Myo-optogenetics: optogenetic stimulation and electrical recording in skeletal muscles.

Complex movements involve highly coordinated control of local muscle elements. Highly controlled perturbations of motor outputs can reveal insights into the neural control of movements. Here we introduce an optogenetic method, compatible with electromyography (EMG) recordings, to perturb muscles in transgenic mice. By expressing channelrhodopsin in muscle fibers, we achieved noninvasive, focal activation of orofacial muscles, enabling detailed examination of the mechanical properties of optogenetically evoked jaw muscle contractions. We demonstrated simultaneous EMG recording and optical stimulation, revealing the electrophysiological characteristics of optogenetically triggered muscle activity. Additionally, we applied optogenetic activation of muscles in physiologically and behaviorally relevant settings, mapping precise muscle actions and perturbing active behaviors. Our findings highlight the potential of muscle optogenetics to precisely manipulate muscle activity, offering a powerful tool for probing neuromuscular control systems and advancing our understanding of motor control.

Rule-based modulation of a sensorimotor transformation across cortical areas.

Flexible responses to sensory stimuli based on changing rules are critical for adapting to a dynamic environment. However, it remains unclear how the brain encodes and uses rule information to guide behavior. Here, we made single-unit recordings while head-fixed mice performed a cross-modal sensory selection task where they switched between two rules: licking in response to tactile stimuli while rejecting visual stimuli, or vice versa. Along a cortical sensorimotor processing stream including the primary (S1) and secondary (S2) somatosensory areas, and the medial (MM) and anterolateral (ALM) motor areas, single-neuron activity distinguished between the two rules both prior to and in response to the tactile stimulus. We hypothesized that neural populations in these areas would show rule-dependent preparatory states, which would shape the subsequent sensory processing and behavior. This hypothesis was supported for the motor cortical areas (MM and ALM) by findings that (1) the current task rule could be decoded from pre-stimulus population activity; (2) neural subspaces containing the population activity differed between the two rules; and (3) optogenetic disruption of pre-stimulus states impaired task performance. Our findings indicate that flexible action selection in response to sensory input can occur via configuration of preparatory states in the motor cortex.

Tactile processing in mouse cortex depends on action context.

The brain receives constant tactile input, but only a subset guides ongoing behavior. Actions associated with tactile stimuli thus endow them with behavioral relevance. It remains unclear how the relevance of tactile stimuli affects processing in the somatosensory (S1) cortex. We developed a cross-modal selection task in which head-fixed mice switched between responding to tactile stimuli in the presence of visual distractors or to visual stimuli in the presence of tactile distractors using licking movements to the left or right side in different blocks of trials. S1 spiking encoded tactile stimuli, licking actions, and direction of licking in response to tactile but not visual stimuli. Bidirectional optogenetic manipulations showed that sensory-motor activity in S1 guided behavior when touch but not vision was relevant. Our results show that S1 activity and its impact on behavior depend on the actions associated with a tactile stimulus.

Bilateral Whisker Representations in the Primary Somatosensory Cortex in Robo3cKO Mice Are Reflected in the Primary Motor Cortex.

In Robo3cKO mice, midline crossing defects of the trigeminothalamic projections from the trigeminal principal sensory nucleus result in bilateral whisker maps in the somatosensory thalamus and consequently in the face representation area of the primary somatosensory (S1) cortex (Renier et al., 2017; Tsytsarev et al., 2017). We investigated whether this bilateral sensory representation in the whisker-barrel cortex is also reflected in the downstream projections from the S1 to the primary motor (M1) cortex. To label these projections, we injected anterograde viral axonal tracer in S1 cortex. Corticocortical projections from the S1 distribute to similar areas across the ipsilateral hemisphere in control and Robo3cKO mice. Namely, in both genotypes they extend to the M1, premotor/prefrontal cortex (PMPF), secondary somatosensory (S2) cortex. Next, we performed voltage-sensitive dye imaging (VSDi) in the left hemisphere following ipsilateral and contralateral single whisker stimulation. While controls showed only activation in the contralateral whisker barrel cortex and M1 cortex, the Robo3cKO mouse left hemisphere was activated bilaterally in both the barrel cortex and the M1 cortex. We conclude that the midline crossing defect of the trigeminothalamic projections leads to bilateral whisker representations not only in the thalamus and the S1 cortex but also downstream from the S1, in the M1 cortex.

