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CSMR_A_502381
Corresponding author:
V. KHATRI
Title: Whisking in air: Encoding of kinematics by VPM neurons
in awake rats
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Somatosensory and Motor Research, September 2010; 27(3): 1–10
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ORIGINAL ARTICLE
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Whisking in air: Encoding of kinematics by VPM neurons in awake rats
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V. KHATRI1, R. BERMEJO2, J. C. BRUMBERG3, & H. P. ZEIGLER3
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Department of Biology, City College of New York, New York, NY, USA, 2Department of Psychology, Hunter College,
New York, NY, USA, and 3Department of Psychology, Queens College, Flushing, NY, USA
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Abstract
Rodent whisking behavior generates two types of neural signals: one produced by whisker contact with objects; the other by
movements in air. While kinematic signals generated by contact reliably activate neurons at all levels of the trigeminal
neuraxis, the extent to which the kinematics of whisking in air are reliably encoded at each level remains unclear. Previously,
we showed that the responses of trigeminal ganglion (TG) neurons in awake, head-fixed rats are correlated with whisking
kinematic parameters, but that individual neurons may differ substantially in the reliability of their kinematic encoding. Here,
we extend that analysis to neurons in the ventral posterior medial (VPM) nucleus. Three possible coding strategies were
examined: (1) firing rate across an entire movement; (2) the probability of individual spikes as a function of the instantaneous
movement trajectory; and (3) the coherence between spikes and whisking. While VPM neurons were clearly responsive to
variations in whisker kinematics during whisking in air, the encoding of whisker kinematics by VPM neurons was less
consistent than that of TG neurons. Furthermore, we found that, in VPM as in TG, movement direction is an important
determinant of unit responsiveness during whisking in air.
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Keywords: 2222
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whisker protraction. However, in awake, behaving
rats: (1) TG neurons do not respond consistently to
whisker movements in air; (2) their responses tend to
be temporally dispersed throughout the entire movement; and (3) neurons may display preferences for
either protractions or retractions. Moreover, kinematic analyses indicate that selectivity for whisker
movement direction is strongly modulated by the
amplitude and speed of movements (Khatri et al.
2009). Nonetheless, a majority of TG neurons
(70%) display significant correlations between
firing rate and one or more kinematic parameters
(Leiser and Moxon 2007; Khatri et al. 2009).
Beyond the trigeminal ganglion and brainstem,
whisking signals can be processed by lemniscal and
paralemniscal thalamic neurons. Recently, Masri
et al. (2008) demonstrated that whisker movements
in air do not reliably evoke spikes in the paralemniscal thalamic nucleus posteromedial (POm) or its
lemniscal counterpart, the ventroposterior medial
(VPM) nucleus. However, no previous study has
examined the role of kinematics as we do in the
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Introduction
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Mobile sensors require the brain to differentiate
signals produced by external inputs (exafference) from
those generated by the animal’s own sensor movements (reafference). Because the rodent vibrissae
could provide both types of signals, the vibrissal
array is a useful model for investigating the neural
processing that underlies active sensing. Exafferent
signals, produced by active and passive contacts, are
encoded by neurons throughout the whisker system,
from the trigeminal ganglion (TG) to barrel cortex
(TG: Jones et al. 2004; Stüttgen et al. 2006;
thalamus and cortex: Pinto et al. 2000; Arabzadeh
et al. 2005; von Heimendahl et al. 2007; Stüttgen
and Schwarz 2008; Jadhav et al. 2009). However, the
extent to which central trigeminal neurons reliably
encode reafferent whisking signals is currently
unclear.
Using an ‘‘electrical whisking’’ paradigm in anesthetized rats, Szwed et al. (2003) reported that
TG neurons primarily respond to the onset of a
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Correspondence: V. Khatri, Department of Biology, City College of New York, New York, NY, USA. E-mail: vkhatri@ccny.cuny.edu
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(Received 22 22 22; accepted 22 22 22)
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ISSN 0899–0220 print/ISSN 1369–1651 online ß 2010 Informa Healthcare Ltd.
DOI: 10.3109/08990220.2010.502381
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present study for VPM neurons. Since in addition to
PrV inputs, VPM neurons receive both corticothalamic feedback and modulatory influences from
structures such as the thalamic reticular nucleus,
VPM responses to whisker movements in air could
differ from those of first-order TG neurons. We
therefore examined the responses of VPM neurons
under experimental conditions identical to those
used in our study of TG neurons.
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Methods
All procedures were in accordance with National
Institutes of Health guidelines and were approved by
an institutional animal care and use committee.
