2.1. Surgery and animal preparation
Experiments were conducted on four cases (one right and three left hemispheres) of three adult female cats. All protocols were approved by the University of California, San Francisco Committee on Animal Research in accordance with federal guidelines for care and use of animals in research. Animals were sedated by intramuscular injections of a mixture of ketamine (22 mg/kg) and acepromazine (0.11 mg/kg). After venous cannulation, sodium pentobarbital (15–30 mg/kg) was administered and supplemented as needed throughout the surgical procedure. Following tracheotomy, a craniotomy was performed to expose the ectosylvian gyrus. The dura mater was partially removed, and the cortical surface was covered with viscous silicone oil. Before commencing the electrophysiological recordings, sodium pentobarbital anesthesia was replaced with a continuous intravenous infusion of a mixture of ketamine (2–10 mg/kg/h) and diazepam (0.05–0.2 mg/kg/h) in lactated Ringers (1–3 ml/kg/h). To prevent edema and mucus secretion, dexamethasone (1.2 mg/kg, S.C.) and atropine sulfate (0.04 mg/kg, S.C.) were administered every 12 hours. Since recordings lasted for three to four days, an antibiotic (cephalosporin, 11 mg/kg, I.V.) was administrated to prevent wound infection. Body temperature was monitored and maintained by a water heating pad at 37±1°C. Electrocardiogram and respiration rate were monitored continuously during surgery and recording procedures.
2.2. Acoustic stimuli
Experiments were conducted in a double-walled, anechoic chamber (Industrial Acoustics, Bronx, NY). Stimuli were delivered by a STAX-54 headphone through a sealed tube into the acoustic meatus contralateral to the studied hemisphere. The system frequency transfer function was flat (±6 dB) up to 14 kHz and rolled off 10dB/octave at higher frequencies.
Two different stimuli, pure tone bursts and click trains, were presented for measuring frequency response areas and RRTFs, respectively. Pure tone stimuli of 50ms duration (including 3-ms linear rise and fall time) were generated at intervals of 400–750 ms by a microprocessor (TMS32010, 16 bits resolution and 120 kHz digital-to-analog sampling rate). Frequency response areas were mapped by presenting 675 pseudo-randomized tone bursts at 45 different frequencies (3–5 octave range) and 15 sound levels (70 dB range in 5 dB steps). For RRTFs, click trains (monophasic, rectangular pulses of 200 µs duration; 500 ms train duration) were presented 15 times for repetition rates from 2 to at least 38 Hz (2 or 4 Hz steps) at sound level of 82–102 dB SPL (at peak level) depending on neural threshold (see below). For most sites that still showed phase-locked responses and/or elevated FRs at 38 Hz, higher repetition rates were presented (up to 250 Hz) until no clear evidence for stimulus-driven responsiveness was observed. For analysis, the 2 Hz stimulus was treated as 1 Hz because only one click was presented at an interval of 950 ms during the analysis window (550 ms) for spike counts (see below).
Parylene- or epoxylite-coated tungsten microelectrodes (Micro Probes, Potomac, MD or Frederic Haer & Co., Bowdoinham, ME) with 0.5–4 MΩ impedance at 1 kHz were used for single- and multi-site recordings. Single or double microelectrodes were advanced perpendicular to the cortical surface with a hydraulic microdrive (David Kopf Instruments, Tujunga, CA). A video picture of the cortical surface was captured and digitized with a CCD digital camera (Cohu, San Diego, CA). Each recording site was marked on the digitized picture using Canvas software (Deneva, Miami, FL). The marked sites were used to reconstruct tessellation maps of the recording area (see below). Neuronal activity was obtained in main thalamocortical recipient layers (Huang and Winer, 2000
). Action potentials were amplified and band-pass filtered (0.3–10 kHz; World Precision Instruments, Sarasota, FL, and Axon Instruments, Union City, CA), fed to an oscilloscope, and isolated from background noise with a time/amplitude window discriminator (BAK Electronic, Mount Airy, MD). For frequency response areas and RRTFs, spikes occurring in the first 50 ms or 550ms, respectively, after stimulus onset were recorded at 10 or 100 µs resolution for the analyses.
