A total of 15 rats were implanted with microelectrode arrays and an injection cannula for muscimol delivery. Single-unit activity was recorded simultaneously in S1 and M1, or in M1, VPM and POM while rats performed an active aperture discrimination task (Krupa et al., 2001
; Wiest et al., 2010
) before, during and after pharmacological inactivation of M1 ipsilateral to the recording sites.
A total of 2,575 single S1, VPM, and POM units were recorded in 120 behavioral sessions from the microelectrodes implanted across four different regions (-). illustrates examples of the quality of cluster separation (A) waveforms, (B) ISI distribution, (C-D) as well as cluster related statistics (Nicolelis et al., 2003
) (J3: 2.939±0.08; Pseudo-F: 49773±2507; Davies-Bouldin: 0.1981±0.01; F:1.725± 0.02) (). All these measurements confirmed the high quality of single unit isolation obtained in each of the sampled brain structures. The proportion of units recorded in each region was: 39.42% in S1 (n = 1015 units), 19.57% in VPM (n = 504 units), 17.94% in POM (n = 462 units) and 23.07% in M1 (n = 594 units). In the S1, 40.99% (n=416 units) were recorded from the supragranular layers, 30.44% (n=309 units) were recorded from the granular layer and 28.56% (n=290 units) were recorded from the infragranular layers. Additionally, we recorded single (n= 31 units) and multiunits (n=705 multiunits) from the TG. Note that as the electrode arrays were not moved every session it is possible that the number of single units could be slightly smaller than values reported above. We estimate that approximately ~20% of the neurons recorded remained the same across different sessions.
Ranking of neuronal ensembles reveals extensive anticipatory firing activity in M1, S1, VPM, and POM
Cluster separation and waveform quality in recordings
Overall, statistically significant modulations of firing rates were found in a large proportion (75%) of neurons in all conditions and regions tested (). Specifically, we found patterns of concurrent increased and decreased neuronal activity that varied across different layers of S1 and thalamic nuclei (VPM and POM). In the control condition, anticipatory firing modulations were observed in 40.19% of the S1 units, 49.67% of the VPM units and 37.93% of the POM neurons recorded in this study. The magnitude of anticipatory firing in S1 was 2.72±0.1 spikes/trial and its duration was 195.4±14.62ms. In VPM the magnitude of the anticipatory firing was 2.61±0.3 spikes/trial and its duration was 247.4±19.51ms. In POM, the magnitude of the anticipatory firing was 2.4±0.2 spikes/trial and its duration was 184.0±32.58ms. Such modulation in neuronal firing frequently started several hundred milliseconds before the animals’ facial whiskers made any physical contact with the tactile stimulus ( and ). Characteristic examples of these anticipatory firing modulations in different S1 layers, VPM and POM nuclei can be observed in the PSTHs depicted in . Note that multiple increases and decreases of cortical and thalamic firing occur before the animals break the infrared beam and touch the edges of the bar with their facial whiskers (see ). Different colors in schematically illustrate the relation between the behavioral task and the different analysis periods. The anticipatory epoch corresponds to the period before the whiskers make contact with the discrimination bars (light blue), while the discriminatory period (green) corresponds to the period immediately after the whiskers touch the target bars. The observed cortical and thalamic firing modulations were not restricted to one specific task period but instead occurred during many different time epochs. depict the average performance and average number of trials for each condition studied (see below for detailed description).
