One day prior to surgery, and again on the day of surgery, animals were given a single dose of dexamethasone sodium phosphate (1 mg/kg, i.m.) to reduce the likelihood of edema. On the day of surgery the anesthetic ketamine hydrochloride (20 mg/kg, i.m.) with acepromazine maleate (0.1 mg/kg, i.m.) was administered to animals in their home cage before transport. They were then transported to the surgical suite, intubated for the induction (2–4%) and maintenance (1.5–2%) of gaseous (isoflurane) anesthesia during surgery, and placed in a stereotaxic frame. During surgery expiratory CO2, blood pressure and heart rate were continuously monitored using a digital vital signs monitor (VetSpecs VSM7), and body temperature was maintained at ~37–38°C with a heating pad. A stainless steel recording chamber was placed over a craniotomy to provide access to the SC and attached to the skull with stainless steel screws and dental acrylic (McHaffie & Stein, 1983). After surgery, animals were administered analgesics (buprenorphine 0.005–0.01 mg/kg, i.m., b.i.d. for 3 days), antibiotics (ceftriaxone, 20 mg/kg, i.m., b.i.d. for 7 days) and a dexamethasone ‘taper’ (0.5 mg/kg, i.m., b.i.d. for 3 days; 0.25 mg/kg, i.m., b.i.d. on day 4 and s.i.d. on day 5).
Weekly recording sessions began after at least 7 days of post-surgical recovery. Again, the animal was administered ketamine hydrochloride (20 mg/kg, i.m.) with acepromazine maleate (0.1 mg/kg, i.m.) in the home cage, transported to the experimental room, intubated and ventilated mechanically. It lay in a recumbent position and its head was fixed in orientation by two horizontal stainless steel bars attached to the recording chamber, thereby avoiding the induction of any wounds or pressure points. Respiratory rate and volume were adjusted to keep the end-tidal CO2 at ~4.0%. Expiratory CO2, heart rate and blood pressure were monitored continuously to assess and, if necessary, adjust depth of anesthesia. Neuromuscular blockade was induced with an injection of pancuronium bromide (0.1 mg/kg, i.v.) to preclude movement artifacts, prevent ocular drift and fix the pinnae in place. The pupil of the eye contralateral to the SC under study was dilated with 1% atropine sulfate, and a contact lens focused the eye on a tangent screen in front of the animal. The optic disc of that eye was mapped on the screen and an opaque lens occluded the other eye. Anesthesia, paralysis and hydration were maintained by intravenous infusion of ketamine hydrochloride (5–10 mg/kg/h), pancuronium bromide (0.05 mg/kg/h) and 5% dextrose in sterile saline (2.4–3.6 mL/h). Body temperature was maintained at 37–38°C.
Conventional methods were used for single-neuron electrophysiological recording (Meredith & Stein, 1983; Yu et al., 2009, 2010; Xu et al., 2012). A glass-coated tungsten electrode (tip diameter: 1–3 μm, impedance: 1–3 MΩ at 1 kHz) was lowered to the surface of the SC and then advanced by a hydraulic microdrive to search for single neurons in its multisensory (i.e. deep) layers. Single neurons were studied when they were clearly isolated (i.e. action potential amplitude was at least three times the level of background noise). Single neuron activity was recorded, amplified and routed to an oscilloscope, audio monitor and computer for on-line and off-line analyses.
At the end of the recording session, the animal was injected with 40–50 mL of saline subcutaneously to ensure postoperative hydration, and anesthesia and neuromuscular blockade were terminated. The animal was removed from the head-holder, the endotracheal tube and intravenous lines were removed, and it was returned to its home cage in the noise room once stable respiration and locomotion returned.
Procedures for acute recordings in 3-month-old animals were substantially the same as those described above for the adults albeit their head wells were implanted on the day of recording, they were not treated with dexamethasone and were terminated at the end of the recording session (200 mg/kg sodium pentobarbital, i.v.).
