Department of Anatomy and Cell Biology, University of Bergen, N-5009 Bergen, Norway
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ABSTRACT |
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Hartveit, Espen.
Reciprocal synaptic interactions between rod bipolar cells and amacrine
cells in the rat retina. Reciprocal synaptic transmission between rod bipolar cells and presumed A17 amacrine cells was studied
by whole cell voltage-clamp recording of rod bipolar cells in a rat
retinal slice preparation. Depolarization of a rod bipolar cell evoked
two identifiable types of Ca2+ current, a T-type current
that activated at about 70 mV and a current with L-type pharmacology
that activated at about
50 mV. Depolarization to greater than
or equal to
50 mV also evoked an increase in the frequency of
postsynaptic currents (PSCs). The PSCs reversed at
~ECl (the chloride equilibrium potential), followed changes in ECl, and were blocked by
-aminobutyric acidA (GABAA) and
GABAC receptor antagonists and thus were identified as
GABAergic inhibitory PSCs (IPSCs). Bipolar cells with cut axons displayed the T-type current but lacked an L-type current and depolarization-evoked IPSCs. Thus L-type Ca2+ channels are
placed strategically at the axon terminals to mediate transmitter
release from rod bipolar cells. The IPSCs were blocked by the
non-N-methyl-D-aspartate (non-NMDA) receptor
antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that
non-NMDA receptors mediate the feed-forward bipolar-to-amacrine
excitation. The NMDA receptor antagonist
3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid had no
consistent effect on the depolarization-evoked IPSCs, indicating that
activation of NMDA receptors is not essential for the feedforward
excitation. Tetrodotoxin (a blocker of voltage-gated Na+
channels) reversibly suppressed the reciprocal response in some cells
but not in others, indicating that graded potentials are sufficient for
transmitter release from A17 amacrine cells, but suggesting that
voltage-gated Na+ channels, under some conditions, can
contribute to transmitter release.
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INTRODUCTION |
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Despite detailed morphological information
available about the individual components, it has been difficult to
obtain clear evidence regarding the function of specific reciprocal
synapses in the mammalian retina. Bipolar cell synapses in the inner
plexiform layer occur as dyads where a synaptic ribbon in the bipolar
cell axon terminal is apposed simultaneously to two postsynaptic
processes. A reciprocal synapse occurs when an amacrine cell process
provides a synapse back onto the bipolar cell axon terminal
(Dowling and Boycott 1966; Raviola and Raviola
1967
). For rod bipolar cells, both postsynaptic processes (AI
and AII) are those of amacrine cells, but only one of the postsynaptic
processes (AI) provides a reciprocal synapse onto the rod bipolar
terminal (Kolb and Famiglietti 1974
). In the cat retina,
the AI process has been identified as generally belonging to the type
A17 wide-field amacrine cell, which lacks an axon but has a large
number of thin, radially oriented dendrites of considerable lateral
extension (Nelson and Kolb 1985
). In the rat retina,
A17-like amacrine cells have been described in Golgi-material
(Perry and Walker 1980
), but their involvement in rod
bipolar cell dyads has not been demonstrated directly. However, the
circuitry of the rod pathway in the rat retina has been shown to be
similar to that of other mammals (Chun et al. 1993
; Kim et al. 1998
); thus in this
study, the amacrine cell providing the reciprocal process in rod
bipolar dyads in the rat retina for simplicity will be called the A17 cell.
It generally is thought that the bipolar cell-to-amacrine cell
connection is glutamatergic (Dong and Werblin 1998;
Ehinger et al. 1988
; Tachibana and Okada
1991
) and that the reciprocal amacrine cell-to-bipolar cell
connection is GABAergic (Chun and Wässle 1989
;
Dong and Werblin 1998
; Freed et al.
1987
). In terms of function, one idea is that feedback from
amacrine cells to bipolar cells may expand the operating range of the
bipolar cells (e.g., Tachibana and Kaneko 1988
). Little
is known, however, about the functional characteristics of this
reciprocal synaptic interaction in the mammalian retina. It is not
known, for example, whether transmitter release from a single rod
bipolar axon terminal is sufficient to trigger a feedback response onto
the same bipolar terminal from which transmitter was released, whether
synaptic release of GABA from A17 amacrine cells activates both
GABAA and GABAC receptors on rod bipolar
terminals, or whether GABA is released from A17 amacrine cells by a
conventional vesicular mechanism or a nonvesicular,
transporter-dependent mechanism as has been suggested for some amacrine
cells (O'Malley et al. 1992
; Vardi and Auerbach
1995
).
In the present study, the functional properties of the synaptic
transmission between rod bipolar cells and A17 amacrine cells were
examined directly by whole cell voltage-clamp recordings from rod
bipolar cells in the rat retinal slice preparation. The main findings
were that rod bipolar cells display both T- and L-type voltage-gated
Ca2+ currents, transmitter release is elicited from the
axon terminals by depolarization greater than or equal to 50 mV, and
transmitter release is mediated by the L-type Ca2+ current.
Furthermore synaptic input from rod bipolar cells to A17 amacrine cells
is mediated by ionotropic glutamate receptors and the reciprocal
inhibitory feedback is most likely mediated by vesicular release of
GABA, acting on both GABAA and GABAC receptors on the rod bipolar axon terminals. A brief account of some of these
findings has been published in abstract form (Hartveit
1997b
).
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METHODS |
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The methods have been described previously in detail
(Hartveit 1996, 1997a
). Albino rats (4-8 wk postnatal)
were anesthetized deeply with halothane in oxygen and killed by
cervical dislocation. After the retina was dissected free, vertical
slices were cut by hand.
Electrophysiology and infrared video microscopy
During experiments, slices were viewed with infrared differential interference contrast (Nomarski) video microscopy and epifluorescence optics. The extracellular perfusing solution was bubbled continuously with 95% O2-5% CO2 and had the following composition (in mM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 2.5 CaCl2, 1 MgCl2, 1.25 NaH2PO4, and 10 glucose, pH 7.4 (18-22°C). In all but a few experiments, 0.5 µM strychnine was added to the extracellular solution. In experiments where Co2+ was added to the extracellular solution (replacing Ca2+), NaH2PO4 was omitted.
All recordings were made in the whole cell configuration of the
patch-clamp technique. The electrode resistance was 5-6 M except
for a few experiments where the resistance was increased to 7-9 M
.
