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. 2003 Feb 7;548(Pt 1):219–232. doi: 10.1113/jphysiol.2002.036285

Developmental changes in membrane excitability and morphology of neurons in the nucleus angularis of the chicken

Iwao Fukui 1, Harunori Ohmori 1
PMCID: PMC2342792  PMID: 12576492

Abstract

In order to understand how sound intensity information is extracted and processed in the auditory nuclei, we investigated the neuronal excitability in the nucleus angularis (NA) of the chicken (P0–5) and the chicken embryo (E16–21). In embryos, neurons fired basically in three patterns in response to current injections: the onset pattern (19 %), the tonic pattern (52 %) and the pause pattern (29 %). After hatching, neurons fired either in the tonic pattern (83 %) or in the onset pattern (17 %). In both pre- and post-hatch periods, multiple firing neurons (tonic and pause) increased the maximum rate of rise of the action potential 2.6-fold, the fall 3.9-fold, and the maximum firing frequency 4-fold, and shifted the threshold potential to be more negative. After hatching, the firing frequency of tonic neurons reached a maximum at about 650 Hz. Application of TEA (1 mm) reduced the firing frequency, broadened action potentials and reduced the maximum rate of fall, but the threshold current was not changed. Dendrotoxin-I (DTX, 100 nm) reduced the threshold current. Application of DTX induced the onset neuron to fire repetitively. Branching patterns of auditory nerve fibres (ANFs) in NA were visualized by labelling with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Di-I) placed within the cochlea. Di-I placed near the apex of the cochlea labelled the ventral part of the NA, and Di-I placed in the base labelled the dorso-lateral part. Tonic neurons labelled with biocytin extended dendrites in parallel with the projection of ANFs in the nucleus after hatching. ANF activity of a limited range of characteristic sound frequencies is thought to be extracted by tonic neurons and encoded into firing frequencies proportional to the strength of the input.

In the avian cochlear nuclei, the auditory nerve fibre (ANF) makes a bifurcation, with one branch innervating the nucleus angularis (NA) and the other the nucleus magnocellularis (NM). The terminal morphology of the branches of the ANF is different in these two nuclei: bouton-type terminals are found in NA and end-bulb type terminals (the end-bulb of Held) are found in NM (Jhaveri & Morest, 1982a-c). In the barn owl, it is thought that NA extracts and processes sound intensity information, while NM does so for sound temporal information (Sullivan & Konishi, 1984; Takahashi et al. 1984; Takahashi & Konishi, 1988a,b). NA neurons make projections to the lateral lemniscal nuclei and to the inferior colliculus (Takahashi & Konishi, 1988a). The posterior division of the dorsal nucleus of the lateral lemniscus (LLDp, formerly VLVp) is the first site where the bilateral sound intensity information is compared (Moiseff & Konishi, 1983; Manley et al. 1988; Mogdans & Knudsen, 1994). The sound timing information is extracted in NM and is transmitted to the nucleus laminaris (NL), where the interaural timing differences are first computed. These two parallel pathways encoding the level and the phase of sound information converge in the external nucleus of the inferior colliculus (Knudsen & Konishi, 1978; Brainard et al. 1992), where the two sound features are utilized as cues to detect the sound source location.

Warchol & Dallos (1990) investigated firing properties of NA neurons in an in vivo chicken preparation (10–28 postnatal days, P10–28). They demonstrated three types of post-stimulus time histograms (PSTHs): chopper, primary-like and onset. The chopper (52 %) and the primary-like (40 %) PSTHs were dominant. Morphological studies of NA neurons have recently been made in the pigeon (Häusler et al. 1999), in the barn owl (Soares & Carr, 2001) and in the embryonic chicken (Soares et al. 2002). In the barn owl, NA contained four major types of neurons: planar, radiate, vertical and stubby (Soares & Carr, 2001); in the pigeon five major types of neurons were identified: large, medium, small multipolar, medium bipolar and stubby (Häusler et al. 1999). In the late chicken embryo (embryonic days 16–19, E16–19), Soares et al. (2002) studied intrinsic firing patterns in relation to the morphology of the neurons, classifying the firing patterns into three major types: one-spike cells, damped cells and tonic cells. Tonic cells were further divided into three subtypes, I, II and III. These cells showed some characteristic morphology: one-spike cells were generally stubby, damped cells were generally planar, tonic I and III cells were generally radiate and tonic II cells were generally vertical.

In this paper, we report changes in the firing patterns of NA neurons during development in late chicken embryos (E16–21) and in chickens after hatching (P0–5). In neurons of post-hatch chickens, the effects of potassium channel blockers on firing properties are described. We also discuss the branching patterns of neurons in relation to Di-I-labelled projection patterns of ANFs in the NA.

Methods

Slice preparation

Thin slices of the brainstem were prepared from the chicken embryo (E16–21) and from the post-hatch chicken (P0–5). Chickens were deeply anaesthetized with halothane (fluothain, Takeda, Osaka, Japan) and embryos were anaesthetized by ice cooling the egg before decapitation. These procedures conformed to the guiding principles for the care and use of animals in the field of physiological sciences set by the Japanese Physiological Society. Brains were quickly removed and placed in ice-cold oxygenated (95 % O2−5 % CO2) dissection saline containing (mm): 250 sucrose, 2.5 KCl, 0.5 CaCl2, 5 MgSO4, 26 NaHCO3, 1.25 NaH2PO4 and 17 d-glucose. Transverse sections (300–400 μm) containing NA and NM were obtained by a tissue slicer (Zero-1, Dosaka, Kyoto, Japan). These slices were then placed in oxygenated artificial cerebrospinal fluid (ACSF) at 37 °C, and incubated for at least 1 h before experiments. ACSF contained (mm): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2PO4 and 17 d-glucose.

Electrophysiological recording

Slices were transferred to a recording chamber on the stage of an upright microscope (BX50WI, Olympus, Japan) and were perfused continuously with ACSF at the rate of 2–4 ml min−1 by a peristaltic pump (P-3, Pharmacia, USA). The temperature of the bathing medium filling the recording chamber was monitored by a thermistor probe and controlled by a water-cooled Peltier system (DTC-300, Dia Medical, Tokyo, Japan), and was maintained during the experiment at about the body temperature of the chicken (40–41 °C). This degree of temperature control was possible because we used an aluminum-made recording chamber coated with Teflon to facilitate heat conduction. A cover glass was glued to the bottom of the chamber with silicone resin (Sylgard, Dow Corning). Neurons in NA were visualized with a ×40 objective lens with Nomarski optics equipped with an IR-CCD camera (C5999, Hamamatsu Photonics, Hamamatsu, Japan).

The whole-cell recording pipette solution contained (mm): 130 potassium gluconate, 10 Hepes, 0.2 EGTA, 10 KCl, 5 NaCl, 1 MgCl2, 5 ATP, 5 creatine phosphate, with 1 mg ml−1 biocytin (Sigma, pH adjusted to 7.2). The electrode resistance was 2–5 MΩ in the bath, and was 6–15 MΩ during recording. Current clamp was achieved by using an Axopatch 200B amplifier (Axon Instruments, USA) in the fast mode. Data were filtered at 5 kHz with a 4-pole low-pass Bessel filter and were sampled at 20–100 kHz. The liquid junction potential (10 mV) was corrected. Tetraethylammonium chloride (TEA, Nakalai, Japan) and dendrotoxin-I (DTX, Alomone Laboratories, USA) were added to the ACSF in some experiments.

