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.