Developmental changes in mouse motor skill learning and cortical circuitry.

Learning motor skills requires plasticity in the primary motor cortex (M1), including changes in inhibitory circuitry. But how inhibitory synaptic connections change during skill acquisition and whether this varies over development is not fully understood. This study assesses the normal developmental trajectory of motor learning and then addresses inhibitory connectivity changes after motor learning. We trained mice of both sexes to run on a custom accelerating rotarod at ages from postnatal day (P) 20 to P120, tracking paw position and quantifying time to fall and changes in gait pattern. Performance improved most rapidly between P30-60, while paw position and gait patterns change with learning, though differently between age groups. To address circuit changes, we labeled task-active and task-inactive pyramidal cells with CaMPARI2, a genetically encoded activity marker. We then evoked inhibitory responses (IPSCs) from two major interneuron types: parvalbumin-expressing (PV+) interneurons and somatostatin-expressing (SOM+) interneurons. After one training day, PV-mediated inhibition is greater in task-active cells, while SOM-mediated inhibition is not different. These results suggest early changes in PV-mediated inhibition may support motor skill acquisition in mice. Whether PV-mediated inhibitory changes persist or changes in SOM+ interneuron connections arise later in training remains to be tested.

Symmetry in Frontal But Not Motor and Somatosensory Cortical Projections.

The neocortex and striatum are topographically organized for sensory and motor functions. While sensory and motor areas are lateralized for touch and motor control, respectively, frontal areas are involved in decision-making, where lateralization of function may be less important. This study contrasted the topographic precision of cell-type-specific ipsilateral and contralateral cortical projections while varying the injection site location in transgenic mice of both sexes. While sensory cortical areas had strongly topographic outputs to the ipsilateral cortex and striatum, they were weaker and not as topographically precise to contralateral targets. The motor cortex had somewhat stronger projections but still relatively weak contralateral topography. In contrast, frontal cortical areas had high degrees of topographic similarity for both ipsilateral and contralateral projections to the cortex and striatum. Corticothalamic organization is mainly ipsilateral, with weaker, more medial contralateral projections. Corticostriatal computations might integrate input outside closed basal ganglia loops using contralateral projections, enabling the two hemispheres to act as a unit to converge on one result in motor planning and decision-making.

Dopamine-mediated plasticity preserves excitatory connections to direct pathway striatal projection neurons and motor function in a mouse model of Parkinson's disease.

The cardinal symptoms of Parkinson's disease (PD) such as bradykinesia and akinesia are debilitating, and treatment options remain inadequate. The loss of nigrostriatal dopamine neurons in PD produces motor symptoms by shifting the balance of striatal output from the direct (go) to indirect (no-go) pathway in large part through changes in the excitatory connections and intrinsic excitabilities of the striatal projection neurons (SPNs). Here, we report using two different experimental models that a transient increase in striatal dopamine and enhanced D1 receptor activation, during 6-OHDA dopamine depletion, prevent the loss of mature spines and dendritic arbors on direct pathway projection neurons (dSPNs) and normal motor behavior for up to 5 months. The primary motor cortex and midline thalamic nuclei provide the major excitatory connections to SPNs. Using ChR2-assisted circuit mapping to measure inputs from motor cortex M1 to dorsolateral dSPNs, we observed a dramatic reduction in both experimental model mice and controls following dopamine depletion. Changes in the intrinsic excitabilities of SPNs were also similar to controls following dopamine depletion. Future work will examine thalamic connections to dSPNs. The findings reported here reveal previously unappreciated plasticity mechanisms within the basal ganglia that can be leveraged to treat the motor symptoms of PD.

Long-range inhibitory neurons mediate cortical neurovascular coupling.

