Assessing the role of Ih channels in synaptic transmission and mossy fiber LTP

Abstract

Hyperpolarization-activated nonselective cation channels (Ih channels) play an important role in the control of membrane excitability and rhythmic neuronal activity. The functional relevance of presynaptic Ih channels in regulating synaptic function, however, is not well established. Recently, it has been proposed [Mellor, J., Nicoll, R. A. & Schmitz, D. (2002) Science 295, 143-147] that presynaptic Ih channels are necessary for hippocampal mossy fiber long-term potentiation (LTP). This observation challenges an alternative model that suggests presynaptic forms of LTP are caused by a direct modification of the transmitter release machinery. Here, we assess the role of Ih in hippocampal mossy fiber LTP as well as cerebellar parallel fiber LTP, forms of potentiation that share common mechanisms. Our results show that after Ih blockade neither mossy fiber LTP nor parallel fiber LTP are affected. Furthermore, Ih does not significantly modify basal excitatory synaptic transmission in the hippocampus, whereas the organic Ih blockers ZD7288 and DK-AH 269 induce a large Ih-independent depression of synaptic transmission. In summary, our results indicate that Ih-mediated persistent changes in presynaptic excitability do not underlie presynaptic forms of LTP.

Figure 1

Depression of basal synaptic transmission by organic Ih channel blockers. (a) Summary graph showing the effect of 1, 10, and 50 μM ZD7288 (n = 3, 3, and 4 slices, respectively) on field excitatory postsynaptic potentials (fEPSP) recorded from the CA1 area of the hippocampus. (b) Effects of 50 μM ZD7288 on the fiber volley (FV) amplitude measured in the continuous presence of 10 μM NBQX. Representative FV traces (averages of 10 individual responses) in the absence of NBQX (Inset) are superimposed before (Control), 90 min after ZD7288 bath application and after increasing stimulus strength to match the initial FV amplitude. (c and d) Summary graphs demonstrating that the depression of synaptic transmission (fEPSPs, open circles) induced by both 50 μM ZD7288 (c, n = 4) and 100 μM DK-AH 269 (d, n = 3) was associated with an increase in the PPF magnitude (filled triangles). Superimposed averaged traces before and after application of the organic Ih blockers are shown above. These synaptic responses are also shown normalized (right traces above on each c and d).

Figure 2

Basal excitatory synaptic transmission is not modulated by Ih. (a Left) Single experiment from a CA1 pyramidal cell in which the total current (I leak + Ih) induced by a voltage step, from −50 mV to −90 mV, and the amplitude of EPSCs were simultaneously recorded and plotted over time. Bath application of 50 μM ZD7288 completely blocked Ih within 15–20 min, whereas the EPSC amplitude remained virtually unchanged. (Right) Sample traces of the total current recorded during the hyperpolarization step (Upper) and the EPSC (Lower) are shown before and after ZD7288 application. (b and c) Summary graphs of experiments performed as in a in 50 μM ZD7288 (b, n = 4) and 100 μM DK-AH 269 (c, n = 4).

Figure 3

Synaptic depression induced by ZD7288 and DK-AH 269 is not mimicked by Cs⁺. (a) Bath-application of 1 mM Cs⁺, which effectively blocked Ih, did not depress synaptic transmission but rather induced a slight increase of synaptic responses. Superimposed sample traces before and after Cs⁺ application are shown on the right side of the panel. (b and c) In the same slices, after synaptic responses were stabilized in presence of cesium, subsequent application of 50 μM ZD7288 (b) or 100 μM DK-AH 269 (c) markedly depressed synaptic transmission. Sample traces before and after Ih blockers application are shown on the right.

Figure 4

Organic Ih channel blockers do not block mossy fiber LTP, despite significant depression of basal synaptic transmission. (a Left) Scheme of the arrangement of electrodes for stimulating and recording. (Right) Representative experiment in which synaptic responses were induced by alternatively stimulating one of these pathways at 0.05 Hz (values are the mean of three successive points). ZD7288 (50 μM) was bath applied during 40 min before tetanizing one pathway. At the end of each experiment, 1 μM DCG-IV blocked transmission confirming that these were mossy fiber synaptic responses. Sample traces taken during baseline, in presence of ZD7288, 1hr after tetanus and in presence of DCG-IV are shown above. (b Left) Summary graph of 7 experiments performed as in a. (Right) Summary graph showing the magnitude of the LTP for each individual experiment that was estimated as the ratio of synaptic responses between the tetanized (Tet.) and nontetanized (Control) synapses. (c) DK-AH 269 (100 μM) also depressed synaptic transmission in 7 experiments but did not affect mossy fiber LTP.

Figure 5

Mossy fiber LTP is independent of Ih channels. (a) Mossy fiber LTP was also normal in 7 slices that were incubated in 1 μM ZD7288 for 3–6 h and also applied during the experiment at the same dose. Sample traces from a single experiment during baseline, 50–60 min after tetanus and in 1 μM DCG-IV are shown on the right. (b) Summary graph of 4 experiments performed in presence of 2 mM Cs⁺. The LTP-inducing tetanus was applied at least 1 h after Cs⁺ bath application and 20 min stable baseline. Sample traces before and after tetanus are shown on the right. (c) Ih currents recorded from granule cells (induced by voltage steps from −50 to −100 mV) were blocked in presence of 50 μM ZD7288, 100 μM DK-AH 269, or 1 mM cesium.

Figure 6

Blocking Ih has no effect on the forskolin-induced enhancement of mossy fiber synaptic responses. (a) Forksolin (10 μM; FSK, filled circles) was bath-applied during 20 min (horizontal bar) prior slice incubation in 10 μM ZD7288 for 2–4 h (5 slices). The rundown effect of ZD7288 application on mossy fiber synaptic transmission was estimated from 4 interleaved slices (Control, open circles). Forskolin-induced potentiation is also shown as the ratio between synaptic responses in forskolin-treated slices and control slices (Inset). (b) Forskolin enhancement of mossy fiber synaptic responses is identical in slices incubated for 1 h in 1–2 mM cesium (filled circles, n = 5) and in control slices (open circles, n = 3).

Figure 7

Ih blockade did not affect LTP or forskolin-induced potentiation of transmission at parallel fiber-Purkinje synapses. (a) Experimental paradigm to stimulate and record two independent sets of parallel fibers in the cerebellar cortex. (b) Summary graph of EPSCs recorded in Purkinje cells after at least 2 h of incubation in 10 μM ZD7288 (also applied during the experiment). A single tetanus (100 pulses at 10 Hz) was given at time 0 (arrow) induced normal LTP (n = 4). Average traces from a representative experiment before and 30 min after tetanus are illustrated above. (c) Summary graph of the LTP magnitude that was calculated as the EPSC ratio between the tetanized (Tet.) and nontetanized (Control) synapses for each individual experiment. (d) Forskolin-enhancement of synaptic transmission after at least 1 h of incubation in 2 mM Cs⁺ (also applied during the experiment, n = 4) is identical to that observed in control (n = 3).