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Neuronal activity evokes a vascular response that is essential to sustain brain function. We show that neurovascular coupling (NVC) is mediated by long-range projecting GABAergic neurons that express Tacr1. Whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feed-forward excitatory pathways. Optogenetic activation of Tacr1 neurons elicited vasodilation, whereas inhibition significantly reduced whisker-evoked hemodynamic responses. Moreover, vasodilation was preceded by capillary pericyte activity, demonstrating a mechanism for NVC.

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 2-channel circuit mapping to excite cortical and thalamic inputs to PV+ interneurons and Pyr neurons in female and male mouse motor cortex. 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 neurons are embedded in local subnetworks. 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.SIGNIFICANCE STATEMENTIncoming sensory information to motor cortex (M1) excites neurons to plan and control movements. This input also recruits feedforward inhibition. Whether inhibition indiscriminately suppresses cortical excitation or forms specific subnetworks is unclear. Specific differences in connectivity in circuits promoting different movements might assist in motor control. We show that input to connected pairs of pyramidal (Pyr) excitatory neurons and parvalbumin (PV+) inhibitory interneurons is more strongly correlated than non-connected pairs, suggesting the integration of interneurons into specific cortical subnetworks. Despite sparse connections between these cells, pyramidal neurons are vastly more likely (3x) to excite PV+ cells connected to them. Thus, inhibition integrates into specific circuits in motor cortex, suggesting that separate, specific circuits exist for recruitment of feedforward inhibition.

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.

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 STATEMENTM1 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.

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 brightfield 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.

Neocortical circuits sit hierarchically atop somatosensory, visual, and auditory sensory processing in the mammalian brain. These circuits are complicated because each cortical area contains a vast range of excitatory and inhibitory cell types and each cell type integrates a variety of local and long-range input. Unraveling this circuitry is essential to understanding how the neocortex works, but this process has been slow due to the absence of means to reliably identify, visualize, record, and manipulate distinct cell types in most species. Furthermore, because the function of different areas across the cortical surface varies, relating cortical activity to function has been challenging. Primary somatosensory cortex has emerged as one model with clear functional representation of incoming information about touch, along with clearly differentiated cellular components in different layers. The connections of these elements and how they develop is the subject of this chapter. This chapter begins by describing what is known about the adult connectivity of the circuitry. It then describes the birth and migration of excitatory and inhibitory neurons of cortex and proceeds to address thalamic innervation, the main ascending input to sensory cortex. A description of function and anatomy of the barrel cortex, the rodent whisker sensory processing area, is added, as many of the rules for development and plasticity of circuits are known from this model system. A description of critical period plasticity in these circuits follows. The chapter closes with a description of adult plasticity.

The basal ganglia constitute a forebrain system associated with affecting motor and other behaviors that involve the cerebral cortex. The principal input nucleus is the striatum, which receives excitatory projections from the cerebral cortex and the intralaminar thalamus as well as neuromodulatory input from midbrain dopamine neurons. Output of the basal ganglia arises from GABAergic inhibitory neurons in the substantia nigra and internal segment of the globus pallidus that project to the thalamus and midbrain motor circuits. Two systems connect the striatum to the output nuclei: a direct pathway from D1 dopamine receptor expressing striatal neurons that project directly to the output nuclei, and an indirect pathway from D2 expressing striatal neurons that project to the external segment of the globus pallidus and connect indirectly to the output nuclei through the subthalamic nucleus. The direct and indirect striatal pathways, respectively, promote and constrain behaviors affected by the basal ganglia.

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.

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.

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.

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 non-cholinergic, cortically-projecting neurons. In other words, they form the pallido-cortical arm of the cortico-pallido-cortical loop. Our data further described 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 pre-existing tools. While 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—i.e., PV+neurons and Npas1+neurons.Significance statementThe poor understanding of the neuronal composition in the GPe undermines our ability to interrogate its precise behavioral and disease involvements. In this study, twelve different genetic crosses were used, hundreds of neurons were electrophysiologically-characterized, and over 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.

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.

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 AAV-Cre dependent vectors and for mapping inputs to such neurons using retrograde trans-synaptic tracing with modified rabies viral vectors.

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.

