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.

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.

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.

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.

The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, we mapped the developmental origin of 160 interneurons from seven bilateral neural progenitors (neuroblasts), and identify them in a synapse-scale TEM reconstruction of the larval CNS. We find that lineages concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, we show that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, we demonstrate that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system.

Drosophila show innate olfactory-driven behaviours that are observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate these behaviours are genetically programmed. Despite the numerical simplicity of the fly nervous system, features of the anatomical organization of the fly brain often confound the delineation of these circuits. Here we identify a neural circuit responsive to cVA, a pheromone that elicits sexually dimorphic behaviours. We have combined neural tracing using an improved photoactivatable green fluorescent protein (PA-GFP) with electrophysiology, optical imaging and laser-mediated microlesioning to map this circuit from the activation of sensory neurons in the antennae to the excitation of descending neurons in the ventral nerve cord. This circuit is concise and minimally comprises four neurons, connected by three synapses. Three of these neurons are overtly dimorphic and identify a male-specific neuropil that integrates inputs from multiple sensory systems and sends outputs to the ventral nerve cord. This neural pathway suggests a means by which a single pheromone can elicit different behaviours in the two sexes.

The extraction of directional motion information from changing retinal images is one of the earliest and most important processing steps in any visual system. In the fly optic lobe, two parallel processing streams have been anatomically described, leading from two first-order interneurons, L1 and L2, via T4 and T5 cells onto large, wide-field motion-sensitive interneurons of the lobula plate. Therefore, T4 and T5 cells are thought to have a pivotal role in motion processing; however, owing to their small size, it is difficult to obtain electrical recordings of T4 and T5 cells, leaving their visual response properties largely unknown. We circumvent this problem by means of optical recording from these cells in Drosophila, using the genetically encoded calcium indicator GCaMP5 (ref. 2). Here we find that specific subpopulations of T4 and T5 cells are directionally tuned to one of the four cardinal directions; that is, front-to-back, back-to-front, upwards and downwards. Depending on their preferred direction, T4 and T5 cells terminate in specific sublayers of the lobula plate. T4 and T5 functionally segregate with respect to contrast polarity: whereas T4 cells selectively respond to moving brightness increments (ON edges), T5 cells only respond to moving brightness decrements (OFF edges). When the output from T4 or T5 cells is blocked, the responses of postsynaptic lobula plate neurons to moving ON (T4 block) or OFF edges (T5 block) are selectively compromised. The same effects are seen in turning responses of tethered walking flies. Thus, starting with L1 and L2, the visual input is split into separate ON and OFF pathways, and motion along all four cardinal directions is computed separately within each pathway. The output of these eight different motion detectors is then sorted such that ON (T4) and OFF (T5) motion detectors with the same directional tuning converge in the same layer of the lobula plate, jointly providing the input to downstream circuits and motion-driven behaviours.

Automatic 3D digital reconstruction (tracing) of neurons embedded in noisy microscopic images is challenging, especially when the cell morphology is complex.