1. Chase Hellmer1 ; Bozena E. Fyk-Kolodziej1 ; Tomomi Ichinose1,2
Intracellular Ca2+ Shapes mGluR6 Signaling in a Subset of Rod and Cone Bipolar Cells in the Mouse Retina
A0075
Chase Hellmer1 ; Bozena E. Fyk-Kolodziej1 ; Tomomi Ichinose1,2
Departments of 1. Anatomy & Cell Biology and 2.Ophthalmology, Wayne State University School of Medicine, Detroit, MI
INTRODUCTION
RESULTS
Representative CPPG Puff Response of ONBCs
Rise Times of ONBC CPPG Puff Response
Retinal bipolar cells (BCs) encode various aspects of visual signaling separately among different subtypes. BCs transmit visual signals primarily by transient and/or sustained graded potentials, although how they shape these signals is not well understood. Previous studies show that Ca2+ influx desensitizes visual signals in rod BCs (RBCs). We investigated whether desensitization shapes mGluR6 signaling in a BC subtype dependent manner in the mouse retina. 3 6
METHODS
Whole-cell recordings were made from RBCs and ON-responding cone bipolar cells (ONBCs) of mouse retinal slices. CPPG (mGluR6 antagonist) puff-evoked responses were recorded either in voltage-clamp or current-clamp mode. L-AP4 (mGluR6 agonist) was continuously perfused to mimic dark conditions for ONBCs. EGTA (0.5 mM, weak Ca2+ chelator) or BAPTA (10 mM, strong Ca2+ chelator) was included in the pipette solution. Network effects were blocked with GABA and glycine receptor antagonists. Morphological subtypes were identified by including sulforhodamine B and neurobiotin in the pipette along with ChAT staining by immunolabeling. The peak amplitude and rise time were measured and a transient-to-sustained ratio was also calculated. 1
Figure 6 Intracellular EGTA or BAPTA did not affect the rise times for ONBCs. T5 cells appeared to respond quicker in voltage clamp with intracellular BAPTA respond than cells with intracellular EGTA (p=0.07). It is likely that non-responding T5 cells (see fig 5a) which were not seen in BAPTA recordings were included here.
Figure 3 CPPG evoked responses of ONBC subtypes. A group of Type 5 BCs with intracellular EGTA responded minimally to CPPG stimulation. All other ONBC subtypes responded with sustained responses to CPPG puff stimulation in all conditions.
Figure 1 A whole cell patch clamp configuration was made on BC somas. Subsequently, CPPG was applied to the BC dendrites and cell responses were recorded.
Transient/Sustained Ratio of CPPG Puff Responses 4
CONCLUSION
mGluR6 evoked currents are desensitized by intracellular Ca2+ in a subset of RBCs. Intracellular Ca2+ speeds up the rise time kinetics for this subset of RBC mGluR6 evoked currents.
There appear then to be at least two distinct physiological subtypes of RBCs based on voltage clamp recording. They are not morphologically distinguishable.
Voltage-gated channels mask the effects of intracellular Ca2+ on mGluR6 signaling.
Intracellular Ca2+ does not affect the rise time nor cause response desensitization of ONBCs as well as one subtype of RBC.
mGluR6 response amplitudes are suppressed by intracellular Ca2+ in a subset of T5 BCs and XBCs.
Intracellular Ca2+ desensitizes mGluR6-evoked responses in a subset of RBCs as well as T5 and XBC ONBCs.
RESULTS
CPPG Response Yields Two Physiological RBC Types B 2A
Figure 4 Ratios of plateau amplitude to peak amplitude were used to determine Ca2+ desensitization. BAPTA increased the T/S ratio only for RBCs (p=0.002). RBCs under current clamp mode showed sustained responses with either EGTA or BAPTA present (p=0.25). BAPTA appeared to increase the T/S ratio for T7 (p=0.058). XBC, T5, T6, and T7 then all responded in a sustained fashion with either EGTA or BAPTA. C
Amplitude of ONBC CPPG Puff Response
Figure 5 XBC amplitude appeared to increase with the addition of BAPTA (p=0.08). In addition, a subset of XBC cells with EGTA produced only minimal responses to CPPG stimulation in voltage or current-clamp mode. Similarly, a subgroup of T5 BCs with EGTA also produced minimal responses in both voltage and current clamp modes (N=4, N=4). 5
Figure 2 A. CPPG evoked responses of RBC subtypes. In voltage clamp mode with EGTA, two physiological types of RBCs were seen. One type responded transiently to CPPG stimulation while another showed a sustained response (N=5, N=4 respectively). The two types were not morphologically distinguishable. B. Based on rise time, two types of RBC were observed in voltage clamp mode. This occurred in the presence of either EGTA or BAPTA (p=0.02, p=0.001 respectively). There was no significant difference in rise times among voltage responses (p=0.24). C. For the EGTA RBC current responses, the fast rise times correlated with transient type RBCs and slow rise times correlated with sustained type RBCs.
ACKNOWLEDGEMENT
This work was supported by NIH R01 EY020533, WSU Startup Fund, RPB, and Midwest Eye Bank grants.

