Shanghai Institute of Physiology and Key Laboratory of Neurobiology, Chinese Academy of Sciences; and the Institute of Neurobiology, Fudan University, Shanghai 200031, China
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ABSTRACT |
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Du, Jiu-Lin and
Xiong-Li Yang.
Subcellular Localization and Complements of GABAA and
GABAC Receptors on Bullfrog Retinal Bipolar Cells.
J. Neurophysiol. 84: 666-676, 2000.
-Aminobutyric acid (GABA) receptors on retinal bipolar cells (BCs)
are highly relevant to spatial and temporal integration of visual
signals in the outer and inner retina. In the present work, subcellular
localization and complements of GABAA and GABAC receptors on BCs were investigated by whole cell recordings and local
drug application via multi-barreled puff pipettes in the bullfrog
retinal slice preparation. Four types of the BCs (types 1-4) were
identified morphologically by injection of Lucifer yellow. According to
the ramification levels of the axon terminals and the responses of
these cells to glutamate (or kainate) applied at their dendrites, types
1 and 2 of BCs were supposed to be OFF type, whereas types
3 and 4 of BCs might be ON type. Bicuculline (BIC), a
GABAA receptor antagonist, and imidazole-4-acetic acid (I4AA), a GABAC receptor antagonist, were used to
distinguish GABA receptor-mediated responses. In all BCs tested, not
only the axon terminals but also the dendrites showed high GABA
sensitivity mediated by both GABAA and GABAC
receptors. Subcellular localization and complements of
GABAA and GABAC receptors at the dendrites and
axon terminals were highly related to the dichotomy of OFF and ON BCs. In the case of OFF BCs,
GABAA receptors were rather evenly distributed at the
dendrites and axon terminals, but GABAC receptors were
predominantly expressed at the axon terminals. Moreover, the relative
contribution of GABAC receptors to the axon terminals was
prevalent over that of GABAA receptors, while the situation
was reversed at the dendrites. In the case of ON BCs,
GABAA and GABAC receptors both preferred to be
expressed at the axon terminals; relative contributions of these two
GABA receptor subtypes to both the sites were comparable, while
GABAC receptors were much less expressed than
GABAA receptors. GABAA, but not
GABAC receptors, were expressed clusteringly at axons of a
population of BCs. In a minority of BCs, I4AA suppressed the
GABAC responses at the dendrites, but not at the axon
terminal, implying that the GABAC receptors at these two
sites may be heterogeneous. Taken together, these results suggest that
GABAA and GABAC receptors may play different
roles in the outer and inner retina and the differential complements of
the two receptors on OFF and ON BCs may be
closely related to physiological functions of these cells.
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INTRODUCTION |
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Recent experimental and model
studies have clearly demonstrated that for a specific kind of ion
channels, not only their characteristics but also their subcellular
spatial distribution are of utmost relevance to physiological functions
of the cells (for review, see Magee et al. 1998;
Nusser and Somogyi 1997
; Safronov 1999
). Bipolar cells (BCs) are second-order neurons, which are involved in
information processing in the outer and inner plexiform layers (OPL and
IPL) of the vertebrate retina (Dowling 1987
).
-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter
that modulates synaptic transmission at both synaptic layers
(Barnstable 1993
; Lukasiewicz and Shields
1998a
; Wu 1992
; Yazulla 1986
). It
is generally thought that BCs receive input from GABAergic horizontal
cells in the OPL, which may mediate the surround response of the
receptive field of BCs via feedback inhibition onto photoreceptors
(Wu 1992
) and/or feedforward inhibition onto BC
dendrites (Dowling and Werblin 1969
; Hare and
Owen 1992
; Lasansky 1973
, 1980
; Yang and
Wu 1991
). Furthermore GABAergic amacrine cells are supposed to
feedback to BC axon terminals through the reciprocal synapses, which
may be responsible for temporal modulation of BC output signals
(Dong and Werblin 1998
; Du and Yang
1999a
; Hartveit 1999
). It is evident, therefore
that spatial distribution of GABA receptors on BCs is crucial for
spatial and temporal integration of visual signals in the outer and
inner retina. The results concerning subcellular localization of GABA
receptors on BCs seem to be inconsistent. While GABA receptor have been
demonstrated morphologically (Enz et al. 1995
, 1996
;
Fletcher et al. 1998
; Koulen et al. 1997
,
1998
; for review, see Wässle et al. 1998
)
and physiologically (Euler and Wässle 1998
;
Feigenspan et al. 1993
; Karschin and Wässle 1990
; Lukasiewicz and Wong 1997
;
Lukasiewicz et al. 1994
; Maple and Wu
1996
; Tachibana and Kaneko 1987
) to be localized
exclusively on BC axon terminals in most vertebrate retinas, GABA
receptor-mediated currents were reported to be induced from both
dendrites and axon terminals of BCs in several species (Qian and
Dowling 1995
; Suzuki et al. 1990
). Our first
goal was to re-examine this issue, using a bullfrog retinal slice preparation.
