Whisking motor output in awake and freely moving rat is investigated with optogenetic excitation/inhibition of the vibrissae motor cortex (vMCx) layer V. The goal of the study is to establish the direct causal relationship between the cortical activity and the whisking output using optical stimulation, excitatory or inhibitory, with different frequencies. Progression and reduction of the whisking frequency was obtained; however, the whisking frequency did not necessarily followed the entrainment stimulus. Based on our observations, the excitation of the vMCx doubled and inhibition reduced the whisking frequency to half, compared to control, at all stimulus frequencies. This result is an empirical evidence that the cortex exerted control through a central pattern generator structure since complete inhibition was not obtained and the frequency of the response was different from that of the stimulus. We suggest that the use of the optogenetic approach, which enabled us to perform the bidirectional modulation and direct readout from vMCx, has brought valid evidence for the causal connection between cortical activity and whisking motor output.
© 2012 OSA
The study of motor control output in general and of specific systems like whisking in particular, has been a permanent case for investigation in neurophysiology. Fast rhythmic vibrissae movements are used by rodents to explore the environment . The so called active exploratory whisking is a combination of protraction followed by retraction movement within an antero-posterior horizontal plane, in air and over objects at frequencies of 4 to 15Hz recorded by electrodes [2, 3, 4]. Rhythmic movements similar to exploratory whisking and small vibrissa retractions can be triggered by electric low intensity stimulation of the medial areas of the motor cortex [5, 6, 8]. This area is considered as the vibrissa motor cortex (vMCx) which affects whisking via producing reciprocal projections with the barrel cortex [9, 10, 11] and premotoneuron networks that forms a central pattern generator (CPG) distributed throughout the mid- and hindbrain [13, 14, 15]. In addition, vMCx projects directly to facial motorneurons providing a route to control vibrissae movements , the coordinator [6, 7, 17, 18], and also a part of the CPG [19, 20, 21].
More profound understanding in the field could be generated using new experimental paradigms in conjunction with recent methodologies. Such a recent method is optogenetics [22, 23, 24] which has opened the possibility for selective targeting of neuronal subpopulation to stimulate or inhibit neural activities by exposing the cells to appropriate wavelengths. For this purpose, genetically targeted neurons express opsins, such as channelrhodopsin 2 (ChR2) and halorhodopsin (eNpHR) fused in frame with enhanced yellow fluorescent protein (EYFP), under the control of a neuron type specific promoter, e.g., Ca2+/calmodulin dependent proteinkinase II α (CaMKIIα) which mostly targets the excitatory pyramidal neurons. Expression of ChR2 enables the neuron to be depolarized by blue light exposure at 473nm and eNpHR to hyperpolarize by yellow light with peak sensitivity at 593nm. Using the optogenetic toolbox, by co-expression of both opsins, one can interrogate the vMCx using this novel method which is different from the electrical intracortical microstimulation (ICMS) which known to be the classical approach in neurophysiology. Taking advantage of an integrated dual laser single optical fiber device [26, 25] that is developed in our lab and can be used for both excitation and detection of fluorescence simultaneously, we developed an optogenetic paradigm for the study of the modulation of whisking motor output through the vMCx in the awake, unconditioned freely moving rat. We hypothesize that after co-expression of ChR2 and eNpHR under control of CaMKIIα promoter in the pyramidal neurons of the layer V in the vMCx of the rat , we should be able to manipulate the whisking behavior and retrieve characteristic parameters of the process.
2. Materials and Methods
Experiments were carried out in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Experimental protocols were approved by the IACUC of the University of Wisconsin-Milwaukee.
This study reports the experiments performed on six Sprague Dawley female rats, all being surgically instrumented for implantation of optical neural interface. Of these, four were stereotactically injected with lentivirus mixture for co-expression of ChR2 and eNpHR and two with phosphate buffered saline (PBS) as controls. The animals have been only gentled, not trained or conditioned for any task. Experiments started after 30 days allowing the expression level for the ChR2 and eNpHR to attain the acceptable values for experiments.
