Search

Hydra is genus of freshwater cnidarian polyp found in lakes, ponds, and streams all over the world. Its remarkable ability to regenerate missing body parts, and even its whole body from fragments, has made Hydra a model system for regeneration and development for over 250 years. Despite their anatomical simplicity, much about Hydra’s basic biology and extraordinary regeneration caption remains mysterious. The simplicity of caring for and doing experiments on Hydra makes it an accessible subject for undergraduate research, high school lab activities, or even a home hobbyist. This collection of protocols describes low-cost methods for maintaining Hydra cultures, including step-by-step instructions for preparing media, growing brine shrimp for feeding to Hydra, and caring for your Hydra colonies. We’ve focused on equipment that can be found in most high school and college labs, or consumer products that can be purchased online. Experiments on Hydra can be incredibly simple. Much can be learned from dissecting a Hydra and observing its regeneration. Hydra’s response to varying environmental conditions can also be explored in a classroom setting with minimal resources. These simple experiments can be a bridge to cutting-edge molecular biology and stem cell research. Sequencing of the Hydra genome has opened the door for genetic manipulations and large-scale bioinformatics studies. Transgenic lines with fluorescently-labeled cell types and proteins, including neuron and muscle activity reporters, are available. References: Contents: 1: Storing brine shrimp cysts 2: Brine shrimp hatching medium 3: Hatch brine shrimp 4: Prepare stock solutions 5: Prepare Hydra medium 6: Harvest brine shrimp 7: Feed Hydra 8: Routine Hydra tank cleaning 9: Full cleaning of Hydra tanks 10: Monitor Hydra health 11: Emergency antibiotic treatment

This is a series of introductory lab activities for BIOL309-03: Research Methods. The purpose is to master vocabulary related to Hydra anatomy, become familiar with compound microscopes and dissecting microscopes, and practice microdissections and micropipette use. The second section sets up a head and tentacle regeneration experiment that will be documented a week later to provide quantitative data for a statistics activity.

The freshwater cnidarian Hydra has been a model system for regeneration and developmental biology for over 250 years, but much remains unknown about their biodiversity and global distribution. As a citizen scientist, you can contribute to our understanding of Hydra in the wild by becoming a "Hydra Hunter". All it takes is a few simple materials and a little patience. Collecting Hydra in the wild can be challenging. You will certainly not find them everywhere you look. Keep in mind that NOT finding Hydra is still useful information because this will help us understand the environmental factors that effect their distribution. Metadata submission form: https://forms.gle/cAZCiiRCyE922G5t5 Please contact chyland@sandiego.edu for more information or to receive a Hydra collecting kit. Hydra collecting kits were purchased with a grant to Kimberly Sladek from the University of San Diego Associated Students Government. Thank you to Rob Steele for helpful feedback on this protocol. References: Campbell, R. D. (1983). Hydra Collecting. In H. M. Lenhoff (Ed.). Hydra: Research Methods. New York: Springer Science + Business Media. Martínez, D. E., et al. (2010). Phylogeny and biogeography of Hydra (Cnidaria: Hydridae) using mitochondrial and nuclear DNA sequence. Molecular Phylogenetics and Evolution, 57, 403-410. doi:10.1016/j.ympev.2010.06.016

Hydra, is genus of freshwater cnidarian polyp found in freshwater ponds and streams all over the world. Its remarkable ability to regenerate missing body parts, and even its whole bodies from fragments, has made Hydra a model system for regeneration and development for over 250 years. Despite their anatomical simplicity, much about Hydra’s basic biology and extraordinary regeneration caption remains mysterious. The simplicity of caring for and doing experiments on Hydra makes it an accessible subject for undergraduate research, high school lab activities, or even a home hobbyist. This collection of protocols describes low-cost methods for maintaining Hydra cultures, including step-by-step instructions for preparing media, growing brine shrimp for feeding to Hydra, and caring for your Hydra colonies. We’ve focused on equipment that can be found in most high school and college labs, or consumer products that can be purchased online. Experiments on Hydra can be incredibly simple. Much can be learned from dissecting a Hydra and observing its regeneration. Hydra’s response to varying environmental conditions can also be explored in a classroom setting with minimal resources. These simple experiments can be a bridge to cutting-edge molecular biology and stem cell research. Sequencing of the Hydra genome has opened the door for genetic manipulations and large-scale bioinformatics studies. Transgenic lines with fluorescently-labeled cell types and proteins, including neuron and muscle activity reporters, are available. References: Contents: 1: Storing brine shrimp cysts 2: Brine shrimp hatching medium 3: Hatch brine shrimp 4: Prepare stock solutions 5: Prepare Hydra medium 6: Harvest brine shrimp 7: Feed Hydra 8: Routine Hydra tank cleaning 9: Full cleaning of Hydra tanks 10: Monitor Hydra health 11: Emergency antibiotic treatment