In addition to GABAA and
GABAB receptors, a novel
GABAC receptor has recently been identified
(Feigenspan et al. 1993; Qian and Dowling
1993
). GABAA and
GABAC receptors are both ionotropic receptors
incorporating chloride channels, but they have distinct kinetics and
may mediate signal transfer in different time domains in the retina
(Han and Yang 1999
; Lukasiewicz and Shields
1998a
). A second goal of the present work was to study
complements of GABAA and
GABAC receptors on BCs to gain an insight into
the physiological roles of these receptor subtypes for information
processing of BCs.
In both mammalian and nonmammalian retinas, distinct subtypes of BCs
have been identified morphologically and physiologically. Several lines
of evidence indicate that OFF-type BCs, which are hyperpolarized by illumination of the receptive field center, have
telodendria ramifying in the distal IPL (sublamina a), while ON-type BCs, which are depolarized by illumination of their
receptive field centers, have telodendria ramifying in the proximal IPL (sublamina b) (Dowling 1987; Euler and
Wässle 1995
; Hare et al. 1986
).
Furthermore these two subtypes show different responses to glutamate,
mediated by distinct subtypes of glutamate receptors (for review, see
Massey and Maguire 1995
; Shiells and Falk
1995
; Wilson 1994
). A final purpose of this work
was to determine how relative contributions of
GABAA and GABAC receptors
were related to the dichotomy of OFF and ON BCs
in the bullfrog retina. Part of this study has appeared in abstract
form (Du and Yang 1998
).
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METHODS |
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Retinal slice preparation
Adult bullfrogs (Rana catesbeiana), maintained in
tanks at 4°C on a 12-h light/12-h dark cycle, were used in the
present work. Retinal slices were prepared following the procedures
reported previously (Werblin 1978; Wu
1987
) with minor modifications (Du and Yang
1999b
). In brief, prior to an experiment an animal was dark-adapted for at least 1 h. After the animal was pithed and decapitated, an eye was enucleated, and the cornea and lens were removed. The eyecup thus formed was immersed with the standard bullfrog
Ringer solution (see Solutions and drug application for the
composition) and quantisected. A small piece of the eyecup was placed
vitreal-side down onto a piece of Millipore filter (pore size: 0.45 µm), and the sclera was removed. The retina attached to the filter
was cut into 100-µm-thick slices using a Mcilawain tissue chopper
(Mickle Lab Engineering Co. LTD, Gomshall, UK). The slices were then
transferred into a glass-bottomed recording chamber with the cut side
up and anchored on two petroleum jelly (Vaseline) strips painted on the
bottom, and they were further held mechanically in place by a grid of
parallel nylon strings glued onto a U-shape frame of platinum wire. All
these procedures were performed under dim red illumination.
Optical setup and recording chamber
The recording chamber was placed on a fixed stage microscope (Zeiss, ACM, Germany), which was equipped with epifluorescence illumination and Hoffman modulation contrast optics (Hoffman Modulation Optics, Greenvale, NY). A water-immersion objective with a working distance of 2.9 mm was used (Zeiss, ×40, 0.75 NA), with its metal parts being coated with Epon to avoid corrosion and contact voltages. During the experiments, the cells were imaged by an infra-red video camera and visualized on a TV monitor. Additional magnification was obtained by a ×3 lens (Zeiss Optovar) inserted in the imaging pathway.
The recording chamber had a volume of ~0.8 ml and was continuously perfused with the oxygen-bubbled extracellular solution. The solution was fed in and out of the recording chamber with a peristaltic pump (Minipulse 3, Gilson Medical Electronics, Villiers-le-Bel, France) at a rate of 2-3 ml/min.
Whole cell recordings
Recording patch electrodes were pulled from borosilicate glass
(Shanghai Brain Research Institute, Shanghai, China) with a two-stage
vertical puller (PB-7, Narishige, Japan). The resistance of the
electrodes was 8-12 M in the bathing medium when filled with the
intracellular solution whose composition is given in the following
text. Connected to the amplifier (CEZ-2300, Narishige, Japan) via an
Ag/AgCl wire, the recording electrode was mounted on a mechanical
micromanipulator (MMN-9, Narishige, Japan). To get a large angle
between the recording electrode and the slice, the pipette was visually
bent for ~30-50° at 0.5-0.8 mm away from the tip by a heating
platinum wire under microscopy before the experiments. Fine adjustment
of the electrode was made possible with the aid of a hydraulic
micromanipulator (MHW-3, Narishige, Japan). The reference electrode was
an Ag/AgCl wire connected to the recording chamber. The liquid junction
potential of the recording electrode was measured (Neher
1992
) and routinely corrected.
