Selective dendritic localization of mRNA in Drosophila mushroom body output neurons.

Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process requires the visualization of the relevant mRNAs within these neuronal compartments. Here, we used single-molecule fluorescence in situ hybridization to localize mRNAs at subcellular resolution in the adult Drosophila brain. mRNAs for subunits of nicotinic acetylcholine receptors and kinases could be detected within the dendrites of co-labeled mushroom body output neurons (MBONs) and their relative abundance showed cell specificity. Moreover, aversive olfactory learning produced a transient increase in the level of CaMKII mRNA within the dendritic compartments of the γ5ß'2a MBONs. Localization of specific mRNAs in MBONs before and after learning represents a critical step towards deciphering the role of dendritic translation in the neuronal plasticity underlying behavioral change in Drosophila.

Microscope-Cockpit: Python-based bespoke microscopy for bio-medical science.

We have developed "Microscope-Cockpit" (Cockpit), a highly adaptable open source user-friendly Python-based Graphical User Interface (GUI) environment for precision control of both simple and elaborate bespoke microscope systems. The user environment allows next-generation near instantaneous navigation of the entire slide landscape for efficient selection of specimens of interest and automated acquisition without the use of eyepieces. Cockpit uses "Python-Microscope" (Microscope) for high-performance coordinated control of a wide range of hardware devices using open source software. Microscope also controls complex hardware devices such as deformable mirrors for aberration correction and spatial light modulators for structured illumination via abstracted device models. We demonstrate the advantages of the Cockpit platform using several bespoke microscopes, including a simple widefield system and a complex system with adaptive optics and structured illumination. A key strength of Cockpit is its use of Python, which means that any microscope built with Cockpit is ready for future customisation by simply adding new libraries, for example machine learning algorithms to enable automated microscopy decision making while imaging.

Microscope-AOtools: a generalised adaptive optics implementation.

Aberrations arising from sources such as sample heterogeneity and refractive index mismatches are constant problems in biological imaging. These aberrations reduce image quality and the achievable depth of imaging, particularly in super-resolution microscopy techniques. Adaptive optics (AO) technology has been proven to be effective in correcting for these aberrations, thereby improving the image quality. However, it has not been widely adopted by the biological imaging community due, in part, to difficulty in set-up and operation of AO. The methods for doing so are not novel or unknown, but new users often waste time and effort reimplementing existing methods for their specific set-ups, hardware, sample types, etc. Microscope-AOtools offers a robust, easy-to-use implementation of the essential methods for set-up and use of AO elements and techniques. These methods are constructed in a generalised manner that can utilise a range of adaptive optics elements, wavefront sensing techniques and sensorless AO correction methods. Furthermore, the methods are designed to be easily extensible as new techniques arise, leading to a streamlined pipeline for new AO technology and techniques to be adopted by the wider microscopy community.

Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains.

RNA in situ hybridization is a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary nascent transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3'UTR, and introns, we show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. Our simple and rapid protocol can be used to co-visualise a variety of different transcripts and proteins in neuronal stem cells as well as deep brain structures such as mushroom body neuropils, using conventional confocal microscopy. Finally, we introduce the use of smFISH as a sensitive alternative to immunofluorescence for labelling specific neural stem cell populations in the brain.

Light Activated Escape Circuits: A Behavior and Neurophysiology Lab Module using Drosophila Optogenetics.

The neural networks that control escape from predators often show very clear relationships between defined sensory inputs and stereotyped motor outputs. This feature provides unique opportunities for researchers, but it also provides novel opportunities for neuroscience educators. Here we introduce new teaching modules using adult Drosophila that have been engineered to express csChrimson, a red-light sensitive channelrhodopsin, in specific sets of neurons and muscles mediating visually guided escape behaviors. This lab module consists of both behavior and electrophysiology experiments that explore the neural basis of flight escape. Three preparations are described that demonstrate photo-activation of the giant fiber circuit and how to quantify these behaviors. One of the preparations is then used to acquire intracellular electrophysiology recordings from different flight muscles. The diversity of action potential waveforms and firing frequencies observed in the flight muscles make this a rich preparation to study the ionic basic of cellular excitability. By activating different cells within the giant fiber pathway we also demonstrate principles of synaptic transmission and neural circuits. Beyond conveying core neurobiological concepts it is also expected that using these cutting edge techniques will enhance student motivation and attitudes towards biological research. Data collected from students and educators who have been involved in development of the module are presented to support this notion.