Immediately after the start of whole-cell recordings, the firing patterns of neurons were tested using current injections, and then several basic membrane properties were measured. Most neurons fired stably for more than 10 min. Data were only included in the statistics if the resting potential was more negative than −50 mV. Data were recorded from 136 NA neurons (77 from the late chicken embryos and 59 from chickens after hatching). Data were presented as means ±s.e.m. (n = number of cells). The following basic parameters were measured. The membrane resistance was measured from the slope of the I-V relationship obtained by applying small hyperpolarizing current injections. The membrane time constant was measured by injecting a hyperpolarizing current of between −0.05 and −0.1 nA. The spike threshold potential was taken as the voltage at which the rapid upstroke of the action potential began. The threshold current was obtained by giving stepwise current injections in 0.05–0.1 nA increments. The spike amplitude was measured as the peak voltage of the action potential from the spike threshold. The spike half-width was measured as the spike duration measured at the spike half-amplitude, as marked in Fig. 4A. The spike after-hyperpolarization amplitude was measured as the difference between the peak voltage of the hyperpolarization potential and the spike threshold. The maximum rate of rise and fall, spike amplitude, spike half-width and spike after-hyperpolarization amplitude were measured from the first spike in a burst. For these measurements, action potentials were induced by injecting a slightly (0.1–0.2 nA) larger current than the threshold intensity. Statistical evaluations (P) were made by Welch's unpaired t test or Student's paired t test.

Figure 4. Development of membrane excitability.

Figure 4

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A, first action potentials of tonic neurons generated by the threshold intensity of current injection, recorded from an embryo (E16) and a chicken after hatching (P1), are superimposed. The thresholds of these neurons were −43 mV for E16 and −54 mV for P1. Vertical bars show the spike half-amplitude. B, the spike half-width decreased during development and reached about 0.2 ms in the chicken after hatching. The number of cells is indicated in parentheses and is the same for C and D. C, the maximum rate of rise (MRR) and fall (MRF) increased during development. □, onset neurons; ▪, multiple firing neurons in B and C. D, ratios of MRR/MRF were plotted during three stages of development for multiple firing neurons (•) and onset neurons (○). *P < 0.05; **P < 0.01; n.s. no significant difference (P ≥ 0.05).

Di-I labelling

Chickens (P2) were anaesthetized with ketamine, and crystals of Di-I were placed in the cochlear organ. The cochlear organ was approached from the oval window after removing the stapes. After placement of Di-I, the oval window and the affected part of the middle ear were sealed with Sylgard. After 2 days, the projections to the NA were observed in slices.

Intracelluler labelling with biocytin

Slices containing biocytin-filled cells were fixed overnight in the solution containing 4 % formalin, 0.003 % glutaraldehyde and 75 % picric acid. These slices were then processed by washing 3 times with PBS (5 min); then with 50 % methanol-50 % acetone (5 min, −20 °C); and then 3 times with 0.2 % Triton X-100 in PBS (5 min). PBS contained (mm): 137 NaCl, 2.7 KCl, 8.1 Na2HPO4 and 1.5 KH2PO4. The slices were then incubated first in avidin-horseradish peroxidase ABC solution (Vector, USA) for 2 h, and then in a 3,3′-diaminobenzidine (DAB; Wako, Japan) solution containing 0.2 % DAB, 0.02 % NiCl2, 0.001 % H2O2 and 50 mm Tris (pH 7.4). The slices were mounted on gelatin-coated glass slides and were dried for 1 day, and then dehydrated with ethanol, cleared with xylene and cover-slipped. The soma size of neurons was measured using NIH Image software. Seventy-three neurons were stained with biocytin, of which 21 were from P0–5 chickens, and 52 were from E16–21 chicken embryos. The aspect ratio of dendritic projections was calculated as the ratio of the length of dendrites parallel to ANF to the length of dendrites perpendicular to it. The orientation of ANF projections in the NA was shown by Di-I labelling (Fig. 10).

Figure 10. Branching pattern of ANFs in NA.

Figure 10

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A, projection of ANFs was visualized as Di-I fluorescence in the NA. The continuous lines show the right dorsal corner of the transverse hemi-section of the brainstem. The outline of NA is indicated as dashed lines for the rostral (1–3) and caudal (4–6) planes. The intensity of fluorescence was reversed to show black on a white background. Projection pairs 1 and 4, and 3 and 6 were obtained from the same chicken and the projection pair 2 and 5 were from different animals. Regions of auditory nerve projection in the NA varied according to the where Di-I was placed in the cochlear organ. B, summary of projection patterns in A. Shaded areas indicate thick projections of ANFs, and dashed lines traced the boundaries of Di-I-positive regions. Similar results were obtained from 2 other chickens (data not shown, total 6 chickens).

Results

Changes in the firing properties of NA neurons during development

When NA neurons were injected with depolarizing currents of long duration, they fired either repetitively during the current injection (multiple firing neurons) or with a single spike at the onset of current injections (onset neurons). Figure 1A shows the firing patterns found after hatching (P2), and Fig. 1B shows the firing patterns found during embryonic days E16–17. After hatching, all multiple firing neurons generated action potentials continuously during current injections (tonic neurons). However, in chicken embryos, there were two firing patterns among multiple firing neurons (tonic and pause neurons).

Figure 1. Development of firing properties.

Figure 1

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Current-clamp recordings from a NA neuron in response to depolarizing current pulses. Sample traces show the typical firing patterns recorded in a chicken after hatching (A) and in a chicken embryo (B). In the chicken after hatching, NA neurons fired either tonically (Aa) or at the onset of current injections (Ab). In the embryo, neurons fired in three patterns: tonic (Ba and Bb), onset (Bd) and pause (Bc). Some tonic neurons showed a damped firing when current intensity was high (Bb). Injected currents and resting potentials are indicated to the left of each voltage response.

Tonic neurons generated a single or a few action potentials when the current intensity was low, and repetitive firing occurred with a slight increase in current intensity (Fig. 1Aa, Ba and Bb). There was no noticeable reduction in spike height during burst firing in the post-hatch chicken (Fig. 1Aa). However, in the embryo, action potential amplitude progressively decreased in most tonic neurons (Fig. 1Ba), and in some only a damped oscillation remained in the late phase when the current intensity was high (Fig. 1Bb).

Pause neurons generated action potentials with an initial delay or a pause after the first spike during current injection (Fig. 1Bc). Pause neurons also generated multiple action potentials in bursts, as did tonic neurons when the current intensity was high. Onset neurons generated one or a few action potentials at the onset of current injection even at higher current intensities (Fig. 1Ab and Bd).

Figure 2 illustrates the percentage of cells showing each firing pattern found at three developmental stages: E16–17 (n = 30 cells), E19–21 (n = 47 cells) and P0–5 (n = 59 cells). In the E16–17 chicken embryos, the pause neurons (33.3 %) and the tonic neurons (46.7 %) were dominant. During development, the percentage of tonic neurons increased and no pause neurons were found in chickens after hatching (P0–5), when tonic neurons comprised 83.1 % of the total. The percentage of onset neurons was nearly constant during development: 20.0 % for E16–17, 19.1 % for E19–21 and 16.9 % for P0–5.

