Contribution of the external urethral sphincter to urinary void size in unanesthetized unrestrained rats.

AIMS: In anesthetized rats, voiding is typically associated with phasic activation (bursting) of the external urethral sphincter (EUS). During spontaneous voiding in unanesthetized, unrestrained rats, EUS bursting is the most common form of EUS activity exhibited, but it is not necessary for productive voiding to occur. The aim of the present study was to determine which aspects of EUS activity contributed to void size during bursting and non-bursting voiding in conscious, freely moving rats. METHODS: Female rats were implanted with electrodes adjacent to the EUS for recording electromyographic activity (EMG). EUS EMG recordings were performed during 24-hr sessions in a metabolic cage while voided urine was continuously collected and weighed. RESULTS: Void size was positively correlated with the duration of the intra-burst silent and active periods and variables reflecting the overall intensity and duration of bursting, particularly at lower frequencies within the 3-10 Hz range of EUS bursting. In addition, void size was inversely related to the frequency of bursting and to the average EMG amplitude during voiding, both in voids with and without bursting. CONCLUSIONS: EUS bursting contributes to productive voiding when bursting is present. Lower bursting frequencies elicit more productive voiding than do higher frequencies. In the absence of bursting, the association of increased void size with smaller average EUS EMG amplitude suggests that conscious rats can perform synergic voiding (i.e., bladder contraction with EUS relaxation) that is comparable to that seen in humans and other typically non-bursting species. Neurourol. Urodynam. 35:696-702, 2016. © 2015 Wiley Periodicals, Inc.

Long-term recording of external urethral sphincter EMG activity in unanesthetized, unrestrained rats.

The external urethral sphincter muscle (EUS) plays an important role in urinary function and often contributes to urinary dysfunction. EUS study would benefit from methodology for longitudinal recording of electromyographic activity (EMG) in unanesthetized animals, but this muscle is a poor substrate for chronic intramuscular electrodes, and thus the required methodology has not been available. We describe a method for long-term recording of EUS EMG by implantation of fine wires adjacent to the EUS that are secured to the pubic bone. Wires pass subcutaneously to a skull-mounted plug and connect to the recording apparatus by a flexible cable attached to a commutator. A force transducer-mounted cup under a metabolic cage collected urine, allowing recording of EUS EMG and voided urine weight without anesthesia or restraint. Implant durability permitted EUS EMG recording during repeated (up to 3 times weekly) 24-h sessions for more than 8 wk. EMG and voiding properties were stable over weeks 2-8. The degree of EUS phasic activity (bursting) during voiding was highly variable, with an average of 25% of voids not exhibiting bursting. Electrode implantation adjacent to the EUS yielded stable EMG recordings over extended periods and eliminated the confounding effects of anesthesia, physical restraint, and the potential for dislodgment of the chronically implanted intramuscular electrodes. These results show that micturition in unanesthetized, unrestrained rats is usually, but not always, associated with EUS bursting. This methodology is applicable to studying EUS behavior during progression of gradually evolving disease and injury models and in response to therapeutic interventions.

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.

A major new dimension in the problem of brain injury.

Evidence that neurohormones contribute to the contralateral effects of unilateral brain injury challenges a fundamental assumption of basic neuroscience and clinical neurology.

Brain-computer interface-based assessment of color vision.

Objective.Present methods for assessing color vision require the person's active participation. Here we describe a brain-computer interface-based method for assessing color vision that does not require the person's participation.Approach.This method uses steady-state visual evoked potentials to identify metamers-two light sources that have different spectral distributions but appear to the person to be the same color.Main results.We demonstrate that: minimization of the visual evoked potential elicited by two flickering light sources identifies the metamer; this approach can distinguish people with color-vision deficits from those with normal color vision; and this metamer-identification process can be automated.Significance.This new method has numerous potential clinical, scientific, and industrial applications.

External urethral sphincter motoneuron properties in adult female rats studied in vitro.

