Kinematics of ICMS-evoked whisking
The vibrissa representation of the motor cortex (vMCx) has been divided into two functional subregions based on movements evoked by intracortical microstimulation (ICMS; Haiss and Schwarz 2005
; Sanderson et al. 1984
). In awake rats, stimulation within the retraction-face (RF) subregion evokes vibrissal retractions and concurrent activation of other facial muscles. Stimulation within the rhythmic-whisking (RW) subregion evokes rhythmic protractions of the vibrissae that mimic natural whisking (Haiss and Schwarz 2005
). To investigate the mechanisms of cortical regulation of the whisking CPG in a mechanically stable platform, we developed an anesthetized preparation in which ICMS in the RW subregion evokes rhythmic whisking. A representative example of an ICMS-evoked whisking epoch in an anesthetized rat is shown in . The bottom trace
shows the EMG recorded from the intrinsic muscle of a single vibrissa, with the power spectral density (PSD) of the whisking epoch shown above. As we frequently observed during ICMS-evoked whisking, the rhythmic EMG activity outlasted the stimulation (see also , , , and ). The spectral features of the EMG (5–15 Hz) mimic those recorded from behaving animals (Berg and Kleinfeld 2003b
; Hattox et al. 2003
), indicating that ICMS in the anesthetized rat evokes rhythmic vibrissae movements that closely resemble exploratory whisking.
FIG. 1 Intracortical microstimulation (ICMS)–evoked whisking in the anesthetized rat. A: power spectral density (PSD, top) of the intrinsic EMG (bottom)—evoked by ICMS of vibrissa motor cortex (vMCx)—shows a peak at 9 Hz. ICMS parameters: (more ...)
FIG. 3 Whisking kinematics are accurately predicted by the intrinsic electromyogram (EMG). A, top traces: spectrograms of ICMS-evoked whisking in response to 3 stimulation intensities. Below the spectrograms are recordings of vibrissa position as a function (more ...)
FIG. 4 Decomposition of EMGs into constituent motor units reveals motoneuron activity during ICMS-evoked rhythmic whisking. A: representative example of EMG decomposition, where 5 motor units were isolated from the EMG. Rasters for each motor unit are shown (more ...)
FIG. 6 Activity of vibrissa motoneurons (vMNs) during ICMS-evoked whisking. A: intrinsic EMG (top) and extracellular recordings (bottom) from isolated vMNs show similar firing patterns as observed in motor unit recordings: firing single action potentials (unit (more ...)
FIG. 7 Serotonin receptor antagonist, metergoline, suppresses motor unit firing rates and whisking. A: ICMS-evoked vibrissal EMG recorded before (#1) and after (#2–#5) application of metergoline to vMNs. ICMS epochs (1-s duration) indicated by horizontal (more ...)
To quantify ICMS-evoked whisking amplitudes and velocities we used a CCD device to track the movements of individual vibrissae (see METHODS). In 513 whisks monitored by the CCD device whisk amplitudes ranged from 0.01 to 3.7 mm (approximately 0.04–14°). Larger whisks were generated during higher-frequency whisking epochs ().
The relationship between protraction and retraction velocities is shown in , where protraction velocities are plotted as a function of the subsequent retraction velocity. The color of each data point indicates the amplitude of the corresponding whisk. At low whisk amplitudes, most retractions were slower than the preceding protractions (see unity line ). As whisk amplitudes increased, retraction velocities exceeded protraction velocities, mimicking observations from voluntarily whisking rats (Bermejo et al. 1998
; Carvell and Simons 1990
). Whisk amplitudes and velocities were highly correlated (P <
0.0001), but these correlations were higher for retractions (r
= 0.97) compared with protractions (r
= 0.88; ).
Protraction set points often changed during ICMS-evoked whisking epochs, such that rhythmic whisking occurred on top of a sustained protraction (see and ). We never observed a net retraction of the vibrissa past the initial set point.
Stimulation of vibrissa motor cortex activates the whisking CPG
Epochs of ICMS-evoked whisking were consistently preceded by relatively long onset latencies. Further, whisking occurred at frequencies (5–15 Hz) that were significantly different from the stimulation frequencies (50–90 Hz). Both of these characteristics varied with the stimulation intensity. Onset latency decreased exponentially with increasing stimulation intensity (); on average, the shortest latency (evoked by the highest stimulation intensity) was 200 ± 100 ms. As a result, the duration of whisking was proportional to the ICMS intensity.
