The Mind of a Mouse.

Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.

A Map-like Micro-Organization of Grid Cells in the Medial Entorhinal Cortex.

How the topography of neural circuits relates to their function remains unclear. Although topographic maps exist for sensory and motor variables, they are rarely observed for cognitive variables. Using calcium imaging during virtual navigation, we investigated the relationship between the anatomical organization and functional properties of grid cells, which represent a cognitive code for location during navigation. We found a substantial degree of grid cell micro-organization in mouse medial entorhinal cortex: grid cells and modules all clustered anatomically. Within a module, the layout of grid cells was a noisy two-dimensional lattice in which the anatomical distribution of grid cells largely matched their spatial tuning phases. This micro-arrangement of phases demonstrates the existence of a topographical map encoding a cognitive variable in rodents. It contributes to a foundation for evaluating circuit models of the grid cell network and is consistent with continuous attractor models as the mechanism of grid formation.

Geometry of abstract learned knowledge in the hippocampus.

Hippocampal neurons encode physical variables1-7 such as space1 or auditory frequency6 in cognitive maps8. In addition, functional magnetic resonance imaging studies in humans have shown that the hippocampus can also encode more abstract, learned variables9-11. However, their integration into existing neural representations of physical variables12,13 is unknown. Here, using two-photon calcium imaging, we show that individual neurons in the dorsal hippocampus jointly encode accumulated evidence with spatial position in mice performing a decision-making task in virtual reality14-16. Nonlinear dimensionality reduction13 showed that population activity was well-described by approximately four to six latent variables, which suggests that neural activity is constrained to a low-dimensional manifold. Within this low-dimensional space, both physical and abstract variables were jointly mapped in an orderly manner, creating a geometric representation that we show is similar across mice. The existence of conjoined cognitive maps suggests that the hippocampus performs a general computation-the creation of task-specific low-dimensional manifolds that contain a geometric representation of learned knowledge.

Detecting and correcting false transients in calcium imaging.

Population recordings of calcium activity are a major source of insight into neural function. Large datasets require automated processing, but this can introduce errors that are difficult to detect. Here we show that popular time course-estimation algorithms often contain substantial misattribution errors affecting 10-20% of transients. Misattribution, in which fluorescence is ascribed to the wrong cell, arises when overlapping cells and processes are imperfectly defined or not identified. To diagnose misattribution, we develop metrics and visualization tools for evaluating large datasets. To correct time courses, we introduce a robust estimator that explicitly accounts for contaminating signals. In one hippocampal dataset, removing contamination reduced the number of place cells by 15%, and 19% of place fields shifted by over 10 cm. Our methods are compatible with other cell-finding techniques, empowering users to diagnose and correct a potentially widespread problem that could alter scientific conclusions.

Specialized coding of sensory, motor and cognitive variables in VTA dopamine neurons.

There is increased appreciation that dopamine neurons in the midbrain respond not only to reward1 and reward-predicting cues1,2, but also to other variables such as the distance to reward3, movements4-9 and behavioural choices10,11. An important question is how the responses to these diverse variables are organized across the population of dopamine neurons. Whether individual dopamine neurons multiplex several variables, or whether there are subsets of neurons that are specialized in encoding specific behavioural variables remains unclear. This fundamental question has been difficult to resolve because recordings from large populations of individual dopamine neurons have not been performed in a behavioural task with sufficient complexity to examine these diverse variables simultaneously. Here, to address this gap, we used two-photon calcium imaging through an implanted lens to record the activity of more than 300 dopamine neurons from the ventral tegmental area of the mouse midbrain during a complex decision-making task. As mice navigated in a virtual-reality environment, dopamine neurons encoded an array of sensory, motor and cognitive variables. These responses were functionally clustered, such that subpopulations of neurons transmitted information about a subset of behavioural variables, in addition to encoding reward. These functional clusters were spatially organized, with neighbouring neurons more likely to be part of the same cluster. Together with the topography between dopamine neurons and their projections, this specialization and anatomical organization may aid downstream circuits in correctly interpreting the wide range of signals transmitted by dopamine neurons.

Novel stimuli evoke excess activity in the mouse primary visual cortex.

To explore how neural circuits represent novel versus familiar inputs, we presented mice with repeated sets of images with novel images sparsely substituted. Using two-photon calcium imaging to record from layer 2/3 neurons in the mouse primary visual cortex, we found that novel images evoked excess activity in the majority of neurons. This novelty response rapidly emerged, arising with a time constant of 2.6 ± 0.9 s. When a new image set was repeatedly presented, a majority of neurons had similarly elevated activity for the first few presentations, which decayed to steady state with a time constant of 1.4 ± 0.4 s. When we increased the number of images in the set, the novelty response's amplitude decreased, defining a capacity to store ∼15 familiar images under our conditions. These results could be explained quantitatively using an adaptive subunit model in which presynaptic neurons have individual tuning and gain control. This result shows that local neural circuits can create different representations for novel versus familiar inputs using generic, widely available mechanisms.

