Tomohiko Irie

Cartwheel interneurons of the dorsal cochlear nucleus (DCN) potently suppress multisensory signals that converge with primary auditory afferent input, and thus regulate auditory processing. Noradrenergic fibers from locus coeruleus project to the DCN, and α2-adrenergic receptors inhibit spontaneous spike activity but simultaneously enhance synaptic strength in cartwheel cells, a dual effect leading to enhanced signal- to-noise for inhibition. However, the ionic mechanism of this striking modulation is unknown. We generated a glycinergic neuron-specific knockout of the Na+ leak channel NALCN, and found that its presence was required for spontaneous firing in cartwheel cells. Activation of α2-adrenergic receptors inhibited both NALCN and spike generation, and this modulation was absent in the NALCN knockout. Moreover, α2-dependent enhancement of synaptic strength was also absent in the knockout. GABAB receptors mediated inhibition through NALCN as well, acting on the same population of channels as α2 receptors, suggesting close apposition of both receptor subtypes with NALCN. Thus, multiple neuromodulatory systems determine the impact of synaptic inhibition by suppressing the excitatory leak channel, NALCN.

Cartwheel interneurons of the dorsal cochlear nucleus (DCN) potently suppress multisensory signals that converge with primary auditory afferent input, and thus regulate auditory processing. Noradrenergic fibers from locus coeruleus project to the DCN, and α2-adrenergic receptors inhibit spontaneous spike activity but simultaneously enhance synaptic strength in cartwheel cells, a dual effect leading to enhanced signal- to-noise for inhibition. However, the ionic mechanism of this striking modulation is unknown. We generated a glycinergic neuron-specific knockout of the Na ⁺ leak channel NALCN, and found that its presence was required for spontaneous firing in cartwheel cells. Activation of α2-adrenergic receptors inhibited both NALCN and spike generation, and this modulation was absent in the NALCN knockout. Moreover, α2-dependent enhancement of synaptic strength was also absent in the knockout. GABA _B receptors mediated inhibition through NALCN as well, acting on the same population of channels as α2 receptors, suggesting close apposition of both receptor subtypes with NALCN. Thus, multiple neuromodulatory systems determine the impact of synaptic inhibition by suppressing the excitatory leak channel, NALCN.

Among all voltage-gated potassium (Kv) channels, Kv2 channels are the most widely expressed in the mammalian brain. However, studying Kv2 in neurons has been challenging because of a lack of high-selective blockers. Recently, a peptide toxin, guangxitoxin-1E (GxTX), has been identified as a specific inhibitor of Kv2, thus facilitating the study of Kv2 in neurons. The mammalian dorsal cochlear nucleus (DCN) integrates auditory and somatosensory information. In the DCN, cartwheel inhibitory interneurons receive excitatory synaptic inputs from parallel fibers conveying somatosensory information. The activation of parallel fibers drives action potentials in the cartwheel cells up to 130 Hzin vivo, and the excitation of cartwheel cells leads to the strong inhibition of principal cells. Therefore, cartwheel cells play crucial roles in monaural sound localization and cancelling detection of self-generated sounds. However, how Kv2 controls the high-frequency firing in cartwheel cells is unknown. In this study, we performed immunofluorescence labeling with anti-Kv2.1 and anti-Kv2.2 antibodies using fixed mouse brainstem slice preparations. The results revealed that Kv2.1 and Kv2.2 were largely present on the cartwheel cell body membrane but not on the axon initial segment (AIS) nor the proximal dendrite. Whole-cell patch-clamp recordings using mouse brainstem slice preparation and GxTX demonstrated that blockade of Kv2 induced failure of parallel fiber-induced action potentials when parallel fibers were stimulated at high frequencies (30–100 Hz). Thus, somatic Kv2 in cartwheel cells regulates the action potentials in a frequency-dependent manner and may play important roles in the DCN function.

Small-conductance Ca²⁺-activated K⁺ (SK) and large-conductance voltage- and Ca²⁺-activated K⁺ (BK) channels are Ca²⁺-activated K⁺ channels that control action potential firing in diverse neurons in the brain. In cartwheel cells of the dorsal cochlear nucleus, blockade of either channel type leads to excessive production of spike bursts. In the same cells, P/Q-type Ca²⁺ channels in plasma membrane and ryanodine receptors in endoplasmic reticulum supply Ca²⁺ to BK channels through Ca²⁺ nanodomain signaling. In this study, voltage-clamp experiments were performed in cartwheel cells in mouse brain slices to examine the Ca²⁺ signaling pathways underlying activation of SK channels. As with BK channels, SK channels required the activity of P/Q-type Ca²⁺ channels. However, this signaling occurred across Ca²⁺ micro- rather than nanodomain distances and was independent of Ca²⁺ release from endoplasmic reticulum. These differential modes of activation may lead to distinct time courses of the two K⁺ currents and therefore control excitability of auditory neurons across different timescales. NEW & NOTEWORTHY This study has shown for the first time that in cartwheel cells of the dorsal cochlear nucleus, small-conductance Ca²⁺-activated K⁺ (SK) channels were triggered by the activation of P/Q-type Ca²⁺ channels in which SK–P/Q-type coupling is mediated within the Ca²⁺ microdomains (loose coupling). Although Ca²⁺-induced Ca²⁺ release is able to activate large-conductance voltage- and Ca²⁺-activated K⁺ (BK) channels in cartwheel cells, it did not contribute to SK activation.

The metabolism of 4-methyl-2-mercaptobenzimidazole (4-MeMBI), 5-methyl-2-mercaptobenzimidazole (5-MeMBI), and 2-mercaptobenzimidazole (MBI) was examined in vitro in rat liver microsomes. The test chemicals were incubated in the presence of liver microsomes from male Sprague-Dawley rats, and their metabolism was analyzed by HPLC. The metabolism amount increased in an incubation time-dependent manner, and was similar among the test chemicals. SKF-525A, a non-selective inhibitor of cytochrome P450 (CYP) enzymes, decreased the metabolic rate of all the test chemicals, indicating the involvement of liver microsomal CYP enzymes. When liver microsomes from rats treated with CYP-inducers (β-naphthoflavone, phenobarbital, and isoniazid) were used, 4-MeMBI was more decreased than 5-MeMBI, particularly in the phenobarbital-treated group. These results, together with the reported inducibility of the drug-metabolizing activity by the test chemicals, partly explained the counteraction in the toxic effects between 4-MeMBI and 5-MeMBI in the in vivo study.