In vivo evidence for the cellular basis of central hypoventilation of Rett syndrome and pharmacological correction in the rat model.

Rett syndrome (RTT) is a neurodevelopmental disorder caused mostly by mutations in the MECP2 gene. RTT patients show periodical hypoventilation attacks. The breathing disorder contributing to the high incidence of sudden death is thought to be due to depressed central inspiratory (I) activity via unknown cellular processes. Demonstration of such processes may lead to targets for pharmacological control of the RTT-type hypoventilation. We performed in vivo recordings from medullary respiratory neurons on the RTT rat model. To our surprise, both I and expiratory (E) neurons in the ventral respiratory column (VRC) increased their firing activity in Mecp2-null rats with severe hypoventilation. These I neurons including E-I phase-spanning and other I neurons remained active during apneas. Consistent with enhanced central I drive, ectopic phrenic discharges during expiration as well as apnea were observed in the Mecp2-null rats. Considering the increased I neuronal firing and ectopic phrenic activity, the RTT-type hypoventilation does not seem to be caused by depression in central I activity, neither reduced medullary I premotor output. This as well as excessive E neuronal firing as shown in our previous studies suggests inadequate synaptic inhibition for phase transition. We found that the abnormal respiratory neuronal firing, ectopic phrenic discharge as well as RTT-type hypoventilation all can be corrected by enhancing GABAergic inhibition. More strikingly, Mecp2-null rats reaching humane endpoints with severe hypoventilation can be rescued by GABAergic augmentation. Thus, defective GABAergic inhibition among respiratory neurons is likely to play a role in the RTT-type hypoventilation, which can be effectively controlled with pharmacological agents.

Novel method for action potential measurements from intact cardiac monolayers with multiwell microelectrode array technology.

The cardiac action potential (AP) is vital for understanding healthy and diseased cardiac biology and drug safety testing. However, techniques for high throughput cardiac AP measurements have been limited. Here, we introduce a novel technique for reliably increasing the coupling of cardiomyocyte syncytium to planar multiwell microelectrode arrays, resulting in a stable, label-free local extracellular action potential (LEAP). We characterized the reliability and stability of LEAP, its relationship to the field potential, and its efficacy for quantifying AP morphology of human induced pluripotent stem cell derived and primary rodent cardiomyocytes. Rise time, action potential duration, beat period, and triangulation were used to quantify compound responses and AP morphology changes induced by genetic modification. LEAP is the first high throughput, non-invasive, label-free, stable method to capture AP morphology from an intact cardiomyocyte syncytium. LEAP can accelerate our understanding of stem cell models, while improving the automation and accuracy of drug testing.

Mecp2 Disruption in Rats Causes Reshaping in Firing Activity and Patterns of Brainstem Respiratory Neurons.

People with Rett Syndrome (RTT), a neurodevelopmental disorder caused by mutations in the MECP2 gene, have breathing abnormalities manifested as periodical hypoventilation with compensatory hyperventilation, which are attributable to a high incidence of sudden death. Similar breathing abnormalities have been found in animal models with Mecp2 disruptions. Although RTT-type hypoventilation is believed to be due to depressed central inspiratory activity, whether this is true remains unknown. Here we show evidence for reshaping in firing activity and patterns of medullary respiratory neurons in RTT-type hypoventilation without evident depression in inspiratory neuronal activity. Experiments were performed in decerebrate rats in vivo. In Mecp2-null rats, abnormalities in breathing patterns were apparent in both decerebrate rats and awake animals, suggesting that RTT-type breathing abnormalities take place in the brainstem without forebrain input. In comparison to their wild-type counterparts, both inspiratory and expiratory neurons in Mecp2-null rats extended their firing duration, and fired more action potentials during each burst. No changes in inspiratory or expiratory neuronal distributions were found. Most inspiratory neurons started firing in the middle of expiration and changed their firing pattern to a phase-spanning type. The proportion of post-inspiratory neurons was reduced in the Mecp2-null rats. With the increased firing activity of both inspiratory and expiratory neurons in null rats, phrenic discharges shifted to a slow and deep breathing pattern. Thus, the RTT-type hypoventilation appears to result from reshaping of firing activity of both inspiratory and expiratory neurons without evident depression in central inspiratory activity.

Micromachining on and of Transparent Polymers for Patterning Electrodes and Growing Electrically Active Cells for Biosensor Applications.

We report on microfabrication and assembly process development on transparent, biocompatible polymers for patterning electrodes and growing electrically active cells for in vitro cell-based biosensor applications. Such biosensors are typically fabricated on silicon or glass wafers with traditional microelectronic processes that can be cost-prohibitive without imparting necessary biological traits on the devices, such as transparency and compatibility for the measurement of electrical activity of electrogenic cells and other biological functions. We have developed and optimized several methods that utilize traditional micromachining and non-traditional approaches such as printed circuit board (PCB) processing for fabrication of electrodes and growing cells on the transparent polymers polyethylene naphthalate (PEN) and polyethylene terephthalate (PET). PEN-based biosensors are fabricated utilizing lithography, metal lift-off, electroplating, wire bonding, inkjet printing, conformal polymer deposition and laser micromachining, while PET-based biosensors are fabricated utilizing post-processing technologies on modified PCBs. The PEN-based biosensors demonstrate 85⁻100% yield of microelectrodes, and 1-kHz impedance of 59.6 kOhms in a manner comparable to other traditional approaches, with excellent biofunctionality established with an ATP assay. Additional process characterization of the microelectrodes depicts expected metal integrity and trace widths and thicknesses. PET-based biosensors are optimized for a membrane bow of 6.9 to 15.75 µm and 92% electrode yield on a large area. Additional qualitative optical assay for biomaterial recognition with transmitted light microscopy and growth of rat cortical cells for 7 days in vitro (DIV) targeted at biological functionalities such as electrophysiology measurements are demonstrated in this paper.