Proportions and type of firing modulations by region
Neural ensemble activity across multiple thalamocortical loops during active tactile discrimination
Each panel of shows the normalized firing activity (relative to the maximum firing rate of each neuron) of all the cortical and thalamic neurons recorded during the execution of the tactile discrimination task in control conditions, and after saline or muscimol injections in M1. Continuous changes in neuronal activity occurred before and after stimulus contact within all cortical and thalamic regions sampled during the animals’ performance of the tactile discrimination task ( and ). Anticipatory activity, i.e. prior to any whisker contact with aperture edges, was represented by both increases and/or decreases in neuronal firing. In VPM and granular layer of S1, anticipatory activity was mostly associated with a decrease in firing. In POM and S1 infragranular layers, the pattern of anticipatory activity followed the opposite trend (i.e. firing rate increased immediately before tactile discrimination). Based on previous published studies from our laboratory (Krupa et al., 2004
; Wiest et al., 2010
), the presence of these different patterns of neuronal firing modulations, within and between different structures, suggested that cortical and thalamic neuronal anticipatory firing was fundamental for task performance. To demonstrate the relation between the animal’s behavior during a trial and the diversity of neuronal firing modulations observed across multiple cortical and thalamic structures sampled in this study, depicts neuronal activity rank ordered by time, from -2.0 to 2 seconds. The multiple PSTHs presented show peaks of increased and decreased activation in all cortical and thalamic regions throughout a trial. The sequential order by which these peaks appear suggests the hypothesis that active tactile discrimination relies significantly on top down effects that cannot be explained by the classic feedforward model of tactile information processing. This hypothesis is supported by the finding that TG neurons (see bottom PSTH of ) only start modulating their firing rate after the rat’s whiskers touch the aperture edges. In , the fraction of neurons with significant increases or decreases in responses is shown by cortical area or thalamic nucleus. As noted above, virtually identical patterns of anticipatory firing activity occurred in VPM and in the granular layer of S1. Conversely, the patterns of anticipatory firing increases in the POM, M1 and infragranular layers of S1 also look similar.
Histological analysis () was used to locate the thalamic recordings sites. Different functional compartments (), coincident with different depths of recording, have been recently described for the VPM, namely the “head” and “core” of the barreloids (Urbain and Deschenes, 2007a
). Thus, we further investigated whether neural anticipatory modulations during tactile discrimination were restricted to a specific VPM depth. Solely for this analysis, we pooled data from all the control and saline sessions reported here and added 99 units recorded in VPM and 168 units recorded from POM (n=4 animals in 10 sessions from a different study that utilized the same task; these animals were either control subjects or injected with 500nl of saline in S1). illustrates that the depths of the recordings coincident with the “head” (starting at -5.2mm) and “core” (starting at -5.4mm) of the barreloids in VPM are associated with fundamentally different physiological properties, as previously reported in anesthetized animals (Urbain and Deschenes, 2007a
). The region of the “head” of the barreloids was characterized by anticipatory activity coincident with the major periods of increased activity in POM, M1 and S1 infragranular layers. On the other hand, the “core” of the barreloids was coincident with the pattern of decreased-increased-decreased activity found in layer IV of S1 (see ). These two subregions of the VPM nucleus exhibited different proportions of anticipatory firing increases (VPM “head”: 31/64 units; VPM “core”: 54/256 units; Chi Square = 18.25, df = 1, P
<0.0001) and decreases (VPM “head”: 4/64 units; VPM “core”: 107/256 units; Chi Square = 29.2, df = 1, P
<0.0001). Despite these differences, cells with anticipatory increased activity were found at all depths studied.
Both “head” and “core” of barreloids in VPM present anticipatory neural activity
Lastly, due to the proximity of POM and VPM “head” regions (see ), we compared the physiological properties of neurons recorded from these two areas. Clear differences were found in the proportion of significant increased responses in POM in the anticipatory period corresponding to the rat entering the inner chamber (VPM “head”: 6/31 responses; POM: 50/125 responses; Chi Square = 3.75, df =1, P = 0.05) in the magnitude of decreased neural activity (VPM “head”: 1.68±0.1 spikes/trial; POM 1.44±0.1 spikes/trial; Mann-Whitney U = 4616; P = 0.0187), and in the duration of increased neural responses (VPM “head”: 153.0±25.31 ms; POM 182.4±9.94 ms; Mann-Whitney U = 12540; P = 0.05). Altogether these results show that distinct compartments associated with “head” and “core” of the VPM exhibit anticipatory increased and decreased neural activity prior to whisker contact with a discriminanda.
