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.

Using the MS2 system for labeling mRNA, in this issue, Gallardo et al. (2011) find that telomere lengthening depends on a stable accumulation of multiple telomerase complexes in late S phase and that this process is temporally regulated by Rif1/2 proteins.

Mechanosensation, one of the fastest sensory modalities, mediates diverse behaviors including those pertinent for survival. It is important to understand how mechanical stimuli trigger defensive behaviors. Here, we report that Drosophila melanogaster adult flies exhibit a kicking response against invading parasitic mites over their wing margin with ultrafast speed and high spatial precision. Mechanical stimuli that mimic the mites' movement evoke a similar kicking behavior. Further, we identified a TRPV channel, Nanchung, and a specific Nanchung-expressing neuron under each recurved bristle that forms an array along the wing margin as being essential sensory components for this behavior. Our electrophysiological recordings demonstrated that the mechanosensitivity of recurved bristles requires Nanchung and Nanchung-expressing neurons. Together, our results reveal a novel neural mechanism for innate defensive behavior through mechanosensation.

SIGNIFICANCE STATEMENT: We discovered a previously unknown function for recurved bristles on the Drosophila melanogaster wing. We found that when a mite (a parasitic pest for Drosophila) touches the wing margin, the fly initiates a swift and accurate kick to remove the mite. The fly head is dispensable for this behavior. Furthermore, we found that a TRPV channel, Nanchung, and a specific Nanchung-expressing neuron under each recurved bristle are essential for its mechanosensitivity and the kicking behavior. In addition, touching different regions of the wing margin elicits kicking directed precisely at the stimulated region. Our experiments suggest that recurved bristles allow the fly to sense the presence of objects by touch to initiate a defensive behavior (perhaps analogous to touch-evoked scratching; Akiyama et al., 2012).

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.