Cells on the surface of the slices could be easily visualized with the
aid of the optical setup. The action of the fluid stream coming out of
the electrode tip driven by a positive pressure applied to the
electrode was sufficient for seal formation, and no extra cleaning of
cell surface was needed. In most cases, whole cell recordings
(Hamill et al. 1981) were only made from cells located
at, or a little below, the surface of the slices. Gigaohm (2-10 G
)
sealing between the electrode tip and the cell membrane was obtained by
positioning the electrode tip onto the cell surface, releasing the
positive pressure, and applying careful suction. The whole cell
configuration was established by rupturing the membrane with brief
pulses of suction and/or in combination with brief voltage transients
applied to the electrode. The series resistance of the recording
electrode estimated from the peak amplitude of the capacitative current
was reduced to ~10 M
with resistance compensation, which would
produce a holding voltage error of <2 mV when the currents recorded
were typically <200 pA. Capacitative currents caused by the electrode
and cell capacitance were partially cancelled by the circuit of the
amplifier. pClamp 6.0.4 (Axon instruments, Foster City, CA) was used to
generate voltage command outputs, acquire data, and trigger drug
application puff via a DigiData 1200A (Axon instruments) interface on
an IBM-compatible personal computer. The data were low-pass filtered
with a fourth Bessel filter at 1 kHz and digitized at 2.5 kHz. Data
statistical analysis was performed using Student's paired test.
To classify BCs morphologically, the patch electrodes were filled with Lucifer yellow (0.1%), which could diffuse into the BCs during the recordings when the whole cell configuration was established so that both cell bodies and fine processes were clearly visible by epifluorescent illumination under microscopy. The experiments were performed under room illumination and the slices were very likely light adapted.
Solutions and drug application
The standard intracellular solution with an osmolality of 290 mOsm consisted of (in mM) 100.5 cesium fluoride, 40 cesium chloride, 3 sodium chloride, 0.4 magnesium chloride, 0.1 calcium chloride, 10 HEPES, 1 EGTA, 3 Mg1.5ATP, and 0.5 Na3GTP, adjusted to pH 7.7 with cesium hydroxide.
The standard bath medium, which was only used for the preparation of
retinal slices, contained (in mM) 150 sodium chloride, 2 potassium
chloride, 2 calcium chloride, 2 magnesium chloride, 10 HEPES, and 10 glucose, adjusted to pH 7.8 with sodium hydroxide, with an osmolality
of ~310 mOsm. Since cobalt ions can substantially suppress the
responses mediated by GABA receptors (Kaneda et al.
1997; Kaneko and Tachibana 1986
), we instead
used 20 mM magnesium chloride, substituting for an osmotically
equivalent amount of sodium chloride and 2 mM calcium chloride to block
synaptic transmission (Lukasiewicz and Werblin 1994
;
Lukasiewicz et al. 1994
) in all the experiments reported in this paper. In the high Mg solution, neither spontaneous
excitatory/inhibitory postsynaptic current (EPSC/IPSC) nor calcium
currents could be recorded from BCs, proving that these cells received
no synaptic inputs. Glutamate (GLU), kainate (KA),
6-cyanoquinoxaline-2,3-dione (CNQX),
-aminobutyric acid (GABA),
picrotoxin (PTX), bicuculline (BIC), and imidazole-4-acetic acid (I4AA)
were obtained from Research Biochemicals (RBI, MA). All other chemicals
were purchased from Sigma Chemicals (St. Louis).
When subcellular localization of GABA receptors was studied, drugs were locally applied to a specific subcellular site of the recorded BC using seven-barreled pneumatic puff pipettes with a hydraulic micromanipulator (MO-330, Narishige, Japan). The puff pipettes were pulled from borosilicate glass (CG-17, Shanghai Brain Research Institute, Shanghai, China) with a multi-stage vertical puller (Shanghai Institute of Physiology, Shanghai, China), and the tip of each pipette was 3-5 µm in diameter. These pipettes were positioned in parallel with the longitude axis of the retinal slice and in a direction opposite to the bath medium flow in the chamber. Drugs were pressure ejected by nitrogen gas with a pressure of 4-8 psi via a Picospritzer II (General Valve, Fairfield, NJ), which was triggered by the pClamp software. In control experiments, all seven barrels were filled with the same solution to make sure that each barrel was positioned correctly to optimize consistency of drug delivery between barrels, and differences of <10% in whole cell currents were found with drug delivery given by different barrels. When no pressure was applied to the pipettes, small amount of the bath medium was continuously sucked into the pipettes by capillary attraction, which prevented the test solutions from leaking out. The concentrations of drugs given throughout the text refer to the concentrations in barrels of the puff pipettes and were chosen in reference to the results of preliminary experiments. They were 1 mM for GLU, 0.2 mM for KA, 0.05 mM for CNQX, 1 mM for GABA, 0.5 mM for PTX, 0.5 mM for BIC, and 2 mM for I4AA. The actual concentrations at the cell membrane were surely much lower because of bulk flow, diffusion, and potent uptake systems in the retina.
