Methods for automated delineation and assessment of EMG responses evoked by peripheral nerve stimulation in diagnostic and closed-loop therapeutic applications.

Objective.Surface electromyography measurements of the Hoffmann (H-) reflex are essential in a wide range of neuroscientific and clinical applications. One promising emerging therapeutic application is H-reflex operant conditioning, whereby a person is trained to modulate the H-reflex, with generalized beneficial effects on sensorimotor function in chronic neuromuscular disorders. Both traditional diagnostic and novel realtime therapeutic applications rely on accurate definitions of the H-reflex and M-wave temporal bounds, which currently depend on expert case-by-case judgment. The current study automates such judgments.Approach.Our novel wavelet-based algorithm automatically determines temporal extent and amplitude of the human soleus H-reflex and M-wave. In each of 20 participants, the algorithm was trained on data from a preliminary 3 or 4 min recruitment-curve measurement. Output was evaluated on parametric fits to subsequent sessions' recruitment curves (92 curves across all participants) and on the conditioning protocol's subsequent baseline trials (∼1200 per participant) performed nearHmax. Results were compared against the original temporal bounds estimated at the time, and against retrospective estimates made by an expert 6 years later.Main results.Automatic bounds agreed well with manual estimates: 95% lay within ±2.5 ms. The resulting H-reflex magnitude estimates showed excellent agreement (97.5% average across participants) between automatic and retrospective bounds regarding which trials would be considered successful for operant conditioning. Recruitment-curve parameters also agreed well between automatic and manual methods: 95% of the automatic estimates of the current required to elicitHmaxfell within±1.4%of the retrospective estimate; for the 'threshold' current that produced an M-wave 10% of maximum, this value was±3.5%.Significance.Such dependable automation of M-wave and H-reflex definition should make both established and emerging H-reflex protocols considerably less vulnerable to inter-personnel variability and human error, increasing translational potential.

Isolation and Electrophysiology of Murine Sympathetic Postganglionic Neurons in the Thoracic Paravertebral Ganglia.

The thoracic paravertebral sympathetic chain postganglionic neurons (tSPNs) represent the predominant sympathetic control of vascular function in the trunk and upper extremities. tSPNs cluster to form ganglia linked by an interganglionic nerve and receive multisegmental convergent and divergent synaptic input from cholinergic sympathetic preganglionic neurons of the spinal cord (Blackman and Purves, 1969; Lichtman et al., 1980 ). Studies in the past have focused on cervical and lumbar chain ganglia in multiple species, but few have examined the thoracic chain ganglia, whose location and diminutive size make them less conducive to experimentation. Seminal studies on the integrative properties of preganglionic axonal projections onto tSPNs were performed in guinea pig (Blackman and Purves, 1969; Lichtman et al., 1980 ), but as mice have become the accepted mammalian genetic model organism, there is need to reproduce and expand on these studies in this smaller model. We describe an ex vivo approach that enables electrophysiological, calcium imaging, and optogenetic assessment of convergence, divergence, and studies on pre- to postganglionic synaptic transmission, as well as whole-cell recordings from individual tSPNs. Preganglionic axonal connections from intact ventral roots and interganglionic nerves across multiple segments can be stimulated to evoke compound action potential responses in individual thoracic ganglia as recorded with suction electrodes. Chemical block of synaptic transmission differentiates spiking of preganglionic axons from synaptically-recruited tSPNs. Further dissection, including removal of the sympathetic chain, enables whole-cell patch clamp recordings from individual tSPNs for characterization of cellular and synaptic properties.

Dramatically Amplified Thoracic Sympathetic Postganglionic Excitability and Integrative Capacity Revealed with Whole-Cell Patch-Clamp Recordings.

Thoracic paravertebral sympathetic postganglionic neurons (tSPNs) comprise the final integrative output of the distributed sympathetic nervous system controlling vascular and thermoregulatory systems. Considered a non-integrating relay, what little is known of tSPN intrinsic excitability has been determined by sharp microelectrodes with presumed impalement injury. We thus undertook the first electrophysiological characterization of tSPN cellular properties using whole-cell recordings and coupled results with a conductance-based model to explore the principles governing their excitability in adult mice of both sexes. Recorded membrane resistance and time constant values were an order of magnitude greater than values previously obtained, leading to a demonstrable capacity for synaptic integration in driving recruitment. Variation in membrane resistivity was the primary determinant controlling cell excitability with vastly lower currents required for tSPN recruitment. Unlike previous microelectrode recordings in mouse which observed inability to sustain firing, all tSPNs were capable of repetitive firing. Computational modeling demonstrated that observed differences are explained by introduction of a microelectrode impalement injury conductance. Overall, tSPNs largely linearly encoded injected current magnitudes over a broad frequency range with distinct subpopulations differentiable based on repetitive firing signatures. Thus, whole-cell recordings reveal tSPNs have more dramatically amplified excitability than previously thought, with greater intrinsic capacity for synaptic integration and with the ability for maintained firing to support sustained actions on vasomotor tone and thermoregulatory function. Rather than acting as a relay, these studies support a more responsive role and possible intrinsic capacity for tSPNs to drive sympathetic autonomic function.

Use of electric field sensors for recording respiration, heart rate, and stereotyped motor behaviors in the rodent home cage.

