Alternative splicing of clock transcript mediates the response of circadian clocks to temperature changes.

Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. In Drosophila, seasonal adaptations and temperature compensation are regulated by temperature-sensitive alternative splicing (AS) of period (per) and timeless (tim) genes that encode key transcriptional repressors of clock gene expression. Although clock (clk) gene encodes the critical activator of clock gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. We therefore sought to investigate whether clk exhibits AS in response to temperature and the functional changes of the differentially spliced transcripts. We observed that clk transcripts indeed undergo temperature-sensitive AS. Specifically, cold temperature leads to the production of an alternative clk transcript, hereinafter termed clk-cold, which encodes a CLK isoform with an in-frame deletion of four amino acids proximal to the DNA binding domain. Notably, serine 13 (S13), which we found to be a CK1α-dependent phosphorylation site, is among the four amino acids deleted in CLK-cold protein. Using a combination of transgenic fly, tissue culture, and in vitro experiments, we demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing CLK occupancy at clock gene promoters. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature, which can be explained by the higher amounts of CLK-cold isoforms that lack S13 residue. This study provides new insights into the complex collaboration between AS and phospho-regulation in shaping temperature responses of the circadian clock.

Positive skill transfer in balance and speed control from balance bike to pedal bike in adults: A multiphase intervention study.

BACKGROUND: Learning to cycle can be challenging for adults who did not acquire the necessary skills during childhood. Balance bikes have been used to teach children how to cycle, but it was unclear whether this approach could also be effective for adults. PURPOSE: To address this, a multi-phase intervention study was conducted to investigate whether healthy adults could be taught to cycle independently through the use of a balance bike. METHODS: In Phase 1, a case-control observational study was conducted in which 13 cyclists and 8 non-cyclists completed balance bike tests. Based on the findings, an 8-session intervention pre- and post-test study was conducted in Phase 2, using an 8 × 20-minute balance bike training programme to improve cycling postural stability and control. Another 11 non-cyclists completed the novel programme. The time taken to complete the balance bike tests was compared before and after the program, while their cycling confidence was recorded in each session. To assess the effectiveness of the programme, participants were invited to cycle on a pedal bike to evaluate their ability to cycle independently. RESULTS: The results in Phase 1 showed that cyclists performed better on the balance bike than non-cyclists, with Bayes factor analyses providing evidence of this difference, BF01 = 0.228 in the 15 m sprint test and BF01 = 0.138 in the two-turn curved sprint test. The novel training programme in Phase 2 demonstrated remarkable effectiveness in improving their balance bike riding performance, as evidenced by the Bayes factor for completion times in the repeated measures being BF01 < 0.001. All participants were able to cycle independently with confidence after the programme. CONCLUSIONS: This study sheds light on the idea that it's never too late for adults to learn how to ride a bike. It provides evidence that healthy adults can learn to ride independently with the help of a balance bike, a tool that's commonly used for teaching children. The study identifies five key principles for effective balance bike training in adults, including focusing on riding speed, gliding to turn, building cycling confidence, engaging high motor skills, and using a dual-task approach. Our evidence-based training programme offers a safe, enjoyable, and effective way for adults to develop the skills and confidence they need to ride, even if they've never ridden before.

Associations of Device-Measured Physical Activity, Sedentary Behavior, and Executive Function in Preadolescents: A Latent Profile Approach.

PURPOSE: This study investigated the associations between physical activity (PA), sedentary behavior, and executive function in preadolescents. METHODS: One hundred and twenty preadolescents were recruited from 2 Hong Kong primary schools. PA and sedentary behavior were recorded for 7 consecutive days by accelerometer. Executive function performance, including inhibition (Stroop task and Flanker task) and working memory (Sternberg paradigm task), were measured. Body mass index and cardiorespiratory fitness (multistage fitness test) were tested. Latent profile analysis explored the profiles of PA and sedentary behavior in preadolescents. RESULTS: Three distinct profiles were identified: low activity, average activity, and high activity. Participants in low activity performed worse in the accuracy of Stroop task (vs average activity, P = .03; vs high activity, P < .01), Flanker task (vs average activity, P = .02; vs high activity, P < .001), and Sternberg paradigm task (vs average activity, P < .01; vs high activity, P < .01). No significant difference was observed between participants with average and high activities. No significant association was observed for profiles on body mass index and cardiorespiratory fitness. CONCLUSION: Supplementing the consensus of the literature that moderate to vigorous PA benefits cognition, the authors conclude that light PA may also enhance preadolescents' executive functioning.