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RESULTS |
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Morphological and functional identification and classification of bullfrog BCs
With intracellular Lucifer yellow staining, four types of BCs
could be distinguished according to the ramification levels of their
axons within the IPL and, for brevity, they are referred to as types
1-4 of BCs in the present work. The classification scheme is shown in
Fig. 1, and the cells are numbered
according to the stratification levels (from outer to inner IPL) of
their axons. The axons of types 1 and 2 terminate in sublamina
a (distal 40% of the IPL), whereas the axons of types 3 and
4 terminate in sublamina b (proximal 60% of the IPL). These
cells were tentatively classified as OFF and ON
BCs, respectively (Dowling 1987; Euler and
Wässle 1995
; Euler et al. 1996
;
Hare et al. 1986
; Kolb 1994
; Wu
and Maple 1998
). Sublamina a and b were
further subdivided in two strata of equal width respectively (labeled
1-4). The axon of type 1 branches into two layers of finer processes
ramifying in strata 1 and 2, respectively. In contrast, the axons of
types 2-4 have no branches before terminating in fine processes at a single stratum (2-4, respectively). In most cases, BCs have a single
dendritic trunk originating from the soma and branching into multiple
finer processes near the OPL, with one ascending into the outer nuclear
layer (ONL), sometimes terminating in a Landolt's club, and the others
extending horizontally for 10-30 µm in the OPL. Their axon terminals
extend horizontally in the IPL for 10-50 µm with fine branches
ending into small swellings, different from the large knob-shaped
swellings characteristic of ON BCs in carp and goldfish retinae
(Han et al. 1997
; Matthews et al. 1994
).
In addition, there are small pearl-like varicosities along the
processes of most BC dendrites and axons. The BC somata were ~8 × 5 µm (length × width) in dimensions.
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The preceding morphological classification of OFF and
ON BCs was further confirmed by examining the responses
induced by the application of glutamate to BC dendrites. It is
generally thought that at their dendrites, OFF BCs express
ionotropic glutamate receptors (iGluR), whereas ON BCs
express metabotropic glutamate receptors (mGluRs) in a variety of
vertebrate retinas (Euler et al. 1996; for review, see
Massey and Maguire 1995
; Shiells and Falk
1995
; Wilson 1994
).
When glutamate receptor agonists were locally applied to the dendrites
of BCs, two types of responses were observed that were closely related
to the stratification levels of the axon terminals of these cells:
sublamina a or b. Whole cell recordings from a type 1 BC and a type 3 BC identified morphologically are shown in Fig.
2. Dendritic puff application of kainate
(200 µM), an agonist of iGluRs, to the type 1 cell (labeled I in Fig.
2A) evoked an inward sustained current with a reversal
potential of about +1 mV, which could be completely blocked in the
presence of CNQX (50 µM), a
non-N-methyl-D-aspartate (NMDA) receptor
antagonist (Fig. 2B). Similar results were obtained with
glutamate application, except that the glutamate-induced currents were
more transient (data not shown). Such currents were recorded from all
types 1 and 2 of BCs tested (18/18) without exception, which further
strengthened the suggestion that types 1 and 2 were OFF
BCs. For the type 3 BC (labeled II in Fig. 2A), glutamate (1 mM) evoked an outward current (Fig. 2C) with a negative
slope resistance and a reversal potential of about 5 mV. Following
co-application of CNQX, the current only slightly decreased in size,
suggesting that the current was mainly mediated by mGluRs. Similar
results were observed in 5 of 11 type 3 and 4 BCs examined. In the
remaining six cells, glutamate elicited no currents at all; this may be
due to the rundown of mGluR responses of ON BCs during the
whole cell recordings, as often reported in previous work
(Lasansky 1992
; Lukasiewicz and Werblin
1994
).
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Spatial profile of GABA sensitivity of BCs
Spatial distribution of GABA receptors on bullfrog BCs was further explored by applying GABA locally at different subcellular sites via single-barreled puff pipettes with a fine tip (<1 µm ID; Fig. 3A). As shown in Fig. 3B, currents could be elicited from this type 3 BC when 1 mM GABA was puffed either at the dendrites (D), soma (S), axon (A), or axon terminal (T). The GABA-induced currents were relatively large at the dendrites (36 pA) and axon terminal (27 pA) but less at the axon (18 pA). The current was even detectable at the soma (6 pA). It may be argued that the current elicited by puffing GABA at the soma was simply a consequence of the activation of the GABA receptors at the dendrites and axon terminal by GABA diffusion. To clarify this issue, the soma and processes of the cell were gently elevated with the patch electrode (position II in Fig. 3A), but without destroying the whole cell recording configuration, to avoid possible diffusion effects as much as possible. Under such a condition, the response to local GABA application at the soma disappeared (Fig. 3C), while the other three responses to local GABA application to D, A, and T remained almost unchanged. This result implies that no or few GABA receptors are expressed on the soma. Similar experiment conducted in eight other BCs (3 OFF, 5 ON) yielded comparable results. In agreement with this suggestion, it was found that GABA failed to elicit any observable currents from outside-out membrane patches of the BC soma (n = 10, data not shown). All the responses to application of 1 mM GABA to the dendrites, axon, and axon terminal were completely blocked by 0.5 mM PTX (Fig. 3D), a classical blocker of chloride channels, in all BCs tested.