2.4. Data analysis
Data were analyzed using the MATLAB (Mathwork, Natick, MA) platform. StatView (SAS Institute, Cary, NC) was used for statistical analysis. Spectral receptive field parameters (RFPs) such as characteristic frequency (CF), minimum threshold, quality factors, and latency were obtained. Threshold was defined as minimum excitatory SPL, and estimated at 5 dB resolution. CF was defined as the frequency at which a single neuron or neuron cluster produced sound-evoked spikes at threshold sound level. Spectral bandwidths were calculated as CF divided by excitatory bandwidth at 10 dB (Q10) or 40 dB (Q40) above threshold; the higher the Q-value, the more sharply tuned are the neurons. Latency was determined as the minimum value in the averaged latency-level function at CF and the two adjacent test-frequencies (CF +/− 1/15 to 1/9 octaves). Results for spectral RFP distributions were presented elsewhere (Imaizumi et al., 2004
For RRTFs, spike occurrence to the first click was discarded except for the 1 Hz stimulus. Spikes were counted from the second click onset to 550 ms after the first click onset (for 1 Hz stimulus, spikes occurring between the first click onset and 550 ms were used). VS and FR were used to measure temporal following activity (Eggermont, 1998
; Joris et al., 2004
). VS measures how well spikes are phase-locked to the clicks:
is the total number of spikes, t
is time of spike occurrence, and T
is the inter-click interval (Goldberg and Brown, 1969
). Significance of synchronization was examined by a Rayleigh test (p
< 0.001) (Mardia, 1972
). SR was measured by multiplying VS and FR (Eggermont, 1998
), and is equivalent to the un-normalized VS or the un-normalized first Fourier component of the PSTH spectrum (Joris et al., 2004
). illustrates two examples of the SR construction and the main measurements to characterize RRTFs. For illustration only, a polynomial cubic spline was used to connect data points. For analysis, however, a linear interpolation was employed to measure the RRTF parameters. Filled (or opened) circles represent data points with statistically significant (or non-significant) response synchrony to a given repetition rate (). Best repetition rate (BRR) was determined as the click repetition rate at which a neuron or neuron cluster produced the highest SR (e.g., 1 Hz in and 18 Hz in ). Q50% values were calculated as BRR divided by bandwidth at 50% SRs (BW50% in ) (Liang et al., 2002
). For Q-values of low-pass responses (e.g., ; ~9% of the total sites: 32/368), the lowest repetition rate of 1 Hz was used as low cutoff repetition rate. High cutoff repetition rate (HRR) was estimated from the 50% response. For 40 (out of 369) sites, 38 Hz (their highest tested repetition rate) was taken as HRR because the 50% reduction in SR was not reached. For these sites, the HRR may be an underestimate.
Fig. 1 Measurement method of temporal receptive fields by repetition rate transfer function (RRTF). A, D) Vector strength (VS) as a function of repetition rates. Data points were connected by a polynomial cubic spline fit for illustration. For analysis, a linear (more ...)
2.5. Classification of RRTF properties
RRTFs were classified into three temporal filter types, and were considered band-pass when the response peak was flanked by troughs in which the responses dropped <75% of the peak activity (Tian and Rauschecker, 1994
). If one of the response troughs did not reach the criterion, RRTFs were considered either as low- or high-pass type.
2.6. Voronoi-Dirichlet tessellation map
To reconstruct the spatial distribution of RRTF parameters across the cortical surface, tessellation maps were calculated by Voronoi-Dirichlet tessellation (Kilgard and Merzenich, 1998b
). The polygon surrounding each electrode penetration in the tessellation map characterizes the area assigned to the functional parameter at the recording site. Borders between neighboring polygons were determined from the midpoints of a straight line between adjacent recording points. The value of each RFP or RRTF parameter in the cortical surface map is illustrated by color codes.
2.7. Spatial organization analysis
Two types of spatial organization analysis were performed complementarily. To investigate global spatial organization, spatial autocorrelation was used to estimate a measure of redundancy by determining Geary’s C coefficient (Cliff and Ord, 1973
). A Geary’s C value is computed by autocorrelation of spectral RFPs or RRTF parameters between two adjacent recording sites:
is the number of recording sites, wij
is a spatial weight at recording sites i
is sum of all wij
is the value of the variable, and
is mean of x
. C values are based on value differences between pairs of observations for the whole map and quantify whether adjacent observations of the same phenomenon are correlated. They can vary between 0 and 2. A C value of 2 indicates perfect positive spatial correlation (high spatial uniformity and maximum neighbor similarity), while a C value of 0 corresponds to negative correlations (maximal dispersion and high value contrast between neighbors). A null hypothesis of random spatial distribution results in a C value of 1. In a Monte-Carlo analysis, the statistical significance of the experimental C values was derived from the C-value distribution of 10,000 randomized map versions.
To investigate local spatial organization, the value similarity between each polygon and its direct neighbors was examined. Statistical significance was obtained by Monte-Carlo analysis by which the value similarity was compared with 10,000 randomly redistributed neighboring polygon values. The statistical significance of the number of polygons with similar direct neighbors in each experimental map was determined in relation to those found in 1,000 randomized maps. This estimates the proportion of local parameter clusters.