Overall, cortical and thalamic neuronal firing preceding the tactile stimulus could have originated from three possible sources: (i) whiskers contacting chamber walls or floor surface during the interval from the door opening and the aperture beam break; (ii) whisker movements producing sensory reafference that triggered thalamic activity; or (iii) top down neuronal afferents that induced anticipatory firing unrelated to whisker contact or movement. The first two possibilities have been mostly ruled out in previous studies conducted in our laboratory that demonstrated that whisker movements or early whisker contacts with the chamber walls are not the basis for anticipatory activity observed in S1 (Krupa et al., 2001
; Krupa et al., 2004
; Wiest et al., 2010
). To rule out these possibilities once and for all, we conducted two additional control experiments.
Neurons in the trigeminal ganglion are not modulated before any contact with the tactile stimulus
To control for the possibility of early whisker contacts with the chamber walls or floor, we simultaneously recorded neuronal activity from TG, S1 and VPM in the same subjects while rats performed the same tactile discrimination task. TG is the main recipient of primary afferent inputs from the whiskers and thus, the presence of neuronal responses in this ganglion provides a very reliable indicator of any mechanical displacement of the animal’s facial vibrissae. depicts a sample of PSTHs to illustrate the characteristic TG neuron firing modulations during execution of the tactile discrimination task. Analysis of these TG neurons’ firing rate modulations revealed three main periods of increased activation corresponding to: whisker contacts with the chamber’s door, whisker contact with the aperture edges (immediately after beam break) and whisker contacts with the center nosepoke. These accounted for the sensory evoked responses of 81.73% (528/736 single or multiunits) of the TG neurons recorded. The firing patterns of all TG neurons recorded in this study (31 single units and 705 multiunits) are depicted in . Neurons in the TG exhibited clear sensory evoked responses just after Time=0 (Beam Break), indicating that these first order cells fired maximally immediately after the whiskers contacted the aperture edges. Interestingly, a large percentage (68.42%, 442/736) of TG neurons also exhibited significant decreases of activity as rats run through the corridor that separated the door from the beam break. These modulations can be explained somewhat by the fact that during the period used to collect baseline firing data (before the door opens) the rat’s whiskers often made contact with the surface of the closed doors.
Trigeminal ganglion activity is phase locked to the tactile stimulus contact and does not appear during the anticipatory firing period
Using the same PSTH analysis employed for examining the cortical and thalamic data, we observed that a meager 5.02% of the neurons (37/736 of the units/multiunits) showed increased activations around the 250ms prior to beam break. Careful analysis of the trials in which these 37 TG neurons fired revealed that such sensory evoked responses were due to late whisker contacts with the chamber doors. In another words, these sensory evoked responses did not occur during the anticipatory period. Restricting the time window to [-0.2 -0.05 ] seconds to omit such occasional late whisker contacts with the doors reduced the number of excitatory responses in the anticipatory period to 1.902% (14/736 of the multiunits). Comparison of the activity occurring in the interval between [-0.3; 0] seconds () further showed that the TG presented a period of increased activity coinciding with the animal’s whiskers contacting door. Again, this epoch did not match the period of increased anticipatory activity observed in VPM and S1. This point is highlighted even further when individual PSTHs of simultaneously recorded TG, VPM, and S1 neurons in three different rats are plotted together (). This plot shows that after TG neurons respond to the whiskers contacting the doors, their firing rate tends to decrease rapidly to almost zero. Thus, S1 and VPM increases in anticipatory firing tend to occur precisely during the period in which TG neurons are virtually quiet. However, when the animal’s whiskers touched the aperture edges, immediately after the beam break (see BB at the bottom of the PSTHs), neurons in all three regions (TG, VPM, and S1) produced vigorous firing increases.
In conclusion, our control data, involving the largest sample to date of TG neurons recorded in behaving rats, clearly indicates that the anticipatory activity observed in S1, VPM, and POM during the period the animal crosses the corridor that separates the door from the aperture edge cannot be explained by peripheral activation of first order TG neurons.