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Complements of GABAA and GABAC receptors at BC dendrites and axon terminals
Since GABAA and GABAC
receptors both are chloride channels, our next questions were to
determine relative contributions of the currents mediated by
GABAA and GABAC receptors
to the total responses to GABA application at BC dendrites and axon
terminals and how the current fractions were related to different BC
types. For these purposes, BIC, a competitive
GABAA receptor antagonist, and I4AA, a presumably
competitive antagonist of GABAC receptors (Han et al. 1997; Kusama et al. 1993
;
Picaud et al. 1998
; Qian and Dowling 1994
,
1995
), were used to suppress the GABAA
and GABAC response components, respectively, so
that the fractions of these components could be determined. For a more
quantitative estimate of these current fractions, two cautions should
be considered. First, in consideration of the different affinities of
GABAA and GABAC receptors
for GABA (for review, see Feigenspan and Bormann 1998
;
Lukasiewicz and Shields 1998a
), a saturating
concentration of GABA should be used so that all GABA receptors are
fully activated. It was found that the GABA-induced currents were no
longer increased in amplitude with the increase of GABA concentration
when it exceeded 0.5 mM in the puff pipette. Second, we must be sure
that BIC or I4AA, co-applied with GABA, could fully suppress the
GABAA or GABAC components
of GABA-induced responses. We have found that suppression of the
GABAA or GABAC components
was not increased with a further increase of BIC or I4AA concentration
when it was 0.5 mM for BIC and 2 mM for I4AA. Actually, as shown in
Fig. 4, at the axon terminal of a type 2 BC, 1 mM GABA induced-current (bottom,
) is precisely the
algebraic sum (bottom, - - -) of the currents
respectively induced by co-application of 1 mM GABA with 2 mM I4AA
(GABAA component, top) and with 0.5 mM
BIC (GABAC component, middle). The
ratio of the algebraic sum to the total GABA current was 0.92 ± 0.12 (mean ± SD; n = 8, P > 0.05). In consequence, 1 mM GABA, 2 mM I4AA, and 0.5 mM BIC (all in the puff pipettes) were used as "standard" concentrations for this kind
of analysis.
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In most of BCs tested (87/90), GABAA and
GABAC currents could be elicited from both
dendrites and axon terminals. An example recorded from a type 1 BC is
shown in Fig. 5. Two seven-barreled puff
pipettes were, respectively, directed toward the dendrites and axon
terminal of the cell, which was voltage-clamped at 60 mV.
Co-application of GABA and I4AA at the dendrites and axon terminal
elicited comparable inward currents (GABAA
responses, 38 pA vs. 46 pA; Fig. 5A). When GABA was
co-applied with BIC, the current (GABAC response)
elicited from the axon terminal (74 pA) was significantly larger than
that from the dendrites (11 pA; Fig. 5B). It is noteworthy
that the GABAC responses elicited from both the
dendrites and axon terminal were more sustained than the
GABAA responses; this is in agreement with
previous reports (Han et al. 1997
; Qian and
Dowling 1993
, 1995
; for review, see Feigenspan and
Bormann 1998
; Lukasiewicz and Shields 1998a
).
This may be due to the slower desensitization and deactivation of
GABAC receptors than the
GABAA receptors. The GABA responses at the two
sites were almost completely blocked by co-application of I4AA and BIC
(Fig. 5C). These results clearly demonstrate that GABAA and GABAC receptors
are expressed at both the dendrites and axon terminals of the bullfrog
BCs.
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We have made this kind of analysis in 19 OFF (12 type 1 and 7 type 2), and 17 ON (7 type 3 and 10 type 4) BCs. Due to qualitative similarity, we did not made distinction between the data obtained from types 1 and 2 or types 3 and 4. All the data concerning the peak currents mediated by GABAA and GABAC receptors elicited from the dendrites and axon terminals of these cells are summarized in Table 1. It is evident that the relative contributions of the GABAA and GABAC receptors to dendrites and axon terminals varied with the cell types: OFF or ON types.
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In Fig. 6A, the ratios of the
currents elicited from the dendrites and axon terminals (D/A ratios)
are represented by black () and white (
) bars, respectively for
the GABAA and GABAC
responses. In the case of OFF BCs, D/A ratio was 1.12 ± 0.53 (mean ± SD) for the GABAA currents,
indicating that the GABAA currents elicited from
the dendrites and axon terminals were almost equal (P > 0.05). The GABAC currents of the dendrites
were much smaller than those of the axon terminals (D/A = 0.14 ± 0.12, P < 0.001). In the case of
ON BCs, D/A ratio was 0.48 ± 0.23 for the
GABAA currents and 0.34 ± 0.21 for the
GABAC currents, showing that
GABAA and GABAC receptors
both prefer to be expressed at their axon terminals (P < 0.001).