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., How corticostriatal connections of different pyramidal cell types are organized, particularly in convergent circuits, has not been evaluated in detail. Here, cell type-specific Cre-driver mice reveal that pyramidal tract-type corticostriatal projections, though broadly similar to intratelencephalic-type projections from the same cortical region, are generally more restricted and variable in their topographic termination patterns.

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. Long-range corticostriatal projections from sensory and motor cortex are somatotopic, with a decreasing somatotopic specificity as injections move from sensory to motor and frontal areas. Somatotopic organization differs between IT-type and PT-type neurons, including injections in the same site, with IT-type neurons having higher somatotopic stereotypy than PT-type neurons. Furthermore, IT-type projections from interconnected cortical areas have stronger correlations in corticostriatal targeting than PT-type projections do. Thus, as predicted by a long-standing basal ganglia model, corticostriatal projections of interconnected cortical areas form parallel circuits in basal ganglia-thalamus-cortex loops.

The mammalian nervous system is constructed of many neuronal cell types, but the principles underlying this diversity are poorly understood. To begin to assess brain-wide transcriptional diversity, we sequenced the transcriptomes of the largest collection of genetically and/or anatomically identified neuronal classes from throughout the central nervous system. Using improved expression metrics that distinguish information content from signal-to-noise-ratio, we found that homeobox transcription factors contain the highest information about cell types and have the lowest noise. Non-transcription factors that contribute the most to neuronal diversity tend to be long, due to large introns, and are enriched in genes specifically involved in neuronal function. Genome accessibility measurements reveal that long genes have more candidate regulatory elements arrayed in more distinct patterns. These candidate regulatory elements frequently overlap interspersed repeats and the pattern of repeats is predictive of gene expression. Elongation of neuronal genes by insertions of mobile elements and the resulting new regulatory sites may be an evolutionary force enhancing nervous system complexity.

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 as a 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.

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.

In the mammalian visual system, sensory experience is widely thought to sculpt cortical circuits during a precise critical period. In contrast, subcortical regions, such as the thalamus, were thought to develop at earlier ages in a vision-independent manner. Recent studies at the retinogeniculate synapse, however, have demonstrated an influence of vision on the formation of synaptic circuits in the thalamus. In mice, dark rearing from birth does not alter normal developmental maturation of the connection between retina and thalamus. However, deprivation 20 d after birth [postnatal day 20 (p20)] resulted in dramatic weakening of synaptic strength and an increase in the number of retinal inputs that innervate a thalamic relay neuron. Here, by quantifying changes in synaptic strength and connectivity in response to different time windows of deprivation, we find that several days of vision after eye opening is necessary for triggering experience-dependent plasticity. Shorter periods of visual experience do not permit similar experience-dependent synaptic reorganization. Furthermore, changes in connectivity are rapidly reversible simply by restoring normal vision. However, similar plasticity did not occur when shifting the onset of deprivation to p25. Although synapses still weakened, recruitment of additional retinal inputs no longer occurred. Therefore, synaptic circuits in the visual thalamus are unexpectedly malleable during a late developmental period, after the time when normal synapse elimination and pruning has occurred. This thalamic sensitive period overlaps temporally with experience-dependent changes in the cortex, suggesting that subcortical plasticity may influence cortical responses to sensory experience.

Several psychiatric disorders are associated with white matter defects, suggesting that oligodendrocyte (OL) abnormalities underlie some aspects of these diseases. Neuregulin 1 (NRG1) and its receptor, erbB4, are genetically linked with susceptibility to schizophrenia and bipolar disorder. In vitro studies suggest that NRG1-erbB signaling is important for OL development. To test whether erbB signaling contributes to psychiatric disorders by regulating the structure or function of OLs, we analyzed transgenic mice in which erbB signaling is blocked in OLs in vivo . Here we show that loss of erbB signaling leads to changes in OL number and morphology, reduced myelin thickness, and slower conduction velocity in CNS axons. Furthermore, these transgenic mice have increased levels of dopamine receptors and transporters and behavioral alterations consistent with neuropsychiatric disorders. These results indicate that defects in white matter can cause alterations in dopaminergic function and behavior relevant to neuropsychiatric disorders.