Yet, since well-trained animals would typically protract their whiskers to perform this task, one could argue that in some trials whisker contacts could have occurred slightly earlier than the beam break. To additionally test if primary afferent neuron activation could occur during the time required for the animals to cross the corridor that separated the door and the aperture, we reanalyzed video recordings presented elsewhere (Wiest et al., 2010
) and calculated the difference between whisker contacts and beam break in 24 sessions. The video analysis directly showed that the rat’s whiskers had no contact with any surface prior to the moment they touched the aperture edges. Also, the distribution of the timing, within a trial, of whisker contacts with the bar showed that typical whisker contacts (43.0% of the trials) occurred at frame 0 (the video frame of contact is the same as the frame of beam break) or at -20ms (44.2% of the trials) (the video frame of whisker contact immediately precedes the frame of the beam break). Since the onset of neuronal anticipatory firing activity in M1, S1, VPM and POM typically starts at -250ms, even if the onset of TG activity was further corrected for the possibility of whisker contacts at -40ms (which would include 96.8% of the trials analyzed), we would still observe clear peaks of anticipatory neuronal activity in M1, S1, VPM, and POM that cannot be explained at all by early whisker contacts.
Altogether, these two control experiments, as well as extensive data already published (Krupa et al., 2001
; Krupa et al., 2004
; Wiest et al., 2010
) rule out the hypothesis that increased S1, VPM, and POM anticipatory activity before the beam break is in any way related to early whisker mechanical stimulation by spurious whisker contacts with the chamber walls or floor.
Anticipatory firing in the S1 and thalamic nuclei are not due to reafference of whisking signals
Having excluded the possibility of early whisker contacts, we tested the possibility that increased neuronal activity before contact with the tactile discriminanda could be due to some other form of whisker movements that led to sensory reafference. Using video recordings, we have repeatedly observed that no whisking of any sort occurred as rats perform this tactile discrimination task (Krupa et al., 2001
; Krupa et al., 2004
; Wiest et al., 2010
). Instead, well trained animals tend to spread their whiskers, which seems to improve their tactile perception of approaching objects (Krupa et al., 2001
; Krupa et al., 2004
; Wiest et al., 2010
). We refer to this type of whisker positioning as object-detection mode. This behavior which is present in very well trained rats moving at a high speed, has been described only recently (Arkley et al., 2011
). In our experiments, animals also tended to perform the task at a high locomotion speed as well, and sample the tactile discriminanda for a very small amount of time (Wiest et al., 2010
). Yet, to test for the possibility that the anticipatory increases in S1 and thalamic neuronal activity could be related to reafferent peripheral inputs produced by some other type of whisker positioning, we further conducted recordings in three rats (two implanted in the VPM and one rat implanted in both VPM and POM) with bilateral facial nerve lesions. In the same animals, we also recorded EMGs from the whisker pad as a control for facial musculature activation. By simultaneously recording EMG activity and neuronal activity from thalamic nuclei, we were able to measure directly whether anticipatory neuronal activity was related to whisking. Overall, we found that bilateral facial nerve lesions prevented the animals from positioning their whiskers in the object detection-mode as well as from making large exploratory movements. EMG recordings allowed detection of small facial muscle contractions or artifacts associated with the possibility of wall contacts.
After recovery from surgery, these animals quickly learned that chewing or sniffing allowed them to make small whisker movements, although they could no longer make the large exploratory whisking movements or position their whiskers in the object-detection mode. These small whisker movements were easily detected by the EMG activity. shows the EMG activity of one of these animals in an open field. Different frequencies of EMG events were found for exploring, sniffing and grooming in an open field.
Examples of rectified EMG activity recorded from a rat with bilateral facial nerve lesion in an open field during three typical behaviors
We then recorded neural and EMG activity while the animals performed the tactile discrimination task (N=4 sessions). We found that, on average, the animals displayed detectable EMG activity during the anticipatory period in only 7.64 ±3.4 % of the trials. This value is below the ~15% previously reported by us (Wiest et al., 2010
) possibly due to the bilateral facial nerve lesions. After removing the trials where EMG events were present, analysis of neural activity showed that anticipatory modulations were present in 40.0% (26/65) of the thalamic (VPM and POM) units and multiunits recorded, a value that is virtually identical to those found in our control experiments. In we show examples of several trials with clear anticipatory thalamic activity in the absence of any EMG events. In all the trials shown in this figure, thalamic anticipatory firing began more than 100ms before the beam break occurred, excluding the possibility of early whisker contacts (which as demonstrated above can be ruled out with an extremely conservative measure of up to 40ms). Trials 2 and 4 in also show that EMG activity (red triangles) did not necessarily evoke any increases in neuronal firing either during the anticipatory period or after the beam break.