Rule-based modulation of a sensorimotor transformation across cortical areas.

Flexible responses to sensory stimuli based on changing rules are critical for adapting to a dynamic environment. However, it remains unclear how the brain encodes rule information and uses this information to guide behavioral responses to sensory stimuli. Here, we made single-unit recordings while head-fixed mice performed a cross-modal sensory selection task in which they switched between two rules in different blocks of trials: licking in response to tactile stimuli applied to a whisker while rejecting visual stimuli, or licking to visual stimuli while rejecting the tactile stimuli. Along a cortical sensorimotor processing stream including the primary (S1) and secondary (S2) somatosensory areas, and the medial (MM) and anterolateral (ALM) motor areas, the single-trial activity of individual neurons distinguished between the two rules both prior to and in response to the tactile stimulus. Variable rule-dependent responses to identical stimuli could in principle occur via appropriate configuration of pre-stimulus preparatory states of a neural population, which would shape the subsequent response. We hypothesized that neural populations in S1, S2, MM and ALM would show preparatory activity states that were set in a rule-dependent manner to cause processing of sensory information according to the current rule. This hypothesis was supported for the motor cortical areas by findings that (1) the current task rule could be decoded from pre-stimulus population activity in ALM and MM; (2) neural subspaces containing the population activity differed between the two rules; and (3) optogenetic disruption of pre-stimulus states within ALM and MM impaired task performance. Our findings indicate that flexible selection of an appropriate action in response to a sensory input can occur via configuration of preparatory states in the motor cortex.

Simultaneous recordings of pupil size variation and locus coeruleus activity in mice.

An extensive literature describes how pupil size reflects neuromodulatory activity, including the noradrenergic system. Here, we present a protocol for the simultaneous recording of optogenetically identified locus coeruleus (LC) units and pupil diameter in mice under different conditions. We describe steps for building an optrode, performing surgery to implant the optrode and headpost, searching for opto-tagged LC units, and performing dual LC-pupil recording. We then detail procedures for data processing and analysis. For complete details on the use and execution of this protocol, please refer to Megemont et al.1.

Elephant trunks: Strength and dexterity from mini-fascicles.

Muscular hydrostats, such as the elephant trunk, can perform precise motor actions. A new study has revealed that the elephant trunk contains a dense network of tiny muscle fascicles, suggesting that muscle miniaturization may be a key toward understanding how soft organs achieve both strength and dexterity.

Low- and high-level coordination of orofacial motor actions.

Orofacial motor actions are movements that, in rodents, involve whisking of the vibrissa, deflection of the nose, licking and lapping with the tongue, and consumption through chewing. These actions, along with bobbing and turning of the head, coordinate to subserve exploration while not conflicting with life-supporting actions such as breathing and swallowing. Orofacial and head movements are comprised of two additive components: a rhythm that can be entrained by the breathing oscillator and a broadband component that directs the actuator to the region of interest. We focus on coordinating the rhythmic component of actions into a behavior. We hypothesize that the precise timing of each constituent action is continually adjusted through the merging of low-level oscillator input with sensory-derived, high-level rhythmic feedback. Supporting evidence is discussed.

Trigeminal innervation and tactile responses in mouse tongue.

The mammalian tongue is richly innervated with somatosensory, gustatory and motor fibers. These form the basis of many ethologically important functions such as eating, speaking and social grooming. Despite its high tactile acuity and sensitivity, the neural basis of tongue mechanosensation remains largely mysterious. Here we explored the organization of mechanosensory afferents in the tongue and found that each lingual papilla is innervated by Piezo2 + trigeminal neurons. Notably, each fungiform papilla contained highly specialized ring-like sensory neuron terminations that circumscribe the taste buds. Myelinated lingual afferents in the mouse lingual papillae did not form corpuscular sensory end organs but rather had only free nerve endings. In vivo single-unit recordings from the trigeminal ganglion revealed two types of lingual low-threshold mechanoreceptors (LTMRs) with conduction velocities in the Aδ range or above and distinct response properties: intermediately adapting (IA) units and rapidly adapting (RA) units. IA units were sensitive to static indentation and stroking, while RA units had a preference for tangential forces applied by stroking. Lingual LTMRs were not directly responsive to rapid cooling or chemicals that can induce astringent or numbing sensations. Genetic labeling of lingual afferents in the tongue revealed at least two types of nerve terminal patterns, involving dense innervation of individual fungiform papillae by multiple putatively distinct afferents, and relatively sparse innervation of filiform papillae. Together, our results indicate that fungiform papillae are mechanosensory structures, while suggesting a simple model that links the functional and anatomical properties of tactile sensory neurons in the tongue.

