Hiroshi Kuba

Presynaptic activity regulates Na+ channel distribution at the axon initial segment

Deprivation of afferent inputs in neural circuits leads to diverse plastic changes in both pre- and postsynaptic elements that restore neural activity. The axon initial segment (AIS) is the site at which neural signals arise, and should be the most efficient site to regulate neural activity. However, none of the plasticity currently known involves the AIS. We report here that deprivation of auditory input in an avian brainstem auditory neuron leads to an increase in AIS length, thus augmenting the excitability of the neuron. The length of the AIS, defined by the distribution of voltage-gated Na+ channels and the AIS anchoring protein, increased by 1.7 times in seven days after auditory input deprivation. This was accompanied by an increase in the whole-cell Na+ current, membrane excitability and spontaneous firing. Our work demonstrates homeostatic regulation of the AIS, which may contribute to the maintenance of the auditory pathway after hearing loss. Furthermore, plasticity at the spike initiation site suggests a powerful pathway for refining neuronal computation in the face of strong sensory deprivation.

Axonal site of spike initiation enhances auditory coincidence detection

Neurons initiate spikes in the axon initial segment or at the first node in the axon. However, it is not yet understood how the site of spike initiation affects neuronal activity and function. In nucleus laminaris of birds, neurons behave as coincidence detectors for sound source localization and encode interaural time differences (ITDs) separately at each characteristic frequency (CF). Here we show, in nucleus laminaris of the chick, that the site of spike initiation in the axon is arranged at a distance from the soma, so as to achieve the highest ITD sensitivity at each CF. Na+ channels were not found in the soma of high-CF (2.5–3.3 kHz) and middle-CF (1.0–2.5 kHz) neurons but were clustered within a short segment of the axon separated by 20–50 μm from the soma; in low-CF (0.4–1.0 kHz) neurons they were clustered in a longer stretch of the axon closer to the soma. Thus, neurons initiate spikes at a more remote site as the CF of neurons increases. Consequently, the somatic amplitudes of both orthodromic and antidromic spikes were small in high-CF and middle-CF neurons and were large in low-CF neurons. Computer simulation showed that the geometry of the initiation site was optimized to reduce the threshold of spike generation and to increase the ITD sensitivity at each CF. Especially in high-CF neurons, a distant localization of the spike initiation site improved the ITD sensitivity because of electrical isolation of the initiation site from the soma and dendrites, and because of reduction of Na+-channel inactivation by attenuating the temporal summation of synaptic potentials through the low-pass filtering along the axon.

Calcium-dependent Phospholipid Scramblase Activity of TMEM16 Protein Family Members

Background: TMEM16A and 16B work as Cl− channel, whereas 16F works as phospholipid scramblase. The function of other TMEM16 members is unknown. Results: Using TMEM16F−/− cells, TMEM16C, 16D, 16F, 16G, and 16J were shown to be lipid scramblases. Conclusion: Some TMEM16 members are divided into two Cl− channels and five lipid scramblases. Significance: Learning the biochemical function of TMEM16 family members is essential to understand their physiological role. Asymmetrical distribution of phospholipids between the inner and outer plasma membrane leaflets is disrupted in various biological processes. We recently identified TMEM16F, an eight-transmembrane protein, as a Ca2+-dependent phospholipid scramblase that exposes phosphatidylserine (PS) to the cell surface. In this study, we established a mouse lymphocyte cell line with a floxed allele in the TMEM16F gene. When TMEM16F was deleted, these cells failed to expose PS in response to Ca2+ ionophore, but PS exposure was elicited by Fas ligand treatment. We expressed other TMEM16 proteins in the TMEM16F−/− cells and found that not only TMEM16F, but also 16C, 16D, 16G, and 16J work as lipid scramblases with different preference to lipid substrates. On the other hand, a patch clamp analysis in 293T cells indicated that TMEM16A and 16B, but not other family members, acted as Ca2+-dependent Cl− channels. These results indicated that among 10 TMEM16 family members, 7 members could be divided into two subfamilies, Ca2+-dependent Cl− channels (16A and 16B) and Ca2+-dependent lipid scramblases (16C, 16D, 16F, 16G, and 16J).

