In vivo quantification of excitation and kilohertz frequency block of the rat vagus nerve.

OBJECTIVE: There is growing interest in treating diseases by electrical stimulation and block of peripheral autonomic nerves, but a paucity of studies on the excitation and block of small-diameter autonomic axons. We conducted in vivo quantification of the strength-duration properties, activity-dependent slowing (ADS), and responses to kilohertz frequency (KHF) signals for the rat vagus nerve (VN). APPROACH: We conducted acute in vivo experiments in urethane-anaesthetized rats. We placed two cuff electrodes on the left cervical VN and one cuff electrode on the anterior subdiaphragmatic VN. The rostral cervical cuff was used to deliver pulses to quantify recruitment and ADS. The caudal cervical cuff was used to deliver KHF signals. The subdiaphragmatic cuff was used to record compound action potentials (CAPs). MAIN RESULTS: We quantified the input-output recruitment and strength-duration curves. Fits to the data using standard strength-duration equations were qualitatively similar, but the resulting chronaxie and rheobase estimates varied substantially. We measured larger thresholds for the slowest fibres (0.5-1 m s-1), especially at shorter pulse widths. Using a novel cross-correlation CAP-based analysis, we measured ADS of ~2.3% after 3 min of 2 Hz stimulation, which is comparable to the ADS reported for sympathetic efferents in somatic nerves, but much smaller than the ADS in cutaneous nociceptors. We found greater ADS with higher stimulation frequency and non-monotonic changes in CV in select cases. We found monotonically increasing block thresholds across frequencies from 10 to 80 kHz for both fast and slow fibres. Further, following 25 s of KHF signal, neural conduction could require tens of seconds to recover. SIGNIFICANCE: The quantification of mammalian autonomic nerve responses to conventional and KHF signals provides essential information for the development of peripheral nerve stimulation therapies and for understanding their mechanisms of action.

Modeling the response of small myelinated axons in a compound nerve to kilohertz frequency signals.

OBJECTIVE: There is growing interest in electrical neuromodulation of peripheral nerves, particularly autonomic nerves, to treat various diseases. Electrical signals in the kilohertz frequency (KHF) range can produce different responses, including conduction block. For example, EnteroMedics' vBloc® therapy for obesity delivers 5 kHz stimulation to block the abdominal vagus nerves, but the mechanisms of action are unclear. APPROACH: We developed a two-part computational model, coupling a 3D finite element model of a cuff electrode around the human abdominal vagus nerve with biophysically-realistic electrical circuit equivalent (cable) model axons (1, 2, and 5.7 µm in diameter). We developed an automated algorithm to classify conduction responses as subthreshold (transmission), KHF-evoked activity (excitation), or block. We quantified neural responses across kilohertz frequencies (5-20 kHz), amplitudes (1-8 mA), and electrode designs. MAIN RESULTS: We found heterogeneous conduction responses across the modeled nerve trunk, both for a given parameter set and across parameter sets, although most suprathreshold responses were excitation, rather than block. The firing patterns were irregular near transmission and block boundaries, but otherwise regular, and mean firing rates varied with electrode-fibre distance. Further, we identified excitation responses at amplitudes above block threshold, termed 're-excitation', arising from action potentials initiated at virtual cathodes. Excitation and block thresholds decreased with smaller electrode-fibre distances, larger fibre diameters, and lower kilohertz frequencies. A point source model predicted a larger fraction of blocked fibres and greater change of threshold with distance as compared to the realistic cuff and nerve model. SIGNIFICANCE: Our findings of widespread asynchronous KHF-evoked activity suggest that conduction block in the abdominal vagus nerves is unlikely with current clinical parameters. Our results indicate that compound neural or downstream muscle force recordings may be unreliable as quantitative measures of neural activity for in vivo studies or as biomarkers in closed-loop clinical devices.

Topography of spinal neurons active during hindlimb withdrawal reflexes in the decerebrate cat.

