1Department of Physiology, 2Department of Ophthalmology, and 3Department of Physical Sciences/Division of Biophysics, University of Oulu, FIN-90220 Oulu, Finland
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ABSTRACT |
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Uusitalo, R. O. and M. Weckström. Potentiation in the First Visual Synapse of the Fly Compound Eye. J. Neurophysiol. 83: 2103-2112, 2000. In the first visual synapse of the insect compound eye, both the presynaptic and postsynaptic signals are graded, nonspiking changes in membrane voltage. The synapse exhibits tonic transmitter release (even in dark) and strong adaptation to long-lasting light backgrounds, leading to changes also in the dynamics of signal transmission. We have studied these adaptational properties of the first visual synapse of the blowfly Calliphora vicina. Investigations were done in situ by intracellular recordings from the presynaptic photoreceptors, photoreceptor axon terminals, and the postsynaptic first order visual interneurons (LMCs). The dark recovery, the shifts in intensity dependence, and the underlying processes were studied by stimulating the visual system with various adapting stimuli while observing the recovery (i.e., dark adaptation). The findings show a transient potentiation in the postsynaptic responses after intense light adaptation, and the underlying mechanisms seem to be the changes in the equilibrium potential of the transmitter-gated conductance (chloride) of the postsynaptic neurons. The potentiation by itself serves as a mechanism that after light adaptation rapidly recovers the sensitivity loss of the visual system. However, this kind of mechanism, being an intrinsic property of graded potential transmission, may be quite widespread among graded synapses, and the phenomenon demonstrates that functional plasticity is also a property of graded synaptic transmission.
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INTRODUCTION |
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The anatomy and electrophysiological properties of
the neurons in the visual system of the fly make it an excellent model for studying the neural signal processing with graded potentials in the
first visual synapse (Meinertzhagen and Frölich
1983; Shaw 1984
; Strausfeld 1976
,
1984
). In the compound eye of the blowfly Calliphora
vicina, six photoreceptors synapse with three first order visual
interneurons (LMCs) according to the neural superposition principle
(Kirschfeld 1967
; Uusitalo et al. 1995b
; van Hateren 1986
) forming the first synaptic complex
with multiple synapses (Shaw 1984
). In this synapse the
graded light-induced depolarization of the presynaptic photoreceptors
is inverted, made more transient, and amplified (Autrum et al.
1970
; Juusola et al. 1995a
, 1996
;
Laughlin 1987
). The photoreceptor transmitter histamine
(Hardie 1987
, 1989
) opens ligand-gated
Cl
channels in postsynaptic LMCs (Hardie
1989
; Zettler and Straka 1987
) causing a
hyperpolarization in response to light intensity increments (light-on
response). The transmitter release has been proposed to be tonic even
in dark (Laughlin et al. 1987
; Uusitalo et al.
1995a
). Similar tonically active graded potential synapses are
also present in other visual systems (vertebrate rods and cones,
Baylor and Fettiplace 1971
; Dowling and Ripps
1973
).
In the fly compound eye, strong light adaptation causes the membrane
potential of the photoreceptor soma to hyperpolarize by the activation
of an electrogenic Na+/K+
ATPase (Hamdorf et al. 1988; Jansonius
1990
). This hyperpolarization could conceivably reduce the
tonic transmitter release causing an imbalance in the influx and efflux
of the Cl
ions into the postsynaptic LMCs. This
would lead to increased light-on responses in the LMCs (Uusitalo
et al. 1995a
). The main hypothesis in this paper is that the
presynaptic Na+/K+
transporter during light adaptation hyperpolarizes the axon terminals thereby decreasing the tonic transmitter release. During this hyperpolarization, the postsynaptic tonic Cl
conductance is decreased whereas the
Cl
-extrusion mechanism remains operational.
This chain of events leads to decreased intracellular
Cl
concentration and to real postsynaptic potentiation.