Cortical cells integrate synaptic input from multiple sources, but how these different inputs are distributed across individual neurons is largely unknown. Differences in input might account for diverse responses in neighboring neurons during behavior. We present a strategy for comparing the strengths of multiple types of input onto the same neuron. We developed methods for independent dual-channel photostimulation of synaptic inputs using ChR2 together with ReaChR, a red-shifted channelrhodopsin. We used dual-channel photostimulation to probe convergence of sensory information in the mouse primary motor cortex. Input from somatosensory cortex and thalamus converges in individual neurons. Similarly, inputs from distinct somatotopic regions of the somatosensory cortex are integrated at the level of single motor cortex neurons. We next developed a ReaChR transgenic mouse under the control of both Flp- and Cre-recombinases that is an effective tool for circuit mapping. Our approach to dual-channel photostimulation enables quantitative comparison of the strengths of multiple pathways across all length scales of the brain.

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.

SummarySensory experience and spontaneous activity play important roles in development of sensory circuits; however, their relative contributions are unclear. Here, we test the role of different forms of activity on remodeling of the mouse retinogeniculate synapse. We found that the bulk of maturation occurs without patterned sensory activity over 4 days spanning eye opening. During this early developmental period, blockade of spontaneous retinal activity by tetrodotoxin, but not visual deprivation, retarded synaptic strengthening and inhibited pruning of excess retinal afferents. Later in development, synaptic remodeling becomes sensitive to changes in visually evoked activity, but only if there has been previous visual experience. Synaptic strengthening and pruning were disrupted by visual deprivation following 1 week of vision, but not by chronic deprivation from birth. Thus, spontaneous activity is necessary to drive the bulk of synaptic refinement around the time of eye opening, while sensory experience is important for the subsequent maintenance of connections.

GABAergic terminals of chandelier cells exclusively innervate the axon initial segment (AIS) of excitatory neurons. Although the anatomy of these synapses has been well-studied in several brain areas, relatively little is known about their physiological properties. Using vesicular γ-aminobutyric acid transporter-channelrhodopsin 2-enhanced yellow fluorescence protein (VGAT-ChR2-YFP)-expressing mice and a novel fibreoptic 'laserspritzer' approach that we developed, we investigated the physiological properties of axo-axonic synapses (AASs) in brain slices from the piriform cortex (PC) of mice. AASs were in close proximity to voltage-gated Na(+) (NaV) channels located at the AIS. AASs were selectively activated by a 5 μm laserspritzer placed in close proximity to the AIS. Under a minimal laser stimulation condition and using whole-cell somatic voltage-clamp recordings, the amplitudes and kinetics of IPSCs mediated by AASs were similar to those mediated by perisomatic inhibitions. Results were further validated with channelrhodopsin 2-assisted circuit mapping (CRACM) of the entire inhibitory inputs map. For the first time, we revealed that the laserspritzer-induced AAS-IPSCs persisted in the presence of TTX and TEA but not 4-AP. Next, using gramicidin-based perforated patch recordings, we found that the GABA reversal potential (EGABA) was -73.6 ± 1.2 mV when induced at the AIS and -72.8 ± 1.1 mV when induced at the perisomatic site. Our anatomical and physiological results lead to the novel conclusions that: (1) AASs innervate the entire length of the AIS, as opposed to forming a highly concentrated cartridge, (2) AAS inhibition suppresses action potentials and epileptiform activity more robustly than perisomatic inhibitions, and (3) AAS activation alone can be sufficient to inhibit action potential generation and epileptiform activities in vitro.

Neurophotonics methods offer powerful ways to access neuronal signals and circuits. We highlight recent advances and current themes in this area, emphasizing tools for mapping, monitoring, and manipulating excitatory projection neurons and their synaptic circuits in mouse motor cortex.

