Max-Planck-Institut für Hirnforschung, Neuroanatomische Abteilung, D-60528 Frankfurt am Main, Germany
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ABSTRACT |
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Frech, Moritz J.,
Jorge Pérez-León,
Heinz Wässle, and
Kurt H. Backus.
Characterization of the Spontaneous Synaptic Activity of Amacrine
Cells in the Mouse Retina.
J. Neurophysiol. 86: 1632-1643, 2001.
Amacrine cells are a heterogeneous class
of interneurons that modulate the transfer of the light signals through
the retina. In addition to ionotropic glutamate receptors, amacrine
cells express two types of inhibitory receptors,
GABAA receptors (GABAARs) and glycine receptors (GlyRs). To characterize the functional contribution of these different receptors, spontaneous postsynaptic currents (sPSCs) were recorded with the whole cell
configuration of the patch-clamp technique in acutely isolated slices
of the adult mouse retina. All amacrine cells investigated
(n = 47) showed spontaneous synaptic activity. In six
amacrine cells, spontaneous excitatory postsynaptic currents could be
identified by their sensitivity to kynurenic acid. They were
characterized by small amplitudes [mean: 13.7 ± 1.5 (SE) pA] and rapid decay kinetics (mean
: 1.35 ± 0.16 ms). In contrast, the reversal potential of sPSCs characterized by slow
decay kinetics (amplitude-weighted time constant,
w, >4 ms) was dependent on the intracellular
Cl
concentration (n = 7),
indicating that they were spontaneous inhibitory postsynaptic currents
(sIPSCs). In 14 of 34 amacrine cells sIPSCs were blocked by bicuculline
(10 µM), indicating that they were mediated by
GABAARs. Only four amacrine cells showed glycinergic sIPSCs that were inhibited by strychnine (1 µM). In one
amacrine cell, sIPSCs mediated by GABAARs and
GlyRs were found simultaneously. GABAergic sIPSCs could be
subdivided into one group best fit by a monoexponential decay function
and another biexponentially decaying group. The mean amplitude of
GABAergic sIPSCs (
42.1 ± 5.8 pA) was not significantly
different from that of glycinergic sIPSCs (
28.0 ± 8.5 pA).
However, GlyRs (mean T10/90: 2.4 ± 0.08 ms) activated significantly
slower than GABAARs (mean T10/90: 1.2 ± 0.03 ms). In addition, the decay kinetics of monoexponentially decaying
GABAARs (mean
w:
20.3 ± 0.50), biexponentially decaying GABAARs (mean
w:
30.7 ± 0.95), and GlyRs (mean
w = 25.3 ± 1.94) were significantly different. These differences in
the activation and decay kinetics of sIPSCs indicate that amacrine
cells of the mouse retina express at least three types of functionally
different inhibitory receptors: GlyRs and possibly two subtypes of
GABAARs.
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INTRODUCTION |
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GABA and glycine are the most
abundant inhibitory neurotransmitters in the mammalian CNS. By
activating specific receptors, GABA and glycine can modulate the
membrane resistance by controlling a
Cl/HCO
; Möhler et al.
1996a
). Recently the distribution of these subunits in the
mammalian nervous system was investigated with in situ hybridization and immunohistochemical studies (Fritschy and Möhler
1995
; Laurie et al. 1992
; Persohn et al.
1992
; Wisden et al. 1992
). With
electrophysiological experiments, some pharmacological and biophysical
properties of several recombinant receptor subtypes have been
characterized (Mihic et al. 1995
; Möhler et
al. 1996b
). However, our knowledge of the molecular composition
and the functional significance of endogenous
GABAAR and GlyR subtypes is still poor. To
characterize the functional differences of these receptors and their
physiological roles, we used retinal amacrine cells as a model system.
Amacrine cells are a heterogeneous class of 20-40 types of
interneurons that modulate the transfer of light signals in the
mammalian retina (MacNeil and Masland 1998
; Vaney
1990
). About one half of the amacrine cell population is known
to release GABA while the other half was found to be glycinergic.
GABAergic and glycinergic amacrine cells provide multiple inhibitory
inputs to retinal bipolar cells and ganglion cells, thereby mediating
some of the most important functions in light signal processing such as
lateral inhibition and direction selectivity (Cook and
McReynolds 1998
; Taylor et al. 2000
;
Wässle and Boycott 1991
). In addition, amacrine
cells themselves receive synaptic input from other amacrine cells and express GABAARs and GlyRs (Greferath et
al. 1994a
,b
, 1995
; Sassoè-Pognetto and
Wässle 1997
; Sassoè-Pognetto et al.
1994
; Wässle et al. 1998
). Thus the
reciprocal inhibition mediated by GABAARs and GlyRs between amacrine cells might play an important role in the fine
tuning of the inhibition directed to bipolar cells and ganglion cells.
To investigate the functional contributions of
GABAARs and GlyRs in the inhibitory network
formed by amacrine cells, we have applied the patch-clamp technique in
acutely isolated slices of the adult mouse retina to record the
spontaneous inhibitory postsynaptic currents (sIPSCs) of amacrine
cells. Besides rapidly decaying spontaneous excitatory postsynaptic
currents (sEPSCs), slowly activating glycinergic and fast activating
GABAergic sIPSCs were found. At least two types of GABAergic sIPSCs
could be identified that significantly differed with respect to their
decay kinetics. A portion of these results has been presented in
abstract form (Frech and Backus 2000).