BACKGROUND: Numerous environmental and genetic factors can contribute significantly to behavioral and cardiorespiratory variability observed experimentally. Affordable technologies that allow for noninvasive home cage capture of physio-behavioral variables should enhance understanding of inter-animal variability including after experimental interventions. NEW METHOD: We assessed whether EPIC electric field sensors (Plessey Semiconductors) embedded within or attached externally to a rodent's home cage could accurately record respiration, heart rate, and motor behaviors. COMPARISON WITH EXISTING METHODS: Current systems for quantification of behavioral variables require expensive specialty equipment, while measures of respiratory and heart rate are often provided by surgically implanted or chronically affixed devices. RESULTS: Sensors accurately encoded imposed sinusoidal changes in electric field tested at frequencies ranging from 0.5-100Hz. Mini-metronome arm movements were easily detected, but response magnitude was highly distance dependent. Sensors accurately reported respiration during whole-body plethysmography. In anesthetized rodents, PVC tube-embedded sensors provided accurate mechanical detection of both respiratory and heart rate. Comparable success was seen in naturally behaving animals at rest or sleeping when sensors were attached externally. Video-verified motor behaviors (sniffing, grooming, chewing, and rearing) were detectable and largely separable by their characteristic voltage fluctuations. Larger movement-related events had comparably larger voltage dynamics that easily allowed for a broad approximation of overall motor activity. Spectrograms were used to quickly depict characteristic frequencies in long-lasting recordings, while filtering and thresholding software allowed for detection and quantification of movement-related physio-behavioral events. CONCLUSIONS: EPIC electric field sensors provide a means for affordable non-contact home cage detection of physio-behavioral variables.

Assembly and operation of the autopatcher for automated intracellular neural recording in vivo.

Whole-cell patch clamping in vivo is an important neuroscience technique that uniquely provides access to both suprathreshold spiking and subthreshold synaptic events of single neurons in the brain. This article describes how to set up and use the autopatcher, which is a robot for automatically obtaining high-yield and high-quality whole-cell patch clamp recordings in vivo. By following this protocol, a functional experimental rig for automated whole-cell patch clamping can be set up in 1 week. High-quality surgical preparation of mice takes ∼1 h, and each autopatching experiment can be carried out over periods lasting several hours. Autopatching should enable in vivo intracellular investigations to be accessible by a substantial number of neuroscience laboratories, and it enables labs that are already doing in vivo patch clamping to scale up their efforts by reducing training time for new lab members and increasing experimental durations by handling mentally intensive tasks automatically.

A novel AX+/BX- paradigm to assess fear learning and safety-signal processing with repeated-measure designs.

One of the core symptoms of anxiety disorders, such as post-traumatic stress disorder, is the failure to overcome feelings of danger despite being in a safe environment. This deficit likely stems from an inability to fully process safety signals, which are cues in the environment that enable healthy individuals to over-ride fear in aversive situations. Studies examining safety signal learning in rodents, humans, and non-human primates currently rely on between-groups designs. Because repeated-measure designs reduce the number of subjects required, and facilitate a broader range of safety signal studies, the current project sought to develop a repeated-measures safety-signal learning paradigm in non-human primates. Twelve healthy rhesus macaques of both sexes received three rounds of auditory fear-potentiated startle training and testing using an AX+/BX- design with all visual cues. Cue AX was paired with an aversive blast of air, whereas the same X cue in compound with another B cue (BX) signaled the absence of an air blast. Hence, cue B served as a safety signal. Once animals consistently discriminated between the aversive (AX+) and safe (BX-) cues, measured by greater startle amplitude in the presence of AX vs. BX, they were tested for conditioned inhibition by eliciting startle in the presence of a novel ambiguous combined cue (AB). Similar to previous AX+/BX- studies, healthy animals rapidly learned to discriminate between the AX+ and BX- cues as well as demonstrate conditioned inhibition in the presence of the combined AB cue (i.e. lower startle amplitude in the presence of AB vs. AX). Additionally, animals performed consistently across three rounds of testing using three new cues each time. The results validate this novel method that will serve as a useful tool for better understanding the mechanisms for the regulation of fear and anxiety.

Rapid, quantitative, reverse transcription PCR in a polymer microfluidic chip.

Quantitative PCR (qPCR) techniques have become invaluable, high-throughput tools to study gene expression. However, the need to measure gene expression patterns quickly and affordably, useful for applications such as stem cell biomanufacturing requiring real-time observation and control, has not been adequately met by rapid qPCR instrumentation to date. We report a reverse transcription, microfluidic qPCR system and its application to DNA and RNA amplification measurement. In the system, an environmental control fixture provides mechanical and thermal repeatability for an infrared laser to achieve both accurate and precise open-loop temperature control of 1 µl reaction volumes in a low-cost polymer microfluidic chip with concurrent fluorescence imaging. We have used this system to amplify serial dilutions of λ-phage DNA (10(5)-10(7) starting copies) and RNA transcripts from the GAPDH housekeeping gene (5.45 ng total mouse embryonic stem cell RNA) and measured associated standard curves, efficiency (57%), repeatability (~1 cycle threshold), melting curves, and specificity. This microfluidic qRT-PCR system offers a practical approach to rapid analysis (~1 h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easy setup.