Mouse visual cortex is subdivided into multiple distinct, hierarchically organized areas that are interconnected through feedforward (FF) and feedback (FB) pathways. The principal synaptic targets of FF and FB axons that reciprocally interconnect primary visual cortex (V1) with the higher lateromedial extrastriate area (LM) are pyramidal cells (Pyr) and parvalbumin (PV)-expressing GABAergic interneurons. Recordings in slices of mouse visual cortex have shown that layer 2/3 Pyr cells receive excitatory monosynaptic FF and FB inputs, which are opposed by disynaptic inhibition. Most notably, inhibition is stronger in the FF than FB pathway, suggesting pathway-specific organization of feedforward inhibition (FFI). To explore the hypothesis that this difference is due to diverse pathway-specific strengths of the inputs to PV neurons we have performed subcellular Channelrhodopsin-2-assisted circuit mapping in slices of mouse visual cortex. Whole-cell patch-clamp recordings were obtained from retrobead-labeled FFV1→LM- and FBLM→V1-projecting Pyr cells, as well as from tdTomato-expressing PV neurons. The results show that the FFV1→LMpathway provides on average 3.7-fold stronger depolarizing input to layer 2/3 inhibitory PV neurons than to neighboring excitatory Pyr cells. In the FBLM→V1pathway, depolarizing inputs to layer 2/3 PV neurons and Pyr cells were balanced. Balanced inputs were also found in the FFV1→LMpathway to layer 5 PV neurons and Pyr cells, whereas FBLM→V1inputs to layer 5 were biased toward Pyr cells. The findings indicate that FFI in FFV1→LMand FBLM→V1circuits are organized in a pathway- and lamina-specific fashion.

SummaryDuring development, neurons are constantly refining their connections in response to changes in activity. Experience-dependent plasticity is a key form of synaptic plasticity, involving changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) accumulation at synapses. Here, we report a critical role for the AMPAR auxiliary subunit stargazin in this plasticity. We show that stargazin is functional at the retinogeniculate synapse and that in the absence of stargazin, the refinement of the retinogeniculate synapse is specifically disrupted during the experience-dependent phase. Importantly, we found that stargazin expression and phosphorylation increased with visual deprivation and led to reduced AMPAR rectification at the retinogeniculate synapse. To test whether stargazin plays a role in homeostatic plasticity, we turned to cultured neurons and found that stargazin phosphorylation is essential for synaptic scaling. Overall, our data reveal an important role for stargazin in regulating AMPAR abundance and composition at glutamatergic synapses during homeostatic and experience-dependent plasticity.

Determining how long-range synaptic inputs engage pyramidal neurons in primary motor cortex (M1) is important for understanding circuit mechanisms involved in regulating movement. We used channelrhodopsin-2-assisted circuit mapping to characterize the long-range excitatory synaptic connections made by multiple cortical and thalamic areas onto pyramidal neurons in mouse vibrissal motor cortex (vM1). Each projection innervated vM1 pyramidal neurons with a unique laminar profile. Collectively, the profiles for different sources of input partially overlapped and spanned all cortical layers. Specifically, orbital cortex (OC) inputs primarily targeted neurons in L6. Secondary motor cortex (M2) inputs excited neurons mainly in L5B, including pyramidal tract neurons. In contrast, thalamocortical inputs from anterior motor-related thalamic regions, including VA/VL (ventral anterior thalamic nucleus/ventrolateral thalamic nucleus), targeted neurons in L2/3 through L5B, but avoided L6. Inputs from posterior sensory-related thalamic areas, including POm (posterior thalamic nuclear group), targeted neurons only in the upper layers (L2/3 and L5A), similar to inputs from somatosensory (barrel) cortex. Our results show that long-range excitatory inputs target vM1 pyramidal neurons in a layer-specific manner. Inputs from sensory-related cortical and thalamic areas preferentially target the upper-layer pyramidal neurons in vM1. In contrast, inputs from OC and M2, areas associated with volitional and cognitive aspects of movements, bypass local circuitry and have direct monosynaptic access to neurons projecting to brainstem and thalamus.

Over the first few postnatal weeks, extensive remodeling occurs at the developing murine retinogeniculate synapse, the connection between retinal ganglion cells (RGCs) and the visual thalamus. Although numerous studies have described the role of activity in the refinement of this connection, little is known about the mechanisms that regulate glutamate concentration at and around the synapse over development. Here we show that interactions between glutamate transporters and metabotropic glutamate receptors (mGluRs) dynamically control the peak and time course of the excitatory postsynaptic current (EPSC) at the immature synapse. Inhibiting glutamate transporters by bath application of TBOA (dl- threo-β-benzyloxyaspartic acid) prolonged the decay kinetics of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) currents at all ages. Moreover, at the immature synapse, TBOA-induced increases in glutamate concentration led to the activation of group II/III mGluRs and a subsequent reduction in neurotransmitter release at RGC terminals. Inhibition of this negative-feedback mechanism resulted in a small but significant increase in peak NMDAR EPSCs during basal stimulation and a substantial increase in the peak with coapplication of TBOA. Activation of mGluRs also shaped the synaptic response during high-frequency trains of stimulation that mimic spontaneous RGC activity. At the mature synapse, however, the group II mGluRs and the group III mGluR7-mediated response are downregulated. Our results suggest that transporters reduce spillover of glutamate, shielding NMDARs and mGluRs from the neurotransmitter. Furthermore, mechanisms of glutamate clearance and release interact dynamically to control the glutamate transient at the developing retinogeniculate synapse.

