Molecular Architecture of the Mouse Nervous System.

The mammalian nervous system executes complex behaviors controlled by specialized, precisely positioned, and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse and were grouped by developmental anatomical units and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission, and membrane conductance. We discovered seven distinct, regionally restricted astrocyte types that obeyed developmental boundaries and correlated with the spatial distribution of key glutamate and glycine neurotransmitters. In contrast, oligodendrocytes showed a loss of regional identity followed by a secondary diversification. The resource presented here lays a solid foundation for understanding the molecular architecture of the mammalian nervous system and enables genetic manipulation of specific cell types.

Specialized astrocytes mediate glutamatergic gliotransmission in the CNS.

Multimodal astrocyte-neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4-7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8-10. However, the existence of this mechanism has been questioned11-13 owing to inconsistent data14-17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18-21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.

A transcriptomic axis predicts state modulation of cortical interneurons.

Transcriptomics has revealed that cortical inhibitory neurons exhibit a great diversity of fine molecular subtypes1-6, but it is not known whether these subtypes have correspondingly diverse patterns of activity in the living brain. Here we show that inhibitory subtypes in primary visual cortex (V1) have diverse correlates with brain state, which are organized by a single factor: position along the main axis of transcriptomic variation. We combined in vivo two-photon calcium imaging of mouse V1 with a transcriptomic method to identify mRNA for 72 selected genes in ex vivo slices. We classified inhibitory neurons imaged in layers 1-3 into a three-level hierarchy of 5 subclasses, 11 types and 35 subtypes using previously defined transcriptomic clusters3. Responses to visual stimuli differed significantly only between subclasses, with cells in the Sncg subclass uniformly suppressed, and cells in the other subclasses predominantly excited. Modulation by brain state differed at all hierarchical levels but could be largely predicted from the first transcriptomic principal component, which also predicted correlations with simultaneously recorded cells. Inhibitory subtypes that fired more in resting, oscillatory brain states had a smaller fraction of their axonal projections in layer 1, narrower spikes, lower input resistance and weaker adaptation as determined in vitro7, and expressed more inhibitory cholinergic receptors. Subtypes that fired more during arousal had the opposite properties. Thus, a simple principle may largely explain how diverse inhibitory V1 subtypes shape state-dependent cortical processing.

Tracking neurons across days with high-density probes.

Neural activity spans multiple time scales, from milliseconds to months. Its evolution can be recorded with chronic high-density arrays such as Neuropixels probes, which can measure each spike at tens of sites and record hundreds of neurons. These probes produce vast amounts of data that require different approaches for tracking neurons across recordings. Here, to meet this need, we developed UnitMatch, a pipeline that operates after spike sorting, based only on each unit's average spike waveform. We tested UnitMatch in Neuropixels recordings from the mouse brain, where it tracked neurons across weeks. Across the brain, neurons had distinctive inter-spike interval distributions. Their correlations with other neurons remained stable over weeks. In the visual cortex, the neurons' selectivity for visual stimuli remained similarly stable. In the striatum, however, neuronal responses changed across days during learning of a task. UnitMatch is thus a promising tool to reveal both invariance and plasticity in neural activity across days.

Striatal activity topographically reflects cortical activity.

The cortex projects to the dorsal striatum topographically1,2 to regulate behaviour3-5, but spiking activity in the two structures has previously been reported to have markedly different relations to sensorimotor events6-9. Here we show that the relationship between activity in the cortex and striatum is spatiotemporally precise, topographic, causal and invariant to behaviour. We simultaneously recorded activity across large regions of the cortex and across the width of the dorsal striatum in mice that performed a visually guided task. Striatal activity followed a mediolateral gradient in which behavioural correlates progressed from visual cue to response movement to reward licking. The summed activity in each part of the striatum closely and specifically mirrored activity in topographically associated cortical regions, regardless of task engagement. This relationship held for medium spiny neurons and fast-spiking interneurons, whereas the activity of tonically active neurons differed from cortical activity with stereotypical responses to sensory or reward events. Inactivation of the visual cortex abolished striatal responses to visual stimuli, supporting a causal role of cortical inputs in driving the striatum. Striatal visual responses were larger in trained mice than untrained mice, with no corresponding change in overall activity in the visual cortex. Striatal activity therefore reflects a consistent, causal and scalable topographical mapping of cortical activity.

