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. Author manuscript; available in PMC: 2008 Aug 7.
Published in final edited form as: Cereb Cortex. 2007 Oct 19;18(6):1314–1325. doi: 10.1093/cercor/bhm163

Sensory inputs from whisking movements modify cortical whisker maps visualized with functional magnetic resonance imaging

Benito de Celis Alonso 1, Andrew S Lowe 1,*, John P Dear 2, Kalok C Lee 3, Steven C R Williams 1, Gerald T Finnerty 1
PMCID: PMC2492395  EMSID: UKMS1119  PMID: 17951597

Abstract

Rodents vary the frequency of whisking movements during exploratory and discriminatory behaviours. The effect of whisking frequency on whisker cortical maps was investigated by simulating whisking at physiological frequencies and imaging the whisker representations with blood-oxygen-level-dependent (BOLD) functional magnetic resonance imaging (fMRI). Repetitive deflection of many right-sided whiskers at 10 Hz evoked a positive BOLD response that extended across contralateral primary somatosensory cortex (SI) and secondary somatosensory cortex (SII). In contrast, synchronous deflection of two adjacent whiskers (right C1 and C2) at 10 Hz evoked separate positive BOLD responses in contralateral SI and SII that were predominantly located in upper cortical layers. The positive BOLD responses were separated and partially surrounded by a negative BOLD response that was mainly in lower cortical layers. Two-whisker representations varied with the frequency of simulated whisking. Positive BOLD responses were largest with 7 Hz deflection. Negative BOLD responses were robust at 10 Hz, but were weaker or absent with 7 Hz or 3 Hz deflection. Our findings suggest that sensory inputs attributable to the frequency of whisking movements modify whisker cortical representations.

Keywords: cortex, fMRI, negative BOLD, rat, somatosensory, whiskers

Much of the cortex is given over to maps of sensory inputs. These cortical maps are not static representations of the sensory periphery. For example, stimulation of one whisker evokes firing that is first recorded in the barrel column of the stimulated whisker. Neural activity then propagates across multiple barrel columns over tens of milliseconds (Moore and Nelson 1998; Zhu and Connors 1999; Brecht and others 2003) and can involve the whole barrel field (Petersen and others 2003). It has been suggested that this cross-columnar signaling may be important for sensory processing during active touch (Carvell and Simons 1995).

Rats move their whiskers back and forth (commonly termed whisking) at frequencies of 4 - 12 Hz when investigating their environment (Welker 1964; Carvell and Simons 1990). The frequency of whisking is not random, but can be modulated during active touch. Typically, exploratory behaviours are associated with whisking at 6 - 9 Hz whereas discriminatory behaviours involve whisking at frequencies above 9 Hz (Carvell and Simons 1995; Harvey and others 2001). It has been proposed that whisker representations change with the sensory task that is being performed. The hypothesis is that the whisker sensory system is set to detect contact when whiskers are stationary, but is retuned by whisking behaviour (Fanselow and Nicolelis 1999; Moore 2004). In primary somatosensory cortex (SI), whisker representations become more focused with alertness (Castro-Alamancos 2004) and with whisker deflection in the upper part of the whisking frequency range (Kleinfeld and Delaney 1996; Sheth and others 1998). Furthermore, the frequency of whisking modifies the response in SI evoked by passive whisker stimulation (Fanselow and Nicolelis 1999; Crochet and Petersen 2006; Ferezou and others 2006). These findings suggest that whisker representations exhibit frequency-dependent modifications.

Several techniques have been used to image whisker representations evoked by repetitive deflection. Intrinsic signal imaging (Masino and others 1993; Devor and others 2005) and voltage sensitive dye-based imaging (Kleinfeld and Delaney 1996; Petersen and others 2003) give high resolution images of whisker representations in SI. However, both techniques favour signals from upper cortical layers. Electrophysiological recordings indicate that individual cortical layers may behave differently because artificially-induced whisking at 5 Hz evokes neuronal depression in cortical layer 2/3 and neuronal activation in layer 5a (Derdikman and others 2006).

Functional magnetic resonance imaging (fMRI) with contrast based on either blood oxygen-level-dependent (BOLD) signals (Ogawa and others 1990), cerebral blood flow (Kwong and others 1992; Williams and others 1992) or cerebral blood volume (Mandeville and others 1998) offers several approaches for depth-independent imaging of whisker maps. Here, we use BOLD fMRI to image whisker representations. We simulated whisking at physiological frequencies in anaesthetized rats to isolate the sensory input attributable to large-amplitude whisker movements from the effects of inputs from whisker-related motor centres (Fee and others 1997; Hentschke and others 2006). Neural activity in upper layers of SI adapts with a few cycles of whisking (Moore 2004). Therefore, we aimed to image ‘steady-state’ representations evoked by simulated whisking at different frequencies. We show that whisker representations imaged with BOLD fMRI vary with the number of deflected whiskers and exhibit frequency-dependent changes in the physiological whisking range. The results suggest that the frequency of whisking movements modifies whisker cortical maps.

Materials and Methods

Animal Preparation

All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. Anaesthesia of male Sprague Dawley rats (200-250 g) was induced with 4% isoflurane in 50% O2 and maintained initially with 2% isoflurane in a 1:1 mixture of O2: air for 5 - 10 minutes. A tail vein was cannulated, a bolus of α-chloralose (65 mg/kg) was injected and an infusion of α-chloralose (30 mg/kg/hour) started. Isoflurane anaesthesia was discontinued 3 minutes after the bolus injection. The rat’s head was restrained in a custom-made stereotaxic frame, which was isolated from the whisker stimulators to minimise head motion artefacts. Respiratory rate (pre-scanning, 73 ± 1 breaths per min; post-scanning (3 hours later) 65 ± 1 breaths per min, n = 6 animals) and rectal temperature were monitored during scanning (SA Instruments Inc, Stony Brook, NY). Rectal temperature was maintained at 37.0 ± 0.5 °C by a temperature-controlled hot air supply (SA Instruments Inc, Stony Brook, NY). We restricted image acquisition to a 2.25 hr period commencing 45 mins after the α-chloralose bolus was given to ensure that anaesthesia was stable during scanning (Austin and others 2005).

