Source Paper
Theta Rhythms Coordinate Hippocampal–Prefrontal Interactions in a Spatial Memory Task
Matthew W Jones, Matthew A Wilson
PLoS Biology • 2005
Materials & Equipment Checklist
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Materials2
Software3
M. A. Wilson
M. A. Wilson
MathWorks, Natick, Massachusetts, United States
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Protocol Steps
1
Animal preparation and food deprivation
Six male Long-Evans rats aged 2-6 months were mildly food-deprived to maintain body weight at 85% of free-feeding weight
Note: Food deprivation used to motivate task performance
View evidence from paper
“Six male Long-Evans rats (2–6 mo) were mildly food-deprived (to 85% of free-feeding body weight)”
2
Spatial-alternation task training
Rats trained to run continuous spatial-alternation task on narrow 6 cm track. Each trial comprised forced-turn epoch (rat forced to turn in one direction) followed by choice epoch (rat must alternate direction to win reward). Forced-turn direction varied randomly with no more than three consecutive trials in one direction. Relative location of forced-turn end varied between animals
12-14 days
Note: Analysis epochs excluded reward points and turning points. Running constrained to overlapping linear trajectories using narrow track
View evidence from paper
“trained to run a continuous spatial-alternation task... Each trial comprised distinct sample and test epochs. The contingency was set such that, for example, a rat forced to turn to his right during the forced-turn epoch had to choose a left-hand turn to win reward during the subsequent choice epoch. Forced-turn direction was varied randomly, with no more than three consecutive trials in one direction. The relative location of the forced-turn end of the maze was varied between animals. Every effort was made to constrain running to overlapping linear trajectories by using a narrow track (6 cm)”
3
Training to asymptotic performance
Rats trained until reaching asymptotic performance criterion of at least 80% choice-correct on two consecutive days
12-14 days total training period
Note: Criterion must be met before surgical implantation
View evidence from paper
“Each rat was trained to asymptotic performance (two consecutive days of at least 80% choice-correct) over a period of 12–14 d before surgery”
4
Surgical implantation of tetrode arrays
Rats implanted with arrays of adjustable tetrode recording electrodes. Primary targets: mPFC at +3.2 mm anterior, +0.6 mm lateral from bregma, and ipsilateral dorsal CA1 at -3.6 mm anterior, +2.2 mm lateral. In two rats, additional tetrodes targeted to ventral CA1 at -6.3 mm anterior, +6.2 mm lateral
Climbing Test - Measures Vertical Activity in Rodents
Matches: Tetrode recording electrodes
$6,595View
Note: Surgery performed after reaching asymptotic performance. Local reference electrodes placed in white matter or cortical regions without spiking activity
View evidence from paper
“implanted with arrays of adjustable tetrode recording electrodes targeted to the mPFC (+3.2 mm, +0.6 mm from bregma) and ipsilateral dorsal CA1 (−3.6 mm, +2.2 mm). In two rats, tetrodes were also targeted to ventral CA1 (−6.3 mm, +6.2 mm)”
5
Electrophysiological recording setup
Differential recordings made using Keithley Instruments DAS-1802HC acquisition boards. Action potentials sampled at 31.25 kHz per channel, filtered between 600 Hz and 6 kHz. Continuous LFP sampled at 3.125 kHz per channel, filtered between 1 and 475 Hz
Note: Local reference electrodes positioned in overlying white matter (for dorsal CA1), adjacent white matter (for ventral CA1), or proximal cortical region without spiking activity 2.4-2.7 mm below pial surface (for mPFC)
View evidence from paper
“Differential recordings of extracellular action potentials (sampled at 31.25 kHz per channel, filtered between 600 Hz and 6 kHz) and continuous LFP (sampled at 3.125 kHz per channel, filtered between 1 and 475 Hz) were made using Keithley Instruments acquisition boards (DAS-1802HC)”
6
Electrolytic lesion marking
Electrolytic lesions performed at end of each experiment to establish tetrode tip positions
Note: Lesion positions used to verify recording electrode locations
View evidence from paper
“Electrolytic lesions established tetrode tip positions at the end of each experiment”
7
Action potential clustering and neuron identification
Extracellular action potentials assigned to individual neurons by off-line, manual clustering using Xclust software