Effects of Caffeine on Performances of Simulated Match, Wingate Anaerobic Test, and Cognitive Function Test of Elite Taekwondo Athletes in Hong Kong.

This study aimed to investigate the effects of caffeine on performances of simulated match, Wingate Anaerobic Test (WAnT), and cognitive function test of elite taekwondo athletes. Ten elite taekwondo athletes in Hong Kong volunteered to participate in two main trials in a randomized double-blinded crossover design. In each main trial, 1 h after consuming a drink with caffeine (CAF) or a placebo drink without caffeine (PLA), the participants completed two simulated taekwondo match sessions followed by the WAnT. The participants were instructed to complete three cognitive function tests, namely the Eriksen Flanker Test (EFT), Stroop Test, and Rapid Visual Information Processing Test, at baseline, before exercise, and immediately after the simulated matches. They were also required to wear functional near-infrared spectroscopy equipment during these tests. Before exercise, the reaction time in the EFT was shorter in the CAF trial than in the PLA trial (PLA: 494.9 ± 49.2 ms vs. CAF: 467.9 ± 38.0 ms, p = 0.035). In the WAnT, caffeine intake increased the peak power and mean power per unit of body weight (by approximately 13% and 6%, respectively, p = 0.018 & 0.042). The performance in the simulated matches was not affected by caffeine intake (p = 0.168). In conclusion, caffeine intake enhances anaerobic power and may improve certain cognitive functions of elite taekwondo athletes in Hong Kong. However, this may not be enough to improve the simulated match performance.

Effect of game-based high-intensity interval training program on the executive function of children with ADHD: Protocol of a randomized controlled trial.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a common developmental disorder in childhood, with a 5%-6% worldwide prevalence. Children with ADHD often demonstrate impaired executive function, which is closely related to the development of the commonly observed behavioral problems such as inattention, impaired inhibition, and hyperactivity. The purpose of this study is to examine whether a game-based high-intensity interval training (HIIT) program can improve the executive function of children with ADHD, compared with a traditional structured aerobic exercise program and a non-treatment control group. METHODS/DESIGN: A total of 42 children with ADHD will be recruited to participate in this three-arm school-based randomized controlled trial. An 8-week specially designed game-based HIIT (GameHIIT) program and a traditional game-based structured aerobic exercise (GameSAE) program will be delivered to those children randomly assigned to these two intervention groups, while the children in the control group will maintain their regular physical activity over the same period. A number of outcome measures including executive function, cerebral hemodynamic response, physical activity, physical fitness, and enjoyment and adherence to the intervention will be assessed for both groups at baseline (T0), immediately after the intervention period (T1), and after the follow-up period (T2). DISCUSSION: HIIT has recently emerged as a feasible and efficacious strategy for increasing physical health outcomes and cognitive function, including executive function, in healthy young people. However, research has yet to investigate whether the executive function of children with ADHD can be effectively enhanced through HIIT. If, as hypothesized, GameHIIT program improves outcomes for children with ADHD, the present research will inform the development of targeted exercise programs that can be more broadly used with this particular population.

Site directed spin labeling to elucidating the mechanism of the cyanobacterial circadian clock.

Electron Paramagnetic Resonance (EPR) is a spectroscopic technique that provides structural and dynamic information on unpaired spins and their surrounding environments. Introduction of exogenous spin labels via site directed spin labeling (SDSL) enables characterization of systems of interests lacking intrinsic unpaired spins. This chapter describes the use of SDSL in quantifying KaiB-KaiC binding in the cyanobacterial circadian clock (Kai Clock), exploiting the changes in mobility of the local environment around the spin label on KaiB-KaiC interactions. While the Kai system serves as our model system to demonstrate SDSL-EPR utility in quantifying protein-protein interactions, this technique is readily amenable to other systems of interest whenever specific protein-protein interactions need to be isolated. We first present a protocol for spin labeling KaiB. Then, we detail the sample preparation and acquisition processes to maximize signal-to-noise for downstream analysis. We close this chapter by highlighting recent advances in SDSL technology to incorporate spin labels into proteins of interest and in EPR technology to improve detection sensitivity that may allow greater flexibilities to the types of experiments possible.

A Night-Time Edge Site Intermediate in the Cyanobacterial Circadian Clock Identified by EPR Spectroscopy.

