Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft

Abstract

Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²⁺ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation may be a widespread phenomenon.

Figure 1. Feedback consists of a fast and a slow component.

(A) Feedback response measured in a cone in goldfish retina. Cones were clamped at E_Cl (−50 mV) and saturated with a small spot of light. HCs were then hyperpolarized by a 500 ms full-field white light stimulus (4,500 µm). This induced a feedback inward current. The mean amplitude was 11.0±1.1 pA (n = 23). The onset of the feedback response could be fitted best by the sum of two exponentials (red line, sum of the exponential fits; blue lines, individual exponential fits). (B) The amplitude and time constants of the two exponential functions. The fast feedback component (τ_f = 29±3 ms; n = 23) had an amplitude (A_f) that constituted 64±6% of the total feedback response. The remaining 36±6% of the feedback response (A_s) was mediated by a process with a time constant (τ_s) of 189±25 ms (n = 19). (C) Feedback responses in cones from wild-type (WT, black) and Cx55.5^−/− mutant zebrafish (red) were also best fitted by a double exponential function. (D) Compared to goldfish, WT zebrafish had a similar fast component to slow component amplitude ratio (gray: A_f, 68±4%; A_s, 32±4%; n = 13), whereas it shifted in Cx55.5^−/− mutants in favor of the slow feedback component (red: A_f, 52±7%; A_s, 48±7%; n = 9). The amplitude of the slow component of feedback in Cx55.5^−/−mutants (1.62±0.39 pA; n = 9) did not significantly differ from WT (1.87±0.39 pA; n = 13; p = 0.67), showing that the slow component is independent of Cx55.5 hemichannels. Note that the amplitude axes for the WT and mutants are scaled such that it reflects the total reduction of feedback in the mutant relative to WT. The time constant of the fast component (τ_f) did not differ significantly between WT and Cx55.5^−/− mutants (WT, 29.3±3.3 ms; n = 13; Cx55.5^−/−, 40.6±9.6 ms; n = 9; p = 0.21) and was similar to goldfish. The time constant of the slow component (τ_s) in WT and Cx55.5^−/− mutants did not differ significantly (WT, 300.2±52.3 ms; n = 13; Cx55.5^−/−, 408.5±169.7; n = 9; p = 0.49) and was larger than in goldfish.

Figure 2. An ATP release mechanism and the enzymes needed to hydrolyze ATP and degrade adenosine are present in the synaptic complex of cones.

(A) Fluorescent images of zebrafish retina showing Panx1-IR (green) and a nuclear stain (red) (from Prochnow et al. [11]). Panx1-IR is present in characteristic horseshoe-shaped structures indicative for processes invaginating the cone synaptic terminal. (B) Electronmicrograph of a cone synaptic terminal in zebrafish retina. Immunolabeling is restricted to HC dendrites flanking the synaptic ribbons (R). (C) Higher magnification of a HC dendrite lateral of the synaptic ribbon (R). Note that one HC dendrite is devoid of Panx1 labeling. (D) NTPDase1-IR (green) and a nuclear stain (red). NTPDase1-IR is also present in horseshoe-shaped structures characteristic for localization in HC dendrites. (E) Double labeling of NTPDase1 antibody with an antibody against GluR2 (green), a marker for HC dendrites ,, indicated that NTPDase-1 (red) was specifically expressed on HC dendrites. (F) ADA-IR (green) was present in horseshoe-shaped structures. (G) ADA-IR colocalized with the GluR2-IR (green), indicating that ADA (red) is expressed on HC dendrites. Expression of ecto-ATPases in the photoreceptor synaptic terminal has been observed previously in rat , mouse, and zebrafish . Scale bars in panels A, D, E, F, and G indicate 5 µm. Scale bar in panels B and C indicate 500 nm and 250 nm, respectively.

Figure 3. HCs release ATP.

(A) Dissociated goldfish HC. This preparation consisted of about 90% of HCs. The remaining material was mostly debris. Scale bar, 20 µm. (B) The mean ± sem whole-cell IV relations of six dissociated HCs in conditions where potassium currents were inhibited by Cs. Application of 500 µM probenecid (red trace) reduces the conductance of the HCs. The green trace is the IV relation of the probenecid blocked current. This current has similar characteristics as the Panx1 current described by Prochnow et al. . (C) Change in ATP release as measured with the luminescence assay. Depolarization of dissociated HCs by AMPA led to an increase in ATP release, whereas inhibition with probenecid decreased the release significantly. In probenecid, the ATP release was significantly lower than baseline, indicating that HCs in control conditions release ATP.

Figure 4. Pharmacological profile of the slow component of feedback.