There exists a spatial organization of receptive fields and a modular organization of the flexion withdrawal reflex system. However, the three dimensional location and organization of interneurons interposed in flexion reflex pathways has not been systematically examined. We determined the anatomical locations of spinal neurons involved in the hindlimb flexion withdrawal reflex using expression of the immediate early gene c-fos and the corresponding FOS protein. The flexion withdrawal reflex was evoked in decerebrate cats via stimulation of the tibial or superficial peroneal nerve. Animals that received stimulation had significantly larger numbers of cells expressing FOS-like immunoreactivity (42.7+/-2.3 cells/section, mean+/-standard error of the mean) than operated unstimulated controls (18.6+/-1.4 cells/section). Compared with controls, cells expressing FOS-like immunoreactivity were located predominantly on the ipsilateral side, in laminae IV-VI, at L6 and rostral L7 segments, and between 20% and 60% of the distance from the midline to the lateral border of the ventral gray matter. Labeled neurons resulting from tibial nerve stimulation were medial to neurons labeled following superficial peroneal nerve stimulation in laminae I-VI, but not VII. The mean mediolateral positions of labeled neurons from both nerves shifted medially as the transverse plane in which they were viewed was moved from rostral to caudal and as the coronal plane in which they were viewed was moved from dorsal to ventral. The mediolateral separation between populations of labeled cells was consistent with primary afferent projections and the location of reflex encoders. This topographical segregation corresponding to different afferent inputs is a possible anatomical substrate for a modular organization of the flexion withdrawal reflex system.

Non-invasive measurement of the input-output properties of peripheral nerve stimulating electrodes.

A non-invasive method was developed to determine the input-output (I/O) properties of peripheral nerve stimulating electrodes. An apparatus was fabricated to measure the 3-dimensional (3-D) isometric torque generated at the cat ankle joint by electrical activation of the sciatic nerve. The performance of the apparatus was quantified, and the utility of the method was demonstrated by measuring the recruitment properties of multiple contact nerve cuff electrodes. Torque-twitch waveforms, recruitment curves of peak torque as a function of stimulus current amplitude, and 2-D joint torque vectors were used to analyze the recruitment properties of the cuff. The peak of the twitch torque was an accurate measure of excitation even for muscles having fibers with varying speeds of contraction. The evoked twitch waveforms and torque vectors generated by selective stimulation of individual nerve branches with a hook electrode were compared to those produced by stimulation of the nerve trunk with the cuff electrode. These data allowed determination of the regions of the nerve trunk that were activated by different electrode geometries and stimulus parameters. The positional stability of electrode recruitment properties could be quantified by measuring I/O characteristics at different limb positions. The methods described are useful for characterization of neural stimulating electrodes and for studies of motor system physiology.

Optimal filtering of whole nerve signals.

Electroneurographic recordings suffer from low signal to noise (S/N) ratios. The S/N ratio can be improved by different signal processing methods including optimal filtering. A method to design two types of optimal filters (Wiener and Matched filters) was developed for use with neurographic signals, and the calculated filters were applied to nerve cuff recordings from the cat S1 spinal root that were recorded during the activation of cutaneous, bladder, and rectal mechanoreceptors. The S1 spinal root recordings were also filtered using various band-pass (BP) filters with different cut-off frequencies, since the frequency responses of the Wiener and Matched filters had a band-pass character. The mean increase in the S/N ratio across all recordings was 54, 89, and 85% for the selected best Wiener, Matched, and band-pass filters, respectively. There were no statistically significant differences between the performance of the selected filters when all three methods were compared. However, Matched filters yielded a greater increase in S/N ratio than Wiener filters when only two filtering techniques were compared. All three filtering methods have in most cases also improved the selectivity of the recordings for different sensory modalities. This might be important when recording nerve activity from a mixed nerve innervating multiple end-organs to increase the modality selectivity for the nerve fibers of interest. The mean Modality Selectivity Indices (MSI) over different receptor types and for the same selected filters as above were 1.12, 1.27, and 1.29, respectively, and indicate increases in modality selectivity (MSI>1). Improving the S/N ratio and modality selectivity of neurographic recordings is an important development to increase the utility of neural signals for understanding neural function and for use as feedback or control signals in neural prosthetic devices.

Bladder and urethral pressures evoked by microstimulation of the sacral spinal cord in cats.