We report on wide-field optically detected magnetic resonance imaging of nitrogen-vacancy centers (NVs) in type IIa polycrystalline diamond. These studies reveal a heterogeneous crystalline environment that produces a varied density of NV centers, including preferential orientation within some individual crystal grains, but preserves long spin coherence times. Using the native NVs as nanoscale sensors, we introduce a three-dimensional strain imaging technique with high sensitivity (, Allen Institute for Brain Science, Howard Hughes Medical Institute

Fatigue damage in a mica-containing glass-ceramic is examined using Hertzian contact tests. For the material in its base glass state, such tests indicate that fatigue occurs solely by chemically enhanced cone crack extension. In the glass-ceramic, fatigue is evident as an expansion of a macroscopic subsurface microfracture zone. Comparative observations of the subsurface damage in static and cyclic loading, and tests in different environments, indicate that the fatigue in the glass-ceramic is mechanical in origin, although it is enhanced by moisture. This result is reinforced by load-point-displacement data, which reveal significant hysteresis in the glass-ceramic but not in the base glass. Flexure tests on Hertz-indented glass-ceramic specimens show only a slight loss of strength, 5cycles. This contrasts with the base glass which, although of higher laboratory strength, is subject to abrupt and severe strength degradation from cone crack pop-in. High magnification examination of the subsurface damage in the glass-ceramic suggests the underlying cause of the mechanical fatigue mechanism to be attrition of frictional tractions at closed microcrack interfaces.

Flaw-tolerance and associated toughness-curve ( T -curve) characteristics in SiC/glass particle/matrix composites are studied. Two glass compositions, chosen to produce composites at extremes of high (H) and low (L) thermal expansion mismatch relative to the SiC particles, are investigated. In-situ observations of crack extension from indentation flaws reveal widely different responses: in the L composite the path is relatively undistorted from the planar geometry, with trans-particle fractures; in the H composite the path deflects strongly around the particles, with consequent interfacial bridge formation and activity in the crack wake. Surface fracture patterns produced by spherical indenters confirm the implied transition from trans-particle to inter-particle fracture with increasing internal residual stress, and point to a potential degradation in short-crack properties like wear and fatigue. Indentation-strength measurements also show different characteristics in the two composites: minor flaw tolerance in the L material, consistent with a single-valued, ‘rule of mixtures’ toughness; major tolerance in the H material, consistent with a pronounced T -curve. The T -curves themselves are deconvoluted from the indentation-strength data for each composite and analyzed.

Cortical neurons form specific circuits, but the functional structure of this microarchitecture and its relation to behaviour are poorly understood. Two-photon calcium imaging can monitor activity of spatially defined neuronal ensembles in the mammalian cortex. Here we applied this technique to the motor cortex of mice performing a choice behaviour. Head-fixed mice were trained to lick in response to one of two odours, and to withhold licking for the other odour. Mice routinely showed significant learning within the first behavioural session and across sessions. Microstimulation and trans-synaptic tracing identified two non-overlapping candidate tongue motor cortical areas. Inactivating either area impaired voluntary licking. Imaging in layer 2/3 showed neurons with diverse response types in both areas. Activity in approximately half of the imaged neurons distinguished trial types associated with different actions. Many neurons showed modulation coinciding with or preceding the action, consistent with their involvement in motor control. Neurons with different response types were spatially intermingled. Nearby neurons (within approximately 150 mum) showed pronounced coincident activity. These temporal correlations increased with learning within and across behavioural sessions, specifically for neuron pairs with similar response types. We propose that correlated activity in specific ensembles of functionally related neurons is a signature of learning-related circuit plasticity. Our findings reveal a fine-scale and dynamic organization of the frontal cortex that probably underlies flexible behaviour.

