Receptor and transmitter release properties set the time course of retinal inhibition

Abstract

Synaptic inhibition is determined by the properties of postsynaptic receptors, neurotransmitter release, and clearance, but little is known about how these factors shape sensation-evoked inhibition. The retina is an ideal system to investigate inhibition because it can be activated physiologically with light, and separate inhibitory pathways can be assayed by recording from rod bipolar cells that possess distinct glycine, GABA(A), and GABA(C) receptors (R). We show that receptor properties differentially shape spontaneous IPSCs, whereas both transmitter release and receptor properties shape light-evoked (L) IPSCs. GABA(C)R-mediated IPSCs decayed the slowest, whereas glycineR- and GABA(A)R-mediated IPSCs decayed more rapidly. Slow GABA(C)Rs determined the L-IPSC decay, whereas GABA(A)Rs and glycineRs, which mediated rapid onset responses, determined the start of the L-IPSC. Both fast and slow inhibitory inputs distinctly shaped the output of rod bipolar cells. The slow GABA(C)Rs truncated glutamate release, making the A17 amacrine cell L-EPSCs more transient, whereas the fast GABA(A)R and glycineRs reduced the initial phase of glutamate release, limiting the peak amplitude of the L-EPSC. Estimates of transmitter release time courses suggested that glycine release was more prolonged than GABA release. The time course of GABA release activating GABA(C)Rs was slower than that activating GABA(A)Rs, consistent with spillover activation of GABA(C)Rs. Thus, both postsynaptic receptor and transmitter release properties shape light-evoked inhibition in retina.

Figure 1.

GABA_C receptors prolong rod bipolar cell L-IPSCs. A, L-IPSCs evoked by brief light stimuli (30 ms; thick dark gray bar) were smaller and decayed faster in GABA_CR null mice (gray trace) than in WT mice (black trace). Calibration: 5 pA, 200 ms. B, Blocking GABA_C receptors with TPMPA decreased and shortened L-IPSCs (gray trace) compared with control conditions (black trace). C, The Q in GABA_CR null mice was significantly smaller than in WT mice (*p < 0.05). The Q in rod bipolar cells after TPMPA application was significantly smaller than in control retinas (*p < 0.05). D, The L-IPSC decay (D₃₇) in GABA_CR mice was significantly briefer than for WT mice (*p < 0.001). Similarly, the L-IPSC decay measured in the presence of TPMPA also was briefer than that measured in control conditions (*p < 0.05).

Figure 2.

Glycine, GABA_A, and GABA_C receptors mediated L-IPSCs, with different properties. A1 and A2 show pharmacologically isolated and peak-scaled L-IPSCs evoked by a 30 ms light stimulus (dark gray bar at bottom of trace) mediated by GABA_A, GABA_C, and glycine receptors. GABA_C receptors mediated L-IPSCs with a slower decay time than those mediated by GABA_A or glycine receptors. Calibration, 200 ms. B1, The D₃₇ was measured from L-IPSCs mediated by isolated glycine (black), GABA_A (white), and GABA_C (gray) receptors. GABA_A L-IPSCs had a significantly briefer D₃₇ than either glycine receptors or GABA_C receptors; *p < 0.01. Glycine receptor-mediated L-IPSCs were on average briefer than GABA_C receptor-mediated L-IPSCs (p < 0.08). B2, GABA_C receptor-mediated L-IPSCs had a longer time-to-peak than either glycine or GABA_A receptor-mediated L-IPSCs; *p < 0.01. There was no significant difference in time-to-peak between GABA_A and glycine L-IPSCs (p = 0.8). C1, GABA_C receptor-mediated L-IPSCs had significantly larger Q than either GABA_A or glycine L-IPSCs (p < 0.01). There was no significant difference in Q between GABA_A and glycine L-IPSCs (p = 0.3). C2, GABA_A receptor-mediated L-IPSCs on average had a smaller peak value than GABA_C and glycine L-IPSCs (p < 0.1).