The external urethral sphincter (EUS) muscle plays a crucial role in lower urinary tract function: its activation helps maintain continence, whereas its relaxation contributes to micturition. To determine how the intrinsic properties of its motoneurons contribute to its physiological function, we have obtained intracellular current-clamp recordings from 49 EUS motoneurons in acutely isolated spinal cord slices from adult female rats. In all, 45% of EUS motoneurons fired spontaneously and steadily (average rate = 12-27 pulses/s). EUS motoneurons were highly excitable, having lower rheobase, higher input resistance, and smaller threshold depolarization than those of rat hindlimb motoneurons recorded in vitro. Correlations between these properties and afterhyperpolarization half-decay time are consistent with EUS motoneurons having characteristics of both fast and slow motor unit types. EUS motoneurons with a slow-like spectrum of properties exhibited spontaneous firing more often than those with fast-like characteristics. During triangular current ramp-induced repetitive firing, recruitment typically occurred at lower current levels than those at derecruitment, although the opposite pattern occurred in 10% of EUS motoneurons. This percentage was likely underestimated due to firing rate adaptation. These findings are consistent with the presence of a basal level of persistent inward current (PIC) in at least some EUS motoneurons. The low EUS motoneuron current and voltage thresholds make them readily recruitable, rendering them well suited to their physiological role in continence. The expression of firing behaviors consistent with PIC activation in this highly reduced preparation raises the possibility that in the intact animal, PICs contribute to urinary function not only through neuromodulator-dependent but also through neuromodulator-independent mechanisms.

Plasticity from muscle to brain.

Recognition that the entire central nervous system (CNS) is highly plastic, and that it changes continually throughout life, is a relatively new development. Until very recently, neuroscience has been dominated by the belief that the nervous system is hardwired and changes at only a few selected sites and by only a few mechanisms. Thus, it is particularly remarkable that Sir John Eccles, almost from the start of his long career nearly 80 years ago, focused repeatedly and productively on plasticity of many different kinds and in many different locations. He began with muscles, exploring their developmental plasticity and the functional effects of the level of motor unit activity and of cross-reinnervation. He moved into the spinal cord to study the effects of axotomy on motoneuron properties and the immediate and persistent functional effects of repetitive afferent stimulation. In work that combined these two areas, Eccles explored the influences of motoneurons and their muscle fibers on one another. He studied extensively simple spinal reflexes, especially stretch reflexes, exploring plasticity in these reflex pathways during development and in response to experimental manipulations of activity and innervation. In subsequent decades, Eccles focused on plasticity at central synapses in hippocampus, cerebellum, and neocortex. His endeavors extended from the plasticity associated with CNS lesions to the mechanisms responsible for the most complex and as yet mysterious products of neuronal plasticity, the substrates underlying learning and memory. At multiple levels, Eccles' work anticipated and helped shape present-day hypotheses and experiments. He provided novel observations that introduced new problems, and he produced insights that continue to be the foundation of ongoing basic and clinical research. This article reviews Eccles' experimental and theoretical contributions and their relationships to current endeavors and concepts. It emphasizes aspects of his contributions that are less well known at present and yet are directly relevant to contemporary issues.

Spinal Transection Alters External Urethral Sphincter Activity during Spontaneous Voiding in Freely Moving Rats.

The rat is a commonly used model for the study of lower urinary tract function before and after spinal cord injury. We have previously reported that in unanesthetized freely moving rats, although phasic external urethral sphincter (EUS) activity (bursting) is most common during micturition, productive voiding can occur in the absence of bursting, which differs from results seen in anesthetized or unanesthetized restrained animals. The purpose of the present study was to characterize EUS behavior in unanesthetized, freely moving rats before and after mid-thoracic (T8) or thoraco-lumbar (T13-L1) spinal transection to determine how EUS behavior after spinal cord injury differs from that seen in anesthetized or unanesthetized restrained rats. Several abnormalities became evident that were comparable after transection at either level, including the following: repetitive non-voiding EUS contractions; increased prevalence, intensity, and duration of EUS bursting; decreased rate of urine evacuation during bursting; increased void size and decreased number of daily voids; shorter inter-burst silent period and increased frequency of bursting; and loss of the direct linear relationships that are evident in intact animals between void size and bursting silent period. These data suggest that transection-induced delayed initiation of EUS bursting allows co-contraction of the bladder and the EUS that prevents or limits urine evacuation, resulting in a detrusor-sphincter dyssynergia-like phenomenon. In addition, the higher-than-normal frequency at which EUS bursting occurs after transection is associated with shorter silent periods during which urine typically flows, which interferes with voiding by slowing the rate of urine evacuation. That results were comparable after either transection suggests that the central pattern generator responsible for EUS bursting is located caudal to the L1 spinal segment.

Long-term spinal reflex studies in awake behaving mice.

