Article Figures & Data
Figures
- Fig. 1.
GABAA receptors predominate on bipolar cell dendrites. A, Current responses evoked by puffing GABA (200 μm pipette concentration) onto bipolar cell dendrites. Horizontal bar above the current traces denotes the duration of the puff in this and all subsequent figures. The control current had a time-to-peak of 128 msec and a D37 of 226 msec. Bicuculline (200 μm) reduced the response charge transfer to 18% of control (Bic). 3-APMPA (300 μm) decreased the bicuculline-resistant component to 2% of control (Bic & 3A).B, Effects of bicuculline and 3-APMPA on a mixed population of bipolar cells. On average, bicuculline (Bic) reduced the charge transfer to 14 ± 2% (n = 15) of control. The bicuculline-resistant component was reduced to 2.3 ± 0.8% of control by the subsequent addition of 3-APMPA (Bic/3A;n = 12). Currents recovered to 82 ± 6% of control levels upon washout of antagonist (Wash;n = 15).
- Fig. 2.
Time course of GABAergic currents varies among distinct classes of bipolar cells. A, Line drawings from photomicrographs of cells included in the recorded rod (1, 2), ON cone (3,4), and OFF cone (5–7) bipolar cell populations. Scale bar, 20 μm. B, Currents evoked by puffing GABA (200 μm pipette concentration) onto the terminals of an OFF cone and a rod bipolar cell. The current recorded from the OFF cone bipolar had aD37 of 284 msec and a time-to-peak of 71 msec (thick trace, OFF). TheD37 and time-to-peak of the response measured in the rod bipolar were 1044 and 213 msec, respectively (thin trace, Rod). C, Average D37 and time-to-peak data for populations of rod, ON cone, and OFF cone bipolar cells. The averageD37 values (white bars) were 808 ± 92 msec (n = 18) for rod (Rod), 638 ± 77 msec (n = 18) for ON cone (ON), and 434 ± 44 msec (n = 18) for OFF cone (OFF) bipolar cells. There was a significant difference in the averageD37 between OFF and ON cone bipolars (p = 0.01) and between OFF and rod bipolars (p < 0.001), but not between ON cone and rod bipolar cells (p = 0.08). The average time-to-peak values (black bars) were 168 ± 20 msec (n = 18; Rod), 202 ± 25 msec (n = 18; ON), and 150 ± 20 msec (n = 18;OFF). No significant differences in the time-to-peak were found between classes of bipolar cells.
- Fig. 3.
GABAA and GABACreceptor-mediated currents have distinct time courses.A, Current responses evoked by puffing GABA (200 μm pipette concentration) onto the axon terminals of a single rod bipolar cell in the presence of either bicuculline (200 μm) (thin trace,GABAC ) or 3-APMPA (300 μm) (thick trace,GABAA ). For comparison, thetrace marked GABAA has been scaled to the amplitude of the trace markedGABAC . Vertical calibration bar: GABAC, 50 pA; GABAA, 27 pA. In the presence of 3-APMPA, the GABAA receptor-mediated current had aD37 of 112 msec and a time-to-peak of 58 msec. The GABAC component, recorded in the presence of bicuculline, had a D37 of 1037 msec and a time-to-peak of 170 msec. B, C, Time course data for currents evoked at the dendrites or axon terminals in a mixed population of bipolar cells. B, The averageD37 of currents evoked at the dendrites (black bar) was 244 ± 24 msec (n = 16). For responses evoked at the terminals (white bars), the average D37of currents mediated by GABAA receptors (labeledA) was 225 ± 20 msec (n = 36) and by GABAC receptors (labeled C) was 677 ± 66 msec (n = 40). The averageD37 of GABAA receptors on the axon terminals was significantly different from GABACreceptors on the terminals (p < 0.001), but not from GABA receptors on the dendrites (p= 0.3). C, Currents evoked at the dendrites (black bar) reached peak amplitude in an average of 100 ± 13 msec (n = 16). For currents evoked at the axon terminals (white bars), the average time-to-peak of the GABAA component (labeledA) was 120 ± 12 msec (n = 36) and of the GABAC component (labeled C) was 192 ± 15 msec (n = 40). The average time-to-peak of GABAA receptors on the terminals was significantly different from GABAC receptors on the terminals (p < 0.001), but not from dendritic GABA receptors (p = 0.1).