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METHODS |
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Preparation of retinal slices, visualization and identification of amacrine cells
Retinal slices were prepared as previously described
(Boos et al. 1993; Euler et al. 1996
).
Adult mice (older than 7 wk) were killed by cervical dislocation. The
eyes were enucleated and opened by an encircling cut along the ora
serrata. The vitreous body was removed, and the retina was carefully
dissected free and cut into four pieces. Vertical slices (thickness ca.
200-300 µm) of the retina were cut with a scalpel or a microslicer
(DSK, Kyoto, Japan), stored at room temperature in
extracellular saline, and bubbled with 95%
O2-5% CO2.
Electrophysiological experiments were started 30 min after preparation
of the slices. Retinal neurons were visualized by differential
interference contrast microscopy using a ×40 water-immersion objective
and a digital camera (PCO Computer Optics GmbH, Kelheim, Germany)
mounted on an upright microscope (Axioskop FS; Zeiss, Oberkochen,
Germany). Recordings were taken from visually identified amacrine cells
in the inner nuclear layer (INL) of the retina. All cells were filled
with Lucifer yellow during recording to confirm their identity as
amacrine cells based on their cell body position in the INL and on
morphological criteria such as the dendritic ramification pattern (Fig.
1A). However, we did not find
any correlation between any morphological characteristics of the
amacrine cells investigated and the properties of sEPSCs or sIPSCs
analyzed. In addition, electrical membrane properties were checked
(Fig. 1B). Voltage-dependent Na+
currents were found in only one amacrine cell, which was not included
in our sample. Bipolar cells also did not express significant voltage-dependent inward currents but could be easily distinguished by
their characteristic morphology. Amacrine cells located in the ganglion
cell layer (displaced amacrine cells) were not included in this study.
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Solutions and chemicals
Slices were continuously superfused with a physiological
extracellular saline that contained (in mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 25 glucose, 2 CaCl2, and 1 MgCl2. To
maintain the extracellular pH at 7.4, the saline was bubbled with 95%
O2 and 5% CO2. The patch
pipette solution contained (in mM): 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES,
and 11 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); pH adjusted to 7.2 with KOH. Stock solutions of strychnine (1 mM in distilled water) and bicuculline (10 mM in distilled water) were prepared shortly before the
experiments and added to the extracellular saline in defined concentrations. To block sEPSCs mediated by ionotropic glutamate receptors, 1 mM kynurenic acid was added to the extracellular saline in
some experiments.
Patch-clamp recordings
Patch pipettes were pulled from borosilicate glass tubing (2.0 mm OD, 0.5-mm wall thickness; Hilgenberg, Malsfeld, Germany). When
filled with internal solution, they had a resistance of 6-8 M.
Amacrine cells were approached under visual control by maintaining a
moderate positive pressure in the patch pipette. Membrane currents were
recorded in the whole cell configuration of the patch-clamp technique
using an EPC-9 amplifier (Heka, Lambrecht, Germany). Patch pipette
capacitance and cell capacitance were canceled and series resistance
was compensated by about 80% using the internal compensation circuits
of the amplifier. Recordings were done at a holding potential
(Vh) of
60 mV. If the holding
current exceeded ±20 pA, the recording was discarded. The sampling
frequency was 10 kHz. Currents were filtered at 2 kHz using the
internal low-pass filter of the amplifier. Data were digitized and
stored on-line using the Pulse software (Heka). All recordings were
made at room temperature (20-24°C).
Analysis of postsynaptic currents and statistics
Spontaneous postsynaptic currents (sPSCs) were detected
(threshold 8-10 pA) by using the MiniAnalysis software (Synaptosoft, Leonia, NJ). Peak amplitudes, rise times, and decay time constants were
estimated for single sPSCs and further analyzed on a Pentium-based personal computer using the MiniAnalysis, Igor (Wavemetrics, Lake Oswego, OR), and Origin software (MicroCal, Northampton, MA). Mean
amplitudes and frequencies of sPSCs were computed from all sIPSCs and
all sEPSCs observed in an amacrine cell. Only events that did not show
any signs of multiple peaks (i.e., contamination of rise or decay
phases by subsequent events) were selected for analysis of the kinetics
and for exponential fitting. The rise times of the sPSCs were
determined by calculating the time in which the current increased from
10 to 90% of the peak amplitude denoted as T10/90. To compare our
T10/90 values with the activation kinetics of recombinant
GABAARs from another study (Zhu et al. 1998), we converted their 20-80% rise time values (T20/80)
into T10/90 values by assuming a monoexponential rise, which gives T10/90 = T20/80 * 1.585. Decay kinetics of single sIPSCs were determined by least-square fits of the decay phase after the peak current using a monoexponential and a biexponential decay function. The
number of exponentials necessary for a good fit of the data were
determined by visual inspection. To allow the comparison of events best
fitted with a different number of exponentials, the weighted time
constant,
w = (A1
1 + A2
2)/(A1 + A2), was calculated, where
A1 and
1 are
the amplitude and the time constant of the fast component and
A2 and
2 are
the amplitude and the time constant of the slow component of the
biexponential fit, respectively. To calculate the decay kinetics of
sEPSCs, at least 50 sEPSCs were randomly selected, averaged, and fitted
using a monoexponential decay function.
