Adult barn owls (Tyto alba) of both sexes were anesthetized by intramuscular injections of ketamine hydrochloride (20 mg/kg; Ketaset) and xylazine (4 mg/kg; Anased) and given prophylactic antibiotics (oxytetracycline; 20 mg/kg; Phoenix Pharmaceuticals). Anesthesia was maintained with supplemental injections of ketamine and xylazine during the experiment. A heating pad (k-20-f; American Medical Systems, Cincinnati, OH) was used to maintain the body temperature and the wings were restrained with a soft leather jacket. A stainless-steel plate was placed on the skull using dental acrylic to hold the head in subsequent experiments, and a reference pin was implanted on the midline point of the interaural plane. Recording sessions began a week after this initial surgery. The owl was anesthetized and placed into a custom-built stereotaxic device that held the head such that the ventral surface of the palatine ridge was at 30° from the horizontal plane. We opened a small hole on the skull area overlying either Ov or ICx and cut a slit in the duramater for electrode insertion. At the end of the experiment, we covered the craneotomy with gelfoam and dental cement and closed the skin incision. Owls were returned to their individual cages and monitored for recovery. Depending on the owl’s weight and recovery conditions, experiments were repeated every 7–10 days for a period of several weeks. These procedures comply with guidelines set forth by the National Institutes of Health and Caltech Institute’s Animal Care and Use Committee.
Custom software was used to generate sound stimuli, data collection and analysis. Acoustic stimuli were delivered by a stereo analog interface (DD1; Tucker Davis Technologies) through a calibrated earphone assembly. Tonal, broadband, and narrowband noise stimuli (50-ms duration, 5-ms linear rise/fall time) were presented once per second. Broadband stimuli had a band-pass of 1–12 kHz. Narrowband signals were 1-kHz bandwidth noise.
Spikes were recorded during a time window set to start 100 ms before the stimulus onset and ending 200 ms after stimulus offset. ITD was varied in steps of 30 µs, ILD in steps of 5 dB, and frequency in steps of 100 Hz. We averaged the response ≥10 randomized repetitions of the same stimulus. The stimulus intensity could be varied independently for each ear using a pair of digitally controlled attenuators (PA4, Tucker Davis Technologies).
All recordings were performed in a double-walled sound-attenuating chamber. Each earphone consisted of a speaker (Knowles 1914) and a microphone (Knowles 1319) encased in a custom-made metal delivery piece (5 mm long and 7 mm diam) that fits the owl’s ear canal. The gaps between the earphone assembly and the ear canal were filled with silicone impression material (Gold Velvet II, All American Laboratory). Simultaneous measurement of sound with both the B&K and the Knowles microphones made it possible to translate the voltage output of the Knowles into sound intensity in dB SPL. The Knowles microphones were then used to calibrate the earphone assemblies at the beginning of each experiment. The calibration data contained the amplitudes and phase angles measured in steps of 100 Hz. The computer automatically smoothed irregularities in amplitude and phase of the frequency response of each earphone from 0.5 to 12 kHz.
The activity of single neurons in Ov and ICx was recorded extra-cellularly with tungsten electrodes (1MΩ, 0.005-in, A-M Systems). Action potentials were amplified, filtered (Amplifier System, µA-200, Beckman Electronic Shop), and converted to transistor-transistor logic (TTL) pulses with a spike discriminator (SD1, Tucker Davis Technologies). The data were stored in a computer via a time converter (ET1, Tucker Davis Technologies) and an A/D converter (DD1, Tucker Davis Technologies) with a sampling rate of 48 kHz and 16-bit resolution.
Ov was localized using stereotaxic coordinates and locating its tonotopically organized region (Proctor 1993
; Proctor and Konishi 1997
). ICx was localized stereotaxically and by its physiological response properties (Knudsen and Konishi 1978
; Peña and Konishi 2000
). The electrodes were advanced with a microdrive (Motion Controller, Model PMC 100, Newport) in steps of 100 µm until the nucleus was reached. The size of the steps was then reduced to 2–4 µm to search and isolate single units. The neurons were recorded every approximately 100 µm in the dorsoventral plane.
