Unique applications of cultured neuronal networks in pharmacology, toxicology, and basic neuroscience

This dissertation research explored the capabilities of neuronal networks grown on substrate integrated microelectrode arrays in vitro with emphasis on utilizing such preparations in three specific application domains: pharmacology and drug development, biosensors and neurotoxicology, and the study of burst and synaptic mechanisms. Chapter 1 details the testing of seven novel AChE inhibitors, demonstrating that neuronal networks rapidly detect small molecular differences in closely related compounds, and reveal information about their probable physiological effects that are not attainable through biochemical characterization alone. Chapter 2 shows how neuronal networks may be used to classify and characterize an unknown compound. The compound, trimethylol propane phosphate (TMPP) elicited changes in network activity that resembled those induced by bicuculline, a known epileptogenic. Further work determined that TMPP produces its effects on network activity through a competitive inhibition of the GABAA receptor. This demonstrates that neuronal networks can provide rapid, reliable warning of the presence of toxic substances, and from the manner in which the spontaneous activity changes provide information on the class of compound present and its potential physiological effects. Additional simple pharmacological tests can provide valuable information on primary mechanisms involved in the altered neuronal network responses. Chapter 3 explores the effects produced by a radical simplification of synaptic driving forces. With all synaptic interactions pharmacologically limited to those mediated through the NMDA synapse, spinal cord networks exhibited an extremely regular burst oscillation characterized by a period of 2.9 ± 0.3 s, with mean coefficients of variation of 3.7, 4.7, and 4.9 % for burst rate, burst duration, and inter-burst interval, respectively (16 separate cultures). The reliability of expression of this oscillation suggests that it may represent a fundamental mechanism of importance during periods of NMDA receptor dominated activity, such as embryonic and early postnatal development. NMDA synapse mediated activity produces a precise oscillatory state that allows the study of excitatory-coupled network dynamics, burst mechanisms, emergent network properties, and structure-function relationships

Botulinum Toxin Suppression of CNS Network Activity In Vitro

The botulinum toxins are potent agents which disrupt synaptic transmission. While the standard method for BoNT detection and quantification is based on the mouse lethality assay, we have examined whether alterations in cultured neuronal network activity can be used to detect the functional effects of BoNT. Murine spinal cord and frontal cortex networks cultured on substrate integrated microelectrode arrays allowed monitoring of spontaneous spike and burst activity with exposure to BoNT serotype A (BoNT-A). Exposure to BoNT-A inhibited spike activity in cultured neuronal networks where, after a delay due to toxin internalization, the rate of activity loss depended on toxin concentration. Over a 30 hr exposure to BoNT-A, the minimum concentration detected was 2 ng/mL, a level consistent with mouse lethality studies. A small proportion of spinal cord networks, but not frontal cortex networks, showed a transient increase in spike and burst activity with exposure to BoNT-A, an effect likely due to preferential inhibition of inhibitory synapses expressed in this tissue. Lastly, prior exposure to human-derived antisera containing neutralizing antibodies prevented BoNT-A induced inhibition of network spike activity. These observations suggest that the extracellular recording from cultured neuronal networks can be used to detect and quantify functional BoNT effects

Early Interfaced Neural Activity from Chronic Amputated Nerves

Direct interfacing of transected peripheral nerves with advanced robotic prosthetic devices has been proposed as a strategy for achieving natural motor control and sensory perception of such bionic substitutes, thus fully functionally replacing missing limbs in amputees. Multi-electrode arrays placed in the brain and peripheral nerves have been used successfully to convey neural control of prosthetic devices to the user. However, reactive gliosis, micro hemorrhages, axonopathy and excessive inflammation currently limit their long-term use. Here we demonstrate that enticement of peripheral nerve regeneration through a non-obstructive multi-electrode array, after either acute or chronic nerve amputation, offers a viable alternative to obtain early neural recordings and to enhance long-term interfacing of nerve activity. Non-restrictive electrode arrays placed in the path of regenerating nerve fibers allowed the recording of action potentials as early as 8 days post-implantation with high signal-to-noise ratio, as long as 3 months in some animals, and with minimal inflammation at the nerve tissue-metal electrode interface. Our findings suggest that regenerative multi-electrode arrays of open design allow early and stable interfacing of neural activity from amputated peripheral nerves and might contribute towards conveying full neural control and sensory feedback to users of robotic prosthetic devices

Noise Characterization, Modeling, and Reduction for In Vivo Neural Recording

Studying signal and noise properties of recorded neural data is critical in developing more efficient algorithms to recover the encoded information. Important issues exist in this research including the variant spectrum spans of neural spikes that make it difficult to choose a globally optimal bandpass filter. Also, multiple sources produce aggregated noise that deviates from the conventional white Gaussian noise. In this work, the spectrum variability of spikes is addressed, based on which the concept of adaptive bandpass filter that fits the spectrum of individual spikes is proposed. Multiple noise sources have been studied through analytical models as well as empirical measurements. The dominant noise source is identified as neuron noise followed by interface noise of the electrode. This suggests that major efforts to reduce noise from electronics are not well spent. The measured noise from in vivo experiments shows a family of 1/f^x spectrum that can be reduced using noise shaping techniques. In summary, the methods of adaptive bandpass filtering and noise shaping together result in several dB signal-to-noise ratio (SNR) enhancement

Blood Flow Restriction Training for the Rotator Cuff: A Randomized Controlled Trial

Context: Blood flow restriction (BFR) training utilizes a tourniquet, applied to the proximal portion of one or more extremities, to occlude blood flow during exercise. Significant gains in strength and cross-sectional area can be achieved in muscles, both distal and proximal to BFR cuff application. Purpose: To compare strength gains of the rotator cuff and changes in tendon size in subjects who performed side-lying external-rotation exercise with or without BFR. Methods: Forty-six subjects (mean age 25.0 [2.2] y) were randomized to either a BFR + exercise group or to the exercise-only group. Subjects performed 4 sets of the exercise (30/15/15/15 repetitions) at 30% 1-repetition maximum 2 days per week for 8 weeks. Results: Subjects in both groups experienced strength gains in the supraspinatus and the external rotators (P = .000, P = .000). However, there was no difference in strength gains between groups for the supraspinatus (P = .750) or the external rotators (P = .708). Subjects in both groups experienced increases in supraspinatus tendon thickness (BFR P = .041, exercise only P = .011). However, there was no difference between groups (P = .610). Conclusions: Exercise with BFR applied to the proximal upper extremity did not augment rotator cuff strength gains or tendon thickness when compared with subjects who only exercised. This study did demonstrate that performing multiple sets of high repetitions at a low load led to significant increases in rotator cuff strength and tendon size in the dominant upper extremity

Mass balance of nitrogen and phosphorus in an agricultural watershed : the shallow groundwater component

Partially funded by the Water Quality Strategic Research Initiative, Council on Food and Agricultural ResearchOpe

The Golden Age of European Cabaret

Program for the second annual RISD Cabaret held in the Cellar in the Pit. Design and layout by Anne Johnson, Susan Sellers and Georgie Stout.https://digitalcommons.risd.edu/liberalarts_cabaret_programs/1001/thumbnail.jp