2.1. Lentivirus production and titering:
The expression plasmids pLenti-CaMKIIα-ChR2(H134R)-EYFP-WPRE (courtesy of Dr. Karl Deisseroth, Stanford University) and pLenti-CaMKIIα-eNpHR-EYFP-WPRE (Addgene, Cambridge, MA) were each co-transfected with the plasmids pCMVR8.74 and pCMV-VSV-G (Addgene, Cambridge, MA) via calciumphosphate (Clontech, Mountain View, CA) of HEK 293FT cells (Invitrogen, Grand Island, NY) high titer lentivirus (> 106 i.u./ml) was then packaged. Then, 24h post-transfection, HEK 293FT cells were switched to serum-free medium Optimem (Invitrogen, Grand Island, NY); the supernatant was collected 24h later and concentrated 1000x by ultrafiltration (Pall Co, Ann Arbor, MI). The resulting lentiviral concentrate titering was performed in HEK293 cells that were grown in 24-well plates and inoculated with 5-fold serial dilutions. After 4 days, cultures were resuspended in PBS and counted for EYFP expression in hemacytometer (Hausser Sci, Horsham, PA). The titer of the virus was determined by: [(% of infected cells) × (total number of cells in well) × (dilution factor)]/ (volume of inoculum added to cells) = infectious units (i.u.)/ml. The titer of concentrated lentivirus for in-vivo injection was 109 i.u. /ml. An equal titer mixture 1 : 1 of concentrated lentiviruses was used for the co-expression of ChR2-EYFP and eNpHR-EYFP.
2.2. Implantation of optical neural interface and stereotaxic injection into the brain:
Female rats 250g – 350g were the subjects of these experiments. Concentrated lentivirus solution was stereotactically injected into the rat vibrissae motor cortex (anterior-posterior = 1.5mm from bregma; lateral = 1.5mm; ventral = 1.5mm) corresponding to layer V.
All surgeries were performed under aseptic conditions. The animals were anesthetized with isoflurane 1.5 – 2% isoflurane in 95% oxygen. The head was shaved, cleaned with 70% ethanol and betadine and then placed in a digital lab standard stereotaxic apparatus (Stoelting Co, Wood Dale, IL). Ophthalmic ointment was applied to prevent eye drying. A midline scalp incision was made and then a small craniotomy was performed using a drill mounted on the stereotaxic apparatus (Foredom Electric Co, Bethel, CT). A cannula guide C313G (PlasticsOne, Roanoke, VA) was then inserted through the craniotomy to a depth of 1mm (800μm across frontal bone and 200μm into the brain). Three plastic skull screws 00 – 96x3/32 (PlasticsOne, Roanoke, VA) were placed in the skull surrounding the cannula guide pedestal, and cranioplastic cement (Pearson Dental, Sylmar, CA) was used to anchor the cannula guide system to the skull screws. The virus was delivered using a 10μl syringe and a thin 35 gauge blunt metal needle passing through the cannula guide; the injection volume and flow rate (1μl at 0.1μl/min) was controlled with an injection pump from (World Precision Instruments, Sarasota, FL). After injection the needle was left in place for 10 additional minutes for diffusion of the lentivirus, and then slowly withdrawn and a dummy cannula C313G (PlasticsOne, Roanoke, VA) was inserted to keep the cannula guide open. The free edge of the scalp was brought to the base of the cranioplastic cement using sutures 3 – 0 silk (Ethicon, Somerville, NJ) and tissue adhesive Vetbond (3M, St. Paul, MN). The animal was kept on a heating pad until it recovered from anesthesia. Lidocaine 0.5% gel was applied on the scalp following the surgical procedure to minimize discomfort. The cannula guide also functions as the optical fiber guide. In order to be positioned at the desired depth in the brain, the tip of the optical fiber has a glued stopper so that when inserted into the guide it extends exactly 1.5mm from the base of the guide and 0.5mm out of the guide. Before gluing the stopper, the fiber is passed through a hole in a plastic dust cap, which glides freely on the fiber and screwing it to the pedestal of the fiber guide, secures the optical interface to the skull of the rat stabilizing the tip of the optical fiber at 1.5mm depth measured from the surface of the skull. The tip of the fiber is positioned at the 700μm depth within the motor cortex which corresponds to layer V.