For standard experiments with high intracellular chloride concentration, the recording electrodes were filled with a solution of
the following composition (in mM): 125 CsCl, 1 MgCl2, 15 tetraethylammonium (TEA) chloride, 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 0.1 ethylene
glycol-O,O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), 12 phosphocreatine, and 4 ATP, pH 7.3 with CsOH. EGTA was
added at a low concentration to reduce the contamination of
Ca2+ in the pipette solution, while at the same time
minimally disturb the endogenous Ca2+ buffering systems. In
some experiments, the pipette solution also contained 10 mM CsOH and 10 mM glutamic acid, replacing an equimolar amount of CsCl. A high
internal chloride concentration was used to enhance GABA-gated chloride
currents at a holding potential (Vh) of
70 mV
(chloride equilibrium potential (ECl) = +2.1 mV;
+0.3 mV when 10 mM Cs-glutamate replaced an equivalent amount of CsCl
intracellularly). Because the recordings depended on a high internal
chloride concentration, it was not feasible to use the perforated-patch
technique. In some recordings, the extracellular chloride concentration
was reduced from 134.5 to 20.5 mM by substituting 114.5 mM NaCl with an
equimolar amount of sodium isethionate. This recording condition was
used to enhance GABA-gated chloride currents at a
Vh of
20 or
30 mV
(ECl = +49.6 mV). For experiments with low
intracellular chloride concentration, the solution had the following
composition (in mM): 125 CsOH, 125 gluconic acid, 1 MgCl2,
15 TEA, 10 HEPES, 0.1 EGTA, and 4 ATP, pH 7.3 with CsOH. Lucifer yellow
was added at a concentration of 1 mg/ml to all intracellular solutions.
Theoretical liquid junction potentials of extracellular solutions
against the internal solutions were calculated with the computer
program JPCalcW (Axon Instruments, Foster City, CA) according to the
generalized Henderson equation (Barry and Lynch 1991
).
The software (Pulse; HEKA elektronik, Lambrecht/Pfalz, Germany)
controlling the amplifier (EPC-9; HEKA Elektronik) automatically
corrected the holding potentials for the liquid junction potentials
on-line.
To reduce electrode capacitance, some electrodes were coated with
dental wax (Kerr's sticky wax) and gently fire polished immediately
before usage. Seal resistances were in the gigaohm range (10 G
for
electrodes with heat-polishing, 2-6 G
for electrodes without
heat-polishing). Series resistances were usually between 10 and 30 M
and were in about one-third of the recordings compensated by 50-90%,
with a lag of 2 or 10 µs. Because of the small magnitude of the
evoked currents, the steady-state voltage error at the soma caused by
the series resistance is likely to have been small. Nevertheless, I
sometimes observed sustained tail currents after depolarization-evoked
Ca2+ currents, indicating considerable escape from dynamic
voltage control (cf. Matsui et al. 1998
). These cells
are excluded from the material reported here. Depending on the
experimental protocol, the digital sampling interval was varied between
10 µs and 1.1 ms. Before sampling, the signal was low-pass filtered
(analog 3- and 4-pole Bessel filters in series) with a corner frequency (
3 dB) automatically adjusted to 1/3 of the sampling frequency. Capacitative currents caused by the recording pipette and the cell
membrane were measured with the automatic capacitance neutralization network feature of the EPC-9 amplifier (Sigworth et al.
1995
). With this method, the average cell capacitance was
3.23 ± 0.07 (SE) pF (n = 152). This is likely to
be an underestimate of the true capacitance, as values obtained from
charging transients (Mennerick et al. 1997
), after
disabling the Cslow capacitance neutralization
circuitry of the EPC-9, result in higher capacitance values (M. Veruki
and E. Hartveit, unpublished results).
In experiments where Ca2+ currents were isolated
pharmacologically, potassium currents were blocked by recording with
the intracellular solutions above and by replacing 20 mM NaCl by 20 mM
TEA in the extracellular solution. The extracellular solution also
contained picrotoxin (1 mM). In all but a few recordings of
voltage-gated Ca2+ currents, the concentration of EGTA in
the intracellular solution was increased to 5 mM, with Ca2+
added at a concentration of 1 mM (as CaCl2).
Ca2+ currents were generally evoked from a holding
potential of 60 mV or a 1-s conditioning hyperpolarization to
100
mV either by voltage ramps (to +40 mV; ramp speed 100 mV/s) or by
stepping the membrane potential for 30-150 ms to potentials between
90 and +40 mV. The interpulse interval was 15-20 s. For some
measurements of Ca2+ currents, linear leak and capacitative
currents were subtracted by a P/N protocol.
Drugs were dissolved in a HEPES-buffered extracellular solution
(Hartveit 1996) and applied by pressure from a five- or
seven-barrelled pipette complex. The concentrations of the drugs were
as follows (obtained from Tocris Cookson, Bristol, UK, unless stated
otherwise): 500 µM 3-aminopropyl(methyl)phosphinic acid (3-APMPA);
200 µM baclofen; 20 or 100 µM bicuculline methchloride; 50 µM
3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP); 25 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); 200 µM
-aminobutyric acid (GABA); 10 µM nicardipine (Research Biochemicals, Natick, MA); 100 µM nifedipine (Research Biochemicals); 10, 100, 200, or 400 µM niflumic acid (Sigma, St. Louis, MO); 500 µM or 1 mM picrotoxin (Research Biochemicals); and 1 µM
tetrodotoxin (Research Biochemicals). CNQX, nicardipine, nifedipine,
and niflumic acid were dissolved in dimethylsulfoxide and diluted to
the final concentration in the HEPES-buffered extracellular solution by sonication. In other experiments, niflumic acid was applied directly in
the extracellular solution used to perfuse the slices. Solutions were
either made up freshly for each experiment or were prepared from
aliquots stored at
25°C.
After each recording, epifluorescent illumination was used to confirm
the identity of the cell. Each cell was drawn by hand and for most
cells, infrared differential interference contrast video images were
digitized (ImageGrabber24, Neotech, Hampshire, UK) and stored on
computer. In addition, some cells were photographed at a series of
focal planes on high speed film (Kodak Tmax 400) and the negatives
subsequently were scanned and used to construct a montage (Adobe
Photoshop, Adobe Systems, Mountain View, CA). For details, see
Hartveit (1997a).
General data analysis
Recordings were analyzed off-line with the use of the computer
programs PulseFit (HEKA elektronik), AxoGraph (Axon Instruments), Igor
Pro (Wavemetrics, Lake Oswego, OR), and GB-Stat (Dynamic Microsystems,
Silver Spring, MD). Curve-fitting with polynomial functions employed a
singular value decomposition algorithm (Igor Pro). Curve-fitting with
exponential functions employed a Levenberg-Marquardt algorithm (Igor
Pro) or a Chebyshev algorithm (AxoGraph) with iterative sum-of-squares
minimization. Equilibrium potentials were calculated according to the
Nernst equation with no correction for ionic activities. Statistical
analyses were performed with the use of Student's two-tailed or paired
t-test with a level of significance of P < 0.05. Data are presented as means ± SE (n = number of cells) and percentages are presented as percentage of
control. For illustration purposes, most records were low-pass filtered
at 0.5 or 1 kHz (3 dB; digital nonlagging Gaussian filter).
