HCN Channel-mediated Neuromodulation Can Control Action ... - ELife

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To investigate a modulation of conduction velocity by physiological neuromodulators, we focused on the cerebellar parallel fibers, in which the velocity could be most accurately measured, and then applied several modulators known to act via cAMP-dependent pathways (Figure 2). Application of 200 µM norepinephrine (NE) resulted in a relatively fast increase in conduction velocity (1.9 ± 0.8%; n = 6; see Figure 2A and C–E; see legend for statistical testing), consistent with the existence of β-adrenergic receptors in the cerebellar cortex (Nicholas et al., 1993) that increase the cAMP concentration via Gs-proteins. On the other hand, the application of either 200 µM serotonin (–3.5 ± 0.5%; n = 11), 200 µM dopamine (–5.0 ± 0.7%; n = 13) or 200 µM adenosine (–7.2 ± 0.6%; n = 5) resulted in a continuous decrease of the conduction velocity (Figure 2B and C–E), consistent with the existence of Gi-coupled receptors for serotonin, dopamine, and adenosine in the molecular layer of the cerebellum (Geurts et al., 2002; Schweighofer et al., 2004), which decrease the cAMP concentration. Although we used high concentrations of the agonists and off-target effects cannot be excluded (e.g., the activation of dopamine receptors by NE [Sánchez-Soto et al., 2016]), these data nevertheless indicate that physiological neuromodulators can both increase and decrease action potential conduction velocity, depending on the type of neuromodulator and receptor.

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Neuromodulators differentially regulate conduction velocity via HCN channels.

(A) Example of compound action potentials recorded in parallel fibers. Each trace is an average of signals recorded over a period of 1 min, before (at −5 min, black line) and after application of 200 µM NE (at 15 min, blue line). The delays between the peak of the proximal action potential and the distal compound action potential are indicated by horizontal bars. Traces are aligned to the peak of the compound action potentials recorded with the proximal electrode. (B) Example of compound action potentials as shown in panel (A) with the application of 200 µM adenosine. (C) Average normalized conduction velocity in cerebellar parallel fibers during the application of various neuromodulators that are known to act via cAMP-dependent pathways. (D) Average relative change in conduction velocity measured from 1 to 6 min after the start of neuromodulator application (bracket marked D in panel (C)). PANOVA = 9*10−10. PKruskal-Wallis = 3*10−8. The individual P values of the Dunnett test for multiple comparisons with the control are indicated. (E) Average relative change in conduction velocity measured from 10 to 15 min after the start of application of the neuromodulators (bracket marked E in panel (C)). PANOVA = 3*10−7. PKruskal-Wallis = 3*10−7. The individual P values of the Dunnett test for multiple comparisons with the control are indicated. (F) Average normalized conduction velocity in parallel fibers. 30 µM ZD7288 was applied 25 min before the start of the application of the neuromodulators. ZD7288 remained in the solution during recording to ensure continuously blocked HCN channels. At t = 0 min, 8-Br-cAMP, adenosine or NE was added to the solution. (G) Average relative change in conduction velocity measured 10 to 15 min after the start of application of the neuromodulators (bracket marked G in panel (F)). PANOVA = 0.91. PKruskal-Wallis = 0.77. The individual P values of the Dunnett test for multiple comparisons with the control are indicated.

https://doi.org/10.7554/eLife.42766.004

In addition to HCN channels, some voltage-gated Na+, K+, and Ca2+ channels can be modulated by the intracellular cAMP-pathway (Burke et al., 2018; Yang et al., 2013; Yin et al., 2017). To address the contribution of other channels on the neuromodulation of the conduction velocity, we performed a set of experiments in which HCN channels were first blocked by 30 µM ZD7288 before we applied three modulatory substances that had significantly increased or decreased conduction velocity in previous experiments. With ZD7288 continuously present in the recording solution, the conduction velocity of parallel fibers decreased over the course of 20 min (Figure 2F; see Materials and methods). Compared with control conditions (i.e. only ZD7288), adding 8Br-cAMP (500 µM), adenosine (200 µM) or NE (100 µM) at t = 0 min (i.e. 25 min after application of ZD7288) did not change the conduction velocity. The average conduction velocity between t = 10 and 15 min was decreased by –3.3 ± 2.4% for cAMP (n = 9), –4.6 ± 1.6% for adenosine (n = 9) and –3.7 ± 1.2% for NE (n = 7) when compared to the average velocity between t = 0 and 5 min in the baseline recording. This was not significantly different from the decrease measured in the presence of ZD7288 alone (control, –3.3 ± 1.4%; n = 7, see Figure 2G), indicating that the previously shown effects of cAMP and neuromodulators on conduction velocity are mainly mediated by HCN channels.

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