Animal preparation
Data was collected from 5 female Sprague–Dawley
rats (200–300 g), fitted, under anesthesia, first, with a
head mount permitting head fixation during behavioral testing (details in Bermejo et al. 1998). A
stainless-steel ground screw was inserted into the
skull to serve as a reference for neural recordings.
Three animals, in addition to the five that provided
data, pulled off their headmounts and were killed
prior to electrophysiological recording. Prior to
neural recording, a 2.0 mm 2.0 mm cranial
window was made in the skull above VPM (relative
to bregma: 2.0–4.0 mm posterior, 2.0–4.0 mm lateral), leaving the dura intact and covered with moist
cotton and a thin layer of dental acrylic. These were
removed at the start of each session and replaced
at its end.
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Behavioral training and monitoring of whisker
movements. To reduce habituation and elicit periods of sustained whisking in air, whisker movements
were tracked in the anterior–posterior plane by a
laser emitter–detector system and reinforced with a
sweet liquid (Yoo-hoo) accorded to a fixed-response
(FR) schedule. To visualize a single whisker, a light
(55 mg) self-adhesive foam marker was attached to
its side at 13–20 mm from the base. Despite the
presence of the marker, the whisker’s appearance was
unaltered. Interruption of the laser beam by the
marked whisker results in a voltage shift in a subset of
CCDs, thus enabling the location of the whisker to
be tracked by a downstream comparator circuit and
passed onto a microprocessor for the computation of
trajectories.
It has previously been shown that the marker does
not affect whisking kinematics and that, during
whisking in air, vibrissae in different rows and
columns (e.g., B1 and C3) on one side of the face
move synchronously, with amplitudes differing by
1 (Bermejo et al. 1998, 2005). The C2 whisker
was therefore used to monitor the entire whisker
array on the ipsilateral side of the face. Movements
were recorded with high spatiotemporal precision
(7 mm, 500 Hz) and smoothed with a 4 ms moving
window. The nth bin of the movement record was
replaced with the average of bins N and N þ 1. A
calibration procedure (see Bermejo et al. 1998) was
performed for each rat so that millimeter measurements could be converted to degrees. Our whisker
position measurements are in head-centered coordinates. For example, a value of 90 corresponds to the
whisker pointing straight out from the rat’s head.
Increases in whisker angle correspond to protractions, decreases to retractions. Individual whisker
movements (‘‘whisks’’) were monitored in real time
and required to be above an amplitude of at least
5 mm. Movements surpassing the criterion amplitude were signaled to the rat by a brief flashed light.
A series of ten such whisks were required for the
delivery of a liquid reward. Food deprivation was
performed to keep the animals at 85% of the initial
body weight.
For kinematic analyses, individual whisker movements were identified using an algorithm that defines
three critical points: protraction onset, peak amplitude, and retraction end (see Figure 2B). Protraction
onset was identified by locating an increase between
consecutive position measurements (every 2 ms) that
was 40.14 . Similarly, the end of a retraction was
defined as the first time point, after the peak, where
the decrease was 50.14 . To minimize the possibility
of including movements produced passively during
non-whisking behaviors (e.g., jaw opening), only
whisker movements with well-defined protractions
and retractions, and with minimum amplitudes of 4 ,
were included in the analyses. Due to the limited
range of our CCD device, the monitored whisker
would sometimes move out of range. Individual
movements were only analyzed if the whisker was in
range throughout the movement. The largest detectable movements ranged from 80 to 110 , varying
with the individual calibration of each rat. Because
‘‘in-range’’ movements 480 were rare, they were
not included in our analyses.
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Electrophysiological recording from VPM. Single
VPM neurons were recorded while rats generated
whisker movements in the operant task (Khatri et al.
2009). Using a manual stereotaxic microdrive, an
electrode (FHC, stainless steel, 3–5 M
, 250 mm
shank) was inserted through the dura and slowly
lowered down to VPM. Electrophysiological activity
was band-pass filtered from 1 to 10 kHz and then
acquired with a sampling rate of 20 kHz. Entry into
the thalamus was identified by manual stimulation of
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the vibrissal array. Penetrations into VPM (4.5 mm
in depth) could be distinguished from other thalamic
structures, during the experiments, by predominantly single-whisker responses and the usual progression of topographically represented whiskers as
the electrode was moved deeper into the brain.
During the last recording session an electrolytic
lesion was placed near VPM to assist in reconstruction of the electrode penetration (2 M
stimulating
electrode, pulse amplitude ¼ 0.500 mA, pulse duration ¼ 1 s, number of pulses ¼ 10). Inspection of
histological preparations confirmed our recordings
were targeted to VPM.