All data are given as means ± SE. The significance of the
difference between the mean values of two samples was determined using
the two-tailed Student's t-test for unpaired data. The
correlation between two samples, i.e., between
w and T10/90, was determined by linear
regression analysis. If not stated otherwise, data were denoted as
statistically significant when P < 0.01.
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RESULTS |
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We have used the whole cell configuration of the
patch-clamp-technique to characterize the sPSCs in retinal amacrine
cells. Amacrine cells were filled with Lucifer yellow during the
recordings to identify them by the position of their cell bodies, their
arborization pattern and other morphological criteria (Fig.
1A). Amacrine cells also expressed a characteristic set of
outwardly rectifying K+ currents (Fig.
1B) as previously described (Boos et al.
1993; Menger and Wässle 2000
). The sPSCs
recorded from 34 amacrine cells could be subdivided due to
pharmacological criteria (see following text) into sEPSCs
mediated by ionotropic glutamate receptors and into sIPSCs mediated by
GABAARs and GlyRs (Fig. 1C).
EPSCs
In 6 of 34 amacrine cells, we observed sPSCs that could be clearly
separated into a group that was characterized by small amplitudes and
rapid decay kinetics (Fig. 1C, ) and another group with
significantly higher amplitudes and slower decay kinetics (Fig.
1C). The rapid sPSCs persisted in the presence of 1 µM
strychnine and 10 µM bicuculline but completely disappeared in the
presence of 1 mM kynurenic acid (not shown), indicating that they were sEPSCs mediated by ionotropic glutamate receptors. The other group of
sPSCs could be blocked by bicuculline or strychnine, indicating that
they were sIPSCs mediated by GABAARs and GlyRs
(described in detail in the following text). The appearance of the
sEPSCs was very consistent. Their amplitude distributions showed a
typical narrow distribution ranging from 5 to 25 pA (Fig.
2A, inset) with a
mean peak amplitude of
13.7 ± 1.5 pA (n = 6).
sEPSCs occurred at a mean frequency of 0.49 ± 0.26 Hz
(n = 4) and decayed with a mean
of 1.35 ± 0.16 ms (n = 6). sEPSCs and sIPSCs could be easily
distinguished by the size of their amplitudes (Fig. 2B) and,
in particular, by their different decay kinetics. Superimposed and
averaged traces of sEPSCs and the corresponding cumulative fraction
plot were compared with sIPSCs recorded in the same amacrine cell (Fig.
2, C and D), showing that the decay kinetics of
sEPSCs and sIPSCs were significantly different.
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Unfortunately, the use of kynurenic acid to block sEPSCs also strongly
decreased the frequency of sIPSCs. However, the distinctive properties
of sEPSCs and sIPSCs allowed us to perform most experiments in the
absence of kynurenic acid. In addition, events characterized by a
w < 4 ms were discarded while analyzing sIPSCs.
IPSCs
To resolve currents mediated by Cl
permeable GABAARs and GlyRs at a
Vh close to the resting potential of
amacrine cells (Bloomfield 1992
; Boos et al.
1993
; Zhou and Fain 1995
), a pipette solution was used that contained 144 mM Cl
(ECl
= 3 mV). The
reversal potential of sIPSCs was determined by recording sIPSCs at
different Vh values and plotting their
mean peak amplitudes against Vh. With
the 144 mM Cl
-containing pipette solution,
sIPSCs reversed their polarity at +3.7 mV (n = 6; Fig.
3C) as approximated by linear
regression, a value close to the
ECl
of 3 mV. When a
low-Cl
-containing pipette solution was used (14 mM Cl
;
ECl
=
57 mV; Fig.
3B), the reversal potential of the sIPSCs was
47.1 mV
(n = 7; Fig. 3D), thus significantly shifted
toward the new ECl
, indicating
that sIPSCs were dependent on the intracellular
Cl
concentration as expected for currents
mediated by GABAARs and GlyRs. The deviation of
the estimated reversal potential from ECl
is likely due to a
significant HCO
; Frech et al. 1999
; Kaila
1994
).
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GABAergic sIPSCs
To characterize the properties of the inhibitory receptor types
mediating sIPSCs, we utilized the selective antagonists of GABAARs, bicuculline, and of GlyRs, strychnine. A
typical experiment is presented in Fig.
4, where sIPSCs mediated by
GABAARs could be pharmacologically isolated.
Figure 4A shows the variation of the peak amplitudes of
sIPSCs as a function of the recording time in an amacrine cell that did
not exhibit sEPSCs. In the absence of any antagonist (control, Fig.
4B), sIPSCs were observed at a frequency of 3.1 Hz. The
corresponding amplitude distribution of these sIPSCs showed several
peaks and was skewed toward lower values (Fig. 4C) with a
mean amplitude of 46.3 ± 0.6 pA (n = 2,177).
