Peripheral and Central Nutrient Sensing Underlying Appetite Regulation

The precise regulation of fluid and energy homeostasis is essential for survival. It is well appreciated that ingestive behaviors are tightly regulated by both peripheral sensory inputs and central appetite signals. With recent neurogenetic technologies, considerable progress has been made in our understanding of basic taste qualities, the molecular and/or cellular basis of taste sensing, and the central circuits for thirst and hunger. In this review, we first highlight the functional similarities and differences between mammalian and invertebrate taste processing. We then discuss how central thirst and hunger signals interact with peripheral sensory signals to regulate ingestive behaviors. We finally indicate some of the directions for future research

Peripheral and Central Nutrient Sensing Underlying Appetite Regulation

The precise regulation of fluid and energy homeostasis is essential for survival. It is well appreciated that ingestive behaviors are tightly regulated by both peripheral sensory inputs and central appetite signals. With recent neurogenetic technologies, considerable progress has been made in our understanding of basic taste qualities, the molecular and/or cellular basis of taste sensing, and the central circuits for thirst and hunger. In this review, we first highlight the functional similarities and differences between mammalian and invertebrate taste processing. We then discuss how central thirst and hunger signals interact with peripheral sensory signals to regulate ingestive behaviors. We finally indicate some of the directions for future research

An Automated Microfluidic Multiplexer for Fast Delivery of C. elegans Populations from Multiwells

Automated biosorter platforms, including recently developed microfluidic devices, enable and accelerate high-throughput and/or high-resolution bioassays on small animal models. However, time-consuming delivery of different organism populations to these systems introduces a major bottleneck to executing large-scale screens. Current population delivery strategies rely on suction from conventional well plates through tubing periodically exposed to air, leading to certain disadvantages: 1) bubble introduction to the sample, interfering with analysis in the downstream system, 2) substantial time drain from added bubble-cleaning steps, and 3) the need for complex mechanical systems to manipulate well plate position. To address these concerns, we developed a multiwell-format microfluidic platform that can deliver multiple distinct animal populations from on-chip wells using multiplexed valve control. This Population Delivery Chip could operate autonomously as part of a relatively simple setup that did not require any of the major mechanical moving parts typical of plate-handling systems to address a given well. We demonstrated automatic serial delivery of 16 distinct C. elegans worm populations to a single outlet without introducing any bubbles to the samples, causing cross-contamination, or damaging the animals. The device achieved delivery of more than 90% of the population preloaded into a given well in 4.7 seconds; an order of magnitude faster than delivery modalities in current use. This platform could potentially handle other similarly sized model organisms, such as zebrafish and drosophila larvae or cellular micro-colonies. The device’s architecture and microchannel dimensions allow simple expansion for processing larger numbers of populations.The authors would like to thank the National Institutes of Health (www.nih.gov) for its generous support of this research. Specifically, the grants that made this work possible are the NIH Director's Transformative Award (NIH R01 AG041135), NIH R21 NS067340, and NIH R01 NS060129. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biomedical EngineeringElectrical and Computer EngineeringMechanical Engineerin

Hierarchical neural architecture underlying thirst regulation

Neural circuits for appetites are regulated by both homeostatic perturbations and ingestive behaviour. However, the circuit organization that integrates these internal and external stimuli is unclear. Here we show in mice that excitatory neural populations in the lamina terminalis form a hierarchical circuit architecture to regulate thirst. Among them, nitric oxide synthase-expressing neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Conversely, a distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of GLP1R-expressing MnPO neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. Our study reveals dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids and the consequent drinking behaviour to maintain internal water balance

Parallel and serial microfluidic platforms for femtosecond laser axotomy in Caenorhabditis elegans for nerve regeneration studies