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Data analysis
In addition to the VPM responses recorded in this
study, data from TG neurons obtained in our earlier
study were subjected to additional spike-triggered
analyses. All of the analyses described below were
performed on individual whisks except for coherence
analysis that was performed on continuous bouts of
whisking.
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Comparison of responses to stationary and moving
whiskers. As Figure 1 shows, bursts of whisking
were interspersed with stationary periods. For each
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neuron, we calculated firing rates for periods of
whisker movement (protractions and retractions) and
periods during which whisker position did not
change. The average durations of stationary periods
in milliseconds for VPM and TG neurons were
1487 176 (SEM) and 1145 183 (SEM). The
significance of differences in firing rate between
the whisking and stationary periods was assessed with
the Kolmogorov–Smirnov (K–S) test.
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Correlations between firing rate and whisker movement
kinematics. Response magnitude was quantified for
each neuron by determining the firing rate during the
protraction and retraction portion of each movement. Firing rate was then correlated with the
kinematic measures (protraction amplitude, protraction speed, retraction amplitude, and retraction
speed), using Spearman’s rho.
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Interaction
between
kinematics
and
direction
preferences. Since angular tuning to whisker deflections is a robust property of the whisker-to-barrel
pathway (e.g., Lichtenstein et al. 1990; Bruno et al.
2003; Minnery and Simons 2003; Jacob et al. 2008)
we asked whether directional preferences were
influenced by the kinematics of the whisks.
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Figure 1. Simultaneous whisker tracking and single-unit recording in VPM and TG. (A) An example of a VPM neuron that
displayed an increase in spiking activity during periods of vigorous whisking (indicated by solid black line above whisker
trace). Stationary periods (flat whisker trace) were selected from the periods between vigorous whisking. Note that periods
between vigorous whisking could contain small-amplitude whisker movements, but only flat periods were utilized for
stationary periods. (B) An example of a TG neuron that also increased its spiking during vigorous whisking. Unlike the VPM
neuron, ongoing spiking activity is negligible between whisking bouts.
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We performed a linear regression of the amplitude or
speed difference between the protraction and retraction of an individual whisker movement against the
corresponding firing rate difference. The coefficient
of determination (R2) quantifies the ability of speed
or amplitude to modulate a directional preference.
Using the y-intercept from the regression analysis,
we determined whether the observed direction preference is maintained even when protractions and
retractions are of equal amplitude or speed.
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Spike-triggered kinematics. Similarly to de Kock and
Sakmann (2009), we determined the whisker position and speed that occurred simultaneously with
each spike to form spike-triggered cumulative distributions. For each neuron, K–S tests were used to
determine whether spikes occurred preferentially at
certain positions or speeds. The distributions of
spike-triggered positions and speeds were compared
against all positions and speeds collected for that
neuron. However, we also took the direction of the
movement into account, to determine whether spikes
were evoked by positions or speeds in a directionspecific manner.
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Coherence between spiking and ongoing whisking. We
also assessed the relation between the spike timing
and movement data using a coherence analysis
(Chronux toolbox for Matlab). Whisker movement
bouts at least 1 s in duration were selected and the
multitaper technique was used to estimate spectra of
bouts and their corresponding spiking activity
(bandwidth ¼ 2 Hz; tapers ¼ 3). Coherence between
the movement and spike data was computed for the
peak movement frequency. Coherence was considered significant if it was in excess of the value of the
95% jackknife error bar. Forty-one neurons qualified
for this analysis, in that movement data for at least
five bouts was collected.
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Results
We examined the responses of 47 VPM neurons from
5 rats (6 penetrations per rat) during whisker
movements in air and compared them to those of TG
neurons obtained in an earlier study (Khatri et al.
2009). The median number of whisks acquired per
VPM neuron was 69 (range ¼ 19–248 whisks). The
following kinematics were derived from movements
obtained when recording from VPM and TG neurons (mean SD): protraction amplitude (21.51 7.12 degrees), protraction speed (387.37 103.47
degrees/s),
retraction
amplitude
(21.82 7.12 degrees), retraction speed (678.21 209.57
degrees/s). Like the TG neurons, all VPM neurons
responded to manual deflection of the whiskers,
being predominantly driven by a single whisker as
is characteristic of neurons within the core of a
thalamic barreloid (Bokor et al. 2008).