As the only circadian oscillator that can be reconstituted in vitro with its constituent proteins KaiA, KaiB, and KaiC using ATP as an energy source, the cyanobacterial circadian oscillator serves as a model system for detailed mechanistic studies of day-night transitions of circadian clocks in general. The day-to-night transition occurs when KaiB forms a night-time complex with KaiC to sequester KaiA, the latter of which interacts with KaiC during the day to promote KaiC autophosphorylation. However, how KaiB forms the complex with KaiC remains poorly understood, despite the available structures of KaiB bound to hexameric KaiC. It has been postulated that KaiB-KaiC binding is regulated by inter-KaiB cooperativity. Here, using spin labeling continuous-wave electron paramagnetic resonance spectroscopy, we identified and quantified two subpopulations of KaiC-bound KaiB, corresponding to the "bulk" and "edge" KaiBC sites in stoichiometric and substoichiometric KaiBiC6 complexes (i = 1-5). We provide kinetic evidence to support the intermediacy of the "edge" KaiBC sites as bridges and nucleation sites between free KaiB and the "bulk" KaiBC sites. Furthermore, we show that the relative abundance of "edge" and "bulk" sites is dependent on both KaiC phosphostate and KaiA, supporting the notion of phosphorylation-state controlled inter-KaiB cooperativity. Finally, we demonstrate that the interconversion between the two subpopulations of KaiC-bound KaiB is intimately linked to the KaiC phosphorylation cycle. These findings enrich our mechanistic understanding of the cyanobacterial clock and demonstrate the utility of EPR in elucidating circadian clock mechanisms.

Monitoring Protein-Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy.

The cyanobacterial circadian clock in Synechococcus elongatus consists of three proteins, KaiA, KaiB, and KaiC. KaiA and KaiB rhythmically interact with KaiC to generate stable oscillations of KaiC phosphorylation with a period of 24 h. The observation of stable circadian oscillations when the three clock proteins are reconstituted and combined in vitro makes it an ideal system for understanding its underlying molecular mechanisms and circadian clocks in general. These oscillations were historically monitored in vitro by gel electrophoresis of reaction mixtures based on the differing electrophoretic mobilities between various phosphostates of KaiC. As the KaiC phospho-distribution represents only one facet of the oscillations, orthogonal tools are necessary to explore other interactions to generate a full description of the system. However, previous biochemical assays are discontinuous or qualitative. To circumvent these limitations, we developed a spin-labeled KaiB mutant that can differentiate KaiC-bound KaiB from free KaiB using continuous-wave electron paramagnetic resonance spectroscopy that is minimally sensitive to KaiA. Similar to wild-type (WT-KaiB), this labeled mutant, in combination with KaiA, sustains robust circadian rhythms of KaiC phosphorylation. This labeled mutant is hence a functional surrogate of WT-KaiB and thus participates in and reports on autonomous macroscopic circadian rhythms generated by mixtures that include KaiA, KaiC, and ATP. Quantitative kinetics could be extracted with improved precision and time resolution. We describe design principles, data analysis, and limitations of this quantitative binding assay and discuss future research necessary to overcome these challenges.

On the incompatibility of lithium-O2 battery technology with CO2.

When solubilized in a hexacarboxamide cryptand anion receptor, the peroxide dianion reacts rapidly with CO2 in polar aprotic organic media to produce hydroperoxycarbonate (HOOCO2-) and peroxydicarbonate (-O2COOCO2-). Peroxydicarbonate is subject to thermal fragmentation into two equivalents of the highly reactive carbonate radical anion, which promotes hydrogen atom abstraction reactions responsible for the oxidative degradation of organic solvents. The activation and conversion of the peroxide dianion by CO2 is general. Exposure of solid lithium peroxide (Li2O2) to CO2 in polar aprotic organic media results in aggressive oxidation. These findings indicate that CO2 must not be introduced in conditions relevant to typical lithium-O2 cell configurations, as production of HOOCO2- and -O2COOCO2- during lithium-O2 cell cycling will lead to cell degradation via oxidation of organic electrolytes and other vulnerable cell components.

Effects of postexercise ice-water and room-temperature water immersion on the sensory organization of balance control and lower limb proprioception in amateur rugby players: A randomized controlled trial.