To meet the high energy demands of brain function, cerebral blood flow (CBF) parallels changes in neuronal activity by a mechanism known as neurovascular coupling (NVC). However, which neurons play a role in mediating NVC is not well understood. Here, we identify in mice and humans a specific population of cortical GABAergic neurons that co-express neuronal nitric oxide synthase and tachykinin receptor 1 (Tacr1). Through whole-tissue clearing, we demonstrate that Tacr1 neurons extend local and long-range projections across functionally connected cortical areas. We show that whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feedforward excitatory pathways. Additionally, through optogenetic experiments, we demonstrate that Tacr1 neurons are instrumental in mediating CBF through the relaxation of mural cells in a similar fashion to whisker stimulation. Finally, by electron microscopy, we observe that Tacr1 processes contact astrocytic endfeet. These findings suggest that Tacr1 neurons integrate cortical activity to mediate NVC.

Symmetry in frontal but not motor and somatosensory corticocortical and corticostriatal circuitry.

Neocortex and striatum are topographically organized by cortical areas representing sensory and motor functions, where primary cortical areas are generally used as models for other cortical regions. But different cortical areas are specialized for distinct purposes, with sensory and motor areas lateralized for touch and motor control, respectively. Frontal areas are involved in decision making, where lateralization of function may be less important. This study contrasted the topographic precision of ipsilateral and contralateral projections from cortex based on the injection site location. While sensory cortical areas had strongly topographic outputs to ipsilateral cortex and striatum, they were weaker and not as topographically strong to contralateral targets. Motor cortex had somewhat stronger projections, but still relatively weak contralateral topography. In contrast, frontal cortical areas had high degrees of topographic similarity for both ipsilateral and contralateral projections to cortex and striatum. This contralateral connectivity reflects on the pathways in which corticostriatal computations might integrate input outside closed basal ganglia loops, enabling the two hemispheres to act as a single unit and converge on one result in motor planning and decision making.

Correlated Somatosensory Input in Parvalbumin/Pyramidal Cells in Mouse Motor Cortex.

In mammalian cortex, feedforward excitatory connections recruit feedforward inhibition. This is often carried by parvalbumin (PV+) interneurons, which may densely connect to local pyramidal (Pyr) neurons. Whether this inhibition affects all local excitatory cells indiscriminately or is targeted to specific subnetworks is unknown. Here, we test how feedforward inhibition is recruited by using two-channel circuit mapping to excite cortical and thalamic inputs to PV+ interneurons and Pyr neurons to mouse primary vibrissal motor cortex (M1). Single Pyr and PV+ neurons receive input from both cortex and thalamus. Connected pairs of PV+ interneurons and excitatory Pyr neurons receive correlated cortical and thalamic inputs. While PV+ interneurons are more likely to form local connections to Pyr neurons, Pyr neurons are much more likely to form reciprocal connections with PV+ interneurons that inhibit them. This suggests that Pyr and PV ensembles may be organized based on their local and long-range connections, an organization that supports the idea of local subnetworks for signal transduction and processing. Excitatory inputs to M1 can thus target inhibitory networks in a specific pattern which permits recruitment of feedforward inhibition to specific subnetworks within the cortical column.

Organization of Cortical and Thalamic Input to Inhibitory Neurons in Mouse Motor Cortex.