Detection of synaptic events
The occurrence of spontaneous postsynaptic currents (PSCs) was
detected by a computer program (AxoGraph) employing a variable amplitude event detection algorithm (Clements and Bekkers
1997). A simulated transient with the width and time course of
a typical synaptic event, was moved along the recorded data trace one
point at a time. At each position, this transient (template) was scaled optimally and offset to fit the data and a detection criterion (the
template scaling factor divided by the goodness-of-fit at each
position) was calculated. An event was detected when this criterion
exceeded a threshold and reached a sharp maximum. The template function
used to approximate the time course of spontaneous synaptic events
consisted of a flat baseline region followed by an exponential rise and
decay,
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RESULTS |
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A total of 179 rod bipolar cells are included in this study. They were all investigated electrophysiologically and were identified morphologically as rod bipolar cells after the recording by including Lucifer yellow in the recording pipette. An example of a rod bipolar cell with characteristic large, knob-shaped swellings at the axon terminal in the proximal part of the inner plexiform layer is shown in Fig. 1.
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Voltage-gated Ca2+ currents in rod bipolar cells
A depolarizing voltage ramp command to +40 mV from a 1-s
conditioning potential of 100 mV evoked a low-amplitude,
low-threshold inward current at about
70 mV (Fig.
2A). An additional,
high-amplitude inward current was evoked at about
50 mV (Fig.
2A). To plot the current-voltage (I-V)
relationship of the voltage-activated currents, the linear leak current
first was subtracted by fitting a line to the initial linear segment of
the current trace (typically 0-0.3 s). After extrapolation, the
linear curve was subtracted from the raw current trace and the
difference current was plotted against the corresponding voltage for
each point in time (Fig. 2B). The activation threshold for
the current was defined as the intersection of a line representing the
average
2 SDs of the baseline at hyperpolarized potentials with a
fifth-order polynomial fit of the relevant segment of the
I-V curve. The low-threshold current activated at
66.2 ± 1.3 mV (n = 15). The high-threshold current was studied in isolation by starting the voltage ramp command
from a holding potential of
60 or
55 mV, thereby inactivating the
low-threshold current (Fig. 2, A and B). The
high-threshold current activated between
50 and
40 mV (
47.7 ± 1.4 mV) and reached maximum amplitude between
30 and
20 mV
(
22.3 ± 0.9 mV; n = 10; Fig. 2, A
and B). The difference in time course of the low- and
high-threshold current components was studied with discrete
depolarizing voltage pulses (Fig. 2C). Depolarization to
20 mV from a 1-s conditioning potential of
100 mV evoked both a
transient and a sustained component. Depolarization to
20 mV from a
holding potential of
60 mV inactivated the transient component and
left intact a more sustained component. Subtracting the two current
traces from each other displayed the transient current in isolation
(Fig. 2C). Corresponding depolarizations from conditioning
potentials up to
50 mV did not further alter the time course (not
shown). Similar results were seen for six other cells tested. The
transient, low-threshold current usually ran down faster than the
sustained, high-threshold current. The I-V relationships
generated from currents evoked by discrete depolarizing voltage steps
were very similar to those generated by voltage ramps
(n = 3; data not shown).
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During these recordings, K+ currents were blocked by
Cs+ and TEA+ in the intracellular solution and
by TEA+ in the extracellular solution. The voltage-gated,
inward currents were identified as Ca2+ currents because
they were blocked completely by adding Co2+ (3 mM) to the
extracellular solution (Fig. 2B; n = 10). The
block was reversible on washout of Co2+ (not shown). The
Na+ channel blocker tetrodotoxin (1 µM) did not suppress
either the transient low-threshold component (Fig. 2D;
n = 5) or the sustained high-threshold component (Fig.
2E; n = 3). Application of the dihydropyridine
Ca2+ channel antagonists nicardipine (10 µM) or
nifedipine (100 µM) incompletely blocked the Ca2+ current
evoked from a holding potential of 60 mV (Fig. 2F). The
average suppression was 71 ± 3% (n = 6). In two
of these cells, a weak recovery was observed after washout of
antagonist and before complete rundown of the Ca2+ current
(Fig. 2F). Little effect of the dihydropyridine antagonists was observed on Ca2+ currents evoked by depolarizations
from hyperpolarized potentials (
100 mV). These results suggest that
rod bipolar cells express (at least) two types of Ca2+
currents, a transient low-threshold T-type current of relatively low
amplitude and a sustained high-threshold L-type current of larger amplitude.
For cells with cut axons, most likely severed during cutting of the
slices (Hartveit 1996, 1997a
), depolarization (ramps or voltage steps) from
100 mV evoked the low-threshold but not the high-threshold Ca2+ current (Fig. 2G). The
low-threshold current activated at
70.3 ± 1.1 mV
(n = 5), not significantly different from the
corresponding values for intact cells (P = 0.09). It
was blocked completely and reversibly by 3 mM Co2+ (not
shown). Depolarization from
60 mV evoked no Ca2+ current
at all (n = 17; Fig. 2G). These results
suggest that the high-threshold L-type current is expressed exclusively
in the axon terminal region, whereas the low-threshold T-type current is expressed predominantly, possibly exclusively, in the soma-dendritic region.
For the high-threshold Ca2+ current, the threshold and
maximum amplitude were located at more negative membrane potentials
than found for isolated large-terminal goldfish bipolar cells
(Burrone and Lagnado 1997; Heidelberger and
Matthews 1992
; Tachibana et al. 1993
). This
could be a genuine species difference, but the series resistance
imposed by the axon in the present somatic end recordings also may be
expected to cause a measurable voltage error during activation of
currents in the axon terminal (Mennerick et al. 1997
;
Protti and Llano 1998
). A low-threshold Ca2+
current has not been reported in goldfish bipolar cells.
