Specific connectivity optimizes learning in thalamocortical loops.

Thalamocortical loops have a central role in cognition and motor control, but precisely how they contribute to these processes is unclear. Recent studies showing evidence of plasticity in thalamocortical synapses indicate a role for the thalamus in shaping cortical dynamics through learning. Since signals undergo a compression from the cortex to the thalamus, we hypothesized that the computational role of the thalamus depends critically on the structure of corticothalamic connectivity. To test this, we identified the optimal corticothalamic structure that promotes biologically plausible learning in thalamocortical synapses. We found that corticothalamic projections specialized to communicate an efference copy of the cortical output benefit motor control, while communicating the modes of highest variance is optimal for working memory tasks. We analyzed neural recordings from mice performing grasping and delayed discrimination tasks and found corticothalamic communication consistent with these predictions. These results suggest that the thalamus orchestrates cortical dynamics in a functionally precise manner through structured connectivity.

Motor cortical output for skilled forelimb movement is selectively distributed across projection neuron classes.

The interaction of descending neocortical outputs and subcortical premotor circuits is critical for shaping skilled movements. Two broad classes of motor cortical output projection neurons provide input to many subcortical motor areas: pyramidal tract (PT) neurons, which project throughout the neuraxis, and intratelencephalic (IT) neurons, which project within the cortex and subcortical striatum. It is unclear whether these classes are functionally in series or whether each class carries distinct components of descending motor control signals. Here, we combine large-scale neural recordings across all layers of motor cortex with cell type-specific perturbations to study cortically dependent mouse motor behaviors: kinematically variable manipulation of a joystick and a kinematically precise reach-to-grasp. We find that striatum-projecting IT neuron activity preferentially represents amplitude, whereas pons-projecting PT neurons preferentially represent the variable direction of forelimb movements. Thus, separable components of descending motor cortical commands are distributed across motor cortical projection cell classes.

A genetically defined insula-brainstem circuit selectively controls motivational vigor.

The anterior insular cortex (aIC) plays a critical role in cognitive and motivational control of behavior, but the underlying neural mechanism remains elusive. Here, we show that aIC neurons expressing Fezf2 (aICFezf2), which are the pyramidal tract neurons, signal motivational vigor and invigorate need-seeking behavior through projections to the brainstem nucleus tractus solitarii (NTS). aICFezf2 neurons and their postsynaptic NTS neurons acquire anticipatory activity through learning, which encodes the perceived value and the vigor of actions to pursue homeostatic needs. Correspondingly, aIC → NTS circuit activity controls vigor, effort, and striatal dopamine release but only if the action is learned and the outcome is needed. Notably, aICFezf2 neurons do not represent taste or valence. Moreover, aIC → NTS activity neither drives reinforcement nor influences total consumption. These results pinpoint specific functions of aIC → NTS circuit for selectively controlling motivational vigor and suggest that motivation is subserved, in part, by aIC's top-down regulation of dopamine signaling.

Genetically identified amygdala-striatal circuits for valence-specific behaviors.

The basolateral amygdala (BLA) plays essential roles in behaviors motivated by stimuli with either positive or negative valence, but how it processes motivationally opposing information and participates in establishing valence-specific behaviors remains unclear. Here, by targeting Fezf2-expressing neurons in the BLA, we identify and characterize two functionally distinct classes in behaving mice, the negative-valence neurons and positive-valence neurons, which innately represent aversive and rewarding stimuli, respectively, and through learning acquire predictive responses that are essential for punishment avoidance or reward seeking. Notably, these two classes of neurons receive inputs from separate sets of sensory and limbic areas, and convey punishment and reward information through projections to the nucleus accumbens and olfactory tubercle, respectively, to drive negative and positive reinforcement. Thus, valence-specific BLA neurons are wired with distinctive input-output structures, forming a circuit framework that supports the roles of the BLA in encoding, learning and executing valence-specific motivated behaviors.

Disrupting cortico-cerebellar communication impairs dexterity.