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Rate-based coding
Neuronal
activity
in
the
absence
of
movement. Figure 1 compares the activity of VPM
(panel A) and TG (panel B) neurons during whisking
and stationary periods. In contrast to TG neurons,
VPM neurons tend to be active both during whisker
movements and stationary periods. A K–S test
applied to the data demonstrated significantly
(p50.01) greater activity in VPM than TG neurons
during stationary periods (VPM: median ¼ 7.02 Hz,
mean SEM ¼ 9.83 1.41 Hz;
TG:
median ¼
0.21 Hz, mean SEM ¼ 9.4 4.19 Hz).
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Differentiating moving and stationary whiskers. To
evaluate the ability of VPM neurons to detect the
occurrence of a whisker movement (i.e., a change in
whisker position), we first computed the firing rates
during protractions and retractions (see Figure 2A,
C). For each neuron, the protraction and retraction
firing rate distributions were then separately compared to the corresponding firing rate distributions
obtained for stationary periods with K–S tests (see
Figure 2C, D). For example, if a neuron’s protraction firing rate was greater than that of the stationary
period, then it was classified as displaying an
increase. The majority of VPM neurons (34/47 or
72%) did not differentiate movements (protractions
or retractions) from stationary periods. Relative to
stationary periods, 13% of VPM neurons displayed
higher firing rates during movement, and 15% were
inhibited. In contrast, 11/21 (52%) of TG neurons
produced more spikes during movement than during
stationary periods. Thus, TG neurons are better than
VPM neurons at differentiating whisker movements
and stationary periods.
Next, we determined whether VPM or TG neurons were biased towards protractions or retractions
(see Figure 2E, F). There was a tendency for VPM
neurons to respond more during retractions than
protractions (Wilcoxon signed-rank test, p ¼ 0.05).
In contrast TG neurons responded similarly to both
protractions and retractions (p ¼ 0.52).
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Evaluating the encoding of kinematics. Here we ask
whether VPM neurons reliably encode the kinematics of whisker movements in air. Each whisker
movement was separated into its protraction and
retraction components. For each direction, we calculated the amplitude and speed of the movement.
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Figure 2. Movement-related neural activity. (A) ‘‘Whisks’’ were extracted from the movement trace and separated into their
forward and backward components (protractions and retractions). See Methods for details. (B) A VPM neuron for which
spiking (top) and simultaneous whisks (bottom) were obtained. To allow an examination of phase-locking, spikes have been
plotted relative to a normalized whisk (aligned to start and peak with retraction onset being set to 2/3 of the whisk). Each line
of spikes corresponds to a single whisk. For the sample neuron (100 whisks), spiking is biased towards retractions
(protraction FR: 4.82 1.10 Hz; retraction FR: 11.93 1.86 Hz). Only retraction FRs differed from stationary FRs (K–S
test, p50.05). (C) Plot of protraction FR vs stationary period FR. Points above the unity line represent neurons that increase
their firing rate during a protraction. Similarly, points below the unity line represent decreases. Such comparisons are the
basis for panel D. (D) Fraction of neurons for which the FRs evoked by movements were significantly different from
stationary periods (K–S tests, p50.05). Significantly more TG neurons responded to a movement by a change in FR during
either protraction or retractions (K–S test, p50.001). (E and F) Plots of retraction FR vs protraction FR for VPM and TG
neurons illustrate whether a neuron preferred a direction of movement. Points above the unity line (solid black) represent
neurons that prefer retractions and points below unity signify neurons that prefer protractions.
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PROTRACTION
AMPLITUDE
PROTRACTION
VELOCITY
RETRACTION
AMPLITUDE
RETRACTION
AMPLITUDE
þ
þ
þ
þ
33%
21%
0%
6%
43%
9%
0%
11%
53%
21%
0%
2%
53%
21%
0%
0%
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Table I. Percentage of each neuronal sample (TG/VPM) with positive or negative correlations between kinematics and firing rate.
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TG
VPM
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Speed was defined as the slope from 20 to 80% of
the peak amplitude of the whisk. Correlations were
computed between the firing rate of the neuron and
the following kinematic parameters: protraction
amplitude, protraction speed, retraction amplitude,
and retraction speed. The significance of a correlation was evaluated with Spearman’s rho. Table I
indicates the percentage of VPM neurons with
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Figure 3. Correlating firing rate and kinematics for protractions and retractions. (A) Significant Spearman’s rho values for
the relationship between kinematics and firing rate. Each point corresponds to a single neuron, which can be represented
in more than one kinematic category if a significant correlation is present. Arrows indicate group medians. TG neuron
correlations differed from VPM neurons only for retraction amplitude (Mann–Whitney test, p50.05). (B) Cumulative
distribution functions collapsed across kinematics for TG (gray line) and VPM (black line) neurons. Based upon the
correlations plotted in A. Correlations were significantly larger in TG neurons (K–S test, p ¼ 0.005).