BACKGROUND: This single-blinded, three-armed randomized controlled trial aimed to compare the effects of postexercise ice-water immersion (IWI), room-temperature water immersion (RWI), and no water immersion on the balance performance and knee joint proprioception of amateur rugby players. METHODS: Fifty-three eligible amateur rugby players (mean age ±â€Šstandard deviation: 21.6 ±â€Š2.9 years) were randomly assigned to the IWI group (5.3 °C), RWI group (25.0 °C), or the no immersion control group. The participants in each group underwent the same fatigue protocol followed by their allocated recovery intervention, which lasted for 1 minute. Measurements were taken before and after the fatigue-recovery intervention. The primary outcomes were the sensory organization test (SOT) composite equilibrium score (ES) and the condition-specific ES, which were measured using a computerized dynamic posturography machine. The secondary outcome was the knee joint repositioning error. Two-way repeated measures analysis of variance was used to test the effect of water immersion on each outcome variable. RESULTS: There were no significant within- and between-group differences in the SOT composite ESs or the condition-specific ESs. However, there was a group-by-time interaction effect on the knee joint repositioning error. It seems that participants in the RWI group had lower errors over time, but those in the IWI and control groups had increased errors over time. The RWI group had significantly lower error score than the IWI group at postintervention. CONCLUSION: One minute of postexercise IWI or RWI did not impair rugby players' sensory organization of balance control. RWI had a less detrimental effect on knee joint proprioception to IWI at postintervention.

Sensory organisation and reactive balance control of amateur rugby players: A cross-sectional study.

PURPOSE: This study compared the sensory organisation and reactive balance control of amateur rugby players and a control group. METHODS: Forty-one amateur rugby players (22 males: 19 females; mean height ± SD = 168.8 ± 8.8 cm; mean weight ± SD = 63.9 ± 12.5 kg) and 31 control participants (22 males: 9 females; mean height ± SD = 171.5 ± 10.3 cm; mean weight ± SD = 63.8 ± 10.3 kg) completed the study. Their sensory organisation and standing balance performance were evaluated using a sensory organisation test (SOT), and their reactive balance performance was quantified using a motor control test (MCT). The SOT equilibrium scores (ES) and sensory ratios and the MCT motor response latencies were the major outcome measures. RESULTS: The results revealed that compared to the controls, amateur rugby players had lower SOT ESs under different sensory environments (P < .001, [Formula: see text] = 0.142-0.254) and prolonged reactive motor response times in the MCT (P < .001, d = 0.890). The vestibular and visual ratios were also lower in the rugby group (P = .005, [Formula: see text] = 0.107 and 0.108, respectively). No significant difference was found in the somatosensory ratio (P = .853, [Formula: see text] < 0.001) between the two groups. CONCLUSIONS: Amateur rugby players demonstrated inferior standing balance performance compared to their non-trained counterparts. They relied less heavily on vestibular and visual inputs to maintain standing balance under different sensory environments. In addition, they reacted more slowly to postural disturbance, reflecting their suboptimal reactive balance ability in standing.

Effects of Ving Tsun Chinese Martial Art Training on Upper Extremity Muscle Strength and Eye-Hand Coordination in Community-Dwelling Middle-Aged and Older Adults: A Pilot Study.

Objectives. To evaluate the effects of Ving Tsun (VT) martial art training on the upper extremity muscle strength and eye-hand coordination of middle-aged and older adults. Methods. This study used a nonequivalent pretest-posttest control group design. Forty-two community-dwelling healthy adults participated in the study; 24 (mean age ± SD = 68.5 ± 6.7 years) underwent VT training for 4 weeks (a supervised VT session twice a week, plus daily home practice), and 18 (mean age ± SD = 72.0 ± 6.7 years) received no VT training and acted as controls. Shoulder and elbow isometric muscle strength and eye-hand coordination were evaluated using the Lafayette Manual Muscle Test System and a computerized finger-pointing test, respectively. Results. Elbow extensor peak force increased by 13.9% (P = 0.007) in the VT group and the time to reach peak force decreased (9.9%) differentially in the VT group compared to the control group (P = 0.033). For the eye-hand coordination assessment outcomes, reaction time increased by 2.9% in the VT group and decreased by 5.3% in the control group (P = 0.002). Conclusions. Four weeks of VT training could improve elbow extensor isometric peak force and the time to reach peak force but not eye-hand coordination in community-dwelling middle-aged and older adults.

Comparison between smartphone pedometer applications and traditional pedometers for improving physical activity and body mass index in community-dwelling older adults.