FIG. 2 ICMS parameters affect whisking kinematics. A: whisking onset latency decreases with stimulation intensity. Results for a single stimulation series (4 repetitions at each intensity) are shown. Inset: group data for 124 trials, normalized to the shortest (more ...)
Evoked whisking frequency increased with stimulation intensity (). In contrast to the effects of stimulation intensity, stimulation frequency had little effect on the evoked whisking frequency. This is evidenced by the large degree of overlap in the three traces in , representing trials with stimulation at 50, 70, and 90 Hz.
Together, the relatively long onset latencies, the lack of a correlation between stimulation and whisking frequencies, and the ability of whisking to outlast the stimulation suggest that ICMS of vMCx is activating a CPG to evoke rhythmic whisking. These findings also suggest that vMCx activity regulates the output from the whisking CPG.
Activity of vibrissae motor units
Whisking, whether voluntary or evoked, is ultimately produced by the activity of vibrissae motor units, which consist of a motoneuron and the muscle fibers it innervates. Motor unit action potentials (MUAPs) can be extracted from the EMG by decomposition to reveal the activity of motoneurons contributing to a particular movement (Merletti and Parker 2004
). Because motoneuron behavior reflects the drive they receive, analysis of their activity provides insights into the composition of the whisking CPG. If the EMG is also correlated with whisker movements, as suggested by Carvell et al. (1991)
, then the EMG can be used as a single metric to investigate both the drive from the whisking CPG and the conversion of this drive into movements by vibrissa motor units.
To ascertain that the EMG accurately reflects whisking kinematics we monitored ICMS-evoked movements of a single vibrissa with a CCD device, simultaneously with the corresponding vibrissal EMG from the intrinsic muscles (see METHODS). An example of such simultaneous recordings is shown in , where we plot both the output from the CCD and the EMG records. Note that the EMG envelope—computed by rectifying and smoothing the raw EMG signal—closely resembles the output from the CCD. Rectifying the EMG is justified because ICMS did not produce retractions beyond the set point of each whisk (see above).
To quantify the relationship between vibrissa position and EMG recordings, we plotted the peak vibrissa protraction and retraction amplitudes measured by the CCD device against those measured from the corresponding EMG envelope. Both protraction and retraction peak amplitudes, determined from the two normalized signals, were significantly correlated (). Similarly, peak vibrissa velocities and accelerations calculated from the two signals were also significantly correlated (). These results indicate that the vibrissal EMG provides an accurate representation of whisking kinematics and that the activity of motor units extracted from the EMG can be reliably correlated with whisking kinematics.
To investigate the correlation between the activity of vibrissa motor units and whisking kinematics, we decomposed vibrissal EMGs into their constituent motor units (see METHODS). A representative example of EMG decomposition analysis is shown in , where the MUAP waveforms for five motor units isolated from the EMG are shown to the right. Below the EMG are the MUAP rasters where each tick represents a discharge by that particular motor unit within the EMG. In this example, the epoch of rhythmic whisking was followed by a sustained protraction, and the latter was followed by several rhythmic protractions. During these events, a single motor unit was active in the EMG (motor unit A), suggesting that a single motor unit firing a single MUAP can produce measurable vibrissa movements. This conclusion is also supported by examining the spike-triggered average—triggered by unit A— of the sustained protraction phase of the EMG (′).
Movements during the period of rhythmic whisking showed a gradual increase in amplitude and, during this period, motor unit activity changed in two mutually reinforcing ways: some motor units increased the number of MUAPs fired per whisk (e.g., motor unit A), whereas additional, previously silent motor units were recruited. We quantified this behavior by plotting the mean (n = 70 whisks) peak protraction amplitudes against the number of active motor units (), revealing a direct correlation between these variables. To explore the relationship between firing rate and whisk amplitude we plot data from an experiment (371 whisks) in which EMG activity was dominated by a single motor unit (); there was a significant, positive correlation between these variables. We observed similar relationships between whisking amplitudes and both the number of active units and their firing rates in all whisking epochs examined (n = 94).