639
similar analysis done by linearly regressing directional response differences with amplitude differences, indicated that nine neurons preferred
retractions and four preferred protractions. Thus,
even after accounting for linear effects of amplitude
or speed, some VPM neurons could display significant but weak direction preferences. After removing
the linear effects of speed but not amplitude, VPM
and TG neurons had significantly different direction
preferences (Mann–Whitney test, p ¼ 0.002) with
VPM neurons preferring retractions and TG neurons
preferring protractions (Figure 4C).
By comparing the regression coefficients, we were
able to determine whether kinematics modulated
direction preferences more in VPM or TG neurons.
As Figure 4D indicates, amplitude and speed
influenced direction-selectivity more in TG than
VPM neurons (Mann–Whitney test, p50.001).
645
significant correlations (values of p50.05) between
firing rate and one or more of the kinematic
parameters. Data for TG neurons are presented for
comparison. For most of the VPM neurons, as for
all TG neurons, the majority of correlations were
positive (19/47, 40%) rather than negative (8/47,
17%). However, the positive correlations for
retraction amplitude (Figure 3A) were substantially
weaker for VPM than for TG neurons. Furthermore,
after compiling positive correlations across kinematics, correlations were weaker for VPM than TG
neurons (K–S test, p ¼ 0.009) (VPM: mean SEM ¼ 0.31 0.02;
TG:
mean SEM ¼
0.43 0.03) (Figure 3B). Two of 47 VPM neurons
displayed positive correlations for one direction of
motion, and negative correlations for the opposite
direction.
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Dissociating directional preferences from effects of
kinematics. Having previously found that the directional preferences (protractions vs retractions) of TG
neurons could be modulated by the speed or
amplitude of the movement we asked whether the
directional preferences of VPM neurons are similarly
modulated. For example, Figure 4A shows a neuron
that appears to spike preferentially at the initiation
of retractions. Would this VPM neuron continue to
show a preference for retractions if they were the
same speed as protractions? For each whisk (unnormalized), the relative speed (protraction speed
minus retraction speed) was linearly regressed
against the directional response difference (protraction firing rate minus retraction firing rate). The
neuron of Figure 4A was classified as having a
significant direction preference (independent of
speed) since the y-intercept differed significantly
from zero (see Figure 4B). This analysis identified
nine neurons that preferred retractions and five that
preferred protractions independently of speed. A
Single spike probability
Our previous study of TG neurons suggested a lack of
temporal fidelity between spiking and whisker movement trajectories. Here, we re-examined spike time
precision in both TG and VPM neurons, employing
spike-triggered analyses. By dividing each whisk into
its protraction and retraction components we could
assess the influence of movement direction on the
probability that particular positions (rostral or caudal
relative to all whisk positions) or speeds (slow or fast
relative to all whisk speeds) would trigger spikes (see
Figure 5A). For each neuron, a K–S test compared
the spike-triggered distributions with all position and
speed values, to determine if spikes were evoked by
particular kinematics or randomly distributed
throughout the movement.
Spike-triggered distributions demonstrated that,
for both TG and VPM neurons, spikes could be
biased toward particular positions or speeds.
However, tuning was broad in that spikes were not
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Figure 4. Evaluating the influences of amplitude and speed on preferences for movement direction. (A) Spiking activity for
a VPM neuron with its whisker movement normalized. Note the bias to spike during the initiation of retractions.
(B) Differential contributions of movement direction and movement speed to firing rate of the VPM neuron whose
(normalized) responses are shown in A. The linear regression’s slope and regression coefficient were not significant, but the
y-intercept was significantly different from zero and this indicates that the neuron had a significant preference for retractions
even after eliminating the effect of speed. (C) Neurons with significant y-intercepts from the amplitude and speed
regressions, or in other words, significant direction preferences independent of amplitude and speed, respectively. After
removing effects of speed, TG neurons are biased towards protractions, but VPM neurons prefer retractions. VPM neurons
also preferred retractions independently of amplitude differences. (D) Significant linear regression coefficients, both
amplitude and speed, for TG and VPM neurons (from those with significant correlations in panel 3 A). Some neurons are
represented twice if they had significant regression coefficients for both amplitude and speed. The smaller regression
coefficients indicate that kinematics were significantly less effective in modulating the directional response differences of
VPM than TG neurons.
biased to a specific position or speed, but instead,
to a relatively large range of positions and speeds (see
Figure 5B). Furthermore, in both TG and VPM
neurons, movement direction could determine the
presence of a significant kinematic bias. For example,
the TG neuron of Figure 5B displayed a bias for
relatively high speeds during retractions but not
protractions. Similarly, the same TG neuron displayed a bias for caudal whisker positions during
protractions, but not retractions.