To control reaching, the nervous system must generate large changes in muscle activation to drive the limb toward the target, and must also make smaller adjustments for precise and accurate behavior. Motor cortex controls the arm through projections to diverse targets across the central nervous system, but it has been challenging to identify the roles of cortical projections to specific targets. Here, we selectively disrupt cortico-cerebellar communication in the mouse by optogenetically stimulating the pontine nuclei in a cued reaching task. This perturbation did not typically block movement initiation, but degraded the precision, accuracy, duration, or success rate of the movement. Correspondingly, cerebellar and cortical activity during movement were largely preserved, but differences in hand velocity between control and stimulation conditions predicted from neural activity were correlated with observed velocity differences. These results suggest that while the total output of motor cortex drives reaching, the cortico-cerebellar loop makes small adjustments that contribute to the successful execution of this dexterous movement.

A cerebellar-thalamocortical pathway drives behavioral context-dependent movement initiation.

Executing learned motor behaviors often requires the transformation of sensory cues into patterns of motor commands that generate appropriately timed actions. The cerebellum and thalamus are two key areas involved in shaping cortical output and movement, but the contribution of a cerebellar-thalamocortical pathway to voluntary movement initiation remains poorly understood. Here, we investigated how an auditory "go cue" transforms thalamocortical activity patterns and how these changes relate to movement initiation. Population responses in dentate/interpositus-recipient regions of motor thalamus reflect a time-locked increase in activity immediately prior to movement initiation that is temporally uncoupled from the go cue, indicative of a fixed-latency feedforward motor timing signal. Blocking cerebellar or motor thalamic output suppresses movement initiation, while stimulation triggers movements in a behavioral context-dependent manner. Our findings show how cerebellar output, via the thalamus, shapes cortical activity patterns necessary for learned context-dependent movement initiation.

Neuropixels 2.0: A miniaturized high-density probe for stable, long-term brain recordings.

Measuring the dynamics of neural processing across time scales requires following the spiking of thousands of individual neurons over milliseconds and months. To address this need, we introduce the Neuropixels 2.0 probe together with newly designed analysis algorithms. The probe has more than 5000 sites and is miniaturized to facilitate chronic implants in small mammals and recording during unrestrained behavior. High-quality recordings over long time scales were reliably obtained in mice and rats in six laboratories. Improved site density and arrangement combined with newly created data processing methods enable automatic post hoc correction for brain movements, allowing recording from the same neurons for more than 2 months. These probes and algorithms enable stable recordings from thousands of sites during free behavior, even in small animals such as mice.

Parvalbumin+ and Npas1+ Pallidal Neurons Have Distinct Circuit Topology and Function.

The external globus pallidus (GPe) is a critical node within the basal ganglia circuit. Phasic changes in the activity of GPe neurons during movement and their alterations in Parkinson's disease (PD) argue that the GPe is important in motor control. Parvalbumin-positive (PV+) neurons and Npas1+ neurons are the two principal neuron classes in the GPe. The distinct electrophysiological properties and axonal projection patterns argue that these two neuron classes serve different roles in regulating motor output. However, the causal relationship between GPe neuron classes and movement remains to be established. Here, by using optogenetic approaches in mice (both males and females), we showed that PV+ neurons and Npas1+ neurons promoted and suppressed locomotion, respectively. Moreover, PV+ neurons and Npas1+ neurons are under different synaptic influences from the subthalamic nucleus (STN). Additionally, we found a selective weakening of STN inputs to PV+ neurons in the chronic 6-hydroxydopamine lesion model of PD. This finding reinforces the idea that the reciprocally connected GPe-STN network plays a key role in disease symptomatology and thus provides the basis for future circuit-based therapies.SIGNIFICANCE STATEMENT The external pallidum is a key, yet an understudied component of the basal ganglia. Neural activity in the pallidum goes awry in neurologic diseases, such as Parkinson's disease. While this strongly argues that the pallidum plays a critical role in motor control, it has been difficult to establish the causal relationship between pallidal activity and motor function/dysfunction. This was in part because of the cellular complexity of the pallidum. Here, we showed that the two principal neuron types in the pallidum have opposing roles in motor control. In addition, we described the differences in their synaptic influence. Importantly, our research provides new insights into the cellular and circuit mechanisms that explain the hypokinetic features of Parkinson's disease.

