Volume Electron Microscopic Analyses in the Larval Zebrafish

The goal of this work was two-fold: 1) To apply serial block-face electron microscopy (SBEM) to the spinal cord of a larval zebrafish, in order to gain a mechanistic understanding of motoneuron (MN) recruitment, based on a reconstruction of the wiring between spinal interneurons and MNs and 2) to implement technological improvements to SBEM that would allow datasets to be acquired at much higher speed, in order to acquire a dataset of a complete larval zebrafish brain. The spinal cord of vertebrates contains a neural circuit known as a central pattern generator (CPG), which can generate the rhythmic muscle contractions underlying locomotion independently of the brain. In fish, the rhythm consists of muscle contractions that alternate between the left and right side of the tail and that travel down the length of the fish, from head to tail. When swimming fast, such as during escapes, the rhythm has a high frequency and muscles contract vigorously. During slow, routine swimming, the rhythm has a low frequency and muscles contract with less strength. The MNs in the spinal cord, which elicit the contractions of the tail musculature, are recruited to different degrees during these different behaviors. With increasing contraction strength, more and larger MNs are activated. This phenomenon is called orderly recruitment. The rhythmic excitation that recruits MNs is provided by Circumferential Descending (CiD) interneurons located in the spinal cord. These interneurons also follow a specific recruitment pattern: During weak swimming, ventral cells are active exclusively and dorsal cells are silent. As swims increase in vigor, the activity in these cells shifts towards more dorsal cells, with more ventral cells becoming inactive. The aim of the first part of this thesis was to reconstruct the MNs along with the CiDs that excite them, using a high resolution SBEM dataset of the spinal cord, to identify the pattern of connectivity between these types of neurons and distinguish between competing hypotheses of orderly MN recruitment. Conceptually, orderly recruitment could either be implemented with unspecific connectivity, in which case it would be a consequence of the interplay of size-dependent biophysical properties (in particular the input resistance) with the strengths of the synapses driving them. Alternatively, the wiring pattern could be specific and the CiDs could select the subset of MNs to activate by making synapses with just those cells. MNs in the larval zebrafish spinal cord clustered into distinct subtypes, depending on their size: Small, intermediate and large. The small MNs received almost no synaptic inputs and appeared to be immature. CiDs differentially innervated the intermediate and large MNs: Ventrally located CiDs did not differentiate between the two subtypes, but the dorsal CiDs made synapses onto large MNs with high specificity. Since dorsal CiDs are active only during the fastest swims, this finding can be interpreted as a labeled line specifically recruiting the strongest MNs during the most vigorous behaviors. During weaker behaviors, when the dorsal CiDs are inactive and the more ventral ones are active exclusively, differences in MN excitability due to size would encode the recruitment order. The second objective was to improve SBEM technology to acquire a whole larval zebrafish brain in a relatively short period of time. Due to the very high resolution required to trace small neurites and to identify synapses, even very small brains, such as the brain of a larval zebrafish, would take many months to acquire using a typical SBEM setup. Two main techniques were used to increase net speed. First, line-scanning of individual image tiles was implemented, where the electron beam scans the image in one axis only and the other axis is scanned by moving the stage. This allows larger individual images to be taken, greatly reducing the number of motor moves between images. Second, dynamic adaptation of the image tile mosaic to the shape of the sample was used to avoid scanning the blank plastic regions surrounding an irregularly shaped sample. These improvements allowed the complete brain of a 5 day old larval zebrafish to be imaged in less than 30% of the time than would have been required previously. In a collaborative project with Dr. Fumi Kubo, two-photon calcium imaging was performed prior to EM imaging, revealing pretectal cells active during optokinetic stimulation. The two-photon dataset was successfully registered to the EM data and a functionally identified pretectal cell could be traced. This dataset will be used to reconstruct the complete neural networks that compute the optokinetic response

Machine learning based analyses on metabolic networks supports high-throughput knockout screens