Intracortical inhibition in motor cortex (M1) regulates movement and motor learning. If cortical and thalamic inputs target different inhibitory cell types in different layers, then these afferents may play different roles in regulating M1 output. Using mice of both sexes, we quantified input to two main classes of M1 interneurons, parvalbumin+ (PV+) cells and somatostatin+ (SOM+) cells, using monosynaptic rabies tracing. We then compared anatomic and functional connectivity based on synaptic strength from sensory cortex and thalamus. Functionally, each input innervated M1 interneurons with a unique laminar profile. Different interneuron types were excited in a distinct, complementary manner, suggesting feedforward inhibition proceeds selectively via distinct circuits. Specifically, somatosensory cortex (S1) inputs primarily targeted PV+ neurons in upper layers (L2/3) but SOM+ neurons in middle layers (L5). Somatosensory thalamus [posterior nucleus (PO)] inputs targeted PV+ neurons in middle layers (L5). In contrast to sensory cortical areas, thalamic input to SOM+ neurons was equivalent to that of PV+ neurons. Thus, long-range excitatory inputs target inhibitory neurons in an area and a cell type-specific manner, which contrasts with input to neighboring pyramidal cells. In contrast to feedforward inhibition providing generic inhibitory tone in cortex, circuits are selectively organized to recruit inhibition matched to incoming excitatory circuits.SIGNIFICANCE STATEMENT M1 integrates sensory information and frontal cortical inputs to plan and control movements. Although inputs to excitatory cells are described, the synaptic circuits by which these inputs drive specific types of M1 interneurons are unknown. Anatomical results with rabies tracing and physiological quantification of synaptic strength shows that two main classes of inhibitory cells (PV+ and SOM+ interneurons) both receive substantial cortical and thalamic input, in contrast to interneurons in sensory areas (where thalamic input strongly prefers PV+ interneurons). Further, each input studied targets PV+ and SOM+ interneurons in a different fashion, suggesting that separate, specific circuits exist for recruitment of feedforward inhibition.

Wireless Optogenetic Modulation of Cortical Neurons Enabled by Radioluminescent Nanoparticles.

While offering high-precision control of neural circuits, optogenetics is hampered by the necessity to implant fiber-optic waveguides in order to deliver photons to genetically engineered light-gated neurons in the brain. Unlike laser light, X-rays freely pass biological barriers. Here we show that radioluminescent Gd2(WO4)3:Eu nanoparticles, which absorb external X-rays energy and then downconvert it into optical photons with wavelengths of ∼610 nm, can be used for the transcranial stimulation of cortical neurons expressing red-shifted, ∼590-630 nm, channelrhodopsin ReaChR, thereby promoting optogenetic neural control to the practical implementation of minimally invasive wireless deep brain stimulation.

Circuitry Underlying Experience-Dependent Plasticity in the Mouse Visual System.

Since the discovery of ocular dominance plasticity, neuroscientists have understood that changes in visual experience during a discrete developmental time, the critical period, trigger robust changes in the visual cortex. State-of-the-art tools used to probe connectivity with cell-type-specific resolution have expanded the understanding of circuit changes underlying experience-dependent plasticity. Here, we review the visual circuitry of the mouse, describing projections from retina to thalamus, between thalamus and cortex, and within cortex. We discuss how visual circuit development leads to precise connectivity and identify synaptic loci, which can be altered by activity or experience. Plasticity extends to visual features beyond ocular dominance, involving subcortical and cortical regions, and connections between cortical inhibitory interneurons. Experience-dependent plasticity contributes to the alignment of networks spanning retina to thalamus to cortex. Disruption of this plasticity may underlie aberrant sensory processing in some neurodevelopmental disorders.

Npas1+-Nkx2.1+ Neurons Are an Integral Part of the Cortico-pallido-cortical Loop.

