Okamoto K , Emura N , Sato H , Fukatsu Y , Saito M , Tanaka C , Morita Y , Nishimura K , Kuramoto E , Xu Yin D , Furutani K , Okazawa M , Kurachi Y , Kaneko T , Maeda Y , Yamashiro T , Takada K , Toyoda H , Kang Y .

	Figure 1. TASK1 and TASK3 mRNA expressions in small and large MNs in the dl-TMN. Aa, Toluidine blue staining of the brainstem slice including the dl-TMN. Ab, Ac, Arrowheads showing small MNs (Ab) and large MNs (Ac). A margin of the large MN depicted with a green line (Ac). Ad, An image of the same section as in Ac after laser microdissection. B, TASK1 and TASK3 mRNA expression in large MNs revealed by standard RT-PCR. Ca, Cb, Quantitative real-time PCR analysis of TASK1 and TASK3 mRNA expression compared with GAPDH mRNA in large MNs (Ca) and small MNs (Cb). The ΔRn was plotted against the number of cycles. The mRNA copy number of TASK3 was slightly higher than that of TASK1 in larger MNs, while the former was apparently larger than the latter in small MNs. D, The mean expression levels of TASK1 and TASK3 mRNAs relative to GAPDH mRNA in 80 large MNs and in 400 small MNs (n = 7 and n = 7 samples, respectively). ‡p < 0.001, paired t test; †p < 0.001, unpaired t test. E, The relationships between cell numbers and CT values of GAPDH mRNA obtained from 10, 50, and 80 large MNs (n = 7, n = 5, and n = 5 samples, respectively) and those obtained from 50, 200, and 400 small MNs (n = 6, n = 6, and n = 5 samples, respectively). F, The expression levels of TASK1 and TASK3 mRNAs in a single large MN and those in a single small MN. The respective mRNA expression levels were normalized to that of TASK1 mRNA in a single large MN. ‡p < 0.001, paired t test; †p < 0.001, unpaired t test.
	Figure 2. Complementary distribution of TASK1 and TASK3 channels in MNs in the dl-TMN. Aa–Ad, HEK cells transfected with TASK1 and ZsGreen1 displaying immunoreactivity along the plasma membrane and partly in the cytoplasm (Aa); nuclei staining with DAPI (Ab); TASK1 transfection ensured with ZsGreen1 (Ac); and a merged fluorescence image (Ad). Ba–Bd, HEK cells transfected with mock and ZsGreen1 displaying no immunoreactivity (Ba); nuclei staining with DAPI (Bb); mock transfection ensured with ZsGreen1 (Bc); and a merged fluorescence image (Bd). C, Confocal photomicrographs showing immunoreactivity for ChAT (green) and TASK1 (red) in MNs. As revealed in the merged image, the TASK1 immunoreactivity was seen in somata (filled arrowheads) but not in dendrites. D, Confocal photomicrographs showing the elimination of TASK1 immunoreactivity in ChAT (green)-positive MNs following the absorption of anti-TASK1 antibody by preincubating with the antigen peptide of TASK1. E, Confocal photomicrographs showing immunoreactivity for ChAT (green) and TASK3 (red) in MNs. As revealed in the merged image, the TASK3 immunoreactivity was primarily seen in dendrites but not in somata (filled arrowheads). Scale bars: A–E, 50 μm.
	Figure 3. Relationships between the sizes of MNs in the dl-TMN and IRs, resting membrane potentials, or spike thresholds. Aa, Ba, Small (19 × 17 μm) and large (30 × 21 μm) MNs filled with biocytin (Aa and Ba, respectively). The minor and major axes are indicated with open and filled arrowheads, respectively. Ab, Bb, Membrane potential responses to depolarizing current pulses applied to small and large MNs at the resting membrane potential (Ab and Bb, respectively). C, The inverse relationship between the sizes of MNs and IRs (n = 33). D, The inverse relationship between the sizes of MNs and resting membrane potentials (n = 33). E, The positive relationship between the sizes of MNs and spike thresholds (n = 33). F, The inverse relationship between the IRs and spike thresholds (n = 33).
	Figure 4. Membrane potential responses to depolarizing current pulses in smaller and larger MNs in the dl-TMN. Aa, A brainstem slice including the dl-TMN. A stimulation electrode was placed just dorsal to the TMN. PrV, Trigeminal principle nucleus; VIIn, facial nerve; sV, sensory root of the trigeminal nerve. Scale bar, 500 μm. Ab, A simultaneous recording obtained from a pair of smaller and larger MNs that were filled with Lucifer yellow. Scale bar, 100 μm. Ac, A Lucifer yellow image of the smaller and larger MNs after fixation with paraformaldehyde (open and filled arrowheads, respectively). Scale bar, 100 μm. B, C, Membrane potential responses to depolarizing current pulses applied to the smaller and larger MNs (Bb and Cb, respectively) from the same baseline membrane potential of −86 mV brought about by negative DC current injection of −62 and −70 pA, respectively (Ba and Ca, respectively). The apparent spike threshold was lower in the smaller MN (open arrow) than in the larger MN (filled arrow).