Figure 3.

Presynaptic GABA_C receptors make L-EPSCs from A17 amacrine cells more transient. A, Shown are L-EPSCs (30 ms light stimulus; dark gray bay) from A17 amacrine cells from WT and GABA_CR null mice. The absence of GABA_C receptors in GABA_CR null mice causes the L-EPSC to have a longer decay and larger charge transfer. B, A similar effect was observed in WT mice when TPMPA was added to block GABA_C receptors. C, 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.

Figure 4.

Presynaptic glycine and GABA_A receptors decrease the peak response of L-EPSCs from A17 amacrine cells. A, The peak amplitude of L-EPSCs (30 ms light stimulus; dark gray bar) from A17 amacrine cells from GABA_CR null mice was increased by the addition of strychnine to block glycine receptors, but the decay time of the response was unaffected. B, 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_A receptors, but the response decay was unaffected. C, 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.

Figure 5.

Glycine, GABA_A, and GABA_C receptors mediate sIPSCs with distinct kinetics in rod bipolar cells. A, Examples of isolated glycine receptor sIPSCs that were measured in the presence of bicuculline and TPMPA (left). The τ_decay histogram distribution (normalized to the total number of events) for all glycine receptor-mediated sIPSCs recorded are shown in the right, and the inset shows the average glycine receptor-mediated sIPSC. Calibration:2 pA, 5 ms. B, Examples of isolated GABA_A receptor sIPSCs that were measured in the presence of strychnine and TPMPA (left). The normalized τ_decay histogram distribution for all GABA_A receptor-mediated sIPSCs recorded are shown in the right, and the inset shows the average GABA_A receptor-mediated sIPSC. Calibration: 2 pA, 5 ms. GABA_A receptor-mediated sIPSCs had a significantly shorter τ_decay than glycine receptor-mediated sIPSCs (K–S, p < 0.0001). C, Examples of isolated GABA_C receptor sIPSCs, measured in the presence of kainate, strychnine, and bicuculline (left). The normalized τ_decay histogram distribution for all GABA_C receptor-mediated sIPSCs recorded is shown in the right, and the inset shows the average GABA_C receptor-mediated sIPSC. Calibration: 2 pA, 50 ms. GABA_C receptor-mediated sIPSCs had a significantly longer τ_decay than glycine (K–S, p < 0.0001) and GABA_A (K–S, p < 0.0001) receptor-mediated sIPSCs. D, Average normalized sIPSCs mediated by glycine, GABA_A, and GABA_C receptors. GABA_C receptor-mediated sIPSCs have the longest decay time.

Figure 6.

GABA_A and GABA_C receptor-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_A and GABA_C receptor-mediated L-IPSCs (supplemental data, available at www.jneurosci.org as supplemental material). GABA_A receptor-mediated L-IPSCs have a much larger release function than GABA_C receptors, likely because of the much smaller Q of GABA_A sIPSCs versus GABA_C sIPSCs. The GABA_C release function has a prolonged tail not shown by the GABA_A release function. Calibration: 0.05 quanta/ms, 200 ms. B, To determine how much of the difference between GABA_A and GABA_C L-IPSC kinetics was attributable to receptor properties, we used the GABA_A calculated release function to simulate L-IPSCs with the GABA_A and GABA_C sIPSCs. Shown is the normalized GABA_C L-IPSC, using the GABA_A release function, that had a slower rise and decay time than the normalized GABA_A L-IPSC, as a result of the slower kinetics of the GABA_C sIPSCs. The L-IPSC simulated with the GABA_A release function and sIPSC matched the properties of the average GABA_A L-IPSC from Table 1. Calibration, 200 ms. C, Shown are the normalized GABA_C L-IPSCs using the calculated GABA_A and GABA_C release rates. The prolonged tail of the estimated GABA_C release rate slows the kinetics of the GABA_C L-IPSCs. Again, the L-IPSC simulated with the GABA_C release function and sIPSC matched the properties of the average GABA_C L-IPSC from Table 1. Calibration, 200 ms. D, If GABA_A, GABA_C, and glycine receptor sIPSCs are activated by a brief (square wave, lasting 10 ms) burst of neurotransmitter, then the distinct receptor properties filter the response, as shown by these normalized responses. Calibration, 200 ms.