2.3. Stimulation with light of pyramidal neurons in layer V of vibrissa motor cortex:
Stimulation of ChR2+/eNpHR+ neurons was accomplished using a multimode optical fiber (NA =0.22) with a 200μm silica core FG200UGG (Thorlabs, Newton, NJ) coupled to a 473nm blue laser LRS-0473 Series of Diode-Pumped Solid-State (DPSS) and a 593nm yellow laser LRS-0594 DPSS (Laserglow Technologies, Toronto, ON, Canada). The measured light intensity emanating from the tip of the fiber was 160mW / mm2. The distal end of the fiber was polished and the jacket was stripped; the fiber was inserted into the fiber guide and advanced until flush with the fiber guide end. The animal was then allowed to habituate to the setup.
2.4. Whisker movement recording, frequency and mean angular deflection measurement:
Animals were awake and freely moving. Whiskers contralateral to the fiber guide implantation were video recorded from above the head while the rat is placed inside the arena. The arena in this experiment is a plastic uncovered rat cage illuminated from the bottom through a thin white paper sheet which helps to gain diffuse background lighting. This setup allows to see clearly the vibrissae particularly in contrast inverted images produced by ImageJ (NIH, Bethesda, MA). We used a digital camera, Power Shot SX230 HS (Canon, Lake Success, NY), with 30 frames per second temporal resolution and spatial resolution of 100 – 200μm, to monitor the motion of the vibrissae along the anterior-posterior axis only. ImageJ was used to track movement in the video. For this purpose, one linear vector of pixels in the image in the vicinity of the chosen whisker and parallel to its direction was selected in the captured frames and the value of the associated pixels were extracted for all frames along the time-laps. This vector of pixels function as twitching detectors. Whenever the whisker moves the intensity of these pixels drops which indicates a whisker twitching event. This algorithm can be applied for the case when animal does not move in the arena during the period we record the signal. In case animal moves or the head of the animal moves that data should be discarded. Also using ImageJ angle tool, we measured whiskers mean deflection angle for different frequencies of light stimulus for each of the two wavelengths: 473nm and 593nm. Experiments were initiated once the animal stopped exploring the experimental arena and did not move the head. During an experimental sweep, 20s of pre-stimulus data, 20s of intra-stimulus data (20s pulse of blue or yellow light) and 20s of post-stimulus data were recorded. We noticed that 20s period is relatively good timing over which we can frequently find the animal staying with almost no movement in a part of arena. Therefore, 20s period was reasonable to record the data when animal was free to move but not moving.
2.5. Slice preparation and epi-fluorescence imaging:
For preparation of brain slices, one rat was sacrificed 9 days after viral injection. Acute coronal brain slices (100μm) were prepared in ice-cold cutting solution (64 mM NaCl, 25 mM NaHCO3, 10 mM Glucose, 120 mM Sucrose, 2.5 mM KCl, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 7 mM MgCl2) using a vibratome (Lancer Series 1000; Vibratome Company, St. Louis, MO). The slices were then fixed for one hour in 4% paraformaldehyde, washed with PBS and mounted on microscope slides. Images of EYFP fluorescent neurons were recorded with a cooled CCD camera Exi Aqua (Qimaging, Surrey, BC, Canada) on an upright BX51WI Olympus microscope (Olympus America Inc., Center Valley, PA) and a EYFP filter set: excitation filter BP490-500HQ, dichroic DM505HQ (Olympus America Inc., Center Valley, PA).
2.6. In-vivo measurement of EYFP fluorescence as a measure of the level of opsin expression:
Because the standard histological methods require sacrificing the animal, thus terminating experiments, it would be desirable to determine expression levels in-vivo while experiments are ongoing.
