the calcium signal in CH cells.
Comparison of purely postsynaptic dendrites with pre- and postsynaptic dendrites
The observed differences of motion-induced calcium accumulation in
CH and HS cells are summarized in Table
1. Although there is a number of
anatomically known input elements to the lobula plate (Douglass
and Strausfeld 1995, 1996
, 1998
;
Strausfeld 1976
), the physiological identity of the
input to CH and HS cells is still a matter of speculation. Yet, there
are several lines of evidence supporting the view that CH and HS cells
receive similar ipsilateral input. 1) The arborizations of
the two CH and the three HS cells cover the same area in the lobula
plate (Eckert and Dvorak 1983
; Hausen
1982a
) and are located in close vicinity (Strausfeld et
al. 1995
, their Fig. 2). 2) In their ipsilateral receptive field, both cell classes share the same directional selectivity, both on a global (Gauck et al. 1997
;
Hausen 1982b
) and on a local scale (H. Krapp, personal
communication). 3) Retinotopic calcium acccumulation in both
cell classes indicates retinotopic input from local small field
elements (Egelhaaf et al. 1993
; Egelhaaf and
Borst 1995
). An earlier hypothesis (e.g., Hausen
1984
) according to which HS cells give ipsilateral input to CH
cells could be ruled out because only a contralateral motion stimulus
causes calcium accumulation in the putative HS-CH input region
(Egelhaaf et al. 1993
, Fig. 4). 4) Finally,
detailed analysis of the local motion detector inputs yielded similar
results for both cell classes (Kondoh et al. 1995
).
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Because the neuronal substrate underlying the similar input is still unknown, further experiments are needed to tell intrinsic from extrinsic contributions to the described differences. If synaptic input to CH and HS cells were the same, the described differences in calcium accumulation would be determined entirely by the biophysical properties of the dendrites. An alternative explanation would be that CH and HS cells pool information from different sets of input elements, or at least differ in the number and distribution of their postsynaptic sites.
The computational task of CH cells is to relay retinotopic information
within the lobula plate via inhibitory dendritic synapses. The
presented spatiotemporal properties of calcium accumulation in CH cells
imply that postsynaptic neurons to CH cells, such as the FD1 cell
(Warzecha et al. 1993), receive a spatially low-pass filtered inhibitory signal acting with a time constant no longer than
1 s. It is a plausible assumption that dendritic intracellular calcium in CH cells causes transmitter release in their presynaptic terminals. However, the extent to which the measured calcium
accumulation actually reflects the activation of synaptic transmitter
release is yet to be demonstrated. In HS cells, dendritic intracellular calcium has only postsynaptic functions as a charge carrier and possible modulatory roles on membrane proteins, including ion channels.
Here, calcium accumulation is more than five times slower than in CH
cells and remains locally confined for time periods of several seconds.
Because of these characteristics, it has been suggested that a possible
modulatory role of intracellular calcium in HS cells may be in motion
adaptation (Dürr and Egelhaaf 1998
). Of course,
the same modulatory role cannot be excluded to exist in CH cells too,
yet the different
voltage-[Ca2+]i
relationships between both cell classes should lead to marked differences in the voltage dependency of any modulatory mechanism and
thus offers a set of testable predictions.
Because dendrodendritic synapses are a common feature of vertebrate and
invertebrate nervous systems, e.g., in the retina and olfactory bulb of
vertebrates (Nakanishi 1995) or in stomatogastric and
thoracic ganglia of arthropods (Graubard 1978
;
Laurent 1993
), the described fast low-pass-filtered
relaying of an excitation pattern may prove to be a common
computational feature of this synaptic design.
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ACKNOWLEDGMENTS |
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We thank R. Kurtz and A.-K. Warzecha for helpful comments on the manuscript.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG).
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FOOTNOTES |
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Present address and address for reprint requests: V. Dürr, Abteilung Biokybernetik und Theoretische Biologie, Fakultät für Biologie, Universität Bielefeld, Postfach 10 01 31, D-33501 Bielefeld, Germany.
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 19 March 1999; accepted in final form 6 August 1999.
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REFERENCES |
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