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At the dendrites or axon terminal of a single BC, the fractions of
GABAA and GABAC
receptor-mediated currents were found to be closely related to the cell
types (Fig. 6B). For the OFF BCs, the relative
GABAC:GABAA ratio was
~0.23 ± 0.07 at the dendrites (left ), indicating
that the dendrites of these cells may express GABAC receptors much less than
GABAA receptors (P < 0.001). In contrast, as indicated by the high
GABAC:GABAA ratio
(2.46 ± 1.14) for the axon terminals (left
),
GABAC receptors may be much more expressed at
this site (P < 0.001). For the ON BCs,
however, the GABAC:GABAA
ratios were much less than 1.0 at both the dendrites (0.15 ± 0.05; right
) and axon terminals (0.33 ± 0.09;
right
). In other words, expression of
GABAA receptors at both dendrites and axon
terminals prevailed over that of GABAC receptors
for the ON BCs (P < 0.001).
GABAA receptors at BC axons
As shown in Fig. 3, local application of GABA at the BC axon could evoke currents. One may argue that the currents induced from the axon were simply because GABA locally applied at the axon might have activated GABA receptors located at the axon terminal. To rule out this possibility, whole cell recordings were made from the BCs with soma and dendrites intact but axon terminals lost. For identification of the cell type that lost axon terminals, we also examined the properties of their responses to glutamate. In all these BCs (n = 9), GABAA and GABAC currents were elicited at their dendrites (Fig. 7, A and B, top), which were similar to the results obtained from the BCs with axon terminals intact (compare with Fig. 5). In four of nine cells (2 OFF and 2 ON BCs), neither GABAA nor GABAC currents were elicited at their axons (Fig. 7A, bottom). However, in the remaining five cells (1 OFF and 4 ON BCs), GABAA responses were clearly evoked, while no GABAC currents were detectable (Fig. 7B, bottom). These results demonstrate that GABAA, but not GABAC receptors, exist at the axons of a population of bullfrog BCs.
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GABAA receptors were not evenly distributed along BC axons. In the BCs (n = 3) with axon terminals lost, 1 mM GABA with 2 mM I4AA was applied, using fine single-barreled pipettes, onto four different locations, separated by equal distance (~15 µm), along the axons (Fig. 8A), and the currents were recorded and compared. In the example shown in Fig. 8B, the current induced at location A1 was relatively large, with a short delay and a fast rising phase. As the location was moved away from A1 and closer to the GCL, the induced currents became smaller, with the delay longer and the rising phase slower. The results obtained from the two other BCs were qualitatively similar, though the location at which the largest and fastest current was elicited was not necessarily the same. These results suggest that GABAA receptors may be clustered on the BC axons. It was also noted from Fig. 8 that the current elicited from the soma was smaller than those elicited from A1 and A2. This observation eliminated the possibility that the currents induced by co-application of GABA and I4AA to the axon might be due to a result of the activation of GABAA receptors on the dendrites by GABA diffusion.
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Heterogeneity of GABAC receptors
In most cases (58/62), I4AA acted as an effective antagonist of GABAC receptors of bullfrog BCs. However, in four BCs (2 OFF and 2 ON), it was found that I4AA failed to effectively suppress the GABAC responses. Such an example is shown in Fig. 9. For this ON BC, addition of I4AA (2 mM) completely abolished the GABA current (GABAC response) remaining after co-application of BIC at the dendrites, but only slightly suppressed that at the axon terminal. This result raised a possibility that GABAC receptors at these two subcellular sites may have different pharmacological characteristics in a minority of bullfrog BCs.
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DISCUSSION |
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In the present work, subcellular localization and complements of GABAA and GABAC receptors on morphologically and physiologically identified bullfrog retinal BCs were systematically examined. Our results showed that GABA receptors were expressed at both BC dendrites and axon terminals. We further examined subcellular localization and complements of GABAA and GABAC receptors at the dendrites and axon terminals. As seen in Figs. 4, 5, 7, and 9, the delay and the rate of rise of cell responses showed a relatively large variation. Such variation might be caused by the presence of intrinsic diffusion barriers of slice preparation, such as narrow extracellular spaces and uptake systems, and three-dimensional distribution of GABA receptors on BC processes, etc. Thus we used the peak amplitude of responses as an index for the estimation of relative contributions of these receptors. It was found that relative contributions of GABAA and GABAC receptors were clearly related to the dichotomy of OFF and ON BCs. In the case of OFF BCs, GABAA receptors were rather evenly distributed at the dendrites and axon terminals, but expression of GABAC receptors showed a bias in favor of the axon terminals; the relative contribution of GABAC receptors to the axon terminals was prevalent over that of GABAA receptors, while the situation was reversed for the dendrites. In the case of ON BCs, GABAA and GABAC receptors both preferred to be expressed at the axon terminals, and their relative contributions to either dendrites or axon terminals were comparable. Furthermore the relative contribution of GABAC receptors to both sites was much less than that of GABAA receptors. The results are summarized in the cartoon of Fig. 10.