Reciprocal postsynaptic currents are evoked by depolarization of single rod bipolar cells
To test whether depolarization of a rod bipolar cell might evoke a
feedback response to the same cell, 25- to 140-ms-long (typically 50 ms), 10- to 90-mV depolarizing voltage steps from a holding potential
of 70 mV were applied to single rod bipolar cells. With the standard
(high) intracellular chloride concentration, the chloride equilibrium
potential (ECl) was close to 0 mV (+2.1 mV). At
negative holding potentials, this creates a strong, outwardly directed
driving force for chloride ions. A representative example of the
responses evoked by applying a series of depolarizing voltage steps to
a rod bipolar cell is shown in Fig. 3. At
a command potential of
60 mV, there was no response during or after
the voltage step. At command potentials greater than or equal to
40
mV, an inward current occurred during the voltage step and a
long-lasting inward tail current immediately followed the termination
of the voltage step. The tail current was accompanied by a transient
increase in the frequency of discrete, as well as partially
overlapping, postsynaptic currents (PSCs; Fig. 3, A and
B). The discrete PSCs were characterized by a rapid rise and
a slower decay. The threshold for the response suggested that it
depended on activation of the L-type Ca2+ current. Similar
responses were evoked in the large majority of cells tested (103/116).
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Depolarization of rod bipolar cells from a conditioning
hyperpolarization of 100 mV (n = 7) did not alter the
threshold for evoked PSCs, suggesting that the T-type Ca2+
current is not involved in mediating transmitter release. Application of Co2+-containing extracellular solution blocked both the
tail current and the depolarization-evoked PSCs. A clear increase in
the frequency of PSCs after a depolarizing voltage step rarely was
observed >10-12 min after establishing the whole cell recording
configuration. In general, the rundown occurred after 6-8 min. The
voltage-gated Ca2+ current was also subject to rundown, but
it ran down more slowly than the synaptic transmission. A difference in
the time course of rundown between voltage-gated Ca2+
current and transmitter release or capacitance increase has also been
observed in goldfish bipolar cells (Sakaba et al. 1997
;
von Gersdorff and Matthews 1994
).
The baseline frequency of PSCs was always low enough that the depolarization-evoked increase in frequency could be detected clearly. Under the standard recording conditions, spontaneous PSCs were detected in 11 of 12 cells during observation periods that ranged from 30 to 180 s. To estimate the frequency of spontaneous PSCs, they were detected and captured by a variable amplitude event detection algorithm (see METHODS). The frequency of PSCs, averaged over the entire observation period, varied from 0.03 to 0.8 Hz (0.27 ± 0.08 Hz; n = 11).
To quantify the magnitude of the depolarization-evoked response, a
series of points first were positioned by eye on presumed baseline
segments of the long-lasting tail current, from 0.0025 to 1 s
after return of the membrane potential to 70 mV after a depolarizing
voltage-step (Fig. 4). Next a double
exponential function was fit to the series of points, thereby
constituting a hypothetical baseline from which the PSCs arose. Finally
the double exponential function was subtracted from the raw current trace to yield an estimate of the postsynaptic current (Fig. 4). Integration of the postsynaptic current over the 1-s period yielded the
charge flowing across the cell membrane. This method of quantification, which will be called the "fit-and-subtract" method, excluded any contribution from a Ca2+-activated chloride current (see
following text). Figure 5A
shows the relation between the command potential and the averaged
synaptic feedback response, measured as charge, for nine rod bipolar
cells. The responses in each cell first were normalized to the cell's maximum response, then values for all cells were averaged. At the most
depolarized command potentials (>0 mV), the response clearly was
reduced, suggesting that the Ca2+ influx during the
depolarizing voltage step, and not the Ca2+ tail current
evoked by repolarization to the holding potential, was primarily
responsible for evoking transmitter release (cf. Mennerick and
Matthews 1996
). The overall shape of the charge-voltage relation is relatively similar to the voltage dependence of transmitter release and capacitance increase observed in goldfish bipolar cells
(Tachibana and Okada 1991
; von Gersdorff and
Matthews 1994
). The rise in synaptic response with increasing
depolarization (Fig. 5A) was more abrupt, however, than the
accompanying increase in Ca2+ current (Fig. 2B).
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It is likely that a Ca2+-activated chloride current
contributed to the long-lasting tail current observed on repolarization to 70 mV (Fig. 3, A and B). This current
previously was detected in presynaptic terminals of bipolar cells in
goldfish retina (Okada et al. 1995
). When the total
current was integrated during a period of ~1 s (Fig. 3A,
), the measurements were dominated by the putative Ca2+-activated chloride current. Nevertheless, the shape of
the corresponding charge-voltage relation was similar to that for the
postsynaptic current (Fig. 5B). Unfortunately, niflumic
acid, which has been reported to block Ca2+-activated
chloride currents in other preparations (e.g., Okada et al.
1995
), suppressed not only the long-lasting tail current, but
the voltage-activated Ca2+ current as well (10-400 µM;
n = 17). No further attempts were made to isolate
pharmacologically the postsynaptic response from other
Ca2+-activated processes.
Reversal potential of evoked PSCs
For some cells, evoked PSCs could be discriminated clearly during
several of the depolarizing voltage steps, allowing a reversal of
current close to 0 mV to be observed (Fig.
6A). This is the value
expected for a GABA-evoked current mediated by chloride ions
(ECl = +2.1 mV). The Erev
of GABA-evoked currents in rod bipolar cells was confirmed by drug
application at different holding potentials (Fig. 6B) or
during voltage ramps (100 to +40 mV; 100 mV/s). The average
Erev was
6.5 ± 0.7 mV (n = 5). When ECl was changed to
46.9 mV
(n = 3) by recording with a low intracellular chloride
concentration, outwardly directed PSCs could be observed during
(longer-lasting) voltage steps to 0 mV (Fig. 6C). This indicated that Erev was more negative than 0 mV
and thus followed the change in ECl, suggesting
that the depolarization-evoked PSCs were mediated by chloride ions.
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Depolarization-evoked PSCs are GABAergic inhibitory PSCs (IPSCs)
The frequency of depolarization-evoked PSCs was strongly
suppressed, and in some cases even blocked, by ionotropic
GABAA and/or GABAC receptor antagonists. An
example of the effect of the competitive GABAA receptor
antagonist bicuculline (100 µM) on the response of a rod bipolar cell
to a depolarizing voltage step (to 30 mV) is shown in Fig.
7A. In this case, the response
clearly recovered after bicuculline was washed away. For some cells it
was difficult, however, to observe a clear recovery, due to the rapid
rundown of the synaptic response. This also made it difficult to test more than one drug on each cell. To quantify the suppression, the
response was measured by the fit-and-subtract method during the 1-s
time period after return of the membrane potential to
70 mV after a
depolarizing voltage step (see above). The response was measured for
each sweep in the control condition, during drug application and after
washout. The degree of suppression was calculated as (CONTROL
DRUG)/CONTROL * 100% where CONTROL is the response in the control
condition and DRUG is the response during drug application. To correct
for drug-independent rundown of the response, a theoretical control
value was estimated by fitting a line through the response values
before and after drug application. For a few cells, a single
exponential gave a better fit. The effect of bicuculline was variable
from one cell to another (range of suppression 10-96%), and for one
cell the postsynaptic charge actually increased almost 100%. A
possible explanation for this result is that bicuculline primarily
disinhibited the A17 amacrine cell(s) (cf. Zhang et al.
