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. 2016 Jun 29;36(26):6988-7001.
doi: 10.1523/JNEUROSCI.4190-15.2016.

The Motor Cortex Is Involved in the Generation of Classically Conditioned Eyelid Responses in Behaving Rabbits

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The Motor Cortex Is Involved in the Generation of Classically Conditioned Eyelid Responses in Behaving Rabbits

Claudia Ammann et al. J Neurosci. .
Free PMC article

Abstract

Classical blink conditioning is a well known model for studying neural generation of acquired motor responses. The acquisition of this type of associative learning has been related to many cortical, subcortical, and cerebellar structures. However, until now, no one has studied the motor cortex (MC) and its possible role in classical eyeblink conditioning. We recorded in rabbits the activity of MC neurons during blink conditioning using a delay paradigm. Neurons were identified by their antidromic activation from facial nucleus (FN) or red nucleus (RN). For conditioning, we used a tone as a conditioned stimulus (CS) followed by an air puff as an unconditioned stimulus (US) that coterminated with it. Conditioned responses (CRs) were determined from the electromyographic activity of the orbicularis oculi muscle and/or from eyelid position recorded with the search coil technique. Type A neurons increased their discharge rates across conditioning sessions and reached peak firing during the CS-US interval, while type B cells presented a second peak during US presentation. Both of them project to the FN. Type C cells increased their firing across the CS-US interval, reaching peak values at the time of US presentation, and were activated from the RN. These three types of neurons fired well in advance of the beginning of CRs and changed with them. Reversible inactivation of the MC during conditioning evoked a decrease in learning curves and in the amplitude of CRs, while train stimulation of the MC simulated the profile and kinematics of conditioned blinks. In conclusion, MC neurons are involved in the acquisition and expression of CRs.

Significance statement: Classical blink conditioning is a popular experimental model for studying neural mechanisms underlying the acquisition of motor skills. The acquisition of this type of associative learning has been related to many cortical, subcortical, and cerebellar structures. However, until now, no one has studied the motor cortex (MC) and its possible role in classical eyeblink conditioning. Here, we report that the firing activities of MC neurons, recorded in behaving rabbits, are related to and preceded the initiation of conditioned blinks. MC neurons were identified as projecting to the red or facial nuclei and encoded the kinematics of conditioned eyelid responses. The timed stimulation of recording sites simulated the profile of conditioned blinks. MC neurons play a role in the acquisition and expression of these acquired motor responses.

Keywords: associative learning; delay conditioning; motor cortex; rabbits; unitary recordings.