A Genetically Defined Compartmentalized Striatal Direct Pathway for Negative Reinforcement.

The striosome compartment within the dorsal striatum has been implicated in reinforcement learning and regulation of motivation, but how striosomal neurons contribute to these functions remains elusive. Here, we show that a genetically identified striosomal population, which expresses the Teashirt family zinc finger 1 (Tshz1) and belongs to the direct pathway, drives negative reinforcement and is essential for aversive learning in mice. Contrasting a "conventional" striosomal direct pathway, the Tshz1 neurons cause aversion, movement suppression, and negative reinforcement once activated, and they receive a distinct set of synaptic inputs. These neurons are predominantly excited by punishment rather than reward and represent the anticipation of punishment or the motivation for avoidance. Furthermore, inhibiting these neurons impairs punishment-based learning without affecting reward learning or movement. These results establish a major role of striosomal neurons in behaviors reinforced by punishment and moreover uncover functions of the direct pathway unaccounted for in classic models.

Cell-Type-Specific Outcome Representation in the Primary Motor Cortex.

Adaptive movements are critical for animal survival. To guide future actions, the brain monitors various outcomes, including achievement of movement and appetitive goals. The nature of these outcome signals and their neuronal and network realization in the motor cortex (M1), which directs skilled movements, is largely unknown. Using a dexterity task, calcium imaging, optogenetic perturbations, and behavioral manipulations, we studied outcome signals in the murine forelimb M1. We found two populations of layer 2-3 neurons, termed success- and failure-related neurons, that develop with training, and report end results of trials. In these neurons, prolonged responses were recorded after success or failure trials independent of reward and kinematics. In addition, the initial state of layer 5 pyramidal tract neurons contained a memory trace of the previous trial's outcome. Intertrial cortical activity was needed to learn new task requirements. These M1 layer-specific performance outcome signals may support reinforcement motor learning of skilled behavior.

Cortical pattern generation during dexterous movement is input-driven.

The motor cortex controls skilled arm movement by sending temporal patterns of activity to lower motor centres1. Local cortical dynamics are thought to shape these patterns throughout movement execution2-4. External inputs have been implicated in setting the initial state of the motor cortex5,6, but they may also have a pattern-generating role. Here we dissect the contribution of local dynamics and inputs to cortical pattern generation during a prehension task in mice. Perturbing cortex to an aberrant state prevented movement initiation, but after the perturbation was released, cortex either bypassed the normal initial state and immediately generated the pattern that controls reaching or failed to generate this pattern. The difference in these two outcomes was probably a result of external inputs. We directly investigated the role of inputs by inactivating the thalamus; this perturbed cortical activity and disrupted limb kinematics at any stage of the movement. Activation of thalamocortical axon terminals at different frequencies disrupted cortical activity and arm movement in a graded manner. Simultaneous recordings revealed that both thalamic activity and the current state of cortex predicted changes in cortical activity. Thus, the pattern generator for dexterous arm movement is distributed across multiple, strongly interacting brain regions.

Publisher Correction: A repeated molecular architecture across thalamic pathways.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A repeated molecular architecture across thalamic pathways.

The thalamus is the central communication hub of the forebrain and provides the cerebral cortex with inputs from sensory organs, subcortical systems and the cortex itself. Multiple thalamic regions send convergent information to each cortical region, but the organizational logic of thalamic projections has remained elusive. Through comprehensive transcriptional analyses of retrogradely labeled thalamic neurons in adult mice, we identify three major profiles of thalamic pathways. These profiles exist along a continuum that is repeated across all major projection systems, such as those for vision, motor control and cognition. The largest component of gene expression variation in the mouse thalamus is topographically organized, with features conserved in humans. Transcriptional differences between these thalamic neuronal identities are tied to cellular features that are critical for function, such as axonal morphology and membrane properties. Molecular profiling therefore reveals covariation in the properties of thalamic pathways serving all major input modalities and output targets, thus establishing a molecular framework for understanding the thalamus.

Reconstruction of 1,000 Projection Neurons Reveals New Cell Types and Organization of Long-Range Connectivity in the Mouse Brain.