Figure 2. Proportions of firing patterns during development.

Figure 2

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The percentages of each firing pattern are indicated for E16–17, E19–21 and P0–5 chickens. The number of cells is indicated in parentheses. The percentage of pause neurons decreased with development and in the chicken after hatching no pause neurons were found (P0–5).

We will describe in detail later in this paper the firing properties of tonic neurons and onset neurons found in the chicken after hatching.

Changes in basic membrane properties of neurons during development

Figure 3 summarizes the resting membrane properties and the spike threshold during development. The resting membrane potential became more negative with development (Fig. 3A; between E16–17 and P0–5, P < 0.01); however, the difference was not significant between neurons with different firing patterns (Fig. 3A; •, multiple firing cells; ○, onset cells; P > 0.3). The spike threshold of the chicken was lower than that of the embryo (Fig. 3A, P < 0.01 between E16–17 and E19–21, and between E19–21 and P0–5). The difference in spike threshold between onset and multiple firing neurons was significant in the chicken after hatching (−53.8 ± 0.8 mV for multiple firing neurons, n = 49; −48.3 ± 1.2 mV for onset neurons, n = 10, P < 0.01), but not in the embryos (P > 0.07).

Figure 3. Development of basic membrane properties.

Figure 3

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A, both resting potential (circles) and threshold potential (triangles) decreased during development. Open symbols indicate onset neurons and filled symbols multiple firing neurons. The number of cells is indicated in parentheses and is the same in all panels. The membrane resistance (B) and the membrane time constant (C) decreased during development. □, onset neurons; ▪, multiple firing neurons. **P < 0.01; n.s. no significant difference (P ≥ 0.05).

The membrane resistance and membrane time constant at near-resting membrane potential decreased during development. Figure 3B shows the membrane resistance and Fig. 3C the time constant for multiple firing neurons (▪) and onset neurons (□). The membrane resistance of onset neurons was consistently smaller than that of multiple firing neurons, and the difference was statistically significant at E19–21 and P0–5 (P < 0.01). However, the difference was not significant at E16–17 (P > 0.2). Onset neurons had a significantly shorter membrane time constant than multiple firing neurons in the late E19–21 embryo and after hatching (P0–5) (P < 0.05), but the difference was not significant at E16–17 (P > 0.5). There were no significant differences in the resting potential, threshold potential, membrane resistance and membrane time constant between pause and tonic neurons in the embryo (P > 0.07).

Development of the capability of action potential generation

Figure 4A shows the first action potential of tonic neurons from the embryo (E16) and the chicken (P1). Action potentials were superimposed by matching the threshold and the time in order to facilitate comparison. The action potential was faster in the chicken than in the embryo. The spike amplitude of tonic neurons in the chicken after hatching (48.0 ± 1.6 mV, n = 49) was slightly larger than that of multiple firing neurons in the embryo (37.7 ± 1.3 mV for E16–17, n = 24, P < 0.001). Several properties of action potentials were measured in neurons of different firing patterns and their averages at four stages of development are shown in Fig. 4. The spike half-width decreased during development (Fig. 4B). The maximum rates of rise and fall (MRR and MRF) increased during development (Fig. 4C).

The relative size of MRR and MRF was calculated in each multiple firing neuron and plotted against age in Fig. 4D (•). MRR of the multiple firing neurons was consistently larger than MRF, and the ratio decreased with development. This indicates that both the inward current (MRR) and the outward current (MRF) increased with development (Fig. 4C); however, the increase in outward current may occur at a relatively late phase of development. There was no significant difference in the MRR and MRF between pause and tonic neurons (P > 0.3 for E16–17, P > 0.2 for E19–21).

During the early post-hatch days, we could not find any differences in the nature of firing of tonic neurons between P0–2 and P3–5 (Fig. 4, P > 0.3). There were also few differences in the maximum firing frequencies (P > 0.3): 667 ± 19 Hz (P0–2, n = 36) and 637 ± 28 Hz (P3–5, n = 13). However, both MRR and MRF of multiple firing neurons increased steeply during the late embryonic days (Fig. 4C), and the maximum firing frequency increased with age: 162 ± 15 Hz (n = 24) for E16–17 and 406 ± 15 Hz (n = 38) for E19–21 (P < 0.0001). These differences were statistically significant between E19–21 and P0–2 (P < 0.02), indicating that the properties of action potential generation have attained a mature form by the time of hatching.

Onset neurons had smaller MRR and MRF at all stages of development (Fig. 4C), and tended to have a larger spike half-widths (Fig. 4B) and a smaller spike amplitudes (41.0 ± 4.2 mV (n = 10) for P0–5 and 32.4 ± 2.4 mV (n = 15) for E16–21) than multiple firing cells at each stage of development (48.0 mV for P0–5 and 37.7 mV for E16–17). Both MRR and MRF of the onset neuron increased with development (Fig. 4C), and the ratio MRR/MRF decreased after hatching (Fig. 4D).

Firing characteristics of tonic neurons in the chicken after hatching

Figure 5A shows the voltage responses of tonic neurons in response to a current injection in a chicken after hatching. At the near-threshold current level, a single spike was generated at the beginning of current injection. A slightly larger depolarizing current generated persistent spikes, and firing frequency increased with an increase in current intensity. Firing frequency, calculated by dividing the number of spikes by current duration (150 ms), was plotted against current intensity for five different neurons (Fig. 5B, different symbols indicate different neurons). The discharge rate of all tonic neurons increased monotonically and was almost saturated at a current level of 1.5–3 nA (657 ± 16 Hz, n = 49). The threshold current was 0.26 ± 0.02 nA (range 0.05–0.6 nA, n = 49).

Figure 5. Firing properties of tonic neurons after hatching.

Figure 5

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A, potential responses of a tonic neuron were demonstrated by changing current intensity: ‘+’ for depolarizing current and ‘−’ for hyperpolarizing current. Action potentials were generated in a tonic manner during current injections, and the firing frequency increased with an increase in current intensity. B, the firing frequency plotted here and in all subsequent frequency-current plots was calculated during 150 ms of current injection. Data from five tonic neurons are plotted to show the monotonic increase in firing frequency as a function of injected current. Different symbols indicate different cells, and filled circles are from the cell recorded in A. C, instantaneous firing frequencies were calculated from the inter-spike intervals and are plotted at the timing of the second spike peak of the pair. Steep adaptation occurred during the initial 20–30 ms of current injection.

Steep adaptation occurred during the initial 20–30 ms of current pulse (Fig. 5A, top traces). Figure 5C shows the rapid decrease in the instantaneous frequency followed by constant firing in one tonic neuron. The instantaneous frequency was calculated from the inter-spike interval, and was plotted at the time of the second spike peak of the pair (Fig. 5C). The maximum instantaneous frequency was 814 ± 24 Hz (n = 49) between the first and the second spikes. The degree of adaptation was estimated using an adaptation index, defined here as the firing frequency near the pulse end (100–150 ms) divided by the first instantaneous frequency. Adaptation was dependent on the firing frequency, but was saturated at a relatively low frequency. The adaptation index (0.77 ± 0.02) at the firing frequency when adaptation first emerged (189 ± 10 Hz, pulse end) was the same as that (0.77 ± 0.01, n = 49) at the highest frequency attained in each neuron (Fig. 5C).