1997
) and that most of the GABAergic input to this rod bipolar cell was mediated by GABAC receptors. When this cell was
excluded from the analysis, the average suppression was 63 ± 15%
(n = 6).
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GABAC receptors in rat retina are characterized by
insensitivity to bicuculline (Feigenspan et al. 1993) as
well as being relatively insensitive to concentrations of picrotoxin
that will block GABAC receptors in other species
(Qian and Dowling 1993
). They can be antagonized by
3-APMPA (Pan and Lipton 1995
). An example of strong,
reversible suppression of the depolarization-evoked PSCs by 500 µM
3-APMPA is shown in Fig. 7B. As for bicuculline, the degree
of suppression by 3-APMPA varied from one cell to another (range
38-100%), suggesting that the proportion of GABAA and
GABAC receptors varies between the rod bipolar cells
recorded here (cf. Euler and Wässle 1998
;
Feigenspan et al. 1993
; Qian and Dowling 1995
; Yeh et al. 1996
). Overall, 3-APMPA reduced
the evoked response, measured as integrated synaptic current, by
65 ± 6% (n = 9). Due to rapid rundown of the
evoked response, it was not possible to compare the relative
suppression evoked by application of GABAA and
GABAC receptor antagonists for individual cells. However, when bicuculline and 3-APMPA were co-applied, the depolarization-evoked PSCs were strongly suppressed in most cells tested (range of
suppression 66-100%). An example of a reversible block is shown in
Fig. 8A. The average
suppression by co-application of bicuculline and 3-APMPA was 90 ± 5% (n = 10). Furthermore application of a high
concentration of picrotoxin (1 mM), presumably antagonizing both
GABAA and GABAC receptors, also was able to
strongly suppress the depolarization-evoked PSCs (Fig. 8B).
In the case of picrotoxin, the synaptic response only partially
recovered (before rundown) in one cell. On average, picrotoxin reduced
the evoked response, measured as integrated synaptic current by 83 ± 3% (n = 5; range 80-95%). These results indicate
that the evoked PSCs are GABAergic IPSCs mediated by a variable
combination of GABAA and GABAC receptors on the
rod bipolar cells.
|
Because 3-APMPA is also a potent GABAB receptor agonist
(Seabrook et al. 1990), the inhibition of the
depolarization-evoked IPSCs observed with this drug might have been
mediated by the activation of putative GABAB receptors on
the rod bipolar axon terminal (which could suppress the voltage-gated
Ca2+ current and depolarization-evoked release of
glutamate) (cf. Maguire et al. 1989
). There was no
significant difference, however, in the peak amplitude of
voltage-dependent Ca2+ currents between control recordings
(
49.2 ± 3.8 pA) and recordings with application of the
selective GABAB agonist baclofen (
50.0 ± 2.9 pA; n = 4; P = 0.70; paired
t-test). This is consistent with previous evidence
that GABAB receptors are not expressed by rod bipolar cells
(Koulen et al. 1998b
; Pan and Lipton
1995
; Yeh et al. 1990
). Alternatively, 3-APMPA
might act on putative presynaptic GABAB receptors on the
A17 amacrine cell(s) with consequent reduction of GABA release. The
effect of baclofen was tested on depolarization-evoked IPSCs on four
rod bipolar cells. For three of the cells, there was a small,
reversible reduction in the response (15 ± 5%). For the last
cell, baclofen evoked a reversible increase of the response (25%; data
not shown). This suggests that release of GABA from the amacrine cells
can be modulated by presynaptic GABAB receptors, but that
this mechanism is not sufficient to account for the inhibitory effect
of 3-APMPA on depolarization-evoked IPSCs in these experiments.
Depolarization-evoked IPSCs depend on glutamate release from rod bipolar cells
If the transmission from rod bipolar cells to A17 amacrine cells
is mediated by glutamate, it should be possible to block the
depolarization-evoked IPSCs by antagonists of glutamate receptors. This
was examined by application of the competitive non-NMDA receptor antagonist CNQX. As predicted, CNQX evoked a strong but reversible suppression of the depolarization-evoked IPSCs (Fig.
9A). CNQX reduced the average
response, measured as integrated synaptic current, by 88 ± 5%
(n = 10; range 62-93%). These results strongly suggest that activation of non-NMDA receptors on A17 amacrine cells
evokes the GABAergic feedback onto rod bipolar cells. Functional (i.e.,
conductance increasing) ionotropic non-NMDA receptors are not expressed
by rod bipolar cells (Hartveit 1996), excluding the
possibility that CNQX acts directly on these cells.
|
To examine whether NMDA receptors also are involved in the generation of the feedback response, depolarizing voltage steps were applied in the presence of the competitive NMDA receptor antagonist CPP. There was no consistent effect of CPP on the feedback response. The effect ranged from a 30% decrease to a 10% increase of the response relative to control (0.5 ± 6%; n = 8; Fig. 9B). Although it is difficult to exclude any involvement of NMDA receptors, it seems safe to conclude that under the present conditions, the GABAergic feedback mainly is driven by activation of non-NMDA receptors on the A17 amacrine cells.
In some of the pharmacological tests (Figs. 8B and 9A), it cannot be excluded that the Ca2+-activated chloride current was suppressed weakly by some of the receptor antagonists used. An alternative explanation is that there was overlap of IPSCs in the control recordings (e.g., Fig. 9A; Control, Wash). For responses with such overlap, the fit-and-subtract method would underestimate the synaptic current integral. This would only weakly influence the quantitative estimate of the degree of suppression, except in cases of moderate suppression where it would be underestimated.
Time course of the feedback response
The period of increased frequency of IPSCs after 50- to 150-ms
depolarizing voltage steps seemed to last <1 s for most cells (Figs. 3
and 4). This was analyzed in more detail by extending the sampling
period to 10 s after the voltage step. The postsynaptic current
was measured by the fit-and-subtract method. A single epoch of
recording from a representative cell is shown in Fig. 10A. The curve in Fig.
10B displays the average time course of the postsynaptic
response. It was calculated by averaging 17 individual epochs of
depolarization-evoked postsynaptic currents from nine cells. The
response peaked immediately after the end of the voltage step and then
decayed to a relatively constant level in <2 s. The decay could be fit
well with a single exponential ( = 215 ms; Fig. 10B).
|
It is not clear whether the time course of the feedback response is
determined primarily by the rod bipolar cell or the A17 amacrine
cell(s). If the decay of the synaptic output from the rod bipolar cell
follows a time course similar to that of the GABAergic feedback
response, it means that transmitter release must continue for some time
after closure of the Ca2+ channels in the rod bipolar cell.
