Role of motor cortex in goal-directed forelimb movement

The generation of voluntary movements can be described as a sequence of neuronal processes, including decision making, motor planning and execution. How the motor cortex contributes to movement production is still debated. This thesis focuses on the role of two murine motor cortical subregions in forelimb movements: the caudal forelimb area (CFA) and rostral forelimb area (RFA). To causally link neuronal activity to behavior, we transiently silenced these regions while mice were performing a delayed two-choice joystick task. Both subregions were essential for choosing and executing movements, yet, choice and execution were sequentially processed. Interestingly, CFA showed a prevalent role in dexterous movements. Surprisingly, during the delay period only combined inactivation of CFA and RFA affected choice. Our findings suggest a sequential and partially distributed neural processing of choice and execution across different subregions of the motor cortex

The role of forelimb motor cortex areas in goal directed action in mice

Mammalian motor cortex consists of several interconnected subregions thought to play distinct roles in voluntary movements, yet their specific role in decision making and execution is not completely elucidated. Here we used transient optogenetic inactivation of the caudal forelimb area (CFA) and rostral forelimb area (RFA) in mice as they performed a directional joystick task. Based on a vibrotactile cue applied to their forepaw, mice were trained to push or pull a joystick after a delay period. We found that choice and execution are temporally segregated processes. CFA and RFA were both essential during the stimulus delivery for correct choice and during the answer period for motor execution. Fine, distal motor deficits were restricted to CFA inactivation. Surprisingly, during the delay period neither area alone, but only combined inactivation was able to affect choice. Our findings suggest transient and partially distributed neural processing of choice and execution across different subregions of the motor cortex

Procedures for behavioral experiments in head-fixed mice.

The mouse is an increasingly prominent model for the analysis of mammalian neuronal circuits. Neural circuits ultimately have to be probed during behaviors that engage the circuits. Linking circuit dynamics to behavior requires precise control of sensory stimuli and measurement of body movements. Head-fixation has been used for behavioral research, particularly in non-human primates, to facilitate precise stimulus control, behavioral monitoring and neural recording. However, choice-based, perceptual decision tasks by head-fixed mice have only recently been introduced. Training mice relies on motivating mice using water restriction. Here we describe procedures for head-fixation, water restriction and behavioral training for head-fixed mice, with a focus on active, whisker-based tactile behaviors. In these experiments mice had restricted access to water (typically 1 ml/day). After ten days of water restriction, body weight stabilized at approximately 80% of initial weight. At that point mice were trained to discriminate sensory stimuli using operant conditioning. Head-fixed mice reported stimuli by licking in go/no-go tasks and also using a forced choice paradigm using a dual lickport. In some cases mice learned to discriminate sensory stimuli in a few trials within the first behavioral session. Delay epochs lasting a second or more were used to separate sensation (e.g. tactile exploration) and action (i.e. licking). Mice performed a variety of perceptual decision tasks with high performance for hundreds of trials per behavioral session. Up to four months of continuous water restriction showed no adverse health effects. Behavioral performance correlated with the degree of water restriction, supporting the importance of controlling access to water. These behavioral paradigms can be combined with cellular resolution imaging, random access photostimulation, and whole cell recordings

Supplementing water rewards with sucrose increases the number of trials performed by mice.

<p>A. Example experiment, with water (black circles) and sucrose (red circles) rewards provided on alternating sessions. B. The number of trials is 23% larger with sucrose (p<0.001 in two mice; n.s. in the third). C. The number of rewards per session is larger (p<0.001 in two mice; n.s. in the third). D. The discriminability index is unchanged.</p

Mice with one or more indicators of stress or pain are placed on detailed health assessment.

<p>Activity levels, grooming, and indicators of eating and drinking are scored daily in a health assessment sheet. The total aggregate health score determines if mice are supplied with additional water (see flowchart in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone-0088678-g002" target="_blank">Figure 2</a>).</p

Mouse weight and health during water restriction.

<p>All mice were trained in a lick/no-lick object location discrimination task using a single whisker (same mice as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone-0088678-g002" target="_blank">Figures 2</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone-0088678-g003" target="_blank">3</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-OConnor2" target="_blank">[18]</a>). Rewards consisted of approximately 8 µl of water per trial. A. Experimental time-course for one example mouse, from the beginning of water restriction to the end of electrophysiological recordings. An 85 day old mouse (25.4 g) was put on water restriction for eight days, followed by training (starting on day 9) and recording (starting on day 28). B. Body weight as a function of time. Same mouse as in A. The dashed line indicates 30% weight loss. C. Water consumed per day. After start of training mice mostly received their water during the training session. A larger number of correct trials will lead to more consumed water. Same mouse as in A. D. Health score as a function of time. A health score larger than 3 (dashed line) triggers more detailed evaluation and possibly water supplements. Same mouse as in A. E. Experimental time-course for a group of 5 mice. Same format as A. F. Average body weight of 5 mice (black line) and 2 mice with free access to water (grey line). Shading indicates standard deviation. Experimental time-course for all mice was similar, but not identical to A. G. Average water consumed. H. Average health score.</p

Performance of the lick-left/lick-right object location discrimination task with a delay epoch (data from Figure S1 [7]).

<p>A. Schematic of time-course of experiments. B. Learning curves showing the performance. Thin lines correspond to individual mice. Thick lines, average. Colors correspond to whisker trimming. Vertical dashed line indicates when the delay epoch was introduced. The four mice were from the same litter (2 males and 2 females). Same as Figure S1B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-Guo1" target="_blank">[7]</a>. C. Learning curves showing the discriminability index, d'. D. Bias: performance of lick-right trials minus performance of lick-left trials. Same as Figure S1C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-Guo1" target="_blank">[7]</a>. E. The fraction of trials with licking responses during the sample or delay epoch. Same as Figure S1D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-Guo1" target="_blank">[7]</a>. F. Water consumed. G. Trials per session. H. Health score. A health score larger than 3 (dashed line) triggers more detailed evaluation and possibly water supplements. I. Health score for four mice that were under water restriction for four months. A health score larger than 3 (dashed line) triggers more detailed evaluation and possibly water supplements.</p

Normalized weight of 5 female mice after the initial water restriction (left) and after one day of free access to water (dotted line, day 0).

<p>Normalized weight of 5 female mice after the initial water restriction (left) and after one day of free access to water (dotted line, day 0).</p