Brain-wide neural activity underlying memory-guided movement.

Behavior relies on activity in structured neural circuits that are distributed across the brain, but most experiments probe neurons in a single area at a time. Using multiple Neuropixels probes, we recorded from multi-regional loops connected to the anterior lateral motor cortex (ALM), a circuit node mediating memory-guided directional licking. Neurons encoding sensory stimuli, choices, and actions were distributed across the brain. However, choice coding was concentrated in the ALM and subcortical areas receiving input from the ALM in an ALM-dependent manner. Diverse orofacial movements were encoded in the hindbrain; midbrain; and, to a lesser extent, forebrain. Choice signals were first detected in the ALM and the midbrain, followed by the thalamus and other brain areas. At movement initiation, choice-selective activity collapsed across the brain, followed by new activity patterns driving specific actions. Our experiments provide the foundation for neural circuit models of decision-making and movement initiation.

A midbrain-thalamus-cortex circuit reorganizes cortical dynamics to initiate movement.

Motor behaviors are often planned long before execution but only released after specific sensory events. Planning and execution are each associated with distinct patterns of motor cortex activity. Key questions are how these dynamic activity patterns are generated and how they relate to behavior. Here, we investigate the multi-regional neural circuits that link an auditory "Go cue" and the transition from planning to execution of directional licking. Ascending glutamatergic neurons in the midbrain reticular and pedunculopontine nuclei show short latency and phasic changes in spike rate that are selective for the Go cue. This signal is transmitted via the thalamus to the motor cortex, where it triggers a rapid reorganization of motor cortex state from planning-related activity to a motor command, which in turn drives appropriate movement. Our studies show how midbrain can control cortical dynamics via the thalamus for rapid and precise motor behavior.

A comparison of neuronal population dynamics measured with calcium imaging and electrophysiology.

Calcium imaging with fluorescent protein sensors is widely used to record activity in neuronal populations. The transform between neural activity and calcium-related fluorescence involves nonlinearities and low-pass filtering, but the effects of the transformation on analyses of neural populations are not well understood. We compared neuronal spikes and fluorescence in matched neural populations in behaving mice. We report multiple discrepancies between analyses performed on the two types of data, including changes in single-neuron selectivity and population decoding. These were only partially resolved by spike inference algorithms applied to fluorescence. To model the relation between spiking and fluorescence we simultaneously recorded spikes and fluorescence from individual neurons. Using these recordings we developed a model transforming spike trains to synthetic-imaging data. The model recapitulated the differences in analyses. Our analysis highlights challenges in relating electrophysiology and imaging data, and suggests forward modeling as an effective way to understand differences between these data.

Functional clustering of dendritic activity during decision-making.

The active properties of dendrites can support local nonlinear operations, but previous imaging and electrophysiological measurements have produced conflicting views regarding the prevalence and selectivity of local nonlinearities in vivo. We imaged calcium signals in pyramidal cell dendrites in the motor cortex of mice performing a tactile decision task. A custom microscope allowed us to image the soma and up to 300 µm of contiguous dendrite at 15 Hz, while resolving individual spines. New analysis methods were used to estimate the frequency and spatial scales of activity in dendritic branches and spines. The majority of dendritic calcium transients were coincident with global events. However, task-associated calcium signals in dendrites and spines were compartmentalized by dendritic branching and clustered within branches over approximately 10 µm. Diverse behavior-related signals were intermingled and distributed throughout the dendritic arbor, potentially supporting a large learning capacity in individual neurons.

Shared and distinct transcriptomic cell types across neocortical areas.

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.

Distinct descending motor cortex pathways and their roles in movement.

Activity in the motor cortex predicts movements, seconds before they are initiated. This preparatory activity has been observed across cortical layers, including in descending pyramidal tract neurons in layer 5. A key question is how preparatory activity is maintained without causing movement, and is ultimately converted to a motor command to trigger appropriate movements. Here, using single-cell transcriptional profiling and axonal reconstructions, we identify two types of pyramidal tract neuron. Both types project to several targets in the basal ganglia and brainstem. One type projects to thalamic regions that connect back to motor cortex; populations of these neurons produced early preparatory activity that persisted until the movement was initiated. The second type projects to motor centres in the medulla and mainly produced late preparatory activity and motor commands. These results indicate that two types of motor cortex output neurons have specialized roles in motor control.