The increasing availability of genetic variants of mice has facilitated studies of the roles of specific molecules in specific behaviors. The contributions of such studies could be strengthened and extended by correlation with detailed information on the patterns of motor commands throughout the course of specific behaviors in freely moving animals. Previously reported methodologies for long-term recording of electromyographic activity (EMG) in mice using implanted electrodes were designed for intermittent, but not continuous operation. This report describes the fabrication, implantation, and utilization of fine wire electrodes for continuous long-term recordings of spontaneous and nerve-evoked EMG in mice. Six mice were implanted with a tibial nerve cuff electrode and EMG electrodes in soleus and gastrocnemius muscles. Wires exited through a skin button and traveled through an armored cable to an electrical commutator. In mice implanted for 59-144 days, ongoing EMG was monitored continuously (i.e., 24 h/day, 7 days/week) by computer for 18-92 days (total intermittent recording for 25-130 days). When the ongoing EMG criteria were met, the computer applied the nerve stimulus, recorded the evoked EMG response, and determined the size of the M-response (MR) and the H-reflex (HR). It continually adjusted stimulation intensity to maintain a stable MR size. Stable recordings of ongoing EMG, MR, and HR were obtained typically 3 weeks after implantation. This study demonstrates the feasibility of long-term continuous EMG recordings in mice for addressing a variety of neurophysiological and behavioral issues.

Temporal transformation of multiunit activity improves identification of single motor units.

This report describes a temporally based method for identifying repetitive firing of motor units. This approach is ideally suited to spike trains with negative serially correlated inter-spike intervals (ISIs). It can also be applied to spike trains in which ISIs exhibit little serial correlation if their coefficient of variation (COV) is sufficiently low. Using a novel application of the Hough transform, this method (i.e. the modified Hough transform (MHT)) maps motor unit action potential (MUAP) firing times into a feature space with ISI and offset (defined as the latency from an arbitrary starting time to the first MUAP in the train) as dimensions. Each MUAP firing time corresponds to a pattern in the feature space that represents all possible MUAP trains with a firing at that time. Trains with stable ISIs produce clusters in the feature space, whereas randomly firing trains do not. The MHT provides a direct estimate of mean firing rate and its variability for the entire data segment, even if several individual MUAPs are obscured by firings from other motor units. Addition of this method to a shape-based classification approach markedly improved rejection of false positives using simulated data and identified spike trains in whole muscle electromyographic recordings from rats. The relative independence of the MHT from the need to correctly classify individual firings permits a global description of stable repetitive firing behavior that is complementary to shape-based approaches to MUAP classification.

Reflex conditioning: a new strategy for improving motor function after spinal cord injury.

Spinal reflex conditioning changes reflex size, induces spinal cord plasticity, and modifies locomotion. Appropriate reflex conditioning can improve walking in rats after spinal cord injury (SCI). Reflex conditioning offers a new therapeutic strategy for restoring function in people with SCI. This approach can address the specific deficits of individuals with SCI by targeting specific reflex pathways for increased or decreased responsiveness. In addition, once clinically significant regeneration can be achieved, reflex conditioning could provide a means of reeducating the newly (and probably imperfectly) reconnected spinal cord.

An in vitro protocol for recording from spinal motoneurons of adult rats.

In vitro slice preparations of CNS tissue are invaluable for studying neuronal function. However, up to now, slice protocols for adult mammal spinal motoneurons--the final common pathway for motor behaviors--have been available for only limited portions of the spinal cord. In most cases, these preparations have not been productive due to the poor viability of motoneurons in vitro. This report describes and validates a new slice protocol that for the first time provides reliable intracellular recordings from lumbar motoneurons of adult rats. The key features of this protocol are: preexposure to 100% oxygen; laminectomy prior to perfusion; anesthesia with ketamine/xylazine; embedding the spinal cord in agar prior to slicing; and, most important, brief incubation of spinal cord slices in a 30% solution of polyethylene glycol to promote resealing of the many motoneuron dendrites cut during sectioning. Together, these new features produce successful recordings in 76% of the experiments and an average action potential amplitude of 76 mV. Motoneuron properties measured in this new slice preparation (i.e., voltage and current thresholds for action potential initiation, input resistance, afterhyperpolarization size and duration, and onset and offset firing rates during current ramps) are comparable to those recorded in vivo. Given the mechanical stability and precise control over the extracellular environment afforded by an in vitro preparation, this new protocol can greatly facilitate electrophysiological and pharmacological study of these uniquely important neurons and other delicate neuronal populations in adult mammals.

Spinal and supraspinal effects of long-term stimulation of sensorimotor cortex in rats.