We show that plasticity of the postsynaptic responses is also a
property of a graded potential synapse. During the dark recovery the
short-term plasticity resembles the potentiation seen in action potential synapses (Zucker 1989). However, the overall
function of the potentiation in this visual system may be linked to
dark recovery and could serve as a mechanism restoring the sensitivity loss after strong light adaptation and may also be linked to structural changes in the synapse during illumination (Meinertzhagen
1989
; Pyza and Meinertzhagen 1995
). We show that
in the graded synapse the active regulation of the reversal potential
of the transmitted mediated postsynaptic conductance may also
contribute to the process of visual coding.
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METHODS |
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Adult blowflies (Calliphora vicina) obtained from a
frequently refreshed stock were used for the intracellular in situ
recordings according to established procedures (e.g., Laughlin
and Hardie 1978). All recordings were performed after an
initial 30-min dark adaptation at room temperature (21 ± 1°C). A
small hole filled with high vacuum grease was put into the back of the
head capsule or to the marginal line of the cornea to penetrate the
neuronal tissue. The animal was fixed to a holder and grounded with a
Ag/AgCl wire via the hemolymph. The visual neurons were penetrated with sharp microelectrodes pulled from fiber-filled glass capillaries (Clark
Electromedical, UK) with a microelectrode puller (Sutter Instruments).
A piezoelectric micromanipulator (Burleigh PZ-550) connected to a
cardan arm system was used to advance the microelectrode in tissue with
20-nm steps. The electrodes were filled with 2 M potassium acetate with
5 mM KCl (tip resistance 100-150 M
). A Xenon light source
(Hamamatsu, Japan) connected to a shutter (Uniblitz 132, Germany) and a
filter set (Schott, Germany) was used to light adapt the visual
neurons. The test stimulus used was produced with a Xenon flash unit
(Cathodeon, UK). The recorded signal was amplified with an
intracellular amplifier (NPI-Electronics, Germany), filtered with an
adjustable filter (dual channel elliptic filter, Kemo), and stored to a
DAT-tape (Biologic, France). An IBM-compatible computer using an ASYST
program (Keithley, Juusola 1993
) was used to produce the
stimulus and store the signals used. Two oscilloscopes (Tektronix
5A20A) were used to monitor the compensation of the electrode
capacitance and to visualize the voltage responses of the neurons.
Power spectra of intracellularly recorded voltage samples were
calculated via fast Fourier transform using standard methods (Bendat and Piersol 1971; Juusola et al.
1994
) with Blackman-Harris 4-term window (Harris
1978
). Current clamp experiments using discontinuous (switched)
current injections were done as reported earlier (Finkel and
Redman 1984
; Juusola and Weckström 1993
;
Laughlin and Osorio 1989
). The switching frequency was 3 kHz and the time constant of the electrode (after critical capacitance
compensation) was ~5 µs (Weckström et al.
1992b
).
The ionophoretical injections were done by filling the electrode with 3 M potassium chloride (Re 100-150
M) The Cl
ions were injected with negative
DC current of different magnitudes from the recording electrode
(Uusitalo and Weckström 1994
). The injection was
started well before (~1 min) the light adaptation and the recovery
observed for
2 min. The ionophoretical experiments started with zero
current and went up to
2.5 nA in 0.5-nA steps while the capacitance
was critically monitored.
The best available method for studying the synapse between neurons is
to try and make intracellular recordings from both pre- and
postsynaptic neurons simultaneously. This, however, would be extremely
difficult in the case of this synapse because the small diameter of the
neurons involved (~1-2 µm) makes simultaneous pre- and
postsynaptic recordings extremely rare. At present the possible method
is to make intracellular recordings from the pre- and postsynaptic
neurons separately and to use a set of strict electrophysiological
criteria to rule out recordings that are from injured or poorly
penetrated cells. We mainly used the electrophysiological criteria we
reported earlier (Uusitalo and Weckström 1994).
The criteria for the good penetrations of the photoreceptor axon
terminals were a low resting potential (r.p.) (from
50 to
60 mV), a
large Rin (60-110 M
), and the presence of
the fast-depolarizing transient (Weckström et al.
