Relative contributions of rod and cone bipolar cell inputs to AII amacrine cell light responses in the mouse retina

Abstract

AII amacrine cells (AIIACs) are crucial relay stations for rod-mediated signals in the mammalian retina and they receive synaptic inputs from depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) as well as from other amacrine cells. Using whole-cell voltage-clamp technique in conjunction with pharmacological tools, we found that the light-evoked current response of AIIACs in the mouse retina is almost completely mediated by two DBC synaptic inputs: a 6,7-dinitro-quinoxaline-2,3-dione (DNQX)-resistant component mediated by cone DBCs (DBC(C)s) through an electrical synapse, and a DNQX-sensitive component mediated by rod DBCs (DBC(R)s). This scheme is supported by AIIAC current responses recorded from two knockout mice. The dynamic range of the AIIAC light response in the Bhlhb4-/- mouse (which lacks DBC(R)s) resembles that of the DNQX-resistant component, and that of the connexin36 (Cx36)-/- mouse resembles the DNQX-sensitive component. By comparing the light responses of the DBC(C)s with the DNQX-resistant AIIAC component, and light responses of the DBC(R)s with the DNQX-sensitive AIIAC component, we obtained the input-output relations of the DBC(C)-->AIIAC electrical synapse and the DBC(R)-->AIIAC chemical synapse. Similar to other glutamatergic chemical synapses in the retina, the DBC(R)-->AIIAC synapse is non-linear. Its highest voltage gain (approximately 5) is found near the dark membrane potential, and it saturates for presynaptic signals larger than 5.5 mV. The DBC(C)-->AIIAC electrical synapse is approximately linear (voltage gain of 0.92), consistent with the linear junctional conductance found in retinal electrical synapses. Moreover, relative DBC(R) and DBC(C) contributions to the AIIAC response at various light intensity levels are determined.

Figure 1. Effects of AP4 and DNQX on AII amacrine cells

A, an AII amacrine cell (AIIAC) in a retinal slice filled with Lucifer yellow. The smaller cell above the AIIAC (arrow) is a dye-coupled depolarizing cone bipolar cell (DBC_C) soma. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. B, light-evoked current responses the AIIAC to a −3 (700 R*rod⁻¹ s⁻¹) 2.5 s light step at various holding potentials. C, light-evoked current responses the AIIAC to the same light step (as in B) at various holding potentials in the presence of 20 μml-2-amino-4-phosphonobutyric acid (l-AP4). D, wash 1 shows that l-AP4 actions were reversible. E, light-evoked current responses the AIIAC to the same light step (as in B) at various holding potentials in the presence of 100 μm DNQX. F, wash 2 shows DNQX actions were reversible. G, current–voltage relations of the light-evoked responses under control conditions (○), in 20 μml-AP4 (•), and in 100 μm DNQX (▴).

Figure 2. DNQX-sensitive and DNQX-resistant components of AIIAC responses

Current responses of an AIIAC to 2.5 s light steps of increasing intensities (−7.5 to −3.5, or 0.022–221 R*rod⁻¹ s⁻¹) at holding potential −60 mV in control medium (A), and in 100 μm DNQX (B). C, the difference responses (control − DNQX) were obtained by subtracting responses in B from responses in A at each intensity. D, average (±s.d.n=8) response-intensity (ΔI − log I) of the current responses in control medium, in DNQX and control − DNQX. Curves were fitted by eqn (1) in Methods. The response thresholds and dynamic ranges of the control response (dash–dot curve) are −8.7 (0.0014 R*rod⁻¹ s⁻¹) and 3.97, respectively, for the DNQX-sensitive component (control − DNQX, continuous curve) they are −8.5 (0.0022 R*rod⁻¹ s⁻¹) and 2.56, and for the DNQX-resistant component (DNQX, dashed curve) they are −8.7 (0.0014 R*rod⁻¹ s⁻¹) and 4.27. E, ratio of the postsynaptic response of the depolarizing rod bipolar cell (DBC_R) inputs (control AIIAC response − AIIAC response in DNQX, continuous curve in D) and that of the DBC_C inputs (AIIAC response in DNQX, dashed curve in D) at various light intensities.