For each kinematic parameter (protraction position, protraction speed, retraction position, retraction speed) (see Figure 6), we determined whether
equivalent numbers of TG and VPM neurons
displayed significant spike-triggered curves (chisquare tests). For position (protraction/retraction)
equivalent numbers of TG and VPM neurons had
significant spike-triggered distributions. However,
for speed (protraction/retraction) there were significantly fewer VPM neurons showing such distributions (values of p50.001). Thus, specific kinematic
parameters are less effective at triggering spikes in
VPM than TG neurons.
Coherence between spiking and ongoing whisking
A coherence analysis was performed to determine
whether spiking activity during bouts of whisking
was entrained by the dominant bout frequency (see
Methods for details). On average for each neuron,
only a very small percentage of their bouts (about
11% in VPM neurons and 17%) showed significant
coherence (p50.05). Only one neuron displayed
coherence in more than 50% of its bouts (see
Figure 4A: 67% of bouts). As a whole, the VPM
population displayed significantly less coherence
than TG neurons (Mann–Whitney test, p ¼ 0.015).
The small number of bouts with significant coherence in both VPM and TG neurons indicates that
they are not firing reliably at a particular whisk
phase.
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Discussion
Active sensing requires the brain to differentiate
external inputs (exafference) from the animal’s
own
self-generated
movements
(reafference).
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Figure 5. Spike-triggered position and speed analyses as a function of movement direction. (A) Whisker position and speed
at the time of spikes were determined independently for protractions (under red bar) and retractions (under blue bar). (B) TG
neuron demonstrating that the presence of significant spike-triggered position/speed preferences (K–S tests, p50.05) depend
on movement direction. The red traces are spike-triggered values and the black traces represent all whisk positions or speeds.
For protractions, this TG neuron had a preference for caudal whisker positions and no speed preference. However, for
retractions, there was no position preference but higher speeds were preferred. Notably, even when significant differences were
present, tuning was broad in that a wide range of positions and speeds triggered spikes. (C) ‘‘Complex tuning’’. Only 5/47
VPM neurons generated such a spike-triggered position or speed distribution in which no clear preference can be determined.
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Reafferent signals may also allow an animal to rapidly
fine-tune the movements of its mobile sensor
(Kleinfeld et al. 2006). Reliable encoding of whisker
contacts by the lemniscal pathway has been well
documented in the trigeminal ganglion and barrel
cortex (Hentschke et al. 2006; Stüttgen et al. 2006;
Jadhav et al. 2009). However, little is known about
the reliability with which the kinematics of whisker
movements in air are represented by neural signals
at levels above the trigeminal ganglion. One possible
strategy is for neurons to process exafferent and
reafferent signals in different channels (e.g., paralemniscal vs lemniscal), as was suggested by Ahissar
and his colleagues (Yu et al. 2006). However, Masri
et al. (2008) found no consistent relationship
between the occurrence of spikes in POm and
EMG activity from whisker-related musculature.
To determine whether the trigeminal lemniscal
pathway reliably conveys reafferent information to
the cortex, we have examined the responses of VPM
neurons in awake rodents during whisking in air.
Here we report that VPM neurons are even less
reliable in encoding such signals than are neurons
in the trigeminal ganglion (Khatri et al. 2009). This
seems to be the case not only for the encoding of
whisker movement kinematics, but even for the
detection of a change from a stationary to a moving
whisker. Moreover, these studies, carried out in
awake, behaving animals, fail to confirm the findings
of previous studies employing the ‘‘electrical whisking’’ paradigm in anesthetized animals (Szwed et al.
2003; Yu et al. 2006) which observed significant
phase-locking to movement onset. While we have
observed some weak phase-locking in both TG and
VPM neurons, responses were not consistently
triggered by the onset of protractions or retractions
and tended to be distributed throughout the movement. Taken together, the results of our studies
examining firing rate and single spike probability,
suggest that reafferent signals in single neurons do not
provide a reliable representation of whisker movements in the lemniscal pathway. Coding by population signals provides a likely alternative (see below).
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Cortical responses during whisker movements in air
As in TG and VPM, cortical responses during
whisking in air tend to be broadly distributed
throughout the whisker movement though they can
be biased towards particular phases of the movement
(Fee et al. 1997; Crochet and Petersen 2006). Curtis
and Kleinfeld (2009) recorded from a large number
of cortical neurons and found that ‘‘a select population’’ of 20%, responded during whisking in air.
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Figure 6. Summary of spike-triggered position and speed analyses. Distributions of tuning preferences for TG (A) and VPM
neurons (B). Significantly more TG neurons preferred faster speeds (F) for both protractions and retractions, while VPM
neurons exhibited a significant bias for faster speeds only during retractions. Abbreviations: R ¼ rostral, C ¼ caudal,
S ¼ slow, F ¼ fast, CT ¼ complex tuning.