A modular architecture for organizing, processing and sharing neurophysiology data.

We describe an architecture for organizing, integrating and sharing neurophysiology data within a single laboratory or across a group of collaborators. It comprises a database linking data files to metadata and electronic laboratory notes; a module collecting data from multiple laboratories into one location; a protocol for searching and sharing data and a module for automatic analyses that populates a website. These modules can be used together or individually, by single laboratories or worldwide collaborations.

Spatial connectivity matches direction selectivity in visual cortex.

The selectivity of neuronal responses arises from the architecture of excitatory and inhibitory connections. In the primary visual cortex, the selectivity of a neuron in layer 2/3 for stimulus orientation and direction is thought to arise from intracortical inputs that are similarly selective1-8. However, the excitatory inputs of a neuron can have diverse stimulus preferences1-4,6,7,9, and inhibitory inputs can be promiscuous10 and unselective11. Here we show that the excitatory and inhibitory intracortical connections to a layer 2/3 neuron accord with its selectivity by obeying precise spatial patterns. We used rabies tracing1,12 to label and functionally image the excitatory and inhibitory inputs to individual pyramidal neurons of layer 2/3 of the mouse visual cortex. Presynaptic excitatory neurons spanned layers 2/3 and 4 and were distributed coaxial to the preferred orientation of the postsynaptic neuron, favouring the region opposite to its preferred direction. By contrast, presynaptic inhibitory neurons resided within layer 2/3 and favoured locations near the postsynaptic neuron and ahead of its preferred direction. The direction selectivity of a postsynaptic neuron was unrelated to the selectivity of presynaptic neurons, but correlated with the spatial displacement between excitatory and inhibitory presynaptic ensembles. Similar asymmetric connectivity establishes direction selectivity in the retina13-17. This suggests that this circuit motif might be canonical in sensory processing.

High-dimensional geometry of population responses in visual cortex.

A neuronal population encodes information most efficiently when its stimulus responses are high-dimensional and uncorrelated, and most robustly when they are lower-dimensional and correlated. Here we analysed the dimensionality of the encoding of natural images by large populations of neurons in the visual cortex of awake mice. The evoked population activity was high-dimensional, and correlations obeyed an unexpected power law: the nth principal component variance scaled as 1/n. This scaling was not inherited from the power law spectrum of natural images, because it persisted after stimulus whitening. We proved mathematically that if the variance spectrum was to decay more slowly then the population code could not be smooth, allowing small changes in input to dominate population activity. The theory also predicts larger power-law exponents for lower-dimensional stimulus ensembles, which we validated experimentally. These results suggest that coding smoothness may represent a fundamental constraint that determines correlations in neural population codes.

Distributed coding of choice, action and engagement across the mouse brain.

Vision, choice, action and behavioural engagement arise from neuronal activity that may be distributed across brain regions. Here we delineate the spatial distribution of neurons underlying these processes. We used Neuropixels probes1,2 to record from approximately 30,000 neurons in 42 brain regions of mice performing a visual discrimination task3. Neurons in nearly all regions responded non-specifically when the mouse initiated an action. By contrast, neurons encoding visual stimuli and upcoming choices occupied restricted regions in the neocortex, basal ganglia and midbrain. Choice signals were rare and emerged with indistinguishable timing across regions. Midbrain neurons were activated before contralateral choices and were suppressed before ipsilateral choices, whereas forebrain neurons could prefer either side. Brain-wide pre-stimulus activity predicted engagement in individual trials and in the overall task, with enhanced subcortical but suppressed neocortical activity during engagement. These results reveal organizing principles for the distribution of neurons encoding behaviourally relevant variables across the mouse brain.

NMR crystallization: in situ NMR strategies for monitoring the evolution of crystallization processes.