Sensorimotor cortex (SMC) modifies spinal cord reflex function throughout life and is essential for operant conditioning of the H-reflex. To further explore this long-term SMC influence over spinal cord function and its possible clinical uses, we assessed the effect of long-term SMC stimulation on the soleus H-reflex. In freely moving rats, the soleus H-reflex was measured 24 h/day for 12 wk. The soleus background EMG and M response associated with H-reflex elicitation were kept stable throughout. SMC stimulation was delivered in a 20-day-on/20-day-off/20-day-on protocol in which a train of biphasic 1-ms pulses at 25 Hz for 1 s was delivered every 10 s for the on-days. The SMC stimulus was automatically adjusted to maintain a constant descending volley. H-reflex size gradually increased during the 20 on-days, stayed high during the 20 off-days, and rose further during the next 20 on-days. In addition, the SMC stimulus needed to maintain a stable descending volley rose steadily over days. It fell during the 20 off-days and rose again when stimulation resumed. These results suggest that SMC stimulation, like H-reflex operant conditioning, induces activity-dependent plasticity in both the brain and the spinal cord and that the plasticity responsible for the H-reflex increase persists longer after the end of SMC stimulation than that underlying the change in the SMC response to stimulation.

Recovery of electromyographic activity after transection and surgical repair of the rat sciatic nerve.

The recovery of soleus (SOL), gastrocnemius (GAS), and tibialis anterior (TA) electromyographic activity (EMG) after transection and surgical repair of the sciatic nerve was studied in Sprague-Dawley rats using chronically implanted stimulation and recording electrodes. Spontaneous EMG activity in SOL and GAS and direct muscle (M) responses to posterior tibial nerve stimulation persisted for < or =2 days after sciatic nerve transection, but SOL and GAS H-reflexes disappeared immediately. Spontaneous EMG activity began to return 2-3 wk after transection, rose nearly to pretransection levels by 60 days, and persisted for the duration of the study period (120 days). Recovery of stimulus-evoked EMG responses began about 30 days after sciatic nerve transection as multiple small responses with a wide range of latencies. Over time, the latencies of these fractionated responses shortened, their amplitudes increased, and they merged into a distinct short-latency component (the putative M response) and a distinct long-latency component (the putative H-reflex). The extent of recovery of stimulation-evoked EMG was modest: even 100 days after sciatic nerve transection, the responses were still much smaller than those before transection. Similar gradual development of responses to posterior tibial nerve stimulation was also seen in TA, suggesting that some regenerating fibers sent branches into both tibial and common peroneal nerves.

Diurnal H-reflex variation in mice.

Mice exhibit diurnal variation in complex motor behaviors, but little is known about diurnal variation in simple spinally mediated functions. This study describes diurnal variation in the H-reflex (HR), a wholly spinal and largely monosynaptic reflex. Six mice were implanted with tibial nerve cuff electrodes and electrodes in the soleus and gastrocnemius muscles, for recording of ongoing and nerve-evoked electromyographic activity (EMG). Stimulation and recording were under computer control 24 h/day. During a 10-day recording period, HR amplitude varied throughout the day, usually being larger in the dark than in the light. This diurnal HR variation could not be attributed solely to differences in the net ongoing level of descending and segmental excitation to the spinal cord or stimulus intensity. HRs were larger in the dark than in the light even after restricting the evoked responses to subsets of trials having similar ongoing EMG and M-responses. The diurnal variation in the HR was out of phase with that reported previously for rats, but was in phase with that observed in monkeys. These data, supported by those in other species, suggest that the supraspinal control of the excitability of the HR pathway varies throughout the day in a species-specific pattern. This variation should be taken into account in experimental and clinical studies of spinal reflexes recorded at different times of day.

Sensorimotor cortex ablation prevents H-reflex up-conditioning and causes a paradoxical response to down-conditioning in rats.

Operant conditioning of the H-reflex, a simple model for skill acquisition, requires the corticospinal tract (CST) and does not require other major descending pathways. To further explore its mechanisms, we assessed the effects of ablating contralateral sensorimotor cortex (cSMC). In 22 Sprague-Dawley rats, the hindlimb area of left cSMC was ablated. EMG electrodes were implanted in the right soleus muscle and a stimulating cuff was placed around the right posterior tibial nerve. When EMG remained in a specified range, nerve stimulation just above the M response threshold elicited the H-reflex. In control mode, no reward occurred. In conditioning mode, reward occurred if H-reflex size was above (HRup mode) or below (HRdown mode) a criterion value. After exposure to the control mode for > or = 10 days, each rat was exposed for another 50 days to the control mode, the HRup mode, or the HRdown mode. In control and HRup rats, final H-reflex size was not significantly different from initial H-reflex size. In contrast, in HRdown rats, final H-reflex size was significantly increased to an average of 136% of initial size. Thus like recent CST transection, cSMC ablation greatly impaired up-conditioning. However, unlike recent CST transection, cSMC produced a paradoxical response to down-conditioning: the H-reflex actually increased. These results confirm the critical role of cSMC in H-reflex conditioning and suggest that this role extends beyond producing essential CST activity. Its interactions with ipsilateral SMC or other areas contribute to the complex pattern of spinal and supraspinal plasticity that underlies H-reflex conditioning.