Tonotopic Specialization of Auditory Coincidence Detection in Nucleus Laminaris of the Chick

The interaural time difference (ITD) is a cue for localizing a sound source along the horizontal plane and is first determined in the nucleus laminaris (NL) in birds. Neurons in NL are tonotopically organized, such that ITDs are processed separately at each characteristic frequency (CF). Here, we investigated the excitability and coincidence detection of neurons along the tonotopic axis in NL, using a chick brainstem slice preparation. Systematic changes with CF were observed in morphological and electrophysiological properties of NL neurons. These properties included the length of dendrites, the input capacitance, the conductance of hyperpolarization-activated current, and the EPSC time course. In contrast to these gradients, the conductance of low-threshold K+ current and the expression of Kv1.2 channel protein were maximal in the central (middle-CF) region of NL. As a result, the middle-CF neuron had the smallest input resistance and membrane time constant, and consequently the fastest EPSP, and exhibited the most accurate coincidence detection. The specialization of middle-CF neurons as coincidence detectors may account for the high resolution of sound-source localization in the middle-frequency range observed in avians.

Short- and Long-Term Plasticity at the Axon Initial Segment

The axon initial segment (AIS) is a highly specialized neuronal subregion that is the site of action potential initiation and the boundary between axonal and somatodendritic compartments. In recent years, our understanding of the molecular structure of the AIS, its maturation, and its multiple fundamental roles in neuronal function has seen major advances. We are beginning to appreciate that the AIS is dynamically regulated, both over short timescales via adaptations in ion channel function, and long timescales via activity-dependent structural reorganization. Here, we review results from this emerging field highlighting how structural and functional plasticity relate to the development of the initial segment, and to neuronal disorders linked to AIS dysfunction.

Synaptic depression improves coincidence detection in the nucleus laminaris in brainstem slices of the chick embryo

Neurons in the nucleus laminaris detect the coincidence of binaural signals, and are the first neurons to calculate the interaural time difference for the sound source localization in birds. In this paper, we have studied contributions of synaptic depression to the coincidence detection in the nucleus laminaris in a slice preparation of the chick embryo (E16–18), using the whole‐cell patch recording technique. Under voltage clamp, EPSCs decreased progressively in their amplitude during the course of tetanic stimuli. This synaptic depression was primarily ascribed to the reduction of transmitter release from the presynaptic terminal, because the depression was decreased by reducing transmitter release with 2.5 µm Cd2+ but was not affected by reducing desensitization of postsynaptic AMPA receptors with 20 µm cyclothiazide. Under current clamp, trains of 10 stimuli of 100 Hz were applied bilaterally with changing the time intervals systematically between both sides. Response window, defined as the time interval corresponding to the half‐maximum firing probability, was narrowed during the course of the stimulus train, and this occurred in parallel with a decrease in the EPSP amplitude. In addition, the reduction of the EPSP amplitude due to 2.5 µm Cd2+ or 2 µm CNQX improved the accuracy of coincidence detection. These results indicate that the synaptic depression may improve the coincidence detection in the chick laminaris neurons.

Sound-Intensity-Dependent Compensation for the Small Interaural Time Difference Cue for Sound Source Localization

Interaural time difference (ITD) is a major cue for sound source localization. However, animals with small heads experience small ITDs, making ITD detection difficult, particularly for low-frequency sound. Here, we describe a sound-intensity-dependent mechanism for compensating for the small ITD cues in the coincidence detector neurons in the nucleus laminaris (NL) of the chicken aged from 3 to 29 d after hatching. The hypothesized compensation mechanisms were confirmed by simulation. In vivo single-unit recordings revealed an improved contrast of ITD tuning in low-best-frequency (<1 kHz) NL neurons by suppressing the firing activity at the worst ITD, whereas the firing rate was increased with increasing sound intensity at the best ITD. In contrast, level-dependent suppression was so weak in the middle- and high-best-frequency (≥1 kHz) NL neurons that loud sounds led to increases in firing rate at both the best and the worst ITDs. The suppression of firing activity at the worst ITD in the low-best-frequency neurons required the activation of the superior olivary nucleus (SON) and was eliminated by electrolytic lesions of the SON. The frequency-dependent suppression reflected the dense projection from the SON to the low-frequency region of NL. Thus, the small ITD cues available in low-frequency sounds were partly compensated for by a sound-intensity-dependent inhibition from the SON.