Visual system circuitry, a canonical model system for the study of experience-dependent development, matures before and following the onset of vision. Sensory experience or deprivation during an early critical period results in substantial plasticity and is a crucial factor in establishing the mature circuitry. In adulthood, plasticity has been thought to be reduced or absent. However, recent studies point to the potential for change in neuronal circuits within the mature brain, raising the possibility that aberrant circuit function can be corrected. In this review, we will discuss recent exciting findings in the field of experience-dependent plasticity that advance our understanding of mechanisms underlying the activation, expression, and closure of critical periods in the visual system.

How the developing brain specifies precise neural connectivity has long interested neuroscientists. Because of the immense number of cells and synapses in the CNS, it seems unlikely that each individual cell’s identity and connectivity is intrinsically or genetically specified. Both neural activity and molecular cues provide possible mechanisms by which network properties of the developing brain can emerge from relative disorder. Emphasizing the visual system as a model for synaptic development, we review the role of various factors in synaptic maturation, including sensory activity. Furthermore, we elucidate why the mouse visual system could prove advantageous for investigation of mechanisms governing circuit development.

Optical and electrophysiological tools were used to map out the neural circuits within and between cortical layers in three different brain regions, and the results suggest regional specializations for sensory versus motor information processing., Rodents move their whiskers to locate and identify objects. Cortical areas involved in vibrissal somatosensation and sensorimotor integration include the vibrissal area of the primary motor cortex (vM1), primary somatosensory cortex (vS1; barrel cortex), and secondary somatosensory cortex (S2). We mapped local excitatory pathways in each area across all cortical layers using glutamate uncaging and laser scanning photostimulation. We analyzed these maps to derive laminar connectivity matrices describing the average strengths of pathways between individual neurons in different layers and between entire cortical layers. In vM1, the strongest projection was L2/3→L5. In vS1, strong projections were L2/3→L5 and L4→L3. L6 input and output were weak in both areas. In S2, L2/3→L5 exceeded the strength of the ascending L4→L3 projection, and local input to L6 was prominent. The most conserved pathways were L2/3→L5, and the most variable were L4→L2/3 and pathways involving L6. Local excitatory circuits in different cortical areas are organized around a prominent descending pathway from L2/3→L5, suggesting that sensory cortices are elaborations on a basic motor cortex-like plan., Author Summary The neocortex of the mammalian brain is divided into different regions that serve specific functions. These include sensory areas for vision, hearing, and touch, and motor areas for directing aspects of movement. However, the similarities and differences in local circuit organization between these areas are not well understood. The cortex is a layered structure numbered in an outside-in fashion, such that layer 1 is closest to the cortical surface and layer 6 is deepest. Each layer harbors distinct cell types. The precise circuit wiring within and between these layers allows for specific functions performed by particular cortical regions. To directly compare circuits from distinct cortical areas, we combined optical and electrophysiological tools to map connections between layers in different brain regions. We examined three regions of mouse neocortex that are involved in active whisker sensation: vibrissal motor cortex (vM1), primary somatosensory cortex (vS1), and secondary somatosensory cortex (S2). Our results demonstrate that excitatory connections from layer 2/3 to layer 5 are prominent in all three regions. In contrast, strong ascending pathways from middle layers (layer 4) to superficial ones (layer 3) and local inputs to layer 6 were prominent only in the two sensory cortical areas. These results indicate that cortical circuits employ regional specializations when processing motor versus sensory information. Moreover, our data suggest that sensory cortices are elaborations on a basic motor cortical plan involving layer 2/3 to layer 5 pathways.

SummaryIn the rodent vibrissal system, active sensation and sensorimotor integration are mediated in part by connections between barrel cortex and vibrissal motor cortex. Little is known about how these structures interact at the level of neurons. We used Channelrhodopsin-2 (ChR2) expression, combined with anterograde and retrograde labeling, to map connections between barrel cortex and pyramidal neurons in mouse motor cortex. Barrel cortex axons preferentially targeted upper layer (L2/3, L5A) neurons in motor cortex; input to neurons projecting back to barrel cortex was particularly strong. Barrel cortex input to deeper layers (L5B, L6) of motor cortex, including neurons projecting to the brainstem, was weak, despite pronounced geometric overlap of dendrites with axons from barrel cortex. Neurons in different layers received barrel cortex input within stereotyped dendritic domains. The cortico-cortical neurons in superficial layers of motor cortex thus couple motor and sensory signals and might mediate sensorimotor integration and motor learning.