1992a
). The criteria for the postsynaptic LMCs were as follows:
resting potential (
40 to
60 mV),
Rin (15-40 M
), and light-on
response (greater than
35 mV; Hardie and Weckström 1990
). To be qualified, the recorded neurons had to fulfill all the criteria above.
To study the process of dark recovery in the first visual synapse we
stimulated the photoreceptors axon terminals with a long-lasting light-adapting pulse to which a 30-µs test flash was superimposed. The intensity of the adapting stimulus was varied from 2 × 105 to 2 × 108
effective photons/s and the duration was varied from 100 ms to 40 s. The test stimulus had a constant duration of 30 µs with a
intensity from 73 to 1.5 × 105 effective
photons/flash and a interstimulus period of 300 ms. The light output of
the Xenon light source was calibrated in terms of effective light
quanta by counting, after prolonged (>60 min) dark adaptation, the
discrete small responses evoked by single photons (Lillywhite
1977) occurring in photoreceptors during dim illumination.
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RESULTS |
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Dark recovery
To study the mechanisms behind dark recovery we recorded light-evoked responses from photoreceptor somata, axons terminals, and postsynaptic LMCs following different light-adaptational states (typical experiments shown in Fig. 1). Altogether 45 photoreceptors somata, 15 photoreceptor axon terminal recordings, and 40 LMCs were used for the results.
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After a strong light-adapting stimulus (2 × 108 effective photons/s) the photoreceptor soma
recordings showed a marked hyperpolarization of the resting potential
that reached a maximum of 21.7 ± 2.9 (SD) mV (n = 10). This hyperpolarization that was found in the photoreceptor axon
recordings (Fig. 1; note that here the responses to test flashes are
still depolarization transients) depended on the duration and intensity
of the adapting light. Using exactly the same protocol, after the same
stimulus, the postsynaptic LMCs depolarized by 4 ± 2 mV
(n = 10) and showed a marked decrease in the
conspicuous dark noise (Fig. 1 and Fig. 4, inset). The decrease in the dark noise was seen as a decrease in membrane fluctuation. Concomitantly with this, the LMC responses to a test stimulus showed a clear enhancement, or potentiation, that reached a
maximum of 9 ± 0.2 mV (n = 10; Fig. 1), as shown
before (Uusitalo et al. 1995a
). The response enhancement
lasted ~10 s after light adaptation. The amplitude and time course of
the postsynaptic potentiation were found to depend on the intensity and
duration of the adapting stimulus, in the same manner as the
pump-induced afterhyperpolarization in the presynaptic axon recordings
(see below, e.g., Fig. 6B). The presynaptic pump potential
and the postsynaptic potentiation could also be elicited by a
high-frequency train (120 Hz) of short (30 µs) saturating light-on
stimuli to photoreceptors (data not shown).
Light-evoked responses in presynaptic photoreceptor somata, axons, and postsynaptic neurons after light adaptation
The amplitude of the test flash responses (duration 30 µs)
increased in the photoreceptor somata during the afterhyperpolarization when compared with responses to the same test flash during dark-adapted conditions. This was maximally 6 ± 1.2 mV (n = 20, typical single responses, enlargements from experiments of the type
in Fig. 1 are shown in Fig.
2A). The increase can readily
be interpreted to have been caused by the hyperpolarization of the
resting potential by the Na+ pump potential. This
increased the driving force for the light-gated current and also
reduced the effects of the voltage-gated potassium conductance
(Juusola and Weckström 1993;
Weckström and Laughlin 1995
;
Weckström et al. 1991
). In the axons, the
responses to the test stimulus increased and the transient
characteristics were enhanced (Fig. 2B). The increase of the
fast depolarizing transient was larger than the increase in the soma
recordings (10 ± 2.5 mV; n = 15) and depended on
the hyperpolarization (i.e., the pump potential). Mimicking this, the
increase in the fast depolarizing transient amplitude could be seen by
hyperpolarizing the resting potential of the axon terminal by current
clamp (see also Weckström et al. 1992a
).
