Picton LD , Sillar KT , Zhang HY .

Wellcome Trust , BB/JO1446X/1 Biotechnology and Biological Sciences Research Council , BB/M024946/1 Biotechnology and Biological Sciences Research Council

	Figure 1. Properties of dINs in Stage 42 Larval Xenopus Tadpoles(A) The anatomy of a dIN. The axon courses ventrally and then caudally. Arrows on the image indicate the direction of the axon. FB, forebrain; HB, hindbrain; MB, midbrain; OC, otic capsule; PE, pineal eye; SC, spinal cord.(B) Responses of a dIN to depolarizing current pulses of increasing amplitude. Unlike non-dINs, suprathreshold pulses at all amplitudes generate only a single spike.(C) During swimming, dINs also only fire a single spike per swim cycle. VR, ventral root.(D) Unlike all other neuron types in the Xenopus spinal motor circuit, dINs never display a usAHP in response to a protocol inducing repetitive spiking (Di) or after swimming (Dii). Note that action potentials in (Di) have been truncated.(E) The action potential shape of larval dINs is different from non-dINs. Examples of a dIN and motoneuron (MN) action potential in response to a suprathreshold depolarizing pulse (Ei) are shown. Pooled action potential width of dINs and non-dINs measured at 0 mV (Eii; n = 10; ∗∗∗p < 0.001; median with 50% interquartile range (IQR) displayed as box-and-whisker plots).(F) The intrinsic properties of dINs (n = 28) differ from non-dINs (n = 31) and display a significantly more depolarized RMP (Fi) and significantly lower input resistance (Fii; median with 50% IQR displayed as box-and-whisker plots). ∗∗∗p < 0.001. See also Figure S1.
	Figure 2. Ih Is Present in All dINs and in Some Non-dINs and Is Blocked by ZD7288(A) An example of a sag potential in a non-dIN (Ai) with and without ZD7288 (50 μM). The peak and steady-state membrane potential responses are indicated by dashed lines. (Aii) An example response of a dIN to hyperpolarizing current pulses (3-s duration). ZD7288 (10 μM) hyperpolarized the RMP (see also Figure 3) and abolished the large sag potentials observed at hyperpolarized membrane potentials. Arrow, post-inhibitory depolarization seen in control; arrow head, the membrane potential rebound was absent in the presence of ZD7288. (Aiii) Pooled data showing a comparison of the sag amplitude between −80 and −90 mV in dINs (n = 27) and non-dINs (n = 34; ∗∗∗p < 0.001; median with 50% IQR displayed as box-and-whisker plots).(B) The voltage-current (V-I) relationship for a non-dIN (Bi) and a dIN (Bii) in response to hyperpolarizing current pulses (3-s duration) of increasing amplitude (10 pA incremental steps) and the effect of 10–50 μM ZD7288. For both control (closed circles) and in ZD7288 (open circles), the peak (p) and the steady-state (ss) membrane potential change are plotted against each injected current amplitude.(C) The amplitude of the sag potentials in a non-dIN (Ci) and a dIN (Cii) was plotted against the membrane potential with (open circles) or without (closed circles) ZD7288.(D) Pooled data showing that ZD7288 (10–50 μM) significantly abolished the sag potential observed around −80 mV. Data are expressed as median with 50% IQR and displayed as box-and-whisker plots with individual data points. (Di): 3 non-dINs, p = 0.0048; (Dii): 23 dINs, p < 0.001; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S2.
	Figure 3. Ih Current Is Active at Rest and Contributes to the Intrinsic Properties of dINs(A) When dINs were held depolarized (∼−48 mV), hyperpolarizing pulses revealed a sag response around the original RMP.(B) Raw trace on a slow time base showing a clear membrane hyperpolarization of approximately 10 mV following the application of ZD7288 (10 μM). Blockade of Ih hyperpolarized all dINs.(C) Resting membrane potential (RMP) and input resistance plotted against experiment time for the experiment shown in (B). The hyperpolarization was accompanied by an increase in input resistance. Dashed line indicates the change of IR of the dIN in (B).(D) Pooled paired data showing that (Di) the membrane potential of dINs was significant hyperpolarized by ZD7288 (n = 23; p < 0.001; median with 50% IQR displayed as box-and-whisker plots). (Dii) Pooled paired data show a significant increase in input resistance in ZD7288 (n = 23; p < 0.001; median with 50% IQR displayed as box-and-whisker plots). ∗∗∗p < 0.001. See also Figure S2.