Whisker stimulation

Rats have five rows of whiskers commonly denoted A to E (Welker and Woolsey 1974). The two-whisker protocol comprised trimming all whiskers except for the two most caudal whiskers in the C row (C1 and C2) acutely to the level of the facial hair on both sides of the rat’s snout (Fig. 1A). The right C1 and C2 whiskers were placed in the teeth of a comb located 1 cm away from the rat’s snout (Fig. 1B). The multiple whisker-row protocol comprised placing 3 - 4 whiskers from each of the B - E rows into the comb. The remaining rostral whiskers in these rows were too short to reach the comb. The A row of whiskers and the outlying α, β, γ and δ whiskers were cut to prevent the comb from intermittently contacting them (Fig. 2B).

Figure 1.

Figure 1

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Imaging whisker representations with fMRI. (A) Schematic of the whiskers on the right side of a rat’s snout. Whisker rows are labelled A - E whereas whisker arcs (running dorso-ventrally) are numbered. Outlying whiskers, which are found between whisker rows at the caudal end of the whisker pad, have not been labelled for simplicity. Filled red circles denote the C1 and C2 whiskers. (B) Orientation of the whisker actuator in the scanner. A transmit and receive coil lies on top of the rat’s head. (C) Schematic illustrating the relative position of SI, SII and parietal ventral (PV) whisker representations in a coronal slice approximately 2.5 mm caudal to bregma that cuts through the left C1/C2 whisker barrels in SI (adapted from Chapin and Lin 1984; Hoffer and others 2003; Benison and others 2007). Whisker barrel rows run in and out of the plane of the slice. Red ellipses denote the positions of the cortical columns in SI and SII receiving principal whisker input from C1/C2 whiskers. The relative positions of the whisker barrel columns (A - E) are given for SI (SI wh) and SII (SII wh). SI f/tr denotes the forelimb/trunk representation that lies medial to the SI whisker map. rhf signifies the rhinal fissure.

Figure 2.

Figure 2

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BOLD signal evoked by 10 Hz deflection of either two whiskers or many whiskers in multiple whisker rows in the same animal. (Ai) Schematic shows whisker trim used for two-whisker protocol. Red circles denote the follicles of deflected whiskers. Empty circles indicate trimmed whiskers. Filled circles represent untrimmed whiskers that were not deflected. (Aii) Single-animal map of the BOLD responses evoked by two-whisker deflection. Pseudocoloured voxels have a positive BOLD (red) or negative BOLD (blue) signal that is significantly different from baseline. The pseudocolour scale bars apply to both Aii and Bii. Numbers denote the rostro-caudal distance of each slice from bregma. (Bi) Schematic shows whisker trim used for multiple whisker-row protocol. Whisker colour code as per A. (Bii) Map of the BOLD responses evoked by multiple whisker-row deflection for the animal used in A. Pseudocoloured voxels have a positive BOLD (red) or negative BOLD (blue) signal that is significantly different from baseline. For simplicity, only slices with positive or negative BOLD signal are shown in Aii and Bii.

The comb was moved rostro-caudally at 3 Hz, 7 Hz or 10 Hz (see MRI and fMRI methods for details of randomized block design used for experiments) by a pneumatic system controlled by a custom-written program (Labview, National Instruments, Austin, TX). We refer to the whisker displacement device as an actuator. The operation of the actuator was videoed outside the scanner with a high-speed camera (Phantom IV, Photo-sonics Inc, Burbank, CA, USA) at 1000 frames per second (pixel, 0.16 mm × 0.16 mm in specimen plane) to measure the kinetics of whisker deflection. During a deflection cycle, whisker displacement was 2.7 mm rostro-caudally with a maximum velocity of 0.15 m/s. The kinetics of the actuator during a deflection cycle were similar when operated at 3, 7 and 10 Hz. Operation of the actuator did not induce susceptibility artefacts when a phantom was imaged with our usual setup. Analysis of the phantom data set did not introduce artefactual negative or positive BOLD responses into our results.

Magnetic Resonance Imaging and fMRI methods

Imaging was performed in a horizontal bore 9.4T magnet (Oxford Instruments, Oxford, UK) interfaced with a Varian Inova console (Palo Alto, CA, USA) and equipped with a gradient set (ID 130 mm, 210 mT/m, 160 μs rise time). A 25 mm diameter surface coil (Varian, Palo Alto, CA, USA) was used to transmit and receive the radiofrequency signal. The junction of the optic nerves at the optic chiasm, which is approximately at bregma along the rostro-caudal axis (Paxinos and Watson 1982), was used as an anatomical landmark in our scout scans. This served to determine the rostro-caudal location of the C1 and C2 whisker barrels columns, which are 2 - 3.5 mm caudal to bregma (Chapin and Lin 1984; Remple and others 2003; Benison and others 2007). Coronal anatomical scans were created from a spin-echo sequence (repetition time [TR] / echo time [TE] = 1000/20 ms) with 0.5 mm thick slices, a field of view of 32 × 32 mm, a matrix size of 192 × 192 and four signals averaged. The 0.5 mm slice thickness approximates to the diameter of one barrel column in SI.