Sleep is a conserved neurobehavioral state observed in animals with sufficiently complex nervous systems and is critical for survival. While the exact function of sleep remains unknown, the lack of sleep can have a range of physiological and behavioral effects. Studies in invertebrates and vertebrates have identified conserved neural mechanisms and cellular pathways in control of sleep, wakefulness and arousal. Methodologies to measure sleep have ranged from EEG recordings in humans and rodents to in-depth analysis of locomotor patterns in flies, fish and worms. Here we focus on sleep measurements using activity monitoring in the highly versatile experimental model system, Drosophila melanogaster, which is amenable to a number of genetic, physiological and behavioral manipulations. Further, we also describe methods used to manipulate sleep and wakefulness to understand the neural regulation of sleep and how organisms balance sleep, wakefulness and behavioral arousal. Sleep as a behavioral state is regulated by a number of factors including food, environmental conditions, and genetic background. The methodologies described here provide, a high-throughput approach to study neural regulation of sleep and factors that affect this complex behavior.

Sleep is a conserved neurobehavioral state observed in animals with sufficiently complex nervous systems and is critical for survival. While the exact function of sleep remains unknown, the lack of sleep can have a range of physiological and behavioral effects. Studies in invertebrates and vertebrates have identified conserved neural mechanisms and cellular pathways in control of sleep, wakefulness and arousal. Methodologies to measure sleep have ranged from EEG recordings in humans and rodents to in-depth analysis of locomotor patterns in flies, fish and worms. Here we focus on sleep measurements using activity monitoring in the highly versatile experimental model system, Drosophila melanogaster, which is amenable to a number of genetic, physiological and behavioral manipulations. Further, we also describe methods used to manipulate sleep and wakefulness to understand the neural regulation of sleep and how organisms balance sleep, wakefulness and behavioral arousal. Sleep as a behavioral state is regulated by a number of factors including food, environmental conditions, and genetic background. The methodologies described here provide, a high-throughput approach to study neural regulation of sleep and factors that affect this complex behavior.

Hydra, a simple freshwater animal famous for its regenerative capabilities, must tear a hole through its epithelial tissue each time it opens its mouth. The feeding response of Hydra has been well-characterized physiologically and is regarded as a classical model system for environmental chemical biology. However, due to a lack of in vivo labeling and imaging tools, the biomechanics of mouth opening have remained completely unexplored. We take advantage of the availability of transgenic Hydra lines to perform the first dynamical analysis, to our knowledge, of Hydra mouth opening and test existing hypotheses regarding the underlying cellular mechanisms. Through cell position and shape tracking, we show that mouth opening is accompanied by changes in cell shape, but not cellular rearrangements as previously suggested. Treatment with a muscle relaxant impairs mouth opening, supporting the hypothesis that mouth opening is an active process driven by radial contractile processes (myonemes) in the ectoderm. Furthermore, we find that all events exhibit the same relative rate of opening. Because one individual can open consecutively to different amounts, this suggests that the degree of mouth opening is controlled through neuronal signaling. Finally, from the opening dynamics and independent measurements of the elastic properties of the tissues, we estimate the forces exerted by the myonemes to be on the order of a few nanoNewtons. Our study provides the first dynamical framework, to our knowledge, for understanding the remarkable plasticity of the Hydra mouth and illustrates that Hydra is a powerful system for quantitative biomechanical studies of cell and tissue behaviors in vivo.

Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

Growth cones of elongating neurites exert force against the external environment, but little is known about the role of force in outgrowth or its relationship to the mechanical organization of neurons. We used traction force microscopy to examine patterns of force in growth cones of regenerating Aplysia bag cell neurons. We find that traction is highest in the peripheral actin-rich domain and internal stress reaches a plateau near the transition between peripheral and central microtubule-rich domains. Integrating stress over the area of the growth cone reveals that total scalar force increases with area but net tension on the neurite does not. Tensions fall within a limited range while a substantial fraction of the total force can be balanced locally within the growth cone. Although traction continuously redistributes during extension and retraction of the peripheral domain, tension is stable over time, suggesting that tension is a tightly regulated property of the neurite independent of growth cone dynamics. We observe that redistribution of traction in the peripheral domain can reorient the end of the neurite shaft. This suggests a role for off-axis force in growth cone turning and neuronal guidance., Scientific Reports, 4, ISSN:2045-2322

We have investigated Aplysia hemolymph as a source of endogenous factors to promote regeneration of bag cell neurons. We describe a novel synergistic effect between substrate-bound hemolymph proteins and laminin. This combination increased outgrowth and branching relative to either laminin or hemolymph alone. Notably, the addition of hemolymph to laminin substrates accelerated growth cone migration rate over ten-fold. Our results indicate that the active factor is either a high molecular weight protein or protein complex and is not the respiratory protein hemocyanin. Substrate-bound factor(s) from central nervous system-conditioned media also had a synergistic effect with laminin, suggesting a possible cooperation between humoral proteins and nervous system extracellular matrix. Further molecular characterization of active factors and their cellular targets is warranted on account of the magnitude of the effects reported here and their potential relevance for nervous system repair., Scientific Reports, 4, ISSN:2045-2322