Figure 7.

GABA_C receptor-mediated L-IPSCs are enhanced by blocking GABA uptake, but GABA_A receptor-mediated L-IPSCs are not. A, GABA_C receptor-mediated L-IPSCs (30 ms light) are enhanced by NO-711 (5 μm), which inhibits the GAT-1 GABA transporter. Calibration: 2 pA, 200 ms. B, GABA_A receptor-mediated L-IPSCs (30 ms light) are minimally changed by the addition of NO-711. C, GABA_C L-IPSCs Q is significantly increased by NO-711 (*p < 0.05), whereas the GABA_A L-IPSC Q is decreased by a small amount (*p < 0.01). D, The D₃₇ of GABA_C receptor-mediated L-IPSCs was increased by NO-711 (*p < 0.01), whereas the D₃₇ of GABA_A receptor-mediated L-IPSCs was unchanged (p = 0.3).

Figure 8.

Eliminating GABA_C receptors reduced the decay time of rod bipolar cell L-IPSCs as a function of light intensity. A, L-IPSCs from a WT rod bipolar cell in response to increasing intensities of light (30 ms light stimulus). Calibration: 5 pA, 500 ms. B, L-IPSCs from a GABA_CR null rod bipolar cell in response to increasing intensities of light. The increases in the magnitude and decay of the response are smaller for the GABA_CR null mice. C, The average L-IPSC D₃₇ values from WT (n = 10) and GABA_CR null (n = 6) mice are plotted as a function of light intensity (log relative intensity). The curves are the best fits to a logistic function. The maximum D₃₇ of the GABA_CR null curve was 35% of the WT maximum. The L_20–80 values of the fits were not significantly changed (WT, 2.1 ± 0.8; null, 2.4 ± 0.8), but the GABA_CR null curve had a lower EC₅₀ (WT, −3.4 ± 0.2; null, −2.7 ± 0.2).

Figure 9.

WT and GABA_CR null mice L-IPSCs respond differently to increasing light duration. A, Example L-IPSCs from WT rod bipolar cells in response to 10 ms (dark gray trace) and 1000 ms (black trace) light stimulation. The two L-IPSCs do not have drastically different charge transfers. B, Example L-IPSCs from GABA_CR null rod bipolar cells in response to 10 ms (dark gray trace) and 1000 ms (black trace) light stimulation. The 10 ms L-IPSC is much smaller than that 1000 ms L-IPSC. GABA_CR null L-IPSCs increase significantly more when the light duration is increased (p < 0.01).

Figure 10.

L-IPSCs differentially shape A17 L-EPSCs in WT and GABA_CR null mice. A, Example L-EPSCs from WT A17 amacrine cells in response to 10 ms (dark gray traces) and 1000 ms (black traces) light stimulation. The 10 ms L-EPSC is smaller than that 1000 ms L-EPSC. B, Example L-EPSCs from GABA_CR null A17 amacrine cells in response to 10 and 1000 ms light stimulation. The two L-EPSCs do not have as large a difference in charge transfers as the WT L-EPSCs. WT L-EPSCs increase significantly more when the light duration is increased (p < 0.01).

Figure 11.

The properties of inhibitory synaptic transmission are controlled by many independent factors. Shown is an example of three amacrine cell terminals releasing GABA onto synapses on a bipolar cell. The terminal on the left is activating GABA_A receptors that have a short time course, and the terminal on the right is activating GABA_C receptors that have a prolonged time course. The terminal in the middle has not released GABA, but the GABA_C receptors underlying it are being activated by spillover from neighboring synapses, which respond with a small, delayed current. GABA is taken up by GAT-1 transporters, which control the amount of GABA that leaves the synapse.