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In the postsynaptic LMCs, both unsaturated and saturated responses to
the test flash were potentiated, maximally by ~9 mV after light
adaptation. The period of the potentiation after light adaptation
coincided with the period of the presynaptic pump potential. During
potentiation the hyperpolarizing responses were narrower than in
dark-adapted conditions (Fig. 2C) and thus resembled mostly the transient component (the fast depolarizing transient) of the presynaptic axon responses during this same period (Fig.
2B). The more transient LMC responses were seen during the
potentiation in every experiment. The oscillations often observed in
the LMCs (e.g., Juusola et al. 1995a; van Hateren
1986
) and the light-off depolarization were not present. The
responses recovered when the potentiation effect also disappeared,
~10 s after its initiation.
The voltage versus light intensity functions (V/Log
I functions, see Laughlin et al. 1987) of
photoreceptor somata in dark-adapted conditions and after light
adaptation (Fig. 3A) showed
that the responses increased from the test flash intensity of
~103 effective photons/flash upwards. In the
presynaptic axons (Fig. 3B) this change was bigger and was
present with lower stimulus intensities. The V/Log
I functions from the postsynaptic LMCs revealed that
although both subsaturated and saturated LMC responses were
potentiated, the near saturating responses were potentiated the most
(Fig. 3C). Small (<7 mV) LMC responses to stimuli with <100 photons/s were actually smaller than in dark-adapted conditions. Thus only the LMC responses from ~7 mV upwards were enhanced. The
slope of the V/Log I function was steeper after
light adaptation than under dark-adapted conditions and was shifted
toward lower intensities during the potentiation. The slopes of the
normalized functions were very nearly equal, suggesting that the
sensitivity of transmitter release to the number of
Cl
channel openings did not change (for
discussion of this see Laughlin et al. 1987
).
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Mechanism behind potentiation
Frequency domain analysis of the LMC responses under
potentiation-inducing conditions, during the (now) depolarized resting potential, revealed that the power spectrum of the noise showed attenuation especially at the low-frequency band (Fig.
4, inset, shows a typical
recording trace; note the absence of test flashes in this type of
experiment). The input resistance Rin
of the photoreceptor axons increased 8% from 62 ± 5 M
(n = 3) in dark-adapted conditions to 68 ± 4 M
(n = 3) with a hyperpolarization of ~20 mV.
Concomitantly also the Rin of the
postsynaptic LMCs increased by 7.4 ± 2.9 M
(n = 5; i.e., by 33% during the potentiation). This showed that during
the attenuation of the membrane noise the total conductance of the
cells as well as the LMCs is reduced.
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In the postsynaptic LMCs, all of the changes (reduced noise levels, depolarized period of the resting potential, and period of the increased Rin) recovered to normal at a certain time point after the end of the adapting light pulse. The voltage of the presynaptic photoreceptor axons at this point was below the normal resting potential by 11 ± 1.5 mV (n = 10). We can argue that this result represents the value for the voltage in presynaptic axons below which the postsynaptic membrane noise was significantly decreased. The time that the presynaptic voltage spent below this voltage (during the pump potential) matched the period of depolarized r.p. and the decreased noise level in the postsynaptic LMCs.
Involvement of the postsynaptic Cl transport in the
potentiation
Because the depolarization, noise reduction, and increased
Rin of the LMCs during potentiation
obviously point toward the possibility of reduced transmitter release,
we tested if the postsynaptic Cl transport
contributes to the overall potentiation of the postsynaptic responses.
The Cl
pump (extruding
Cl
) speeds up as the intracellular
Cl
concentration is increased (Uusitalo
and Weckström 1994
). Accordingly, the potentiation should
be bigger if the intracellular Cl
concentration
is increased. We introduced a constant Cl
load
into the LMCs prior to the potentiation-inducing light stimulus using
ionophoretic injections while observing the dark recovery. Consistent
with earlier reports, a large Cl
load (
2.5
nA) was able to cause a reversal of the light-on response (the
hyperpolarizing transient shown in Fig.