Johnson-Kendall-Robert (JKR) theory is the basis of modern contact mechanics. It describes how two deformable objects adhere together, driven by adhesion energy and opposed by elasticity. However, it does not include solid surface tension, which also opposes adhesion by acting to flatten the surface of soft solids. We tested JKR theory to see if solid surface tension affects indentation behaviour. Using confocal microscopy, we characterised the indentation of glass particles into soft, silicone substrates. While JKR theory held for particles larger than a critical, elastocapillary lengthscale, it failed for smaller particles. Instead, adhesion of small particles mimicked the adsorption of particles at a fluid interface, with a size-independent contact angle between the undeformed surface and the particle given by a generalised version of Young's law. A simple theory quantitatively captures this behaviour, and explains how solid surface tension dominates elasticity for small-scale indentation of soft materials., 6 pages, 5 figures

PKC activation enhances myosin II contractility in the central growth cone domain while decreasing actin density and increasing actin network flow rates in the peripheral domain. This dual mode of action has mechanistic implications for interpreting reported effects of PKC on growth cone guidance and neuronal regeneration., Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase–potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.

Numerous cell types have shown a remarkable ability to detect and move along gradients in stiffness of an underlying substrate—a process known as durotaxis. The mechanisms underlying durotaxis are still unresolved, but generally believed to involve active sensing and locomotion. Here, we show that simple liquid droplets also undergo durotaxis. By modulating substrate stiffness, we obtain fine control of droplet position on soft, flat substrates. Unlike other control mechanisms, droplet durotaxis works without imposing chemical, thermal, electrical, or topographical gradients. We show that droplet durotaxis can be used to create large-scale droplet patterns and is potentially useful for many applications, such as microfluidics, thermal control, and microfabrication.

Loss of the RNA-binding protein fragile X mental retardation protein (FMRP) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na+-activated K+channels (KNa), producing an enhancement of channel activity. We have now usedAplysiabag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were colocalized at the periphery of isolated BC neurons, and the two proteins could be reciprocally coimmunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to inside-out patches containing nativeAplysiaSlack channels increased channel opening and, in current-clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that, in BC neurons, Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel–FMRP interactions may link changes in neuronal firing to changes in protein translation.

5-HT promotes neurite growth via IP3-dependent Ca2+ release in neuronal growth cones. Outgrowth depends on increased rates of actin array treadmilling mediated by Ca2+-calcineurin–dependent cofilin activation. This mode of growth contrasts with substrate-dependent responses, for which retrograde actin flow and advance rates have been inversely correlated., Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca2+; however, mechanistic roles for Ca2+ in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca2+ → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca2+ release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT–dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.

To understand how the mechanical properties of tissues emerge from interactions of multiple cells, we measure traction stresses of cohesive colonies of 1-27 cells adherent to soft substrates. We find that traction stresses are generally localized at the periphery of the colony and the total traction force scales with the colony radius. For large colony sizes, the scaling appears to approach linear, suggesting the emergence of an apparent surface tension of order 1E-3 N/m. A simple model of the cell colony as a contractile elastic medium coupled to the substrate captures the spatial distribution of traction forces and the scaling of traction forces with the colony size., 5 pages, 3 figures

Colloidal coatings, such as paint, are all around us. However, we know little about the mechanics of the film-forming process because the composition and properties of drying coatings vary dramatically in space and time. To surmount this challenge, we extend traction force microscopy to quantify the spatial distribution of all three components of the stress at the interface of two materials. We apply this approach to image stress near the tip of a propagating interface crack in a drying colloidal coating and extract the stress intensity factor.

Activated mononucleotides oligomerize in the presence of montmorillonite clay to form RNA oligomers. In the present study, effects of salts, temperature and pH on the clay-catalyzed synthesis of RNA oligomers were investigated. This reaction is favored by relatively high concentration of salts, such as 1 M NaCl. It was shown that the presence of divalent cations was not required for this reaction. High concentrations of NH4+ and HCO3- and 0.01 M HPO4(2-) inhibit the reaction. The yields of RNA oligomers decreased as the temperature was raised from 4 degrees C to 50 degrees C. A5' ppA was the major product at pH's below 6. The catalytic activity of a variety of minerals and three meteorites were investigated but none of them except galena catalyzed the oligomerization. ATP was generated from ADP but it was due to the presence of HEPES buffer and not due to the minerals. Meteorites catalyzed the hydrolysis of the pyrophosphate bonds of ATP. The results suggest that oligomers of RNA could have formed in pH 7-9 solutions of alkali metal salts in the presence of montmorillonite clay.