	Figure 5. IR-dependent activation/recruitment of MNs in the dl-TMN. A, B, Membrane potential responses to hyperpolarizing current pulses and those evoked by 100 Hz stimulation of presumed Ia afferents in the smaller and larger MNs (A and B, respectively). C, D, The enlarged traces of the responses (gray area in A and B, respectively) in the smaller and larger MNs (C and D, respectively). E, The relationship between IRs and latencies to the first spikes in five pairs of MNs of different sizes when the difference in the latency to the first spike between the pair of smaller and larger MNs became maximum. F, The relationship between IRs and spike thresholds in five pairs of MNs of different sizes.
	Figure 6. Modulation of IRs by 8-Br-cGMP in small and large MNs in the dl-TMN. Aa, Ab, Sample traces of responses to depolarizing and hyperpolarizing current pulses obtained before application (Aa) and during application (Ab) of 8-Br-cGMP in a small MN. Ac, The I–V relationships obtained before and during application of 8-Br-cGMP in a small MN. The I–V relationships measured at the peak sag potentials are almost linear (blue and red circles), while those measured just before the offset of current pulses are inwardly rectifying (blue and red crosses). Ba, Bb, Sample traces of responses to depolarizing and hyperpolarizing current pulses obtained before application (Ba) and during application (Bb) of 8-Br-cGMP in a large MN. Bc. The I–V relationships obtained before and during the application of 8-Br-cGMP in a large MN. C, The relationship between IRs of MNs and changes in IRs by 8-Br-cGMP (n = 14). D, EPSPs evoked by stimulation just dorsal to the TMN obtained before and during the application of 8-Br-cGMP in a large MN. E, The relationship between IRs and changes in EPSP amplitudes by 8-Br-cGMP (n = 13).
	Figure 7. 8-Br-cGMP differentially modulates spike-onset latencies between smaller and larger MNs in the dl-TMN. A, B, Membrane potential responses to hyperpolarizing current injections and those evoked by 100 Hz stimulation of presumed Ia afferents in smaller and larger MNs (A and B, respectively) obtained before (green traces) and during the application of 8-Br-cGMP (red traces). C, The relationship between the IRs and the latencies to first spikes obtained before and after the application of 8-Br-cGMP in four pairs of MNs of different sizes. The blue and red double-headed arrows represent the recruitment time, calculated as the difference in the mean latency to the first spike between the smaller and larger MNs obtained before and after the application of 8-Br-cGMP, respectively. *p < 0.05, Wilks’ lambda; ‡p < 0.05, paired t test.
	Figure 8. 8-Br-cGMP inhibits cloned TASK3 channels. A, Superimposed TASK3 currents evoked by voltage steps ranging between −150 and +60 mV in 30 mV steps applied at a holding potential of −90 mV at pH 8.4 and 7.4. The respective current traces were obtained at the time points indicated with *1 and *2 in D. B, Superimposed TASK3 currents obtained at pH 7.4 before (blue traces) and during (red traces) the application of 8-Br-cGMP. The respective current traces were obtained at the time points indicated with *2 and *3 in D. C, Superimposed TASK3 currents obtained at pH 8.4 before (green traces) and during the application of 8-Br-cGMP (orange traces). The respective current traces were obtained at the time points indicated with *1 and *4 in D. D, Plots of TASK3 currents at 0, +30, and +60 mV against time. Gray horizontal bars represent the timing and duration of perfusion of the extracellular solutions at respective pH, and the black horizontal bar represents the timing and duration of the addition of 100 μm 8-Br-cGMP. Arrowheads indicate the time points at which the respective current responses shown in A–C were obtained. E, The normalized TASK3 currents recorded before and during the application of 8-Br-cGMP at pH 7.4 (blue and red columns, respectively) and those recorded before and during the application of 8-Br-cGMP at pH 8.4 (green and orange columns, respectively; n = 8). All of the TASK3 currents are normalized by that obtained before the application of 8-Br-cGMP at pH 8.4. #p < 0.05, two-way ANOVA followed by Fisher’s PLSD. F, The normalized TASK1 currents recorded before and during the application of 8-Br-cGMP at pH 7.4 (blue and red columns, respectively) and those recorded before and during the application of 8-Br-cGMP at pH 8.4 (green and orange columns, respectively; n = 5). All of the TASK1 currents are normalized by that obtained before the application of 8-Br-cGMP at pH 8.4. #p < 0.05, two-way ANOVA followed by Fisher’s PLSD.
