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www.sciencemag.org/cgi/content/full/science.1216567/DC1
Supplementary Materials for
Neural Correlates of a Magnetic Sense Le-Qing Wu and J. David Dickman*
*To whom correspondence should be addressed. E-mail: [email protected]
Published 26 April 2012 on Science Express DOI: 10.1126/science.1216567
This PDF file includes:
Materials and Methods Fig. S1 References (25–30)
Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/science.1216567/DC1)
Movie S1
Supporting Online Materials
Materials and Methods:
Subjects and stimulation
All methods were approved by the Institutional Animal Care and Use Committee
and were in accordance with the National Institutes of Health Guidelines. Seven homing
pigeons (Columba livia) were surgically implanted with a plastic head restraint stud in
order to maintain a stable head position during stimulation (25). Single cell neural
recordings were obtained from vestibular brainstem neurons during magnetic field
stimulation while the pigeon was awake, in the dark (to reduce visual stimulation), and
placed head-fixed (to reduce vestibular stimulation) in the center of a magnetic field coil
system. An artificial magnetic field was delivered using a direct current source through
three pairs of Helmholtz coils (61 cm cube, 38 turns on each side of 1.09 mm copper wire)
driven by a computer interface (Power 1401; Cambridge Electronic Design) and custom
script software (Spike2). The ambient geomagnetic field was first measured (inclination
and amplitude) using a 3-axis magnetometer (HMC2003, Honywell) mounted directly
beneath the birds’ head, then was actively canceled to a net zero amplitude. An artificial
magnetic field vector of 100 µTesla (~2x intensity of the home laboratory) was then
generated and rotated through 360º at 10º steps (100 ms/step, total period = 3.6
s/revolution) along each of four different great planes (45 deg increments, Fig. 3).
Elevation and azimuth values were derived from Cartesian coordinates, where positive x,
y, and z axes corresponded to nasal, left ear, and vertex, respectively. Zero degrees
elevation and azimuth corresponded to a magnetic field vector directed along the positive
X axis; positive elevation and azimuth values corresponded to upward and leftward
angles, respectively (Fig. 3). Both CW (increasing direction angle, 0 – 360) and CCW
(decreasing angle, 360 – 0) magnetic vector rotation directions were alternated, with a
minimum of 10 repetitions for each presented. To examine intensity functions, four
different magnetic field amplitudes of the rotating magnetic vector including 20, 50, 100,
and 150 µT were also delivered in each of the great circle planes, for a subset of neurons.
1
Neural recording and analyses
Single cell neural activity was obtained using epoxy-coated tungsten
microelectrodes (5 – 10 MΩ; FHC, Inc.) placed in guide tubes (28 gauge) and driven
vertically with a remote microdrive. All materials used for implants and recordings (head
stud, electrodes, guide-tubes) were non-magnetic to eliminate possible conductive
artifacts. Neural activity was band-pass filtered (100 – 5 KHz, Bessel), displayed on an
oscilloscope (Tektronix model 5311dn), then digitized (20KHz) and stored on computer.
From the stored data, single cells were identified by template match of waveform shape,
amplitude, and latency (Spike 2, CED, Cambridge England). Spike times were
temporally assorted into 100ms bins for each response to magnetic stimulation in each
great plane. To test for a significant response, the number of spikes per bin was compared
across all bins for the four stimulation great planes and two magnetic field rotation
directions (dependent variables). To be considered a response, the temporal modulation
in firing rate for at least one stimulation plane in either the CW or CCW rotation direction
must have been significantly different (ANOVA, p<.001) from baseline (26). Cells with
no significant modulation in any plane were considered non-responsive and not analyzed
further. As a final control, neural responses were recorded from: (1) a saline solution
substituted for a live pigeon and (2) a euthanized (non-live) pigeon brain while magnetic
stimulation was delivered, with no recorded electrical responses being observed.
Peri-stimulus time histograms (PSTHs) were made by calculating the mean firing
rate for each 100ms time bin for each stimulus plane (Fig. 3). The sensitivity and tuning
direction values for each MR cell were determined for the four great plane responses in
both the CW and CCW rotation directions. Cosine curve fits were applied to each
response using the form FR = Smax x cos(x1 • x2 + y1 • y2 + z1 • z2) + DC, where FR is
the firing rate of the cell, Smax is the maximum sensitivity response, x1;y1;z1 is the
measured response vector from which FR was obtained, x2;y2;z2 is the preferred vector,
and DC is the spontaneous firing rate. The 3D preferred direction was then calculated in
spherical coordinates from the cosine curve fits and plotted as unit vectors. A re-
sampling analysis was performed to assess whether the distribution of preferred
directions was significantly different from uniformity (27). First, the sum squared error
(across bins) between the measured distribution and an ideal uniform distribution were
2
calculated. Next, the sum squared error between the ideal distribution and a random re-
sampled (1000 repetitions) distribution was calculated. If the measured-ideal sum
squared error exceeded the 99% confidence interval of the re-sampled-ideal error, then
the measured distribution was considered to be significantly different from uniform. For
non-uniform distributions, a multimodality test was performed based on the kernel
density estimate to determine the number of modes. A von Mises method was used as
the kernel function for circular data and a Gaussian function for noncircular data. A
goodness-of-fit statistic was calculated to obtain the critical p value (Pk) of k modes
through a bootstrapping procedure (21). If Pk < 0.05 was obtained for k number of
modes, we considered the distribution to be significantly different from the next lowest
modality. As a control, the preferred directions obtained for 50 and 100 µT stimulation
amplitudes were found to be equivalent (pair-wise Sign test, p= 0.51).