Note: Manual clustering performed after data collection
View evidence from paper
“Action potentials were assigned to individual neurons by off-line, manual clustering using Xclust software (M. A. Wilson)”
8
Burst detection and Complex Spike Index calculation
Firing with inter-spike intervals between 2 and 15 ms defined as bursting (minimum inter-burst interval 150 ms). Complex Spike Index calculated combining bursting measure with measure of amplitude reduction in later spikes within bursts
Note: Complex Spike Index used to identify complex spike cells
View evidence from paper
“Firing with inter-spike intervals of between 2 and 15 ms was defined as bursting (minimum inter-burst interval 150 ms). The Complex Spike Index combined a measure of bursting with a measure of the likelihood that spikes later in bursts were smaller in amplitude than spikes earlier in bursts”
9
Spatial information analysis
Spatial information calculated according to Skaggs et al. method. Analysis restricted to neurons firing at least 50 spikes in both choice and forced-turn epochs
Note: Analyses compared firing on central three-quarters section of central arm in two running directions
View evidence from paper
“Spatial information was calculated according to Skaggs et al. Most analyses compared firing on the central three-quarters section of the central arm in the two running directions, and were restricted to neurons that fired at least 50 spikes in both choice and forced-turn epochs”
10
Theta phase analysis
LFPs down-sampled to 600 Hz and band pass filtered between 4 and 12 Hz. Maxima and minima detected with thresholds established to extract theta peak and trough times. Only peaks or troughs greater than one standard deviation from mean amplitude included. Each spike assigned theta phase between 0-360° by linear interpolation relative to enveloping peak (180°) and trough (0° or 360°) times
Note: Approximately 63% ± 6.6% of maxima included during choice epochs, 61% ± 9.5% during forced-turn epochs
View evidence from paper
“LFPs were down-sampled (to 600 Hz) and band pass filtered between 4 and 12 Hz, then maxima and minima detected and thresholds established to extract theta peak and trough times. Only peaks or troughs greater than one standard deviation from the mean amplitude of the filtered LFP were included (63% ± 6.6% of all maxima during choice epochs, 61% ± 9.5% during forced-turn epochs)”
11
Theta phase uniformity testing
Rayleigh test of uniformity used to assess theta phase distributions for deviations from circular uniform distribution. Circular statistics calculated according to Fisher
Note: Tests whether spike timing relative to theta phase is non-random
View evidence from paper
“The Rayleigh test of uniformity was used to assess the resulting phase distributions for deviations from the circular uniform distribution. Circular statistics were calculated according to Fisher”
12
Spectral analysis and coherence calculation
Multi-taper spectral analysis used to calculate power spectra and coherence for LFP data. Technique uses short-time-window Fourier analysis to reduce artifacts from non-stationary elements. Significance of trial-by-trial coherence magnitude during central-arm crossings calculated according to Jarvis and Mitra with coherence values greater than 2/√[(number of trials) × (number of tapers)] considered significant at p = 0.05
Note: Multi-taper approach assumes data stationarity within short sliding time-windows
View evidence from paper
“Multi-taper spectral analysis was used to calculate power spectra and coherence for LFP data. This technique takes advantage of short-time-window Fourier analysis to reduce artifacts caused by non-stationary elements in the data (since data can be assumed to be stationary within the short sliding time-windows). The significance of trial-by-trial magnitude of the coherence during central-arm crossings was calculated according to Jarvis and Mitra”
13
Statistical comparison of task conditions
Statistical comparisons between forced-turn, choice-correct, and choice-error conditions performed on two groups of animal means using Wilcoxon rank sum tests
Note: Data presented as mean ± standard error of the mean
View evidence from paper
“Statistical comparisons between forced-turn, choice-correct, and choice-error conditions were performed on the two groups of animal means using Wilcoxon rank sum tests”
Subjects / Specimens
- Species
- rat
- Strain
- Long-Evans
- Age
- 2-6 months
- Sex
- male
- Count
- 6
Mildly food-deprived to 85% of free-feeding body weight