Motor units also showed a stereotypical recruitment order such that, within a whisk, units with low-amplitude MUAPs tended to be recruited earlier than units with larger MUAPs. A representative example of this recruitment order is shown in for five motor units from nine consecutive ICMS-evoked whisking epochs. Motor units are ranked according to the size of their MUAP with motor unit “A” having the smallest peak-to-peak amplitude and motor unit “E” having the largest. (left panel, “Order”) indicates which motor units were active during a whisk along with the order of their appearance. The diamonds above this panel represent the relative size of each motor unit. With only a single exception (whisk 6), motor units A, C, D, and E were recruited in order according to the size of their respective MUAPs. Motor unit B, however, was an exception to this recruitment order and was often recruited after larger motor units. summarizes the motor unit recruitment order for 73 whisks in which two to five motor units were active. Recruitment order obeyed the size principle in 73% of the whisks. This figure was highest (94%) when only two motor units participated. In the majority of the remaining trials (involving three to five motor units) no more than one of the units was recruited out of order ().
FIG. 5 Recruitment order vibrissa motor units. A: behavior of 5 motor units (abscissa) isolated from EMG recorded during 24 whisks (ordinate). Diamonds at the top represent the relative peak-to-peak amplitudes of the motor units. Left panel (“Order”) (more ...)
Motor units with larger-amplitude MUAPs were associated with larger-amplitude whisks. This relationship is seen in (right panel, “Amplitude”) as an increase in whisk amplitude as the larger motor units (D and E) were recruited. Group data for 73 whisks () show the same relationship: as larger motor units were recruited, whisk amplitude increased. These results indicate that whisking amplitude is regulated through both the recruitment of motor units and the modulation of the motor unit firing frequencies within each whisk.
Activity of vibrissa motoneurons
We observed similar firing patterns in extracellular recordings from vibrissa motoneurons (vMNs; n = 7) during ICMS-evoked whisking. To our knowledge, these data constitute the first recordings obtained from vMNs in vivo. Representative examples of vMN activity during ICMS-evoked whisking are shown in . As we observed in motor units isolated from the EMG (), during each whisk vMNs fired either a single action potential or bursts of action potentials (). The interspike or interburst intervals recorded from vMNs correlated significantly with interwhisk intervals (), confirming that the firing rates of vMNs determine the whisking frequency. We found similar, high correlations (r > 0.99) in all seven vMNs. The firing rate of vMNs within each whisk was significantly correlated with protraction amplitudes in six of seven vMNs (C; r = 0.36–0.45, P < 0.05). This finding is consistent with the conclusion (see above) that whisking amplitude is modulated by the firing frequency of motor units.
Frequency of ICMS-evoked whisking depends on serotonin
Previous work in our laboratory demonstrated that serotonin (5HT), acting through 5HT2
receptors, is necessary and sufficient for rhythmic whisking and that, in vitro, 5HT evokes rhythmic firing in vMNs at whisking frequencies (Hattox et al. 2003
). These findings suggest that serotonergic drive from a component of the whisking CPG could establish the whisking frequency through its actions on vMNs. To test whether ICMS-evoked whisking operates through a similar mechanism, we infused the 5HT receptor antagonist metergoline onto vMNs in the lateral facial nucleus (LVII) during repeated epochs of ICMS-evoked whisking.
In eight of nine trials from five rats, metergoline (100 μM to 1 mM) caused a reversible suppression or a complete inhibition of ICMS-evoked whisking (). The effects of metergoline were manifest as a sharp reduction in spectral power of whisking frequencies (). At higher drug concentrations (500 μM or 1 mM) suppression averaged 99 ± 1% (n = 3, range 98–100%, P < 0.012). At lower concentrations (100–200 μM, n = 5) the suppression averaged 77 ± 7% (range 66–94%, P < 0.011).
Before complete suppression, metergoline occasionally caused a reduction in the whisking frequency (, immediately after drug infusion). This trend was particularly evident at the lower antagonist concentrations. Similarly, recovery to control whisking frequencies was preceded by a gradual increase in evoked whisking frequencies ().
To determine the effects of metergoline on vMN firing, we isolated motor units from the EMG, as described above. Analysis of the two motor units that could be reliably isolated from the EMG in reveals that metergoline caused a gradual and reversible suppression of their firing rates (). We observed similar effects in all eight trials in which metergoline had an effect on evoked whisking.
Control infusions of buffered saline (n = 4) or placement of the probe without injection (n = 1) had no effect on evoked whisking.