Most neurons of the central complex belong to 10 secondary (larvally produced) lineages. In the late larva, undifferentiated axon tracts of these lineages form a primordium in which all of the compartments of the central complex can be recognized as discrete entities. Four posterior lineages (DPMm1, DPMpm1, DPMpm2, and CM4) generate the classes of small-field neurons that interconnect the protocerebral bridge, fan-shaped body, noduli, and ellipsoid body. Three lineages located in the anterior brain, DALv2, BAmv1, and DALcl2, form the large-field neurons of the ellipsoid body and fan-shaped body, respectively. These lineages provide an input channel from the optic tubercle and connect the central complex with adjacent anterior brain compartments. Three lineages in the posterior cortex, CM3, CP2, and DPMpl2, connect the posterior brain neuropil with specific layers of the fan-shaped body. Even though all of the compartments of the central complex are prefigured in the late larval brain by the axon tracts of the above-mentioned lineages, the neuropil differentiates during the first 2 days of the pupal period when terminal branches and synapses of secondary neurons are formed. During this phase the initially straight horizontal layers of the central complex bend in the frontal plane, which produces the characteristic shape of the fan-shaped and ellipsoid body. Our analysis provides a comprehensive picture of the lineages that form the central complex, and will facilitate future studies that address the structure or function of the central complex at the single cell level.

Decoding the wiring diagram of the retina requires simultaneous observation of activity in identified neuron populations. Available recording methods are limited in their scope: electrodes can access only a small fraction of neurons at once, whereas synthetic fluorescent indicator dyes label tissue indiscriminately. Here, we describe a method for studying retinal circuitry at cellular and subcellular levels combining two-photon microscopy and a genetically encoded calcium indicator. Using specific viral and promoter constructs to drive expression of GCaMP3, we labeled all five major neuron classes in the adult mouse retina. Stimulus-evoked GCaMP3 responses as imaged by two-photon microscopy permitted functional cell type annotation. Fluorescence responses were similar to those measured with the small molecule dye OGB-1. Fluorescence intensity correlated linearly with spike rates >10 spikes/s, and a significant change in fluorescence always reflected a significant change in spike firing rate. GCaMP3 expression had no apparent effect on neuronal function. Imaging at subcellular resolution showed compartment-specific calcium dynamics in multiple identified cell types.

Thermosensation is an indispensable sensory modality. Here, we study temperature coding in Drosophila, and show that temperature is represented by a spatial map of activity in the brain. First, we identify TRP channels that function in the fly antenna to mediate the detection of cold stimuli. Next, we identify the hot-sensing neurons and show that hot and cold antennal receptors project onto distinct, but adjacent glomeruli in the Proximal-Antennal-Protocerebrum (PAP) forming a thermotopic map in the brain. We use two-photon imaging to reveal the functional segregation of hot and cold responses in the PAP, and show that silencing the hot- or cold-sensing neurons produces animals with distinct and discrete deficits in their behavioral responses to thermal stimuli. Together, these results demonstrate that dedicated populations of cells orchestrate behavioral responses to different temperature stimuli, and reveal a labeled-line logic for the coding of temperature information in the brain.

A large number of degrees of freedom are required to produce a high quality focus through random scattering media. Previous demonstrations based on spatial phase modulations suffer from either a slow speed or a small number of degrees of freedom. In this work, a high speed wavefront determination technique based on spatial frequency domain wavefront modulations is proposed and experimentally demonstrated, which is capable of providing both a high operation speed and a large number of degrees of freedom. The technique was employed to focus light through a strongly scattering medium and the entire wavefront was determined in 400 milliseconds,  three orders of magnitude faster than the previous report.

Understanding the structure and function of neural circuits are central questions in neuroscience research. To address these questions, new genetically encoded tools have been developed for mapping, monitoring, and manipulating neurons. Essential to implementation of these tools is their selective delivery to defined neuronal populations in the brain. This has been facilitated by recent improvements in cell type-specific transgene expression using recombinant adeno-associated viral vectors. Here, we highlight these developments and discuss areas for improvement that could further expand capabilities for neural circuit analysis.

