Layer 5 Intratelencephalic Neurons in the Motor Cortex Stably Encode Skilled Movement.

The primary motor cortex (M1) and the dorsal striatum play a critical role in motor learning and the retention of learned behaviors. Motor representations of corticostriatal ensembles emerge during motor learning. In the coordinated reorganization of M1 and the dorsal striatum for motor learning, layer 5a (L5a) which connects M1 to the ipsilateral and contralateral dorsal striatum, should be a key layer. Although M1 L5a neurons represent movement-related activity in the late stage of learning, it is unclear whether the activity is retained as a memory engram. Here, using Tlx3-Cre male transgenic mice, we conducted two-photon calcium imaging of striatum-projecting L5a intratelencephalic (IT) neurons in forelimb M1 during late sessions of a self-initiated lever-pull task and in sessions after 6 d of nontraining following the late sessions. We found that trained male animals exhibited stable motor performance before and after the nontraining days. At the same time, we found that M1 L5a IT neurons strongly represented the well-learned forelimb movement but not uninstructed orofacial movements. A subset of M1 L5a IT neurons consistently coded the well-learned forelimb movement before and after the nontraining days. Inactivation of M1 IT neurons after learning impaired task performance when the lever was made heavier or when the target range of the pull distance was narrowed. These results suggest that a subset of M1 L5a IT neurons continuously represent skilled movement after learning and serve to fine-tune the kinematics of well-learned movement.SIGNIFICANCE STATEMENT Motor memory persists even when it is not used for a while. IT neurons in L5a of the M1 gradually come to represent skilled forelimb movements during motor learning. However, it remains to be determined whether these changes persist over a long period and how these neurons contribute to skilled movements. Here, we show that a subset of M1 L5a IT neurons retain information for skilled forelimb movements even after nontraining days. Furthermore, suppressing the activity of these neurons during skilled forelimb movements impaired behavioral stability and adaptability. Our results suggest the importance of M1 L5a IT neurons for tuning skilled forelimb movements over a long period.

Arm movements induced by noninvasive optogenetic stimulation of the motor cortex in the common marmoset.

Optogenetics is now a fundamental tool for investigating the relationship between neuronal activity and behavior. However, its application to the investigation of motor control systems in nonhuman primates is rather limited, because optogenetic stimulation of cortical neurons in nonhuman primates has failed to induce or modulate any hand/arm movements. Here, we used a tetracycline-inducible gene expression system carrying CaMKII promoter and the gene encoding a Channelrhodopsin-2 variant with fast kinetics in the common marmoset, a small New World monkey. In an awake state, forelimb movements could be induced when Channelrhodopsin-2-expressing neurons in the motor cortex were illuminated by blue laser light with a spot diameter of 1 mm or 2 mm through a cranial window without cortical invasion. Forelimb muscles responded 10 ms to 50 ms after photostimulation onset. Long-duration (500 ms) photostimulation induced discrete forelimb movements that could be markerlessly tracked with charge-coupled device cameras and a deep learning algorithm. Long-duration photostimulation mapping revealed that the primary motor cortex is divided into multiple domains that can induce hand and elbow movements in different directions. During performance of a forelimb movement task, movement trajectories were modulated by weak photostimulation, which did not induce visible forelimb movements at rest, around the onset of task-relevant movement. The modulation was biased toward the movement direction induced by the strong photostimulation. Combined with calcium imaging, all-optical interrogation of motor circuits should be possible in behaving marmosets.

Motor learning requires myelination to reduce asynchrony and spontaneity in neural activity.

Myelination increases the conduction velocity in long-range axons and is prerequisite for many brain functions. Impaired myelin regulation or impairment of myelin itself is frequently associated with deficits in learning and cognition in neurological and psychiatric disorders. However, it has not been revealed what perturbation of neural activity induced by myelin impairment causes learning deficits. Here, we measured neural activity in the motor cortex during motor learning in transgenic mice with a subtle impairment of their myelin. This deficit in myelin impaired motor learning, and was accompanied by a decrease in the amplitude of movement-related activity and an increase in the frequency of spontaneous activity. Thalamocortical axons showed variability in axonal conduction with a large spread in the timing of postsynaptic cortical responses. Repetitive pairing of forelimb movements with optogenetic stimulation of thalamocortical axon terminals restored motor learning. Thus, myelin regulation helps to maintain the synchrony of cortical spike-time arrivals through long-range axons, facilitating the propagation of the information required for learning. Our results revealed the pathological neuronal circuit activity with impaired myelin and suggest the possibility that pairing of noninvasive brain stimulation with relevant behaviors may ameliorate cognitive and behavioral abnormalities in diseases with impaired myelination.