Experiments that compared the two-whisker protocol with the multiple whisker-row protocol in the same animal comprised one scan period divided into two contiguous sessions. One session compared 10 Hz deflection of the right C1 and C2 whiskers with no whisker deflection. The other session compared 10 Hz deflection of whiskers in the B - E rows on the left side of the snout with no whisker deflection. The order of the protocols during experiments was randomized. Experiments investigating the effect of deflection frequency were also divided into two contiguous sessions. The first session compared no whisker movement with deflection of the right C1 and C2 whiskers at one frequency chosen from 3 Hz, 7 Hz and 10 Hz. The second session used one of the two remaining deflection frequencies for ON blocks. This protocol was favoured over randomization of all three stimulation frequencies for each rat to ensure adequate averaging of images within the time constraint for stable anaesthesia (Austin and others 2005). These animals were used to prepare the group maps in Fig. 6. An additional four data sets comprising 3 Hz and 7 Hz whisker deflection in randomized order were acquired to reduce the difference in number of animals tested with 3 Hz or 7 Hz compared with 10 Hz thereby improving the power of statistical tests performed on single-animal data. Mock fMRI sequences were run for both two-whisker and multiple whisker-row protocols prior to putting the rat into the scanner to ensure that the actuator did not touch whiskers too short to reach the comb or facial hairs during movement and that deflection of whiskers in the actuator comb did not cause the whiskers to pull on their respective follicles and cause pain.

The fMRI experiments used a randomized block design. Both sessions in an experiment comprised 120 blocks with 60 blocks ON (whisker movement continues throughout block) and 60 blocks OFF (no whisker movement throughout block). A volume of data was acquired throughout each block. Each volume comprised 12 slices of 0.5 mm thickness. A multi-echo gradient echo imaging sequence was custom-written for fMRI experiments to improve the contrast to noise of the BOLD signal (Posse and others 1999). Imaging parameters were: flip angle, 31 degrees; TR = 340 ms; TE = 4, 8, 12, 16, 20 ms; FOV, 32 × 32 mm; matrix, 96 × 96; acquisition time per volume, 32.6 seconds; total scan time per fMRI session, 1 hour 5 minutes. ON and OFF periods were co-ordinated with the scanning protocol by a transistor - transistor logic (TTL) pulse sent from the imaging console to the Labview program. Pulsations due to the cardiac or respiratory cycles introduce coloured noise into functional images, which was reduced by randomization of the ON and OFF blocks with the conditions that: 1) the protocol always begins with an OFF; and 2) a maximum of two ONs occur in succession.

Data analysis

All fMRI data was converted from the Varian to ANALYZE format using custom-written software. Multi-echo GE data sets were converted into single-echo images (effective TE = 12 ms) by summing the magnitude images generated from each echo (Posse and others 1999). Images from each scan session, which form a time series, were co-registered and aligned using the rigid body transformation algorithm in SPM99 (http://www.fil.ion.ucl.ac.uk/spm/). This corrects for motion along three translation and three rotation axes. A precise mask was applied to delineate the brain in the realigned data. Any rat that exhibited head motion exceeding 0.5 mm along x or y axes or more than 0.75 mm in the z direction (along B0) was excluded from further analysis.

We minimized BOLD signal attributable to large draining veins and vascular inflow (Menon and Goodyear 2001) by constructing a coefficient of variation map of the BOLD signal and eliminating voxels with coefficients of variation greater than 15% (Hlustik and others 1998).

The BOLD signal recorded from the brain commonly includes structured (coloured) noise, e.g. due to pulsations attributable to the cardiac or respiratory cycles, and non-structured (white) noise, which reduce BOLD contrast. We reduced noise in our functional images by performing a probabilistic independent component analysis (PICA) on 4D data sets using MELODIC 2.0 (http://www.fmrib.ox.ac.uk/fsl/) (Thomas and others 2002). The resulting components were compared with the stimulus paradigm using a Pearson Correlation test. Components that had a correlation coefficient with a P value > 0.1, i.e. were not correlated with the stimulus paradigm, were removed by linear regression to form a denoised data set. Further data processing and analysis were performed in Statistical Parametric Mapping (SPM). Data were smoothed with a Gaussian kernel to conform to the expectation in SPM that the data approximate a random Gaussian field (Worsley and Friston 1995). The full width half maximum of the kernel (0.99 mm) was chosen to approximate the distributions of the electrophysiological and haemodynamic responses evoked by whisker deflection in SI and SII, thereby, optimize signal detection (Worsley and others 1996; Triantafyllou and others 2006) in single-animal data sets.

The BOLD signal in scalp muscle overlying somatosensory cortex fulfilled two roles in our experiments. Firstly, it was examined to ensure that movement of the actuator had not induced artefactual BOLD signals, which were not found. Secondly, we used the mean BOLD signal within scalp muscle as an estimate of global effects during experiments (Shoaib and others 2004; Lowe and others 2007). The mean scalp muscle BOLD signal and the stimulation paradigm were independent at all frequencies (mean probability for null hypothesis that correlation coefficient = 0: 3 Hz, 0.47 ± 0.08; 7 Hz, 0.50 ± 0.10; 10 Hz, 0.41 ± 0.09; n = 8 for each group, P = 0.790, one-way ANOVA). Therefore, we included both the mean BOLD signal in scalp muscle and the motion parameters from the head motion correction (Friston and others 1996) as covariates of no interest in the statistical model of the BOLD signal. Addition of scalp muscle BOLD signal to statistical models did not introduce artefactual separation of the positive BOLD responses in SI and SII. Implicit mean image intensity normalization was not used during image processing.