Figure 3. Immunostainings of mouse retinas with Cx36 and PKCa antibodies

Vertical retinal sections of WT (a), Cx36−/− (b) and Bhlhb4−/− (c) mice immunostained with antibodies against Cx36 (A) and protein kinase C (PKC) (B). OPL, outer plexiform layer; IPL, inner plexiform layer; and GCL, ganglion cell layer.

Figure 4. Electroretinograms of wt.Cx36−/− and Bhlhb4−/− mice

A, scotopic ERG b-wave responses to 500 nm light of increasing intensities (−6.3 to 2.6 log unit attenuation, or 0.35–1758 R*rod⁻¹ s⁻¹) recorded from WT (A), Cx36−/− (B) and Bhlhb4−/− (C) mice. B, average (±s.d.,n=8) response–intensity plots of the scotopic b-waves of the three types of mice over the intensity span from −7 to −2 (500 nm, 0.07–7000 R*rod⁻¹ s⁻¹). WT, black; Cx36−/−, red; Bhlhb4−/−, green; WT PII, blue (Saszik et al. 2002); and difference b-wave (WT − WT PII), purple.

Figure 5. Light-evoked currents of AIIACs in Cx36−/− and Bhlhb4−/− mice

Top, an AIIAC in a Cx36−/− retinal slice (Aa) and an AIIAC in a Bhlhb4−/− retinal slice (Ba) filled with Lucifer yellow. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Middle, light-evoked current responses at various holding potentials of the AIIAC in Cx36−/− mouse (Ab) and in Bhlhb4−/− mouse (Bb). Bottom, current–voltage relations of the AIIAC in Cx36−/− mouse (Ac) and in Bhlhb4−/− mouse (Bc).

Figure 6. AIIAC response-intensity relations in Cx36−/− and Bhlhb4−/− mice

Current responses of an AIIAC in the Bhlhb4−/− mouse retinal slice (A) and in the Cx36−/− mouse retinal slice (B) to 2.5 s light steps of increasing intensities (−7.5 to −3.5, or 0.022–221 R*rod⁻¹ s⁻¹) at holding potential −60 mV. The average (±s.d.) responses of nine Cx36−/− AIIACs (•, thick continuous curve (fitted by eqn (1)) normalized against the maximum average response amplitude of the DNQX-sensitive component of the WT AIIACs) with an average response threshold and dynamic range of −8.7 (0.0014 R*rod⁻¹ s⁻¹) and 2.87. The ΔI − log I of the DNQX-sensitive component of the WT AIIACs (from Fig. 2D) is shown as the thin dashed curve. The average (±s.d.) responses of eight Bhlhb4−/− AIIACs (♦, thick dashed curve (fitted by eqn (1)) normalized against the maximum average response amplitude of the DNQX-resistant component of the WT AIIACs) with an average response threshold and dynamic range of −8.5 (0.0022 R*rod⁻¹ s⁻¹) and 4.19. The ΔI − log I of the DNQX-resistant component of the WT AIIACs (from Fig. 2D) is shown as the thin dashed curve.

Figure 7. Input-out relations

A, current input–output relation of the DBC_R→AIIAC synapse (continuous curve) obtained by plotting the average DBC_R response versus the average DNQX-sensitive AIIAC response (continuous curve in Fig. 2D), and current input–output relation of the DBC_C→AIIAC synapse (dashed curve) obtained by plotting the average DBC_C responses against the average DNQX-resistant AIIAC responses (dashed curve in Fig. 2D). B, voltage input–output relation of the DBC_R→AIIAC synapse (dash–dot curve) and the DBC_C→AIIAC synapse (dash–double-dot curve). The voltage responses were estimated by multiplying the current responses by the average input resistance (R) of the DBC_Rs, DBC_Cs and AIIACs (R_DBCR, 1.1 GΩ; R_DBCC, 1.0 GΩ; and R_AIIAC, 0.4 GΩ).