Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H(+), Na(+), K(+) and anions have been discovered or engineered, Ca(+)-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca(+) permeability and improve their Ca(+) affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca(+) permeation at negative voltage and low extracellular Ca(+) concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca(+) concentrations. Moreover, CapChR2 can robustly trigger Ca(+) signalling in hippocampal neurons. When expressed together with genetically encoded Ca(+) indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca(+) entry in brain explants

Analysis of Adhesion Molecules and Basement Membrane Contributions to Synaptic Adhesion at the Drosophila Embryonic NMJ

Synapse formation and maintenance crucially underlie brain function in health and disease. Both processes are believed to depend on cell adhesion molecules (CAMs). Many different classes of CAMs localise to synapses, including cadherins, protocadherins, neuroligins, neurexins, integrins, and immunoglobulin adhesion proteins, and further contributions come from the extracellular matrix and its receptors. Most of these factors have been scrutinised by loss-of-function analyses in animal models. However, which adhesion factors establish the essential physical links across synaptic clefts and allow the assembly of synaptic machineries at the contact site in vivo is still unclear. To investigate these key questions, we have used the neuromuscular junction (NMJ) of Drosophila embryos as a genetically amenable model synapse. Our ultrastructural analyses of NMJs lacking different classes of CAMs revealed that loss of all neurexins, all classical cadherins or all glutamate receptors, as well as combinations between these or with a Laminin deficiency, failed to reveal structural phenotypes. These results are compatible with a view that these CAMs might have no structural role at this model synapse. However, we consider it far more likely that they operate in a redundant or well buffered context. We propose a model based on a multi-adaptor principle to explain this phenomenon. Furthermore, we report a new CAM-independent adhesion mechanism that involves the basement membranes (BM) covering neuromuscular terminals. Thus, motorneuronal terminals show strong partial detachment of the junction when BM-to-cell surface attachment is impaired by removing Laminin A, or when BMs lose their structural integrity upon loss of type IV collagens. We conclude that BMs are essential to tie embryonic motorneuronal terminals to the muscle surface, lending CAM-independent structural support to their adhesion. Therefore, future developmental studies of these synaptic junctions in Drosophila need to consider the important contribution made by BM-dependent mechanisms, in addition to CAM-dependent adhesion

Interactions among Drosophila larvae before and during collision

In populations of Drosophila larvae, both, an aggregation and a dispersal behavior can be observed. However, the mechanisms coordinating larval locomotion in respect to other animals, especially in close proximity and during/after physical contacts are currently only little understood. Here we test whether relevant information is perceived before or during larva-larva contacts, analyze its influence on behavior and ask whether larvae avoid or pursue collisions. Employing frustrated total internal reflection-based imaging (FIM) we first found that larvae visually detect other moving larvae in a narrow perceptive field and respond with characteristic escape reactions. To decipher larval locomotion not only before but also during the collision we utilized a two color FIM approach (FIM(2c)), which allowed to faithfully extract the posture and motion of colliding animals. We show that during collision, larval locomotion freezes and sensory information is sampled during a KISS phase (german: Kollisions Induziertes Stopp Syndrom or english: collision induced stop syndrome). Interestingly, larvae react differently to living, dead or artificial larvae, discriminate other Drosophila species and have an increased bending probability for a short period after the collision terminates. Thus, Drosophila larvae evolved means to specify behaviors in response to other larvae

Assembling the presynaptic active zone.

Rapid neurotransmission depends on the structural and functional integrity of synaptic connections. How synapses assemble is currently being intensely investigated to help our understanding of neuronal development and synaptic plasticity. Here we focus on the assembly of the presynaptic active zone, which regulates the synaptic vesicle exo/endo-cycle and is characterized by ultrastructural specializations and large scaffold proteins. While genetic and biochemical studies from rodents, Caenorhabditis elegans and Drosophila have started to identify proteins organizing active zone assembly, drawing a coherent picture remains challenging, with genetically established hierarchies and protein-protein interactions still to be placed into spatio-temporal and functional context. Recent advances in light and electron microscopy, together with in vivo imaging of protein traffic, will help to tackle this challenge

Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics

When imaging through tissue, the optical inhomogeneities of the sample generate aberrations that can prevent effective Stimulated Emission Depletion (STED) imaging. This is particularly problematic for 3D-enhanced STED. We present here an adaptive optics implementation that incorporates two adaptive optic elements to enable correction in all beam paths, allowing performance improvement in thick tissue samples. We use this to demonstrate 3D STED imaging of complex structures in Drosophila melanogaster brains

Aversive learning and appetitive motivation toggle feed-forward inhibition in the Drosophila mushroom body

In Drosophila, negatively reinforcing dopaminergic neurons also provide the inhibitory control of satiety over appetitive memory expression. Here we show that aversive learning causes a persistent depression of the conditioned odor drive to two downstream feed-forward inhibitory GABAergic interneurons of the mushroom body, called MVP2, or mushroom body output neuron (MBON)-γ1pedc>α/β. However, MVP2 neuron output is only essential for expression of short-term aversive memory. Stimulating MVP2 neurons preferentially inhibits the odor-evoked activity of avoidance-directing MBONs and odor-driven avoidance behavior, whereas their inhibition enhances odor avoidance. In contrast, odor-evoked activity of MVP2 neurons is elevated in hungry flies, and their feed-forward inhibition is required for expression of appetitive memory at all times. Moreover, imposing MVP2 activity promotes inappropriate appetitive memory expression in food-satiated flies. Aversive learning and appetitive motivation therefore toggle alternate modes of a common feed-forward inhibitory MVP2 pathway to promote conditioned odor avoidance or approach

Neural correlates of water reward in thirsty Drosophila.

Drinking water is innately rewarding to thirsty animals. In addition, the consumed value can be assigned to behavioral actions and predictive sensory cues by associative learning. Here we show that thirst converts water avoidance into water-seeking in naive Drosophila melanogaster. Thirst also permitted flies to learn olfactory cues paired with water reward. Water learning required water taste and <40 water-responsive dopaminergic neurons that innervate a restricted zone of the mushroom body γ lobe. These water learning neurons are different from those that are critical for conveying the reinforcing effects of sugar. Naive water-seeking behavior in thirsty flies did not require water taste but relied on another subset of water-responsive dopaminergic neurons that target the mushroom body β' lobe. Furthermore, these naive water-approach neurons were not required for learned water-seeking. Our results therefore demonstrate that naive water-seeking, learned water-seeking and water learning use separable neural circuitry in the brain of thirsty flies