Experiments were conducted to measure the bladder and urethral pressures evoked by intraspinal microstimulation of the sacral segments (S1-S2) in neurologically intact, chloralose anesthetized adult male cats. The bladder pressure was measured with a superpubic catheter and the urethral pressure was measured simultaneously at the level of the urethral sphincter and at the level of the penis using a two-element micromanometer. Intraspinal stimuli (typically 1 s, 20 Hz, 100 microA, 100 microseconds) were applied with activated iridium microwire electrodes in ipsilateral segments and intersegmental boundaries with a 250 micrometer mediolateral resolution and a 200 micrometer dorsoventral resolution. Increases in bladder pressures were generated by microstimulation in the intermediolateral region, in the lateral and ventrolateral ventral horn, and around the central canal. Simultaneous increases in urethral pressure were evoked by microstimulation in the ventrolateral ventral horn, but not at the other locations. Small reductions in urethral pressure (<10 cm H(2)O) were evoked at locations in the intermediate laminae and around the central canal. The magnitude of these pressure reductions was weakly dependent on the stimulus parameters. Stimulation around the central canal produced bladder contractions with either no change or a reduction in urethral pressure and voiding of small amounts of fluid. These results demonstrate that regions are present in the spinal intact anesthetized cat where microstimulation generates selective contraction of the bladder without increases in urethral pressure and that regions are present where microstimulation generates small reductions in urethral pressure.

Identification of the spinal neural network involved in coordination of micturition in the male cat.

In these studies, we used the expression of Fos protein to identify cells within the spinal cord that regulate micturition in male cats. The immediate early gene c-fos that encodes the Fos protein can be induced rapidly and transiently in neurons by increased electrical activity. Animals were anesthetized with alpha-chloralose, and received one of four stimulus protocols: electrical stimulation of the pelvic nerve, electrical stimulation of the pudendal nerve, a period of isometric micturition (induced by ligating the proximal urethra and infusing saline into the bladder), or electrical stimulation of Barrington's nucleus. After the period of stimulation, the animals were perfused and neurons expressing Fos-like immunoreactivity (FLI) were visualized with immunocytochemical methods. Stimulation with each protocol resulted in a substantially larger number of neurons expressing FLI than in operated but unstimulated controls, which exhibited few Fos-positive neurons localized to the superficial dorsal horn. In animals undergoing isometric micturition or stimulation of Barrington's nucleus, neurons exhibiting FLI were found bilaterally in the sacral (S1-S3) spinal cord and were localized to the lateral portion of the superficial dorsal horn (laminae I and II), in the intermediolateral region (lateral laminae V-VII), and around the central canal (lamina X and medial laminae V-VII). The intermediolateral region appeared to contain two populations of cells exhibiting FLI: a group of large multipolar cells and a group of small round cells. Few Fos-immunoreactive nuclei were observed in the medial portion of the superficial dorsal horn, and FLI was not observed in ventral horn neurons. Electrical stimulation of the pudendal or pelvic nerves resulted in fewer numbers of cells exhibiting FLI, with a less widespread spatial distribution. These results identify spinal neurons that are active during the micturition cycle, and demonstrate that a behaviorally relevant stimulus (isometric micturition) generated more widespread and greater intensity of Fos expression than repetitive electrical stimulation of the component peripheral nerves.

Extended survival time following pseudorabies virus injection labels the suprapontine neural network controlling the bladder and urethra in the rat.

The central neurons that are involved in control of the urinary bladder and proximal urethra in adult male rats were identified by retrograde transport of the viral transneural tracer pseudorabies virus (PRV, Bartha strain). At 5 days post-injection, PRV-infected neurons were found in suprapontine central nervous system nuclei including ventrolateral periaqueductal gray, magnocellular division of the red nucleus, lateral hypothalamus and paraventricular nucleus, retrochiasmic region and suprachiasmatic nucleus. At days 6 and 7 PRV-infected neurons were observed in the amygdala, lateral septal nucleus, hippocampus, and frontal motor, piriform, and perirhinal cortices. These results identify the supraspinal neural networks that are involved in control of the lower urinary tract, and demonstrate the utility of long survival times to label higher-order neurons with PRV.

Neural network classification of nerve activity recorded in a mixed nerve.