The addition of 1 µM strychnine to the extracellular saline did not
inhibit the sIPSCs but increased their frequency to 4.7 Hz (Fig. 4,
A and B). The corresponding amplitude histogram
(Fig. 4C) was also skewed toward lower values with a mean
amplitude of
33.9 ± 0.3 pA (n = 3,406) that was
significantly different from the mean amplitude in the absence of
strychnine as confirmed by the cumulative amplitude fraction plot in
Fig. 4D. The application of 1 µM strychnine in combination
with 10 µM bicuculline resulted in a nearly complete inhibition of
the sIPSC activity in this amacrine cell (Fig. 4, A and
B). Thereafter, bicuculline was applied alone and no
recovery of the sIPSCs was observed, indicating that these sIPSCs were
mediated by GABAARs. The analysis of the decay kinetics using monoexponential fits (see METHODS section)
revealed two similar
values in the absence and presence of
strychnine (control:
= 14.2 ms; in strychnine: 16.3 ms;
P > 0.05; Fig. 4, E and F),
suggesting that they were mediated by the same type of receptors,
namely GABAARs. It is also possible that
strychnine blocked a subclass of GlyRs with kinetics similar to the
GABAARs expressed in this cell. However, sIPSCs
observed in the presence of strychnine were mediated by
GABAARs. The increase in sIPSCs frequency
observed in the presence of strychnine was most likely due to
disinhibition of some GABAergic neurons that receive inhibitory inputs
from glycinergic amacrine cells. The decrease in amplitude (Fig.
4A), which was observed in a few amacrine cells, may be the
result of a rundown of the sIPSCs during the experiment or caused by a
desensitization of GABAARs due to accumulated
GABA in the synaptic cleft.
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sIPSCs mediated by GABAARs, because they were
blocked by bicuculline in the presence of strychnine, were isolated in
seven amacrine cells. The mean peak amplitude of these GABAergic sIPSCs was 41.5 ± 8.6 pA, and their mean rise time (T10/90; see
METHODS) was 1.4 ± 0.2 ms. In addition, in another
seven amacrine cells, sIPSCs that were blocked by bicuculline in the
absence of strychnine showed a mean peak amplitude of
39.8 ± 7.7 pA and a mean rise time of 1.3 ± 0.2 ms. The mean peak
amplitudes and mean rise times of these sIPSCs were not significantly
different from those recorded in the presence of strychnine.
To characterize the kinetics of these sIPSCs, the current decays were
fitted with one or two exponentials. sIPSCs could be subdivided into
two groups: one group that comprised sIPSCs best fitted with a
monoexponential decay function (monoexponential group) and another
group, comprising sIPSCs best fitted biexponentially (biexponential
group) characterized by a fast (1) and a slow (
2) decay time constant. Strychnine-resistant,
bicuculline-sensitive sIPSCs best fitted monoexponentially were
characterized by a
of 18.3 ± 3.3 ms and those best fitted
biexponentially by a
1 of 6.7 ± 1.0 ms
and
2 of 62.7 ± 12.1 ms.
Bicuculline-sensitive sIPSCs recorded in the absence of strychnine best
fitted monoexponentially were characterized by a
of 17.7 ± 3.2 ms and those best fitted biexponentially by a
1 of 6.9 ± 0.9 ms and
2 of 58.1 ± 9.7 ms. Like the mean peak
amplitudes and the rise times (see preceding text), all corresponding
time constants were not significantly different from each other
indicating that the bicuculline-sensitive sIPSCs recorded in the
presence, and those recorded in the absence of strychnine were not
different from each other. Therefore these samples were pooled as
GABAergic sIPSCs.
To allow a comparison of the monoexponential and the biexponential
groups, the amplitude-weighted time constants
(w; see METHODS) were calculated.
In 4 of 14 amacrine cells, the monoexponential group of sIPSCs was not
different from the biexponential group (mean
w = 21.3 ± 4.7 ms; pooled data from the monoexponential and
biexponential group). However, in 10 of 14 amacrine cells, the
w values of the monoexponential sIPSC group
(
w = 19.4 ± 2.3) were significantly
different from those of the biexponential group
(
w = 35.0 ± 3.6 ms). The following
findings are shown for one representative amacrine cell in Fig.
5. In this amacrine cell, the
monoexponential group was characterized by a unimodal narrow ranged
w distribution (Fig. 5, A and
C), whereas the
w values of the
biexponential sIPSC group showed a polymodal distribution (Fig. 5,
B and C). In addition, we found that the
1 values and the
2
values of the biexponential group were bimodally distributed. The
distribution histograms were fit with two Gaussians showing peaks at
2.8 and 5.7 ms for
1 (Fig. 5D) and
at 20.2 and 79.1 ms for
2 (Fig.
5E), respectively. With respect to the Gaussian fits shown
in Fig. 5D, the
1 values were
subdivided further into the
1 values <4.5 ms
(Fig. 5D,
) and the
1 values
4.5 ms (Fig. 5D,
). When the
2
values were plotted against the
1 values (Fig.
5F), we found that the
1 values
<4.5 ms were paired with fast
2 values (see
Fig. 5F,
) and the
1 values
4.5 were paired with slow
2 values (Fig.
5F, +), suggesting that the biexponential group is composed
of fast and slowly decaying subgroups.
