Through the course of evolution, the gastrointestinal (GI) tract has been modified to maximize nutrient absorption, forming specialized segments that are morphologically and functionally distinct. Here we show that the GI tract of the Mexican tetra, Astyanax mexicanus, has distinct regions, exhibiting differences in morphology, motility, and absorption. We found that A. mexicanus populations adapted for life in subterranean caves exhibit differences in the GI segments compared to those adapted to surface rivers. Cave-adapted fish exhibit bi-directional churning motility in the stomach region that is largely absent in river-adapted fish. We investigated how this motility pattern influences intestinal transit of powdered food and live prey. We found that powdered food is more readily emptied from the cavefish GI tract. In contrast, the transit of live rotifers from the stomach region to the midgut occurs more slowly in cavefish compared to surface fish, consistent with the presence of churning motility. Differences in intestinal motility and transit likely reflect adaptation to unique food sources available to post-larval A. mexicanus in the cave and river environments. We found that cavefish grow more quickly than surface fish when fed ad libitum, suggesting that altered GI function may aid in nutrient consumption or absorption. We did not observe differences in enteric neuron density or smooth muscle organization between cavefish and surface fish. Altered intestinal motility in cavefish could instead be due to changes in the activity or patterning of the enteric nervous system. Exploring this avenue will lead to a better understanding of how the GI tract evolves to maximize energy assimilation from novel food sources.

The role of the microbes in gut homeostasis and its interaction with the nervous system has gained immense attention in recent years, owing to its implications on health and in a diverse range of disease states such as neurodegeneration and autism spectrum disorders. [1][2][3] The initial point of contact between the microbiota and the host is the intestinal epithelium, which is in turn densely innervated by sensory neurons and secretomotor neurons from the underlying enteric nervous system (ENS). This is an extensive neural network organized in two ganglionated plexuses-the submucosal and myenteric plexus. The submucosal plexus is mainly responsible for regulating fluid secretion, while the myenteric plexus is critical for the control of intestinal motility. Contained within these plexuses are sensory,

Epilepsy is a common disorder of the brain characterized by spontaneous recurrent seizures, which develop gradually during a process called epileptogenesis. The mechanistic processes underlying the changes of brain tissue and networks toward increased seizure susceptibility are not fully understood. In rodents, injection of kainic acid (KA) ultimately leads to the development of spontaneous epileptic seizures, reflecting similar neuropathological characteristics as seen in patients with temporal lobe epilepsy (TLE). Although this model has significantly contributed to increased knowledge of epileptogenesis, it is technically demanding, costly to operate and hence not suitable for high-throughput screening of anti-epileptic drugs (AEDs). Zebrafish, a vertebrate with complementary advantages to rodents, is an established animal model for epilepsy research. Here, we generated a novel KA-induced epilepsy model in zebrafish larvae that we functionally and pharmacologically validated. KA was administered by pericardial injection at an early zebrafish larval stage. The epileptic phenotype induced was examined by quantification of seizure-like behavior using automated video recording, and of epileptiform brain activity measured via local field potential (LFP) recordings. We also assessed GFP-labeled GABAergic and RFP-labeled glutamatergic neurons in double transgenic KA-injected zebrafish larvae, and examined the GABA and glutamate levels in the larval heads by liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). Finally, KA-injected larvae were exposed to five commonly used AEDs by immersion for pharmacological characterization of the model. Shortly after injection, KA induced a massive damage and inflammation in the zebrafish brain and seizure-like locomotor behavior. An abnormal reorganization of brain circuits was observed, a decrease in both GABAergic and glutamatergic neuronal population and their associated neurotransmitters. Importantly, these changes were accompanied by spontaneous and continuous epileptiform brain discharges starting after a short latency period, as seen in KA rodent models and reminiscent of human pathology. Three out of five AEDs tested rescued LFP abnormalities but did not affect the seizure-like behavior. Taken together, for the first time we describe a chemically-induced larval zebrafish epilepsy model offering unique insights into studying epileptogenic processes in vivo and suitable for high-throughput AED screening purposes and rapid genetic investigations.

Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules that fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin in neurons both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers like guanosine triphosphate (GTP)-tubulin. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin velocity and density on microtubules consisting of elongated dimers add to the increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin contributes to the regulation of mitochondrial motility and as such to the local distribution of mitochondria along axons.

