Persistent and ramping neural activity in the frontal cortex anticipates specific movements. Preparatory activity is distributed across several brain regions, 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 movement; however, roles for this structure in cognitive processes have also been proposed. In humans, cerebellar damage can cause defects in planning and working memory. 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 regions, and that cerebellar computations extend beyond online motor control.

Over hundreds of millions of years, evolution has optimized brain design to maximize its functionality while minimizing costs associated with building and maintenance. This observation suggests that one can use optimization theory to rationalize various features of brain design. Here, we attempt to explain the dimensions and branching structure of dendritic arbors by minimizing dendritic cost for given potential synaptic connectivity. Assuming only that dendritic cost increases with total dendritic length and path length from synapses to soma, we find that branching, planar, and compact dendritic arbors, such as those belonging to Purkinje cells in the cerebellum, are optimal. The theory predicts that adjacent Purkinje dendritic arbors should spatially segregate. In addition, we propose two explicit cost function expressions, falsifiable by measuring dendritic caliber near bifurcations.

Fluorescent calcium indicator proteins, such as GCaMP3, allow imaging of activity in genetically defined neuronal populations. GCaMP3 can be expressed using various gene delivery methods, such as viral infection or electroporation. However, these methods are invasive and provide inhomogeneous and nonstationary expression. Here, we developed a genetic reporter mouse, Ai38, which expresses GCaMP3 in a Cre-dependent manner from the ROSA26 locus, driven by a strong CAG promoter. Crossing Ai38 with appropriate Cre mice produced robust GCaMP3 expression in defined cell populations in the retina, cortex, and cerebellum. In the primary visual cortex, visually evoked GCaMP3 signals showed normal orientation and direction selectivity. GCaMP3 signals were rapid, compared with virally expressed GCaMP3 and synthetic calcium indicators. In the retina, Ai38 allowed imaging spontaneous calcium waves in starburst amacrine cells during development, and light-evoked responses in ganglion cells in adult tissue. Our results show that the Ai38 reporter mouse provides a flexible method for targeted expression of GCaMP3.

Intrinsically generated, brainwide neural activity displays macroscopic coordination among large populations of neurons that persists beyond the biophysical timescales of individual neurons1-3. It is not well understood how these macroscopic behaviours arise from microscopic, short-lived interactions between pairs of neurons. Here we show that the eigenvalue spectrum and dynamical properties of large-scale neural recordings in mice are similar to those produced by linear dynamics governed by a random symmetric matrix that is critically normalized. An exception was population activity in hippocampal area CA1, which resembled an efficient, uncorrelated neural code that may be optimized for information storage capacity. High-dimensional, global activity modes emerged in critically normalized artificial networks and persisted under sparse, clustered or spatial connectivity. These dynamics were useful for solving time-dependent tasks such as a zero-shot working memory task.

Mitochondria are highly dynamic organelles that play multiple roles in cells. How mitochondria cooperatively modulate embryonic stem (ES) cell function during development is not fully understood. Global disruption of Ptpmt1, a mitochondrial Pten-like phosphatidylinositol phosphate (PIP) phosphatase, resulted in developmental arrest and postimplantation lethality. Ptpmt1(-/-) blastocysts failed to outgrow, and inner-cell-mass cells failed to thrive. Depletion of Ptpmt1 in conditional knockout ES cells decreased proliferation without affecting energy homeostasis or cell survival. Differentiation of Ptpmt1-depleted ES cells was essentially blocked. This was accompanied by upregulation of cyclin-dependent kinase inhibitors and a significant cell cycle delay. Reintroduction of wild-type but not of catalytically deficient Ptpmt1 C132S or truncated Ptpmt1 lacking the mitochondrial localization signal restored the differentiation capabilities of Ptpmt1 knockout ES cells. Intriguingly, Ptpmt1 is specifically important for stem cells, as ablation of Ptpmt1 in differentiated embryonic fibroblasts did not disturb cellular function. Further analyses demonstrated that oxygen consumption of Ptpmt1-depleted cells was decreased, while glycolysis was concomitantly enhanced. In addition, mitochondrial fusion/dynamics were compromised in Ptpmt1 knockout cells due to accumulation of PIPs. These studies, while establishing a crucial role for Ptpmt1 phosphatase in embryogenesis, reveal a mitochondrial metabolic stress-activated checkpoint in the control of ES cell differentiation.

Fluorescence microscopy applications often require specialized instruments that are optimized for different experimental goals. Here, we present a reconfigurable microscopy module that integrates highly inclined swept tile (HIST) illumination for high-sensitivity single-molecule imaging and line-scanning confocal microscopy for rapid and optically sectioned volumetric acquisition. The system shares major hardware components, including lasers, scanning optics, and detection hardware, while employing unique beam shaping pathways to enable rapid switching between modalities without realignment. We characterize the module performance by measuring the excitation beam profiles, the point spread functions (PSF), and the optical transfer functions (OTF) across 40x, 60x, and 100x magnifications and demonstrate imaging applications including diffraction-limited fixed and live-cell volumetric imaging, fluorescence recovery after photobleaching, and super-resolution DNA-PAINT and single particle tracking (SPT). We also demonstrate the capability to execute multimodal imaging workflows by performing confocal imaging for chromatin density classification correlated with SPT data of nuclear proteins with diverse functions. Together, these results demonstrate a versatile imaging platform capable of supporting complementary fluorescence imaging modalities within a single instrument.

When used in combination, self-labelling protein tags such as Halo, SNAP, and CLIP allow for the simultaneous visualization of proteins across a wide fluorescence spectrum. However, the combination of cell type, ligand binding and fluorescent dye chemistry introduces several variables that need to be determined to achieve orthogonal labelling. The Janelia Cell Culture Shared Resource in collaboration with a Research Scientist, and the Lavis Lab have developed a high throughput cytometry-based assay to determine optimal conditions for various combinations of cell type, ligand and JF dyes.

Dendrites transform local electrical activity into intracellular Ca2+ signals that drive plasticity1,2, yet the voltage→Ca2+ mapping during natural behavior remains poorly defined. Here, we measure this transfer function via simultaneous voltage and Ca2+ imaging throughout the dendritic arbors of hippocampal CA2 pyramidal neurons in behaving mice. Dendritic Ca2+ exhibited a hierarchical activation pattern dominated by back-propagating action potentials: simple spikes primarily drove somatic and proximal Ca2+, whereas complex spikes produced larger somatic Ca2+ signals and propagated farther into distal dendrites, sometimes in a branch-selective manner. Dendrite-restricted co-activation of voltage and Ca2+ without concurrent somatic events was rare. A biophysics-inspired model accurately predicted local Ca2+ transients from local voltage waveforms. Our data and model provide a quantitative understanding of when – and why – dendritic Ca2+ signals in CA2 pyramidal cells arise during behavior.

Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations. VIDEO ABSTRACT.