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To investigate whether GABAAR-mediated sIPSCs
with different decay kinetics were due to dendritic filtering, the
correlations of the peak amplitudes and the decay time constants with
the rise times (T10/90; Fig. 6) were
analyzed. However, neither the peak amplitudes nor the
w values were correlated with their
corresponding T10/90 values (Fig. 6, A and B). In
addition, no correlation of the
1 values with
the corresponding T10/90 values was found, indicating that there was
also no significant influence of dendritic filtering on either the fast
decaying sIPSCs (Fig. 6C,
) nor the slowly decaying
sIPSCs (Fig. 6C,
). In summary, the lack of correlation
between peak current amplitudes and decay times with rise times in any
amacrine cell investigated suggested that dendritic filtering was not
the cause of the occurrence of distinct groups of
GABAARs with different decay kinetics.
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Glycinergic IPSCs
In one amacrine cell, two pharmacologically distinct types of
sIPSCs were observed simultaneously (Fig.
7A). In the absence of
antagonists, one type of sIPSC was characterized by fast decay kinetics
(Fig. 7B, a) and another type by a slow current decay (Fig.
7B, b). In the presence of 10 µM bicuculline, the fast
decaying sIPSCs disappeared completely while the amplitude and the
frequency of the slow decaying sIPSCs increased significantly (Fig. 7,
A and B). When bicuculline was applied in
combination with 1 µM strychnine, the sIPSCs were completely blocked.
These results indicate that the fast decaying, bicuculline-sensitive
sIPSCs were mediated by GABAARs while the slowly
decaying, strychnine-sensitive sIPSCs were mediated by GlyRs. The
comparison of averaged and normalized sIPSCs indicated that the sIPSCs
mediated by GABAARs (Fig. 7C, a) were
characterized by a faster rise time and a faster decay kinetics than
those mediated by GlyRs (Fig. 7C, b). The corresponding
cumulative fraction plots of the T10/90 and values are shown in
Fig. 7D. Since no correlation between decay times and T10/90
values of both GABAergic and glycinergic sIPSCs was found in this
amacrine cell (Fig. 7E), it is concluded that the differences in kinetics of GABAAR- and
GlyR-mediated sIPSCs are not due to an impairment of the recordings by
dendritic filtering. Interestingly, the application of bicuculline
induced an increase in frequency and amplitude of the sIPSCs. When the
sIPSCs in the presence of bicuculline were compared with those in its
absence, a significant difference in mean amplitude and frequency was
found that was likely mediated by disinhibition (Fig. 7A).
|
GlyR-mediated sIPSCs could be pharmacologically isolated in four
amacrine cells (Fig. 8, A and
B). In the absence of any antagonist (control), sIPSCs were
observed at a frequency of 0.59 Hz in this cell. Their peak amplitude
distribution was skewed to lower values characterized by a mean
amplitude of 10.0 ± 0.3 pA (n = 140; Fig.
8C). The application of 10 µM bicuculline induced an
increase of the frequency to 0.88 Hz and a significant increase of the mean amplitude (mean:
11.2 ± 0.2 pA; n = 415;
P < 0.001; Fig. 8, C and D).
When 10 µM bicuculline was applied in combination with 0.5 µM
strychnine, sIPSC frequency was 0.59 Hz, while the mean amplitude
remained significantly increased at
11.2 ± 0.3 pA
(n = 281; P < 0.001; Fig. 8,
A-D) compared with the sIPSCs of the control. However, 1 µM strychnine produced a complete inhibition of sIPSCs (Fig.
8A), indicating that these sIPSCs were mediated by GlyRs.
The analysis of the current decay kinetics revealed similar
w values (Fig. 8E) in control
(
w = 35.8 ms), in 10 µM bicuculline
(
w = 40.5 ms), and in 10 µM bicuculline plus
0.5 µM strychnine (
w = 40.6 ms) that were
not significantly different from each other as indicated by the
corresponding cumulative fraction plots (Fig. 8F). In
summary, glycinergic IPSCs were found in only 4 of 34 amacrine cells.
They were characterized by a mean peak amplitude of
28.0 ± 8.5 pA that was not significantly different from that of
GABAARs-mediated IPSCs. However, the mean rise
times of the sIPSCs mediated by GlyRs (mean T10/90: 2.9 ± 0.6 ms;
n = 4) were significantly slower than those mediated by
GABAARs (mean T10/90: 1.2 ± 0.03 ms;
n = 13; see preceding text). Cumulative fraction plots
of the pooled T10/90 values confirmed this significant difference in
the activation kinetics (GlyRs: mean T10/90 = 2.4 ± 0.08 ms;
n = 336 from 4 amacrine cells;
GABAARs: mean T10/90 = 1.2 ± 0.03 ms;
n = 1933 from 13 cells; P < 0.001;
Fig. 9A).
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|
The w values from all cells with sIPSCs
mediated by GABAARs and those mediated by GlyRs
were also analyzed by comparing the corresponding cumulative fraction
plots. The mean
w values of the glycinergic
sIPSCs (24.3 ± 6.7 ms; n = 4) were not
significantly different from the GABAergic sIPSCs (28.2 ± 3.0 ms;
n = 13). However, the
w values
of GABAergic sIPSCs of the monoexponential group (
w = 20.3 ± 0.5 ms; n = 456; pooled data from 13 cells) and the biexponential group (30.7 ± 0.95 ms; n = 1064; pooled data from 13 cells) were
significantly different from each other and from the
w values of the glycinergic sIPSCs
(
w = 25.3 ± 1.94 ms; n = 183; pooled data from 4 cells; Fig. 9B), indicating that
these groups were functionally different with respect to their decay kinetics.