H-reflex operant conditioning in mice.

Rats, monkeys, and humans can alter the size of their spinal stretch reflex and its electrically induced analog, the H-reflex (HR), when exposed to an operant conditioning paradigm. Because this conditioning induces plasticity in the spinal cord, it offers a unique opportunity to identify the neuronal sites and mechanisms that underlie a well-defined change in a simple behavior. To facilitate these studies, we developed an HR operant conditioning protocol in mice, which are better suited to genetic manipulation and electrophysiological spinal cord study in vitro than rats or primates. Eleven mice under deep surgical anesthesia were implanted with tibial nerve stimulating electrodes and soleus and gastrocnemius intramuscular electrodes for recording ongoing and stimulus-evoked EMG activity. During the 24-h/day computer-controlled experiment, mice received a liquid reward for either increasing (up-conditioning) or decreasing (down-conditioning) HR amplitude while maintaining target levels of ongoing EMG and directly evoked EMG (M-responses). After 3-7 wk of conditioning, the HR amplitude was 133 +/- 7% (SE) of control for up-conditioning and 71 +/- 8% of control for down-conditioning. HR conditioning was successful (i.e., > or =20% change in HR amplitude in the appropriate direction) in five of six up-conditioned animals (mean final HR amplitude = 139 +/- 5% of control HR for successful mice) and in four of five down-conditioned animals (mean final HR amplitude = 63 +/- 8% of control HR for successful mice). These effects were not attributable to differences in the net level of motoneuron pool excitation, stimulation strength, or distribution of HR trials throughout the day. Thus mice exhibit HR operant conditioning comparable with that observed in rats and monkeys.

Conduction velocity is inversely related to action potential threshold in rat motoneuron axons.

Intra-axonal recordings were performed in ventral roots of rats in vitro to study the conduction velocity and firing threshold properties of motoneuron axons. Mean values +/- SD were 30.5+/-5.6 m/s for conduction velocity and 11.6+/-4.5 mV for the depolarization from the resting potential required to reach firing threshold (threshold depolarization). Conduction velocity varied inversely and significantly with threshold depolarization ( P=0.0002 by linear regression). This relationship was evident even after accounting for variation in conduction velocity associated with action potential amplitude, injected current amplitude, or body weight. Conduction velocity also varied inversely with the time to action potential onset during just-threshold current pulse injection. These data suggest that the time course of depolarization leading to action potential initiation contributes to the speed of conduction in motoneuron axons.

Corticospinal tract transection prevents operantly conditioned H-reflex increase in rats.

Operant conditioning of the H-reflex, the electrical analog of the spinal stretch reflex, in freely moving rats is a relatively simple model for studying long-term supraspinal control over spinal cord function. Motivated by food reward, rats can gradually increase (i.e., up-condition) or decrease (i.e., down-condition) the soleus H-reflex. Earlier work showed that corticospinal tract transection prevents acquisition and maintenance of H-reflex down-conditioning while transection of other major spinal cord tracts does not. This study explores the effects on acquisition of up-conditioning of the right soleus H-reflex of mid-thoracic transection of: the right lateral column (LC, five rats) (containing the rubrospinal, vestibulospinal, and reticulospinal tracts); the entire dorsal column (DC, six rats) [containing the main corticospinal tract (CST) and the dorsal ascending tract (DA)]; the CST alone (five rats); or the DA alone (seven rats). After initial (i.e., control) H-reflex amplitude was determined, the rat was exposed for 50 days to the up-conditioning mode in which reward was given when the H-reflex was above a criterion value. H-reflex amplitude at the end of up-conditioning was compared to initial H-reflex amplitude. An increase > or =20% was defined as successful up-conditioning. In intact rats, H-reflex amplitude at the end of up-conditioning averaged 164% (+/-10%, SE), and 81% were successful. In the present study, LC and DA rats were similar to intact rats in final H-reflex amplitude and percent successful. In contrast, results for DC and CST rats were significantly different from those of intact rats. In the six DC rats, final H-reflex amplitude averaged 105% (+/-3)% of control and none was successful; and in the five CST rats, final H-reflex amplitude averaged 94% (+/-3)% and none was successful. The results indicate that the main CST, located in the dorsal column, is essential for H-reflex up-conditioning as it is for down-conditioning, while the dorsal column ascending tract and the ipsilateral lateral column (containing the main rubrospinal, vestibulospinal, and reticulospinal tracts) do not appear to be essential.