In isolated goldfish bipolar cells, Tachibana and Okada
(1991) observed a maintained component of release that
continued for some seconds after closure of Ca2+ channels
(their Fig. 7, see also Burrone and Lagnado 1997
). If this is also the case for rod bipolar cells, one would expect the decay
of the free Ca2+ concentration in the synaptic terminal to
follow an equally slow time course on repolarization to
70 mV.
Interestingly, for seven of the nine cells tested with a 10-s sampling
period, sweeps sampled after rundown of synaptic transmission contained
a robust long-lasting tail-current (most likely a
Ca2+-activated chloride current; see preceding text). The
time course of the tail current could be fit well with the sum of two
exponentials. The average time constant of the slow component was
620 ± 350 ms (n = 7), in the same range as the
time constant of decay of the postsynaptic response in the rod bipolar
cell (Fig. 10B). This is consistent with a hypothesis that
both transmitter release as well as the tail current in the rod bipolar
cell could follow the decay of the cytosolic Ca2+
concentration with similar affinity.
Alternatively, the time course of the feedback response in the rod
bipolar cell could be determined primarily by the time course of
transmitter release from the A17 amacrine cell(s). A long-lasting decay
has been suggested for synaptic transmission between chick amacrine
cells in culture (Gleason et al. 1993, 1994
). To further
examine the release properties of rod bipolar cells and A17 amacrine
cells, in particular, whether they are capable of sustained release
during prolonged depolarization of a rod bipolar cell, 20-s-long
depolarizing voltage steps to either
30 or
20 mV were applied
(n = 11). To compensate for the reduced driving force
for chloride ions at the higher command potential compared with
70
mV, the extracellular chloride concentration was reduced to 20.5 mM by
replacing 114 mM NaCl with an equivalent amount of sodium isethionate,
changing the ECl to + 49.6 mV (+47.8 mV when
cesium glutamate partially replaced CsCl intracellularly). In this
condition, a sustained feedback response with a markedly enhanced
frequency of IPSCs was observed for the duration of the depolarizing
voltage step for all cells examined (Fig. 10C). This suggests that both rod bipolar cells and A17 amacrine cells are capable
of sustained release (for periods up to
20 s). To verify that
Ca2+-influx in rod bipolar cells could be sustained for
correspondingly long periods, cells were tested with similar
depolarizing pulses (to
30 or
20 mV for 20 s) in the presence
of 1 mM picrotoxin (n = 5). The example illustrated in
Fig. 10D indicates that although there is a clear reduction
in the current amplitude, the voltage-gated Ca2+ current in
rod bipolar cells can last
20 s. Similar results were seen for two
cells. In the remaining two cells, the Ca2+ current decayed
more or less completely within the 20-s period.
IPSCs are evoked in the inner plexiform layer
From the results reported so far, it cannot be concluded
unequivocally that the IPSCs are evoked in the inner plexiform layer by
reciprocal connections from A17 amacrine cells onto rod bipolar cell
axon terminals. Alternatively, they could be evoked in the outer
plexiform layer from horizontal cell contacts with rod bipolar cell
dendrites (Yang and Wu 1991). Two sets of observations
argue against this interpretation. First, the depolarizing voltage
steps evoked discrete IPSCs, strongly suggesting vesicular release, whereas transmitter release from horizontal cells is thought to be
nonvesicular (Schwartz 1987
). Second, in recordings from
12 cells with cut axons, all of which presumably were rod bipolar cells
(based on the location of the somata in close approximation to the
outer plexiform layer), a depolarization-evoked feedback response was
never observed (Fig. 11). This
indicates that the response is mediated in the inner plexiform layer.
|
Release of GABA does not require activation of voltage-gated Na+-channels
The experiments described so far suggest that release of GABA from A17 amacrine cells onto rod bipolar terminals depends on a vesicular release mechanism. To examine whether the release is influenced by voltage-gated Na+ currents, possibly mediating spiking behavior in the A17 amacrine cells, tetrodotoxin, a specific blocker of voltage-gated Na+-channels, was applied during the depolarizing voltage step protocol. Tetrodotoxin had no or only moderate effects on the synaptic transmission in 5/8 rod bipolar cells. Compared with the control condition, the change in response varied from a 10% increase to a 25% reduction (7 ± 5%; n = 5). For the three other cells, however, tetrodotoxin evoked a strong, reversible suppression of the reciprocal response (70-90%; Fig. 12). Taken together, these results suggest that activation of voltage-dependent Na+-channels can contribute to, but is not required for, transmitter release from the A17 amacrine cells.
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DISCUSSION |
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Morphological evidence for reciprocal synapses between bipolar
cells and amacrine cells was first reported by Dowling and Boycott (1966). In the same year, Rall et al.
(1966)
published physiological and morphological evidence for a
dendrodendritic synaptic pathway for inhibition in the olfactory bulb,
stressing the morphological similarity between retinal amacrine cells
and olfactory granule cells and suggesting that amacrine cells might provide a similar dendrodendritic inhibitory pathway as granule cells.
In several areas of the CNS, polarity of synaptic transmission has been
inferred from specific ultrastructural criteria: round and flat
synaptic vesicles, shown to correlate with excitatory and inhibitory
functions, respectively. These criteria, unfortunately cannot be used
in the retina. Nevertheless, based on other morphological evidence, it
has been postulated that the bipolar-to-amacrine connection is
excitatory and that the amacrine-to-bipolar connection is inhibitory.
In this study, I have examined the functional characteristics of
the reciprocal synaptic interaction between rod bipolar cells and
amacrine cells in the rat retina. The major findings are that depolarization of a single rod bipolar cell evokes feedback PSCs in the
same cell, that the PSCs are GABAergic IPSCs mediated by chloride ions,
that both GABAA and GABAC receptors can
participate in the feedback response, that the threshold for the
feedback response is approximately 50 mV, indistinguishable from the
threshold for the voltage-gated L-type Ca2+ current in rod
bipolar cells, that this Ca2+ current is localized to the
axon terminal, that the feedforward excitation of the amacrine cells is
mediated primarily by CNQX-sensitive non-NMDA receptors, and finally
that activation of voltage-gated Na+ channels is not
essential for, but can possibly contribute to, release of GABA from the
amacrine cells.
It is well established that the reciprocal synapses do not account for
all of the synaptic inputs to rod bipolar axon terminals (Kim et
al. 1998; Strettoi et al. 1990
) and that some of
the nonreciprocal input might be GABAergic (Kim et al.