This would allow monitoring expression levels over the weeks following the injections and determining the ideal time point for starting the work with the respective animal subject. The fluorescence detection device achieves this goal, which for optimal versatility and minimal tissue damage employing a single fiber optic cable both to deliver source light as well as to detect the EYFP emitted fluorescent light. In this design, the same fiber can be used to deliver light for neural modulation (when used for optogenetic experiments) as well as to deliver light for fluorescence excitation (when used for in-vivo fluorescence measurements). The fiber delivers excitation light pulses to the region of interest, and guides a sample of the corresponding emission signal back to a sensitive low-noise detector.
Parallel to this first photodetector, a second detector is employed to eliminate the effect of input light fluctuations, imperfection of the optical filters, self-fluorescence at the tip of the fiber, and random back-reflection of the excitation or stimulation light within tissue. A set of optical fiber splitters are used to combine the input light that is generated by numerous light sources and to distribute the emission signal in the system [26, 25]. A combination of patterned pulses and a software based time-lens filter is used to further improve the signal to noise ratio. Compared to traditional dichroic based setups, the new system is smaller in size and mobile, robust, and requires minimal alignment. The device has a FC/PC connector which is used to attach an optical fiber that we insert into the vMVx of the animal under test. Fluorescence measurements were performed in-vivo after calibration of the device. To measure the EYFP expression level we used 473nm light for excitation while emission optical filter with center wavelength 545nm in front of the sensitive photodetector.
3.1. Co-expression of ChR2 and eNpHR by dual lentiviral infection in vivo:
Dual lentiviral infection of the pyramidal neurons in layer V of the right vMCx in rat was achieved by stereotaxic injection of 1μL of equal titer lentiviral mixture of p-Lenti-CaMKIIα-ChR2-EYFP and p-Lenti-CaMKIIα-eNpHR-EYFP. The control rat was injected with phosphate buffered saline (PBS). The preference for expression of ChR2 and eNpHR from separate transgenes and not taking advantage of a stoichiometric and co-localized gene-fusion strategy is justified by a more natural and even distribution of the constructs which are already a form of fused structures, carrying EYFP . A morphologic control was necessary in order to make sure we obtained the expression in-vivo at the desired stereotaxic coordinates. After nine days, one lentiviral injected rat was sacrificed, the brain was extracted and fresh coronal slices 100μm were prepared from the region of interest and the level of expression was checked by fluorescence microscopy (Figure 1). Once the success of gene delivery and protein expression was confirmed in the test rat, in our next step, we used our single optical fiber fluorescence probe to evaluate the level of EYFP expression in-vivo 30 days after injection (Figure 2). Details of fluorescence detection is explain in reference .
3.2. Continuous versus chirped pulse stimulation:
Initial test of excitation or inhibition of neurons in layer V of vMCx was done either with continuous stimulus for 20s or 20 chirped pulses (linear chirp pulse where frequency is swept from 0Hz to 100Hz over period of 1s for each pulse 1s). Blue light 473nm, both continuous and chirped stimulus, enhanced the whisking activity significantly compared to pre and post-stimulus (events/s: intra-stimulus continuous 1.6 ± 0.19; pre-stimulus 0.74 ± 0.02; post-stimulus 0.79 ± 0.09, two-tailed t tests p < 0.02 (Figure 3a) and intra-stimulus chirped 1.92 ± 0.23 ; pres-stimulus 0.7 ± 0.07; post-stimulus 0.87 ± 0.05, two-tailed t tests p < 0.02 (Figure 3b)) and also compared to control (two-tailed t tests: p < 0.05 for continuous and p < 0.02 for chirped stimulus, respectively (Figure 3ab). The chirped stimulus seemed to be slightly more efficient compared to continuous stimulus (p = 0.18). Inhibition of whisking activity with yellow light 593nm was achieved with both continuous and chirped stimulus and the change is significant compared to pre and post-stimulus (events/s: intra-stimulus continuous 0.3 ± 0.08; pre-stimulus 0.8 ± 0.07; post-stimulus 0.7 ± 0.08, two-tailed t tests p < 0.02 (Figure 3c) and intra-stimulus chirped 0.30 ± 0.08 ; pre-stimulus 0.76 ± 0.07; post-stimulus 0.7 ± 0.08, two-tailed t tests p < 0.02 (Figure 3d)) and also compared to control (two-tailed t tests: p < 0.05 for continuous and chirped stimulus, respectively (Figure 3c, d)). Similarly, inhibitory chirped pulses are slightly more efficient compared to continuous yellow light exposure (p = 0.38).