Within the basal ganglia circuit, the external globus pallidus (GPe) is critically involved in motor control. Aside from Foxp2+ neurons and ChAT+ neurons that have been established as unique neuron types, there is little consensus on the classification of GPe neurons. Properties of the remaining neuron types are poorly defined. In this study, we leverage new mouse lines, viral tools, and molecular markers to better define GPe neuron subtypes. We found that Sox6 represents a novel, defining marker for GPe neuron subtypes. Lhx6+ neurons that lack the expression of Sox6 were devoid of both parvalbumin and Npas1. This result confirms previous assertions of the existence of a unique Lhx6+ population. Neurons that arise from the Dbx1+ lineage were similarly abundant in the GPe and displayed a heterogeneous makeup. Importantly, tracing experiments revealed that Npas1+-Nkx2.1+ neurons represent the principal noncholinergic, cortically-projecting neurons. In other words, they form the pallido-cortical arm of the cortico-pallido-cortical loop. Our data further show that pyramidal-tract neurons in the cortex collateralized within the GPe, forming a closed-loop system between the two brain structures. Overall, our findings reconcile some of the discrepancies that arose from differences in techniques or the reliance on preexisting tools. Although spatial distribution and electrophysiological properties of GPe neurons reaffirm the diversification of GPe subtypes, statistical analyses strongly support the notion that these neuron subtypes can be categorized under the two principal neuron classes: PV+ neurons and Npas1+ neurons.SIGNIFICANCE STATEMENT The poor understanding of the neuronal composition in the external globus pallidus (GPe) undermines our ability to interrogate its precise behavioral and disease involvements. In this study, 12 different genetic crosses were used, hundreds of neurons were electrophysiologically characterized, and >100,000 neurons were histologically- and/or anatomically-profiled. Our current study further establishes the segregation of GPe neuron classes and illustrates the complexity of GPe neurons in adult mice. Our results support the idea that Npas1+-Nkx2.1+ neurons are a distinct GPe neuron subclass. By providing a detailed analysis of the organization of the cortico-pallidal-cortical projection, our findings establish the cellular and circuit substrates that can be important for motor function and dysfunction.

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.

Automatic navigation system for the mouse brain.

Identification and delineation of brain regions in histologic mouse brain sections is especially pivotal for many neurogenomics, transcriptomics, proteomics, and connectomics studies, yet this process is prone to observer error and bias. Here we present a novel brain navigation system, named NeuroInfo, whose general principle is similar to that of a global positioning system (GPS) in a car. NeuroInfo automatically navigates an investigator through the complex microscopic anatomy of histologic sections of mouse brains (thereafter: "experimental mouse brain sections"). This is achieved by automatically registering a digital image of an experimental mouse brain section with a three-dimensional (3D) digital mouse brain atlas that is essentially based on the third version of the Allen Mouse Brain Common Coordinate Framework (CCF v3), retrieving graphical region delineations and annotations from the 3D digital mouse brain atlas, and superimposing this information onto the digital image of the experimental mouse brain section on a computer screen. By doing so, NeuroInfo helps in solving the long-standing problem faced by researchers investigating experimental mouse brain sections under a light microscope-that of correctly identifying the distinct brain regions contained within the experimental mouse brain sections. Specifically, NeuroInfo provides an intuitive, readily-available computer microscopy tool to enhance researchers' ability to correctly identify specific brain regions in experimental mouse brain sections. Extensive validation studies of NeuroInfo demonstrated that this novel technology performs remarkably well in accurately delineating regions that are large and/or located in the dorsal parts of mouse brains, independent on whether the sections were imaged with fluorescence or bright-field microscopy. This novel navigation system provides a highly efficient way for registering a digital image of an experimental mouse brain section with the 3D digital mouse brain atlas in a minute and accurate delineation of the image in real-time.

Whole mouse brain reconstruction and registration to a reference atlas with standard histochemical processing of coronal sections.

Advances in molecular neuroanatomical tools have expanded the ability to map in detail connections of specific neuron subtypes in the context of behaviorally driven patterns of neuronal activity. Analysis of such data across the whole mouse brain, registered to a reference atlas, aids in understanding the functional organization of brain circuits related to behavior. A process is described to image mouse brain sections labeled with standard histochemical techniques, reconstruct those images into a whole brain image volume and register those images to the Allen Mouse Brain Common Coordinate Framework. Image analysis tools automate detection of cell bodies and quantification of axon density labeling in the structures in the annotated reference atlas. Examples of analysis are provided for mapping the axonal projections of layer-specific cortical neurons using Cre-dependent AAV vectors and for mapping inputs to such neurons using retrograde transsynaptic tracing with modified rabies viral vectors.

Author Correction: Topographic precision in sensory and motor corticostriatal projections varies across cell type and cortical area.

In the original version of this Article, support provided during initiation of the project was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support from Karel Svoboda, members of the Svoboda lab, and members of Janelia's Vivarium staff.