Distinct Functional Modules for Discrete and Rhythmic Forelimb Movements in the Mouse Motor Cortex.

Movements of animals are composed of two fundamental dynamics: discrete and rhythmic movements. Although the movements with distinct dynamics are thought to be differently processed in the CNS, it is unclear how they are represented in the cerebral cortex. Here, we investigated the cortical representation of movement dynamics by developing prolonged transcranial optogenetic stimulation (pTOS) using awake, channelrhodopsin-2 transgenic mice. We found two domains that induced discrete forelimb movements in the forward and backward directions, and these sandwiched a domain that generated rhythmic forelimb movements. The forward discrete movement had an intrinsic velocity profile and the rhythmic movement had an intrinsic oscillation frequency. Each of the forward discrete and rhythmic domains possessed intracortical synaptic connections within its own domain, independently projected to the spinal cord, and weakened the neuronal activity and movement induction of the other domain. pTOS-induced movements were also classified as ethologically relevant movements. Forepaw-to-mouth movement was mapped in a part of the forward discrete domain, while locomotion-like movement was in a part of the rhythmic domain. Interestingly, photostimulation of the rhythmic domain resulted in a nonrhythmic, continuous lever-pull movement when a lever was present. The motor cortex possesses functional modules for distinct movement dynamics, and these can adapt to environmental constraints for purposeful movements. Significance statement: Animal behavior has discrete and rhythmic components, such as reaching and locomotion. It is unclear how these movements with distinct dynamics are represented in the cerebral cortex. We investigated the dynamics of movements induced by long-duration transcranial photostimulation on the dorsal cortex of awake channelrhodopsin-2 transgenic mice. We found two domains causing forward and backward discrete forelimb movements and a domain for rhythmic forelimb movements. A domain for forward discrete movement and a domain for rhythmic movement mutually weakened neuronal activity and movement size. The photostimulation of the rhythmic domain also induced nonrhythmic, lever-pull movement, when the lever was present. Thus, the motor cortex has functional modules with distinct dynamics, and each module retains flexibility for adaptation to different environments.

Spatiotemporal dynamics of functional clusters of neurons in the mouse motor cortex during a voluntary movement.

Functional clustering of neurons is frequently observed in the motor cortex. However, it is unknown if, when, and how fine-scale (<100 µm) functional clusters form relative to voluntary forelimb movements. In addition, the implications of clustering remain unclear. To address these issues, we conducted two-photon calcium imaging of mouse layer 2/3 motor cortex during a self-initiated lever-pull task. In the imaging session after 8-9 days of training, head-restrained mice had to pull a lever for ∼600 ms to receive a water drop, and then had to wait for >3 s to pull it again. We found two types of task-related cells in the mice: cells whose peak activities occurred during lever pulls (pull cells) and cells whose peak activities occurred after the end of lever pulls. The activity of pull cells was strongly associated with lever-pull duration. In ∼40% of imaged fields, functional clusterings were temporally detected during the lever pulls. Spatially, there were ∼70-µm-scale clusters that consisted of more than four pull cells in ∼50% of the fields. Ensemble and individual activities of pull cells within the cluster more accurately predicted lever movement trajectories than activities of pull cells outside the cluster. This was likely because clustered pull cells were more often active in the individual trials than pull cells outside the cluster. This higher fidelity of activity was related to higher trial-to-trial correlations of activities of pairs within the cluster. We propose that strong recurrent network clusters may represent the execution of voluntary movements.

Medial prefrontal cortex suppresses reward-seeking behavior with risk of punishment by reducing sensitivity to reward.

Reward-seeking behavior is frequently associated with risk of punishment. There are two types of punishment: positive punishment, which is defined as addition of an aversive stimulus, and negative punishment, involves the omission of a rewarding outcome. Although the medial prefrontal cortex (mPFC) is important in avoiding punishment, whether it is important for avoiding both positive and negative punishment and how it contributes to such avoidance are not clear. In this study, we trained male mice to perform decision-making tasks under the risks of positive (air-puff stimulus) and negative (reward omission) punishment, and modeled their behavior with reinforcement learning. Following the training, we pharmacologically inhibited the mPFC. We found that pharmacological inactivation of mPFC enhanced the reward-seeking choice under the risk of positive, but not negative, punishment. In reinforcement learning models, this behavioral change was well-explained as an increase in sensitivity to reward, rather than a decrease in the strength of aversion to punishment. Our results suggest that mPFC suppresses reward-seeking behavior by reducing sensitivity to reward under the risk of positive punishment.

Two-color, two-photon uncaging of glutamate and GABA.