These results confirmed that modulation of the whisking behavior in the rat could be done reliably for both excitation and inhibition and that the pulsed stimulus had an apparent greater efficiency. This was also the functional test to prove that the expression of ChR2 and eNpHR was obtained in the pyramidal neurons of layer V in the vMCx.
3.3. Various constant frequency pulsed stimulation:
We decided to test different constant frequency stimuli of: 5, 10, 15, 20, 25, 50, 70 and 90Hz for both excitation (473nm) and inhibition (593nm) light exposure, to evaluate the degree of entrainment of whisking in comparison to the stimulus frequency. The stimulus trains of 20s for both wavelengths were formed by square pulses of 10ms for the frequencies from 5Hz – 25Hz, 5ms for frequencies of 50 and 70Hz and 2ms for 90Hz. The results show that the excitation did not entrain the whisking activity to the stimulus frequency but attained a maximum of approximately 3Hz. Also, we could not achieve the complete inhibition. Based on our observation, under yellow light stimulation the rate of twitching reduced but remained persistently at a value around 0.5Hz. The values of whisking activity in the control rat were between 1Hz to 2Hz under exposure with both wavelengths (Figure 4). The 5Hz light pulses produce neither stimulation at 473nm nor inhibition at 593nm compared to pre-stimulus or post-stimulus and also the control rat data (two-tailed t tests: pre (473nm) p = 0.20, post (473nm) p = 0.10, control (473nm) p = 0.7 and pre (593nm) p = 0.57, post (593nm) p = 0.82, control (593nm) p = 0.37, (Figure 4a, b)). Starting with the frequency of 10Hz and up to 90Hz the stimulus trains over the period of 20s triggered significant stimulation of whisking activity at 473nm or inhibition at 593nm (two-tailed t tests p < 0.05 for all comparisons with: pre-stimulus, post-stimulus and control data (Figure 4c to 4p)). This clear non-entraining response of whisking activity when exciting the vMCx with 473nm light centered in the band of 1Hz – 4Hz and the reduction without cancelation when inhibited with 593nm light in the frequency band of 0Hz – 1Hz led us to analyze the spectral properties of the whisking signal.
We performed a moving window analysis with a Short Time Fourier Transform (STFT) of the whisking signal and focused on the bands centered on the 0.5Hz and 3.0Hz. The data shows that stimulation with 473nm (excitation) increases the power of the signal in both bands 0Hz – 1Hz and 2Hz – 4Hz above a baseline at all stimulation frequencies which was more noticeable in the frequency range of 2Hz – 4Hz compared to 0Hz – 1Hz (two-tailed t tests p < 0.05 (Figure 5a)). The stimulation with 593nm (inhibition) decreases the power of the signal in the frequency range of 2Hz – 4Hz under a baseline at all stimulation frequencies. The scenario was the same for the 0Hz – 1Hz band with the exception for the stimulation frequencies of 15Hz and 20Hz. The power in the band 0Hz – 1Hz compared to the band 2Hz – 4Hz increased significantly at all frequencies (two-tailed t tests p < 0.05 (Figure 5b)). The parameters which we retrieved with the optogentic paradigm are important since they characterize the vibrissa motor cortex and its relation to the hypothetical CPG.