Whole-nerve cuff electrodes can be used to record electrical nerve activity in peripheral nerves and are suitable for chronic implantation in animals or humans. If the whole nerve innervates multiple target organs or muscles then the recorded activity will be the superposition of the activity of different nerve fibers innervating these organs. In certain cases it is desirable to monitor mixed nerve activity and to determine the origin (modality) of the recorded activity. A method using the autocorrelation function of recorded nerve activity and an artificial neural network was developed to classify the modality of nerve signals. The method works in cases where different end organs are innervated by nerve fibers having different diameter distributions. The electrical activity in the cat S1 sacral spinal root was recorded using a cuff electrode during the activation of cutaneous, bladder, and rectal mechanoreceptors. Using the classification method, 87.5% of nerve signals were correctly classified. This result demonstrates the effectiveness of the neural network classification method to determine the modality of the nerve activity arising from activation of different receptors.

Modeling the effects of electric fields on nerve fibers: influence of tissue electrical properties.

The effects of anisotropy and inhomogeneity of the electrical conductivity of extracellular tissue on excitation of nerve fibers by an extracellular point source electrode were determined by computer simulation. Analytical solutions to Poison's equation were used to calculate potentials in anisotropic infinite homogeneous media and isotropic semi-infinite inhomogeneous media, and the net driving function was used to calculate excitation thresholds for nerve fibers. The slope and intercept of the current-distance curve in anisotropic media were power functions of the ratio and product of the orthogonal conductivities, respectively. Excitation thresholds in anisotropic media were also dependent on the orientation of the fibers, and in strongly anisotropic media (sigma z/sigma xy > 4) there were reversals in the recruitment order between different diameter fibers and between fibers at different distances from the electrode. In source-free regions of inhomogeneous media (two regions of differing conductivity separated by a plane boundary), the current-distance relationship of fibers parallel to the interface was dependent only on the average conductivity, whereas in regions containing the source the current-distance relationship was dependent on the individual values of conductivity. Reversals in recruitment order between fibers at different distances from the electrode and between fibers of differing diameter were found in inhomogeneous media. The results of this simulation study demonstrate that the electrical properties of the extracellular medium can have a strong influence on the pattern of neuronal excitation generated by extracellular electric fields, and indicate the importance of tissue electrical properties in interpreting results of studies employing electrical stimulation applied in complex biological volume conductors.

The effect of stimulus pulse duration on selectivity of neural stimulation.

Choice of stimulus parameters is an important consideration in the design of neural prosthetic systems. The objective of this study was to determine the effect of rectangular stimulus pulsewidth (PW) on the selectivity of peripheral nerve stimulation. Computer simulations using a cable model of a mammalian myelinated nerve fiber indicated that shorter PW's increased the difference between the threshold currents of fibers lying at different distances from an electrode. Experimental measurements of joint torque generated by peripheral nerve stimulation demonstrated that shorter PW's generated larger torques before spillover and created a larger dynamic range of currents between threshold and spillover. Thus, shorter PW's allowed more spatially selective stimulation of nerve fibers. Analysis of the response of a passive cable model to different duration stimuli indicated that PW dependent contributions of distributed sources to membrane polarization accounted for the observed differences in selectivity.

Inversion of the current-distance relationship by transient depolarization.

The objective of this research was to develop a technique to excite selectively nerve fibers distant from an electrode without exciting nerve fibers close to the electrode. The shape of the stimulus current waveform was designed based on the nonlinear conductance properties of neuronal sodium channel. Models of mammalian peripheral myelinated axons and experimental measurements on cat sciatic nerve were used to determine the effects of subthreshold polarization on neural excitability and recruitment. Subthreshold membrane depolarization generated a transient decrease in neural excitability and thus an increase in the threshold for stimulation by a subsequent stimulus pulse. The decrease in excitability increased as the duration and amplitude of the subthreshold depolarization were increased, and the increase in threshold was greater for fibers close to the electrode. When a depolarizing stimulus pulse was applied immediately after the subthreshold depolarization, nerve fibers far from the electrode could be stimulated without stimulating fibers close to the electrode. Subthreshold depolarizing prepulses inverted the current-distance relationship and allowed selective stimulation of nerve fibers far from the electrode.

Selective control of muscle activation with a multipolar nerve cuff electrode.