Dual-Channel Photostimulation for Independent Excitation of Two Populations.

Manipulation of defined neurons using excitatory opsins, including channelrhodopsin, enables studies of connectivity and the functional role of these circuit components in the brain. These techniques are vital in the neocortex, where diverse neurons are intermingled, and stimulation of specific cell types is difficult without the spatial, temporal, and genetic control available with optogenetic approaches. Channelrhodopsins are effective for mapping excitatory connectivity from one input type to its target. Attempts to use multiple opsins to simultaneously map multiple inputs face the challenge of partially overlapping light spectra for different opsins. This protocol describes one strategy to independently stimulate two comingled inputs in the same brain area to assess convergence and interaction of pathways in neural circuits. This is highly relevant in the neocortex, where pyramidal neurons integrate excitatory inputs from multiple local cell types and long-range corticocortical and thalamocortical projections. © 2018 by John Wiley & Sons, Inc.

Topographic precision in sensory and motor corticostriatal projections varies across cell type and cortical area.

The striatum shows general topographic organization and regional differences in behavioral functions. How corticostriatal topography differs across cortical areas and cell types to support these distinct functions is unclear. This study contrasted corticostriatal projections from two layer 5 cell types, intratelencephalic (IT-type) and pyramidal tract (PT-type) neurons, using viral vectors expressing fluorescent reporters in Cre-driver mice. Corticostriatal projections from sensory and motor cortex are somatotopic, with a decreasing topographic specificity as injection sites move from sensory to motor and frontal areas. Topographic organization differs between IT-type and PT-type neurons, including injections in the same site, with IT-type neurons having higher topographic stereotypy than PT-type neurons. Furthermore, IT-type projections from interconnected cortical areas have stronger correlations in corticostriatal targeting than PT-type projections do. As predicted by a longstanding model, corticostriatal projections of interconnected cortical areas form parallel circuits in the basal ganglia.

Circuit changes in motor cortex during motor skill learning.

Motor cortex is important for motor skill learning, particularly the dexterous skills necessary for our favorite sports and careers. We are especially interested in understanding how plasticity in motor cortex contributes to skill learning. Although human studies have been helpful in understanding the importance of motor cortex in learning skilled tasks, animal models are necessary for achieving a detailed understanding of the circuitry underlying these behaviors and the changes that occur during training. We review data from these models to try to identify sites of plasticity in motor cortex, focusing on rodents asa model system. Rodent neocortex contains well-differentiated motor and sensory regions, as well as neurons expressing similar genetic markers to many of the same circuit components in human cortex. Furthermore, rodents have circuit mapping tools for labeling, targeting, and manipulating these cell types as circuit nodes. Crucially, the projection from rodent primary somatosensory cortex to primary motor cortex is a well-studied corticocortical projection and a model of sensorimotor integration. We first summarize some of the descending pathways involved in making dexterous movements, including reaching. We then describe local and long-range circuitry in mouse motor cortex, summarizing structural and functional changes associated with motor skill acquisition. We then address which specific connections might be responsible for plasticity. For insight into the range of plasticity mechanisms employed by cortex, we review plasticity in sensory systems. The similarities and differences between motor cortex plasticity and critical periods of plasticity in sensory systems are discussed.

Sensorimotor Convergence in Circuitry of the Motor Cortex.

Scientists and philosophers have long appreciated that active somatosensation requires the sensory and motor systems to exchange information about body the body's movements as well as touch in order to accurately interpret incoming somatosensory information and plan future movements. However, the circuitry underlying this sensory and motor integration is complicated and is difficult to study without tools to label specific cellular components in the various brain regions involved. Here, I review the general pathways that convey ascending sensory and descending motor information, using the rodent whisker system as a model to take advantage of the cell type specificity possible in this model. I then detail the circuits in motor cortex in which incoming information from somatosensory cortex and thalamus is integrated. I close with a brief description of changes in these circuits during motor learning.

High-performance probes for light and electron microscopy.

We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.