Spike timing-based regulation of thalamocortical signaling.

For decades, thalamic burst and tonic spiking modes have been theorized to regulate sensory signaling in the thalamocortical circuit. In this issue of Neuron, Borden et al. demonstrate a timing-based mechanism by which thalamic spiking mode controls sensory responses in the awake cortex.

Layers 3 and 4 Neurons of the Bilateral Whisker-Barrel Cortex.

In Robo3R3-5cKO mouse brain, rhombomere 3-derived trigeminal principal nucleus (PrV) neurons project bilaterally to the somatosensory thalamus. As a consequence, whisker-specific neural modules (barreloids and barrels) representing whiskers on both sides of the face develop in the sensory thalamus and the primary somatosensory cortex. We examined the morphological complexity of layer 4 barrel cells, their postsynaptic partners in layer 3, and functional specificity of layer 3 pyramidal cells. Layer 4 spiny stellate cells form much smaller barrels and their dendritic fields are more focalized and less complex compared to controls, while layer 3 pyramidal cells did not show notable differences. Using in vivo 2-photon imaging of a genetically encoded fluorescent [Ca2+] sensor, we visualized neural activity in the normal and Robo3R3-5cKO barrel cortex in response to ipsi- and contralateral single whisker stimulation. Layer 3 neurons in control animals responded only to their contralateral whiskers, while in the mutant cortex layer 3 pyramidal neurons showed both ipsi- and contralateral whisker responses. These results indicate that bilateral whisker map inputs stimulate different but neighboring groups of layer 3 neurons which normally relay contralateral whisker-specific information to other cortical areas.

Cortical processing of flexible and context-dependent sensorimotor sequences.

The brain generates complex sequences of movements that can be flexibly configured based on behavioural context or real-time sensory feedback1, but how this occurs is not fully understood. Here we developed a 'sequence licking' task in which mice directed their tongue to a target that moved through a series of locations. Mice could rapidly branch the sequence online based on tactile feedback. Closed-loop optogenetics and electrophysiology revealed that the tongue and jaw regions of the primary somatosensory (S1TJ) and motor (M1TJ) cortices2 encoded and controlled tongue kinematics at the level of individual licks. By contrast, the tongue 'premotor' (anterolateral motor) cortex3-10 encoded latent variables including intended lick angle, sequence identity and progress towards the reward that marked successful sequence execution. Movement-nonspecific sequence branching signals occurred in the anterolateral motor cortex and M1TJ. Our results reveal a set of key cortical areas for flexible and context-informed sequence generation.

The Importance of Accounting for Movement When Relating Neuronal Activity to Sensory and Cognitive Processes.

A surprising finding of recent studies in mouse is the dominance of widespread movement-related activity throughout the brain, including in early sensory areas. In awake subjects, failing to account for movement risks misattributing movement-related activity to other (e.g., sensory or cognitive) processes. In this article, we (1) review task designs for separating task-related and movement-related activity, (2) review three "case studies" in which not considering movement would have resulted in critically different interpretations of neuronal function, and (3) discuss functional couplings that may prevent us from ever fully isolating sensory, motor, and cognitive-related activity. Our main thesis is that neural signals related to movement are ubiquitous, and therefore ought to be considered first and foremost when attempting to correlate neuronal activity with task-related processes.

Neural activity in the mouse claustrum in a cross-modal sensory selection task.

The claustrum, a subcortical nucleus forming extensive connections with the neocortex, has been implicated in sensory selection. Sensory-evoked claustrum activity is thought to modulate the neocortex's context-dependent response to sensory input. Recording from claustrum neurons while mice performed a tactile-visual sensory-selection task, we found that neurons in the anterior claustrum, including putative optotagged claustrocortical neurons projecting to the primary somatosensory cortex (S1), were rarely modulated by sensory input. Rather, they exhibited different types of direction-tuned motor responses. Furthermore, we found that claustrum neurons encoded upcoming movement during intertrial intervals and that pairs of claustrum neurons exhibiting synchronous firing were enriched for pairs preferring contralateral lick directions, suggesting that the activity of specific ensembles of similarly tuned claustrum neurons may modulate cortical activity. Chemogenetic inhibition of claustrocortical neurons decreased lick responses to inappropriate sensory stimuli. Altogether, our data indicate that the claustrum is integrated into higher-order premotor circuits recently implicated in decision-making.