A contrast comparing all blocks of stimulus ON with all blocks of stimulus OFF (baseline) was built for each individual experiment and for group analysis. T-maps were co-registered with the spin echo images of the whole brain and then superimposed for presentation. In some cases, line drawings from a rat brain atlas (Paxinos and Watson 1982) have been overlaid onto the images to delineate different cortical regions approximately. The cortical representations of whiskers (Fig. 1C) were used to define regions of interest in five contiguous slices centred around a slice positioned 2.5 mm caudal to bregma. We used the MarsBar tool (MARSeille Boîte À Région d’Intérêt) in SPM99 to measure the size of clusters, the changes in BOLD signal intensity and P values for voxels in the regions of interest. A positive BOLD response or negative BOLD response was considered to be present in single animal and group maps if there was a cluster of 4 or more contiguous voxels that were all statistically significant (P < 0.05, uncorrected) in the region of interest (Forman and others 1995). The volume of BOLD responses is reported as number of voxels. The volume in mm3 can be calculated by multiplying the number of voxels by 0.05445.

The cortical representation of rats’ whiskers may show significant variability in mapping studies (Riddle and Purves 1995; Chen-Bee and Frostig 1996). This has direct ramifications on whether group map or single-subject analysis should be used (Woods 1996; Petersson and others 1999a; Petersson and others 1999b; Thirion and others 2007). Group maps derived from a fixed effects model are useful because they can reveal small-amplitude changes in the BOLD signal. In contrast, single animal studies enable analysis of the variability that is known to occur between subjects. Therefore, group maps were used to identify changes in the BOLD signal and single animal data were used to quantify those changes in more detail.

Statistics

Data are given as mean ± SEM unless noted. We could not identify a suitable transform to stabilize the variance of the amplitudes of the positive BOLD response in SI at 3 Hz, 7 Hz and 10 Hz. Therefore, we made pairwise comparisons of the 3 Hz, 7 Hz data and 10 Hz data and reduced the P value for statistical significance from 0.05 to 0.017 (Bonferroni correction for three possible pairwise comparisons).

Results

BOLD whisker representations depend on the number of deflected whiskers

The response of neurons in somatosensory cortex to deflection of multiple whiskers is affected non-linearly by several factors including the number of whiskers that contribute to the sensory input (Shimegi and others 1999; Mirabella and others 2001). It remains unclear, however, whether varying the number of deflected whiskers modifies the BOLD response. Therefore, we compared the BOLD responses evoked by deflection of the right C1 and C2 whiskers at 10 Hz with the BOLD responses evoked by 10 Hz deflection of multiple whiskers in the left B - E rows of the same animals (see Materials and Methods). Synchronous deflection of the right C1 and C2 whiskers evoked two positive BOLD responses in contralateral neocortex that were centred 2 - 3 mm caudal to bregma and extended over 2 - 5 contiguous slices of single-animal maps (Fig. 2A) and group maps (n = 8 rats; Fig. 3A). The location and rostro-caudal extent of the positive BOLD responses were consistent with previous neuroanatomical and electrophysiological studies of whisker cortical maps (Chapin and Lin 1984; Fabri and Burton 1991; Remple and others 2003; Benison and others 2007). We concluded that the positive BOLD responses represented activations in SI and secondary somatosensory cortex (SII). The positive BOLD responses were separated and partially surrounded by a negative BOLD response. In the group maps, negative BOLD responses were present in right-sided neocortex ipsilateral to the two deflected whiskers and in locations that were homotopic to the positive BOLD responses in contralateral SI and SII (Fig. 3Aii).

Figure 3.

Figure 3

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Whisker representations depend on the number of deflected whiskers. (Ai) Schematic of two-whisker deflection protocol. (Aii) Group map (n = 8) of the BOLD responses evoked by the right C1 and C2 whiskers. Pseudocoloured voxels have a positive BOLD (red) or negative BOLD (blue) signal that is significantly different from baseline. The pseudocolour scale bars apply to both Aii and Bii. Statistical parametric maps in Fig. 2 use the same scale to enable comparison. (Bi) Schematic of multiple whisker-row protocol. (Bii) Group map (n = 8) of the BOLD responses evoked by deflection of multiple whisker rows on the left side of the snout. Pseudocoloured voxels have a positive BOLD (red) or negative BOLD (blue) signal that is significantly different from baseline.

Deflection of 3 - 4 whiskers from each of the B - E whisker rows evoked a positive BOLD response in contralateral cortex that was centred 2 - 3 mm caudal to bregma and extended over 5 - 6 contiguous slices in single-animal (Figs. 2B) and group maps (Fig. 3B). The positive BOLD response was separated into two discrete responses in some single-animal maps (Fig. 2B), but not in the group map (Fig. 3B). In contrast to the two-whisker protocol, there was no negative BOLD response contralateral to the deflected whiskers. Examination of the signal surrounding discrete positive BOLD responses in single-animal maps revealed that the BOLD signal was positive, but had not reached statistical significance. A negative BOLD response was present in somatosensory cortex ipsilateral to the deflected B - E whisker rows at points homotopic to the contralateral positive BOLD responses in both single animal (-0.28 ± 0.06 %, n = 16)(Fig. 2B) and group maps (Fig. 3B).