The number of impulses obtained for specific values of stimulus parameters such as frequency, ITD and ILD constitutes the raw data in this study. The stimulus parameters were randomly varied during the recording of neural responses. For each Ov and ICx neuron we examined the ITD, ILD, and frequency tuning (). We computed: mean firing rate as a function of ITD (ITD curves), varied in 30-µs steps within a range from −300 to 300 µs (negative ITDs indicate ipsilateral ear leading); mean firing rate as a function of ILD (ILD curves), varied in steps of 5 dB in a range from −30 to 30 dB (negative ILDs mean left ear louder); and mean firing rate as a function of the stimulus frequency changed in steps of 100 Hz at a constant sound intensity of 40 dB SPL (iso-intensity frequency tuning curve).
FIG. 2 Response properties in Ov and ICx. We located the position of the recording electrodes by the injection of fluorescent tracers. Four different recording sites (seen as white spots) are shown in a photograph of Ov (A) and one recording site in ICx (B). (more ...)
We first determined that a neuron was “sensitive” to ITD or ILD by visual inspection of the tuning curves obtained with broadband noise stimulation. The ITD tuning was later confirmed by the statistics used in fitting the ITD curves obtained with tonal stimulation. The “best ITD ” and “best ILD” elicited the maximum response in the ITD and ILD curves, respectively. In the case of ITD curves with multiple peaks of similar amplitude, we used the peak closest to 0 µs. We used the best ITD to collect the ILD curves and the best ILD to collect the ITD curves. These ITD and ILD values were then used to obtain the neuron’s frequency tuning curve. An Ov neuron was classified as broadly tuned to frequency when it responded to a frequency band equal to or larger than the median half-height width of the iso-intensity frequency tuning curves in the ICx neurons sample (1.4 kHz). In all neurons that were initially considered tuned to ITD and broadly tuned to frequency, we examined the ITD sensitivity across frequency. We performed the same analysis on space-specific neurons of ICx to compare results obtained under identical experimental conditions.
The tuning to ITD in periodic signals (tones) can be expressed in terms of phase differences between the sound arriving to the left and right ears. The interaural phase difference that elicits the maximum mean response will be called the mean interaural phase (MIP) (Goldberg and Brown 1969
). ITD curves for tones in Ov and ICx do not show the clear sinusoidal shape of lower brain stem neurons that allows MIP computation by fitting the data to cosine functions (Peña et al. 1996
; Viete et al. 1997
). Instead, we obtained MIP by folding the ITD curves into a single period of the stimulating frequency, converting ITD to interaural phase difference (IPD), and fitting the data to a Gaussian function using the least-square method. The center of the Gaussian fit was used as MIP. We used visual inspection of each fit and the χ2
statistical test, based on the residuals between the data and the model, and the degrees of freedom of the fitting equation, to evaluate the goodness of each fit. Only the cells whose fits passed the χ2
< 0.05) were used for further analysis. We then performed a linear regression of the MIP versus stimulating-frequency and quantified the difference between the data and the regression line by computing the mean of the squared differences between each point and the regression line. We carried out the same analysis for Ov and ICx neurons.
We studied the ITD tuning across frequency by examining the relationship between MIP and stimulus frequency. We used the residuals between the data and the regression line to quantify the linearity of this relationship. For linear relationships, this is also the most precise method to determine the characteristic delay (CD) of the neurons (Rose et al. 1966
; Yin and Kuwada 1983
). Neurons that have a CD respond to a value of ITD with the same relative firing rate at all stimulus frequencies. In such cells, plotting the MIP against the stimulating frequency yields a line whose slope is the CD.
The width of the main peaks of the ITD curves for broadband noise and tones were measured at 50% of the distance between the minimum and maximum response level (“half-height width”). The same criteria were used to measure the frequency-tuning width in iso-intensity frequency tuning curves.
The recording sites were marked in the last experiment by iontophoretic injection of tracers [fluorescein (FDA) and tetramethylrhodamine (RDA) conjugated dextran amines] and electrolytic lesions in the previously recorded regions of Ov and ICx (). Four to 6 days after tracer injection, the owls were overdosed with sodium pentobarbital (Nembutal, Abbot Laboratories) and perfused with saline followed by 2% paraformaldehyde (Fisher Scientific). Brains were blocked in the plane of the electrode penetration, removed from the skull and placed in 30% sucrose until they sank. They were then cut in 60-µm sections and mounted on slides to verify the location of the recording site. The electrode locations of previous experiments were extrapolated using records of the stereotaxic coordinates of each recording site.