(A) Normalized mean feedback responses of nine cones in control (black) and in 500 µM probenecid (red). The slow component present in control conditions was reduced by probenecid. (B) Normalized mean feedback responses in nine cones in control (black) and in 100 µM ATP (red). ATP blocked the slow component of feedback, indicating the slow component depends on ATP. Note this concentration of ATP does not block Panx1 channels . (C) Normalized mean feedback responses in seven cones in control (black) and in 50 µM ARL67156, a blocker of NTPDase (red). The slow component is reduced by application of ARL67156, indicating that it is mediated by ATP hydrolysis. Feedback response amplitudes were reduced by ATP application (B), but not by ARL67156 application (C), consistent with the notion that a pH buffer generated by ATP hydrolysis inhibits I_Ca of the cone by keeping the synaptic cleft slightly acidic. (D) Normalized mean feedback responses in six cones in control (pH 7.6; black) and when the superfusate pH was 7.2 (red). The slow component is strongly reduced when the pH gradient between the synaptic and the extrasynaptic compartments is removed. (E–H) The slow component amplitude for individual cells (black) used in (A–D) during control, drug application, and wash. The red line indicates the mean ± sem. In each case, the changes of the slow component amplitude were reversible. The figures also suggest that the slow feedback component may have increased slightly over time. (I) The mean ± sem change in the total feedback amplitude relative to control levels for the results shown in (A–D). Probenecid (p = 0.003), ATP (p = 0.0093), and pH (p = 0.0175) all reduced the feedback amplitude significantly. Only ARL67156 did not change the amplitude significantly (p = 0.2295). (J) The mean ± sem amplitude reduction for the slow component of feedback relative to control values for the results shown in (A–D). In each case, the amplitude reduction of the slow component was significant (probenecid, p = 0.0009; ATP, p = 0.036; ARL67156, p = 0.045; pH, p = 0.010). (K) The change in half activation potentials of I_Ca when 500 µM probenecid, 100 µM ATP, or 50 µM ARL67156 were present in the bath solution or when the extracellular pH was shifted to 7.2. Probenecid did not induce a significant shift of the activation potential of I_Ca (n = 9; p = 0.3375). ATP shifted the half activation potential to more positive potentials (n = 8; p = 0.0277), whereas ARL67156 shifted it to more negative potentials (n = 8; p = 0.0131). Lowering the extracellular pH led to a significant shift of I_Ca to positive potentials (n = 5; p = 0.0022).

Figure 5. HC responses in various pharmacological conditions.

(A) Acidification of the extrasynaptic medium reduces the rollback response in goldfish HCs (arrow) (n = 4) and hyperpolarizes the HC membrane potential (see D) (black, control; red, pH 7.2; green, wash). This indicates that the slow component of feedback contributes to the rollback response in HCs. (B) Application of probenecid also led to a slight reduction of the rollback response (n = 18) and hyperpolarization of the HC membrane potential (see D). The rollback response never disappeared completely (black, control; red, probenecid; green, wash). (C) Application of probenecid in a Ringer's solution containing 28 mM HEPES. In this condition, the HC light responses were almost completely blocked and HC hyperpolarized strongly [see D; black, HEPES; red, HEPES+probenecid; green, HEPES (wash)]. (D) Mean ± sem HC membrane hyperpolarization induced by the various pharmacological manipulations.

Figure 6. Purinergic receptor activation does not underlie feedback from HCs to cones.

(A) A whole-cell IV relation for cones in control condition (black) and with 100 µM ATP (red; n = 5). ATP does not activate a nonspecific cation conductance. (B) A whole-cell IV relation for cones in control condition (black) and 50 µM ARL67156 (red; n = 5). ARL67156 did not lead to the activation of a nonspecific cation conductance. (C) Mean feedback responses in control (black) and in 1 µM ZM 241385 (red; n = 4), a blocker of the A2 receptor. The response amplitude and kinetics of the feedback response were not significantly affected, indicating that feedback is not mediated by A2 receptors.

Figure 7. Schematic drawing of the proposed feedback mechanism.

(A) The ephaptic mechanism. Cones continuously release glutamate in the dark via a ribbon (R) synapse optimized for sustained glutamate release. This release depends on the activity of presynaptic Ca²⁺ channels (red). HC dendrites end laterally to the cone synaptic ribbon. Cx hemichannels (green) and Panx1 channels (blue) are expressed on the dendrites of HCs. Because they are both nonspecific channels and are open at the physiological membrane potentials of HCs, a current will flow into the HCs. HC dendrites invaginate the cone synaptic terminal, which leads to a large extracellular resistance (white resistor). The current that flows into HCs via the Cx hemichannels and Panx1 channels has to pass this resistor, which will induce a slight negativity deep in the synaptic cleft. The result will be that the voltage-gated Ca²⁺ channels sense a slightly depolarized membrane potential. When HCs hyperpolarize, the current through the Cx hemichannels will increase and so will the negativity in the synaptic cleft leading to a further decrease of the potential sensed by the cone Ca²⁺ channels. This is the fast component of feedback. (B) The Panx1/ATP-mediated mechanism. Expanded view of the pre- and postsynaptic membranes of cones and HCs. ATP is released by HCs via Panx1 channels. Through a number of steps ATP is converted into inosine, protons, and a phosphate buffer with a pKa of 7.2. This makes the synaptic cleft acidic relative to the extrasynaptic medium (pH 7.6–7.8), which inhibits Ca²⁺ channels and shifts their activation potential to positive potentials. Closing the Panx1 channels prevents HCs from releasing ATP, thereby stopping the production of phosphate buffer, leading to an alkalization of the synaptic cleft. This alkalization disinhibits the Ca²⁺ channels and shifts their activation potential to negative potentials. This mechanism underlies the slow component of feedback. (C) Schematic representation of I_Ca of cones with and without feedback. The black trace shows the I_Ca of cones in the dark. The red trace shows I_Ca when only the ephaptic feedback is active, whereas the blue trace shows the I_Ca when both the ephaptic and Panx1/ATP-mediated feedback are active.