Methods
Experiments were performed in compliance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health, publication 86-23), the National Research Council’s Guidelines for Care and Use of Mammals in Neuroscience and Behavioral Research (2003), and with approval from the Institutional Animal Care and Use Committee at Virginia Commonwealth University. Details of these procedures are provided in a previous report (Foxworthy et al. , 2013). Ferrets were obtained from a licensed vendor (Marshall Farms, Inc.) and were housed in VCU Department of Animal resources until used. All ferrets were male and their age on the experiment day ranged from P80 – P300. Based on these ages, each ferret was assigned to one of four developmental groups. (1) Infancy: ferrets less than P90 are weaned but have not yet reached sexual maturity (have not yet become adolescents). Next, because onset of sexual maturity can occur as early as 4 months of age, animals aged >P120 are post-pubertal and were considered (2) early adolescent (P120-155); (3) mid-adolescent (P160-200) and (4) late adolescent (P240-P300). There is, as yet, no consensus on when ferrets become neurologically mature adults.
For the recording experiments, the animal was anesthetized (8mg/kg ketamine; 0.03mg/kg dexmedetomidine intramuscularly) and their head were secured in a stereotaxic frame. A craniotomy was made to expose the rostral posterior parietal (PPr) portion of the suprasylvian gyrus. Across this opening a recording well/head supporting device was implanted using stainless steel screws and dental acrylic to support the head without obstructing the eyes or ears. The implant was then secured to a supporting bar. The animals were intubated through the mouth, ventilated (expired CO2: ~4.5%) and immobilized (pancuronium bromide; 0.3 mg/kg initial dose; 0.2 mg/kg h supplement i.p.) to prevent ocular drift or spontaneous limb movements during testing. Fluids (lactated Ringer’s solution) and supplemental anesthetics (4mg/kg h ketamine; 0.5 mg/kg h acepromazine i.p.) were administered continuously with an infusion pump. Heart rate was monitored continuously, and body temperature was monitored and maintained at ~38°C with a heating pad.
Within the craniotomy the dura was opened to identify the location of the PPr (based on gyral patterns) and to insert the recording electrode array. Neuronal activity was recorded using a four shank, 32-channel silicon probe (A4×8-5mm 200–200-413 array; impedance ~1 MΩ; NeuroNexus Technologies, Ann Arbor, MI) as described in previous reports (Allman et al. , 2009; Keniston et al. , 2009; Foxworthy et al. , 2013). Neuronal activity was digitized (rate>25kHz) using a TDT System III Workstation (TuckerDavis Technologies Alchua, FL) using MatLab software and archived for off-line analysis. Spike waveforms were clustered by principal component feature space analysis and sorted into individual units using an automated Bayesian sort-routine. Spikes which exhibited interspike intervals < 2ms were rejected.
Once PPr neurons were identified and templated, their responses to sensory stimulation were assessed. Quantitative sensory testing consisted of somatosensory stimuli produced by a calibrated 1-gram monofilament fiber moved by an electronically-driven, modified shaker that displaced hair or indented the skin. Visual stimulation consisted of a bar or spot of light, whose movement direction, velocity, and amplitude across the visual receptive field was computer-controlled and projected onto the translucent hemisphere. These somatosensory and visual stimuli were presented separately and in combination, and each stimulus or combination was repeated 50 times (randomly interleaved with 3-7 second presentation interval). During combined presentations, the onsets of the stimuli were offset by 40ms (visual preceded tactile) to accommodate for the difference in response latency among these sensory modalities. Attention was given to maintaining the consistency of sensory stimulation between different experiments, such that the somatosensory stimulus was always positioned on the contralateral side of the face and moved at the same velocity and amplitude; visual stimulation always consisted of a moving (150 º/sec) bar (5x20º) of light that crossed ~45º of contralateral visual space in the nasal-to-temporal direction.
Neuronal responses to somatosensory, visual, and combined visual-somatosensory stimuli analyzed using custom software (MatLab; described in (Allman et al. , 2009; Keniston et al. , 2009). A neuronal response was defined as spiking activity which exceeded 3 standard deviations from spontaneous activity, lasted for a minimum of 15ms, and ended when activity returned to baseline for at least 15ms. Neurons showing suprathreshold activation to individual stimuli from more than one sensory modality were defined as bimodal multisensory neurons. Neurons which showed suprathreshold activation by only one modality but exhibited responses that were significantly different in the combined stimulus condition than in the unisensory stimulus condition (determined by t-test) were classified as subthreshold multisensory neurons. However, neurons that were driven exclusively by one modality and did not show change in responses after combined stimulation were categorized as unimodal.
Multisensory (bimodal and subthreshold) neurons were further analyzed to determine if they demonstrated integrated responses to multisensory stimulation. Specifically, responses showing a significantly different (assessed by t-test) activation (mean spikes/trial) to multisensory stimuli versus that elicited by the most effective single modality stimulus were defined as exhibiting multisensory integration (Meredith & Stein, 1983). Significant response increases were termed multisensory response enhancement, while those showing a significantly reduced activation were regarded as demonstrating multisensory response depression (also called multisensory suppression (Keum et al. , 2023)). The magnitude of multisensory integration was calculated according to the method of (Meredith & Stein, 1986) using the formula: (CM-SMmax)/ SMmax x 100 = % Integration. In this equation, SMmax was the neuron’s response to the most effective unisensory stimulus (mean spikes/trial) and CM was the response to the multisensory stimulus. Ultimately, the neuronal response type (unisensory, bimodal, subthreshold) as well as the direction (enhancement, depression) and magnitude (% response change) of multisensory responses were tabulated in relation to the age of the experimental animal.
Once the recording session was completed, the animal was overdosed (Euthasol), perfused intracardially with saline and fixed (4% paraformaldehyde). The brain was blocked stereotaxically and the cortex containing the recording site(s) was processed for histological verification of the recording sites. Recording tracks confirmed within the grey matter of the PPr were included in this study.