	Figure 9. Recruitment of MNs in the dl-TMN caused by the stimulation of presumed Ia afferents. Aa, A bright-field image of a brainstem slice including the dl-TMN. Ab, A resting light intensity image of the dl-TMN. B, C, Sample pseudocolor images of optical responses induced by stimulation of the dorsal part of the TMN before (B) and after (C) the application of 8-Br-cGMP. D, Traces representing averaged temporal profiles obtained before and after the application of 8-Br-cGMP (n = 5). Upward arrows indicate the time points at which the stimuli were applied. Downward arrows indicate the time points at which the respective pseudocolor images were obtained (B, C). E, The normalized peak amplitudes of optical responses induced by the 1st and 10th stimuli before and after the application of 8-Br-cGMP. #p < 0.02, two-way ANOVA followed by Fisher’s PLSD.
	Figure 10. Rank-ordered recruitment of αMNs in the dl-TMN and its modulation by NO inputs. A, During the isometric contraction of JC muscles, αMNs in the dl-TMN are recruited in rank order from smaller to larger αMNs in response to the activation of Ia afferents. B, When NO is released in the TMN by the activity of nitrergic neurons, IRs in small αMNs are decreased, and subsequently increase the spike-onset latencies. By contrast, in relatively large αMNs, NO either hardly changes or slightly increases IRs, subsequently causing no increase or a slight decrease in the spike-onset latencies, while it facilitates recruitment of the largest αMNs. Consequently, NO causes a more synchronous activation of smaller and larger αMNs. C, Alteration of the recruitment of small and large αMNs in the dl-TMN by the activation of NO inputs. T0, Recruitment-onset time; Th, time at which half of αMNs are recruited and from which large αMNs start to be recruited; Tp, time at which all the αMNs are recruited; T’0, recruitment onset time after the activation of NO inputs; T’p, time at which all the αMNs are recruited after the activation of NO inputs. D, Schematic diagram of the slow and fast rank-ordered recruitment observed before and after the activation of NO inputs, respectively. Slow precise and fast ballistic force increases can be achieved through the modulation of TASK1/TASK3 channels by NO inputs.

Abudara, Nitric oxide as an anterograde neurotransmitter in the trigeminal motor pool. 2002, Pubmed

Abudara, Nitric oxide as an anterograde neurotransmitter in the trigeminal motor pool. 2002, Pubmed
Bae, Quantitative ultrastructural analysis of glycine- and gamma-aminobutyric acid-immunoreactive terminals on trigeminal alpha- and gamma-motoneuron somata in the rat. 2002, Pubmed
Bawa, Recruitment order of motoneurons in stretch reflexes is highly correlated with their axonal conduction velocity. 1984, Pubmed
Bayliss, The TASK family: two-pore domain background K+ channels. 2003, Pubmed
Berg, Motoneurons express heteromeric TWIK-related acid-sensitive K+ (TASK) channels containing TASK-1 (KCNK3) and TASK-3 (KCNK9) subunits. 2004, Pubmed
Bredt, Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. 1991, Pubmed
Burke, Group Ia synaptic input to fast and slow twitch motor units of cat triceps surae. 1968, Pubmed
Burke, Motor unit properties and selective involvement in movement. 1975, Pubmed
Card, The motor trigeminal nucleus of the rat: analysis of neuronal structure and the synaptic organization of noradrenergic afferents. 1986, Pubmed
Collins, Heterogeneity of group Ia synapses on homonymous alpha-motoneurons as revealed by high-frequency stimulation of Ia afferent fibers. 1984, Pubmed
CURTIS, Synaptic action during and after repetitive stimulation. 1960, Pubmed
Czirják, Formation of functional heterodimers between the TASK-1 and TASK-3 two-pore domain potassium channel subunits. 2002, Pubmed , Xenbase
Dascal, The use of Xenopus oocytes for the study of ion channels. 1987, Pubmed , Xenbase
Dessem, Morphology of jaw-muscle spindle afferents in the rat. 1989, Pubmed
ECCLES, Electrophysiological studies on gamma motoneurones. 1960, Pubmed
Enyedi, Molecular background of leak K+ currents: two-pore domain potassium channels. 2010, Pubmed
Grimwood, Effects of afferent firing frequency on the amplitude of the monosynaptic EPSP elicited by trigeminal spindle afferents on trigeminal motoneurones. 1995, Pubmed