[Purpose] The effectiveness of a smartphone pedometer application was compared with that of a traditional pedometer for improving the physical activity and weight status of community-dwelling older adults. [Subjects and Methods] This study had a nonequivalent pretest-posttest control group design. Ninety-seven older adults (mean age ± SD, 60.1 ± 5.5 years) joined the smartphone pedometer group and underwent a 2-week walking intervention based on a smartphone pedometer application. Fifty-four older adults (mean age ± SD, 65.3 ± 8.7 years) joined the traditional pedometer group and underwent a 2-week walking intervention based on a traditional pedometer. The participants' physical activity was evaluated using the International Physical Activity Questionnaire-Short Form, and their weight status was quantified by calculating the body mass index. The daily pedometer count was also documented. [Results] No significant time, group, or time-by-group interaction effects were found for any of the outcome variables. However, trends of improvement in physical activity and body mass index were seen only in the smartphone pedometer group. [Conclusion] A smartphone pedometer application might be more favorable than a traditional pedometer in improving physical activity and body mass index in community-dwelling older adults. However, further experimental studies are necessary to confirm the results.

Determinants of sport-specific postural control strategy and balance performance of amateur rugby players.

OBJECTIVES: Postural control strategy and balance performance of rugby players are important yet under-examined issues. This study aimed to examine the differences in balance strategy and balance performance between amateur rugby players and non-players, and to explore training- and injury-related factors that may affect rugby players' balance outcomes. DESIGN: Cross-sectional and exploratory study. METHODS: Forty-five amateur rugby players and 41 healthy active individuals participated in the study. Balance performance and balance strategies were assessed using the sensory organization test (SOT) of the Smart Equitest computerized dynamic posturography machine. Rugby training history and injury history were solicited from the participants. RESULTS: The SOT strategy scores were 1.99-54.90% lower in the rugby group than in the control group (p<0.05), and the equilibrium scores were 1.06-14.29% lower in the rugby group than in the control group (p<0.05). After accounting for age, sex and body mass index, only length of rugby training (in years) was independently associated with the SOT condition 6 strategy score, explaining 15.7% of its variance (p=0.006). There was no association between SOT condition 6 strategy/equilibrium scores and injury history among the rugby players (p>0.05). CONCLUSIONS: Amateur rugby players demonstrated inferior balance strategy and balance performance compared to their non-training counterparts. Their suboptimal balance strategy was associated with insufficient training experience but not with history of injury.

The Diurnal Variation on Cardiovascular Endurance Performance of Secondary School Athlete Student.

BACKGROUND: The previous investigations in diurnal variation of endurance sports performance did not reach a consensus and have been limited. This study would be a valuable resource for endurance sports trainers and event managers to plan their training and competition in a specific time of day. OBJECTIVES: The aim of this study is to find out the diurnal variation in cardiovascular endurance performance in the young athletes. MATERIALS AND METHODS: Thirty five athlete students (15.17 ± 1.62 years) participated in this study. Maximal oxygen uptake (VO2max), post-exercise percentage of maximal heart rate (MHR% post-ex), post-exercise body temperature (BTemppost-ex), and post exercise blood lactic acid level (LApost-ex) were measured in this study. Three non-consecutive testings: A) Morning (09:00-10:00; AM), B) Noon (12:00-13:00; NN) and C) Afternoon (16:00-17:00; PM) were conducted. Participants were required to follow the meal plan and resting schedule for all testing days. RESULTS: VO2max was significantly higher at NN (F2. 68 = 3.29, P < 0.05, η2 = 0.088) in comparison with PM. The MHR%post-ex, BTemppost-ex, LApost-ex was not significantly different among three times of day. CONCLUSIONS: Diurnal effect on endurance performance was found and the highest exercise VO2max was identified at noon. Secondary school students or young athletes are recommended to have sports training related to VO2max at noon for the purpose of maximizing training effectiveness.

From nongelating to gelating: synthesis and structural self-assembling property relationships of a homologous series of oligo(amide-triazole)s.

A homologous series of oligo(amide-triazole)s (OAT) [OAT-CO2H-2 n and OAT-COPrg-(2 n+1)] with an increasing number of primary amide (CONH) and triazole hydrogen-bonding functionalities was prepared by an iterative synthetic procedure. It was found that their self-assembly and thermoreversible gelation strength had a strong correlation to the number of hydrogen-bonding moieties in the oligomers. There also existed a threshold value of the number of CONH units, above which all the oligomers became organogelators. Hence, oligomers with ≤4 CONH units are devoid of intermolecular hydrogen bonding and also non-organogelating, whereas those that contain >4 CONH units show intermolecular association and organogelating properties. For the organogelators, the T(gel) value increases monotonically with increasing number of CONH units. On the basis of FTIR measurements, both the CONH and triazole C-H groups were involved in the hydrogen-bonding process. A mixed xerogel that consisted of a 1:1 weight ratio of two oligomers of different lengths (OAT-CO2H-6 and OAT-CO2H-12) was found to show microphase segregation according to differential scanning calorimetry, thus indicating that oligomers that bear a different number of hydrogen-bonding units exhibited self-sorting to maximize the extent of intermolecular hydrogen bonding in the xerogel state.