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Spatial distribution of GABA receptors on BCs
In all retinae studied immunocytochemically to date (Enz et
al. 1995, 1996
; Fletcher et al. 1998
;
Koulen et al. 1997
, 1998
; Lin and Yazulla
1994
; for review, see Wässle et al. 1998
),
abundant evidence have shown that strong punctuate immunofluorescence
of GABAA and GABAC receptor
subunits was observed at the BC axon terminals but not in the OPL. It
is thus suggested that GABAA and
GABAC receptors are predominantly clustered at
the axon terminals but not at the dendrites. In agreement with the
anatomical results, a lot of electrophysiological work has demonstrated
that GABA failed to induce currents from the BC dendrites, while GABA
responses could be consistently elicited from the BC axon terminals
(Euler and Wässle 1998
; Feigenspan et al.
1993
; Karschin and Wässle 1990
;
Lukasiewicz and Wong 1997
; Lukasiewicz et al.
1994
; Maple and Wu 1996
; Tachibana and
Kaneko 1987
). On the other hand, there are also studies showing
GABA-induced currents from BC dendrites in the retina of mouse
(Suzuki et al. 1990
) and hybrid bass (Qian and
Dowling 1995
). In the bullfrog retina, we have shown that GABA
could induce large currents from the dendrites of most BCs tested that
were comparable in amplitude with those induced from the axon
terminals. This result strongly suggests that GABA receptors indeed
exist on the BC dendrites. Since horizontal cells of many species
(Yazulla 1986
), including bullfrog (Zhang et al.
1999
), are GABAergic and feedforward synapses exist between
horizontal cells and BCs (Dowling and Werblin 1969
;
Hare and Owen 1992
; Lasansky 1973
, 1980
;
Yang and Wu 1991
), it sounds reasonable to postulate that the dendritic GABA receptors may mediate feedforward synaptic inputs from horizontal cells to BCs and participate directly in the
formation of the surround antagonism of the BC receptive fields.
It was of interest to compare our results with a recent work of
Euler and Wässle (1998) on spatial distribution of
GABAA and GABAC receptors
on rat BCs. They recorded GABA receptor-mediated currents from BCs in
the rat retinal slice preparation and found that the contributions of
GABAA and GABAC receptors
were related with the dichotomy of BCs respectively driven by rods and
cones. That is, ~70% of the GABA current was mediated by
GABAC receptors for rod BCs, while the fraction
of the GABAC current was only ~20% for cone
BCs. It should be noted, however, that a much lower standard GABA
concentration (25 µM) was used in their work as compared with 1 mM
used in the present work. For determining the actual receptor
contribution, use of saturating concentration may have been required.
In the present work, to examine the complements of
GABAA and GABAC receptors
on BCs, the saturating concentration (1 mM) of GABA was used to
guarantee that all GABA receptors were fully activated. Since the
GABAA and GABAC response
components were completely suppressed by 0.5 mM BIC and 2 mM I4AA,
respectively, in most cases, the complements of
GABAA and GABAC receptors
determined for a special subcellular site under such experimental
conditions should reflect actual contributions of these two subtype
receptors to the BCs.
The physiological implications of this differential distribution of
GABAA and GABAC receptors
on OFF and ON BCs remain to be explored. Here
we only provide some speculations. First, because GABAC receptors have approximately a 10-fold
higher sensitivity than GABAA receptors (for
review, see Feigenspan and Bormann 1998; Lukasiewicz and Shields 1998a
), their activation may be
associated with illumination-dependent GABA levels in the synaptic
clefts of the retina. It is generally thought that GABA is released
from horizontal and amacrine cells in darkness and that the GABA
release is reduced by illumination (Dowling 1987
;
Yazulla 1986
). Since OFF BCs are activated
when illumination is turned off, which corresponds to a change in GABA
release from a lower to a higher level, the prevalent dominance of
GABAC receptors on the axon terminals of these
cells match such conditions, thus mediating inhibition from amacrine
cells to BCs at low levels of GABA. On the other hand, predominant
existence of GABAA receptors may be beneficial
for ON BCs, which mediate inhibition in a relatively high
background level of GABA since these cells are activated when light is
turned on. Second, GABAC responses are overall
slower than GABAA responses in kinetics. It has
been shown that the time course of inhibition from amacrine cells to
BCs may be differentially shaped by these two receptor subtypes
(Matthews et al. 1994
; Pan and Lipton
1995
). Finally, the differential subcellular localization of
GABAA and GABAC receptors
suggests that GABAC receptors (especially, of OFF BCs) may be mainly involved in visual signal processing
in the inner retina, whereas GABAA receptors are
in both the inner and outer retina.
Spatial distribution of receptors has been demonstrated to be crucial
to their physiological functions (Magee et al. 1998; Nusser and Somogyi 1997
; Safronov 1999
).
