Correcting locomotion dependent observation biases in thermal preference of Drosophila.

Sensing environmental temperatures is essential for the survival of ectothermic organisms. In Drosophila, two of the most used methodologies to study temperature preferences (TP) and the genes involved in thermosensation are two-choice assays and temperature gradients. Whereas two-choice assays reveal a relative TP, temperature gradients can identify the absolute Tp. One drawback of gradients is that small ectothermic animals are susceptible to cold-trapping: a physiological inability to move at the cold area of the gradient. Often cold-trapping cannot be avoided, biasing the resulting TP to lower temperatures. Two mathematical models were previously developed to correct for cold-trapping. These models, however, focus on group behaviour which can lead to overestimation of cold-trapping due to group aggregation. Here we present a mathematical model that simulates the behaviour of individual Drosophila in temperature gradients. The model takes the spatial dimension and temperature difference of the gradient into account, as well as the rearing temperature of the flies. Furthermore, it allows the quantification of cold-trapping and reveals unbiased TP. Additionally, our model reveals that flies have a range of tolerable temperatures, and this measure is more informative about the behaviour than commonly used TP. Online simulation is hosted at http://igloo.uni-goettingen.de . The code can be accessed at https://github.com/zerotonin/igloo .

Neuroligins Nlg2 and Nlg4 Affect Social Behavior in Drosophila melanogaster.

The genome of Drosophila melanogaster includes homologs to approximately one-third of the currently known human disease genes. Flies and humans share many biological processes, including the principles of information processing by excitable neurons, synaptic transmission, and the chemical signals involved in intercellular communication. Studies on the molecular and behavioral impact of genetic risk factors of human neuro-developmental disorders [autism spectrum disorders (ASDs), schizophrenia, attention deficit hyperactivity disorders, and Tourette syndrome] increasingly use the well-studied social behavior of D. melanogaster, an organism that is amenable to a large variety of genetic manipulations. Neuroligins (Nlgs) are a family of phylogenetically conserved postsynaptic adhesion molecules present (among others) in nematodes, insects, and mammals. Impaired function of Nlgs (particularly of Nlg 3 and 4) has been associated with ASDs in humans and impaired social and communication behavior in mice. Making use of a set of behavioral and social assays, we, here, analyzed the impact of two Drosophila Nlgs, Dnlg2 and Dnlg4, which are differentially expressed at excitatory and inhibitory central nervous synapses, respectively. Both Nlgs seem to be associated with diurnal activity and social behavior. Even though deficiencies in Dnlg2 and Dnlg4 appeared to have no effects on sensory or motor systems, they differentially impacted on social interactions, suggesting that social behavior is distinctly regulated by these Nlgs.

A multiaxis device for in-focus manipulation of objects under a dissecting microscope.

An inexpensive specimen holder for objects under a dissecting microscope is described which allows arbitrary orientation and rotation of the object within the visual field and focal plane of the microscope. Based on a spherical cap upon a magnet, this apparatus is easy to construct and permits single-handed manipulation of the specimen from some distance. Precise positioning is demonstrated for halteres of Drosophila under up to 120× magnification. Because of the restricted field of view and depth of focus of a dissecting microscope, it is often difficult to position tiny objects under study in varying orientation, especially if the specimen must simultaneously be dissected with forceps, microscissors or needles. The problem is solved most conveniently with a specimen holder based on a spherical cap which is held by a strong magnet on a steel ring. The specimen tilts vertically in any direction by up to 70° and-even in oblique orientation-rotates through 360° around the sphere's centre in the focal plane. By help of an interleaved plastic foil in a ring carrier, these movements can be controlled unimanually from outside the visual field.

A global in vivo Drosophila RNAi screen identifies a key role of ceramide phosphoethanolamine for glial ensheathment of axons.

Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia.

Sprouting interneurons in mushroom bodies of adult cricket brains.

In crickets, neurogenesis persists throughout adulthood for certain local brain interneurons, the Kenyon cells in the mushroom bodies, which represent a prominent compartment for sensory integration, learning, and memory. Several classes of these neurons originate from a perikaryal layer, which includes a cluster of neuroblasts, surrounded by somata that provide the mushroom body's columnar neuropil. We describe the form, distribution, and cytology of Kenyon cell groups in the process of generation and growth in comparison to developed parts of the mushroom bodies in adult crickets of the species Gryllus bimaculatus. A subset of growing Kenyon cells with sprouting processes has been distinguished from adjacent Kenyon cells by its prominent f-actin labelling. Growth cone-like elements are detected in the perikaryal layer and in their associated sprouting fiber bundles. Sprouting fibers distant from the perikarya contain ribosomes and rough endoplasmic reticulum not found in the dendritic processes of the calyx. A core of sprouting Kenyon cell processes is devoid of synapses and is not invaded by extrinsic neuronal elements. Measurements of fiber cross-sections and counts of synapses and organelles suggest a continuous gradient of growth and maturation leading from the core of added new processes out to the periphery of mature Kenyon cell fiber groups. Our results are discussed in the context of Kenyon cell classification, growth dynamics, axonal fiber maturation, and function.

Activity-dependent suppression of spontaneous spike generation in the Retzius neurons of the leech Hirudo medicinalis L.

We report on factors affecting the spontaneous firing pattern of the identified serotonin-containing Retzius neurons of the medicinal leech. Increased firing activity induced by intracellular current injection is followed by a 'post-stimulus-depression' (PSD) without spiking for up to 23 s. PSD duration depends both on the duration and the amplitude of the injected current and correlates inversely with the spontaneous spiking activity. In contrast to serotonin-containing neurons in mammals, serotonin release from the Retzius cells presumably does not mediate the observed spike suppression in a self-inhibitory manner since robust PSD persists after synaptic isolation. Moreover, single additional spikes elicited at specific delays after spontaneously occurring action potentials are sufficient to significantly alter the firing pattern. Since sub-threshold current injections do not affect the ongoing spiking pattern and PSD persists in synaptically isolated preparations our data suggest that PSD reflects an endogenous and 'spike-dependent' mechanism controlling the spiking activity of Retzius cells in a use-dependent way.