Despite recent interest in reconstructing neuronal networks, complete wiring diagrams on the level of individual synapses remain scarce and the insights into function they can provide remain unclear. Even for Caenorhabditis elegans, whose neuronal network is relatively small and stereotypical from animal to animal, published wiring diagrams are neither accurate nor complete and self-consistent. Using materials from White et al. and new electron micrographs we assemble whole, self-consistent gap junction and chemical synapse networks of hermaphrodite C. elegans. We propose a method to visualize the wiring diagram, which reflects network signal flow. We calculate statistical and topological properties of the network, such as degree distributions, synaptic multiplicities, and small-world properties, that help in understanding network signal propagation. We identify neurons that may play central roles in information processing, and network motifs that could serve as functional modules of the network. We explore propagation of neuronal activity in response to sensory or artificial stimulation using linear systems theory and find several activity patterns that could serve as substrates of previously described behaviors. Finally, we analyze the interaction between the gap junction and the chemical synapse networks. Since several statistical properties of the C. elegans network, such as multiplicity and motif distributions are similar to those found in mammalian neocortex, they likely point to general principles of neuronal networks. The wiring diagram reported here can help in understanding the mechanistic basis of behavior by generating predictions about future experiments involving genetic perturbations, laser ablations, or monitoring propagation of neuronal activity in response to stimulation.

Biotinidase deficiency is the primary enzymatic defect in biotin-responsive, late-onset multiple carboxylase deficiency. Untreated children with profound biotinidase deficiency usually exhibit neurological symptoms including lethargy, hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous symptoms including skin rash, conjunctivitis and alopecia. Although the clinical features of the disorder markedly improve or are prevented with biotin supplementation, some symptoms, once they occur, such as developmental delay, hearing loss and optic atrophy, are usually irreversible. To prevent development of symptoms, the disorder is screened for in the newborn period in essentially all states and in many countries. In order to better understand many aspects of the pathophysiology of the disorder, we have developed a transgenic biotinidase-deficient mouse. The mouse has a null mutation that results in no detectable serum biotinidase activity or cross-reacting material to antibody prepared against biotinidase. When fed a biotin-deficient diet these mice develop neurological and cutaneous symptoms, carboxylase deficiency, mild hyperammonemia, and exhibit increased urinary excretion of 3-hydroxyisovaleric acid and biotin and biotin metabolites. The clinical features are reversed with biotin supplementation. This biotinidase-deficient animal can be used to study systematically many aspects of the disorder and the role of biotinidase, biotin and biocytin in normal and in enzyme-deficient states.

Histochemistry (chemistry in the context of biological tissue) is an invaluable set of techniques used to visualize biological structures. This field lies at the interface of organic chemistry, biochemistry, and biology. Integration of these disciplines over the past century has permitted the imaging of cells and tissues using microscopy. Today, by exploiting the unique chemical environments within cells, heterologous expression techniques, and enzymatic activity, histochemical methods can be used to visualize structures in living matter. This review focuses on the labeling techniques and organic fluorophores used in live cells.

Small molecules that modulate protein-protein interactions are of great interest for chemical biology and therapeutics. Here I present a structure-based approach to predict ’bi-functional’ sites able to bind both small molecule ligands and proteins, in proteins of unknown structure. First, I develop a homology-based annotation method that transfers binding sites of known three-dimensional structure onto protein sequences, predicting residues in ligand and protein binding sites with estimated true positive rates of 98% and 88%, respectively, at 1% false positive rates. Applying this method to the human proteome predicts 8463 proteins with bi-functional residues and correctly recovers the targets of known interaction modulators. Proteins with significantly (p < 0.01) more bi-functional residues than expected were found to be enriched in regulatory and depleted in metabolism functions. Finally, I demonstrate the utility of the method by describing examples of predicted overlap and evidence of their biological and therapeutic relevance. The results suggest that combining the structures of known binding sites with established fold detection algorithms can predict regions of protein-protein interfaces that are amenable to small molecule modulation. Open-source software and the results for several complete proteomes are available at http://pibase.janelia.org/homolobind.

An important open question in biophysics is to understand how mechanical forces shape membrane-bounded cells and their organelles. A general solution to this problem is to calculate the bending energy of an arbitrarily shaped membrane surface, which can include both lipids and cytoskeletal proteins, and minimize the energy subject to all mechanical constraints. However, the calculations are difficult to perform, especially for shapes that do not possess axial symmetry. We show that the spherical harmonics parameterization (SHP) provides an analytic description of shape that can be used to quickly and reliably calculate minimum energy shapes of both symmetric and asymmetric surfaces. Using this method, we probe the entire set of shapes predicted by the bilayer couple model, unifying work based on different computational approaches, and providing additional details of the transitions between different shape classes. In addition, we present new minimum-energy morphologies based on non-linear models of membrane skeletal elasticity that closely mimic extreme shapes of red blood cells. The SHP thus provides a versatile shape description that can be used to investigate forces that shape cells.