5A turned into a depolarizing
one in Fig. 5B) by shifting the
ECl to be more positive than the
resting potential (Hardie 1987
; Uusitalo and
Weckström 1994
; Zettler and Straka 1987
).
Under these conditions, when the response was reversed, the
potentiation also occurred. The peak light response turned back (from a
depolarizing transient) into a hyperpolarization (in Fig.
5C) for a period of time approximately as long as the
potentiation lasted normally. The relative magnitude of the
potentiation in terms of mV, when calculated relative to the
on-response size, was 24 mV, which is larger than during the Cl
load. The dark resting potential was also
depolarized after the Cl
load, apparently
because of a large basal Cl
conductance and the
shift of the ECl to a more positive
value (see also Uusitalo and Weckström 1994
).
After the potentiation was over, the response returned back to the
reversed state (Fig. 5D) These findings are consistent with
the hypothesis that an increase (positive shift) in
ECl was behind the potentiation.
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Dependence of the potentiation on light-adapting stimulus
The magnitude of the pump-induced hyperpolarization in the somata
of the presynaptic photoreceptors has been reported to depend on
intracellular sodium concentration (Jansonius 1990).
Because the potentiation is linked to presynaptic hyperpolarizing pump potential, it is evident that the amount of sodium entering the photoreceptors during light adaptation is of critical importance. We
tested in the following changes that take place in both the presynaptic
pump potential and the postsynaptic potentiation (in terms of size and
duration) by varying the intensity and duration of the light-adapting
stimulus. Thus we also putatively varied the intracellular sodium
concentration at the end of the adapting light pulse in the presynaptic photoreceptors.
When the duration of the light-adaption (intensity 2 × 108 photons/s) was increased, both the
pump-induced presynaptic hyperpolarization and the postsynaptic
potentiation increased in size up to the duration of 2 s (Fig.
6). With longer light adaptation both the pump potential and potentiation started to decline. This seemed to be
correlated with an additional depolarizing afterpotential that was
manifested just after the end of the light-adapting stimuli, both in
the photoreceptor somata and in the axon terminals. The second
afterpotential started to mask the hyperpolarization caused by the
Na+ pump from a duration of 4 s onwards
(data not shown). The afterpotential was also seen after preadaptation
with green (555 nm) stimuli suggesting the noninvolvement of the
prolonged depolarizing afterpotential (e.g., Hamdorf and
Schwemer 1975). After >20 s of light adaptation the
pump-induced presynaptic hyperpolarization did not reach the threshold
for decreased transmitter release (~11 mV below the resting
potential) and no postsynaptic potentiation was seen. The maximum
amplitude of the potentiation corresponded well with the maximum
hyperpolarization caused by the pump potential (Fig. 6).
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Synaptic voltage transfer
We considered it important to characterize the function of the
first visual synapse at voltages below the dark-adapted resting potential because the presynaptic resting potential is relatively depolarized when compared with many other preparations, and in a tonic
synapse these voltages could be used for signal coding (Laughlin
1987). Below the dark-adapted resting potential (i.e., during
the pump potential) the gain of the synapse, as defined from the slope
of the characteristic curve (the postsynaptic response in function of
the presynaptic response, Fig. 7, e.g.,
Laughlin and Hardie 1978
), was larger, especially just
above the presynaptic threshold (
71 mV) for the transmitter release.
During the potentiation the synaptic gain gradually decreased
approaching values normally observed during dark-adapted conditions.
This synaptic gain went even below the initial value from the
presynaptic voltage of
55 mV onwards, when defined on the basis of
presynaptic axon recordings. This was not surprising because the
characteristic curve for the synapse below the dark-adapted resting
potential was studied here immediately after light adaptation (i.e.,
during the potentiation). In contrast, the synaptic gain with small
presynaptic voltages after the light adaptation was astonishingly high.