We present a discussion of the range of NMR techniques that have been utilized for in situ monitoring of crystallization processes, highlighting the opportunities that now exist for exploiting the versatility of NMR techniques to reveal insights into the changes that occur in both the solid phase and the liquid phase as a function of time during crystallization processes from solution. New results are presented from in situ NMR studies of a range of crystallization processes using the CLASSIC NMR strategy and other techniques, specifically covering the following topics: (i) crystallization of glycine from aqueous solution at low temperature, revealing the relatively long-lived existence of a pure phase of the highly meta-stable ß polymorph, (ii) the complementarity of 1H→13C cross-polarization NMR and direct-excitation 13C NMR techniques in probing the evolution of the solid and liquid phases in in situ NMR studies of crystallization processes, (iii) in situ NMR studies of the process of guest exchange between a crystalline host-guest material in contact with the liquid phase of a more favourable type of guest, and (iv) systematic studies of the influence of magic-angle sample spinning on the behaviour of a crystallization system.

Coherent encoding of subjective spatial position in visual cortex and hippocampus.

A major role of vision is to guide navigation, and navigation is strongly driven by vision1-4. Indeed, the brain's visual and navigational systems are known to interact5,6, and signals related to position in the environment have been suggested to appear as early as in the visual cortex6,7. Here, to establish the nature of these signals, we recorded in the primary visual cortex (V1) and hippocampal area CA1 while mice traversed a corridor in virtual reality. The corridor contained identical visual landmarks in two positions, so that a purely visual neuron would respond similarly at those positions. Most V1 neurons, however, responded solely or more strongly to the landmarks in one position rather than the other. This modulation of visual responses by spatial location was not explained by factors such as running speed. To assess whether the modulation is related to navigational signals and to the animal's subjective estimate of position, we trained the mice to lick for a water reward upon reaching a reward zone in the corridor. Neuronal populations in both CA1 and V1 encoded the animal's position along the corridor, and the errors in their representations were correlated. Moreover, both representations reflected the animal's subjective estimate of position, inferred from the animal's licks, better than its actual position. When animals licked in a given location-whether correctly or incorrectly-neural populations in both V1 and CA1 placed the animal in the reward zone. We conclude that visual responses in V1 are controlled by navigational signals, which are coherent with those encoded in hippocampus and reflect the animal's subjective position. The presence of such navigational signals as early as a primary sensory area suggests that they permeate sensory processing in the cortex.

Resiliency among United States Air Force personnel: The direct and interactive influence of cognitive fitness and confidence in social connections.

The United States (U.S.) military has focused on increasing service members' (SM) mental and social fitness to bolster resiliency (successful role performance). The Resiliency Model of Role Performance posits that individual assets and social connections account for SM's differential success in meeting military demands and personal obligations. We used a U.S. Air Force (AF) active-duty dataset to test for a direct, positive relationship between cognitive fitness and both formal and informal social connections, and the impact on successful role performance. We also tested for potential moderating influences of formal and informal social connections on role performance among SMs with low vs. high cognitive fitness. Data were collected from a non-probability purposive sample of AF SMs and civilians (N = 59,094) who completed the Support and Resiliency Inventory between November 4, 2011 and January 7, 2014. We focused on the married active-duty subsample (n = 29,387). We employed multivariate hierarchical regression analysis across three models to explore the direct and interactive influence of cognitive and social fitness on resiliency. Controlling for military demographic characteristics, we found a positive linear relationship between cognitive fitness and resiliency and between informal and formal support and resiliency. Informal social support moderated the association between cognitive fitness and resiliency, compensating for resiliency among SMs with lower cognitive fitness. Study findings support current military resilience-building initiatives and underline the importance of prioritizing informal social support in U.S. military settings.

Probabilistic cell typing enables fine mapping of closely related cell types in situ.

Understanding the function of a tissue requires knowing the spatial organization of its constituent cell types. In the cerebral cortex, single-cell RNA sequencing (scRNA-seq) has revealed the genome-wide expression patterns that define its many, closely related neuronal types, but cannot reveal their spatial arrangement. Here we introduce probabilistic cell typing by in situ sequencing (pciSeq), an approach that leverages previous scRNA-seq classification to identify cell types using multiplexed in situ RNA detection. We applied this method by mapping the inhibitory neurons of mouse hippocampal area CA1, for which ground truth is available from extensive previous work identifying their laminar organization. Our method identified these neuronal classes in a spatial arrangement matching ground truth, and further identified multiple classes of isocortical pyramidal cell in a pattern matching their known organization. This method will allow identifying the spatial organization of closely related cell types across the brain and other tissues.

