A striking aspect of cortical neural networks is the divergence of a relatively small number of input channels from the peripheral sensory apparatus into a large number of cortical neurons, an over-complete representation strategy. Cortical neurons are then connected by a sparse network of lateral synapses. Here we propose that such architecture may increase the persistence of the representation of an incoming stimulus, or a percept. We demonstrate that for a family of networks in which the receptive field of each neuron is re-expressed by its outgoing connections, a represented percept can remain constant despite changing activity. We term this choice of connectivity REceptive FIeld REcombination (REFIRE) networks. The sparse REFIRE network may serve as a high-dimensional integrator and a biologically plausible model of the local cortical circuit.

Insect molting is triggered by ecdysteroids, which are produced in the prothoracic glands (PG). The broad (br) gene is one of the ’early genes’ directly regulated by ecdysteroids. Ectopic expression of the BR-Z3 isoform in early second instar Drosophila larvae (L2) before the rise of the ecdysteroid titer prevented molting to the third instar, but the larvae subsequently formed L2 prepupae after prolonged feeding. When these larvae were fed on diet containing 20-hydroxyecdysone (20E), they formed pharate third instar larvae. The critical weight for normal L3 pupariation of w(1118) larvae was found to be 0.8 mg and that for L2 pupariation was 0.45 mg. We also defined a threshold weight for metamorphosis of 0.3 mg, above which L2 larvae will metamorphose when provided with 20E. BR-Z3 apparently works through the PG cells of the ring gland but not the putative neurosecretory cells that drive ecdysone secretion, because ectopic expression of BR-Z3 specifically in the ring gland caused 53% of the larvae to become permanent first instar larvae. Driving other BR isoforms in the ring gland prevented larval molting or pupariation to varying degrees. These molting defects were rescued by feeding 20E. Overexpression of each of the BR isoforms caused degeneration of the PG cells but on different time courses, indicating that BR is a signal for the degeneration of the PG cells that normally occurs during the pupal-adult transition.

Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.

Selection of appropriate oviposition sites is essential for progeny survival and fitness in generalist insect species, such as Drosophila melanogaster, yet little is known about the mechanisms regulating how environmental conditions and innate adult preferences are evaluated and balanced to yield the final substrate choice for egg-deposition. Female D. melanogaster are attracted to food containing acetic acid (AA) as an oviposition substrate. However, our observations reveal that this egg-laying preference is a complex process, as it directly opposes an otherwise strong, default behavior of positional avoidance for the same food. We show that 2 distinct sensory modalities detect AA. Attraction to AA-containing food for the purpose of egg-laying relies on the gustatory system, while positional repulsion depends primarily on the olfactory system. Similarly, distinct central brain regions are involved in AA attraction and repulsion. Given this unique situation, in which a single environmental stimulus yields 2 opposing behavioral outputs, we propose that the interaction of egg-laying attraction and positional aversion for AA provides a powerful model for studying how organisms balance competing behavioral drives and integrate signals involved in choice-like processes.

How brains are hardwired to produce aggressive behavior, and how aggression circuits are related to those that mediate courtship, is not well understood. A large-scale screen for aggression-promoting neurons in Drosophila identified several independent hits that enhanced both inter-male aggression and courtship. Genetic intersections revealed that 8-10 P1 interneurons, previously thought to exclusively control male courtship, were sufficient to promote fighting. Optogenetic experiments indicated that P1 activation could promote aggression at a threshold below that required for wing extension. P1 activation in the absence of wing extension triggered persistent aggression via an internal state that could endure for minutes. High-frequency P1 activation promoted wing extension and suppressed aggression during photostimulation, whereas aggression resumed and wing extension was inhibited following photostimulation offset. Thus, P1 neuron activation promotes a latent, internal state that facilitates aggression and courtship, and controls the overt expression of these social behaviors in a threshold-dependent, inverse manner.

Live-cell fluorescence light microscopy has emerged as an important tool in the study of cellular biology. The development of fluorescent markers in parallel with super-resolution imaging systems has pushed light microscopy into the realm of molecular visualization at the nanometer scale. Resolutions previously only attained with electron microscopes are now within the grasp of light microscopes. However, until recently, live-cell imaging approaches have eluded super-resolution microscopy, hampering it from reaching its full potential for revealing the dynamic interactions in biology occurring at the single molecule level. Here we examine recent advances in the super-resolution imaging of living cells by reviewing recent breakthroughs in single molecule localization microscopy methods such as PALM and STORM to achieve this important goal.

Different stimulus intensities elicit distinct perceptions, implying that input signals are either conveyed through an overlapping but distinct subpopulation of sensory neurons or channeled into divergent brain circuits according to intensity. In Drosophila, carbon dioxide (CO2) is detected by a single type of olfactory sensory neuron, but information is conveyed to higher brain centers through second-order projection neurons (PNs). Two distinct pathways, PN(v)-1 and PN(v)-2, are necessary and sufficient for avoidance responses to low and high CO2 concentrations, respectively. Whereas low concentrations activate PN(v)-1, high concentrations activate both PN(v)s and GABAergic PN(v)-3, which may inhibit PN(v)-1 pathway-mediated avoidance behavior. Channeling a sensory input into distinct neural pathways allows the perception of an odor to be further modulated by both stimulus intensity and context.

Ultrasound pulse guided digital phase conjugation has emerged to realize fluorescence imaging inside random scattering media. Its major limitation is the slow imaging speed, as a new wavefront needs to be measured for each voxel. Therefore 3D or even 2D imaging can be time consuming. For practical applications on biological systems, we need to accelerate the imaging process by orders of magnitude. Here we propose and experimentally demonstrate a parallel wavefront measurement scheme towards such a goal. Multiple focused ultrasound pulses of different carrier frequencies can be simultaneously launched inside a scattering medium. Heterodyne interferometry is used to measure all of the wavefronts originating from every sound focus in parallel. We use these wavefronts in sequence to rapidly excite fluorescence at all the voxels defined by the focused ultrasound pulses. In this report, we employed a commercially available sound transducer to generate two sound foci in parallel, doubled the wavefront measurement speed, and reduced the mechanical scanning steps of the sound transducer to half.

A parallel wavefront optimization method is demonstrated experimentally to focus light through random scattering media. The simultaneous modulation of multiple phase elements, each at a unique frequency, enables a parallel determination of the optimal wavefront. Compared to a pixel-by-pixel measurement, the reported parallel method uses the target signal in a highly efficient way. With 441 phase elements, a high-quality focus was formed through a glass diffuser with a peak-to-background ratio of \~{}270. The accuracy and repeatability of the system were tested through experiments.

Neural circuits for essential natural behaviors are shaped by selective pressure to coordinate reliable execution of flexible goal-directed actions. However, the structural and functional organization of survival-oriented circuits is poorly understood due to exceptionally complex neuroanatomy. This is exemplified by AGRP neurons, which are a molecularly defined population that is sufficient to rapidly coordinate voracious food seeking and consumption behaviors. Here, we use cell-type-specific techniques for neural circuit manipulation and projection-specific anatomical analysis to examine the organization of this critical homeostatic circuit that regulates feeding. We show that AGRP neuronal circuits use a segregated, parallel, and redundant output configuration. AGRP neuron axon projections that target different brain regions originate from distinct subpopulations, several of which are sufficient to independently evoke feeding. The concerted anatomical and functional analysis of AGRP neuron projection populations reveals a constellation of core forebrain nodes, which are part of an extended circuit that mediates feeding behavior.