In the CNS most neurons possess finer dendrites and axons; this makes
such an analysis difficult. Moreover, since CNS neurons commonly have
long processes, currents recorded from the soma could have been
seriously decayed when drugs were locally applied to dendrites or axon
terminals. In contrast, bullfrog BCs have a compact electrotonic
architecture and their space constant (
) ranges between 0.1 and 0.2 (unpublished observations). As a result, the axon terminal or dendritic
GABA currents would decay no more than 10-20% when they were recorded from the soma (Spruston et al. 1993
). Retinal BCs thus
provide a good model for exploring physiological implication of spatial distribution of a special receptor type.
GABAA receptors at BC axons
When GABA was locally applied to BC axons, currents mediated
by GABAA receptors were recorded from a
population of BCs. It seems unlikely that these currents were induced
by GABA diffusion to the dendrites of these cells, thus activating
GABAA receptors on them. If it was the case,
these currents should have been smaller than those recorded from the
soma of the cell, a site that is closer to the dendrites. But we have
consistently found that the results were reversed (see Fig. 8).
Furthermore these currents were recorded whether or not the axon
terminals were intact, suggesting that they were clearly not produced
by activation of the GABAA receptors on the axon
terminals of these cells due to diffusion of GABA locally applied to
the axons. There are no data available concerning the existence of any
GABAA receptor subunits on BC axons, but subunits were recently found to be clearly clustered in synaptic hot
spots along BC axons in mammalian retinae (Enz et al.
1996
). These results suggest that BCs may receive GABAergic synaptic and/or extrasynaptic inputs along their axons. Since there is
no morphological evidence showing the existence of synapses on BC axons
(Calkins et al. 1998
), the GABA receptors found on BC
axons must be extrasynaptic ones, which can be activated by the buildup
of the bulk concentration of GABA in the retina following sustained
activities of GABAergic cells (horizontal and amacrine cells).
Modulation of BC signals mediated by the GABA receptors along the axons
may be significantly different from that exerted by GABAergic inputs to
the BC dendrites and axon terminals. It was reported in the CNS that
GABA receptors at different subcellular sites may exert distinct
physiological actions. For instance, in pyramidal neurons of
hippocampus (Miles et al. 1996
), the
GABAA receptor-mediated inputs at the soma
control the axonal outputs of the cells, whereas activation of the
dendritic GABAA receptors suppresses
calcium-dependent spikes originating from the dendrites. It sounds
reasonable to speculate that the GABAA receptors
along the BC axons may modulate signal conduction from the soma to the axon terminal. On the other hand, GABAergic inputs at the BC dendrites can strongly influence spatial and temporal integration of the glutamatergic postsynaptic potentials produced by photoreceptors (Du and Yang 1999a
), whereas GABAergic inputs at BC axon
terminals mainly modify transmitter release of BC (Dong and
Werblin 1998
; Hartveit 1999
; Pan and
Lipton 1995
)
Heterogeneity of GABAC receptors
Three types of characteristic subunits of
GABAC receptors 1,
2, and
3 have been
cloned in vertebrate retinas (for review, see Feigenspan and
Bormann 1998
). The multiplicity of
subunits and their
splice variants suggest the existence of heterogeneous GABAC receptors. Evidence for this are the
different PTX and/or I4AA sensitivities of native
GABAC receptors in various animals documented in
recent work. For instance, in fish, salamander, and ferret retinas,
PTX can block completely the GABAC
responses (Lukasiewicz 1996
; Lukasiewicz and Wong
1997
; Lukasiewicz et al. 1994
; Qian
and Dowling 1993
, 1994
), while GABAC
currents of rat BCs were resistant to PTX (Feigenspan and
Bormann 1993
). Moreover, I4AA has been demonstrated to be a
specific antagonist of GABAC receptors in a
variety of retinas (Han et al. 1997
; Kusama et al. 1993
; Picaud et al. 1998
; Qian and
Dowling 1994
, 1995
) but was found recently to be able to
activate GABAC receptors of BCs in the salamander
retina (Lukasiewicz and Shields 1998b
) and recombinant GABAC receptors of the white perch retina
(Qian et al. 1998
). In the present work, PTX could
entirely block both the dendritric and axon terminal
GABAC responses of the bullfrog BCs. For most of
the cell tested, I4AA acted as a potent antagonist of
GABAC receptors but had little effect on the
GABAC receptors at the axon terminals of a
minority of BCs, implying the existence of GABAC
receptor heterogeneity, even in a single bullfrog BC.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China, the National Foundation of Natural Science of China (Grant 39770256), Shanghai Commission of Science and Technology, and Shanghai Institutes of Life Sciences, Chinese Academy of Sciences.
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FOOTNOTES |
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Address for reprint requests: X.-L. Yang, Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue-Yang Rd., Shanghai 200031, China (E-mail: xlyang{at}sunm.shcnc.ac.cn).
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 7 December 1999; accepted in final form 2 April 2000.
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REFERENCES |
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