Effects of potassium channel blockers on the firing properties of tonic neurons

High-threshold potassium currents carried by the K+ channel Kv3 are blocked by 1 mm TEA (Grissmer et al. 1994; Kanemasa et al. 1995; Rathouz & Trussell, 1998; Rudy et al. 1999). These currents are required for neurons firing at high rates (Massengill et al. 1997; Perney & Kaczmarrek, 1997; Wang et al. 1998; Martina et al. 1998; Rudy et al. 1999; Lau et al. 2000). Figure 6A shows the effect of TEA (1 mm) on one tonic neuron, and Fig. 6B shows the firing frequency of two neurons for the control (filled symbols) and after application of TEA (open symbols). TEA decreased the firing frequency, but the threshold current was not affected (Fig. 6B). After TEA application, the rate of increase in firing frequency decreased and the firing frequency attained only 50 % of the control value at the maximal current levels (588 ± 34 Hz in control, 293 ± 14 Hz in TEA, n = 5). The threshold current was not affected in any of the neurons tested (0.22 ± 0.06 nA in control and 0.20 ± 0.04 nA in TEA, n = 5).

Figure 6. Effects of TEA on the firing properties of tonic neurons after hatching.

Figure 6

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A, voltage responses for depolarizing current injections (0.5 and 1 nA) in the control (Aa) and in the presence of 1 mm TEA (Ab). Records were obtained from the same cell. The firing frequency decreased after application of TEA. B, frequency-current plots. Two neurons with different threshold currents are shown in control conditions (filled symbols) and in the presence of TEA (open symbols). The firing frequency decreased at all current levels but the threshold current was not changed by TEA.

Figure 7Aa shows the effect of TEA on spikes and their time differentiated traces on an expanded time scale. The positive peak of the differentiated trace indicates MRR, and the negative peak MRF (Fig. 7Ab). Thick and thin lines are records from a single neuron in the control and in the presence of TEA, respectively. TEA broadened the spike half-width (0.27 ± 0.02 ms in control, 0.41 ± 0.01 ms in TEA, n = 5, P < 0.05), decreased MRF (−166.6 ± 19.9 V s−1 in control, −107.5 ± 9.0 V s−1 in TEA, n = 5, P < 0.01) and increased the spike amplitude (37.0 ± 3.6 mV in control, 42.2 ± 5.3 mV in TEA, n = 5, P < 0.05) (Fig. 7A). However, MRR was not affected (234.4 ± 23.2 V s−1 in control, 237.6 ± 27.1 V s−1 in TEA, n = 5, P > 0.1).

Figure 7. Effects of TEA and DTX on action potentials of tonic neurons after hatching.

Figure 7

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A, action potentials (Aa) and their first derivatives (Ab) in the control (thick lines) and in 1 mm TEA (thin lines). The maximum rate of rise of action potentials was not affected, but the maximum rate of fall (dashed lines in Ab) decreased. B, action potentials (Ba) and their first derivatives (Bb) in the control (thick lines) and in the presence of 100 nm DTX (thin lines). DTX did not affect the maximum rate of rise, the maximum rate of fall, the spike half-width, or the spike amplitude. The amplitude of the after-hyperpolarization was slightly reduced.

In contrast to 1 mm TEA, 100 nm DTX had only minor effects on the shape of action potentials of tonic neurons (Fig. 7B); however, DTX reduced the threshold current (Fig. 8). DTX is known to block the low-threshold K+ currents (Rathouz & Trussell, 1998), probably those passing through the K+ channels Kv1.1, Kv1.2 or Kv1.6 (Hopkins et al. 1994; Harvey, 2001). After applying DTX, tonic firing occurred at a subthreshold intensity in the control (Fig. 8A). The firing frequency increased at all current injection levels; however, the rate of increase in firing frequency was not clearly affected by DTX (Fig. 8B, •, control; ▵, DTX). Arrows in Fig. 8B show the threshold currents of the neuron presented in Fig. 8A; the average was 0.38 ± 0.09 nA in the control and 0.18 ± 0.05 nA in DTX (n = 4). The firing frequency measured during an injected current of 1 nA was 393 ± 47 Hz in the control and 467 ± 63 Hz in DTX (n = 4). The threshold current of one tonic neuron out of four cells tested was small in the control (0.2 nA), and was not affected by DTX (0.2 nA in DTX). The increase in firing frequency in this neuron (1 nA current injection; 333 Hz in control, 347 Hz in DTX) was also smaller than neurons having higher threshold currents. Expression of low-threshold K+ currents might have been small in this neuron. In contrast, TEA did not affect threshold current, even in cases in which the threshold current level was high (0.3 and 0.4 nA, two tonic cells).

Figure 8. Effects of DTX on the firing pattern of tonic neurons after hatching.

Figure 8

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A, voltage responses for current injections (0.28 and 0.48 nA) in the control (left traces) and in 100 nm DTX (right traces). Records were obtained from the same cell. In DTX, repetitive firing was generated by a current of subthreshold intensity in the control. B, frequency-current plots in control (•) and in DTX (▵). The threshold current intensity decreased by application of DTX (arrows).

Figure 7B shows the detailed effects of DTX on spikes on an expanded time scale. Although the threshold current was affected (Fig. 8B), the threshold voltage was unchanged (−52.1 ± 2.2 mV in control, −51.1 ± 3.5 mV in DTX, n = 4). There were no effects on MRR (244.5 ± 58.5 V s−1 in control, 233.8 ± 58.0 V s−1 in DTX, n = 4, P > 0.4), MRF (−213.0 ± 48.8 V s−1 in control, −203.9 ± 52.5 V s−1 in DTX, n = 4, P > 0.5), the spike half-width (0.31 ± 0.07 ms in control, 0.31 ± 0.08 ms in DTX, n = 4), or the spike amplitude (48.4 ± 3.2 mV in control, 47.8 ± 4.2 mV in DTX, n = 4, P > 0.5). However, the amplitude of spike after-hyperpolarization decreased (−13.4 ± 2.6 mV in control, −10.7 ± 2.4 mV in DTX, n = 4, P < 0.05) (Fig. 7B). In contrast to the actions of TEA, DTX reduced threshold current but had no effect on MRF and the spike half-width. Therefore, a TEA-sensitive current is essential for the high frequency firing of the tonic neuron, while a DTX-sensitive low-threshold K+ current is required to set the threshold current level higher.

Firing characteristics of onset neurons after hatching

Ten out of 59 NA neurons (16.9 %) fired only at the onset of current injection (Fig. 9A). There was normally only one action potential, but at a higher current intensity (1 nA or higher) some neurons fired a few action potentials (Fig. 9A, top trace). A further increase in pulse duration (up to 1 s) or current intensity (up to 3 nA) did not generate additional spikes. Onset firing was accompanied with a slow re-polarization during current injection (Fig. 9A, arrowheads). The threshold current was higher than that of tonic neurons (0.93 ± 0.11 nA, n = 10 for onset neurons; 0.26 ± 0.02 nA, n = 49 for tonic neurons). At the onset of a current injection of subthreshold intensity, there was a depolarizing transient potential (Fig. 9A, at 0.7 nA, arrow). This transient potential was extinguished by application of DTX (data not shown).

Figure 9. Firing patterns and effects of DTX and TEA on onset neurons.