Mapping of a non-spatial dimension by the hippocampal-entorhinal circuit.

During spatial navigation, neural activity in the hippocampus and the medial entorhinal cortex (MEC) is correlated to navigational variables such as location, head direction, speed, and proximity to boundaries. These activity patterns are thought to provide a map-like representation of physical space. However, the hippocampal-entorhinal circuit is involved not only in spatial navigation, but also in a variety of memory-guided behaviours. The relationship between this general function and the specialized spatial activity patterns is unclear. A conceptual framework reconciling these views is that spatial representation is just one example of a more general mechanism for encoding continuous, task-relevant variables. Here we tested this idea by recording from hippocampal and entorhinal neurons during a task that required rats to use a joystick to manipulate sound along a continuous frequency axis. We found neural representation of the entire behavioural task, including activity that formed discrete firing fields at particular sound frequencies. Neurons involved in this representation overlapped with the known spatial cell types in the circuit, such as place cells and grid cells. These results suggest that common circuit mechanisms in the hippocampal-entorhinal system are used to represent diverse behavioural tasks, possibly supporting cognitive processes beyond spatial navigation.

Oculomotor plant and neural dynamics suggest gaze control requires integration on distributed timescales.

A fundamental principle of biological motor control is that the neural commands driving movement must conform to the response properties of the motor plants they control. In the oculomotor system, characterizations of oculomotor plant dynamics traditionally supported models in which the plant responds to neural drive to extraocular muscles on exclusively short, subsecond timescales. These models predict that the stabilization of gaze during fixations between saccades requires neural drive that approximates eye position on longer timescales and is generated through the temporal integration of brief eye velocity-encoding signals that cause saccades. However, recent measurements of oculomotor plant behaviour have revealed responses on longer timescales. Furthermore, measurements of firing patterns in the oculomotor integrator have revealed a more complex encoding of eye movement dynamics. Yet, the link between these observations has remained unclear. Here we use measurements from the larval zebrafish to link dynamics in the oculomotor plant to dynamics in the neural integrator. The oculomotor plant in both anaesthetized and awake larval zebrafish was characterized by a broad distribution of response timescales, including those much longer than 1 s. Analysis of the firing patterns of oculomotor integrator neurons, which exhibited a broadly distributed range of decay time constants, demonstrates the sufficiency of this activity for stabilizing gaze given an oculomotor plant with distributed response timescales. This work suggests that leaky integration on multiple, distributed timescales by the oculomotor integrator reflects an inverse model for generating oculomotor commands, and that multi-timescale dynamics may be a general feature of motor circuitry. KEY POINTS: Recent observations of oculomotor plant response properties and neural activity across the oculomotor system have called into question classical formulations of both the oculomotor plant and the oculomotor integrator. Here we use measurements from new and published experiments in the larval zebrafish together with modelling to reconcile recent oculomotor plant observations with oculomotor integrator function. We developed computational techniques to characterize oculomotor plant responses over several seconds in awake animals, demonstrating that long timescale responses seen in anaesthetized animals extend to the awake state. Analysis of firing patterns of oculomotor integrator neurons demonstrates the sufficiency of this activity for stabilizing gaze given an oculomotor plant with multiple, distributed response timescales. Our results support a formulation of gaze stabilization by the oculomotor system in which commands for stabilizing gaze are generated through integration on multiple, distributed timescales.

Volumetric two-photon imaging of neurons using stereoscopy (vTwINS).

Two-photon laser scanning microscopy of calcium dynamics using fluorescent indicators is a widely used imaging method for large-scale recording of neural activity in vivo. Here, we introduce volumetric two-photon imaging of neurons using stereoscopy (vTwINS), a volumetric calcium imaging method that uses an elongated, V-shaped point spread function to image a 3D brain volume. Single neurons project to spatially displaced 'image pairs' in the resulting 2D image, and the separation distance between projections is proportional to depth in the volume. To demix the fluorescence time series of individual neurons, we introduce a modified orthogonal matching pursuit algorithm that also infers source locations within the 3D volume. We illustrated vTwINS by imaging neural population activity in the mouse primary visual cortex and hippocampus. Our results demonstrated that vTwINS provides an effective method for volumetric two-photon calcium imaging that increases the number of neurons recorded while maintaining a high frame rate.

Coarse Graining, Fixed Points, and Scaling in a Large Population of Neurons.

