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.

A Neural Circuit for the Suppression of Pain by a Competing Need State.

Hunger and pain are two competing signals that individuals must resolve to ensure survival. However, the neural processes that prioritize conflicting survival needs are poorly understood. We discovered that hunger attenuates behavioral responses and affective properties of inflammatory pain without altering acute nociceptive responses. This effect is centrally controlled, as activity in hunger-sensitive agouti-related protein (AgRP)-expressing neurons abrogates inflammatory pain. Systematic analysis of AgRP projection subpopulations revealed that the neural processing of hunger and inflammatory pain converge in the hindbrain parabrachial nucleus (PBN). Strikingly, activity in AgRP → PBN neurons blocked the behavioral response to inflammatory pain as effectively as hunger or analgesics. The anti-nociceptive effect of hunger is mediated by neuropeptide Y (NPY) signaling in the PBN. By investigating the intersection between hunger and pain, we have identified a neural circuit that mediates competing survival needs and uncovered NPY Y1 receptor signaling in the PBN as a target for pain suppression.

Neural circuit basis of placebo pain relief.

Placebo effects are notable demonstrations of mind-body interactions1,2. During pain perception, in the absence of any treatment, an expectation of pain relief can reduce the experience of pain-a phenomenon known as placebo analgesia3-6. However, despite the strength of placebo effects and their impact on everyday human experience and the failure of clinical trials for new therapeutics7, the neural circuit basis of placebo effects has remained unclear. Here we show that analgesia from the expectation of pain relief is mediated by rostral anterior cingulate cortex (rACC) neurons that project to the pontine nucleus (rACC→Pn)-a precerebellar nucleus with no established function in pain. We created a behavioural assay that generates placebo-like anticipatory pain relief in mice. In vivo calcium imaging of neural activity and electrophysiological recordings in brain slices showed that expectations of pain relief boost the activity of rACC→Pn neurons and potentiate neurotransmission in this pathway. Transcriptomic studies of Pn neurons revealed an abundance of opioid receptors, further suggesting a role in pain modulation. Inhibition of the rACC→Pn pathway disrupted placebo analgesia and decreased pain thresholds, whereas activation elicited analgesia in the absence of placebo conditioning. Finally, Purkinje cells exhibited activity patterns resembling those of rACC→Pn neurons during pain-relief expectation, providing cellular-level evidence for a role of the cerebellum in cognitive pain modulation. These findings open the possibility of targeting this prefrontal cortico-ponto-cerebellar pathway with drugs or neurostimulation to treat pain.

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: 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.

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.

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.

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

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

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.

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.

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.

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.

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.

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.

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.

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.

A Brainstem-Spinal Cord Inhibitory Circuit for Mechanical Pain Modulation by GABA and Enkephalins.

Pain thresholds are, in part, set as a function of emotional and internal states by descending modulation of nociceptive transmission in the spinal cord. Neurons of the rostral ventromedial medulla (RVM) are thought to critically contribute to this process; however, the neural circuits and synaptic mechanisms by which distinct populations of RVM neurons facilitate or diminish pain remain elusive. Here we used in vivo opto/chemogenetic manipulations and trans-synaptic tracing of genetically identified dorsal horn and RVM neurons to uncover an RVM-spinal cord-primary afferent circuit controlling pain thresholds. Unexpectedly, we found that RVM GABAergic neurons facilitate mechanical pain by inhibiting dorsal horn enkephalinergic/GABAergic interneurons. We further demonstrate that these interneurons gate sensory inputs and control pain through temporally coordinated enkephalin- and GABA-mediated presynaptic inhibition of somatosensory neurons. Our results uncover a descending disynaptic inhibitory circuit that facilitates mechanical pain, is engaged during stress, and could be targeted to establish higher pain thresholds. VIDEO ABSTRACT.