We developed a caged GABA (gamma-aminobutyric acid), which, when combined with an appropriate caged glutamate, allows bimodal control of neuronal membrane potential with subcellular resolution using optically independent two-photon uncaging of each neurotransmitter. We used two-color, two-photon uncaging to fire and block action potentials from rat hippocampal CA1 neurons in brain slices with 720-nm and 830-nm light, respectively. Our method should be generalizable to other chemical messenger pairs.

Dynamic organization of cerebellar climbing fiber response and synchrony in multiple functional components reduces dimensions for reinforcement learning.

Cerebellar climbing fibers convey diverse signals, but how they are organized in the compartmental structure of the cerebellar cortex during learning remains largely unclear. We analyzed a large amount of coordinate-localized two-photon imaging data from cerebellar Crus II in mice undergoing 'Go/No-go' reinforcement learning. Tensor component analysis revealed that a majority of climbing fiber inputs to Purkinje cells were reduced to only four functional components, corresponding to accurate timing control of motor initiation related to a Go cue, cognitive error-based learning, reward processing, and inhibition of erroneous behaviors after a No-go cue. Changes in neural activities during learning of the first two components were correlated with corresponding changes in timing control and error learning across animals, indirectly suggesting causal relationships. Spatial distribution of these components coincided well with boundaries of Aldolase-C/zebrin II expression in Purkinje cells, whereas several components are mixed in single neurons. Synchronization within individual components was bidirectionally regulated according to specific task contexts and learning stages. These findings suggest that, in close collaborations with other brain regions including the inferior olive nucleus, the cerebellum, based on anatomical compartments, reduces dimensions of the learning space by dynamically organizing multiple functional components, a feature that may inspire new-generation AI designs.

Change detection in the primate auditory cortex through feedback of prediction error signals.

Although cortical feedback signals are essential for modulating feedforward processing, no feedback error signal across hierarchical cortical areas has been reported. Here, we observed such a signal in the auditory cortex of awake common marmoset during an oddball paradigm to induce auditory duration mismatch negativity. Prediction errors to a deviant tone presentation were generated as offset calcium responses of layer 2/3 neurons in the rostral parabelt (RPB) of higher-order auditory cortex, while responses to non-deviant tones were strongly suppressed. Within several hundred milliseconds, the error signals propagated broadly into layer 1 of the primary auditory cortex (A1) and accumulated locally on top of incoming auditory signals. Blockade of RPB activity prevented deviance detection in A1. Optogenetic activation of RPB following tone presentation nonlinearly enhanced A1 tone response. Thus, the feedback error signal is critical for automatic detection of unpredicted stimuli in physiological auditory processing and may serve as backpropagation-like learning.

Reinforcing operandum: rapid and reliable learning of skilled forelimb movements by head-fixed rodents.

Stereotaxic head fixation plays a necessary role in current physiological techniques, such as in vivo whole cell recording and two-photon laser-scanning microscopy, that are designed to elucidate the cortical involvement in animal behaviors. In rodents, however, head fixation often inhibits learning and performance of behavioral tasks. In particular, it has been considered inappropriate for head-fixed rodents to be operantly conditioned to perform skilled movements with their forelimb (e.g., lever-press task), despite the potential applicability of the task. Here we have solved this problem conceptually by integrating a lever (operandum) and a rewarding spout (reinforcer) into one ″spout-lever″ device for efficient operant learning. With this device, head-fixed rats reliably learned to perform a pull manipulation of the spout-lever with their right forelimb in response to an auditory cue signal (external-trigger trial, namely, Go trial) within several days. We also demonstrated stable whole cell recordings from motor cortex neurons while the rats were performing forelimb movements in external-trigger trials. We observed a behavior-related increase in the number of action potentials in membrane potential. In the next session, the rats, which had already learned the external-trigger trial, effortlessly performed similar spout-lever manipulation with no cue presentation (internal-trigger trial) additionally. Likewise, some of the rats learned to keep holding the spout-lever in response to another cue signal (No-go trial) in the following session, so that they mastered the Go/No-go discrimination task in one extra day. Our results verified the usefulness of spout-lever manipulation for behavioral experiments employing cutting-edge physiological techniques.

Two-photon uncaging of gamma-aminobutyric acid in intact brain tissue.

We have synthesized a photosensitive (or caged) 4-carboxymethoxy-5,7-dinitroindolinyl (CDNI) derivative of gamma-aminobutyric acid (GABA). Two-photon excitation of CDNI-GABA produced rapid activation of GABAergic currents in neurons in brain slices with an axial resolution of approximately 2 mum and enabled high-resolution functional mapping of GABA-A receptors. Two-photon uncaging of GABA, the main inhibitory neurotransmitter, should allow detailed studies of receptor function and synaptic integration with subcellular precision.