The synaptic characteristic curve calculated from the axon terminals
(Fig. 7B) showed a somewhat smaller synaptic gain increase
with low voltages compared with those obtained on the basis of the soma recordings, probably related to the fast depolarizing transient in the
axons.
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Function of the potentiation
To study if the postsynaptic potentiation could have some
contribution to the sensitivity recovery after light adaptation, we
measured the recovery of subsaturated responses in the LMCs after
saturating light adaptation (2 × 108
photons/s) with conditions where the potentiation was present and
compared these results with those obtained in conditions without the
potentiation. The amplitude of the flash responses was converted into
intensities via the V/Log I curve of the cells
and from those on to sensitivities (in %). The sensitivity after the
light adaptation under conditions without the potentiation (i.e., after
strong light adaptation of 20 s) showed a more reduced sensitivity
than expected. The sensitivity loss was more than tenfold and it
recovered to normal after a period of ~50 s. Under normal conditions
(i.e., without the potentiation) the recovery took place with two slow phases with a faster one in between (Fig.
8). During potentiation the sensitivity
after adaptation was found to be recover to normal within 3 s.
Thereafter it exceeded the normal sensitivity by almost eightfold. The
sensitivity recovery after potentiation resembled the recovery of
responses after mild light adaptation both in photoreceptors and LMCs
(Laughlin and Hardie 1978). The difference in the
sensitivity just after the light adaptation between these two
conditions was 2 log-units in favor of the potentiation (Fig. 8). The
sensitivity recovery should be faster with shorter light stimulation as
demonstrated earlier (Laughlin and Hardie 1978
), but
normally it should not be enhanced beyond the dark-adapted level, which
happens in this experiment. Accordingly, the potentiation could be an
important mechanism in recovering the sensitivity loss after intense
light adaptation.
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DISCUSSION |
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The performance and adaptation of synapses where signals are
transmitted with graded potentials are poorly understood. We have
utilized the first visual synapse in the fly compound eye (see reviews
by Juusola et al. 1996; Laughlin et al.
1987
) to investigate the processes after light adaptation
(i.e., during dark recovery of the synaptic function). The dipteran
visual system, like that of any other diurnal animal, is as frequently
exposed to dark adaptation as it is to light adaptation. The efficiency of the processes underlying dark recovery can be argued to be of high
significance to the synaptic function in general and to strongly
contribute to the overall visual behavioral performance as well. As
this study demonstrates, the light-on responses of LMCs are clearly
enhanced after light adaptation. This enhancement, as judged from the
time course, fulfills the criteria stated for the short-term
potentiation (e.g., Zucker 1989
) and thus can be called
postsynaptic potentiation. The enhancement seems to be causally linked
to tonic transmitter release in the photoreceptor-interneuron synapse
(see Uusitalo et al. 1995a
).
After light adaptation there seems to be two major processes operating
simultaneously determining the DC voltage of the presynaptic photoreceptors. The first is the activation of the electrogenic Na+/K+-ATPase (Fig.
9) that operates under stoichiometry of
~3/2 (Hamdorf et al. 1988; Jansonius
1990
) and, when increasingly activated, hyperpolarizes the
resting potential. The second is the process that depolarizes the
resting potential after light adaptation with longer periods. This
depolarization is likely to be related to the
Na+/Ca+ exchanger
(Gerster et al. 1997
; Hochstrate 1991
;
Minke and Kirschfeld 1984
). It appears that
light-adapting pulses lasting from 100 ms to 2 s cause an increase
of the afterhyperpolarization (Fig. 6), which would be expected when
the intracellular sodium concentration and thus the activation of the
Na+/K+ pump increases
(Jansonius 1990
). When the stimulus duration is increased using the same light intensity, the hyperpolarizing pump-potential starts to decrease, thereby decreasing the potentiation amplitude in the postsynaptic LMCs as well (Fig. 6). The most obvious
explanation for this is that the depolarizing afterpotential is
increasingly masking the
Na+/K+ pump potential,
because the intracellular calcium rises to high levels
(Oberwinkler and Stavenga 1998
). Another process being activated as a result of the hyperpolarization of the resting potential
is the increasing amplitude of the fast depolarizing transient in the
presynaptic axon terminals (Weckström and Laughlin 1995
; Weckström et al. 1992a
). When the
resting potential is lowered, the fast depolarizing transient seems to
be activated more (Fig. 2). Although the exact mechanism of this is
beyond the scope of this paper, we may safely assume (see
Weckström et al. 1992a
) that it is caused by
voltage-gated ion channels, analogously as in the bee photoreceptors
(Vallet and Coles 1993
; Vallet et al.