PSCs remaining in the presence of bicuculline and strychnine
In some amacrine cells, sPSCs that persisted in the presence of 1 µM strychnine and 10 µM bicuculline could be observed. These PSCs
were always characterized by significantly smaller peak amplitudes (less than 20 pA) compared with the peak amplitudes recorded in the
absence of strychnine and bicuculline (control). The analysis of their
kinetics never showed any significant difference to the kinetics of the
control sIPSCs of the same cell. Therefore these sPSCs might have
persisted because of an incomplete exchange of bicuculline and
strychnine. However, because these sPSCs were not studied in detail, it
cannot be excluded that they were mediated by receptors other than
GABAARs or GlyRs.
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DISCUSSION |
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To characterize the functional contribution of the different
synaptic inputs to amacrine cells, their postsynaptic activity was
recorded in the absence and presence of specific receptor antagonists.
Rapidly decaying sPSCs that could be blocked with kynurenic acid were
mediated by ionotropic glutamate receptors. sPSCs with significantly
higher peak amplitudes and slow decay kinetics were identified as
sIPSCs. Based on their pharmacological and kinetic properties, sIPSCs
could also be further subdivided. While sIPSCs sensitive to strychnine
were recognized as glycinergic, those inhibited by bicuculline were
mediated by GABAARs. A few sPSCs that remained in
the presence of strychnine and bicuculline were reduced in amplitude
and showed similar kinetics to control sIPSCs. Since higher
concentration of bicuculline and strychnine have not been used, because
they were reported to affect both receptors (Cohen et al.
1989; Protti et al. 1997
), these sPSCs could
have been mediated by receptors other than
GABAARs or GlyRs. A contribution of sIPSCs
mediated by GABAC receptors is unlikely because
the GABAC receptor-specific
subunits are not
expressed by amacrine cells (Bormann and Feigenspan
1995
; Enz et al. 1996
).
sEPSCs in amacrine cells
In amacrine cells, sEPSCs and sIPSCs were characterized by
significantly different time constants (cf. Fig. 2D) and
could thus be separated for analysis easily. The sEPSCs were
characterized by their small amplitudes and rapid decay kinetics
( = 1.35 ms). The sEPSC amplitudes showed a narrow unimodal
distribution, suggesting that only one or a few vesicles were
spontaneously released at excitatory synapses. Indeed we never found
more than one peak or a significantly skewed amplitude distribution of
sEPSCs in amacrine cells. Since only PSCs were analyzed that did not
show any contamination of subsequent events, it is assumed that a
single PSC resulted from the activation of a single synapse. Thus a
variation in the number of vesicles should produce a different number
of peaks in the amplitude distribution of the EPSCs. However, the lack
of multiple peaks in the amplitude distributions of sEPSCs in amacrine
cells suggests that the number of vesicles released was relatively
constant and/or that the transmitter concentration released was
sufficient to saturate the excitatory postsynaptic receptors.
At present, ionotropic glutamate receptors are subdivided into three
subclasses: the N-methyl-D-aspartate receptors
(NMDARs), the -amino-3-hydroxy-5-methyl-4-isoxazole propionate
receptors (AMPARs), and the kainate receptors. Amacrine cells express
many subunits that are specific for these different subclasses. The subunits GluR6, GluR7, KA2, and
1/2, which are specific for kainate receptors, have been found in amacrine cells of the adult rat retina.
In a subset of adult rat amacrine cells, the NR1, NR2A, and NR2B
subunits of NMDARs were found (Fletcher et al. 2000
), and in many amacrine cells, including AII amacrine cells, the AMPAR-specific subunits GluR2 and GluR4 were present (Qin and Pourcho 1999
). The decay kinetics of sEPSCs in amacrine cells were similar to that of EPSCs mediated by AMPARs in rat retinal ganglion cells (Protti et al. 1997
; Tian et al.
1998
) and in rat hippocampal and cortical pyramidal cells
(Jonas et al. 1993
; Stern et al. 1992
).
Because the decay kinetics of the sEPSCs we recorded in amacrine cells
were also similar to that of recombinant AMPARs (Jonas and
Sakmann 1992
; Mosbacher et al. 1994
) but
significantly faster than that of NMDARs (Barnes-Davies and
Forsythe 1995
; Taschenberger et al. 1995
), we
conclude that the sEPSCs we observed in amacrine cells were probably
mediated by AMPARs. However, NMDA-activated currents have been
previously observed in AII amacrine cells (Hartveit and Veruki
1997
). Therefore it is likely that amacrine cells express NMDA
receptor-mediated EPSCs that were inhibited at the
Vh of
60 mV in the
Mg2+-containing saline used in this study.
sIPSCs
Since kynurenic acid, a broad spectrum antagonist of ionotropic
glutamate receptors, did not affect PSCs with a
w > 4 ms, these PSCs were not mediated by
ionotropic glutamate receptors. All sIPSCs recorded at different
holding potentials were characterized by a reversal potential that
depended on the ECl
. In
addition, the majority of sIPSCs were blocked either by bicuculline or
strychnine. Therefore we conclude that PSCs with a
w > 4 ms in the amacrine cells of this study
were mainly sIPSCs mediated by GABAARs or GlyRs.