A cortico-cerebellar loop for motor planning.

Persistent and ramping neural activity in the frontal cortex anticipates specific movements1-6. Preparatory activity is distributed across several brain regions7,8, but it is unclear which brain areas are involved and how this activity is mediated by multi-regional interactions. The cerebellum is thought to be primarily involved in the short-timescale control of movement9-12; however, roles for this structure in cognitive processes have also been proposed13-16. In humans, cerebellar damage can cause defects in planning and working memory13. Here we show that persistent representation of information in the frontal cortex during motor planning is dependent on the cerebellum. Mice performed a sensory discrimination task in which they used short-term memory to plan a future directional movement. A transient perturbation in the medial deep cerebellar nucleus (fastigial nucleus) disrupted subsequent correct responses without hampering movement execution. Preparatory activity was observed in both the frontal cortex and the cerebellar nuclei, seconds before the onset of movement. The silencing of frontal cortex activity abolished preparatory activity in the cerebellar nuclei, and fastigial activity was necessary to maintain cortical preparatory activity. Fastigial output selectively targeted the behaviourally relevant part of the frontal cortex through the thalamus, thus closing a cortico-cerebellar loop. Our results support the view that persistent neural dynamics during motor planning is maintained by neural circuits that span multiple brain regions17, and that cerebellar computations extend beyond online motor control13-15,18.

Fertilizer and Soil Solubility of Secondary P Sources-The Estimation of Their Applicability to Agricultural Soils.

The demand for phosphorus (P) sources is increasing with the growing world population, while objections to direct agricultural use of waste P sources, such as sewage sludge, are being raised. Therefore, the need arises to employ safe and efficient secondary P fertilizer sources, originating from P-rich wastes. These recycling sources are commonly tested in accordance with the current fertilizer rules, designed originally for conventional apatite-based P fertilizers. The behavior of sewage sludge ash, an inorganic recycling secondary P source, was investigated under soil-like conditions. Standardized soil P tests, including the soil buffering capacity test and the Olsen, the Mehlich3, and water extraction methods, were employed together with standardized fertilizer P-solubility tests by neutral ammonium citrate and 2% citric acid extraction. In addition, total content and the overall soil mobility of selected metallic elements present in sewage sludge ash were investigated. The suitability of standardized soil tests for the evaluation of recycling P sources was shown. An apparent influence of Ca:Al content ratio on sewage sludge ash behavior under different soil-like conditions shows the inadequacy of the current fertilizer test and the necessity to understand soil-like behavior of secondary P sources, when considering these as possible agricultural P bearers (fertilizers).

Low-Dimensional and Monotonic Preparatory Activity in Mouse Anterior Lateral Motor Cortex.

Neurons in multiple brain regions fire trains of action potentials anticipating specific movements, but this "preparatory activity" has not been systematically compared across behavioral tasks. We compared preparatory activity in auditory and tactile delayed-response tasks in male mice. Skilled, directional licking was the motor output. The anterior lateral motor cortex (ALM) is necessary for motor planning in both tasks. Multiple features of ALM preparatory activity during the delay epoch were similar across tasks. First, most neurons showed direction-selective activity and spatially intermingled neurons were selective for either movement direction. Second, many cells showed mixed coding of sensory stimulus and licking direction, with a bias toward licking direction. Third, delay activity was monotonic and low-dimensional. Fourth, pairs of neurons with similar direction selectivity showed high spike-count correlations. Our study forms the foundation to analyze the neural circuit mechanisms underlying preparatory activity in a genetically tractable model organism.SIGNIFICANCE STATEMENT Short-term memories link events separated in time. Neurons in the frontal cortex fire trains of action potentials anticipating specific movements, often seconds before the movement. This "preparatory activity" has been observed in multiple brain regions, but has rarely been compared systematically across behavioral tasks in the same brain region. To identify common features of preparatory activity, we developed and compared preparatory activity in auditory and tactile delayed-response tasks in mice. The same cortical area is necessary for both tasks. Multiple features of preparatory activity, measured with high-density silicon probes, were similar across tasks. We find that preparatory activity is low-dimensional and monotonic. Our study forms a foundation for analyzing the circuit mechanisms underlying preparatory activity in a genetically tractable model organism.