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Like VPM neurons, cortical neurons display a bias
for responding during the retraction component of
a whisker movement (see Figure 4(b) of Kleinfeld
et al. 2006). These investigators also reported that,
during whisking in air, individual cortical units ‘‘tend
to spike at specific phases of the whisk cycle’’ (Curtis
and Kleinfeld 2009, p. 494). However, de Kock and
Sakmann (2009) recently reported that single cortical
spikes are only weakly related to whisker position or
phase—an observation consistent with our findings
for VPM neurons. If single neurons do not contain
reliable position or phase signals, then, for there to be
a useful signal, the aggregate activity of a population
of neurons would have to occur at a preferential
position or phase of whisker movement.
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Whisking-related reafference during whisking in air:
Origin of the reafferent signal
Curtis and Kleinfeld (2009) suggested ‘‘a reafferent
signal encoding phase is present at the level of
primary sensory neurons in the trigeminal ganglion’’
(p. 497). Our data from awake, behaving animals
suggests that the TG signal is only modestly correlated with kinematic parameters (Khatri et al. 2009)
and the present study suggests that the reliability with
which reafferent information from whisking in air
is encoded actually decreases at higher levels of the
neuraxis. One possibility for the relatively weak
encoding of whisking in air at any level may be the
absence of those frequent, slip-evoked, high velocity/
acceleration signals which characterize active whisker
contact (Jadhav et al. 2009) and to which central
trigeminal neurons are so sensitive (e.g., Pinto et al.
2000). Indeed, the fact that retractions generate
higher speeds than protractions might account for
the bias for responding during the retractions,
reported for both VPM and cortical neurons.
Whatever its origin, the fact that rats can actively
modulate the kinematics of both contact (Carvell and
Simons 1990; Harvey et al. 2001) and non-contact
(Gao et al. 2003) whisker movements according to
task demands suggests the presence of some mechanisms for producing a more robust representation of
movement kinematics than is available to a single
neuron. This could involve the convergence of populations of neurons or the contribution of neural
structures containing populations of neurons with
large receptive fields comprising many whiskers such
as the superior colliculus (Hemelt and Keller 2007;
Cohen et al. 2008) and laterodorsal nucleus of the
thalamus (Bezdudnaya and Keller 2008).
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Brainstem-filtering or top-down modulation?
The present study compared VPM and TG responses
to whisking. This was done to determine how afferent
inputs are transformed along the leminscal pathway.
We find that the reliability of whisking responses
decreases from the TG to the VPM barreloids. This
could be due either to response suppression at the
level of the whisker brainstem (e.g., Furuta et al.
2008) or due to corticofugal feedback (e.g.,
Hentschke et al. 2006; Lee et al. 2008). Further
studies are needed to determine the respective roles
of the brainstem and corticofugal feedback on VPM
responses to whisking in air.
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Acknowledgments
We would like to thank V. Lawson for preparing the
animals and assistance in behavioral training. David
Kleinfeld and Asaf Keller provided helpful comments to improve the manuscript. This work was
supported by Grant NS048937 (HPZ) and CUNY
Collaborative Grant 80209 (HPZ and JCB).
Infrastructure support for the Zeigler lab was provided by RCMI Grant RR 03037. JCB was also
supported by Grant NS058758.
Declaration of interest: The authors report no
conflict of interest. The authors alone are responsible
for the content and writing of the paper.
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References
Arabzadeh E, Zorzin E, Diamond ME. 2005. Neuronal encoding
of texture in the whisker sensory pathway. PLoS Biol 3:e17.
Bezdudnaya T, Keller A. 2008. Laterodorsal nucleus of the
thalamus: A processor of somatosensory inputs. J Comp Neurol
507:1979–1989.
Bokor H, Acsády L, Deschênes M. 2008. Vibrissal responses of
thalamic cells that project to the septal columns of the barrel
cortex and to the second somatosensory area. J Neurosci
28:5169–5177.
Bruno RM, Khatri V, Land PW, Simons DJ. 2003.
Thalamocortical angular tuning domains within individual
barrels of rat somatosensory cortex. J Neurosci 23:9565–9574.
Carvell GE, Simons DJ. 1990. Biometric analyses of vibrissal
tactile discrimination in the rat. J Neurosci 10:2638–2648.
Cohen JD, Hirata A, Castro-Alamancos MA. 2008. Vibrissa
sensation in superior colliculus: Wide-field sensitivity and statedependent cortical feedback. J Neurosci 28:11205–11220.