Ca2+ imaging is a widely used microscopy technique to simultaneously study cellular activity in multiple cells. The desired information consists of cell-specific time series of pixel intensity values, in which the fluorescence intensity represents cellular activity. For static scenes, cellular signal extraction is straightforward, however multiple analysis challenges are present in recordings of contractile tissues, like those of the enteric nervous system (ENS). This layer of critical neurons, embedded within the muscle layers of the gut wall, shows optical overlap between neighboring neurons, intensity changes due to cell activity, and constant movement. These challenges reduce the applicability of classical segmentation techniques and traditional stack alignment and regions-of-interest (ROIs) selection workflows. Therefore, a signal extraction method capable of dealing with moving cells and is insensitive to large intensity changes in consecutive frames is needed. Here we propose a b-spline active contour method to delineate and track neuronal cell bodies based on local and global energy terms. We develop both a single as well as a double-contour approach. The latter takes advantage of the appearance of GCaMP expressing cells, and tracks the nucleus’ boundaries together with the cytoplasmic contour, providing a stable delineation of neighboring, overlapping cells despite movement and intensity changes. The tracked contours can also serve as landmarks to relocate additional and manually-selected ROIs. This improves the total yield of efficacious cell tracking and allows signal extraction from other cell compartments like neuronal processes. Compared to manual delineation and other segmentation methods, the proposed method can track cells during large tissue deformations and high-intensity changes such as during neuronal firing events, while preserving the shape of the extracted Ca2+ signal. The analysis package represents a significant improvement to available Ca2+ imaging analysis workflows for ENS recordings and other systems where movement challenges traditional Ca2+ signal extraction workflows.

Live calcium imaging is often used as a proxy for electrophysiological measurements and has been a valuable tool that allows simultaneous analysis of neuronal activity in multiple cells at the population level. In the enteric nervous system, there are two main electrophysiological classes of neurons, AH- and S-neurons, which have been shown to have different calcium handling mechanisms. However, they are rarely considered separately in calcium imaging experiments. A handful of studies have shown that in guinea pig, a calcium transient will accompany a single action potential in AH-neurons, but multiple action potentials are required to generate a calcium transient in S-neurons. How this translates to different modes of cellular depolarisation and whether this is consistent across species is unknown. In this study, we used simultaneous whole-cell patch-clamp electrophysiology together with calcium imaging to investigate how enteric neurons respond to different modes of depolarisation. Using both traditional (4Hz) and also high-speed (1000Hz) imaging techniques, we found that single action potentials elicit calcium transients in both AH-neurons and S-neurons. Sub-threshold membrane depolarisations were also able to elicit calcium transients, although calcium responses were generally amplified if an action potential was present. Further, we identified that responses to nicotinic acetylcholine receptor stimulation can be used to distinguish between AH- and S-neurons in calcium imaging.

Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium.Mitochondrial transport requires and depends on microtubules which fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers, similar to GTP-tubulin, that are more straight and rigid. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin step processivity on microtubules consisting of elongated dimers underlies increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin controls mitochondrial motility and as such locally regulates the distribution of mitochondria along axons.

Ca 2+ imaging is a widely used microscopy technique to simultaneously study cellular activity in multiple cells. The desired information consists of cell-specific time series of pixel intensity values, in which the fluorescence intensity represents cellular activity. For static scenes, cellular signal extraction is straightforward, however multiple analysis challenges are present in recordings of contractile tissues, like those of the enteric nervous system (ENS). This layer of critical neurons, embedded within the muscle layers of the gut wall, shows optical overlap between neighboring neurons, intensity changes due to cell activity, and constant movement. These challenges reduce the applicability of classical segmentation techniques and traditional stack alignment and regions-of-interest (ROIs) selection workflows. Therefore, a signal extraction method capable of dealing with moving cells and is insensitive to large intensity changes in consecutive frames is needed. Here we propose a b-spline active contour method to delineate and track neuronal cell bodies based on local and global energy terms. We develop both a single as well as a double-contour approach. The latter takes advantage of the appearance of GCaMP expressing cells, and tracks the nucleus’ boundaries together with the cytoplasmic contour, providing a stable delineation of neighboring, overlapping cells despite movement and intensity changes. The tracked contours can also serve as landmarks to relocate additional and manually-selected ROIs. This improves the total yield of efficacious cell tracking and allows signal extraction from other cell compartments like neuronal processes. Compared to manual delineation and other segmentation methods, the proposed method can track cells during large tissue deformations and high-intensity changes such as during neuronal firing events, while preserving the shape of the extracted Ca 2+ signal. The analysis package represents a significant improvement to available Ca 2+ imaging analysis workflows for ENS recordings and other systems where movement challenges traditional Ca 2+ signal extraction workflows.