Dimensionality Reduction in Controlling Articulated Snake Robot for Endoscopy Under Dynamic Active Constraints.

This paper presents a real-time control framework for a snake robot with hyper-kinematic redundancy under dynamic active constraints for minimally invasive surgery. A proximity query (PQ) formulation is proposed to compute the deviation of the robot motion from predefined anatomical constraints. The proposed method is generic and can be applied to any snake robot represented as a set of control vertices. The proposed PQ formulation is implemented on a graphic processing unit, allowing for fast updates over 1 kHz. We also demonstrate that the robot joint space can be characterized into lower dimensional space for smooth articulation. A novel motion parameterization scheme in polar coordinates is proposed to describe the transition of motion, thus allowing for direct manual control of the robot using standard interface devices with limited degrees of freedom. Under the proposed framework, the correct alignment between the visual and motor axes is ensured, and haptic guidance is provided to prevent excessive force applied to the tissue by the robot body. A resistance force is further incorporated to enhance smooth pursuit movement matched to the dynamic response and actuation limit of the robot. To demonstrate the practical value of the proposed platform with enhanced ergonomic control, detailed quantitative performance evaluation was conducted on a group of subjects performing simulated intraluminal and intracavity endoscopic tasks.

Identification of potentially damaging amino acid substitutions leading to human male infertility.

There are a number of known genetic alterations found in men with nonobstructive azoospermia, or testicular failure, such as Y microdeletions and cytogenetic abnormalities. However, the etiology of nonobstructive azoospermia is unknown in the majority of men. The aim of this study was to investigate the possibility that unexplained cases of nonobstructive azoospermia are caused by nonsynonymous single-nucleotide polymorphisms (SNPs) in the coding regions of autosomal genes associated with sperm production and fertility. Using a candidate gene approach based on genetics of male infertility in mice, we resequenced nine autosomal genes from 78 infertile men displaying testicular failure using custom-made next-generation resequencing chips. Analysis of the data revealed several novel heterozygous nonsynonymous SNPs in four of nine sequenced genes in 14 of 78 infertile men. Eight SNPs in SBF1, three SNPs in LIMK2, two SNPs in LIPE, and one SNP in TBPL1 were identified. All of the novel mutations were in a heterozygous configuration, suggesting that they may be de novo mutations with dominant negative properties.

Moving live dissociated neurons with an optical tweezer.

The use of an optical tweezer for moving dissociated neurons was studied. The main features of the tweezers are outlined as well as the general principles of its operation. Infrared beams at 980 and 1064 nm were used, focused so as to make a trap for holding neurons and moving them. Absorption by cells at those wavelengths is very small. Experiments were done to evaluate nonsticky substrate coatings, from which neurons could be easily lifted with the tweezers. The maximum speed of cell movement as a function of laser power was determined. Detailed studies of the damage to cells as a function of beam intensity and time of exposure were made. The 980 nm beam was much less destructive, for reasons that are not understood, and could be used to safely move cells through distances of millimeters in times of seconds. An illustrative application of the use of the tweezers to load neurons without damage into plastic cages on a glass substrate was presented. The conclusion is that optical tweezers are an accessible and practical tool for helping to establish neuron cultures of cells placed in specific locations.

Parylene neurocages for electrical stimulation on silicon and glass substrates.

We present a refined method and design for building parylene neurocages for in vitro studies of live neural networks. Parylene neurocages are biocompatible and very robust, making them ideally suited for studying the synaptic connections between individual neurons to gain insight into learning and memory. The neurocage fabrication process incorporates electrodes into the neurocages to allow for stimulation and recording of action potentials. These neurocages can be fabricated on either silicon or glass substrates. The resulting neurocages have a long term cell survival rate of approximately 50%, and have proven to be 99% effective in trapping neurons. Preliminary results demonstrate that current pulses passing through the electrode can stimulate action potentials in the neurons trapped in neurocages.