Environmental Enrichment Sharpens Sensory Acuity by Enhancing Information Coding in Barrel Cortex and Premotor Cortex.

Environmental enrichment (EE) is beneficial to sensory functions. Thus, elucidating the neural mechanism underlying improvement of sensory stimulus discrimination is important for developing therapeutic strategies. We aim to advance the understanding of such neural mechanism. We found that tactile enrichment improved tactile stimulus feature discrimination. The neural correlate of such improvement was revealed by analyzing single-cell information coding in both the primary somatosensory cortex and the premotor cortex of awake behaving animals. Our results show that EE enhances the decision-information coding capacity of cells that are tuned to adjacent whiskers, and of premotor cortical cells.

Locus coeruleus spiking differently correlates with S1 cortex activity and pupil diameter in a tactile detection task.

We examined the relationships between activity in the locus coeruleus (LC), activity in the primary somatosensory cortex (S1), and pupil diameter in mice performing a tactile detection task. While LC spiking consistently preceded S1 membrane potential depolarization and pupil dilation, the correlation between S1 and pupil was more heterogeneous. Furthermore, the relationships between LC, S1, and pupil varied on timescales of sub-seconds to seconds within trials. Our data suggest that pupil diameter can be dissociated from LC spiking and cannot be used as a stationary index of LC activity.

Of mice and monkeys: Somatosensory processing in two prominent animal models.

Our understanding of the neural basis of somatosensation is based largely on studies of the whisker system of mice and rats and the hands of macaque monkeys. Results across these animal models are often interpreted as providing direct insight into human somatosensation. Work on these systems has proceeded in parallel, capitalizing on the strengths of each model, but has rarely been considered as a whole. This lack of integration promotes a piecemeal understanding of somatosensation. Here, we examine the functions and morphologies of whiskers of mice and rats, the hands of macaque monkeys, and the somatosensory neuraxes of these three species. We then discuss how somatosensory information is encoded in their respective nervous systems, highlighting similarities and differences. We reflect on the limitations of these models of human somatosensation and consider key gaps in our understanding of the neural basis of somatosensation.

Spectral hallmark of auditory-tactile interactions in the mouse somatosensory cortex.

To synthesize a coherent representation of the external world, the brain must integrate inputs across different types of stimuli. Yet the mechanistic basis of this computation at the level of neuronal populations remains obscure. Here, we investigate tactile-auditory integration using two-photon Ca2+ imaging in the mouse primary (S1) and secondary (S2) somatosensory cortices. Pairing sound with whisker stimulation modulates tactile responses in both S1 and S2, with the most prominent modulation being robust inhibition in S2. The degree of inhibition depends on tactile stimulation frequency, with lower frequency responses the most severely attenuated. Alongside these neurons, we identify sound-selective neurons in S2 whose responses are inhibited by high tactile frequencies. These results are consistent with a hypothesized local mutually-inhibitory S2 circuit that spectrally selects tactile versus auditory inputs. Our findings enrich mechanistic understanding of multisensory integration and suggest a key role for S2 in combining auditory and tactile information.

Coding of whisker motion across the mouse face.

Haptic perception synthesizes touch with proprioception, the sense of body position. Humans and mice alike experience rich active touch of the face. Because most facial muscles lack proprioceptor endings, the sensory basis of facial proprioception remains unsolved. Facial proprioception may instead rely on mechanoreceptors that encode both touch and self-motion. In rodents, whisker mechanoreceptors provide a signal that informs the brain about whisker position. Whisking involves coordinated orofacial movements, so mechanoreceptors innervating facial regions other than whiskers could also provide information about whisking. To define all sources of sensory information about whisking available to the brain, we recorded spikes from mechanoreceptors innervating diverse parts of the face. Whisker motion was encoded best by whisker mechanoreceptors, but also by those innervating whisker pad hairy skin and supraorbital vibrissae. Redundant self-motion responses may provide the brain with a stable proprioceptive signal despite mechanical perturbations during active touch.