Anticipatory activity is independent of EMG events
Next, in all rats, EMG activity was cross correlated with the beam break and with the EMG events (). While EMG events were surrounded by peaks of EMG activity reflecting the frequencies of behaviors observed (note the repeated peaks at ~6Hz), no clear peak of EMG activity occurs before the beam break. Lastly, comparison of neuronal activity centered at the beam break or at the EMG events () further suggests that the peaks of activity related to anticipatory activity or related to EMG events occur in fundamentally different classes of thalamic neurons. These results demonstrate that the type of anticipatory neuronal activity observed in S1 and thalamic nuclei cannot be caused at all by sensory reafference related to whisker positioning or whisker movements.
Having ruled out the possibility that anticipatory neuronal activity was originated by early whisker contacts or early whisker movements we further analyzed if this anticipatory activity could result from a top-down signal originated from the primary motor cortex.
Anticipatory firing modulations in lemniscal and paralemniscal pathways can be explained by two Principal Components
The multitude of increased and decreased anticipatory neuronal firing modulations found in all thalamic and cortical regions studied here suggests that the TCLs continuously integrate information from both ascending and descending pathways, originating at subcortical and cortical levels. To determine whether the similar patterns of neuronal activity observed at cortical and thalamic levels were the result of largely independent modulations or, alternatively, they reflected wide interregional and correlated modulations across the TCLs, we performed a Principal Components Analysis (PCA) on all increased and decreased firing modulations in M1, VPM, POM, and each S1 layer for the period of [-0.5;1.0]s. This analysis revealed that the first two principal components accounted for 71.03% of the variance of the entire data set (). When a third principal component was added, 93% of the variance of the firing patterns observed across multiple cortical and thalamic structures was explained. This result clearly supports the existence of highly correlated patterns of anticipatory activity across the TCLs. For example, the first component included decreased activity from all S1 layers, M1, POM, and increased activity in VPM, while the second component included increased activity in all S1 layers, VPM, and POM, and decreased activity in VPM and S1 granular layer (see for positive and negative loadings in each component). The presence of such a high portion of variance explained with only three principal components suggests that patterns of anticipatory activity are linearly correlated across cortical and thalamic structures and that three of these neuronal patterns are sufficient to explain most of the response variability found across the TCLs. These concurrent patterns of responses also suggest that active tactile encoding is widely distributed across the multiple TC loops of the trigeminal system and results from large-scale, temporally asynchronous interactions between the many structures that define this circuit (Ghazanfar and Nicolelis, 2001
Principal Components Analysis of increased and decreased activity
Anticipatory activity predicts tactile performance
The presence of both cortical and thalamic neurons exhibiting anticipatory firing activity suggests that all these brain areas are engaged during the time period that precedes whisker contact with the stimulus (see -). Since we have shown that facial whiskers do not move during this pre-contact phase (see above), this finding cannot be explained by the classical feedforward model of tactile processing proposed to account for the main physiological properties of the trigeminal somatosensory system. As a typical increase or decrease in cortical and thalamic neuronal activity started around -250ms relative to the whiskers’ contact with the bars and ended at the time of discrimination, this anticipatory firing was related to the period that separated the crossing of the chamber door and the facial whisker contact with the aperture edges (typically 250ms).