Acute experiments were performed on adult cats to study selective activation of medial gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus with a cuff electrode. A spiral nerve cuff containing twelve "dot" electrodes was implanted around the sciatic nerve and evoked muscle twitch forces were recorded in six experiments. Spatially isolated "dot" electrodes in four geometries: monopolar, longitudinal tripolar, tripolar with four common anodes, and two parallel tripoles, were combined with transverse field steering current(s) from an anode(s) located 180 degrees around from the cathode(s) to activate different regions of the nerve trunk. To quantify the degree of selectivity, a selectivity index was defined as the ratio of the force in one muscle to the force in all four muscles in response to a particular stimulus. The selectivity index was used to construct recruitment curves for a muscle with the optimal degree of selectivity. Physiological responses were correlated with the anatomical structure of the sciatic nerve by identifying the nerve fascicles innervating the four muscles, and by determining the relative positions of the electrodes and the nerve fascicles. The results indicated that the use of transverse field steering current improved selectivity. We also found that tripoles with individual dot anodes were more selective than tripoles with four common dot anodes. Stimulation with two parallel tripoles was effective in activating selectively fascicles that could not be activated selectively with only a single tripole. The multipolar cuff proved an effective method to control selectively and progressively the force in muscles innervated by fascicles that were well defined at the level of the cuff.

Modeling the effects of electric fields on nerve fibers: determination of excitation thresholds.

We have developed a method to predict excitation of axons based on the response of passive models. An expression describing the transmembrane potential induced in passive models to an applied electric field is presented. Two terms were found to drive the polarization of each node. The first was a source term described by the activating function at the node, and the other was an ohmic term resulting from redistribution of current from sources at other nodes. A total equivalent driving function including both terms was then defined. We found that the total equivalent driving function can be used to provide accurate predictions of excitation thresholds for any applied field. The method requires only knowledge of the intracellular strength-duration relationship of the axon, the passive step response of the axon to an intracellular current, and the values of the extracellular potentials. Excitation thresholds for any given applied field can then be calculated using a simple algebraic expression. This method eliminates the errors associated with use of the activating function alone, and greatly reduces the computation required to determine fiber response to applied extracellular fields.

Modelling the effects of electric fields on nerve fibres: influence of the myelin sheath.

The excitation and conduction properties of computer-based cable models of mammalian motor nerve fibres, incorporating three different myelin representations, are compared. The three myelin representations are a perfectly insulating single cable (model A), a finite impedance single cable (model B) and a finite impedance double cable (model C). Extracellular stimulation of the three models is used to study their strength-duration and current-distance (I-X) relationships, conduction velocity (CV) and action potential shape. All three models have a chronaxie time that is within the experimental range. Models B and C have increased threshold currents compared with model A, but each model has slope to the I-X relationship that matches experimental results. Model B has a CV that matches experimental data, whereas the CV of models A and C are above and below the experimental range, respectively. Model C is able to produce a depolarising afterpotential (DAP), whereas models A and B exhibit hyperpolarising afterpotentials. Models A and B are determined to be the preferred models when low-frequency stimulation (< approximately 25 Hz) is used, owing to their efficiency and accurate excitation and conduction properties. For high frequency stimulation (approximately 25 Hz and greater), model C, with its ability to produce a DAP, is necessary accurately to simulate excitation behaviour.

At the interface: convergence of neural regeneration and neural prostheses for restoration of function.

The rapid pace of recent advances in development and application of electrical stimulation of the nervous system and in neural regeneration has created opportunities to combine these two approaches to restoration of function. This paper relates the discussion on this topic from a workshop at the International Functional Electrical Stimulation Society. The goals of this workshop were to discuss the current state of interaction between the fields of neural regeneration and neural prostheses and to identify potential areas of future research that would have the greatest impact on achieving the common goal of restoring function after neurological damage. Identified areas include enhancement of axonal regeneration with applied electric fields, development of hybrid neural interfaces combining synthetic silicon and biologically derived elements, and investigation of the role of patterned neural activity in regulating various neuronal processes and neurorehabilitation. Increased communication and cooperation between the two communities and recognition by each field that the other has something to contribute to their efforts are needed to take advantage of these opportunities. In addition, creative grants combining the two approaches and more flexible funding mechanisms to support the convergence of their perspectives are necessary to achieve common objectives.

Emerging clinical applications of electrical stimulation: opportunities for restoration of function.