Grinvald, Real-time optical imaging of naturally evoked electrical activity in intact frog brain. , Pubmed
Gustafsson, Relations among passive electrical properties of lumbar alpha-motoneurones of the cat. 1984, Pubmed
Gustafsson, An investigation of threshold properties among cat spinal alpha-motoneurones. 1984, Pubmed
Heckman, Computer simulations of the effects of different synaptic input systems on motor unit recruitment. 1993, Pubmed
Heckman, Analysis of effective synaptic currents generated by homonymous Ia afferent fibers in motoneurons of the cat. 1988, Pubmed
Henneman, The size principle and its relation to transmission failure in Ia projections to spinal motoneurons. 1991, Pubmed
HENNEMAN, FUNCTIONAL SIGNIFICANCE OF CELL SIZE IN SPINAL MOTONEURONS. 1965, Pubmed
Honig, Alpha-motoneuron EPSPs exhibit different frequency sensitivities to single Ia-afferent fiber stimulation. 1983, Pubmed
Inanobe, Interaction between the RGS domain of RGS4 with G protein alpha subunits mediates the voltage-dependent relaxation of the G protein-gated potassium channel. 2001, Pubmed , Xenbase
Kang, Involvement of persistent Na+ current in spike initiation in primary sensory neurons of the rat mesencephalic trigeminal nucleus. 2007, Pubmed
Kang, Functional expression of TASK-1/TASK-3 heteromers in cerebellar granule cells. 2004, Pubmed
Kang, Nitric oxide activates leak K+ currents in the presumed cholinergic neuron of basal forebrain. 2007, Pubmed
Karschin, Expression pattern in brain of TASK-1, TASK-3, and a tandem pore domain K(+) channel subunit, TASK-5, associated with the central auditory nervous system. 2001, Pubmed , Xenbase
Lauritzen, K+-dependent cerebellar granule neuron apoptosis. Role of task leak K+ channels. 2003, Pubmed
Leonoudakis, An open rectifier potassium channel with two pore domains in tandem cloned from rat cerebellum. 1998, Pubmed , Xenbase
Lesage, Pharmacology of neuronal background potassium channels. 2003, Pubmed
Limwongse, Cell body locations and axonal pathways of neurons innervating muscles of mastication in the rat. 1977, Pubmed
Lund, Mastication and its control by the brain stem. 1991, Pubmed
Mizuno, Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase. 1975, Pubmed
Moschovakis, The size and dendritic structure of HRP-labeled gamma motoneurons in the cat spinal cord. 1991, Pubmed
Peshori, EPSP amplitude modulation at the rat Ia-alpha motoneuron synapse: effects of GABAB receptor agonists and antagonists. 1998, Pubmed
Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR. 2001, Pubmed
Pose, Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat. 2005, Pubmed
Rokx, Identification of alpha and gamma trigeminal motoneurons by the vibratome paraplast technique for HRP histochemistry. 1987, Pubmed
Rokx, Distribution of innervating neurons of masticatory muscle spindles in the rat: an HRP study. 1985, Pubmed
Sato, Differential columnar processing in local circuits of barrel and insular cortices. 2008, Pubmed
Shigenaga, Morphology of single mesencephalic trigeminal neurons innervating masseter muscle of the cat. 1988, Pubmed
Shigenaga, Two types of jaw-muscle spindle afferents in the cat as demonstrated by intra-axonal staining with HRP. 1990, Pubmed
Talley, Cns distribution of members of the two-pore-domain (KCNK) potassium channel family. 2001, Pubmed
Toyoda, Protein kinase G dynamically modulates TASK1-mediated leak K+ currents in cholinergic neurons of the basal forebrain. 2010, Pubmed
Toyoda, cGMP activates a pH-sensitive leak K+ current in the presumed cholinergic neuron of basal forebrain. 2008, Pubmed
Travers, Neurotransmitter phenotypes of intermediate zone reticular formation projections to the motor trigeminal and hypoglossal nuclei in the rat. 2005, Pubmed
Wenker, Nitric oxide activates hypoglossal motoneurons by cGMP-dependent inhibition of TASK channels and cGMP-independent activation of HCN channels. 2012, Pubmed
Yabuta, Light microscopic observations of the contacts made between two spindle afferent types and alpha-motoneurons in the cat trigeminal motor nucleus. 1996, Pubmed
Yemm, The orderly recruitment of motor units of the masseter and temporal muscles during voluntary isometric contraction in man. 1977, Pubmed