Neuronal cell types are the nodes of neural circuits that determine the flow of information within the brain. Neuronal morphology, especially the shape of the axonal arbor, provides an essential descriptor of cell type and reveals how individual neurons route their output across the brain. Despite the importance of morphology, few projection neurons in the mouse brain have been reconstructed in their entirety. Here we present a robust and efficient platform for imaging and reconstructing complete neuronal morphologies, including axonal arbors that span substantial portions of the brain. We used this platform to reconstruct more than 1,000 projection neurons in the motor cortex, thalamus, subiculum, and hypothalamus. Together, the reconstructed neurons constitute more than 85 meters of axonal length and are available in a searchable online database. Axonal shapes revealed previously unknown subtypes of projection neurons and suggest organizational principles of long-range connectivity.

Molecular Logic of Spinocerebellar Tract Neuron Diversity and Connectivity.

Coordinated motor behaviors depend on feedback communication between peripheral sensory systems and central circuits in the brain and spinal cord. Relay of muscle- and tendon-derived sensory information to the CNS is facilitated by functionally and anatomically diverse groups of spinocerebellar tract neurons (SCTNs), but the molecular logic by which SCTN diversity and connectivity is achieved is poorly understood. We used single-cell RNA sequencing and genetic manipulations to define the mechanisms governing the molecular profile and organization of SCTN subtypes. We found that SCTNs relaying proprioceptive sensory information from limb and axial muscles are generated through segmentally restricted actions of specific Hox genes. Loss of Hox function disrupts SCTN-subtype-specific transcriptional programs, leading to defects in the connections between proprioceptive sensory neurons, SCTNs, and the cerebellum. These results indicate that Hox-dependent genetic programs play essential roles in the assembly of neural circuits necessary for communication between the brain and spinal cord.

Mapping the transcriptional diversity of genetically and anatomically defined cell populations in the mouse brain.

Understanding the principles governing neuronal diversity is a fundamental goal for neuroscience. Here, we provide an anatomical and transcriptomic database of nearly 200 genetically identified cell populations. By separately analyzing the robustness and pattern of expression differences across these cell populations, we identify two gene classes contributing distinctly to neuronal diversity. Short homeobox transcription factors distinguish neuronal populations combinatorially, and exhibit extremely low transcriptional noise, enabling highly robust expression differences. Long neuronal effector genes, such as channels and cell adhesion molecules, contribute disproportionately to neuronal diversity, based on their patterns rather than robustness of expression differences. By linking transcriptional identity to genetic strains and anatomical atlases, we provide an extensive resource for further investigation of mouse neuronal cell types.

Author Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

The version of this paper originally published cited a preprint version of ref. 12 instead of the published version (Proc. Natl. Acad. Sci. USA 115, 5594-5599; 2018), which was available before this Nature Methods paper went to press. The reference information has been updated in the PDF and HTML versions of the article.

Publisher Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors' original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.

Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution.

Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

Developmental pattern and structural factors of dendritic survival in cerebellar granule cells in vivo.

Granule cells (GCs) in the cerebellar cortex are important for sparse encoding of afferent sensorimotor information. Modeling studies show that GCs can perform their function most effectively when they have four dendrites. Indeed, mature GCs have four short dendrites on average, each terminating in a claw-like ending that receives both excitatory and inhibitory inputs. Immature GCs, however, have significantly more dendrites-all without claws. How these redundant dendrites are refined during development is largely unclear. Here, we used in vivo time-lapse imaging and immunohistochemistry to study developmental refinement of GC dendritic arbors and its relation to synapse formation. We found that while the formation of dendritic claws stabilized the dendrites, the selection of surviving dendrites was made before claw formation, and longer immature dendrites had a significantly higher chance of survival than shorter dendrites. Using immunohistochemistry, we show that glutamatergic and GABAergic synapses are transiently formed on immature GC dendrites, and the number of GABAergic, but not glutamatergic, synapses correlates with the length of immature dendrites. Together, these results suggest a potential role of transient GABAergic synapses on dendritic selection and show that preselected dendrites are stabilized by the formation of dendritic claws-the site of mature synapses.