A linear regression analysis of the relationship between the onset of anticipatory firing in cortical and thalamic units and the percentage of correct trials in each session revealed that, both in control and saline conditions, the timing of the onset of anticipatory cortical activity in M1 and S1 was a good predictor of the animal’s task performance (, panels C1-2). Such a correlation between onset of neuronal anticipatory responses and behavioral performance was also observed for VPM (F1,5 = 6.941, P = 0.0463, R2 = 0.58) and POM neurons (F1,14 = 18.69, P = 0.0007, R2 = 0.57) (, panel C4). Therefore, the timing of the onset of anticipatory neuronal firing activity, in both the lemniscal and paralemniscal pathways of the trigeminal system, can predict the animal’s tactile performance in our tactile discrimination task: the earlier the onset of anticipatory firing, the better the animal’s performance. This finding suggests that this type of neural modulation may be functionally significant for sensory-motor integration in a tactile discrimination task that does not require whisker movements.
Timing of anticipatory firing activity predicts animal’s performance
M1 inactivation affects tactile discrimination
Intracortical injection of 500ng of muscimol in 500nl of saline induced a temporary inactivation of M1. This was confirmed by an initial reduction in neural activity, followed by a complete absence of action potentials from M1 neurons recorded by the microelectrodes surrounding the injection cannula (Krupa et al., 1999
; Ghazanfar et al., 2001
; Shuler et al., 2002
). Our M1 inactivation was very localized and did not induce any gross motor impairment such as a reduction in the number of trials performed () (Control: 102.9± 3.15 trials; Saline: 102.0±3.66 trials; Muscimol: 103.5±4.00 trials; one way ANOVA: F2,118
= 0.04142; P
= 0.9594) or reduced locomotion speed (Control: 22.7±1.19 cm/s; Saline: 26.5±1.69 cm/s; Muscimol: 23.7±1.60 cm/s; Kruskal-Wallis statistic = 0.8736; P
= 0.6461). The only behavioral impairment observed in these animals was a decrease in their ability to discriminate with their whiskers a 14mm difference in width between a narrow vs wide aperture (Control: 83.2%±1.01 correct trials; Saline: 82.9%±1.66 correct trials; Muscimol: 70.3%±3.50 correct trials; Kruskal-Wallis statistic = 10.21; P
= 0.0061, post hoc
comparisons with Dunn’s test: Control vs Saline P
> 0.05, n.s.; Control vs Muscimol P
< 0.05)(). As we have repeatedly demonstrated here and elsewhere (see above), whisker movements were not required for rats to discriminate the aperture width with their vibrissae (Krupa et al., 2004
; Wiest et al., 2010
High resolution video analysis of the whisker angles and duration of contact of the whiskers with the stimulus showed no differences across all three different conditions (Control, Saline, and Muscimol). Comparison of whisker angles showed an overall significant effect for face side, suggesting that animals have a natural bias towards larger angles between the right whiskers and the right whisker pad (F1,8 = 77.57; P < 0.0001 non significant for post hoc analysis in all conditions; Control left 32.32± 6.085 degrees; Control right 41.29 ± 4.048 degrees; t6 = 1.227, P = 0.2657 ; Saline left 35.38 ± 4.754 degrees; Saline right 43.10 ± 4.389 degrees, t6 = 1.194, P = 0.2775; Muscimol left 36.30 ± 5.502 degrees, Muscimol right 45.96 ± 6.235 degrees, t4 = 1.161, P = 0.3101). Also, no interaction (F2,8 = 0.7364; P < 0.7364) or experimental condition effects (F2,6 = 0.08416; P = 0.9203) were found. Comparison of the amount of time that the whiskers contacted the tactile stimulus did not differ between conditions (Control: 0.245±16.74 secs; Saline: 0.213±9.81 secs; Muscimol: 0.201±12.44 secs; One Way ANOVA: F2,8 = 2.694; P = 0.1275).