Simultaneous visualization of multiple neuronal properties with single-cell resolution in the living rodent brain.

To understand the fine-scale structures and functional properties of individual neurons in vivo, we developed and validated a rapid genetic technique that enables simultaneous investigation of multiple neuronal properties with single-cell resolution in the living rodent brain. Our technique PASME (promoter-assisted sparse-neuron multiple-gene labeling using in uteroelectroporation) targets specific small subsets of sparse pyramidal neurons in layer 2/3, layer 5 of the cerebral cortex and in the hippocampus with multiple fluorescent reporter proteins such as postsynaptic PSD-95-GFP and GFP-gephyrin. The technique is also applicable for targeting independently individual neurons and their presynaptic inputs derived from surrounding neurons. Targeting sparse layer 2/3 neurons, we uncovered a novel subpopulation of layer 2/3 neurons in the mouse cerebral cortex. This technique, broadly applicable for probing and manipulating neurons with single-cell resolution in vivo, should provide a robust means to uncover the basic mechanisms employed by the brain, especially when combined with in vivo two-photon laser-scanning microscopy and/or optogenetic technologies.

Silent microscopy to explore a brain that hears butterflies' wings.

A silent two-photon laser-scanning microscopy system, which eliminates mechanical vibrations in the audible range, has enabled the detection of auditory cortical neurons with responses at sound pressure levels as low as 5 dB in nonhuman primates.

Transition of distinct context-dependent ensembles from secondary to primary motor cortex in skilled motor performance.

When voluntary movements are executed under different contexts, different context-dependent signals are thought to weaken from secondary motor cortex (M2) to primary motor cortex (M1). However, it is unclear how the different contexts are processed from M2 to M1 to execute skilled movement. We conduct two-photon calcium imaging of M2 and M1 in mice performing internally generated and external-cue-triggered movements. Context dependency is consistently high in M2 L2/3 neurons and consistently low in M1 pyramidal tract neurons. By contrast, context dependency in M2 → M1 axons and M1 L2/3 neurons increases as task performance improves. In addition, the context dependency of M1 L2/3, but not M2 → M1 axons, is associated with fine-movement proficiency. The increase in context dependency correlates with stabilization of the context-dependent population activity and an increase in the neurons that strongly encode contextual and motor information. Thus, emergence of distinct context-dependent ensembles may be necessary for the context-to-motor transformation that facilitates skilled motor performance.

In vivo two-photon uncaging of glutamate revealing the structure-function relationships of dendritic spines in the neocortex of adult mice.

Two-photon (2P) uncaging of caged neurotransmitters can efficiently stimulate individual synapses and is widely used to characterize synaptic functions in brain slice preparations. Here we extended 2P uncaging to neocortical pyramidal neurons in adult mice in vivo where caged glutamate was applied from the pial surface. To validate the methodology, we applied a small fluorescent probe using the same method, and confirmed that its concentrations were approximately homogenous up to 200 µm below the cortical surface, and that the extracellular space of the neocortex was as large as 22%. In fact, in vivo whole-cell recording revealed that 2P glutamate uncaging could elicit transient currents (2pEPSCs) very similar to excitatory postsynaptic currents (EPSCs). A spatial resolution of glutamate uncaging was 0.6-0.8 µm up to the depth of 200 µm, and in vivo 2P uncaging was able to stimulate single identified spines. Automated three-dimensional (3-D) mapping of such 2pEPSCs which covered the surfaces of dendritic branches revealed that functional AMPA receptor expression was stable and proportional to spine volume.Moreover, in vivo 2P Ca2+ imaging and uncaging suggested that the amplitudes of glutamate-induced Ca2+ transients were inversely proportional to spine volume. Thus, the key structure-function relationships hold in dendritic spines in adult neocortex in vivo, as in young hippocampal slice preparations. In vivo 2P uncaging will be a powerful tool to investigate properties of synapses in the neocortex.

Neuronal representations of reward-predicting cues and outcome history with movement in the frontal cortex.

Transformation of sensory inputs to goal-directed actions requires estimation of sensory-cue values based on outcome history. We conduct wide-field and two-photon calcium imaging of the mouse neocortex during classical conditioning with two cues with different water-reward probabilities. Although licking movement dominates the area-averaged activity over the whole dorsal neocortex, the dorsomedial frontal cortex (dmFrC) affects other dorsal frontal cortical activities, and its inhibition extinguishes differences in anticipatory licking between the cues. Many dorsal frontal and medial prefrontal cortical neurons are task related. Subsets of these neurons are more excited by the low-reward-predicting cue or unrewarded outcomes than by the high-reward-predicting cue or rewarded outcomes, respectively. Task-related activities of these neurons and the others are counterbalanced, so that population activity appears dominated by licking. The reward-predicting cue and outcome history are most strongly represented in dmFrC. Our results suggest that dmFrC is crucial for initiating cortical processes to select or inhibit action.