Background: Computational identification of new drug targets is a major goal of pharmaceutical bioinformatics. Results: This paper presents a machine learning strategy to study and validate essential enzymes of a metabolic network. Each single enzyme was characterized by its local network topology, gene homologies and co-expression, and flux balance analyses. A machine learning system was trained to distinguish between essential and non-essential reactions. It was validated by a comprehensive experimental dataset, which consists of the phenotypic outcomes from single knockout mutants of Escherichia coli (KEIO collection). We yielded very reliable results with high accuracy (93%) and precision (90%). We show that topologic, genomic and transcriptomic features describing the network are sufficient for defining the essentiality of a reaction. These features do not substantially depend on specific media conditions and enabled us to apply our approach also for less specific media conditions, like the lysogeny broth rich medium. Conclusion: Our analysis is feasible to validate experimental knockout data of high throughput screens, can be used to improve flux balance analyses and supports experimental knockout screens to define drug targets

The evolution of plasmid-carried antibiotic resistance

BACKGROUND: Antibiotic resistance represents a significant public health problem. When resistance genes are mobile, being carried on plasmids or phages, their spread can be greatly accelerated. Plasmids in particular have been implicated in the spread of antibiotic resistance genes. However, the selective pressures which favour plasmid-carried resistance genes have not been fully established. Here we address this issue with mathematical models of plasmid dynamics in response to different antibiotic treatment regimes. RESULTS: We show that transmission of plasmids is a key factor influencing plasmid-borne antibiotic resistance, but the dosage and interval between treatments is also important. Our results also hold when plasmids carrying the resistance gene are in competition with other plasmids that do not carry the resistance gene. By altering the interval between antibiotic treatments, and the dosage of antibiotic, we show that different treatment regimes can select for either plasmid-carried, or chromosome-carried, resistance. CONCLUSIONS: Our research addresses the effect of environmental variation on the evolution of plasmid-carried antibiotic resistance

Visual recognition of social signals by a tectothalamic neural circuit

Social affiliation emerges from individual-level behavioural rules that are driven by conspecific signals1,2,3,4,5. Long-distance attraction and short-distance repulsion, for example, are rules that jointly set a preferred interanimal distance in swarms6,7,8. However, little is known about their perceptual mechanisms and executive neural circuits3. Here we trace the neuronal response to self-like biological motion9,10, a visual trigger for affiliation in developing zebrafish2,11. Unbiased activity mapping and targeted volumetric two-photon calcium imaging revealed 21 activity hotspots distributed throughout the brain as well as clustered biological-motion-tuned neurons in a multimodal, socially activated nucleus of the dorsal thalamus. Individual dorsal thalamus neurons encode local acceleration of visual stimuli mimicking typical fish kinetics but are insensitive to global or continuous motion. Electron microscopic reconstruction of dorsal thalamus neurons revealed synaptic input from the optic tectum and projections into hypothalamic areas with conserved social function12,13,14. Ablation of the optic tectum or dorsal thalamus selectively disrupted social attraction without affecting short-distance repulsion. This tectothalamic pathway thus serves visual recognition of conspecifics, and dissociates neuronal control of attraction from repulsion during social affiliation, revealing a circuit underpinning collective behaviour.publishe

Automated synapse-level reconstruction of neural circuits in the larval zebrafish brain

This Resource presents a serial block-face EM dataset of the whole larval zebrafish brain, including automated segmentation of neurons, detection of synapses and reconstruction of circuitry for visual motion processing.Dense reconstruction of synaptic connectivity requires high-resolution electron microscopy images of entire brains and tools to efficiently trace neuronal wires across the volume. To generate such a resource, we sectioned and imaged a larval zebrafish brain by serial block-face electron microscopy at a voxel size of 14 x 14 x 25 nm(3). We segmented the resulting dataset with the flood-filling network algorithm, automated the detection of chemical synapses and validated the results by comparisons to transmission electron microscopic images and light-microscopic reconstructions. Neurons and their connections are stored in the form of a queryable and expandable digital address book. We reconstructed a network of 208 neurons involved in visual motion processing, most of them located in the pretectum, which had been functionally characterized in the same specimen by two-photon calcium imaging. Moreover, we mapped all 407 presynaptic and postsynaptic partners of two superficial interneurons in the tectum. The resource developed here serves as a foundation for synaptic-resolution circuit analyses in the zebrafish nervous system