Figure 9

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A, current-clamp records of onset firing at 4 levels of current injection. A depolarizing transient potential was present following current injections of subthreshold intensity (arrow). Arrowheads indicate a gradual repolarization after the action potential. Ba, application of 100 nm DTX induced repetitive firing. Bb, action potentials were broadened by further application of 1 mm TEA. C, frequency-current plots. On application of DTX, the threshold current decreased (arrows) and the firing frequency was increased as in tonic neurons (○). Further application of TEA did not affect the firing frequency (▴). D, voltage responses on an expanded time scale to show the initial portion of the spike responses of an onset neuron in control, DTX and DTX together with TEA. Current strength was 0.8 nA. Spikes broadened after application of TEA. The first spike in TEA + DTX demonstrates a prolonged duration with a reduced maximum rate of fall, but the maximum rate of rise was not affected. E, application of DTX to an NM neuron produced repetitive action potentials.

Effects of potassium channel blockers on onset neurons

Onset firing neurons such as the principal neurons of MNTB and NM neurons generated a train of action potentials after application of 100 nm DTX (Brew & Forsythe, 1995; Rathouz & Trussell, 1998). These neurons also have K+ currents sensitive to a low concentration (1 mm) of TEA (Reyes et al. 1994; Brew & Forsythe, 1995; Wang et al. 1998; Rathouz & Trussell, 1998).

After addition of 100 nm DTX, all onset neurons tested (from E19, E20 and P2, n = 3) fired repetitively (Fig. 9B). In the presence of DTX, the firing frequency increased with an increase in injected current (Fig. 9C). The threshold current decreased from 0.93 ± 0.12 nA in the control, to 0.38 ± 0.08 nA (n = 3, P < 0.05) in DTX (Fig. 9C, arrows), but the threshold potential was not changed (−48.9 ± 3.9 mV in control, −46.6 ± 2.9 mV in DTX, n = 3, P > 0.6). The spike half-width, the spike amplitude, MRR and MRF were not changed significantly in DTX (spike half-width, 0.52 ± 0.10 ms in control, 0.65 ± 0.12 ms in DTX; spike amplitude, 39.3 ± 4.6 mV in control, 37.3 ± 4.9 mV in DTX; MRR, 242 ± 65 V s−1 in control, 203 ± 64 V s−1 in DTX; MRF, −105.0 ± 34.4 V s−1 in control, −83.7 ± 29.5 V s−1 in DTX, n = 3, P > 0.07). The lack of any effect of DTX on the shape of individual action potentials was similar in tonic neurons.

Further application of 1 mm TEA in addition to DTX broadened the action potential (n = 3) (Fig. 9Bb and D). The spike half-width increased (1.63 ± 0.67 ms in DTX and TEA, n = 3) (Fig. 9D). The broadening of the action potential was reflected in a decrease in MRF (−36.6 ± 20.6 V s−1 in DTX and TEA, n = 3, P < 0.05). However, MRR was not changed (203.1 ± 94.2 V s−1 in DTX and TEA, n = 3, P > 0.9). The threshold current was not changed by addition of TEA with DTX (Fig. 9C, left arrow).

The effects of DTX on NA onset neurons were slightly different from those on NM neurons (Fig. 9E). After application of DTX, the spike amplitude of NM neurons was significantly increased (22.6 ± 2.9 mV in control, 39.2 ± 5.9 mV in DTX, n = 6, P < 0.01), and the spike half-width was increased (0.18 ± 0.01 ms in control, 0.42 ± 0.04 ms in DTX, n = 6, P < 0.01). However, the threshold current decreased (1.60 ± 0.24 nA in control, 0.24 ± 0.04 nA in DTX, n = 6, P < 0.01), and MRR and MRF were not changed (MRR, 231.0 ± 25.2 V s−1 in control, 266.8 ± 48.7 V s−1 in DTX; MRF, −225.7 ± 34.4 V s−1 in control, −206.6 ± 56.3 V s−1 in DTX, n = 6, P > 0.3). Thus, DTX-sensitive currents are more dominant in NM neurons than in onset neurons in NA.

Hyperpolarizing responses of NA neurons

Voltage responses to hyperpolarizing current steps demonstrated a pronounced sag in both onset (Fig. 9A, −0.6 nA) and tonic neurons (Fig. 5A, −0.3 nA). The amplitude of the sag relative to the peak hyperpolarization was calculated from the voltage responses that reached −100 to −110 mV and was not different between tonic neurons (33 ± 4 %, n = 6) and onset neurons (39 ± 3 %, n = 5). However, the time constant of the sag of onset neurons (21 ± 3 ms, n = 5) was shorter than that of tonic neurons (37 ± 4 ms, n = 6). This may reflect the smaller time constant of onset neurons at near-resting potential (Fig. 3B).

Projection patterns of auditory nerve fibres in NA

NA is arranged tonotopically and the characteristic frequency is reported as being high in the dorsal region and low in the ventral region in the chicken (Warchol & Dallos, 1990). However, the exact pattern of projection is not clear. In order to understand the relationship between the tonotopic projection of ANFs and the dendritic arborization of individual NA neurons, we examined the projection of Di-I labelled ANFs in NA.

When Di-I crystals were placed near the apex or mid-region of the cochlea, ANFs projecting to the ventral region of NA were labelled (Fig. 10A1 and 4). The dorsal-to-lateral region of NA was labelled by placing Di-I near the base of the cochlea (Fig. 10A3 and 6). These patterns of projections were traced in the rostral and the caudal planes of NA and are summarized in Fig. 10B. Shaded areas indicate thick projections of ANF, and dashed lines trace the boundaries of Di-I-positive regions. These boundaries may reflect the projection of ANFs from the edge of the Di-I-filled region in the cochlea. The orientation of ANF was relatively simple in the ventral region, running from medial to lateral (Fig. 10B, areas 1, 2, 4 and 5), and became gradually slanted in the dorsal region, running from dorsal to ventral (Fig. 10B, areas 3 and 6).

Morphology of NA neurons

During whole cell recordings neurons were intracellularly stained with biocytin, and their firing patterns and locations within the nucleus were recorded. Among 73 biocytin-stained neurons, 60 cells were multiple firing neurons (Fig. 11A, cells without circles) and 13 were onset neurons (Fig.11A, cells within circles). In Fig. 11A, the positions of labelled neurons from E19-P5 were separately plotted in the rostral and caudal planes, because the general shape of the nucleus is different between rostral and caudal planes. The average soma size was 177.5 ± 4.4 μm2 (n = 73), and was not different between multiple firing neurons (175.2 ± 4.7 μm2, n = 60) and onset neurons (188.1 ± 12.1 μm2, n = 13, P > 0.3). Cell sizes were not different between the dorsal region (174.9 ± 6.3 μm2, n = 36) and the ventral region (180.0 ± 6.3 μm2, n = 37, P > 0.5). Typically, NA neurons had two or more dendrites, which often terminated as tufted arborizations (Fig. 11Ba–h). The dendritic length of NA neurons was different from neuron to neuron. Some neurons had dendrites extending more than 100 μm and in others the dendrites extended less than 40 μm, but almost all NA neurons (93 %) had dendrites longer than 40 μm.

Figure 11. Dendritic branching and aspect ratio.