M1 inactivation modulates anticipatory activity across the TCL
After M1 inactivation with muscimol, S1 neuronal firing modulations were widely affected. The proportion of units with increased responses in S1 rose in the anticipatory period (Control = 27.9% (36 units), Saline = 24.0% (29 units) and Muscimol = 46.2% (49 units) (Control vs Saline: Chi Square = 0.32, df = 1, P = 0.57; Control vs Muscimol: Chi Square = 7.68, df = 1, P = 0.0056), and less cells exhibited decreased activity in infragranular layers (Control = 26.88% (24 units), Saline = 17.72% (14 units) and Muscimol = 4.84% (3 units); Chi Square = 18.9, df = 2, P <0.0001). In addition, the magnitude of increased neuronal activity was larger (Control: 1.9±0.1 spikes/trial; Saline: 2.1±0.2 spikes/trial; Muscimol: 2.24±0.2 spikes/trial; Kruskal-Wallis statistic = 6.305; P = 0.0427, post hoc comparisons with Dunn’s test: Control vs Saline P > 0.05, n.s.; Control vs Muscimol P < 0.05). Moreover, the magnitude of neuronal activity reduction was lowered in the infragranular S1 layers (Control: 19.8±2.0 spikes/trial; Saline: 18.0±2.0 spikes/trial; Muscimol: 9.105±1.6 spikes/trial; Kruskal-Wallis statistic = 8.523; P = 0.0141, post hoc comparisons with Dunn’s test: Control vs Saline P > 0.05, n.s.; Control vs Muscimol P < 0.05).
M1 inactivation also led to a reduction in the magnitude of anticipatory activity both in the POM (Control: 2.4±0.2 spikes/trial; Saline: 2.2±0.2 spikes/trial; Muscimol: 1.7±0.1 spikes/trial; Kruskal-Wallis statistic = 13.57, P = 0.0011, post hoc comparisons with Dunn’s test: Control vs Saline P > 0.05, n.s.; Control vs Muscimol P < 0.01) and VPM (Control: 2.7±0.3 spikes/trial; Saline: 1.9±0.1 spikes/trial; Muscimol: 1.3±0.1 spikes/trial; Kruskal-Wallis statistic = 15.05, P < 0.001, post hoc comparisons with Dunn’s test: Control vs Saline P > 0.05, n.s.; Control vs Muscimol P < 0.01), as shown in . The effects of M1 inactivation in a POM unit in three consecutive sessions are shown in . This cell presented similar firing rates, waveforms, ISI and response profiles (increased anticipatory followed by decreased discriminatory activity) in all three sessions. During control and saline conditions the response profile shows a sharp peak of significant increased activity that begins in the anticipatory period and ends when the whiskers make contact with the discrimination bars. After M1 inactivation the peak of activity was not as sharp as in other two conditions; the neuron’s activity remained significantly high after the whiskers made contact with the bars and its period of significant reduced firing activity was longer than in the previous conditions.
To study if the two different compartments recorded in the VPM were differentially affected by M1 inactivation we further analyzed the magnitude of neural anticipatory activity in the “head” and “core” of the barreloids for the animals used in the inactivation experiments. A total of 77 neurons recorded from nine sessions were used for analysis of the VPM “head” of barreloids, while 391 neurons recorded in 33 sessions were used for the analysis of the VPM barreloids “core”. No significant differences were found in the anticipatory activity in the VPM “head” following M1 inactivation. In the core region of VPM, which sends thalamocortical projections to layer IV of S1 cortex, M1 inactivation lowered the magnitude of decreased neural anticipatory activity (Control: 2.8±0.5 spikes/trial; Saline: 1.7±0.1 spikes/trial; Muscimol: 1.0±0.2 spikes/trial; Kruskal Wallis statistic = 18.16, P = 0.0001, post hoc comparisons with Dunn’s test: control vs saline P > 0.05, n.s.; Control vs Muscimol P <0.0001). Also, a non-significant trend was found in the magnitude of increased neural anticipatory activity (Control: 1.8±0.3 spikes/trial; Saline: 2.1±0.2 spikes/trial; Muscimol: 1.3±0.2 spikes/trial; Kruskal Wallis statistic = 9.076, P = 0.0107, post hoc comparisons with Dunn’s test: Control vs Saline P > 0.05, n.s.; Control vs Muscimol P > 0.05, n.s.). Thus, M1 inactivation induced an overall increase in significant neuronal responses in the granular and infragranular layers of S1, before and after the whiskers contacted with the aperture’s edge. At the same time, the same manipulation produced a reduction in anticipatory and discriminatory activity in both the POM and the VPM.