We develop a phenomenological coarse-graining procedure for activity in a large network of neurons, and apply this to recordings from a population of 1000+ cells in the hippocampus. Distributions of coarse-grained variables seem to approach a fixed non-Gaussian form, and we see evidence of scaling in both static and dynamic quantities. These results suggest that the collective behavior of the network is described by a nontrivial fixed point.

Membrane potential dynamics of grid cells.

During navigation, grid cells increase their spike rates in firing fields arranged on a markedly regular triangular lattice, whereas their spike timing is often modulated by theta oscillations. Oscillatory interference models of grid cells predict theta amplitude modulations of membrane potential during firing field traversals, whereas competing attractor network models predict slow depolarizing ramps. Here, using in vivo whole-cell recordings, we tested these models by directly measuring grid cell intracellular potentials in mice running along linear tracks in virtual reality. Grid cells had large and reproducible ramps of membrane potential depolarization that were the characteristic signature tightly correlated with firing fields. Grid cells also demonstrated intracellular theta oscillations that influenced their spike timing. However, the properties of theta amplitude modulations were not consistent with the view that they determine firing field locations. Our results support cellular and network mechanisms in which grid fields are produced by slow ramps, as in attractor models, whereas theta oscillations control spike timing.

Choice-specific sequences in parietal cortex during a virtual-navigation decision task.

The posterior parietal cortex (PPC) has an important role in many cognitive behaviours; however, the neural circuit dynamics underlying PPC function are not well understood. Here we optically imaged the spatial and temporal activity patterns of neuronal populations in mice performing a PPC-dependent task that combined a perceptual decision and memory-guided navigation in a virtual environment. Individual neurons had transient activation staggered relative to one another in time, forming a sequence of neuronal activation spanning the entire length of a task trial. Distinct sequences of neurons were triggered on trials with opposite behavioural choices and defined divergent, choice-specific trajectories through a state space of neuronal population activity. Cells participating in the different sequences and at distinct time points in the task were anatomically intermixed over microcircuit length scales (<100 micrometres). During working memory decision tasks, the PPC may therefore perform computations through sequence-based circuit dynamics, rather than long-lived stable states, implemented using anatomically intermingled microcircuits.

Cellular resolution optical access to brain regions in fissures: imaging medial prefrontal cortex and grid cells in entorhinal cortex.

In vivo two-photon microscopy provides the foundation for an array of powerful techniques for optically measuring and perturbing neural circuits. However, challenging tissue properties and geometry have prevented high-resolution optical access to regions situated within deep fissures. These regions include the medial prefrontal and medial entorhinal cortex (mPFC and MEC), which are of broad scientific and clinical interest. Here, we present a method for in vivo, subcellular resolution optical access to the mPFC and MEC using microprisms inserted into the fissures. We chronically imaged the mPFC and MEC in mice running on a spherical treadmill, using two-photon laser-scanning microscopy and genetically encoded calcium indicators to measure network activity. In the MEC, we imaged grid cells, a widely studied cell type essential to memory and spatial information processing. These cells exhibited spatially modulated activity during navigation in a virtual reality environment. This method should be extendable to other brain regions situated within deep fissures, and opens up these regions for study at cellular resolution in behaving animals using a rapidly expanding palette of optical tools for perturbing and measuring network structure and function.

Intracellular dynamics of hippocampal place cells during virtual navigation.

Hippocampal place cells encode spatial information in rate and temporal codes. To examine the mechanisms underlying hippocampal coding, here we measured the intracellular dynamics of place cells by combining in vivo whole-cell recordings with a virtual-reality system. Head-restrained mice, running on a spherical treadmill, interacted with a computer-generated visual environment to perform spatial behaviours. Robust place-cell activity was present during movement along a virtual linear track. From whole-cell recordings, we identified three subthreshold signatures of place fields: an asymmetric ramp-like depolarization of the baseline membrane potential, an increase in the amplitude of intracellular theta oscillations, and a phase precession of the intracellular theta oscillation relative to the extracellularly recorded theta rhythm. These intracellular dynamics underlie the primary features of place-cell rate and temporal codes. The virtual-reality system developed here will enable new experimental approaches to study the neural circuits underlying navigation.

Magnetic voluntary head-fixation in transgenic rats enables lifespan imaging of hippocampal neurons.