In contrast to the sEPSCs, the amplitude distributions of sIPSCs showed
several peaks and were strongly skewed to lower values (cf. Figs.
2B, 4C, and 8C), indicating a strong
variation in the number of vesicles released. The question arises
whether the amount of transmitter activating an IPSC is sufficient to
saturate the postsynaptic receptors. Our results suggest that this is
not the case in all amacrine cells. Inhibitory inputs onto amacrine
cells originate from other glycinergic or GABAergic amacrine cells. In
five amacrine cells, the application of strychnine or bicuculline
induced a significant increase in frequency and amplitude of sIPSCs
(e.g., Figs. 7A and 8A). This was likely due to a
disinhibition of the presynaptic cell. Indeed, multiple synapses
between amacrine cells have been found in the inner plexiform layer
(Chun and Wässle 1989
; Koontz and
Hendrickson 1990
). Since a single sIPSC results from the
spontaneous activation of a single synapse, the increase of the mean
sIPSC amplitudes evoked by disinhibition is likely due to an increase
in the number of activated receptors caused by an increase in the
transmitter concentration. On-bipolar cells depolarize in response to
light stimuli. Since they are nonspiking interneurons, they produce a
tonic and graded glutamate release onto amacrine cells that is
proportional to the light intensity (Matsui et al.
1998
). A nonsaturating vesicle content combined with a high
number of postsynaptic receptors would broaden the operating range of
the synapses. This would enable amacrine cells to respond to tiny
differences of light signals providing a basis for the fine tuning of
inhibition mediated by these cells.
GABAergic IPSCs
In situ hybridization and immunohistochemical experiments showed a
strong heterogeneity in the expression of GABAAR
subunits in amacrine cells (Greferath et al. 1995).
Since the functional properties of ligand gated channels vary with
their subunit combinations, it was not surprising that amacrine cells
expressed GABAergic sIPSCs with strongly varying properties. For
example, the T10/90 values ranged from a few hundred microseconds to a
few milliseconds with a mean value of 1.2 ms. This is significantly
faster than that of 2.63 ms found in cultured chick amacrine cells
(Gleason et al. 1993
). Rapid GABA application
experiments with recombinant receptors composed of
1
3
2
subunits revealed T10/90 values of 0.4-1.27 ms (Zhu et al.
1998
; calculated from T20/80 = 0.25-0.8 ms; see
METHODS). For receptors composed of
1
1
2, the mean
T10/90 value was 2.1 ms and for
2
1
2 receptors 1.0 ms
(Lavoie et al. 1997
), indicating that different subunit
assemblies result in distinct activation kinetics. Thus the wide range
of the T10/90 distribution of GABAergic sIPSCs in amacrine cells may
reflect the contribution of more than one GABAAR
subtype. The decay kinetics of recombinant
GABAARs was also found to be dependent on the
subunit composition. The decay kinetics of GABAergic sIPSCs of amacrine cells from this study showed a strong variability. In most amacrine cells, sIPSCs could be subdivided into a group best fit
monoexponentially and another best fit biexponentially (Fig.
5C). The
w values of these groups
were significantly different, suggesting the occurrence of different
GABAAR subtypes in amacrine cells. In addition,
the bimodal distributions of the
1 and the
2 values within the biexponential group
suggest that this last group comprises two subgroups, a fast and a
slowly decaying group (Fig. 5, D and E). If these
distributions would be at random, then the
1
values should be randomly paired with
2
values. However, fast
1 values were highly
correlated with fast
2 values and slow
1 values with slow
2
values, indicating that there are two populations of GABAergic sIPSCs
with distinct decay kinetics. Interestingly, the
w value of the monoexponential group is
similar to the
w value of the fast
biexponential group (Fig. 5, A and B), suggesting
that these subpopulations might be similar. Thus at least two types of
differentially decaying GABAergic sIPSCs are present in amacrine cells,
suggesting the expression of more than one GABAAR
subtype within the same cell. The time constants we found in amacrine
cells did not exactly match with those obtained from recombinant
receptors. Thus the question of which subunits contribute to
GABAARs in amacrine cells still remains unsolved.
Immunocytochemical studies have shown that amacrine cells express
different GABAARs subtypes. While cholinergic
amacrine cells likely express GABAARs assembled
from
2,
1,
2/3,
, and possibly
2 subunits, dopaminergic
amacrine cells were found to express
1,
2,
3, and
2
subunits (Greferath et al. 1995
; Gustincich et
al. 1999
). GABAARs composed of
1,
2/3, and
2 are expressed in glycinergic amacrine cells
(Greferath et al. 1995
). The
w
values of the monoexponential group of GABAARs in
amacrine cells of this study was 20.3 ms, which is close to the
w values of 19.3 ms reported for spontaneous
quantal events mediated by GABAARs of cultured
chick amacrine cells (Gleason et al. 1993
), of native
GABAARs expressed in juvenile ICC neurons (
w = 22.4 ms) (Backus et al.
