Cryogenic electron tomography (cryo-ET) has gained increasing interest in recent years due to its ability to image whole cells and subcellular structures in 3D at nanometer resolution in their native environment. However, due to dose restrictions and the inability to acquire high tilt angle images, the reconstructed volumes are noisy and have missing information. Thus, features are unreliable, and precision extraction of the cell boundary is difficult, manual and time intensive. This paper presents an efficient recursive algorithm called BLASTED (Boundary Localization using Adaptive Shape and Texture Discovery) to automatically extract the cell boundary using a conditional random field (CRF) framework in which boundary points and shape are jointly inferred. The algorithm learns the texture of the boundary region progressively, and uses a global shape model and shape-dependent features to propose candidate boundary points on a slice of the membrane. It then updates the shape of that slice by accepting the appropriate candidate points using local spatial clustering, the global shape model, and trained boosted texture classifiers. The BLASTED algorithm segmented the cell membrane over an average of 93% of the length of the cell in 19 difficult cryo-ET datasets.

Dopaminergic neurons in mammals respond to rewards and reward-predicting cues, and are thought to play an important role in learning actions or sensory cues that lead to reward. The anatomical sources of input that drive or modulate such responses are not well understood; these ultimately define the range of behavior to which dopaminergic neurons contribute. Primary rewards are not the immediate objective of all goal-directed behavior. For example, a goal of vocal learning is to imitate vocal-communication signals. Here, we demonstrate activation of dopaminergic neurons in songbirds driven by a basal ganglia region required for vocal learning, area X. Dopaminergic neurons in anesthetized zebra finches respond more strongly to the bird’s own song (BOS) than to other sounds, and area X is critical for these responses. Direct pharmacological modulation of area X output, in the absence of auditory stimulation, is sufficient to bidirectionally modulate the firing rate of dopaminergic neurons. The only known pathway from song control regions to dopaminergic neurons involves a projection from area X to the ventral pallidum (VP), which in turn projects to dopaminergic regions. We show that VP neurons are spontaneously active and inhibited preferentially by BOS, suggesting that area X disinhibits dopaminergic neurons by inhibiting VP. Supporting this model, auditory-response latencies are shorter in area X than VP, and shorter in VP than dopaminergic neurons. Thus, dopaminergic neurons can be disinhibited selectively by complex sensory stimuli via input from the basal ganglia. The functional pathway we identify may allow dopaminergic neurons to contribute to vocal learning.

Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.

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.

Large collections of full-length cDNAs are important resources for genome annotation and functional genomics. We report the creation of a collection of 50 599 full-length cDNA clones from the pea aphid, Acyrthosiphon pisum. Sequencing from 5’ and 3’ ends of the clones generated 97 828 high-quality expressed sequence tags, representing approximately 9000 genes. These sequences were imported to AphidBase and are shown to play crucial roles in both automatic gene prediction and manual annotation. Our detailed analyses demonstrated that the full-length cDNAs can further improve gene models and can even identify novel genes that are not included in the current version of the official gene set. This full-length cDNA collection can be utilized for a wide variety of functional studies, serving as a community resource for the study of the functional genomics of the pea aphid.

Current imaging methods such as Magnetic Resonance Imaging (MRI), Confocal microscopy, Electron Microscopy (EM) or Selective Plane Illumination Microscopy (SPIM) yield three-dimensional (3D) data sets in need of appropriate computational methods for their analysis. The reconstruction, segmentation and registration are best approached from the 3D representation of the data set.

Dopamine is a mediator of the stimulant properties of drugs of abuse, including ethanol, in mammals and in the fruit fly Drosophila. The neural substrates for the stimulant actions of ethanol in flies are not known. We show that a subset of dopamine neurons and their targets, through the action of the D1-like dopamine receptor DopR, promote locomotor activation in response to acute ethanol exposure. A bilateral pair of dopaminergic neurons in the fly brain mediates the enhanced locomotor activity induced by ethanol exposure, and promotes locomotion when directly activated. These neurons project to the central complex ellipsoid body, a structure implicated in regulating motor behaviors. Ellipsoid body neurons are required for ethanol-induced locomotor activity and they express DopR. Elimination of DopR blunts the locomotor activating effects of ethanol, and this behavior can be restored by selective expression of DopR in the ellipsoid body. These data tie the activity of defined dopamine neurons to D1-like DopR-expressing neurons to form a neural circuit that governs acute responding to ethanol.

The pea aphid, Acyrthosiphon pisum, exhibits several environmentally cued, discrete, alternate phenotypes (polyphenisms) during its life cycle. In the case of the reproductive polyphenism, differences in day length determine whether mothers will produce daughters that reproduce either sexually by laying fertilized eggs (oviparous sexual reproduction), or asexually by allowing oocytes to complete embryogenesis within the mother without fertilization (viviparous parthenogenesis). Oocytes and embryos that are produced asexually and develop within the mother develop more rapidly, are yolk-free, and much smaller than oocytes and embryos that are produced sexually. These overt differences suggest that there may be underlying differences in the molecular mechanisms of pattern formation. Indeed, our preliminary comparative gene expression work suggests that there are important differences in the terminal patterning system, involving the Torso pathway, between viviparous and oviparous development. We have so far examined the expression of homologs of torso-like and capicua, members of the Drosophila Torso pathway. We have detected clear differential expression of torso-like and possible differential expression of capicua. Establishing such differences in the expression of patterning genes between these developmental modes is a first step toward understanding how a single genome manages to direct patterning events in such different embryological contexts.

Activation of group I metabotropic glutamate receptors (subtypes mGluR1 and mGluR5) regulates neural activity in a variety of ways. In CA1 pyramidal neurons, activation of group I mGluRs eliminates the post-burst afterhyperpolarization (AHP) and produces an afterdepolarization (ADP) in its place. Here we show that upregulation of Ca(v)2.3 R-type calcium channels is responsible for a component of the ADP lasting several hundred milliseconds. This medium-duration ADP is rapidly and reversibly induced by activation of mGluR5 and requires activation of phospholipase C (PLC) and release of calcium from internal stores. Effects of mGluR activation on subthreshold membrane potential changes are negligible but are large following action potential firing. Furthermore, the medium ADP exhibits a biphasic activity dependence consisting of short-term facilitation and longer-term inhibition. These findings suggest that mGluRs may dramatically alter the firing of CA1 pyramidal neurons via a complex, activity-dependent modulation of Ca(v)2.3 R-type channels that are activated during spiking at physiologically relevant rates and patterns.