Monitoring Neural Activity with Bioluminescence during Natural Behavior

Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca$^{2+}$-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of ~20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior.Molecular and Cellular Biolog

Using G-CaMP 1.6 to Monitor Visually-Evoked Synaptic Activity in Tectal Neurons in vivo

To investigate the visual system of the zebrafish, different techniques were used to express genetically-encoded Ca2+ indicators and test their functionality in vivo. XFPs, G-CaMP 1.6 and troponeons were expressed in a mosaic pattern and in single neurons under different promoters in the whole central nervous system of larval zebrafish. Light-evoked responses to a flickering LED in G-CaMP1.6 expressing tectal neurons were recorded with single photon excitation. Furthermore, changes of intracellular [Ca2+] were measured in separate dendritic structures. For the characterization of expression patterns, repeated two-photon imaging of the whole tectum opticum was performed, allowing the monitoring of single cells in high resolution over time.The analysis of the expression of green fluorescent protein (GFP) showed that nearly all cell types present in the adult, are found as early as 7 dpf (days post fertilisation) in the zebrafish larvae, which was unknown previously. Equally, at 7dpf, the lamination of the tectum opticum was found to be similar to that of the adult. Furthermore, the onset of development of the tectal laminar structure seems to coincidence with the first arborization of retinal axons onto their tectal target (84 hpf).Linking of these morphological aspects, with functional properties of tectal neurons in the larva, gives valuable information about different players in the visual processing in the tectum. The system developed in the course of this study presents an experimental tool for the investigation of the cytoarchitecture of neural circuits with a quasi non-invasive monitoring of network activity throughout early development in an in vivo preparation, which has direct application to complex questions of systems level neuroscience

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field