Crochet S, Petersen CC. 2006. Correlating whisker behavior
with membrane potential in barrel cortex of awake mice. Nat
Neurosci 9:608–610.
Curtis JC, Kleinfeld D. 2009. Phase-to-rate transformations
encode touch in cortical neurons of a scanning sensorimotor
system. Nat Neurosci 12:492–501.
de Kock CPJ, Sakmann B. 2009. Spiking in primary somatosensory cortex during natural whisking in awake head-restrained
rats is cell-type specific. PNAS 106:16446–16450.
Fee MS, Mitra PP, Kleinfeld D. 1997. Central versus peripheral
determinants of patterned spike activity in rat vibrissa cortex
during whisking. J Neurophysiol 78:1144–1149.
Furuta T, Timofeeva E, Nakamura K, Okamoto-Furuta K,
Togo M, Kaneko T, Deschênes M. 2008. Inhibitory gating of
vibrissal inputs in the brainstem. J Neurosci 28:1789–1797.
Gao P, Hattox AM, Jones LM, Keller A, Zeigler HP. 2003.
Whisker motor cortex ablation and whisker movement patterns.
Somatosens Mot Res 20:191–198.
Harvey MA, Bermejo R, Zeigler HP. 2001. Discriminative
whisking in the head-fixed rat: Optoelectronic monitoring
during tactile detection and discrimination tasks. Somatosens
Mot Res 18:211–222.
Hemelt ME, Keller A. 2007. Superior sensation: Superior
colliculus participation in rat vibrissa system. BMC Neurosci
8:12–22.
Hentschke H, Haiss F, Schwarz C. 2006. Central signals rapidly
switch tactile processing in rat barrel cortex during whisker
movements. Cereb Cortex 16:1142–1156.
Jacob V, Cam JL, Ego-Stengel V, Shulz DE. 2008. Emergent
properties of tactile scenes selectively activate barrel cortex
neurons. Neuron 60:1112–1125.
Jadhav SP, Wolfe J, Feldman DE. 2009. Sparse temporal coding
of elementary tactile features during active whisker sensation.
Nat Neurosci 12:792–800.
Jones LM, Depireux DA, Simons DJ, Keller A. 2004. Robust
temporal coding in the trigeminal system. Science 304:1986–
1989.
Khatri V, Bermejo R, Brumberg JC, Keller A, Zeigler HP. 2009.
Whisking in air: Encoding of kinematics by trigeminal ganglion
neurons in awake rats. J Neurophysiol 101:1836–1846.
Kleinfeld D, Ahissar E, Diamond ME. 2006. Active sensation:
Insights from the rodent vibrissa sensorimotor system. Curr
Opin Neurobiol 16:435–444.
Lee S, Carvell GE, Simons DJ. 2008. Motor modulation of
afferent somatosensory circuits. Nat Neurosci 11:1430–1438.
Leiser SC, Moxon KA. 2007. Responses of trigeminal ganglion
neurons during natural whisking behaviors in the awake rat.
Neuron 53:117–133.
Lichtenstein SH, Carvell GE, Simons DJ. 1990. Responses of rat
trigeminal ganglion neurons to movements of vibrissae in
different directions. Somatosens Mot Res 7:47–65.
Masri R, Bezdudnaya T, Trageser JC, Keller A. 2008. Encoding
of stimulus frequency and sensor motion in the posterior medial
thalamic nucleus. J Neurophysiol 100:681–689.
Minnery BS, Simons DJ. 2003. Response properties of whiskerassociated trigeminothalamic neurons in rat nucleus prinicalis.
J Neurophysiol 89:40–56.
Pinto DJ, Brumberg JC, Simons DJ. 2000. Circuit dynamics and
coding strategies
in rodent somatosensory
cortex.
J Neurophysiol 83:1158–1166.
Stüttgen MC, Rüter J, Schwarz C. 2006. Two psychophysical
channels of whisker deflection in rats align with two neuronal
classes of primary afferents. J Neurosci 26:7933–7941.
Stüttgen MC, Schwarz C. 2008. Psychophysical and neurometric
detection performance under stimulus uncertainty. Nat
Neurosci 11:1091–1099.
Szwed M, Bagdasarian K, Ahissar E. 2003. Encoding of vibrissal
active touch. Neuron 40:621–630.
von Heimendahl M, Itskov PM, Arabzadeh E, Diamond ME.
2007. Neuronal activity in rat barrel cortex underlying texture
discrimination. PLoS Biol 5:e305–22.
Yu C, Derdikman D, Haidarliu S, Ahissar E. 2006. Parallel
thalamic pathways for whisking and touch signals in the rat.
PLoS Biol 4:e124–22.
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