Drosophila melanogster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a novel technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball’s motion is tracked at high resolution and can be treated as a proxy for the fly’s own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system (HS) lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in HS-neurons correlated with robust optomotor behavior during walking. Our technique allows an entirely new set of questions to be addressed by monitoring behavior and physiology in identified neurons in a powerful genetic model organism with an extensive repertoire of walking behaviors.

A number of recent studies have provided compelling demonstrations that both mice and rats can be trained to perform a variety of behavioral tasks while restrained by mechanical elements mounted to the skull. The independent development of this technique by a number of laboratories has led to diverse solutions. We found that these solutions often used expensive materials and impeded future development and modification in the absence of engineering support. In order to address these issues, here we report on the development of a flexible single hardware design for electrophysiology and imaging both in brain tissue in vitro. Our hardware facilitates the rapid conversion of a single preparation between physiology and imaging system and the conversion of a given system between preparations. In addition, our use of rapid prototyping machines (“3D printers”) allows for the deployment of new designs within a day. Here, we present specifications for design and manufacturing as well as some data from our lab demonstrating the suitability of the design for physiology in behaving animals and imaging in vitro and in vivo.

The holotype partial skull of Agorophius pygmaeus (the monotypic form for both the genus Agorophius and the Family Agorophiidae) has been missing for approximately 140 years. Since the discovery of Agorophius pygmaeus, many additional taxa and specimens have been placed in the Family Agorophiidae, only to be reclassified and removed later. This has created confusion as to what is and what is not an agorophiid and a lack of clarity as to what characteristics delimit the Agorophiidae. A newly discovered skull of an agorophiid recently collected from an underwater cliff face of the Ashley River, South Carolina, USA, is assigned to Agorophius pygmaeus. It derives from the base of the Ashley Formation (early Oligocene). The new specimen consists of most of the skull and periotics, which are well preserved and described for the first time in an agorophiid. The new specimen provides an opportunity to diagnose the Agorophiidae and place the genus and species within the phylogenetic context of the early odontocete radiation in the Oligocene, along with other taxa such as the Ashleycetidae, Mirocetidae, Patriocetidae, Simocetidae, Waipatiidae, and Xenorophidae. Based on this new understanding, Agorophiidae are known with certainty only from the early Oligocene of South Carolina, with other undescribed, potential agorophiid specimens from the Oligocene of the North Pacific region (Japan, Mexico, and Washington State).

Intracellular recording allows precise measurement and manipulation of individual neurons, but it requires stable mechanical contact between the electrode and the cell membrane, and thus it has remained challenging to perform in behaving animals. Whole-cell recordings in freely moving animals can be obtained by rigidly fixing ('anchoring') the pipette electrode to the head; however, previous anchoring procedures were slow and often caused substantial pipette movement, resulting in loss of the recording or of recording quality. We describe a UV-transparent collar and UV-cured adhesive technique that rapidly (within 15 s) anchors pipettes in place with virtually no movement, thus substantially improving the reliability, yield and quality of freely moving whole-cell recordings. Recordings are first obtained from anesthetized or awake head-fixed rats. UV light cures the thin adhesive layers linking pipette to collar to head. Then, the animals are rapidly and smoothly released for recording during unrestrained behavior. The anesthetized-patched version can be completed in ∼4-7 h (excluding histology) and the awake-patched version requires ∼1-4 h per day for ∼2 weeks. These advances should greatly facilitate studies of neuronal integration and plasticity in identified cells during natural behaviors.