Tb3+-doped fluorescent glass for biology.

Optical investigation and manipulation constitute the core of biological experiments. Here, we introduce a new borosilicate glass material that contains the rare-earth ion terbium(III) (Tb3+), which emits green fluorescence upon blue light excitation, similar to green fluorescent protein (GFP), and thus is widely compatible with conventional biological research environments. Micropipettes made of Tb3+-doped glass allowed us to target GFP-labeled cells for single-cell electroporation, single-cell transcriptome analysis (Patch-seq), and patch-clamp recording under real-time fluorescence microscopic control. The glass also exhibited potent third harmonic generation upon infrared laser excitation and was usable for online optical targeting of fluorescently labeled neurons in the in vivo neocortex. Thus, Tb3+-doped glass simplifies many procedures in biological experiments.

Structural basis of long-term potentiation in single dendritic spines.

Dendritic spines of pyramidal neurons in the cerebral cortex undergo activity-dependent structural remodelling that has been proposed to be a cellular basis of learning and memory. How structural remodelling supports synaptic plasticity, such as long-term potentiation, and whether such plasticity is input-specific at the level of the individual spine has remained unknown. We investigated the structural basis of long-term potentiation using two-photon photolysis of caged glutamate at single spines of hippocampal CA1 pyramidal neurons. Here we show that repetitive quantum-like photorelease (uncaging) of glutamate induces a rapid and selective enlargement of stimulated spines that is transient in large mushroom spines but persistent in small spines. Spine enlargement is associated with an increase in AMPA-receptor-mediated currents at the stimulated synapse and is dependent on NMDA receptors, calmodulin and actin polymerization. Long-lasting spine enlargement also requires Ca2+/calmodulin-dependent protein kinase II. Our results thus indicate that spines individually follow Hebb's postulate for learning. They further suggest that small spines are preferential sites for long-term potentiation induction, whereas large spines might represent physical traces of long-term memory.

Common marmoset as a model primate for study of the motor control system.

The common marmoset (Callithrix jacchus), a New World monkey, is emerging as a promising animal model for biomedical and neuroscience research. This species shares its basic brain architecture, including the organization of the motor cortical areas and the connections between these and other areas, with humans and other primates. Its small and lissencephalic cerebral cortex is suitable for the application of modern biological techniques. Optogenetic stimulation of the motor cortex induces forelimb movements, and two-photon calcium imaging allows detection of forelimb movement-related activity in multiple motor cortical neurons. The common marmoset also has a large repertoire of forelimb-related behaviors and vocal communications. Thus, the common marmoset is a good model for research into voluntary forelimb movements, social behaviors, and their dysfunctions.

Non-action Learning: Saving Action-Associated Cost Serves as a Covert Reward.

"To do or not to do" is a fundamental decision that has to be made in daily life. Behaviors related to multiple "to do" choice tasks have long been explained by reinforcement learning, and "to do or not to do" tasks such as the go/no-go task have also been recently discussed within the framework of reinforcement learning. In this learning framework, alternative actions and/or the non-action to take are determined by evaluating explicitly given (overt) reward and punishment. However, we assume that there are real life cases in which an action/non-action is repeated, even though there is no obvious reward or punishment, because implicitly given outcomes such as saving physical energy and regret (we refer to this as "covert reward") can affect the decision-making. In the current task, mice chose to pull a lever or not according to two tone cues assigned with different water reward probabilities (70% and 30% in condition 1, and 30% and 10% in condition 2). As the mice learned, the probability that they would choose to pull the lever decreased (<0.25) in trials with a 30% reward probability cue (30% cue) in condition 1, and in trials with a 10% cue in condition 2, but increased (>0.8) in trials with a 70% cue in condition 1 and a 30% cue in condition 2, even though a non-pull was followed by neither an overt reward nor avoidance of overt punishment in any trial. This behavioral tendency was not well explained by a combination of commonly used Q-learning models, which take only the action choice with an overt reward outcome into account. Instead, we found that the non-action preference of the mice was best explained by Q-learning models, which regarded the non-action as the other choice, and updated non-action values with a covert reward. We propose that "doing nothing" can be actively chosen as an alternative to "doing something," and that a covert reward could serve as a reinforcer of "doing nothing."