Altered circadian activity and sleep/wake rhythms in the stable tubule only polypeptide (STOP) null mouse model of schizophrenia.

Sleep and circadian rhythm disruptions commonly occur in individuals with schizophrenia. Stable tubule only polypeptide (STOP) knockout (KO) mice show behavioral impairments resembling symptoms of schizophrenia. We previously reported that STOP KO mice slept less and had more fragmented sleep and waking than wild-type littermates under a light/dark (LD) cycle. Here, we assessed the circadian phenotype of male STOP KO mice by examining wheel-running activity rhythms and EEG/EMG-defined sleep/wake states under both LD and constant darkness (DD) conditions. Wheel-running activity rhythms in KO and wild-type mice were similarly entrained in LD, and had similar free-running periods in DD. The phase delay shift in response to a light pulse given early in the active phase under DD was preserved in KO mice. KO mice had markedly lower activity levels, lower amplitude activity rhythms, less stable activity onsets, and more fragmented activity than wild-type mice in both lighting conditions. KO mice also spent more time awake and less time in rapid eye movement sleep (REMS) and non-REMS (NREMS) in both LD and DD conditions, with the decrease in NREMS concentrated in the active phase. KO mice also showed altered EEG features and higher amplitude rhythms in wake and NREMS (but not REMS) amounts in both lighting conditions, with a longer free-running period in DD, compared to wild-type mice. These results indicate that the STOP null mutation in mice altered the regulation of sleep/wake physiology and activity rhythm expression, but did not grossly disrupt circadian mechanisms.

SH-SY5Y and LUHMES cells display differential sensitivity to MPP+, tunicamycin, and epoxomicin in 2D and 3D cell culture.

SH-SY5Y and LUHMES cell lines are widely used as model systems for studying neurotoxicity. Most of the existing data regarding the sensitivity of these cell lines to neurotoxicants have been recorded from cells growing as two-dimensional (2D) cultures on the surface of glass or plastic. With the emergence of 3D culture platforms designed to better represent native tissue, there is a growing need to compare the toxicology of neurons grown in 3D environments to those grown in 2D to better understand the impact that culture environment has on toxicant sensitivity. Here, a simple 3D culture method was used to assess the impact of growth environment on the sensitivity of SH-SY5Y cells and LUHMES cells to MPP+, tunicamycin, and epoxomicin, three neurotoxicants that have been previously used to generate experimental models for studying Parkinson's disease pathogenesis. SH-SY5Y cell viability following treatment with these three toxicants was significantly lower in 2D cultures as compared to 3D cultures. On the contrary, LUHMES cells did not show significant differences between growth conditions for any of the toxicants examined. However, LUHMES cells were more sensitive to MPP+, tunicamycin, and epoxomicin than SH-SY5Y cells. Thus, both the choice of cell line and the choice of growth environment must be considered when interpreting in vitro neurotoxicity data.

Fabrication of thin-layer matrigel-based constructs for three-dimensional cell culture.

Extracellular matrix-based hydrogels such as Matrigel are easy-to-use, commercially available, and offer environments for three-dimensional (3-D) cell culture that mimic native tissue. However, manipulating small volumes of these materials to produce thin-layer 3-D culture systems suitable for analysis is difficult because of air-liquid-substrate interfacial tension effects and evaporation. Here, we demonstrate two simple techniques that use standard liquid-handling tools and nontreated 96-well plates to produce uniform, thin-layer constructs for 3-D culture of cells in Matrigel. The first technique, the floating 3-D cell culture method, uses phase-separating polymers to form a barrier between the dispensed Matrigel, air, and cultureware surface to generate consistently thin hydrogels from volumes as low as 5 µL. These unanchored gels provide a useful assay for investigating airway smooth muscle cell contraction and may have future applications in studying asthma pathophysiology. The second technique, the fixed 3-D cell culture method, provides an anchored gel system for culturing noncontractile cells (e.g., neurons) where 20 µL of Matrigel is dispensed into the bottom of a well filled with culture medium to form a thin gel containing embedded cells. This technique has potential widespread applications as an accessible 3-D culture platform for high-throughput production of disease models for evaluation of novel drug therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2733, 2019.

Emerging Biotechnology Applications of Aqueous Two-Phase Systems.

Liquid-liquid phase separation between aqueous solutions containing two incompatible polymers, a polymer and a salt, or a polymer and a surfactant, has been exploited for a wide variety of biotechnology applications throughout the years. While many applications for aqueous two-phase systems fall within the realm of separation science, the ability to partition many different materials within these systems, coupled with recent advances in materials science and liquid handling, has allowed bioengineers to imagine new applications. This progress report provides an overview of the history and key properties of aqueous two-phase systems to lend context to how these materials have progressed to modern applications such as cellular micropatterning and bioprinting, high-throughput 3D tissue assembly, microscale biomolecular assay development, facilitation of cell separation and microcapsule production using microfluidic devices, and synthetic biology. Future directions and present limitations and design considerations of this adaptable and promising toolkit for biomolecule and cellular manipulation are further evaluated.

Microscopic evaluation of aqueous two-phase system emulsion characteristics enables rapid determination of critical polymer concentrations for solution micropatterning.

Aqueous two-phase systems have emerged as valuable tools for microscale analysis of cell growth and many other biotechnology applications. The most critical step in developing an aqueous two-phase system for a specific application is identifying the critical concentrations at which the polymer solutions phase-separate. Current techniques for determining these critical concentrations rely on laborious methods, highly specialized assays or computational methods that make this step difficult for non-specialists. To overcome these limitations, we present a simplified assay that uses only readily accessible laboratory instruments and consumables (e.g., multichannel micropipettes, 96-well plates and a simple compound microscope) to determine the critical concentrations of aqueous two-phase system-forming polymers. We demonstrate that formulations selected from phase diagrams that describe these critical concentrations can be applied for solution micropatterning of cells.

Region-specific increases in FosB/ΔFosB immunoreactivity in the rat brain in response to chronic sleep restriction.

Using a rat model of chronic sleep restriction (CSR) featuring periodic sleep deprivation with slowly rotating wheels (3h on/1h off), we previously observed that 99h of this protocol induced both homeostatic and allostatic (adaptive) changes in physiological and behavioural measures. Notably, the initial changes in sleep intensity and attention performance gradually adapted during CSR despite accumulating sleep loss. To identify brain regions involved in these responses, we used FosB/ΔFosB immunohistochemistry as a marker of chronic neuronal activation. Adult male rats were housed in motorized activity wheels and underwent the 3/1 CSR protocol for 99h, or 99h followed by 6 or 12days of recovery. Control rats were housed in home cages, locked activity wheels, or unlocked activity wheels that the animals could turn freely. Immunohistochemistry was conducted using an antibody that recognized both FosB and ΔFosB, and 24 brain regions involved in sleep/wake, autonomic, and limbic functions were examined. The number of darkly-stained FosB/ΔFosB-immunoreactive cells was increased immediately following 99h of CSR in 8/24 brain regions, including the medial preoptic and perifornical lateral hypothalamic areas, dorsomedial and paraventricular hypothalamic nuclei, and paraventricular thalamic nucleus. FosB/ΔFosB labeling was at control levels in all 8 brain areas following 6 or 12 recovery days, suggesting that most of the immunoreactivity immediately after CSR reflected FosB, the more transient marker of chronic neuronal activation. This region-specific induction of FosB/ΔFosB following CSR may be involved in the mechanisms underlying the allostatic changes in behavioural and physiological responses to CSR.