Multiple pathways of inhibition shape bipolar cell responses in the retina

Abstract

Bipolar cells (BCs) are critical relay neurons in the retina that are organized into parallel signaling pathways. The three main signaling pathways in the mammalian retina are the rod, ON cone, and OFF cone BCs. Rod BCs mediate incrementing dim light signals from rods, and ON cone and OFF cone BCs mediate incrementing and decrementing brighter light signals from cones, respectively. The outputs of BCs are shaped by inhibitory inputs from GABAergic and glycinergic amacrine cells in the inner plexiform layer, mediated by three distinct types of inhibitory receptors: GABA(A), GABA(C), and glycine receptors. The three main BC pathways receive distinct forms of inhibition from these three receptors that shape their light-evoked inhibitory signals. Rod BC inhibition is dominated by slow GABA(C) receptor inhibition, while OFF cone BCs are dominated by glycinergic inhibition. The inhibitory inputs to BCs are also shaped by serial inhibitory connections between GABAergic amacrine cells that limit the spatial profile of BC inhibition. We discuss our recent studies on how inhibitory inputs to BCs are shaped by receptor expression, receptor properties, and neurotransmitter release properties and how these affect the output of BCs.

Fig. 1

Inhibition to retinal BCs. A cartoon of the retina is shown with the rod (R) and cone (C) photoreceptors in the outer nuclear layer (ONL) that make connections with the BCs in the outer plexiform layer (OPL). BCs and amacrine cells are located in the inner nuclear layer (INL) and make contacts with the ganglion cells (GCL) in the inner plexiform layer (IPL). BCs receive inhibitory inputs from glycinergic (Gly, white) and GABAergic (GAB, dark gray) amacrine cells that have distinct spatial extents in the retina. Rod and OFF cone BCs (OFF) receive inhibitory inputs onto GABA_A, GABA_C, and glycine receptors (R), while ON cone BCs (ON) receive inputs onto GABA_A and GABA_CRs, all of which is direct inhibition. Glutamatergic inputs to BCs are mediated by two distinct types of glutamate receptors, AMPA/kainate receptors in the OFF pathway (A/K R) and mGluR6s in the ON pathway, including rod and ON cone BCs. GABAergic amacrine cells also receive inhibitory inputs from other GABAergic amacrine cells that are mediated by GABA_ARs (serial inhibition). The illustrated pathway shows inhibition onto BCs, but the cone BCs receive similar inhibition (see text).

Fig. 2

The contributions of GABA_A, GABA_C, and glycineR currents vary across BC class. (A) GABA-evoked currents were measured in BCs by focally applying GABA to the BC axon terminal (inset). GABA_AR- or GABA_CR-mediated currents were isolated and measured using (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) (50 μM) or bicuculline (50 μM), respectively in: rod (A1), ON cone (A2), and OFF cone (A3) BCs. (The dark gray bar below each trace indicates the duration of the GABA puff.) (A4) Fractional GABA-evoked current mediated by GABA_AR and GABA_CRs in BC types was calculated by normalizing GABA_AR and GABA_CR charge transfer (Q) to total Q. In rod and ON cone BCs, GABA_CRs (black bars) mediated significantly more of the total response (Q) than GABA_ARs (gray bars; P < 0.001 and 0.005, respectively). In OFF cone BCs, the proportion of GABA_AR and GABA_CR contributions was similar (P = 0.3). The error bars represent the s.e.m.(B) L-IPSCs were recorded from BCs voltage clamped to 0 mV, the reversal potential for excitatory currents mediated by nonselective cation channels, and elicited with a 30-ms full-field stimulus (dark gray bar), as shown in the inset. (B1) Rod BCs have large L-IPSCs mediated by GABA_CRs (in strychnine 500 nM and bicuculline 50 μM) and modest L-IPSCs mediated by glycine (in TPMPA, 50 μM, and bicuculline,50 μM) and GABA_ARs (in TPMPA 50 μM and bicuculline 50 μM). (B2) ON BCs have moderate GABA_CR and GABA_AR L-IPSCs and no glycinergic currents. (B3) OFF cone BCs have large glycinergic L-IPSCs and smaller GABA_AR and GABA_CRL-IPSCs. (B4) The average Q of GABA_AR, GABA_CR, and glycineR L-IPSCs was normalized to the Q of GABA_ARL-IPSCs for each BC type. Rod BCs have proportionately the largest GABA_CR L-IPSCs, and OFF cone BCs have the largest glycinergic L-IPSCs. Portions of this figure were adapted from Eggers et al. (2007), Journal of Physiology.