Figure 11

Open in a new tab

A, biocytin-labelled neurons and their positions in NA, superimposed on the projection pattern of ANFs. Positions of labelled neurons recorded from E19-P5 NA slices were plotted in two planes, rostral and caudal. Onset neurons are encircled. All cells had two or more branched dendrites as shown in B. Most tonic neurons and one onset neuron (labelled with #) extended dendrites parallel to the ANF projection. One tonic neuron with the aspect ratio of less than 1 is marked with §. B, close-up images of the neurons labelled a-h in A. a (P2), b (P1), c (P1), d (P1), e (P1), f (P3) and g (P2) are tonic neurons; h (E19) is an onset neuron. C, a plot of the lengths of dendrites, measured in each neuron (n = 73) in the axes parallel and perpendicular to the ANFs; filled symbols, multiple firing neurons; open symbols the onset neurons; circles, E19-P5; triangles, E16–17. D, aspect ratios were calculated for multiple firing neurons (Da) and onset neurons (Db) and a histogram was plotted with a bin width of 0.4. ▪, E19-P5; □, E16–17.

The length of dendrites was measured in each neuron in two axes, one being parallel to the ANF projection and the other perpendicular to it. These axes were determined by superimposing the neuronal images on the diagram of ANF orientation (Fig. 10B and Fig. 11A). Figure 11C plots lengths of dendrites for the multiple firing neurons (filled symbols) and for the onset neurons (open symbols). Dendrites tended to be longer in the axis parallel to ANF projection in most multiple firing neurons, and these neurons had an aspect ratio of larger than 1 (see Methods). One tonic neuron had longer dendrites in the axis perpendicular to ANF projection (marked with § in Fig. 11A, rostral plane), with an aspect ratio of less than 1. Aspect ratios ranged between 0.45 and 3.82, and the grand average was 2.12 ± 0.09 (n = 60 multiple firing cells, Fig. 11D), being 1.98 ± 0.11 for E16–17 (n = 36), and 2.34 ± 0.15 for E19-P5 (n = 24). These observations indicate that almost all multiple firing neurons have a tendency to project dendrites parallel to ANF (Fig. 11A).

Some onset neurons extended dendrites in the axis parallel to the ANF projection (labelled with # in Fig. 11A, caudal plane). However, onset neurons did not show a clear pattern of dendrite orientation (Fig. 11C and D). This may be partly due to the small number of onset neurons we were able to record. The mean aspect ratio was 1.58 ± 0.26 (n = 13), being 1.79 ± 0.31 (n = 9) for E16–17, and 1.11 ± 0.41 (n = 4) for E19-P5.

Discussion

We have investigated the firing properties of neurons in NA of the chicken embryo (E16–21) and the chicken after hatching (P0–5), and found that neurons fired either multiple action potentials or a single action potential in response to current injections (Fig. 1). After hatching, NA neurons fired either tonically during current injection, or at the onset of the injection (Fig. 1A). This variation in multiple firing patterns in the embryo may reflect the low level of membrane excitability (Fig. 4). The proportion of onset firing neurons did not change during development (Fig. 2). In tonic neurons, the firing frequency increased in proportion to the injected current (Fig. 5). Multiple firing neurons, including the tonic neurons after hatching, extended dendrites nearly parallel to the projection of ANFs (Fig. 11).

NA neurons of the chicken embryo

Soares et al. (2002) classified NA neurons in the chicken embryo (E16–19) according to their firing patterns as one-spike (20 % of cells recorded), damped (35 %) and tonic (45 %)cells; tonic cells were subdivided into three subtypes (I-III). They discussed the correlation between the firing patterns of neurons and their distinct morphologies: stubby cells generally had one-spike firing patterns, planar cells generally had damped spikes, vertical cells generally had tonic II firing patterns, and radiate cells tonic I and III firing patterns. They also calculated the aspect ratios of dendritic arborization as the width of the cell's dendritic axis parallel to the presumed isofrequency axis divided by the axis perpendicular to it. The aspect ratios were 2.11 in the damped cells and 1.9 (I), 0.68 (II) and 1.44 (III) in tonic cells. They concluded that damped cells extend dendrites in parallel to the isofrequency axis, but tonic cells not do so consistently. The relatively low aspect ratios of their tonic neurons might be related to the embryonic age of their chickens, because in our study the aspect ratio tended to be smaller in the multiple firing neurons of E16–17 (1.98) than the tonic firing neurons of E19-P5 (2.34, Fig. 11D), although the difference was not significant statistically (P = 0.06). It might also be a consequence of some mismatch between their presumed tonotopic axes and the actual organization in NA (Fig. 1 of Soares et al. 2002).

We have classified NA neurons of embryos (E16–21) into onset firing neurons and two subtypes of multiple firing neurons (pause and tonic cells). Onset neurons were comparable to the one-spike cells of Soares et al. (2002), and pause neurons to their tonic type II cells. Our embryonic tonic neurons may be further subdivided into damped and tonic cells. However, we believe that these classifications simply reflect one aspect of development of the tonic firing neuron.

Comparison with other birds

Köppl (2001) injected horseradish peroxidase iontophoretically in NA to label ANFs, after identifying their characteristic frequencies in the barn owl, and demonstrated tonotopic frequency representation of ANFs in NA. The pattern of projection of individual ANFs was similar to what we found in the chicken (Fig. 10). Our results are also consistent with reports in the pigeon (Boord & Rasmussen, 1963; Hotta, 1971), the chicken (Warchol & Dallos, 1990) and the house sparrow (Konishi, 1970).

Tonic neurons were the major component (83.1 %) of NA neurons of the chicken after hatching, and tended to extend their dendrites along a plane parallel to the projection of ANFs (Fig. 11D, aspect ratio 2.34). This may indicate that tonic neurons encode sound intensity information of some restricted sound frequency region. The planar cells of the barn owl have a dendritic arborization pattern that is restricted to the isofrequency axis of the NA (Soares & Carr, 2001). However, the percentage of planar cells was only 8.7 % and the dominant cell type (66.2 %) was stubby cells. Because stubby cells have short, thick dendrites, these neurons may also receive synaptic inputs from auditory neurons having a limited range of characteristic frequencies. The combined percentage of planar and stubby cells was 74.9 %, which is similar to the percentage of tonic neurons in our study. It is plausible that most NA neurons in the chicken and the barn owl may receive projections of ANFs responding to a limited range of sound frequencies.

Effects of potassium currents on the firing properties of NA neurons

In the fast-spiking neocortical interneurons, currents associated with Kv3.1 (Massengill et al. 1997; Martina et al. 1998) and Kv3.2 (Lau et al. 2000) allow the rapid repolarization of the membrane, which is a prerequisite for high frequency firing. Computer modelling studies indicate that a selective block of Kv3 currents impairs high frequency firing (Perney et al. 1992; Wang et al. 1998; Erisir et al. 1999). It is probable that Kv3 channels also have similar effects in the mouse MNTB principal neurons (Wang et al. 1998) and rat subthalamic nucleus neurons (Wigmore & Lacey, 2000). Kv3 potassium channels are sensitive to a low concentration (1 mm) of TEA (Grissmer et al. 1994; Kanemasa et al. 1995; Rudy et al. 1999), and TEA reduced the MRF and the maximum firing frequency of the tonic neuron in our study (Fig. 6 and Fig. 7A).

