Whole-cell recording has been used to measure and manipulate a neuron's spiking and subthreshold membrane potential, allowing assessment of the cell's inputs and outputs as well as its intrinsic membrane properties. This technique has also been combined with pharmacology and optogenetics as well as morphological reconstruction to address critical questions concerning neuronal integration, plasticity, and connectivity. This protocol describes a technique for obtaining whole-cell recordings in awake head-fixed animals, allowing such questions to be investigated within the context of an intact network and natural behavioral states. First, animals are habituated to sit quietly with their heads fixed in place. Then, a whole-cell recording is obtained using an efficient, blind patching protocol. We have successfully applied this technique to rats and mice.

Visual motion sensing neurons in the fly also encode a range of behavior-related signals. These nonvisual inputs appear to be used to correct some of the challenges of visually guided locomotion.

Intracellular recording is an essential technique for investigating cellular mechanisms underlying complex brain functions. Despite the high sensitivity of the technique to mechanical disturbances, intracellular recording has been applied to awake, behaving, and even freely moving, animals. Here we summarize recent advances in these methods and their application to the measurement and manipulation of membrane potential dynamics for understanding neuronal computations in behaving animals.

BACKGROUND: The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted.

NEW METHOD: Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently.

RESULTS: iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner.

CONCLUSIONS: Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo.

To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.

The past quarter century has witnessed rapid developments of fluorescence microscopy techniques that enable structural and functional imaging of biological specimens at unprecedented depth and resolution. The performance of these methods in multicellular organisms, however, is degraded by sample-induced optical aberrations. Here I review recent work on incorporating adaptive optics, a technology originally applied in astronomical telescopes to combat atmospheric aberrations, to improve image quality of fluorescence microscopy for biological imaging.

Efforts to map neural circuits have been galvanized by the development of genetic technologies that permit the manipulation of targeted sets of neurons in the brains of freely behaving animals. The success of these efforts relies on the experimenter's ability to target arbitrarily small subsets of neurons for manipulation, but such specificity of targeting cannot routinely be achieved using existing methods. In Drosophila melanogaster, a widely used technique for refined cell-type specific manipulation is the Split GAL4 system, which augments the targeting specificity of the binary GAL4-UAS system by making GAL4 transcriptional activity contingent upon two enhancers, rather than one. To permit more refined targeting, we introduce here the "Killer Zipper" (KZip(+)), a suppressor that makes Split GAL4 targeting contingent upon a third enhancer. KZip(+) acts by disrupting both the formation and activity of Split GAL4 heterodimers, and we show how this added layer of control can be used to selectively remove unwanted cells from a Split GAL4 expression pattern or to subtract neurons of interest from a pattern to determine their requirement in generating a given phenotype. To facilitate application of the KZip(+) technology, we have developed a versatile set of LexAop-KZip(+) fly lines that can be used directly with the large number of LexA driver lines with known expression patterns. The Killer Zipper significantly sharpens the precision of neuronal genetic control available in Drosophila and may be extended to other organisms where Split GAL4-like systems are used.

Summary: State-of-the-art light and electron microscopes are capable of acquiring large image datasets, but quantitatively evaluating the data often involves manually annotating structures of interest. This processis time-consuming and often a major bottleneck in the evaluation pipeline. To overcome this problem, we have introduced the Trainable Weka Segmentation (TWS), a machine learning tool that leveragesa limited number of manual annotations in order to train a classifier and segment the remaining dataautomatically. In addition, TWS can provide unsupervised segmentation learning schemes (clustering) and can be customized to employ user-designed image features or classifiers.

Availability and Implementation: TWS is distributed as open-source software as part of the Fiji image processing distribution of ImageJ at http://imagej.net/Trainable_Weka_Segmentation.

Contact: ignacio.arganda@ehu.eus.

Supplementary information: Supplementary data are available at Bioinformatics online.

Animals use rules to initiate behaviors. Such rules are often described as triggers that determine when behavior begins. However, although less explored, these selection rules are also an opportunity to establish sensorimotor constraints that influence how the behavior ends. These constraints may be particularly significant in influencing success in prey capture. Here we explore this in dragonfly prey interception. We found that in the moments leading up to takeoff, perched dragonflies employ a series of sensorimotor rules that determine the time of takeoff and increase the probability of successful capture. First, the dragonfly makes a head saccade followed by smooth pursuit movements to orient its direction-of-gaze at potential prey. Second, the dragonfly assesses whether the prey's angular size and speed co-vary within a privileged range. Finally, the dragonfly times the moment of its takeoff to a prediction of when the prey will cross the zenith. Each of these processes serves a purpose. The angular size-speed criteria biases interception flights to catchable prey, while the head movements and the predictive takeoff ensure flights begin with the prey visually fixated and directly overhead-the key parameters that underlie interception steering. Prey that do not elicit takeoff generally fail at least one of the criterion, and the loss of prey fixation or overhead positioning during flight is strongly correlated with terminated flights. Thus from an abundance of potential targets, the dragonfly selects a stereotyped set of takeoff conditions based on the prey and body states most likely to end in successful capture.