The peak amplitude of the positive BOLD response in SI evoked by deflecting two whiskers (+0.34 ± 0.04 %) was less than that elicited by the multiple whisker row protocol (+0.91 ± 0.08 %; paired t-test, t = 6.74, n = 8, P < 0.001)(Fig. 4A). Similarly, the peak amplitude of the BOLD signal in SII evoked by two whisker deflection (+0.13 ± 0.05 %) was smaller than that elicited by multiple whisker row deflection (+0.84 ± 0.11 %; paired t-test, t = 8.21, n = 8, P < 0.001)(Fig. 4B). The changes in volumes of the positive BOLD responses mirrored the differences in their amplitudes. The volume of the positive BOLD response in SI evoked by deflection of two whiskers (29 ± 5 voxels) was smaller than that elicited by the multiple whisker row protocol (116 ± 18 voxels; paired t-test, t = 4.31, n = 8, P = 0.004)(Fig. 4C). Similarly, the volume of the positive BOLD response in SII evoked by deflection of two whiskers (16 ± 7 voxels) was less than that evoked by the multiple whisker row protocol (66 ± 16 voxels; paired t-test, t = 2.62, n = 8, P = 0.034)(Fig. 4D). We concluded that the positive BOLD response evoked by deflection of two whiskers is of lower amplitude and is smaller in extent than the positive BOLD response evoked by deflection of many whiskers at 10 Hz.

Figure 4.

Figure 4

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Deflection of many whiskers evokes a larger positive BOLD response in SI and SII than two-whisker deflection. (A) Amplitudes of the positive BOLD responses in SI evoked by the two-whisker and multiple row deflection protocols in the same animals. (B) Amplitudes of the positive BOLD responses in SII evoked by the two-whisker and multiple row deflection protocols in the same animals. (C) Volumes of the positive BOLD responses in SI evoked by the two-whisker and multiple row deflection protocols in the same animals. (D) Volumes of the positive BOLD responses in SII evoked by the two-whisker and multiple row deflection protocols in the same animals.

Whisker representations are frequency-dependent

The greatest decrease in deoxyhaemoglobin levels and increase in cerebral blood volume (CBV) in the contralateral whisker barrel cortex evoked by deflection of one whisker occurs with 10 Hz stimulation (Sheth and others 2003). Hence, BOLD responses evoked by repetitive deflection of two whiskers may vary across the physiological whisking range. We explored whether the cortical representation of the right C1 and C2 whiskers was frequency dependent by deflecting those whiskers synchronously: at 3 Hz, which is just below the physiological whisking range; at 7 Hz to represent exploratory whisking behaviour; or at 10 Hz to exemplify discriminatory whisking behaviour (Carvell and Simons 1995; Harvey and others 2001). Deflection at 10 Hz evoked BOLD responses that were similar to those described earlier. Two positive BOLD responses were centred 2.5 - 3 mm caudal to bregma in contralateral neocortex and were abutted by a negative BOLD response in single animal maps (Fig. 5) and group maps (Fig. 6C). In the group maps, two negative BOLD responses were present in right-sided neocortex ipsilateral to the whisker stimulation in locations that were homotopic to the positive BOLD responses in contralateral SI and SII (Figs. 6C). These data demonstrated that the positive and negative BOLD responses evoked by 10 Hz deflection of two whiskers were reproducible.

Figure 5.

Figure 5

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Single-animal maps of the BOLD signal evoked by right C1 and C2 whisker deflection at 3 Hz (A), 7 Hz (B) and 10 Hz (C). The rostro-caudal position of each slice with respect to bregma is given. Pseudocoloured voxels have a positive BOLD (red) or negative BOLD (blue) signal that is significantly different from baseline. The pseudocolour scale bar applies to all maps.

Figure 6.

Figure 6

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Whisker representations in neocortex depend on deflection frequency. (A) Group map (n = 7) of BOLD responses evoked by 3 Hz deflection of right C1 and C2 whiskers. Numbers denote the rostro-caudal position of each slice with respect to bregma. A line drawing from a rat atlas is superimposed on the slice 2.5 mm caudal to bregma. Pseudocoloured voxels have a positive BOLD (red) or negative BOLD (blue) signal that is significantly different from baseline. (B) Group map (n = 7) of BOLD responses evoked by 7 Hz deflection of right C1 and C2 whiskers arranged as in A. (C) Group map (n = 8) of BOLD responses evoked by 10 Hz deflection of right C1 and C2 whiskers arranged as in A. The pseudocolour scale bar applies to all maps.

Whisker deflection at 3 Hz evoked a single positive BOLD response in a location consistent with SI (n = 7, Fig. 6A). Whisker stimulation at 7 Hz evoked a single positive BOLD response within contralateral neocortex that overlapped the 3 Hz activation, but was larger in volume and extended laterally towards SII (n = 7; Fig. 6B). In contrast to the 10 Hz group map, no negative BOLD response was present in the 3 Hz or 7 Hz group maps contralateral to deflected whiskers. However, we noted that negative BOLD responses between SI and SII were elicited in single-animal maps (3/11 animals) with 7 Hz deflection when the positive BOLD response amplitude was large. Negative BOLD responses between contralateral SI and SII were infrequent in 3 Hz single-animal maps (1/11 animals). This suggested that the mechanisms driving the negative BOLD response during 10 Hz stimulation were also operating with 7 Hz stimulation, but more weakly. A negative BOLD response ipsilateral to the deflected whiskers was evoked in 45 % (5/11) rats with whisker deflection at either 7 Hz (-0.28 ± 0.11 %, n = 11) or 3 Hz (-0.14 ± 0.06 %, n = 11).