1992
). The activation of the fast depolarizing transient is not
directly linked to potentiation, however, because even the saturated
LMC responses were clearly enhanced during the potentiation (see Figs.
1-3) and the increase of the presynaptic depolarization should not
increase the postsynaptic response when the postsynaptic responses have
reached saturation. On the other hand, the more transient nature of the
LMC responses during potentiation may well be caused by the increased
presynaptic fast depolarizing transient (Fig. 2).
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Further exclusions can also be made. The mechanism behind potentiation
cannot be increased sensitivity of the transmitter release mechanism in
the photoreceptors. This increase and the resulting increased
Cl conductance in the already saturated LMCs
are not able to increase the response amplitude without a change in
ECl. Another possibility would be an
additional synaptic input to LMCs that would be activated after strong
light (e.g., Hardie et al. 1989
; Nässel
1987
; Weckström et al. 1989
). This would
increase the conductance to some ions during the responses in the LMCs.
Thus the activation of additional conductances would mean a drop in the
Rin of the LMCs. Contrary to this
hypothesis, we found that in the LMCs the
Rin increased during the potentiation by
33%. Another possibility is that the ~4 mV steady-state
depolarization of the LMC membrane potential could, by
voltage-sensitivity of the histamine-gated channels, account for the
potentiation. We have no evidence for this but it has been shown
previously that the histamine-gated channels have only very weak
voltage sensitivity that could not explain the potentiation
(Hardie 1989
).
As judged from the characteristics of the membrane noise (Fig. 4), the
transmitter release seems to be decreased when the presynaptic axon
terminals hyperpolarize. It is known that the release of the synaptic
vesicles containing the transmitter is caused by activation of
Ca2+ channels. Thus an increase in the release
reflects the increase of the presynaptical intracellular free
Ca2+ (Llinas 1982). The decrease
in the transmitter release could be directly related to this if the
probability of the Ca2+ channels being open is
drastically decreased below the voltage of ~11 mV under the normal
resting potential (
60-65 mV) of the photoreceptor axons. This means
a relative threshold for the Ca2+-channel
activation of approximately
71 to
76 mV. This activation threshold
does not agree with properties of the L-type channels likely to
dominate the Ca2+ current in the presynaptic
membrane (Juusola et al. 1996
) but it agrees with
findings in the axons of barnacle photoreceptors (Hayashi and
Stuart 1993
).
What is causing the enhancement of the postsynaptic LMC responses if
the potentiation mechanism is substantially postsynaptical? The signal
transfer in the photoreceptor-LMC synapse is relatively nonlinear
(Juusola et al. 1995b). As a component of the
nonlinearity, the LMCs have been reported to have an efficient
Cl
-extrusion mechanism to maintain a stable
ionic composition (Uusitalo and Weckström 1994
).
Apparently, during light adaptation the intracellular chloride
accumulation exceeds the capability of the extrusion mechanism for the
ion, resulting in depolarization of the resting potential and a
decrease in the peak amplitude of the hyperpolarizing response
(Uusitalo and Weckström 1994
). It is conceivable
that during the period of reduced transmitter release (the
afterhyperpolarization, presynaptically) the chloride extrusion would
still operate, at least for a relatively short period, as in normal
conditions. This would cause a negative shift of the equilibrium
potential of the histamine-gated conductance. During strong
illumination the extracellular field potential is being depolarized by
up to 25 mV (P. Kettunen, S. B. Laughlin, and M. Weckström,
unpublished observations), rendering the transmembrane potential in the
LMCs more hyperpolarized, which would still accelerate the
Cl
extrusion.
