Elucidating the roles of neuronal cell types for physiology and behavior is essential for understanding brain functions. Perturbation of neuron electrical activity can be used to probe the causal relationship between neuronal cell types and behavior. New genetically encoded neuron perturbation tools have been developed for remotely controlling neuron function using small molecules that activate engineered receptors that can be targeted to cell types using genetic methods. Here we describe recent progress for approaches using genetically engineered receptors that selectively interact with small molecules. Called "chemogenetics," receptors with diverse cellular functions have been developed that facilitate the selective pharmacological control over a diverse range of cell-signaling processes, including electrical activity, for molecularly defined cell types. These tools have revealed remarkably specific behavioral physiological influences for molecularly defined cell types that are often intermingled with populations having different or even opposite functions.

Mating induces pronounced changes in female reproductive behavior, typically including a dramatic reduction in sexual receptivity. In Drosophila, postmating behavioral changes are triggered by sex peptide (SP), a male seminal fluid peptide that acts via a receptor (SPR) expressed in sensory neurons (SPSNs) of the female reproductive tract. Here, we identify second-order neurons that mediate the behavioral changes induced by SP. These SAG neurons receive synaptic input from SPSNs in the abdominal ganglion and project to the dorsal protocerebrum. Silencing SAG neurons renders virgin females unreceptive, whereas activating them increases the receptivity of females that have already mated. Physiological experiments demonstrate that SP downregulates the excitability of the SPSNs, and hence their input onto SAG neurons. These data thus provide a physiological correlate of mating status in the female central nervous system and a key entry point into the brain circuits that control sexual receptivity.

Drosophila melanogaster females respond to male courtship by either rejecting the male or allowing copulation. The neural mechanisms underlying these female behaviors likely involve the integration of sensory information in the brain. Because doublesex (dsx) controls other aspects of female differentiation, we asked whether dsx-expressing neurons mediate virgin female receptivity to courting males. Using intersectional techniques to manipulate the activities of defined subsets of dsx-expressing neurons, we found that activation of neurons in either the pCd or pC1 clusters promotes receptivity, while silencing these neurons makes females unreceptive. Furthermore, pCd and pC1 neurons physiologically respond to the male-specific pheromone cis-vaccenyl acetate (cVA), while pC1 neurons also respond to male courtship song. The pCd and pC1 neurons expressing dsx in females do not express transcripts from the fruitless (fru) P1 promoter. Thus, virgin female receptivity is controlled at least in part by neurons that are distinct from those governing male courtship.

The cricket's auditory system is a highly directional pressure difference receiver whose function is hypothesised to depend on phase relationships between the sound waves propagating through the auditory trachea that connects the left and right hearing organs. We tested this hypothesis by measuring the effect of experimentally constructed phase shifts in acoustic stimuli on phonotactic behavior of Gryllus bimaculatus, the oscillatory response patterns of the tympanic membrane, and the activity of the auditory afferents. The same artificial calling song was played simultaneously at the left and right sides of the cricket, but one sound pattern was shifted in phase by 90 deg (carrier frequencies between 3.6 and 5.4 kHz). All three levels of auditory processing are sensitive to experimentally induced acoustic phase shifts, and the response characteristics are dependent on the carrier frequency of the sound stimulus. At lower frequencies, crickets steered away from the sound leading in phase, while tympanic membrane vibrations and auditory afferent responses were smaller when the ipsilateral sound was leading. In contrast, opposite responses were observed at higher frequencies in all three levels of auditory processing. Minimal responses occurred near the carrier frequency of the cricket's calling song, suggesting a stability at this frequency. Our results indicate that crickets may use directional cues arising from phase shifts in acoustic signals for sound localisation, and that the response properties of pressure difference receivers may be analysed with phase-shifted sound stimuli to further our understanding of how insect auditory systems are adapted for directional processing.

A general method to recognize and track unmarked animals within a population will enable new studies of social behavior and individuality.

The automated tape-collecting ultramicrotome (ATUM) makes it possible to collect large numbers of ultrathin sections quickly-the equivalent of a petabyte of high resolution images each day. However, even high throughput image acquisition strategies generate images far more slowly (at present ~1 terabyte per day). We therefore developed WaferMapper, a software package that takes a multi-resolution approach to mapping and imaging select regions within a library of ultrathin sections. This automated method selects and directs imaging of corresponding regions within each section of an ultrathin section library (UTSL) that may contain many thousands of sections. Using WaferMapper, it is possible to map thousands of tissue sections at low resolution and target multiple points of interest for high resolution imaging based on anatomical landmarks. The program can also be used to expand previously imaged regions, acquire data under different imaging conditions, or re-image after additional tissue treatments.

BACKGROUND: In a series of landmark papers, Kyriacou, Hall, and colleagues reported that the average inter-pulse interval of Drosophila melanogaster male courtship song varies rhythmically (KH cycles), that the period gene controls this rhythm, and that evolution of the period gene determines species differences in the rhythm's frequency. Several groups failed to recover KH cycles, but this may have resulted from differences in recording chamber size.

RESULTS: Here, using recording chambers of the same dimensions as used by Kyriacou and Hall, I found no compelling evidence for KH cycles at any frequency. By replicating the data analysis procedures employed by Kyriacou and Hall, I found that two factors--data binned into 10-second intervals and short recordings--imposed non-significant periodicity in the frequency range reported for KH cycles. Randomized data showed similar patterns.

CONCLUSIONS: All of the results related to KH cycles are likely to be artifacts of binning data from short songs. Reported genotypic differences in KH cycles cannot be explained by this artifact and may have resulted from the use of small sample sizes and/or from the exclusion of samples that did not exhibit song rhythms.

In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA-like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.

The patch-clamp technique and the whole-cell measurements derived from it have greatly advanced our understanding of the coding properties of individual neurons by allowing for a detailed analysis of their excitatory/inhibitory synaptic inputs, intrinsic electrical properties, and morphology. Because such measurements require a high level of mechanical stability they have for a long time been limited to in vitro and anesthetized preparations. Recently, however, a considerable amount of effort has been devoted to extending these techniques to awake restrained/head-fixed preparations allowing for the study of the input-output functions of neurons during behavior. In this chapter we describe a technique extending patch-clamp recordings to awake animals free to explore their environments.

Modern biological research relies heavily on microscopic imaging. The advanced genetic toolkit of Drosophila makes it possible to label molecular and cellular components with unprecedented level of specificity necessitating the application of the most sophisticated imaging technologies. Imaging in Drosophila spans all scales from single molecules to the entire populations of adult organisms, from electron microscopy to live imaging of developmental processes. As the imaging approaches become more complex and ambitious, there is an increasing need for quantitative, computer-mediated image processing and analysis to make sense of the imagery. Bioimage Informatics is an emerging research field that covers all aspects of biological image analysis from data handling, through processing, to quantitative measurements, analysis and data presentation. Some of the most advanced, large scale projects, combining cutting edge imaging with complex bioimage informatics pipelines, are realized in the Drosophila research community. In this review, we discuss the current research in biological image analysis specifically relevant to the type of systems level image datasets that are uniquely available for the Drosophila model system. We focus on how state-of-the-art computer vision algorithms are impacting the ability of Drosophila researchers to analyze biological systems in space and time. We pay particular attention to how these algorithmic advances from computer science are made usable to practicing biologists through open source platforms and how biologists can themselves participate in their further development.