2000
), and of recombinant
1
1
2 receptors
(
w = 20.5 ms) (calculated from Lavoie
et al. 1997
), but faster than that of
2
1
2 receptors
(
w = 198.7 ms) (calculated from Lavoie
et al. 1997
), suggesting the contribution of the
1 subunit
in a subpopulation of GABAARs most likely
expressed in glycinergic amacrine cells.
The activation and decay kinetics of sIPSCs are not exclusively caused by the ligand on- and off-binding rates, which are intrinsic properties of specific receptor subtypes, but also by the transmitter exchange rate at the synapse. Since the GABA exchange rate is dependent on the morphological particularities of a release site, i.e., the width of the synaptic cleft, the GABA reuptake rate, the degree of clustering of postsynaptic receptors and the contribution of glial cells, it cannot be excluded that the different kinetics were caused by one of these factors. Given the fact that we did not find a correlation between amplitudes and decay or rise times, the different kinetics reflect the intrinsic properties of distinct GABAAR subtypes rather than dendritic filtering.
Glycinergic sIPSCs
In some cells, we recorded sIPSCs that were sensitive to
strychnine but persisted in the presence of bicuculline, indicating that they were mediated by GlyRs. Whether amacrine cells also express
different subtypes of GlyRs remains unclear. In situ hybridization experiments showed strong signals for the 2 subunits, whereas only
low amounts of mRNA coding for the
1 subunit was found in the
amacrine cell layer. Transcripts of
subunits were found in the INL
but their distribution could not yet be resolved on the cellular level
(Greferath et al. 1994a
). These findings may suggest the
expression of different GlyRs subtypes in amacrine cells. Although
glycinergic sIPSCs showed some heterogeneity with respect to their
kinetics, the low number of amacrine cells that showed glycinergic
sIPSCs did not allow a more complete analysis. This was surprising
because when glycine was applied by bath application, all amacrine
cells tested responded with an inward current (data not shown) as
previously reported by others (Boos et al. 1993
; Menger and Wässle 2000
). Horizontal, bipolar, and
ganglion cells do not release glycine onto amacrine cells, suggesting
that the most likely presynaptic cells are glycinergic amacrine cells. Most glycinergic amacrine cells are nonspiking neurons because the
sIPSCs recorded in ganglion cells, which were mediated by glycinergic
amacrine cells, were not tetrodotoxin sensitive (Protti et al.
1997
). Thus the spontaneous release of glycine, which depends on the membrane potential and membrane conductance, might have been low
under our experimental light-adapted conditions. In addition, glycinergic amacrine cells have smaller dendritic fields than GABAergic
amacrine cells (Pourcho and Goebel 1985
; Vaney
1990
; Wässle and Boycott 1991
), suggesting
that they provide a lower coverage than GABAergic amacrine cells and
that there is a lower number of glycinergic synapses. Indeed,
immunocytochemical labeling with antibodies specific for
GABAAR and GlyR subunits have shown that there
are four times more GABAergic than glycinergic synapses in the
plexiform layers of the mammalian retina (Fischer et al. 2000
).
Are GABAergic and glycinergic IPSCs functionally different?
The mean amplitudes of GlyRs were not significantly different from
those of GABAergic sIPSCs, but their activation and decay kinetics were
different from those of the presumed GABAARs
subtypes (cf. Fig. 9). In one amacrine cell, the glycinergic sIPSCs
were significantly slower decaying than the GABAergic sIPSCs (Fig. 7),
suggesting that at least a subpopulation of
GABAARs is functionally different from GlyRs. In
addition, the mean T10/90 values of glycinergic sIPSCs were
significantly slower than that of GABAergic sIPSCs. These differences
may be due to different association rate constants for
GABAARs and GlyRs. However, glycinergic synapses
may also differ with respect to several other properties, such as
slower transmitter exchange rate, which could be due to different
morphological properties, different glial ensheathment, or different
transmitter reuptake rate. Finally, glycinergic synapses might be
characterized by a different form of receptor clustering resulting in a
more desynchronized activation of the GlyRs, thus leading to a slower rise time. Differences in rise times have also been observed in recombinant GABAARs subtypes of different subunit
composition (Lavoie et al. 1997) and between
GABAARs and GlyRs in nucleated patches obtained
from neurons of the inferior colliculus (Backus et al.
2000
). Although we have found clear functional differences between GABAARs and GlyRs in amacrine cells, at
present, the physiological significance of these differences remains unknown.
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ACKNOWLEDGMENTS |
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We thank Drs. D. A. Protti and B. O'Brien for critically reading the manuscript. We are grateful to F. Boij for excellent technical assistance.
This study was supported by grants to K. H. Backus [Deutsche Forschungsgemeinschaft (DFG)-Schwerpunkt 1026; BA 1311/6-2] and H. Wässle (DFG-Sonderforschungsbereich 269/B4).
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FOOTNOTES |
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Present address and address for reprint requests: K. H. Backus, Institute of Physiology II, Cellular Neurophysiology, University of Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany (E-mail: backus{at}em.uni-frankfurt.de).
Received 18 December 2000; accepted in final form 13 June 2001.
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REFERENCES |
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