The precise neural mechanisms within the brain that contribute to the remarkable lifetime persistence of memory are not fully understood. Two-photon calcium imaging allows the activity of individual cells to be followed across long periods, but conventional approaches require head-fixation, which limits the type of behavior that can be studied. We present a magnetic voluntary head-fixation system that provides stable optical access to the brain during complex behavior. Compared to previous systems that used mechanical restraint, there are no moving parts and animals can engage and disengage entirely at will. This system is failsafe, easy for animals to use and reliable enough to allow long-term experiments to be routinely performed. Animals completed hundreds of trials per session of an odor discrimination task that required 2-4 s fixations. Together with a reflectance fluorescence collection scheme that increases two-photon signal and a transgenic Thy1-GCaMP6f rat line, we are able to reliably image the cellular activity in the hippocampus during behavior over long periods (median 6 months), allowing us track the same neurons over a large fraction of animals' lives (up to 19 months).

Neural circuit models for evidence accumulation through choice-selective sequences.

Decision making is traditionally thought to be mediated by populations of neurons whose firing rates persistently accumulate evidence across time. However, recent decision-making experiments in rodents have observed neurons across the brain that fire sequentially as a function of spatial position or time, rather than persistently, with the subset of neurons in the sequence depending on the animal's choice. We develop two new candidate circuit models, in which evidence is encoded either in the relative firing rates of two competing chains of neurons or in the network location of a stereotyped pattern ("bump") of neural activity. Encoded evidence is then faithfully transferred between neuronal populations representing different positions or times. Neural recordings from four different brain regions during a decision-making task showed that, during the evidence accumulation period, different brain regions displayed tuning curves consistent with different candidate models for evidence accumulation. This work provides mechanistic models and potential neural substrates for how graded-value information may be precisely accumulated within and transferred between neural populations, a set of computations fundamental to many cognitive operations.

Two-photon excitation of channelrhodopsin-2 at saturation.

We demonstrate that channelrhodopsin-2 (CR), a light-gated ion channel that is conventionally activated by using visible-light excitation, can also be activated by using IR two-photon excitation (TPE). An empirical estimate of CR's two-photon absorption cross-section at lambda = 920 nm is presented, with a value (260 +/- 20 GM) indicating that TPE stimulation of CR photocurrents is not typically limited by intrinsic molecular excitability [1 GM = 10(-50)(cm4 s)/photon]. By using direct physiological measurements of CR photocurrents and a model of ground-state depletion, we evaluate how saturation of CR's current-conducting state influences the spatial resolution of focused TPE photostimulation, and how photocurrents stimulated by using low-power scanning TPE temporally summate. We show that TPE, like visible-light excitation, can be used to stimulate action potentials in cultured CR-expressing neurons.

Multiple timescales of sensory-evidence accumulation across the dorsal cortex.

Cortical areas seem to form a hierarchy of intrinsic timescales, but the relevance of this organization for cognitive behavior remains unknown. In particular, decisions requiring the gradual accrual of sensory evidence over time recruit widespread areas across this hierarchy. Here, we tested the hypothesis that this recruitment is related to the intrinsic integration timescales of these widespread areas. We trained mice to accumulate evidence over seconds while navigating in virtual reality and optogenetically silenced the activity of many cortical areas during different brief trial epochs. We found that the inactivation of all tested areas affected the evidence-accumulation computation. Specifically, we observed distinct changes in the weighting of sensory evidence occurring during and before silencing, such that frontal inactivations led to stronger deficits on long timescales than posterior cortical ones. Inactivation of a subset of frontal areas also led to moderate effects on behavioral processes beyond evidence accumulation. Moreover, large-scale cortical Ca2+ activity during task performance displayed different temporal integration windows. Our findings suggest that the intrinsic timescale hierarchy of distributed cortical areas is an important component of evidence-accumulation mechanisms.

In situ X-ray-assisted electron microscopy staining for large biological samples.

Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high-quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here, we present an in situ time-lapsed X-ray-assisted staining procedure that opens the 'black box' of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method, we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in potassium ferrocyanide reduced osmium solution. X-ray-assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards in silico staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray-assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys, or humans.

Sequential and efficient neural-population coding of complex task information.

Recent work has highlighted that many types of variables are represented in each neocortical area. How can these many neural representations be organized together without interference and coherently maintained/updated through time? We recorded from excitatory neural populations in posterior cortices as mice performed a complex, dynamic task involving multiple interrelated variables. The neural encoding implied that highly correlated task variables were represented by less-correlated neural population modes, while pairs of neurons exhibited a spectrum of signal correlations. This finding relates to principles of efficient coding, but notably utilizes neural population modes as the encoding unit and suggests partial whitening of task-specific information where different variables are represented with different signal-to-noise levels. Remarkably, this encoding function was multiplexed with sequential neural dynamics yet reliably followed changes in task-variable correlations throughout the trial. We suggest that neural circuits can implement time-dependent encodings in a simple way using random sequential dynamics as a temporal scaffold.