We quantified the BOLD responses using regions of interest in the single-animal maps (Fig. 7A) and incorporated the two-whisker data from the study comparing the representations of two whiskers with multiple whisker rows. Whisker deflection at 7 Hz (n = 11) or 10 Hz (n = 16) always evoked a positive BOLD response in SI contralateral to the deflected whiskers. Deflection at 3 Hz elicited a contralateral positive BOLD response in the majority (9/11) of animals. The volume of the positive BOLD response in SI was correlated with the amplitude of the positive BOLD response in SI (r = 0.59, n = 38, P < 0.001)(Fig. 7B). Deflection frequency had a significant effect on the peak amplitude of the SI positive BOLD response with larger responses at 7 Hz compared with either 3 Hz (P = 0.003, t = 3.4, t-test) or with 10 Hz (P = 0.008, T = 209, Mann-Whitney rank sum test)(3 Hz, 0.27 ± 0.05 % (n = 11); 7 Hz, 0.56 ± 0.07 % (n = 11); 10 Hz, 0.36 ± 0.02 % (n = 16); see Materials and Methods)(Fig. 7C). The volume of the positive BOLD response in SI of single animals was greater for 7 Hz whisker deflection compared with 3 Hz (P < 0.05, Dunn’s multiple comparison), but did not attain statistical significance when 7 Hz was compared with 10 Hz deflection (3 Hz, 13 ± 4 voxels (n = 11); 7 Hz, 41 ± 9 voxels (n = 11); 10 Hz, 27 ± 3 voxels (n = 16); P = 0.003, H = 11.8, Kruskal-Wallis one-way ANOVA on ranks).

Figure 7.

Figure 7

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Single-animal analysis of two-whisker BOLD representations. (A) Regions of interest and the line drawing from a rat atlas are superimposed on a brain slice. The colour code (red, positive BOLD; blue, negative BOLD) denotes the principal BOLD response in each region of interest. (B) Volume and amplitude of the positive BOLD response (PBR) are correlated. (C) Change in BOLD signal in SI (filled circles) and SII (open circles) with varying whisker deflection frequency. Error bars denote SEM. (D) Maximum BOLD signal intensities in SI and SII. The dashed line is the unity line. (E) Number of voxels in the SI positive BOLD response and SII positive BOLD response. The dashed line is the unity line.

Positive BOLD responses were present in SII of 36 % (4/11) of rats with 3 Hz stimulation, 54 % (6/11) of rats with 7 Hz stimulation and 50 % (8/16) of rats with 10 Hz stimulation. The peak amplitude of the positive BOLD response in SII did not vary with whisker deflection frequency (3Hz = 0.11 ± 0.05 % (n = 11); 7 Hz = 0.19 ± 0.06 % (n = 11); 10 Hz = 0.14 ± 0.04 % (n = 16): P = 0.507, H = 1.36, one-way ANOVA on ranks)(Fig. 7C) in contrast to our results for SI. However, the amplitudes of BOLD responses in SII were close to the threshold for detection. Hence, the lack of correlation should be treated cautiously.

Our data indicated that the amplitude of the positive BOLD response in SI is greatest at a whisker deflection frequency, 7 Hz, which is more typical of exploratory whisking behaviour. Whisker deflection at higher frequencies to mimic discriminatory whisking behaviour was associated with a decrease in amplitude of the positive BOLD response in SI and the emergence of a robust negative BOLD response that partially surrounded the positive BOLD responses in SI and SII (Fig. 3A, 6C). We concluded that the cortical representation of two whiskers imaged with BOLD fMRI was frequency-dependent in the physiological whisking range.

Positive BOLD responses in contralateral SI and SII

The thalamic inputs to SI and SII differ (Carvell and Simons 1987; Diamond 1995; Kwegyir-Afful and Keller 2004), which raises the possibility that BOLD signals in SI and SII may have distinct relationships with whisker deflection frequency. However, reciprocal intracortical pathways connect SI and SII (Fabri and Burton 1991; Hoffer and others 2003) and these pathways may couple BOLD responses in SI and SII. We found that the amplitude of the positive BOLD response was larger in SI compared with SII (P < 0.001, t = 6.7, n = 38, paired t-test)(Fig. 7D), but the amplitudes of the positive BOLD responses were not correlated (r = 0.18, P = 0.275, n = 38, Pearson correlation)(Fig. 7D). The lack of correlation (r = 0.28, P = 0.093, n = 37, Pearson correlation) persisted after removing one data point that was a possible outlier (3 Hz whisker deflection: SI, 0 %; SII, 0.36 %).

We counted the number of voxels in positive BOLD responses in SI and SII. The positive BOLD response in SI was spatially more extensive than the positive BOLD response in SII (positive BOLD response at 3 Hz, 7 Hz and 10 Hz, median volume: SI = 22 voxels, SII = 5 voxels; n = 38, P < 0.001, signed rank test). The volumes of the positive BOLD responses in SI and SII were correlated (r = 0.46, P = 0.004, n = 38, Pearson correlation; Fig. 7E). We concluded that deflection of two adjacent whiskers evokes a positive BOLD response in SI and SII and that the positive BOLD response in SI is bigger than the positive BOLD response in SII. The lack of correlation between the amplitudes of the positive BOLD responses in SI and SII should be interpreted cautiously because the amplitudes of positive BOLD responses in SII were close to the threshold (approximately 0.15 - 0.2 % signal change) for detection of responses in single animals. However, the results tend to suggest that BOLD responses in SI and SII are not extremely tightly coupled.

Positive and negative BOLD responses have different laminar locations

Inspection of the group maps of BOLD responses evoked by 10 Hz deflection of two whiskers (Figs. 3A, 6C) suggested that the positive and negative BOLD responses were located at different depths in the cortex. We quantified this by dividing the regions of interest in contralateral whisker somatosensory cortex denoted in Fig. 7A by SI and N (negative BOLD response between SI and SII positive BOLD responses) into an upper half, which corresponds approximately to layers 1 - 4 (L1 - 4) and a lower half, which consists of layers 5 - 6 (L5 - 6)(Fig. 8A). We then measured the volumes of the positive and negative BOLD responses in both halves of the SI and N regions of interest evoked by 10 Hz whisker deflection. The majority of the SI positive BOLD response was in the upper cortical layers (L1 - 4 = 31 ± 6 voxels, L5 - 6 = 7 ± 3 voxels; P = 0.004, n = 16, t = 3.4, paired t-test) whereas the largest part of the negative BOLD response was in the lower cortical layers (L1 - 4 = 10 ± 3 voxels, L5 - 6 = 28 ± 7 voxels; P = 0.031, n = 16, t = 2.4, paired t-test)(Fig. 8B). We concluded that the BOLD response showed laminar preference with the positive BOLD response mainly located in upper cortical layers whereas the negative BOLD response was predominantly found in lower cortical layers.