Anatomical and genotype-specific mechanosensory responses in Drosophila melanogaster larvae.

Afferent neurons commonly exhibit a somatotopic arrangement in the central nervous system that organizes spatially discrete sensory input. We are interested in how that spatial input gets integrated into motor commands. With resources for screening genes and neural circuits, and given that the cells and ion channels that transduce tactile stimuli in Drosophila larvae are remarkably well-characterized, larval mechanosensation is an ideal system for investigating how specific behaviors emerge from localized sensory input. We observed that crawling larvae are more reactive to a 20mN tactile stimulus on the head than on the tail or abdomen. Behavioral responses that were evoked by the stimuli also depended on where the stimulus was delivered. Differences in relative sensitivity were observed in different genotypes, e.g., a null white mutant and hypomorphic smn mutant are significantly more reactive to tail touches than Canton-S larvae. Responses were inhibited by silencing chemical transmission in a combination of multidendritic and chordotonal neurons, but not by inhibiting any specific subset of neurons. Extracellular recordings from segmental nerves revealed that sensory-evoked responses exhibit spike-timing dependence at the neural circuit level. Tactile stimuli reduced endogenous firing frequency and increased bursting periods when applied during periods of motor activity. The same stimulus initiated bursts of activity when applied during inactive periods. Together, these data depict the spatial and temporal complexity of mechanosensation as it applies to action selection, and provide a foundation for addressing how neural circuits in the CNS adjust their activity to afferent input.

Analysis of various physiological salines for heart rate, CNS function, and synaptic transmission at neuromuscular junctions in Drosophila melanogaster larvae.

Drosophila serves as a playground for examining the effects of genetic mutations on development, physiological function and behavior. Many physiological measures that address the effects of mutations require semi-intact or cultured preparations. To obtain consistent physiological recordings, cellular function needs to remain viable. Numerous physiological salines have been developed for fly preparations, with emphasis on nervous system viability. The commonly used saline drifts in pH and will cause an alteration in the heart rate. We identify a saline that maintains a stable pH and physiological function in the larval heart, skeletal neuromuscular junction, and ventral nerve cord preparations. Using these common assays, we screened various pH buffers of differing concentrations to identify optimum conditions. Buffers at 25 mM produce a stable heart rate with minimal variation in pH. Excitatory junction potentials evoked directly on larval muscles or through sensory-CNS-motor circuits were unaffected by at buffers at 25 mM. The salines examined did not impede the modulatory effect of serotonin on heart rate or neural activity. Together, our results indicate that the higher buffer concentrations needed to stabilize pH in HL3 hemolymph-like saline do not interfere with the acute function of neurons or cardiac myocytes.

Neural circuit recording from an intact cockroach nervous system.

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

Pharmacological analysis of dopamine modulation in the Drosophila melanogaster larval heart.

Dopamine (DA) and other neurotransmitters affect nonneuronal tissues in insects by circulating in the hemolymph. In several organisms, DA has been shown to modulate distinct aspects of cardiac function but the signal transduction pathways that mediate dopaminergic effects on the heart are not well characterized. Here, we used a semiintact Drosophila melanogaster larva preparation and drugs targeting DA receptors and canonical second messenger pathways to identify signaling cascades that mediate the effect of DA on a myogenic heart. DA has a positive chronotropic effect that is mimicked by SKF38393 (type-1 DA receptor agonist) and quinpirole (type-2 DA receptor agonist). SCH23390 and spiperone (type-1 and type-2 DA receptor antagonists) are moderately effective at inhibiting DA's effect. An adenylate cyclase inhibitor (SQ,22536) is also effective at blocking the stimulatory effect of DA but the drug has its own dose-dependent effect. Activation of protein kinase C with a diacylglycerol analog has a stimulatory effect on heart rate (HR). These results suggest that (1) both DA receptor subtypes are expressed in third instar larva cardiac myocytes to increase HR in response to rising levels of DA in the hemolymph, and (2) canonical second messenger pathways modulate HR in D. melanogaster larvae. Having these disparate signaling cascades converge toward a common modulatory function appears redundant, but in the context of multiple cardioactive chemicals this redundancy is likely to increase the fidelity of signal transduction.

Intracellular recording, sensory field mapping, and culturing identified neurons in the leech, Hirudo medicinalis.

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Proprioception and tension receptors in crab limbs: student laboratory exercises.

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.