Three additional heterologously expressed K+ channels are known to be TEA sensitive: Kv1.1 (Kd∼500 μm), KCNQ2 (90 % blocked by 1 mm TEA) and Ca2+-activated K+ (BK) channels containing proteins of the Slo family (Kd 80–330 μm) (Coetzee et al. 1999). KCNQ2 channels are unlikely to be expressed in the NA neurons, because of the very slow activation and deactivation kinetics (time constants of hundreds of milliseconds to seconds). Iberiotoxin (50 nm), the blocker of BK channels, produced no effect on the spike shape (data not shown). DTX (100 nm) selectively blocks Kv1.1, Kv1.2 and Kv1.6 (Hopkins et al. 1994; Harvey, 2001) and had few measurable effects on the spike shape of tonic neurons, although it reduced after-hyperpolarization (Fig. 7B). However, the threshold current was reduced, and consequently the firing frequency was enhanced (Fig. 8). TEA-sensitivity of tonic neurons is likely to be due to the expression of Kv3 channels, and the neurons may have a small number of Kv1 channels.

Onset neurons were also sensitive to TEA (Fig. 9D), but were also had a high sensitivity to DTX, and their firing pattern changed to repetitive firing after DTX application (Fig. 9). Thus, onset firing was not generated by the inactivation of the sodium channel.

Comparison with electrophysiological experiments performed in vivo

NA neurons coded intensity over a wide range of sound levels, and the maximum discharge rate varied from 50 to 415 spikes s−1 in a P10–28 chicken in vivo (Warchol & Dallos, 1990). This level of maximum firing frequency was lower than the level achieved by tonic neurons. In chickens after hatching, tonic neurons fired near-maximally at a rate of 700 Hz in the steady state and transiently at well over 850 Hz. Therefore, in vitro neurons seem to have a greater capacity for firing than in vivo neurons. Three types of PSTH patterns have been reported in NA neurons of the chicken when tonal stimuli were applied: chopper (52 %), onset (8 %) and primary-like (40 %) (Warchol & Dallos, 1990). The population of neurons with the onset PSTH pattern was smallest, and the proportion was closer to the percentage of onset neurons we observed (16.9 %). The neurons with primary-like and chopper PSTH patterns could be related to our tonic firing neurons; however, further information about synaptic inputs, their locations on dendrites and on soma, and the firing properties of NA neurons when driven via ANF activities is required before these PSTH responses can be elucidated. NM neurons fire at the onset of current injection (Reyes et al. 1994), receive ANF inputs on their soma and show a primary-like PSTH pattern (Warchol & Dallos, 1990). In contrast, stellate cells in the anteroventral cochlear nucleus have a tonic firing pattern (Oertel et al. 1988). A computer simulation showed several patterns of PSTH including primary-like, primary-like-with-notch and chopper (Banks & Sachs, 1991). The pattern and the regularity of chopping depend simply on the location and the number of excitatory synaptic inputs of ANFs.

Coding sound information in NA

Since most tonic neurons extend their dendrites parallel to the plane of ANF projection (Fig. 11A), these neurons are likely to receive synaptic inputs from auditory nerves having a restricted range of characteristic frequencies. Moreover, tonic neurons could fire at a high rate, and the firing frequency could reflect the strength of the injected current and have a wide dynamic range. These properties of tonic neurons are appropriate for extracting sound intensity data from ANFs and encoding it as firing frequencies while at the same time saving the associated sound frequency information. While the proportion of onset neurons is small, their firing properties may indicate that they play an important role in extracting and coding temporal information in the NA, as suggested by the onset pattern of PSTH (Warchol & Dallos, 1990).

Acknowledgments

We express our thanks to Dr K. Koyano for his assistance with early experiments, and M. Fukao for technical assistance. This study was supported by the Grants-in-Aid from the Ministry of Education to H. Ohmori.

