Tonotopic specialization of auditory coincidence detection in nucleus laminaris of the chick

Abstract

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.

Figure 5.

Synaptic responses along the tonotopic axis. A, EPSC (thinner traces, polarity reversed) and EPSP (thicker traces) from the same neurons are size-normalized and superimposed. EPSCs were recorded at -80 mV, and EPSPs were recorded at the resting potential, indicated to the left. a, High-CF neuron; b, middle-CF neuron; c, low-CF neuron. B, C, Ten to 90% rise time (open bars) and half-amplitude width (filled bars) of EPSC (B) and EPSP (C). Between the high- and the low-CF neurons, the difference is statistically significant for the half-amplitude width of EPSC (p < 0.01) and EPSP (p < 0.05). D, Ten to 90% rise time histogram of mEPSC from populations of neurons. a, High-CF region (1479 mEPSCs from 16 cells); b, middle-CF region (2850 mEPSCs from 33 cells); c, low-CF region (2332 mEPSCs from 19 cells). Mean values are indicated by arrows. Inset, Ensemble averaged mEPSC. Note a progressively larger distribution of slower components toward the low-CF neurons.

Figure 7.

Coincidence detection along the tonotopic axis. A, Four superimposed voltage traces in response to bilateral stimuli with three different time intervals between the two sides (Δt in milliseconds) (see Materials and Methods). In A and B, high-CF neuron (a), middle- (b), and low-CF neuron (c). B, Probability of spike generation as a function of Δt calculated from the neurons in A. Ipsi, Ipsilateral; contra, contralateral.

Figure 8.

Coincidence detection is most accurate in the middle-CF neuron. A, Firing probabilities normalized to the maximum value were calculated from 10 cells in the high-CF region (open circles), 15 cells in the middle-CF region (gray triangles), and 6 cells in the low-CF region (filled squares) and were plotted against the time difference (Δt). The firing probabilities from six high-CF cells at Δt of ±1 ms were connected with dotted lines. The time intervals between ±1 and ±0.5 ms were not tested. B, C, Time window (B) and percentage maximum slope (C), the maximum change in firing probability per 10 μs of Δt, were evaluated from each coincidence detection curve and plotted for three CF regions. ^*p < 0.01; ^#p < 0.05.

Figure 6.

Distribution of Kv1.2 channel protein along the tonotopic axis. A, Transverse slice stained with the Kv1.2 antibody. Boxes show the regions of NL magnified in B-D. The left panel in A and bottom panels in E-G show the relative fluorescence intensity of individual NL neurons (see Materials and Methods). Numbers on the abscissa indicate the sector of NL. B-D, Immunoreactivity is strong in the middle region (C) and decreases in both the rostromedial (B) and caudolateral (D) region (p < 0.01 by Mann-Whitney U test among the three CF regions in a slice). E-G, Coronal slices stained with the antibody. R, Rostral; M, medial; D, dorsal. The immunoreactivity increases from sector 2 to sector 3 in slice E (high CF) but decreases in slice F from sectors 7-8 (middle CF) to sector 9 (low CF) and decreases in slice G toward the lower-CF sector (p < 0.01 by Mann-Whitney U test among sectors in each slice). H, This gradient is developed along the tonotopic axis, which is directed from the rostromedial high CF to the caudolateral low CF. Each slice is illustrated by a dotted line in H. I, Relative fluorescence intensity in each sector. Data from four chicks were averaged, and comparisons were made among sectors within the same slices (see Materials and Methods). ^#Statistical significance for p < 0.05.

Figure 1.

Planar projection of NL. Two-dimensional image of NL was constructed on a percentile grid (Rubel and Parks, 1975), and 11 sectors were defined depending on caudal-to-rostral and lateral-to-medial positions within the nucleus. From the linear relationship between CF and rostrocaudal and mediolateral position in NL (Rubel and Parks, 1975), these sectors were classified into three regions, and the characteristic frequency of each region was estimated as follows: high-CF region (open sectors, 2.5-3.3 kHz), middle-CF region (gray sectors, 1-2.5 kHz), and low-CF region (filled sectors, 0.4-1 kHz). Dotted lines indicate isofrequency lines corresponding to 2.5 and 1 kHz, respectively.

Figure 2.

Confocal images of NL neurons. A series of pictures were taken with a confocal microscope with a z-depth of 1 μm and stacked up to one image. A-C, Lucifer yellow-labeled neurons: high-CF (A), middle-CF (B), and low-CF (C) neuron. Length of dendrites increased from high- to low-CF neuron. D, E, Di-I-labeled neurons: high-CF (D) and middle-CF (E) neuron. Note the different scale in C. LY, Lucifer yellow; V, ventral; L, lateral.

Figure 3.

Membrane properties along the tonotopic axis. A, Voltage responses to a small hyperpolarizing current injection of 7 ms duration and 0.04 nA amplitude. a, High-CF neuron; b, middle-CF neuron; c, low-CF neuron. Resting membrane potential is indicated near the trace in this and in the following figures. Thinner lines indicate exponential fittings to the trace, and the time constants are indicated at the bottom left. c, Note two time constants in the low-CF neuron attributable to a double-exponential fitting. B-D, Membrane time constant (B), input resistance (C), and input capacitance (D). Here and in subsequent figures, the numbers in parentheses are the numbers of cells. Statistical significance is evaluated between middle-CF neurons and neurons in the other two regions and is indicated by an asterisk for p < 0.01 in this and the following figures. Between high- and low-CF neurons, the difference is significant for the input resistance and the input capacitance (p < 0.05) but not significant for the membrane time constant (p = 0.58). Here and in the following figures, error bars indicate SE.

Figure 4.

Membrane conductances along the tonotopic axis. A, Voltage responses to depolarizing (4 nA; top) and hyperpolarizing (-2 nA; bottom) current injections of 70 ms duration. a, High-CF neuron; b, middle-CF neuron; c, low-CF neuron. B, Voltage-current relationships measured at the end of current injection from -2 to 4 nA, with a step of 0.2 nA; 35 cells in the high-CF neuron; (open circles), 58 cells in the middle-CF region (gray triangles), and 42 cells in the low-CF region (filled squares). Error bars were so small and were overlaid by symbols. C, Limiting slope conductance. Above (a) and below (b) the resting potential. Open bars, control; filled bars, in the presence of DTX (a) and ZD7288 (b). Limiting slope conductances were measured between 3.6 and 4 nA for a, and between -1.6 and -2 nA for b. After application of DTX or ZD7288 (filled bars), the limiting conductance decreased significantly compared with the control (open bars) (p < 0.01 by unpaired t test), except for the low-CF neurons in DTX (p = 0.1 by unpaired t test). Controls are from neurons in B. For high-, middle-, and low-CF region, records are from 7, 12, and 8 cells in DTX and 7, 7, and 6 cells in ZD7288. D, E, Amplitude (D) and maximum rate of rise (E) of the action potentials, induced by a short current injection of 0.5 ms, 5 nA.