Fully integrated silicon probes for high-density recording of neural activity.

Sensory, motor and cognitive operations involve the coordinated action of large neuronal populations across multiple brain regions in both superficial and deep structures. Existing extracellular probes record neural activity with excellent spatial and temporal (sub-millisecond) resolution, but from only a few dozen neurons per shank. Optical Ca2+ imaging offers more coverage but lacks the temporal resolution needed to distinguish individual spikes reliably and does not measure local field potentials. Until now, no technology compatible with use in unrestrained animals has combined high spatiotemporal resolution with large volume coverage. Here we design, fabricate and test a new silicon probe known as Neuropixels to meet this need. Each probe has 384 recording channels that can programmably address 960 complementary metal-oxide-semiconductor (CMOS) processing-compatible low-impedance TiN sites that tile a single 10-mm long, 70 × 20-µm cross-section shank. The 6 × 9-mm probe base is fabricated with the shank on a single chip. Voltage signals are filtered, amplified, multiplexed and digitized on the base, allowing the direct transmission of noise-free digital data from the probe. The combination of dense recording sites and high channel count yielded well-isolated spiking activity from hundreds of neurons per probe implanted in mice and rats. Using two probes, more than 700 well-isolated single neurons were recorded simultaneously from five brain structures in an awake mouse. The fully integrated functionality and small size of Neuropixels probes allowed large populations of neurons from several brain structures to be recorded in freely moving animals. This combination of high-performance electrode technology and scalable chip fabrication methods opens a path towards recording of brain-wide neural activity during behaviour.

Dopamine Axons in Dorsal Striatum Encode Contralateral Visual Stimuli and Choices.

The striatum plays critical roles in visually-guided decision-making and receives dense axonal projections from midbrain dopamine neurons. However, the roles of striatal dopamine in visual decision-making are poorly understood. We trained male and female mice to perform a visual decision task with asymmetric reward payoff, and we recorded the activity of dopamine axons innervating striatum. Dopamine axons in the dorsomedial striatum (DMS) responded to contralateral visual stimuli and contralateral rewarded actions. Neural responses to contralateral stimuli could not be explained by orienting behavior such as eye movements. Moreover, these contralateral stimulus responses persisted in sessions where the animals were instructed to not move to obtain reward, further indicating that these signals are stimulus-related. Lastly, we show that DMS dopamine signals were qualitatively different from dopamine signals in the ventral striatum (VS), which responded to both ipsilateral and contralateral stimuli, conforming to canonical prediction error signaling under sensory uncertainty. Thus, during visual decisions, DMS dopamine encodes visual stimuli and rewarded actions in a lateralized fashion, and could facilitate associations between specific visual stimuli and actions.SIGNIFICANCE STATEMENT While the striatum is central to goal-directed behavior, the precise roles of its rich dopaminergic innervation in perceptual decision-making are poorly understood. We found that in a visual decision task, dopamine axons in the dorsomedial striatum (DMS) signaled stimuli presented contralaterally to the recorded hemisphere, as well as the onset of rewarded actions. Stimulus-evoked signals persisted in a no-movement task variant. We distinguish the patterns of these signals from those in the ventral striatum (VS). Our results contribute to the characterization of region-specific dopaminergic signaling in the striatum and highlight a role in stimulus-action association learning.

Biogenic Guanine Crystals Are Solid Solutions of Guanine and Other Purine Metabolites.

Highly reflective crystals of the nucleotide base guanine are widely distributed in animal coloration and visual systems. Organisms precisely control the morphology and organization of the crystals to optimize different optical effects, but little is known about how this is achieved. Here we examine a fundamental question that has remained unanswered after over 100 years of research on guanine: what are the crystals made of? Using solution-state and solid-state chemical techniques coupled with structural analysis by powder XRD and solid-state NMR, we compare the purine compositions and the structures of seven biogenic guanine crystals with different crystal morphologies, testing the hypothesis that intracrystalline dopants influence the crystal shape. We find that biogenic "guanine" crystals are not pure crystals but molecular alloys (aka solid solutions and mixed crystals) of guanine, hypoxanthine, and sometimes xanthine. Guanine host crystals occlude homogeneous mixtures of other purines, sometimes in remarkably large amounts (up to 20% of hypoxanthine), without significantly altering the crystal structure of the guanine host. We find no correlation between the biogenic crystal morphology and dopant content and conclude that dopants do not dictate the crystal morphology of the guanine host. The ability of guanine crystals to host other molecules enables animals to build physiologically "cheaper" crystals from mixtures of metabolically available purines, without impeding optical functionality. The exceptional levels of doping in biogenic guanine offer inspiration for the design of mixed molecular crystals that incorporate multiple functionalities in a single material.