Figure 8.

Figure 8

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Relationship between positive and negative BOLD responses in SI. (A) Schematic of an image slice through whisker barrel cortex. A dashed line splits SI into upper (L1 - L4) and lower (L5 - L6) cortical layers. Red, positive BOLD response. Blue, negative BOLD response. (B) Volumes of the positive and negative BOLD responses in L1 - L4 and L5 - L6 of SI.

Discussion

We imaged the steady-state cortical representations evoked by repetitive deflection of whiskers at multiple frequencies. Whisker representations depended on the number of displaced whiskers. Deflection of many whiskers evoked a positive BOLD response extending through contralateral SI and SII. In contrast, deflection of two adjacent whiskers at 10 Hz elicited positive BOLD responses in contralateral SI and SII, which were separated and partially surrounded by a negative BOLD response. The positive and negative BOLD responses showed different laminar specificities; positive BOLD responses were evoked predominantly in cortical layers 1 - 4 whereas negative BOLD responses were mainly found in cortical layers 5 - 6. Varying the whisker deflection frequency in the physiological whisking range modified the amplitude and extent of the evoked BOLD responses. Our findings suggest that alterations in sensory input attributable to whisking movements modify whisker cortical representations.

Positive BOLD responses

Deflection of many whiskers (multi-row protocol) at 10 Hz evoked an extensive positive BOLD response that spread across contralateral SI and contralateral SII as reported previously (Sachdev and others 2003; Lu and others 2004; Kennerley and others 2005). Similarly, focal stimulation of the whisker sensory input by deflecting two adjacent whiskers at 7 Hz resembled the positive BOLD response evoked by 8 Hz deflection of one whisker (Yang and others 1996). In contrast, two-whisker deflection at 10 Hz evoked very different representations with positive BOLD responses in SI and SII that were separated and partially surrounded by a negative BOLD response.

The limited resolution attainable with the BOLD signal (Ugurbil and others 2003) might suggest that the separate positive BOLD responses in SI and SII with 10 Hz deflection were artefactual. It has proven difficult to map representations at the level of cortical columns using the BOLD signal and single-condition maps (Kim and others 2000). However, refinements in functional imaging strategies such as differential mapping using the BOLD signal (Kim and others 2000) or cerebral blood flow fMRI (Duong and others 2001) have generated sub-millimetre resolution maps. Our ability to resolve separate positive BOLD responses in SI and SII was improved by the interspaced negative BOLD response. Furthermore, post-processing of our data facilitated imaging of steady-state whisker representations. In particular, a vascular mask was used to minimize BOLD signal attributable to large draining veins and vascular inflow, which could markedly distort the location and extent of whisker representations (Menon and Goodyear 2001). A similar approach has been implemented to remove signal from surface vessels in optical imaging studies of single-whisker responses (Sheth and others 2004a). Hence, we contend that the positive BOLD responses in SI and SII primarily represent separate foci of cortical activity evoked by whisker deflection.

The amplitude of the positive BOLD response evoked by deflection of many whiskers was larger than that elicited by two-whisker deflection. We believe that this probably reflects a combination of greater thalamocortical input during multiple row whisker deflection and spread of neuronal activity between barrel columns via horizontal intracortical connections (Moore and Nelson 1998; Zhu and Connors 1999; Petersen and others 2003; Brecht and others 2003).

Negative BOLD responses

We imaged a robust negative BOLD response adjacent to positive BOLD responses in contralateral SI and SII with two-whisker deflection at 10 Hz. This negative BOLD response is unlikely to be artefactual because: it was not evoked by deflection of multiple whisker rows in the same animals despite the larger amplitude of the evoked positive BOLD response; our analysis suggests that it was frequency-dependent; and it was not seen in scalp muscle near to somatosensory cortex or in phantoms.

Negative BOLD responses adjacent to positive BOLD responses have been described in visual cortex of cats (Harel and others 2002), humans (Shmuel and others 2002) and monkeys (Shmuel and others 2006), but have not been reported previously in rodent somatosensory cortex to our knowledge. The negative BOLD response that we found typically extends only 1 - 2 mm from the positive BOLD response, which is much less than that described for visual cortex (Harel and others 2002; Shmuel and others 2006). Our data further indicate that the negative BOLD response in somatosensory cortex evoked by whisker deflection is more prominent in lower cortical layers and is frequency dependent.

The mechanisms that underpin negative BOLD responses have attracted considerable debate with suggestions that they result from: diminution of neuronal activity (Raichle 1998); inhibitory synaptic activity (Blankenburg and others 2003; Shmuel and others 2006); neural activity insufficient to drive a vascular response (Nielsen and Lauritzen 2001; Sheth and others 2004b); or a reduction in cerebral blood volume (Harel and others 2002) that may be caused by vascular steal (Kannurpatti and Biswal 2004). Furthermore, there has been discussion about the mechanisms that couple the negative BOLD response to neural activity and the role played by neurotransmitters released from inhibitory interneurons in neurovascular coupling (Attwell and Iadecola 2002; Shulman and others 2004).