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.

Mechanisms underlying a thalamocortical transformation during active tactile sensation.

During active somatosensation, neural signals expected from movement of the sensors are suppressed in the cortex, whereas information related to touch is enhanced. This tactile suppression underlies low-noise encoding of relevant tactile features and the brain's ability to make fine tactile discriminations. Layer (L) 4 excitatory neurons in the barrel cortex, the major target of the somatosensory thalamus (VPM), respond to touch, but have low spike rates and low sensitivity to the movement of whiskers. Most neurons in VPM respond to touch and also show an increase in spike rate with whisker movement. Therefore, signals related to self-movement are suppressed in L4. Fast-spiking (FS) interneurons in L4 show similar dynamics to VPM neurons. Stimulation of halorhodopsin in FS interneurons causes a reduction in FS neuron activity and an increase in L4 excitatory neuron activity. This decrease of activity of L4 FS neurons contradicts the "paradoxical effect" predicted in networks stabilized by inhibition and in strongly-coupled networks. To explain these observations, we constructed a model of the L4 circuit, with connectivity constrained by in vitro measurements. The model explores the various synaptic conductance strengths for which L4 FS neurons actively suppress baseline and movement-related activity in layer 4 excitatory neurons. Feedforward inhibition, in concert with recurrent intracortical circuitry, produces tactile suppression. Synaptic delays in feedforward inhibition allow transmission of temporally brief volleys of activity associated with touch. Our model provides a mechanistic explanation of a behavior-related computation implemented by the thalamocortical circuit.

Maintenance of persistent activity in a frontal thalamocortical loop.

Persistent neural activity maintains information that connects past and future events. Models of persistent activity often invoke reverberations within local cortical circuits, but long-range circuits could also contribute. Neurons in the mouse anterior lateral motor cortex (ALM) have been shown to have selective persistent activity that instructs future actions. The ALM is connected bidirectionally with parts of the thalamus, including the ventral medial and ventral anterior-lateral nuclei. We recorded spikes from the ALM and thalamus during tactile discrimination with a delayed directional response. Here we show that, similar to ALM neurons, thalamic neurons exhibited selective persistent delay activity that predicted movement direction. Unilateral photoinhibition of delay activity in the ALM or thalamus produced contralesional neglect. Photoinhibition of the thalamus caused a short-latency and near-complete collapse of ALM activity. Similarly, photoinhibition of the ALM diminished thalamic activity. Our results show that the thalamus is a circuit hub in motor preparation and suggest that persistent activity requires reciprocal excitation across multiple brain areas.

A Map of Anticipatory Activity in Mouse Motor Cortex.

Activity in the mouse anterior lateral motor cortex (ALM) instructs directional movements, often seconds before movement initiation. It is unknown whether this preparatory activity is localized to ALM or widely distributed within motor cortex. Here we imaged activity across motor cortex while mice performed a whisker-based object localization task with a delayed, directional licking response. During tactile sensation and the delay epoch, object location was represented in motor cortex areas that are medial and posterior relative to ALM, including vibrissal motor cortex. Preparatory activity appeared first in deep layers of ALM, seconds before the behavioral response, and remained localized to ALM until the behavioral response. Later, widely distributed neurons represented the outcome of the trial. Cortical area was more predictive of neuronal selectivity than laminar location or axonal projection target. Motor cortex therefore represents sensory, motor, and outcome information in a spatially organized manner.

Layer 4 fast-spiking interneurons filter thalamocortical signals during active somatosensation.

We rely on movement to explore the environment, for example, by palpating an object. In somatosensory cortex, activity related to movement of digits or whiskers is suppressed, which could facilitate detection of touch. Movement-related suppression is generally assumed to involve corollary discharges. Here we uncovered a thalamocortical mechanism in which cortical fast-spiking interneurons, driven by sensory input, suppress movement-related activity in layer 4 (L4) excitatory neurons. In mice locating objects with their whiskers, neurons in the ventral posteromedial nucleus (VPM) fired in response to touch and whisker movement. Cortical L4 fast-spiking interneurons inherited these responses from VPM. In contrast, L4 excitatory neurons responded mainly to touch. Optogenetic experiments revealed that fast-spiking interneurons reduced movement-related spiking in excitatory neurons, enhancing selectivity for touch-related information during active tactile sensation. These observations suggest a fundamental computation performed by the thalamocortical circuit to accentuate salient tactile information.