The ionophoresis of Cl into the LMCs (Fig. 5)
clearly shifted the ECl to be more
positive than the resting potential thus reversing the polarity of the
responses. The potentiation after light adaptation appeared here with
the usual time course but was clearly enhanced in size. This was a
further indication that the Cl
-extrusion
mechanism was causing the potentiation. The extrusion process speeds up
with increasing intracellular chloride (Uusitalo and
Weckström 1994
). The small 4 mV depolarization observed
normally was not seen after Cl
injection which
was caused by the smaller Cl
driving force when
the ECl was near the resting potential
and when the drop in the resting Cl
influx did
not change the membrane potential.
The recovery of sensitivity by the enhancement of light-on responses
(Fig. 8) is not apparent under all conditions. This takes place in a
"potentiation window" (determined by the preceding stimulation) in
which the responses are enhanced and the sensitivity loss is recovered.
This window extends in time from light-adapting durations from 100 ms
to almost 20 s (Fig. 6). As we do not have enough behavioral
evidence, we can only hypothesize about the conditions in which this
recovery mechanism could be of functional significance. However, the
blowfly is usually operating in daytime and normally moves constantly
with considerable velocity, flying above and among (possibly moving)
objects. For a fast moving animal, an adequate visual performance under
all conditions is of crucial importance. This is true not only in dark-
and light-adapted conditions but also during periods between them when
shadows and sunshine are alternating. Our hypothesis is that the visual
system is operating quite poorly when the fly moves from shadow to full
sunshine, during which the light-adapting stimulus well resembles the
one used here to elicit the potentiation. The loss of visual function is caused at least partially because the light-adapting process takes
time whereas the gain in the first visual synapse remains high. It has
been reported that the light-adapting process after intense light
adaptation can take up to 2 min in the synapse (Juusola et al.
1995a). During this time it would be convenient for the fly to
go back to shadow again to recover the adequate visual performance. The
mechanism presented in this paper would allow the fly to spend a period
of up to 20 s in light-adapted conditions and still recover the
sensitivity very rapidly (Fig. 8) when reaching the shadow again, a
clear behavioral advantage.
Regardless of the possible functional and behavioral significance of
the potentiation, it has to be recognized that the regulation of the
Cl homeostasis in these interneurons is of
great importance. As shown earlier, the Cl
equilibrium potential is depolarized during light adaptation (Uusitalo and Weckström 1994
). It was shown in
this paper that the ECl is being
shifted to a more negative value after light adaptation. Both of these
changes require a role of the Cl
-extrusion
mechanism (Fig. 9) and suggest that modulation of the synaptic
transmission via this Cl
pump is of importance.
The function of neurons is crucially dependent on different ion
conductances and their modulation. The present evidence shows that the
action of ion exchangers opposing the ionic conductances has to be
given greater emphasis, at least in small graded potential neurons
where the changes in intracellular ionic composition is relatively
easily changed. These neurons have difficulties maintaining a constant
ionic composition because they use very large ion fluxes compared with
their size to generate the signals. Whereas this clearly is a problem,
the fly LMCs have also developed a way of taking advantage of this
property, which is to decrease the sensitivity loss after light adaptation.
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ACKNOWLEDGMENTS |
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We thank R. Hardie, S. Laughlin, E. Kouvalainen, E. Warrant, M. Juusola, and P. Tavi for critical help during the course of the work, and S. Leo and A. Vanhala for technical support.
This work was supported by the Finnish Eye Research Society, the foundation Suomen Silmä-ja kudospankkisäätiö, and the Academy of Finland.
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FOOTNOTES |
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Address for reprint requests: M. Weckström, Dept. of Physiology, University of Oulu, Kajaanintie 52a 90220 Oulu, Finland.
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 30 November 1998; accepted in final form 9 November 1999.
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REFERENCES |
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