Negative BOLD responses were evoked in ipsilateral somatosensory cortex by both two whisker and multiple whisker-row deflection protocols. Performance of unilateral motor or sensory tasks in humans also evokes a negative BOLD response in ipsilateral sensorimotor cortex, which has been attributed to inhibition (Allison and others 2000; Stefanovic and others 2004). Such a mechanism could apply to the ipsilateral negative BOLD response that we describe because transcallosal connections linking right and left SI in the rat mediate feed-forward inhibition (Pidoux and Verley 1979). The lack of positive BOLD response in ipsilateral cortex suggests that two-whisker deflection did not induce a significant increase in blood flow to ipsilateral cortex, and, hence, implies that vascular steal is an unlikely cause.

Inhibition may underlie the contralateral negative BOLD response that we report. This proposal is supported by electrophysiological recordings, which indicate that synchronous deflection of two whiskers enhances surround inhibition (Simons and Carvell 1989; Brumberg and others 1996) in addition to increasing neuronal activity in supragranular cortex (Shimegi and others 1999; Mirabella and others 2001). Furthermore, repetitive deflection in the upper part of the physiological whisking range and larger amplitude deflections entrain putative inhibitory interneurons better than excitatory neurons (Simons 1978). Whisker representations can extend outside whisker barrel cortex (Brett-Green and others 2001). However, surround inhibition within whisker barrel cortex has a spatial gradient (Brumberg and others 1996) and it is possible that the underlying inhibitory circuitry could result in the effects of surround inhibition being greater within whisker barrel cortex than in adjacent non-whisker barrel cortex. Hence, two-whisker deflection at 10 Hz may evoke negative BOLD responses because the protocol enables the effects of surround inhibition within whisker barrel cortex to be imaged. This proposal would explain why deflection of many whiskers did not evoke a contralateral negative BOLD response.

Non-fMRI imaging modalities have shown that somatosensory stimulation evokes an area of increased neuronal activity, increased oxygenation and vasodilation in contralateral SI that is surrounded by a region of vasoconstriction and decreased oxygenation (Woolsey and others 1996; Devor and others 2005; Devor and others 2007). Neurons in the surround region are hyperpolarized, which has been attributed to inhibition, but firing is unaffected (Kleinfeld and Delaney 1996; Derdikman and others 2003; Devor and others 2007). In contrast, negative BOLD responses in visual cortex are associated with both decreased cerebral blood flow (Shmuel and others 2002; Harel and others 2002) and diminished neuronal firing (Shmuel and others 2006). The amplitude of the negative BOLD response correlates with the decrease in neuronal firing suggesting that diminished neural firing is an important factor underpinning negative BOLD responses (Shmuel and others 2006).

We make a simple proposal bringing together hypotheses concerning the mechanisms underpinning negative BOLD. This synthesis centres on the balance between excitation and inhibition. A large proportion of excitatory synaptic inputs in neocortex arise from neighbouring pyramidal neurons (Binzegger and others 2004). Excitatory neurotransmitters released by those circuits cause vasodilation (Zonta and others 2003; Wang and others 2006; Takano and others 2006). Therefore, inhibition that is strong enough to dampen firing has two effects, which both promote vasoconstriction. Firstly, inhibition can cause vasoconstriction, possibly through a direct effect on vessels (Cauli and others 2004). Secondly, diminished neural firing results in decreased release of excitatory neurotransmitters from local excitatory circuits and, hence, reduced vasodilatory drive. The combination of inhibition-induced vasoconstriction and reduced vasodilatory drive enhance the reduction in cerebral blood flow. A negative BOLD response would then occur in cortex if the decrease in cerebral blood flow was much greater than the corresponding decrease in oxygen consumption.

Whisker representations are frequency-dependent

The amplitude of the positive BOLD response evoked by two-whisker deflection was frequency-dependent with a peak response at 7 Hz. The mechanisms underpinning the frequency dependence of the positive BOLD response remain unclear. Optical imaging combined with local field potential measurements suggest that single-whisker responses are greatest with 10 Hz deflection (Sheth and others 2003). In contrast, deflection of multiple whiskers evokes maximal synaptic responses (measured as the sum of local field potentials) with air puffs delivered at 5 Hz and this correlates with increased astrocytic calcium levels (Wang and others 2006), which cause vasodilatation (Takano and others 2006). Preliminary reports of the BOLD response evoked by deflection of multiple whiskers have yielded mixed results (Lu and others 2003; Melzer and Ebner 2004). The discrepancies in these studies may be due to configuration of the whisker stimulus, which markedly affects evoked responses (Pinto and others 2000; Petersen and others 2003).

The emergence of a robust negative BOLD response in the 10 Hz group maps elicited by two-whisker deflection suggested that the boundary of the positive BOLD response was modulated by whisker deflection in the upper part of the physiological whisking range. Changes in representation boundaries, referred to as sharpening, have been reported with similar whisker stimulation frequencies (Sheth and others 1998). The whisker deflections that we used were designed to mimic whisking and, therefore, may have had larger amplitudes. However, the boundary changes that we imaged with 10 Hz deflection of two whiskers resemble sharpening of whisker representations suggesting the possibility that the contralateral negative BOLD and sharpening of whisker representations are driven by similar mechanisms.

Acknowledgements

We thank Professor Mick Brammer and Claire Cheetham for helpful comments and Professor Jimmy Bell, Dr Amy Herlihy and Dr Po-Wah So for support with the project. All scans were performed at the MRC Biological Imaging Centre, Imperial College London. Funded by the Wellcome Trust and MRC. GTF is a Wellcome Trust Senior Fellow in the Clinical Sciences.

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