2. Chase Hellmer1 ; Bozena E. Fyk-Kolodziej1 ; Tomomi Ichinose1,2
Intracellular Ca2+ Shapes mGluR6 Signaling in a Subset of Rod and Cone Bipolar Cells in the Mouse Retina
Chase Hellmer1 ; Bozena E. Fyk-Kolodziej1 ; Tomomi Ichinose1,2
Departments of 1. Anatomy & Cell Biology and 2.Ophthalmology, Wayne State University School of Medicine, Detroit, MI
INTRODUCTION
RESULTS
Representative CPPG Puff Response of Mouse DBCs
Rise Times of ONBC CPPG Puff Response
Retinal bipolar cells (BCs) encode various aspects of visual signaling separately among different subtypes. BCs transmit visual signals primarily by transient and/or sustained graded potentials, although how they shape these signals is not well understood. Previous studies show that Ca2+ influx desensitizes visual signals in some depolarizing BCs (DBCs). We investigated whether desensitization shapes mGluR6 signaling in a BC subtype dependent manner in the mouse retina.
METHODS
Whole-cell recordings were made from DBCs of mouse retinal slices. CPPG (mGluR6 antagonist) puff evoked responses were recorded either in voltage-clamp or current-clamp mode. L-AP4 (mGluR6 agonist) was continuously perfused to mimic dark conditions for ONBCs. EGTA (0.5 mM, weak Ca2+ chelator) or BAPTA (10 mM, strong Ca2+ chelator) was included in the pipette solution. Network effects were blocked with GABA and glycine receptor antagonists. Morphological subtypes were identified by including sulforhodamine B and neurobiotin in the pipette along with ChAT staining by immunolabeling. The peak amplitude, charge transfer, and rising time were measured and a transient-to-sustained ratio was calculated.
Figure 3 EPSCs and EPSPs of DBC subtypes. A group of Type 5 BCs with intracellular EGTA did not respond to stimulation. In voltage clamp conditions with EGTA, two physiological types of RBC were seen. One type responded transiently to CPPG stimulation (represented above) while another type showed a sustained response (Not shown. N=5, N=4 respectively).
Figure 6 The addition of either EGTA or BAPTA did not affect the rise times for mouse CBCs. XBC appears to have responded quicker in current clamp than voltage clamp but the difference is not significant (p=0.19). Similarly, T5 cells appear to respond quicker in voltage clamp but the difference is also not significant (p=0.07). It is likely that non-responding T5 cells (see fig 1a) which were not seen in BAPTA recordings were included here.
T/S Ratio of CPPG Puff Responses for DBC Subtypes
CONCLUSION
Figure 1 A whole cell patch clamp configuration was made on BC somas. Subsequently, CPPG was applied to the BC dendrites and EPSCs or EPSPs were recorded.
mGluR6 evoked currents are desensitized by intracellular Ca2+ in a subset of RBCs. Voltage gated channels mask this effect.
Intracellular Ca2+ affects the rise time kinetics of a subset of RBC mGluR6 evoked currents. Voltage gated channels also mask this effect.
Contrary to established retina literature, there appear to be at least two distinct physiological subtypes of RBC based on voltage clamp recording. They are not morphologically distinguishable.
Distinct by T/S ratio as well as rise time kinetics.
Intracellular Ca2+ does not affect the rise time of DBCs.
mGluR6 response amplitudes are suppressed by intracellular Ca2+ in a subset of T5 BCs.
Intracellular Ca2+ does not appear to affect temporal processing of stimuli responses in T6 and T7 ONBCs as well as a subset of T5 ONBCs and RBCs.
Amplitude of DBC CPPG Puff Response
Figure 4 Ratios of plateau amplitude to peak amplitude response for CPPG stimulation were used to determine calcium desensitization. BAPTA increased the T/S ratio only for RBCs (p=0.002). RBCs under current clamp conditions showed sustained responses with both EGTA and BAPTA present (p=0.25). BAPTA appeared to increase the T/S ratio for T7 as well although this was not determined to be significant (p=0.058). XBC, T5, T6, and T7 then all responded in a sustained fashion with both EGTA and BAPTA.
Rise Time of RBC CPPG Puff Response
ACKNOWLEDGEMENT
This work was supported by NIH R01 EY020533, WSU Startup Fund, RPB, and Midwest Eye Bank grants.
Figure 2 XBC amplitude appeared to increase with the addition of BAPTA but this difference was not found to be significant (p=0.08). In addition, 1/4 XBC cells with EGTA did not respond in voltage clamp, while in current clamp, 2/4 XBC cells did not respond to CPPG stimulation. Moreover, a subgroup of T5 BCs did not respond at all to stimulation with EGTA in both voltage and current clamp modes (N=4, N=4).
Figure 5 In the presence of both EGTA and BAPTA, RBC current response rise times provided for both a rapid and slow group (p=0.02, p=0.001 respectively). Moreover, for the EGTA RBC current responses, the fast rise times correlated with transient type RBCs and slow rise times correlated with sustained type RBCs. There was no significant difference in rise times among voltage responses (p=0.24).