Fig. 3

GABA_CR, GABA_AR, and glycineRs mediate L-IPSCs with distinct time courses in all BC classes. L-IPSCs were recorded from BCs voltage clamped to 0 mV, the reversal potential for excitatory currents mediated by nonselective cation channels, and elicited with a 30-ms full-field stimulus (dark gray bar). L-IPSCs were normalized to the peak of the response to illustrate kinetic differences. Receptors were isolated with combinations of antagonists, as in Fig. 2. (A) In rod BCs, GABA_CR L-IPSCs have a slower decay time and rise time than GABA_AR or glycineR L-IPSCs. However, the decay time of glycineR L-IPSCs is slower than GABA_AR L-IPSCs. (B) In ON cone BCs, GABA_AR and GABA_CR L-IPSCs show similar kinetics as in rod BCs. (C) OFF cone BC L-IPSCs show similar kinetics differences as ON cone and rod BCs.

Fig. 4

Spontaneous IPSCs (sIPCs) indicate that GABA_AR, GABA_CR, and glycineRs show distinct biophysical properties. Shown are average sIPSCs from rod, ON cone, and OFF cone BCs (inset). (A) Rod BCs show spontaneous currents mediated by GABA_AR, GABA_CR, and glycineRs, with distinct decay times. (B) ON cone BCs show sIPSCs mediated by only GABA_AR and GABA_CRs, with no currents mediated by glycineRs. GABA_CR currents have a significantly longer decay time than GABA_AR currents. (C) OFF cone BCs show sIPSCs mediated by GABA_AR, GABA_CR, and glycineRs, with distinct decay times. The amplitude of glycineR sIPSCs is significantly larger than other sIPSCs consistent with the primary role of glycinergic inhibition in OFF cone BCs.

Fig. 5

GABA_AR-, GABA_CR-, and glycineR-mediated L-IPSCs have distinct apparent release functions and receptor kinetics, both of which contribute to L-IPSC kinetics. (A) Release functions computed by deconvolving idealized GABA_AR- and GABA_CR-mediated L-IPSCs. GABA_AR-mediated L-IPSCs have a much larger release function than GABA_CRs, likely because of the much smaller Q of GABA_AR sIPSCs versus GABA_CR sIPSCs. The GABA_CR release function has a prolonged tail not shown by the GABA_AR release function. Scale bars are 0.05 quanta/ms and 200 ms. (B) The release functions calculated from the deconvolution of the L-IPSCs and sIPSC traces for GABA_AR and glycineRs are shown. The glycine release function has much slower decay time than the GABA_AR release function. This suggests that part of the differences between GABA_AR- and glycineR-mediated L-IPSCs are due to distinct release kinetics. This figure was adapted from Eggers and Lukasiewicz (2006b), Journal of Neuroscience.

Fig. 6

GABA_CRs shape L-IPSCs in ON cone and rod BCs but not OFF cone BCs. (A–C) Representative L-IPSCs (30-ms light stimulus duration, gray bar) from rod (A), ON cone (B), and OFF cone (C) BCs in WT (black) and GABA_CR null (gray) mice. (D) The histogram plots the average L-IPSCs decay (D₃₇) from GABA_CR null BCs normalized to WT. GABA_CR null L-IPSCs in rod BCs (WT n = 43, null n = 15, P < 0.0001) and ON cone BCs (WT n = 17, null n = 4, P < 0.05) were significantly briefer than WT. There was no significant difference in L-IPSC decays from GABA_CR null and WT OFF cone BCs (WT n = 13, null n = 6, P = 0.7). Error bars in (D) represent propagated s.e.s of the average null to WT values. Adapted from Eggers et al. (2007), Journal of Physiology.

Fig. 7

The decay of combined glycinergic and GABAergic L-IPSCs varies with WT BC class. (A–C) Representative total L-IPSCs (glycinergic + GABAergic) from rod (A), ON cone (B), and OFF cone (C) BCs evoked by a light stimulus (30-ms light stimulus, dark gray bar). (D) The histogram plots the average decay (D₃₇) from each BC class. L-IPSCs from rod BCs were significantly slower than ON cone BCs (rod 5 43, ON cone n = 17, ANOVA P < 0.001, rod vs. ON Scheffe post hoc P < 0.001, *) but similar to OFF cone BCs (n = 13, P = 0.86). The decay of L-IPSCs from OFF cone BCs also was significantly slower than ON cone BCs (P < 0.05, **). Adapted from Eggers et al. (2007), Journal of Physiology.