Development of membrane conductance improves coincidence detection in the nucleus laminaris of the chicken

Coincidence detection at the nucleus laminaris (NL) of a chicken was improved between embryos (embryonic days (E) 16 and 17) and chicks (post‐hatch days (P) 2–7) in slice preparations. Electrical stimuli were applied bilaterally to the projection fibres to the NL at various intervals. The response window corresponding to the temporal separation of electrical stimuli that resulted in half‐maximal firing probability was adopted as the measure of coincidence detection, and was narrower in chicks (1.4 ms) than in embryos (3.9 ms). Between these two ages, the membrane time constant of NL neurons was reduced from 18.4 to 3.2 ms and the membrane conductance was increased 5‐fold, while no difference was measured in the input capacitance. Evoked EPSCs decayed slightly faster in chicks, while the size and the time course of miniature EPSCs were unchanged. Action potentials had lower thresholds and larger after‐hyperpolarization in chicks than in embryos. Dendrotoxin‐I depolarized cells and increased their input resistance significantly at both ages, eliminated the after‐hyperpolarization, and delayed the decay phase of action potentials, indicative of the expression of low‐threshold K+ channels. Cs+ hyperpolarized the cells, increased the input resistance and eliminated sags during hyperpolarization at both ages, while the hyperpolarization sag was affected by neither Ba2+ nor TEA. These data indicate the expression of hyperpolarization‐activated cation channels. Between these two ages, the maximum conductance of low‐threshold K+ channels increased 4‐fold to about 16 nS, and hyperpolarization‐activated channels increased 6‐fold to about 10 nS. Improvement of coincidence detection correlated with the acceleration of the EPSP time course as a result of the increase of these conductances.

Roles of axonal sodium channels in precise auditory time coding at nucleus magnocellularis of the chick

How the axonal distribution of Na+ channels affects the precision of spike timing is not well understood. We addressed this question in auditory relay neurons of the avian nucleus magnocellularis. These neurons encode and convey information about the fine structure of sounds to which they are tuned by generating precisely timed action potentials in response to synaptic inputs. Patterns of synaptic inputs differ as a function of tuning. A small number of large inputs innervate high‐ and middle‐frequency neurons, while a large number of small inputs innervate low‐frequency neurons. We found that the distribution and density of Na+ channels in the axon initial segments varied with the synaptic inputs, and were distinct in the low‐frequency neurons. Low‐frequency neurons had a higher density of Na+ channels within a longer axonal stretch, and showed a larger spike amplitude and whole‐cell Na+ current than high/middle‐frequency neurons. Computer simulations revealed that for low‐frequency neurons, a large number of Na+ channels were crucial for preserving spike timing because it overcame Na+ current inactivation and K+ current activation during compound EPSPs evoked by converging small inputs. In contrast, fewer channels were sufficient to generate a spike with high precision in response to an EPSP induced by a single massive input in the high/middle‐frequency neurons. Thus the axonal Na+ channel distribution is effectively coupled with synaptic inputs, allowing these neurons to convey auditory information in the timing of firing.

Hyperpolarization-Activated Cyclic Nucleotide-Gated Cation Channels Regulate Auditory Coincidence Detection in Nucleus Laminaris of the Chick

Coincidence detection of bilateral acoustic signals in nucleus laminaris (NL) is the first step in azimuthal sound source localization in birds. Here, we demonstrate graded expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels along the tonotopic axis of NL and its role in the regulation of coincidence detection. Expression of HCN1 and HCN2, but not HCN3 or HCN4, was detected in NL. Based on measurement of both subtype mRNA and protein, HCN1 varied along the tonotopic axis and was minimal in high-characteristic frequency (CF) neurons. In contrast, HCN2 was evenly distributed. The resting conductance was larger and the steady-state activation curve of Ih was more positive in neurons of middle to low CF than those of high CF, consistent with the predominance of HCN1 channels in these neurons. Application of 8-Br-cAMP or noradrenaline generated a depolarizing shift of the Ih voltage activation curve. This shift was larger in neurons of high CF than in those of middle CF. The shift in the activation voltage of Ih depolarized the resting membrane, accelerated the EPSP time course, and significantly improved the coincidence detection in neurons of high CF, suggesting that Ih may improve the localization of sound sources.