1998
). Accordingly, it might be argued that some of the
depolarization-evoked IPSCs originated via a hypothetical synaptic
circuit: rod bipolar
AII amacrine
ON-cone bipolar
unknown amacrine
rod bipolar via nonreciprocal synapses
(Strettoi 1990
, 1992
, 1994
). While it is not possible to
exclude a contribution of such a circuit to the evoked responses, it is
likely to have been unimportant in the present study, given its
multisynaptic complexity. With varying patterns of convergence and
divergence at each stage, it is hard to see that an output signal from
a single rod bipolar cell would be transmitted reliably through this
circuit. In contrast, the depolarization-evoked response was clearly
detected in the large majority of cells tested.
Voltage-gated Ca2+ current and transmitter release in rod bipolar cells
Two components of a voltage-gated Ca2+ current were
identified in rat rod bipolar cells. A low-threshold, transient, T-type component activated at about 70 mV and a high-threshold, sustained component activated at about
50 mV. Both currents were blocked reversibly by Co2+. The high-threshold component was
blocked (incompletely) by dihydropyridine antagonists (nicardipine and
nifedipine), suggesting an L-type pharmacology (cf. De la Villa
et al. 1998
; Heidelberger and Matthews 1992
;
Pan and Lipton 1995
; Protti and Llano
1998
; Tachibana et al. 1993
). However, it should
be noted that the incomplete block leaves open the possibility that
other types of Ca2+ currents are present in rod bipolar
cells (N, P/Q, or R type). Cells with cut axons displayed the
low-threshold current but not the high-threshold current. This suggests
that the L-type Ca2+ channels are located at the
(presynaptic) axon terminal (consistent with previous findings)
(De la Villa et al. 1998
; Pan and Lipton 1995
; Protti and Llano 1998
) and that they are
involved directly in mediating transmitter release. A similar role for
an L-type Ca2+ current has been found in goldfish bipolar
cells (Tachibana et al. 1993
) where the dynamics of the
Ca2+ response at the axon terminals correlates with
exocytotic release of neurotransmitters (Tachibana and Okada
1991
; von Gersdorff and Matthews 1994
). It
remains to be determined, however, how well the operating range of the
voltage-gated L-type Ca2+ current is matched to the voltage
excursion produced by a visual stimulus in a rod bipolar cell.
The threshold for the synaptic feedback response was indistinguishable
from the threshold for the voltage-gated L-type Ca2+
current in rod bipolar cells. This indicates that the release properties of rod bipolar cells and A17 amacrine cells are well matched
to each other and that depolarization-evoked transmitter release from a
rod bipolar cell immediately gives rise to an inhibitory feedback
response. In recordings with brief (~50 ms) depolarizations of a rod
bipolar cell, the frequency of feedback IPSCs decayed to the baseline
level with a time constant of ~200 ms. Because the Ca2+
current in the rod bipolar cell would terminate rapidly, the decay may
correspond to a maintained component of release, employing a release
sensor with high Ca2+ affinity. In goldfish bipolar cells,
the presence of asynchronous transmitter release after the termination
of Ca2+ current has not yet been settled (cf.
Burrone and Lagnado 1997; Lagnado et al.
1996
; Sakaba et al. 1997
; Tachibana and
Okada 1991
; von Gersdorff and Matthews 1997
).
Alternatively, the time course of decay of IPSC frequency may be a
property of the A17 amacrine cell(s), consistent with the release
properties of chick amacrine cells, most likely involving a
high-affinity Ca2+ sensor for exocytosis (Gleason et
al. 1993
, 1994
).
In recordings with longer-lasting depolarization of rod bipolar
cells, it was demonstrated that both rod bipolar cells and A17 amacrine
cells are capable of sustained transmitter release. This again could
reflect a role for a release sensor with high Ca2+ affinity
in rod bipolar cells, responding to an increase in the bulk
Ca2+ concentration in the terminal (Lagnado et al.
1996; Sakaba et al. 1997
; von Gersdorff
and Matthews 1997
). However, at least in some rod bipolar
cells, maintained depolarization evoked a sustained Ca2+
current (Fig. 10D), presumably giving rise to strongly
elevated Ca2+ concentrations close to open Ca2+
channels. In that case, a release sensor with low Ca2+
affinity (Heidelberger et al. 1994
; von Gersdorff
and Matthews 1994
) could be involved in mediating sustained
release from the rod bipolar cell. It is possible that paired
recordings from a rod bipolar cell and an A17 (or an AII) amacrine cell
can provide more information concerning the mechanism.
A transient, T-type Ca2+ current has also been reported in
mouse bipolar cells of unspecified type (De la Villa et al.
1998; Kaneko et al. 1989
). In contrast, a recent
investigation of rat rod bipolar cells found a complete absence of a
T-type current and it was suggested that only cone bipolar cells
express such currents (Protti and Llano 1998
). The
present results indicate that rod bipolar cells do indeed express a
T-type current and suggest that the absence of this current in the
study of Protti and Llano (1998)
could be due to its relatively rapid
rundown. Alternatively, the absence of a T-type current, as well as the low frequency of cells with intact synaptic responses and the extremely
low probability of recording from cone bipolar cells (as reported by
the authors) could suggest that methodological differences in the
preparation of the slices may have physiological consequences.
The T-type Ca2+ current in rod bipolar cells does not seem
to be directly involved in transmitter release from the axon terminal. However, the functional role and precise cellular localization of the
corresponding ion channels remains to be determined. Although the
majority of the channels most likely have a soma-dendritic location,
mouse bipolar cells seem to have T-type channels both at the axon
terminal and at the soma (Kaneko et al. 1989).
Neurotransmitter receptors involved in reciprocal synaptic transmission
Previous morphological studies indicate that subunits of both
GABAA and GABAC receptors are localized on the
axon terminals of rod bipolar cells, but it has been difficult to
demonstrate a synaptic localization of the individual subunits
(Greferath et al. 1994; Koulen et al.
1998a
). The results presented here suggest that the transmitter
released from A17 amacrine cells is GABA and that it activates both
GABAA and GABAC receptors at the axon terminals
of rod bipolar cells. This result is well in accord with recent
findings for bipolar-amacrine interactions in the tiger salamander
retina (Dong and Werblin 1998
). It will be important to
determine whether release of a single synaptic vesicle from an A17
amacrine cell can activate both GABAA and GABAC
receptors on rod bipolar terminals (corresponding to a dual-component IPSC) or whether the receptors are strictly segregated to individual synapses (Koulen et al. 1998a
).
Because glycine receptors were blocked pharmacologically, this study
leaves open the possible existence of glycinergic A17 amacrine cells.