During experimentation, visual search stimuli consisted of a moving or flashed bar of light (75 cd/m2) back-projected from an LC 4445 Philips projector onto the tangent screen. Auditory search stimuli consisted of 60–65 dB broadband (20–20 000 Hz) noise bursts and tones delivered via one of 15 stationary speakers positioned around the animal on a hoop whose axis of rotation was in line with the animal's interaural axis. Somatosensory search stimuli consisted of tapping and brushing the hair and skin, as well as manual manipulation of deep tissue and movement of joints. However, visual–auditory neurons were of primary concern for quantitative study and, when such a neuron was identified, its receptive fields were mapped using conventional methods (Meredith & Stein, 1986) and transferred to standardized representations of visual and auditory space (Stein & Meredith, 1993). The unisensory and multisensory responses of these neurons were examined and quantified using randomly interleaved modality-specific and cross-modal stimulus pairs at inter-trial intervals of 5–7 s, 20 trials per condition. The auditory stimuli consisted of brief (100–200 ms) bursts of the broadband noise of varying intensity (55–70 dB) presented against an ambient background of 51.4 dB. Visual stimuli (100–200 ms duration) were rectangular bars of light (6° × 2°) of varying intensity (1.1–13.5 cd/m2) presented against a uniformly dark background (0.86 cd/m2) and moved in the most effective direction and speed for the neuron under study. For cross-modal tests the visual and auditory stimuli were presented within their respective receptive fields and in close spatial and temporal register. In normal animals, this cross-modal stimulus produces a multisensory response that is enhanced relative to that elicited by the best component stimulus (Meredith & Stein, 1983). To identify stimulus intensity values for quantitatively testing a given neuron, a brief series of preliminary tests were conducted in which stimulus intensities were systematically manipulated to quantify its dynamic range. To maximize the integrated multisensory product of the neuron under study, weakly effective individual modality-specific component stimuli were chosen (Kadunce et al., 1997; Perrault et al., 2005; Stanford et al., 2005).
When time permitted, the spatial tuning within a neuron's receptive fields was examined by varying the location in azimuth of a modality-specific stimulus. For examination of visual spatial response profiles, a rectangular moving bar (4° × 1°) was used. Its intensity was 16.5 cd/m2 presented against the uniformly dark background and it was moved at 60°/s through a tightly restricted region (3°) of the visual receptive field. For examination of auditory spatial response profiles, a broadband noise burst 10 dB above threshold was presented multiple times from each of the hoop-mounted speakers in a randomly interleaved fashion. The mean number of impulses evoked by 20 repetitions of each stimulus was used to construct a spatial response profile for each modality. In circumstances in which receptive fields contracted during development to become approximately Gaussian, the tuning profile quantification was improved by fits with Gaussian functions. These made it possible to assess the receptive field center, and the alignment of two receptive fields could be quantified as a t-score:
where Xa and Xv are the locations of the peaks of the Gaussian fits to the auditory spatial and visual spatial tuning profiles, and σa and σv are the standard deviations. The lower the t value, the higher the degree of visual–auditory spatial register.
Three subsets of neurons were subjected to additional testing to examine specific response properties. One subset was tested both with simultaneously presented pairs of visual–visual stimuli (within-modal tests to examine unisensory integration) and pairs of visual–auditory stimuli (cross-modal tests to examine multisensory integration). To evaluate the integrative profiles of these neurons, three stimulus effectiveness levels spanning a neuron's dynamic range were included. In the two other subsets, the spatial and temporal properties of the stimuli were manipulated to determine whether noise-rearing had substantially altered the spatial and temporal principles of multisensory integration rather than precluding its maturation.
Impulse times were recorded for each trial with 1-ms resolution and analysed off-line. The response window was defined using a geometric algorithm based on the cumulative impulse count as described in earlier studies (Rowland et al., 2007). The mean spontaneous firing rate for each condition was calculated in the 500-ms window preceding the stimulus. The magnitude of each response was identified as the mean number of impulses occurring in the response window minus the expected number based on the spontaneous firing rate. The mean response to the stimulus combination was then statistically compared with the response to the most effective single-modality component stimulus (t test, P < 0.05). Multisensory enhancement, the most reliable metric for multisensory integration (Kadunce et al., 1997), was defined as a significant increase in the number of impulses to the combined (cross-modal) stimuli compared with that to the most effective of its individual (modality-specific) component stimuli. The magnitude of multisensory response enhancement (ME) was calculated by the following formula: ME = [(CM − SMmax)/SMmax] × 100, where CM represents the mean magnitude of the multisensory response, and SMmax represents the magnitude of the response evoked by the more effective modality-specific stimulus (Meredith & Stein, 1983). These evaluations were restricted to those samples in which the two unisensory response trains overlapped (i.e. two independent unisensory responses could not be discerned). In cases where enhancement was compared across multiple efficacy levels, including cases where response levels were very low, a contrast index was used in place of ME for stability. The formula for the contrast index is: CME = (CM − SMmax)/(CM + SMmax). The contrast index ranges from −1 to 1, with values near 1 indicating that the multisensory response is far greater than the best unisensory response, and values near −1 indicating the opposite.
Data were compared statistically to determine significant differences using SPSS 11.5, t tests, Wilcoxon's signed rank test, paired t-test, Mann–Whitney rank sum test, and χ2 tests where appropriate. The criterion for statistical significance was P < 0.05.