Figures

Figure 1.
Figure 1.
Experimental design and identification of recorded MC neurons. A, Diagram representing the experimental design. Rabbits were chronically implanted with EMG recording electrodes in the left orbicularis oculi muscle (O.O. EMG). In some animals, eyelid movements were recorded with the magnetic search coil technique. MC neurons contralateral (cMC) to the left eye were recorded (Rec.) with glass micropipettes inserted into the MC area. For proper identification, MC neurons were activated antidromically from the RN or the FN. B, Photomicrographs of MC coronal sections illustrating cannula implantation (top picture and arrow) and electrolytic marks (3 bottom pictures and arrows) made with stimulating metal microelectrodes implanted in selected recording sites. Calibration bars, 1 mm. C, Diagrams illustrating the recording sites following the atlas of Girgis and Shih-Chang (1981). D, Representative examples of the antidromic activation and collision tests of MC neurons from the FN (1) or the RN (2) at threshold-straddling intensities. Arrows indicate stimulus artifacts. E, From top to bottom are illustrated the EMG activity evoked in the O.O. muscle by double (2 ms interval) pulses applied to the cMC (1) and single pulses presented to the contralateral RN (cRN; 2) and the ipsilateral FN (iFN; 3). F, Spike-triggered extracellular activity recorded in the O.O. muscle (2). The triggering action potential corresponded to an identified cMC neuron (1). Average was repeated 1500 times.
Figure 2.
Figure 2.
Confocal photomicrographs of BDA-labeled and ChAT-expressing cells. A, Diagram of BDA tracer injection in the MC. B, BDA-labeled cells located in the MC near the injection site (arrow). Scale bar, 30 μm. The photomicrograph is a 2D projection of 24 consecutive focal planes located 1 μm apart. C, FN Mns expressing ChAT. Scale bar, 100 μm. D–F, ChAT-expressing FN Mns (magenta) and fibers labeled with BDA (green). Arrows indicate axons anterogradely labeled with BDA. Scale bar: (in F) D–F, 50 μm. Insets in DF are enlarged (30%) views of labeled FN Mns and MC projecting axons. Photomicrographs are 2D projections of 29 consecutive focal planes located 1 μm apart. G–I, Same ChAT-expressing cells indicated in D–F with arrowheads. Note the axons anterogradely labeled with BDA near or closely apposed to ChAT-expressing FN Mns. Scale bar: (in I) G–I, 25 μm. Photomicrographs are 2D projections of 22 consecutive focal planes located 0.5 μm apart.
Figure 3.
Figure 3.
Firing activities of MC neurons during classical blink conditioning using a delay conditioning paradigm. A, For conditioning, animals were presented with a tone (600 Hz, 85 dB, 350 ms) as CS and with an air puff (3 kg/cm2, 100 ms) presented to the left cornea as US. The CS coterminated with the US. CRs were recorded with the help of chronically implanted orbicularis oculi (O.O.) EMG electrodes. Contralateral MC neurons (cMC) were recorded (Rec.) across conditioning sessions with glass microelectrodes. B, Evolution of the percentage of CRs across conditioning sessions. Note that animals (n = 10) reached the selected criterion by the seventh or eighth conditioning sessions. MC neurons were recorded during the acquisition process and for >10 sessions after the selected criterion was reached. C, Typical responses of MC neurons (classified as types B and C) to air puff (20 ms, 3 kg/cm2) presentations (left) and during performance of a spontaneous blink. Note the different latencies and time scales. D, Representative example of the firing activity of an MC neuron recorded during a session from a well trained animal. From top to bottom are represented the conditioning paradigm (CS and US presentations), the firing activity of the MC neuron and the EMG activity of the O.O. muscle for a single trial, the raster plot of >60 successive trials, the averaged firing rate, and the rectified EMG activity of the contralateral O.O. muscle. Note that the activation of the MC neuron substantially (>70 ms) preceded the beginning of the eyelid CR.
Figure 4.
Figure 4.
Different types of MC neurons activated during classical blink conditioning with a delay paradigm. A, B, Firing properties of type A neurons. A, From top to bottom are illustrated the conditioning paradigm, the raw activity of a representative type A neuron, the event channel and firing rate of the selected neuron, and the raw EMG activity of the orbicularis oculi (O.O.) muscle during a single CS–US presentation. B, Conditioning paradigm, raster plot of all spikes collected from the same MC neuron during 15 successive trials, and the averaged firing rate. Type A MC neurons were characterized by an increased firing rate before the CR and a noticeable decrease of their firing rates during US presentation. C, D, Same as in A and B for a representative type B MC neuron recorded for 26 trials. Type B neurons were characterized by an initial firing peak preceding the CR and by a second increase of firing rate during US presentation. E, F, Same as in A and B for a representative type C MC neuron recorded for 53 trials. Type C neurons were characterized by a continuous increase in their firing rates preceding the CR and by reaching the maximum peak during US presentation. Time calibration in E is also for A and C. Time calibration in F is also for B and D.
Figure 5.
Figure 5.
Averaged firing rates of representative type A–C MC neurons recorded from well conditioned animals. All recordings were performed after the eighth conditioning session. The averaged firing rates are represented as mean values ± SEM (bin size, 20 ms). A, From top to bottom are illustrated the conditioning paradigm, the averaged firing rate of selected (n = 11) type A neurons, and the average of the rectified EMG responses of the orbicularis oculi (O.O.) muscle. Note that their peak firing rates occurred before US presentation and that they did not respond during the unconditioned response. B, Averaged firing rates and rectified EMG responses collected from n = 15 type B neurons. These MC neurons were characterized by the presence of an initial peak in their firing rates before the CR and then by a second increase of their firing rates during the US. C, Same than as in A for selected (n = 17) type C neurons. These MC neurons presented a continuous increase in their firing rates beginning well before (>90 ms) the start of CRs and reaching the maximum during US presentation—i.e., during the generation of the unconditioned eyelid response.
Figure 6.
Figure 6.
Relationships between changes in firing rates for type A–C neurons and the increase in CRs across conditioning sessions. Analyses were performed for data collected from the third to the seventh conditioning sessions. A, Profiles of firing rates collected from representative type A neurons (inset) across the acquisition process corresponding to the CS–US interval, as indicated in blue. B, Profiles of CRs collected during the conditioning sessions corresponding to data illustrated in A. For A and B, the increase in color intensity indicates the recording order across training. C, Representation of the area of a firing response during the CS–US interval. D, Representation of the area corresponding to the rectified activity of the orbicularis oculi (O.O.) muscle collected during the recording trials for the averaged firing rate illustrated in C. E, Linear relationships between the latency of neuronal responses to CS presentations versus the latency of the conditioned eyelid responses to CS presentations. Note that the three types of MC neurons presented similar relationships with the latency of CRs. F, Linear relationships between integrated neuronal responses during the CS–US interval and the integrated EMG activity of the O.O. muscle corresponding to CRs. The length of the regression lines was restricted to the collected values.
Figure 7.
Figure 7.
Effects of the chemical inactivation or the electrical stimulation of the MC on the generation and kinematics of CRs. A–D, The contralateral MC was perfused with lidocaine (5% solution at a rate of 0.1 μl/min) or control (vehicle injection at the same rate). Three animals per group were injected. Note that lidocaine inactivation of the contralateral MC significantly decreased the percentage of CRs (C) and the integrated amplitude of both CRs and unconditioned responses (UR; D). *p = 0.046; **p = 0.002; ***p = 0.001. E–G, Representative examples of CRs recorded across successive conditioning sessions. Eyelid movements were recorded with the magnetic search coil technique. Note the wave nature of conditioned eyelid responses, and that the number of downward waves increased across conditioning (1, third session; 2, sixth session; 3, eighth session). Note also in F that the spectral power of CR profiles (n = 60) recorded during the same conditioning sessions increased with the number of waves, at ≈10 Hz. The inset in F illustrates spectral powers obtained from the averaged profiles of the firing rates of A–C neurons collected from six animals from the eighth to the 20th conditioning sessions. G, Effects of the electrical stimulation of the contralateral MC on the EMG activity of the orbicularis oculi (O.O.) muscle and on eyelid position. The MC was stimulated with an increasing number of paired pulses (1 ms interpulse interval) at a frequency of 10 Hz. Note how similar the evoked eyelid responses were compared to those presented by actual CRs—i.e., both of them presented a similar ramp-like wavy profile.

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