Fig. 8

Presynaptic GABA_CR limit release, making L-EPSCs from A17 amacrine cells more transient. Glutamate release from rod BCs was monitored by recording L-EPSCs from postsynaptic A17 ACs. (A) The absence of GABA_CRs in GABA_CR null mice causes the L-EPSC to have a longer decay and larger charge transfer. A similar effect was observed in WT mice when TPMPA was added to block GABA_CRs. The decay (D₃₇) of A17 amacrine cells from GABA_CR null mice (P < 0.05) and WT mice in TPMPA (P < 0.01) was significantly longer than WT mice in control conditions. (B) Presynaptic glycine and GABA_ARs decrease the peak response of L-EPSCs from A17 amacrine cells. The peak amplitude of L-EPSCs from A17 amacrine cells from GABA_CR null mice was increased by the addition of strychnine to block glycineRs, but the decay time of the response was unaffected. Similarly, the peak amplitude of L-EPSCs from A17 amacrine cells from GABA_CR null mice was increased by addition of bicuculline to block GABA_ARs, but the response decay was unaffected. The amplitude of L-EPSCs was significantly larger with the addition of both strychnine (P < 0.05, *) and bicuculline (P < 0.05, *). Peak values in bicuculline and strychnine are normalized to control values, represented by the dotted line.

Fig. 9

(A) Serial inhibitory connections limit wide-field but not narrow-field BC L-IPSCs. (A) Wide-field (825 μm, A2) and narrow-field (25 μm, A2) light stimuli (A1, A2, and thick dark gray bar in traces) were applied to BCs. Bicuculline (50 μM) was added to block GABA_ARs. (B) In all BC types (OFF cone BC shown), blocking GABA_ARs increased the charge transfer (Q) of wide-field L-IPSCs (B1), suggesting that serial inhibitory connections between ACs limit wide-field light activated L-IPSCs (rod P < 0.05, ON P < 0.05, OFF P < 0.05). In contrast, blocking GABA_ARs decreased the Q of narrow-field L-IPSCs (B2), suggesting that narrow-field light activated only direct connections (rod n = 10, P < 0.001; ON n = 6, P < 0.01; OFF n = 5, P < 0.05). (C) The spatial responses of BC L-IPSCs are suppressed at large light stimulus sizes in control conditions but not when serial connections are blocked by bicuculline. Light stimuli of 10 different sizes (25–825 μm) were applied to BCs in control and bicuculline, and the Q of each L-IPSC was measured. The average area response function (ARFs) of all BCs recorded were normalized to the maximum light response for each BC and to the light size where the maximum response was elicited. ARFs showed a peak at an intermediate-sized light stimuli for all BC types in control (C1, rod n = 19, ON n = 14, OFF n = 9). When GABA_AR-mediated serial connections are blocked, BC L-IPSCs show no suppression. The average ARFs of all BCs recorded in bicuculline were calculated (C2) and showed an increasing light response with increasing light stimulus size (rod n = 11, ON n = 5, OFF n = 5). This suggests that the spatial tuning of BC L-IPSCs seen in control conditions is limited by serial connections. Adapted from Eggers and Lukasiewicz (2010), Journal of Neurophysiology.

Fig. 10

GABA_AR, GABA_CR, and glycineRs control distinct properties of glutamate release from BCs. (A) GABA_AR and glycineRs mediated light-evoked currents with a fast rise and decay, which primarily limit the peak of glutamate release from BCs. (B) GABA_CRs mediated light-evoked inhibition with slow decay, which primarily limits prolonged glutamate release from BCs. The time course of light-evoked BC inhibition is controlled both by biophysical receptor properties as well as prolonged GABA and glycine release from amacrine cells. (C) The magnitude of inhibition varies between BC pathways. Rod BCs receive large GABA_CR-mediated inhibition and less GABA_AR and glycineR inhibition. ON cone BCs receive moderate amounts of GABA_AR and GABA_CR inhibition with no glycinergic inhibition. OFF cone BCs receive little GABA_CR inhibition, some GABA_AR inhibition, and are dominated by glycinergic inhibition. The magnitude of GABAergic inhibition is also controlled by GABA_AR-mediated synapses between GABAergic amacrine cells that serve to limit inhibition to BCs.