Does [Tf2N]- slither? Equivalence of cation and anion self-diffusion activation volumes in 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide.

New high-pressure self-diffusion data are reported for the ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide ([EMIM][Tf2N]) at pressures up to 363 MPa in the temperature range 288-348 K. The cation and anion activation volumes derived from these are found to be equal at a fixed temperature, within experimental error, in contradiction to a report in the literature that they differ significantly. Self-diffusion activation volumes derived from our earlier high-pressure diffusion studies also show equality for the respective cations and anions of bis(trifluoromethylsulfonyl)amide, tetrafluoroborate and hexafluorophosphate salts with various cations. Stokes-Einstein-Sutherland analysis and density scaling are applied to the [EMIM][Tf2N] self-diffusion measurements and support the conclusion that pressure effects both cation and anion mass (and hence charge) transport in the same way. The density scaling parameters are consistent with the theoretical predictions of Knudsen et al. and agree with that for the viscosity, as for other ionic liquids.

A volumetric and intra-diffusion study of solutions of AlCl3 in two ionic liquids - [C2TMEDA][Tf2N] and [C4mpyr][Tf2N].

Intra-diffusion coefficients (DSi) have been measured for the ionic liquid constituent ions and aluminium-containing species in aluminium chloride (AlCl3) solutions in the ionic liquids 1-(2-dimethyl-aminoethyl)-dimethylethylammonium bis(trifluoromethylsulfonyl)amide ([C2TMEDA][Tf2N]) and N-butyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl)amide ([C4mpyr][Tf2N]), to investigate whether spectroscopically detected interactions between the ions and AlCl3 affect these properties. Such electrolyte solutions are of interest for the electrowinning of aluminium. The temperature, composition and molar volume dependences are investigated. Apparent (Vϕ,1) and partial molar (V1) volumes for AlCl3 have been calculated from solution densities. For [C2TMEDA][Tf2N] solutions, Vϕ,1 increases with increasing solute concentration; for [C4mpyr][Tf2N] solutions, it decreases. In pure [C2TMEDA][Tf2N], the cation diffuses more quickly than the anion, but this changes as the AlCl3 concentration increases. In the [C4mpyr][Tf2N] solutions, the intra-diffusion coefficient ratio remains equal to that for the pure ionic liquid and the aluminium species diffuses at approximately the same rate as the anion at each composition. The intra-diffusion coefficients can be fitted to the Ertl-Dullien free volume power law by superposing the iso-concentration curves with concentration dependent, but temperature independent, molar volume offsets. This suggests that they are primarily dependent on the molar volume and secondarily on a colligative thermodynamic factor due to dilution by AlCl3. AlCl3 complexation by [Tf2N]- and [C2TMEDA]+, confirmed by 27Al, 15N and 19F NMR spectroscopy, seems to play a minor role. Our results indicate that the application of free volume theories might be fruitful in the study of the transport properties of ionic liquid solutions and mixtures.

Thermodynamic or density scaling of the electrical conductivity of molten salts.

Thermodynamic or density scaling of high-pressure conductivities and molar conductivities of the high-temperature molten salts NaOH, and the alkali chlorides, bromides, and nitrates, from Na to Cs, taken from the literature, is found to be consistent with the simulations of Knudsen, Niss, and Bailey (KNB). They used a simple model fluid of point particles interacting through an interionic potential with a repulsive inverse power law part varying as r-9 and an attractive Coulombic part. This yields values between the limits 0.33-3 for the scaling parameter, γ. The Coulombic potential reduces the scaling parameter to values much lower than are normally found for molecular liquids, and KNB used this to explain the low values typically found for ionic liquids. Here, it is shown that the high-temperature molten salts examined behave similarly.