The strength of the directional tuning for each neuron’s preferred response vector
was quantified using a direction discrimination index (DDI), of the
form: ( ) ( )( )MNSSESSSSDDI −÷+−÷−= 2minmaxminmax
where, Smax and Smin are the maximum and minimum cell responses from the
3D cosine fits, SSE is the sum of squared error for the mean response, N is the total
number of repetitions, and M = 8 for the four stimulation planes and CW and CCW
directions (28). DDI values range between 0 – 1, and compares the difference in the
cell’s firing rate between the preferred direction and the minimum (null) directions
against inherent variability. Values close to 1 indicate large response modulations
relative to noise, while values close to zero indicate no modulation.
For all responsive neurons, the sensitivity and tuning direction values were used
to plot Lambert cylindrical equal-area contour maps (29 - 30) illustrating cellular
sensitivity as a function of two stimulus angles: azimuth (0 – 360 degrees) and elevation
(-90 – 90 degrees). In addition, intensity functions were generated by presenting rotating
magnetic field vectors along all four great planes, for both CW and CCW directions, at
four amplitudes; 20, 50, 100, and 150µT for a subset of 9 MR cells. Intensity functions
were examined using a pairwise comparison repeated measures ANOVA and Tukey post-
hoc tests. Exponential curves of the form (f = A(1-exp(-B x X))c) were then fit to the
3
intensity function plots for each cell. Analyses were performed using MATLAB
(Mathworks Inc., Natick, MA) or PYTHON.
Histology
On the final experimental day for each of the seven birds, an electrolytic lesion
was made at the recording site location by passing DC constant current (20µA for 30s)
through the recording electrode. The animal experienced no discomfort during the
current delivery since (1) there are no pain receptors in brain tissue, (2) the lesions were
very small (<30µm diameter) made in regions distant to the dorsal column and
spinothalamic tracts, and (3) no outward signs of animal distress were exhibited. In one
bird, following the electrolytic lesion, magnetic field stimulation was delivered in order
to maximally activate the c-Fos transcription factor using a rotating field vector (150µT)
along each of 36 different elevation angles (12 each for X, Y, and Z axes; 10º increments).
Each magnetic field vector rotation required 2 minutes duration, for a total stimulation
period of 72 (2 x 36) minutes. All birds were then immediately euthanized (500 mg/kg
sodium pentobarbital i.m.) and perfused through the heart with 4% paraformaldehyde in
phosphate buffer. The brains were excised, cut into 50µm sections, and dehydrated with
a graded series of alcohols and xylene. Sections for the one c-Fos activated pigeon were
treated for dark reaction product immunohistochemistry, and were used in conjunction
with an earlier investigation (18). The sections were counterstained (Neutral red) and
photographed with a Nikon Eclipse 600 microscope.
Figure 1S. Neural responses from the representative MR cell shown in Fig. 3, to a single
cycle stimulation for four great circle plane magnetic vector (CW) rotations (gray
shaded), plotted as function of time. Each panel column shows the stimulation plane
(top), the instantaneous firing rate of the MR neuron (second panel, spikes/sec), the
amplified (1000x) and filtered (300 – 5K Hz) neural activity (third panel, MV =
millivolts), and the three magnetometer channels, Gz, Gy, and Gx (µT = micro Tesla).
Movie 1S. Neural activity to a single magnetic stimulation trial. Neural firing rate of
MR cell shown in Fig. 3 and 1S (top) in real time as the rotating magnetic vector (red
4
arrow) is presented in interleaved trials through CW and CCW directions through each of
four great circle planes (bottom, gray shaded). Audio consists of filtered (300 – 5K Hz)
neural activity.
Time (sec)0 1 2 3
Gy
(µT)
Gz
(µT)
−2000
200400600
MV
Firin
g ra
te (s
pks/
s)
0 1 2 3 0 1 2 3 0 1 2 3
10203040
−100
0
100
−100
0
100
−100
0
100
X
Z
YX
Z
YX
Z
YX
Z
Y
Gx
(µT)
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