- Fig. 4.
Effects of bicuculline and 3-APMPA on currents evoked in three morphologically distinct bipolar cells. Thin traces (Control in A) are currents elicited by puffing GABA (200 μm pipette concentration) onto the axon terminals in the absence of GABAergic antagonists. A–C, Compared with control levels, bicuculline (200 μm) (left column,thick traces, Bic) reduced the charge transfer to 82% in a rod (A), 53% in an ON cone (B), and 33% in an OFF cone bipolar cell (C). Relative to control, 3-APMPA (300 μm) (right column, thick traces, 3-A) decreased the charge transfer to 11% in the rod (A), 34% in the ON cone (B), and 57% in the OFF cone bipolar cell (C). The combination of bicuculline and 3-APMPA abolished responses in each of the three bipolar cells (0–6% of control levels; data not shown).
- Fig. 5.
Differential effects of GABAergic antagonists on currents evoked in distinct classes of bipolar cells. A, Effects of bicuculline on the charge transfer of currents evoked by puffing GABA (200 μm pipette concentration) onto the axon terminals. Relative to control, bicuculline (200 μm) reduced the charge transfer to 83 ± 4% (n = 12) in rod (Rod), 71 ± 4% (n = 11) in ON cone (ON), and 54 ± 6% (n = 15) in OFF cone (OFF) bipolar cells. The percent blockade by bicuculline was significantly different between rod and ON cone (p = 0.03), rod and OFF cone (p < 0.001), and ON cone and OFF cone (p = 0.01) bipolar cell populations. B, Relative to control levels, 3-APMPA (300 μm) reduced the charge transfer to 15 ± 3% (n = 9) in rod (Rod), 37 ± 6% (n = 10) in ON cone (ON), and 65 ± 6% (n = 12) in OFF cone (OFF) bipolar cells. The reduction by 3-APMPA was significantly different between populations of rod and ON (p = 0.005), rod and OFF (p < 0.001), and ON and OFF bipolar cells (p = 0.003). In all cells, the combination of bicuculline and 3-APMPA abolished GABA-evoked currents (2.4 ± 0.4% of control levels; n = 50; data not shown).
- Fig. 6.
GABAA and GABAC receptors mediate GABAergic IPSCs. Current responses evoked in an ON cone bipolar cell by puffing kainate (500 μm) in the IPL. Bicuculline (200 μm) decreased the response charge transfer to 31% (Bic), and 3-APMPA (300 μm) reduced the bicuculline-resistant component to 4% of control (Bic & 3A). Upon washout of antagonist, the IPSC charge transfer recovered to 75% of control.
- Fig. 7.
Confocal images of GABAA(A) and GABAC(B) receptor immunostaining in the adult ferret retina. Arrows in A point to two putative bipolar cells that showed dendritic labeling. C, Control section for GABAC staining in which the primary antibody was omitted. Images in B and C were acquired with the same objective (40×) and at the same laser intensity, gain, and black level.
- Fig. 8.
Top. Confocal images (eachpanel represents the maximum intensity projection of a stack of 10–15 images) demonstrating patterns of immunostaining for bipolar cells, GABAA, and GABACreceptors in the adult ferret retina. GABAC receptor immunolabeling is encoded in red, whereas GABAA receptors are in shown in blue. PKC (A, B), recoverin (C,D), and calbindin (E) immunolabeling for bipolar cells are shown in green.
- Fig. 9.
Bottom. High magnification of the maximum intensity projections of short z-stacks of 10–15 images of the IPL showing colabeling of GABAC receptors (red) with bipolar cell axon terminals (green). Bipolar cell terminals were labeled for PKC (A), recoverin (C), and calbindin (E). Silhouettes of the labeled bipolar terminals in A, C, and E are shown in B, D, and F, respectively. Colocalization with GABAC staining is represented by the red profiles, determined after 3-D rotation of the image stacks.
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