To measure whether M1 inactivation, and the consequent changes in anticipatory S1 neuronal activity, affected the prediction of the animal’s tactile performance, a linear regression analysis was carried out between the onset of anticipatory firing in S1 units and the percentage of correct responses after M1 inactivation with muscimol. Although speed remained a good predictor of the performance in the task (F1,18 = 22.19; P = 0.0002, R2 = 0.55), indicating that no major motor deficits were present (consistent with previously unpublished observations), the onset of anticipatory units in S1 (F1,19 = 0.3414; P = 0.79, R2 = 0.075) (see , panel C3) was no longer predictive of the performance in the task. This result clearly indicates that blocking M1 activity affected spatiotemporal patterns of S1 anticipatory neural activity that predicted the animal’s tactile performance.
Encoding of tactile stimulus depends on anticipatory activity
Next, we asked whether single trial alterations in anticipatory activity onset timing in S1 neurons influenced the encoding of the tactile stimulus. To achieve this goal, we first analyzed firing rate changes in neural ensemble activity before the whiskers made contact with the tactile stimulus. Specifically, for each trial, we selected the first bin presenting an ensemble firing rate that was significantly different (at P ≤ 0.05) from baseline. These changes were termed Neural Events of Interest (NEIs, see Methods for details). Note that this analysis was restricted to S1 units and that, to match previous results from our laboratory, we exceptionally employed 50 ms bins only in this analysis.
A similar number of NEIs was found across different conditions (Control: 26.23% of trials; Saline: 31.34% of trials; Muscimol: 27.16% of trials; Control vs Saline: Chi-Square = 2.97, df = 1; P = 0.0849; Control vs Muscimol: Chi-Square = 1.5, df = 1; P = 0.2207), indicating that changes in neural activity occurred in a similar proportion of trials in all conditions. However, comparison of the distribution of anticipatory NEIs between the control condition and during M1 inactivation suggests that blocking M1 activity induced a major disruption in the normal timing pattern of anticipatory activity (). Specifically, the distribution of NEIs did not exhibit a clear peak in the interval of [-400;-200] ms before contact with the stimulus (see ). This suggests that normal M1 activity affects the precise timing of anticipatory activity in S1 neurons.
Trial-by-trial ensemble analysis of anticipatory neural activity
To test whether precise timing of anticipatory activity was related to tactile discrimination performance, we then compared the proportion of NEIs that were present before correct and incorrect trials in early [-500;-200ms] or late anticipatory periods [-200;0ms]. The probability of a correct trial after an NEI was 51.6% of trials in Control sessions and 45.1% of trials in Saline sessions (Chi Square = 0.7921, P = 0.1867). However, after M1 inactivation only 36% of the trials with neural anticipatory NEI were correct (Chi Square = 4.229, P = 0.0199)(). These results suggest that, in the absence of M1 modulation, the late onset of S1 anticipatory activity was associated with tactile discrimination deficits.
Because neurons with anticipatory activity often decreased their firing activity during contact with the tactile stimulus ( and ), we then tested if anticipatory NEIs in [-500; -200] or late anticipatory [-200;0] periods were associated with different ensemble firing rates during the tactile encoding period [0;300ms]. Comparison of the variation between the S1 ensemble firing rate before and after discrimination showed that early anticipatory NEIs were associated with larger decreases in firing rates during the tactile discrimination period in control (Control early: -0.05924±0.0059 spikes/trial; late: -0.03223±0.0093 spikes/trial; Mann-Whitney = 9651, P = 0.0368) and saline conditions (Saline early: -0.03774±0.0063 spikes/trial; late: -0.01483±0.0077 spikes/trial; Mann-Whitney = 7255, P = 0.0276), but not after M1 inactivation with muscimol (Muscimol early: -0.06672 ± 0.0059 spikes/trial; late: -0.05190 ± 0.0098 spikes/trial; Mann-Whitney = 6247, P = 0.4055; n.s.). This finding suggests that, in the absence of M1 modulation, the time onset of anticipatory activity was delayed, possibly affecting the encoding of the tactile stimulus by S1 neuronal ensembles.