Thalamocortical input onto layer 5 pyramidal neurons measured using quantitative large-scale array tomography.

The subcellular locations of synapses on pyramidal neurons strongly influences dendritic integration and synaptic plasticity. Despite this, there is little quantitative data on spatial distributions of specific types of synaptic input. Here we use array tomography (AT), a high-resolution optical microscopy method, to examine thalamocortical (TC) input onto layer 5 pyramidal neurons. We first verified the ability of AT to identify synapses using parallel electron microscopic analysis of TC synapses in layer 4. We then use large-scale array tomography (LSAT) to measure TC synapse distribution on L5 pyramidal neurons in a 1.00 × 0.83 × 0.21 mm(3) volume of mouse somatosensory cortex. We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work. Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 µm compared to a random distribution. Thus, we show that AT is a sensitive and quantitative method to map specific types of synaptic input on the dendrites of entire neurons. We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.

Organization of cortical and thalamic input to pyramidal neurons in mouse motor cortex.

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.

Activity in motor-sensory projections reveals distributed coding in somatosensation.

Cortical-feedback projections to primary sensory areas terminate most heavily in layer 1 (L1) of the neocortex, where they make synapses with tuft dendrites of pyramidal neurons. L1 input is thought to provide 'contextual' information, but the signals transmitted by L1 feedback remain uncharacterized. In the rodent somatosensory system, the spatially diffuse feedback projection from vibrissal motor cortex (vM1) to vibrissal somatosensory cortex (vS1, also known as the barrel cortex) may allow whisker touch to be interpreted in the context of whisker position to compute object location. When mice palpate objects with their whiskers to localize object features, whisker touch excites vS1 and later vM1 in a somatotopic manner. Here we use axonal calcium imaging to track activity in vM1-->vS1 afferents in L1 of the barrel cortex while mice performed whisker-dependent object localization. Spatially intermingled individual axons represent whisker movements, touch and other behavioural features. In a subpopulation of axons, activity depends on object location and persists for seconds after touch. Neurons in the barrel cortex thus have information to integrate movements and touches of multiple whiskers over time, key components of object identification and navigation by active touch.

The functional asymmetry of auditory cortex is reflected in the organization of local cortical circuits.

The primary auditory cortex (A1) is organized tonotopically, with neurons sensitive to high and low frequencies arranged in a rostro-caudal gradient. We used laser scanning photostimulation in acute slices to study the organization of local excitatory connections onto layers 2 and 3 (L2/3) of the mouse A1. Consistent with the organization of other cortical regions, synaptic inputs along the isofrequency axis (orthogonal to the tonotopic axis) arose predominantly within a column. By contrast, we found that local connections along the tonotopic axis differed from those along the isofrequency axis: some input pathways to L3 (but not L2) arose predominantly out-of-column. In vivo cell-attached recordings revealed differences between the sound-responsiveness of neurons in L2 and L3. Our results are consistent with the hypothesis that auditory cortical microcircuitry is specialized to the one-dimensional representation of frequency in the auditory cortex.

Learning-related fine-scale specificity imaged in motor cortex circuits of behaving mice.

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.

The functional properties of barrel cortex neurons projecting to the primary motor cortex.

Nearby neurons, sharing the same locations within the mouse whisker map, can have dramatically distinct response properties. To understand the significance of this diversity, we studied the relationship between the responses of individual neurons and their projection targets in the mouse barrel cortex. Neurons projecting to primary motor cortex (MI) or secondary somatosensory area (SII) were labeled with red fluorescent protein (RFP) using retrograde viral infection. We used in vivo two-photon Ca(2+) imaging to map the responses of RFP-positive and neighboring L2/3 neurons to whisker deflections. Neurons projecting to MI displayed larger receptive fields compared with other neurons, including those projecting to SII. Our findings support the view that intermingled neurons in primary sensory areas send specific stimulus features to different parts of the brain.