Interestingly, however, Kim et al. (1998) found that all
amacrine cell processes with reciprocal connections to rod bipolar
cells in rat retina show immunoreactivity for glutamic acid
decarboxylase (cf. Freed et al. 1987
). Given that GABA
and glycine are not colocalized in the mammalian retina, only some of
the presynaptic, nonreciprocal amacrine cell processes are candidates
for glycinergic input to rod bipolar cells.
The sensitivity of single-cell-evoked feedback IPSCs to the non-NMDA
antagonist CNQX indicates that A17 amacrine cells express ionotropic
non-NMDA receptors and consequently are depolarized by glutamatergic
output from rod bipolar cells (cf. Dong and Werblin 1998). This supports previous evidence that the synaptic
transmission from rod bipolar cells to A17 amacrine cells is
sign-conserving (Raviola and Dacheux 1987
). The evidence
for sustained signal transmission between rod bipolar cells and A17
amacrine cells (Fig. 10C) suggests either that non-NMDA
receptors on A17 amacrine cells undergo little desensitization or that
partially desensitized receptors are sufficient for transmission.
Alternatively, long-lasting depolarization of rod bipolar cells might
activate NMDA receptors on A17 amacrine cells (cf. Matsui et al.
1998
).
Functional characteristics of reciprocal amacrine cells
The present results indirectly reveal some properties of reciprocal (A17) amacrine cells. First, these cells most likely employ a vesicular release mechanism, as evidenced by discrete, depolarization-evoked IPSCs in rod bipolar cells. It is difficult, however, to entirely eliminate the possibility that a nonvesicular release mechanism could generate similar IPSCs. One could imagine that discrete IPSCs might be induced by rapidly desensitizing GABA receptors in rod bipolar cells or by transient depolarization of A17 amacrine cells mediated by non-NMDA receptors. Another potential mechanism for generation of discrete IPSCs, Na+ spike-induced GABA release from A17 amacrine cells, is less likely, given the resistance of some IPSCs to tetrodotoxin.
Second, the threshold for transmitter release from A17 amacrine cells
is low enough that it does not require spiking mediated by
tetrodotoxin-sensitive voltage-gated Na+ channels. However,
tetrodotoxin reversibly suppressed feedback IPSCs in some rod bipolar
cells, suggesting that under some conditions activation of
voltage-gated Na+ channels can enhance transmitter release.
This might occur when an excitatory postsynaptic potential in an A17
varicosity or spine head exceeds a putative threshold for initiation of
regenerative currents (cf. Miller et al. 1985). The
evidence for action potentials in A17 amacrine cells is conflicting
(Bloomfield 1992
, 1996
; Nelson and Kolb
1985
; Raviola and Dacheux 1987
). Alternatively,
A17 amacrine cells in rat retina might be a heterogeneous group of
cells, with different dependence on spiking (mediated by voltage-gated
Na+ channels) for signal transmission.
Comparison between retina and olfactory bulb
The results obtained here for rod bipolar cells and reciprocal
amacrine cells are remarkably similar to those obtained previously for
the mitral cell-granule cell interaction in the olfactory bulb
(Wellis and Kauer 1993; see also Jahr and Nicoll
1982
). Granule cells are axonless GABAergic inhibitory
interneurons that form dendrodendritic synapses with glutamatergic
mitral cells. Application of depolarizing voltage steps to a mitral
cell evokes a prolonged increase in the frequency of GABAergic IPSCs in
the same cell, and the reciprocal synaptic interaction is sensitive to
NMDA and non-NMDA receptor antagonists (mitral cell
granule cell)
and to GABAA antagonists (granule cell
mitral cell)
(Wellis and Kauer 1993
). Jahr and Nicoll
(1982)
found that reciprocal inhibition in the olfactory bulb
persists despite the presence of tetrodotoxin, implying that the
contribution of voltage-gated Na+ channels to the
potentials in granule dendritic spines (Wellis and Kauer
1994
) is not essential for release of transmitter.
Self-inhibition versus lateral inhibition
Although this study has demonstrated that depolarization of a rod
bipolar cell evokes a GABAergic feedback response in itself (self-inhibition), it is unknown whether there is also output to other
rod bipolar cells in contact with the same amacrine cell (lateral
inhibition). Morphological data indicate that the axon terminal of a
single rod bipolar cell is in contact with the dendrites of several
reciprocal amacrine cells (Chun et al. 1993;
Strettoi et al. 1990
), and in cat retina it has been
estimated that a single A17 cell makes reciprocal contacts with
~1,000 rod bipolar axon terminals (Nelson and Kolb
1985
). Lateral inhibition requires that depolarization spreads
from an activated region of an amacrine cell into the neighboring
dendritic tree. This might occur solely by passive, electrotonic spread
of depolarization, but it also could be enhanced by any active or
regenerative properties of the dendritic membrane of the amacrine cell
(cf. Bloomfield 1996
). Even regenerative depolarization
of a nonpropagating nature could facilitate the spread of
depolarization in the amacrine dendritic tree. An alternative view is
that the dendritic varicosities of reciprocal amacrine cells are
isolated electrically (Ellias and Stevens 1980
;
Masland 1988
), implying that each reciprocal synapse is
a locally operating microcircuit. For A17 amacrine cells, however, Nelson and Kolb (1985)
and Bloomfield
(1992)
reported that receptive-field measurements do not
support the notion of isolated dendritic regions. The present study
found that spontaneous IPSCs in rod bipolar cells occur at a low
frequency, suggesting that there is little spontaneous transmitter
release from A17 amacrine cells. This may be because there is little
excitatory drive from rod bipolar cells. Alternatively, an A17 amacrine
cell may continuously receive excitatory input in several regions of
its dendritic tree, except from the rod bipolar cell under study, which
is voltage-clamped at
70 mV and therefore does not release glutamate.
The latter interpretation implies that there is little or no
integration in the dendritic tree of A17 amacrine cells and that
release of GABA is controlled locally. It will be interesting to see
whether dual recordings from pairs of rod bipolar cells can be used to examine whether lateral inhibition, as well as self-inhibition, occurs
in the synaptic transmission between rod bipolar cells and A17 amacrine cells.
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ACKNOWLEDGMENTS |
---|
I am grateful to Dr. Margaret Veruki for many helpful discussions and for critically reading and improving the manuscript. I thank M. Kjøsnes for excellent technical assistance.
This work was supported by the Norwegian Research Council (101085/310, 123485/310), Anders Jahre's Fund, and the Nansen Fund.
Address for correspondence: E. Hartveit, Department of Anatomy and Cell Biology, University of Bergen, Årstadveien 19, N-5009 Bergen, Norway.
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FOOTNOTES |
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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 24 September 1998; accepted in final form 24 February 1999.
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REFERENCES |
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