Emerging clinical application of electrical stimulation in three systems is reviewed. In the bladder, stimulation of sacral posterior roots reduces reflex incontinence and significantly improves bladder capacity. With the combination of anterior and posterior root stimulation, bladder control can be achieved without the need for rhizotomy. Preliminary research demonstrates that bladder contractions may also be generated by stimulation of the urethral sensory branch of the pudendal nerve, even after acute spinal cord transection, while inhibition of the bladder and control of urge incontinence can be achieved by stimulation of the whole pudendal nerve. Spinal cord stimulation can modulate the activity of the intrinsic cardiac nervous system involved in the regulation of regional cardiac function and significantly reduce the pain associated with angina pectoris. Finally in the area of upper airway disorders, functional electrical stimulation has great potential for increasing life support as well as for quality of life in chronic ailments, particularly obstructive sleep apnea and dysphagia.

Neuroprosthetic applications of electrical stimulation.

Neural prostheses are a developing technology that use electrical activation of the nervous system to restore function to individuals with neurological impairment. Neural prostheses function by electrical initiation of action potentials in nerve fibers that carry the signal to an endpoint where chemical neurotransmitters are released, either to affect an end organ or another neuron. Thus, in principle, any end organ under neural control is a candidate for neural prosthetic control. Applications have included stimulation in both the sensory and motor systems and range in scope from experimental trials with single individuals to commercially available devices. Outcomes of motor system neural prostheses include restoration of hand grasp and release in quadriplegia, restoration of standing and stepping in paraplegia, restoration of bladder function (continence, micturition) following spinal cord injury, and electrophrenic respiration in high-level quadriplegia. Neural prostheses restore function and provide greater independence to individuals with disability.

Recording evoked potentials during deep brain stimulation: development and validation of instrumentation to suppress the stimulus artefact.

The clinical efficacy of deep brain stimulation (DBS) for the treatment of movement disorders depends on the identification of appropriate stimulation parameters. Since the mechanisms of action of DBS remain unclear, programming sessions can be time consuming, costly and result in sub-optimal outcomes. Measurement of electrically evoked compound action potentials (ECAPs) during DBS, generated by activated neurons in the vicinity of the stimulating electrode, could offer insight into the type and spatial extent of neural element activation and provide a potential feedback signal for the rational selection of stimulation parameters and closed-loop DBS. However, recording ECAPs presents a significant technical challenge due to the large stimulus artefact, which can saturate recording amplifiers and distort short latency ECAP signals. We developed DBS-ECAP recording instrumentation combining commercial amplifiers and circuit elements in a serial configuration to reduce the stimulus artefact and enable high fidelity recording. We used an electrical circuit equivalent model of the instrumentation to understand better the sources of the stimulus artefact and the mechanisms of artefact reduction by the circuit elements. In vitro testing validated the capability of the instrumentation to suppress the stimulus artefact and increase gain by a factor of 1000 to 5000 compared to a conventional biopotential amplifier. The distortion of mock ECAP (mECAP) signals was measured across stimulation parameters, and the instrumentation enabled high fidelity recording of mECAPs with latencies of only 0.5 ms for DBS pulse widths of 50 to 100 µs/phase. Subsequently, the instrumentation was used to record in vivo ECAPs, without contamination by the stimulus artefact, during thalamic DBS in an anesthetized cat. The characteristics of the physiological ECAP were dependent on stimulation parameters. The novel instrumentation enables high fidelity ECAP recording and advances the potential use of the ECAP as a feedback signal for the tuning of DBS parameters.

Electrical activation of spinal neural circuits: application to motor-system neural prostheses.

Present motor-system neural prostheses use electrical activation of last-order (motor) neurons to restore function. We are pursuing a new approach: restoration of function by electrical activation of higher-order interneurons. Our hypothesis is that electrical activation of spinal neural circuits, rather than direct activation of last-order motoneurons, will simplify generation of complex motor behaviors. We review two approaches to control bladder function and to control skeletal motor function: intraspinal microstimulation for direct activation of spinal neurons and peripheral afferent stimulation for indirect, synaptic activation of spinal neurons. The results demonstrate that electrical activation of spinal neural circuits allows generation of complex motor behaviors including micturition and organized multi-joint motor responses with a single electrode. Electrical activation of spinal neural circuits, and generation of the complex functions they subserve, holds great promise to advance the function of motor system neural prostheses.