Understanding the molecular basis of nerve regeneration can potentiate the development of novel and efficient treatments for neurodegenerative diseases. Severing axons in the small nematode Caenorhabditis elegans (C. elegans) with femtosecond laser surgery and then observing the subsequent axonal regrowth is a promising approach to understand the molecular mechanisms of nerve regeneration in vivo. Effective and reversible immobilization of the nematodes is necessary for both axotomy and follow-up imaging. However, conventional worm handling techniques such as using anesthetics or polystyrene beads are labor-intensive and time-consuming processes, hindering high-throughput. Microfluidic devices enable the manipulation of the nematodes on a single chip with unprecedented throughput and integrity. This dissertation introduces two comprehensive microfluidic systems for femtosecond laser axotomy in C. elegans, offering several advantages over conventional techniques in terms of speed and the ability for automation of the tedious axotomy experiments. The first microfluidic system is an automated serial microfluidic platform for performing femtosecond laser axotomy in C. elegans. The microfluidic platform along with a custom-developed automation program isolates a single nematode from a pre-loaded population, immobilizes the nematode, and performs femtosecond laser axotomy. The full automation of the axotomy process is achieved by combining efficient image analysis methodologies with synchronized valve and flow progression in the microfluidic chip to perform multiple surgeries in a serial and automated manner. The serial automated microfluidic platform reduces the time required to perform axotomies within individual worms to ~ 17 s/worm. The second microfluidic system is a parallelized multitrap microfluidic platform, “worm hospital”, that allows on-chip axotomy, post-surgery housing for recovery, and imaging of nerve regeneration on a single chip. The microfluidic platform features 20 trapping channels for laser axotomy and subsequent post-surgery imaging, and a perfusion area to house the worms after laser axotomy. This microfluidic platform is a single-flow Polydimethylsiloxane (PDMS) layer device and has no active control PDMS layer, which reduces the fabrication and operation complexity of the chip, especially for non-expert users. The roles of neurodevelopmental genes in the Wnt/Frizzled pathway on the nerve regeneration was investigated using the “worm hospital”. In summary, the microfluidic platforms presented in this dissertation enabled performing femtosecond laser axotomy in C. elegans in a fast and repeatable manner with a controllable microenvironment. Both microfluidic platforms offer promising methodologies for prospective large-scale screening of genes involved in nerve regeneration with a high throughput in an automated manner.Electrical and Computer Engineerin

Two-Dimensional MEMS Stage Integrated With Microlens Arrays for Laser Beam Steering

A novel microelectromechanical stage with one uniaxial set of combs capable of 2-D actuation is presented. A polymer microlens array (MLA) is mounted vertically onto the stage. Driven at resonance, the stage deflects 124 mu m out of plane and 34 mu m in plane. Finally, laser beam steering is demonstrated using two cascaded MLAs

A multi-trap microfluidic chip enabling longitudinal studies of nerve regeneration in Caenorhabditis elegans

Several sophisticated microfluidic devices have recently been proposed for femtosecond laser axotomy in the nematode C. elegans for immobilization of the animals for surgery to overcome time-consuming and labor-intensive manual processes. However, nerve regeneration studies require long-term recovery of the animals and multiple imaging sessions to observe the regeneration capabilities of their axons post-injury. Here we present a simple, multi-trap device, consisting of a single PDMS (polydimethylsiloxane) layer, which can immobilize up to 20 animals at the favorable orientation for optical access needed for precise laser surgery and high-resolution imaging. The new device, named "worm hospital" allows us to perform the entire nerve regeneration studies, including on-chip axotomy, post-surgery housing for recovery, and post-recovery imaging all on one microfluidic chip. Utilizing the worm hospital and analysis of mutants, we observed that most but not all neurodevelopmental genes in the Wnt/Frizzled pathway are important for regeneration of the two touch receptor neurons ALM and PLM. Using our new chip, we observed that the cwn-2 and cfz-2 mutations significantly reduced the reconnection possibilities of both neurons without any significant reduction in the regrowth lengths of the severed axons. We observed a similar regeneration phenotype with cwn-1 mutation in ALM neurons only

A fully automated microfluidic femtosecond laser axotomy platform for nerve regeneration studies in C. elegans.

Femtosecond laser nanosurgery has been widely accepted as an axonal injury model, enabling nerve regeneration studies in the small model organism, Caenorhabditis elegans. To overcome the time limitations of manual worm handling techniques, automation and new immobilization technologies must be adopted to improve throughput in these studies. While new microfluidic immobilization techniques have been developed that promise to reduce the time required for axotomies, there is a need for automated procedures to minimize the required amount of human intervention and accelerate the axotomy processes crucial for high-throughput. Here, we report a fully automated microfluidic platform for performing laser axotomies of fluorescently tagged neurons in living Caenorhabditis elegans. The presented automation process reduces the time required to perform axotomies within individual worms to ∼17 s/worm, at least one order of magnitude faster than manual approaches. The full automation is achieved with a unique chip design and an operation sequence that is fully computer controlled and synchronized with efficient and accurate image processing algorithms. The microfluidic device includes a T-shaped architecture and three-dimensional microfluidic interconnects to serially transport, position, and immobilize worms. The image processing algorithms can identify and precisely position axons targeted for ablation. There were no statistically significant differences observed in reconnection probabilities between axotomies carried out with the automated system and those performed manually with anesthetics. The overall success rate of automated axotomies was 67.4±3.2% of the cases (236/350) at an average processing rate of 17.0±2.4 s. This fully automated platform establishes a promising methodology for prospective genome-wide screening of nerve regeneration in C. elegans in a truly high-throughput manner