REFERENCES

  1. Banks MI, Sachs MB. Regularity analysis in a compartmental model of chopper units in the anteroventral cochlear nucleus. J Neurophysiol. 1991;65:606–629. doi: 10.1152/jn.1991.65.3.606. [DOI] [PubMed] [Google Scholar]
  2. Boord RL, Rasmussen GL. Projection of the cochlear and lagenar nerves on the cochlear nuclei of the pigeon. J Comp Neurol. 1963;120:463–475. doi: 10.1002/cne.901200305. [DOI] [PubMed] [Google Scholar]
  3. Brainard MS, Knudsen EI, Esterly SD. Neural derivation of sound source location: resolution of spatial ambiguities in binaural cues. J Acoust Soc Am. 1992;91:1015–1027. doi: 10.1121/1.402627. [DOI] [PubMed] [Google Scholar]
  4. Brew HM, Forsythe ID. Two voltage-dependent K+ conductances with complementary functions in postsynaptic integration at a central auditory synapse. J Neurosci. 1995;15:8011–8022. doi: 10.1523/JNEUROSCI.15-12-08011.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, Rudy B. Molecular diversity of K+ channels. Ann N Y Acad Sci. 1999;868:233–285. doi: 10.1111/j.1749-6632.1999.tb11293.x. [DOI] [PubMed] [Google Scholar]
  6. Erisir A, Lau D, Rudy B, Leonard CS. Function of specific K+ channels in sustained high-frequency firing of fast-spiking neocortical interneurons. J Neurophysiol. 1999;82:2476–2489. doi: 10.1152/jn.1999.82.5.2476. [DOI] [PubMed] [Google Scholar]
  7. Grissmer S, Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA, Karmilowicz MJ, Auperin DD, Chandy KG. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1. 1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol Pharmacol. 1994;45:1227–1234. [PubMed] [Google Scholar]
  8. Harvey AL. Twenty years of dendrotoxins. Toxicon. 2001;39:15–26. doi: 10.1016/s0041-0101(00)00162-8. [DOI] [PubMed] [Google Scholar]
  9. Häusler UH, Sullivan WE, Soares D, Carr CE. A morphological study of the cochlear nuclei of the pigeon (Columba livia) Brain Behav Evol. 1999;54:290–302. doi: 10.1159/000006629. [DOI] [PubMed] [Google Scholar]
  10. Hopkins WF, Allen ML, Houamed KM, Tempel BL. Properties of voltage-gated K+ currents expressed in Xenopus oocytes by mKv1. 1, mKv1.2 and their heteromultimers as revealed by mutagenesis of the dendrotoxin-binding site in mKv1.1. Pflugers Arch. 1994;428:382–390. doi: 10.1007/BF00724522. [DOI] [PubMed] [Google Scholar]
  11. Hotta T. Unit responses from the nucleus angularis in the pigeon's medulla. Comp Biochem Physiol A. 1971;40:415–424. doi: 10.1016/0300-9629(71)90032-6. [DOI] [PubMed] [Google Scholar]
  12. Jhaveri S, Morest DK. Neuronal architecture in nucleus magnocellularis of the chicken auditory system with observations on nucleus laminaris: a light and electron microscope study. Neuroscience. 1982a;7:809–836. doi: 10.1016/0306-4522(82)90045-8. [DOI] [PubMed] [Google Scholar]
  13. Jhaveri S, Morest DK. Sequential alterations of neuronal architecture in nucleus magnocellularis of the developing chicken: a golgi study. Neuroscience. 1982b;7:837–853. doi: 10.1016/0306-4522(82)90046-x. [DOI] [PubMed] [Google Scholar]
  14. Jhaveri S, Morest DK. Sequential alterations of neuronal architecture in nucleus magnocellularis of the developing chicken: an electron microscope study. Neuroscience. 1982c;7:855–870. doi: 10.1016/0306-4522(82)90047-1. [DOI] [PubMed] [Google Scholar]
  15. Kanemasa T, Gan L, Perney TM, Wang LY, Kaczmarek LK. Electrophysiological and pharmacological characterization of a mammalian Shaw channel expressed in NIH 3T3 fibroblasts. J Neurophysiol. 1995;74:207–217. doi: 10.1152/jn.1995.74.1.207. [DOI] [PubMed] [Google Scholar]
  16. Konishi M. Comparative neurophysiological studies of hearing and vocalizations in songbirds. Zeitschrift fur vergleichende Physiologie. 1970;66:257–272. [Google Scholar]
  17. Köppl C. Tonotopic projections of the auditory nerve to the cochlear nucleus angularis in the barn owl. J Assoc Res Otolaryngol. 2001;2:41–53. doi: 10.1007/s101620010027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Knudsen EI, Konishi M. A neural map of auditory space in the owl. Science. 1978;200:795–797. doi: 10.1126/science.644324. [DOI] [PubMed] [Google Scholar]
  19. Lau D, Vega-Saenz de Miera EC, Contreras D, Ozaita A, Harvey M, Chow A, Noebels JL, Paylor R, Morgan JI, Leonard CS, Rudy B. Impaired fast-spiking, suppressed cortical inhibition, and increased susceptibility to seizures in mice lacking Kv3. 2 K+ channel proteins. J Neurosci. 2000;20:9071–9085. doi: 10.1523/JNEUROSCI.20-24-09071.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Manley GA, Köppl C, Konishi M. A neural map of interaural intensity differences in the brain stem of the barn owl. J Neurosci. 1988;8:2665–2676. doi: 10.1523/JNEUROSCI.08-08-02665.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Martina M, Schultz JH, Ehmke H, Monyer H, Jonas P. Functional and molecular differences between voltage-gated K+ channels of fast-spiking interneurons and pyramidal neurons of rat hippocampus. J Neurosci. 1998;18:8111–8125. doi: 10.1523/JNEUROSCI.18-20-08111.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Massengill JL, Smith MA, Son DI, O'Dowd DK. Differential expression of K4-AP currents and Kv3. 1 potassium channel transcripts in cortical neurons that develop distinct firing phenotypes. J Neurosci. 1997;17:3136–3147. doi: 10.1523/JNEUROSCI.17-09-03136.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mogdans J, Knudsen EI. Representation of interaural level difference in the VLVp, the first site of binaural comparison in the barn owl's auditory system. Hear Res. 1994;74:148–164. doi: 10.1016/0378-5955(94)90183-x. [DOI] [PubMed] [Google Scholar]
  24. Moiseff A, Konishi M. Binaural characteristics of units in the owl's brainstem auditory pathway: precursors of restricted spatial receptive fields. J Neurosci. 1983;3:2553–2562. doi: 10.1523/JNEUROSCI.03-12-02553.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oertel D, Wu SH, Hirsch JA. Electrical characteristics of cells and neuronal circuitry in the cochlear nuclei studied with intracellular recordings from brain slices. In: Edelman GM, Gall WE, Cowan WM, editors. Auditory Function. New York: Blackwell Science Inc; 1988. pp. 313–336. [Google Scholar]
  26. Perney TM, Marshall J, Martin KA, Hockfield S, Kaczmarek LK. Expression of the mRNAs for the Kv3. 1 potassium channel gene in the adult and developing rat brain. J Neurophysiol. 1992;68:756–766. doi: 10.1152/jn.1992.68.3.756. [DOI] [PubMed] [Google Scholar]
  27. Perney TM, Kaczmarek LK. Localization of a high threshold potassium channel in the rat cochlear nucleus. J Comp Neurol. 1997;386:178–202. [PubMed] [Google Scholar]
  28. Rathouz M, Trussell L. Characterization of outward currents in neurons of the avian nucleus magnocellularis. J Neurophysiol. 1998;80:2824–2835. doi: 10.1152/jn.1998.80.6.2824. [DOI] [PubMed] [Google Scholar]
  29. Reyes AD, Rubel EW, Spain WJ. Membrane properties underlying the firing of neurons in the avian cochlear nucleus. J Neurosci. 1994;14:5352–5364. doi: 10.1523/JNEUROSCI.14-09-05352.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rudy B, Chow A, Lau D, Amarillo Y, Ozaita A, Saganich M, Moreno H, Nadal MS, Hernandez-Pineda R, Hernandez-Cruz A, Erisir A, Leonard C, Vega-Saenz de Miera E. Contributions of Kv3 channels to neuronal excitability. Ann N Y Acad Sci. 1999;868:304–343. doi: 10.1111/j.1749-6632.1999.tb11295.x. [DOI] [PubMed] [Google Scholar]
  31. Soares D, Carr CE. The cytoarchitecture of the nucleus angularis of the barn owl (Tyto alba) J Comp Neurol. 2001;429:192–205. doi: 10.1002/1096-9861(20000108)429:2<192::aid-cne2>3.0.co;2-5. [DOI] [PubMed] [Google Scholar]
  32. Soares D, Chitwood RA, Hyson RL, Carr CE. Intrinsic neuronal properties of the chick nucleus angularis. J Neurophysiol. 2002;88:152–162. doi: 10.1152/jn.2002.88.1.152. [DOI] [PubMed] [Google Scholar]
  33. Sullivan WE, Konishi M. Segregation of stimulus phase and intensity coding in the cochlear nucleus of the barn owl. J Neurosci. 1984;4:1787–1799. doi: 10.1523/JNEUROSCI.04-07-01787.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Takahashi TT, Konishi M. Projections of the cochlear nuclei and nucleus laminaris to the inferior colliculus of the barn owl. J Comp Neurol. 1988a;274:190–211. doi: 10.1002/cne.902740206. [DOI] [PubMed] [Google Scholar]
  35. Takahashi TT, Konishi M. Projections of nucleus angularis and nucleus laminaris to the lateral lemniscal nuclear complex of the barn owl. J Comp Neurol. 1988b;274:212–238. doi: 10.1002/cne.902740207. [DOI] [PubMed] [Google Scholar]
  36. Takahashi T, Moiseff A, Konishi M. Time and intensity cues are processed independently in the auditory system of the owl. J Neurosci. 1984;4:1781–1786. doi: 10.1523/JNEUROSCI.04-07-01781.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang LY, Gan L, Forsythe ID, Kaczmarek LK. Contribution of the Kv3. 1 potassium channel to high-frequency firing in mouse auditory neurones. J Physiol. 1998;509:183–194. doi: 10.1111/j.1469-7793.1998.183bo.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Warchol ME, Dallos P. Neural coding in the chick cochlear nucleus. J Comp Physiol A. 1990;166:721–734. doi: 10.1007/BF00240021. [DOI] [PubMed] [Google Scholar]
  39. Wigmore MA, Lacey MG. A Kv3-like persistent, outwardly rectifying, Cs+-permeable, K+ current in rat subthalamic nucleus neurones. J Physiol. 2000;527:493–506. doi: 10.1111/j.1469-7793.2000.t01-1-00493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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