4. Bipolar Cell Subtype ChAT / Neurobiotin / Synaptotagmin2 Voltage Clamp
XBC
T -RBC
S -RBC
Type 5
Type 5
Type 6
Type 7
Voltage Clamp
20 pA
EGTA 1s
BAPTA
Current Clamp
EGTA
BAPTA
15 mV 1s
Syt2
IPL
ChAT / Neurobiotin / Synaptotagmin2
ChAT / Neurobiotin

5. For first two columns, p = 0.02 (N=5 and 4)
For second two columns, p = (N=3 and 3)

8. P=0.08 for XBC Current Response
NS technically but interesting…

9. Conclusion
mGluR6 evoked currents are desensitized by intracellular Ca2+ in a subset of RBCs. Voltage gated channels mask this effect.
Intracellular Ca2+ affects the rise time kinetics of a subset of RBC mGluR6 evoked currents. Voltage gated channels also mask this effect.
Intracellular Ca2+ does not affect the rise time of DBCs.
Contrary to established retinal dogma, there appear to be at least two distinct physiological subtypes of RBC based on voltage clamp recording.
Distinct by T/S ratio as well as rise time kinetics.
mGluR6 response amplitudes are suppressed by intracellular Ca2+ in a subset of T5 BCs as well as some XBC BCs
Intracellular Ca2+ does not appear to affect temporal processing of stimuli responses in T6 and T7 ONBCs as well as a subset of T5 ONBCs and RBCs.