3.4. Amplitude of whisker deflection at various frequencies of pulsed optical stimulation:
To develop a more complete picture of the vMCx motor output, we also examined the amplitude of the whisker movement. The video recordings were analyzed frame-by-frame and the maximum angular deflection of the whiskers were measured using the angle tool of ImageJ software. Based on our observations, the optical stimulation of vMCx by 473nm laser in the frequency range of 5Hz to 20Hz caused relatively linear increase in the deflection angle of the contralateral whiskers (paired one-tailed t tests *p < 0.05 (Figure 6a)). The maximum was attained at 25Hz followed by a nonlinear decrease from 25Hz to 90Hz which still remains significantly larger than the pre-stimulus control values (paired one-tailed t tests *p < 0.05 (Figure 6a)). When stimulation was performed with the 593nm wavelength, the angular deflection of the whiskers reduced considerably compared to the pre-stimulus control values at all the frequencies (paired one-tailed t tests *p < 0.05 (Figure 6b)). We noticed a trend of almost linear decrease from 5Hz to 25Hz in the angular amplitude of whisker movements with plateau at 50Hz and a slight raise at 70Hz and 90Hz. The angular amplitude of the whisker movement showed stronger dependence on the frequency of entrainment compared to the frequency of the whisker movement. Interestingly, for the domain of natural frequencies of whisking behavior (5Hz – 20Hz) we noticed a relatively linear dependence of the amplitude of whisking, for both excitation at 473nm and inhibition at 593nm, which points toward the conclusion that vMCx, in this frequency range, controls such aspect of the whisker movement in a more precise manner.
Whisking behavior in rat is one of the most investigated processes that covers from the anatomical substrate to cortical activities. Understanding how the motor cortex imposes control over a simply patterned motor activity, such as whisker twitching, helps us to develop models for more complex movements like limb activities. The voluntary control of a rat on the whisking behavior can be correctly understood in awake, unconditioned, freely moving animal which is instrumented with an appropriate neural interface that allows researchers to modulate the activity of pyramidal neurons in layer V of the vMCx. While the classical ICMS assures precise control of the stimulus delivery, it excites all the neuronal populations in the vicinity of the electrode with no specificity. Although most of the knowledge we presently have in neurophysiology is based on the electric stimulation, with the advent of optogenetic tools we can characterize and investigate the motor cortex using light to specifically target the neuronal population of interest.
As we hypothesized, by optogenetic targeting of pyramidal neurons in layer V of the vMCx in a rat and co-expression of ChR2 and eNpHR, we can study the whisking behavior and retrieve parameters which characterize the dynamics of the motor cortex and the putative CPG through which this activity is controlled. In our attempt to drive the vMCx with blue laser pulses of different frequencies, changing from 5Hz to 90Hz, we obtained a maximum vibrissae response happening at ∼ 3Hz which it is quite different from the stimulation frequencies. This is a piece of evidence that the vibrissa motor cortex acts by modulating a CPG. Nonetheless, the CPG in cause might be the vMCx itself. This is possible since in our paradigm by stimulating only the pyramidal neurons of layer V, and not the inhibitory interneurons as it happens in ICMS, we triggered neocortical synchronized oscillations induced by thalamic disinhibition [29, 30, 31, 32, 36]. The resultant frequency is 3Hz[29, 30] which is apparently generated in the vMCx itself. The second piece of evidence is the fact that the stimulation of the motor cortex by 593nm light stimulus did not completely suppress the whisking activities which continued at a frequency < 1Hz (∼ 0.5Hz).
We obtained the maximum frequency of ∼ 3Hz for vibrissae at any stimulation frequency which was also using the ICMS  or single neuron stimulation in vMCx layer V [6, 7, 33]. The whisking frequency of < 1Hz (∼ 0.5Hz) was observed when the vibrissa motor cortex was inhibited by 593nm light pulses which functionally mimics the ablation of the motor cortex where the whisking frequency drops under 2Hz[34, 35].
Here we presented experimental results where the whisking motor output was optically modulated through the vMCx and we presented some evidences that this activity is controlled by a CPG . We used optogenetic toolbox to extract some fundamental parameters of whisking behavior in awake, unconditioned, freely moving rats.
The author would like to thank the University of Wisconsin-Milwaukee Research Growth Initiative (RGI) program for providing the financial support for this research under grant numbers 101X172, and 101X213.
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