	Figure 4. The Effects of ZD7288 on Xenopus Swim Network Output(A) Two simultaneously recorded raw ventral root traces on the left and right sides showing evoked swim episodes in control (Ai), in the presence of the Ih current blocker ZD7288 (50 μM; Aii), and after washout (Aiii). The right side panels show an expansion of fictive swimming activity.(B) ZD7288 (10–50 μM) significantly shortened episode duration (p = 0.026), and the effect was reversed following washout of ZD7288 (n = 9 complete experiments; p = 0.027; median with 50% IQR displayed as box-and-whisker plots).(C) Time plot showing (Ci) mean swim cycle frequency across 3 evoked episodes in control, ZD7288, and after washout. Note that the swim frequency is lower and more variable. (Cii) ZD7288 (50 μM) caused a significant decrease in cycle frequency (p = 0.0008; n = 9; median with 50% IQR displayed as box-and-whisker plots).(D) Time plot showing (Di) mean burst durations across 3 evoked episodes in control, ZD7288, and following washout. (Dii) ZD7288 (50 μM) caused a significant increase in burst duration (p = 0.0018; n = 9; median with 50% IQR displayed as box-and-whisker plots).∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S3.
	Figure 5. Blocking Ih Currents Alters dIN Firing during Swimming and Affects Rhythm Initiation(A) Example of a dIN firing during swimming.(B) In the presence of ZD7288, the tonic depolarization indicated by the gray area is larger and dINs fire at a more depolarized level. Note the different timescales in (Ai) and (Bi) and the shorter episode duration in the presence of ZD7288. The action potentials shown in (Bii) are smaller than those in (Aii). Arrow indicates a membrane hyperpolarization following the end of the swimming episode.(C) Membrane responses to (Ci) current pulses in control, in the presence of the Ih blocker ZD7288 (10 μM), and in wash. ZD7288 abolished both the sag currents and the PIR action potentials (arrow). Its effect could be partially reversed after a long wash. The post-inhibitory depolarization seen in control disappeared in the presence of ZD7288 and re-appeared during wash (arrow head). (Cii) In the presence of ZD7288, the PIR action potential could be induced when holding RMP to the control level by current injection.(D) The rebound action potential shapes in control condition and in ZD7288.(E) In some dINs (n = 3), (Ei) a single rebound action potential induced by a hyperpolarizing pulse initiated fictive swimming activity (note ventral root [VR] activity). (Eii) The property shown in (Ei) was abolished soon after ZD7288 was applied (approx. 10 min after drug application; n = 3), although PIR firing could still be evoked and the sag potential is only partially blocked. (Eiii) Later in ZD7288 treatment, both the PIR firing and the sag potential were totally abolished.
	Figure 6. Blocking Ih Revealed a Post-swim usAHP(A) Following dIN firing in control, (Ai) the membrane potential repolarized to the baseline. (Aii) In the presence of 10 μM ZD7288, the RMP hyperpolarized (dashed line) and a long-lasting AHP appeared following the end of a swimming episode. (Aiii) The usAHP was abolished after adding 0.5 μM ouabain in the bath.(B) Another dIN displayed a usAHP in the presence of ZD7288 (Bi). Removing K+ ions from saline abolished the usAHP (Bii). After replacing 0K+ saline with control saline, the usAHP reappeared (Biii). Dotted lines in (A) and (B) indicate the resting membrane potentials. Downward deflections in (Bii) are conductance pulses.(C) A train of depolarizing pulses that mimics swimming was applied to dINs following ZD7288 treatment. A small afterhyperpolarization was observed in some dINs (8 out 12), which was never observed in control (see Figure 1Di).(D) Pooled data indicate that the maximum amplitude of usAHP was reduced significantly by 0.5 μM ouabain (left panel; n = 8; ∗∗∗p < 0.001) or 0K+ saline (right panel; n = 5; ∗p = 0.011). Data are expressed as median with 50% IQR and displayed as box-and-whisker plots with individual data points.(E) The input resistance (IR) during the usAHP period was tested and plotted against time. The inset shows an example of current pulses injected during usAHP period. Pooled data indicate that there was no significant change in input resistance during the usAHP (n = 3; one-way ANOVA; p = 0.26; mean ± SD).
	Figure 7. Block of Ih Current Disrupts the Relationship between Swim Interval and Episode Duration and Increases Swimming Failure at Short Intervals(A) Raw traces showing two simultaneously recorded ventral root traces on the left (L-VR) and right (R-VR) sides showing evoked swim episodes using variable inter-episode intervals. In control conditions (Ai), shortening the inter-episode interval reduces episode duration, but swimming can still be reliably evoked at very short intervals (≤2 s). In the presence of ZD7288, swimming initiation failed at short intervals (Aii; crosses, stimuli failing to evoke swimming), an effect which reversed upon drug washout (Aiii).(B) Pooled data illustrating that block of Ih significantly increases the swim failure rate at short inter-swim intervals (n = 6; median with 50% IQR displayed as box-and-whisker plot). ∗∗∗∗p < 0.0001.

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