Emergent epileptiform activity in spinal sensory circuits drives ectopic bursting in afferent axons and sensory dysfunction after cord injury.

ABSTRACT: Spinal cord injury leads to hyperexcitability and dysfunction in spinal sensory processing. As hyperexcitable circuits can become epileptiform, we explored whether such activity emerges in a thoracic spinal cord injury (SCI) contusion model of neuropathic pain. Recordings from spinal sensory axons in multiple below-lesion segmental dorsal roots demonstrated that SCI facilitated the emergence of spontaneous ectopic burst spiking in afferent axons, which were correlated across multiple adjacent dorsal roots. Burst frequency correlated with behavioral mechanosensitivity. The same bursting events were recruited by afferent stimulation, and timing interactions with ongoing spontaneous bursts revealed that recruitment was limited by a prolonged post-burst refractory period. Ectopic bursting in afferent axons was driven by GABAA receptor activation, presumably by conversion of subthreshold GABAergic interneuronal presynaptic axoaxonic inhibitory actions to suprathreshold spiking. Collectively, the emergence of stereotyped bursting circuitry with hypersynchrony, sensory input activation, post-burst refractory period, and reorganization of connectivity represent defining features of an epileptiform network. Indeed, these same features were reproduced in naive animals with the convulsant 4-aminopyridine (fampridine). We conclude that spinal cord injury promotes the emergence of epileptiform activity in spinal sensory networks that promote profound corruption of sensory signaling. This includes hyperexcitability and bursting by ectopic spiking in afferent axons that propagate bidirectionally by reentrant central and peripheral projections as well as sensory circuit hypoexcitability during the burst refractory period. More broadly, the work links circuit hyperexcitability to epileptiform circuit emergence, further strengthening it as a conceptual basis to understand features of sensory dysfunction and neuropathic pain.

Emergent epileptiform activity drives spinal sensory circuits to generate ectopic bursting in intraspinal afferent axons after cord injury.

Spinal cord injury ( SCI ) leads to hyperexcitability and dysfunction in spinal sensory processing. As hyperexcitable circuits can become epileptiform elsewhere, we explored whether such activity emerges in spinal sensory circuits in a thoracic SCI contusion model of neuropathic pain. Recordings from spinal sensory axons in multiple below-lesion segmental dorsal roots ( DRs ) demonstrated that SCI facilitated the emergence of spontaneous ectopic burst spiking in afferent axons, which synchronized across multiple adjacent DRs. Burst frequency correlated with behavioral mechanosensitivity. The same bursting events were recruited by afferent stimulation, and timing interactions with ongoing spontaneous bursts revealed that recruitment was limited by a prolonged post-burst refractory period. Ectopic bursting in afferent axons was driven by GABA A receptor activation, presumably via shifting subthreshold GABAergic interneuronal presynaptic axoaxonic inhibitory actions to suprathreshold spiking. Collectively, the emergence of stereotyped bursting circuitry with hypersynchrony, sensory input activation, post-burst refractory period, and reorganization of connectivity represent defining features of epileptiform networks. Indeed, these same features were reproduced in naïve animals with the convulsant 4-aminopyridine ( 4-AP ). We conclude that SCI promotes the emergence of epileptiform activity in spinal sensory networks that promotes profound corruption of sensory signaling. This corruption includes downstream actions driven by ectopic afferent bursts that propagate via reentrant central and peripheral projections and GABAergic presynaptic circuit hypoexcitability during the refractory period.

Relating Spinal Injury-Induced Neuropathic Pain and Spontaneous Afferent Activity to Sleep and Respiratory Dysfunction.

Abstract Spinal cord injury (SCI) can induce dysfunction in a multitude of neural circuits including those that lead to impaired sleep, respiratory dysfunction, and neuropathic pain. We used a lower thoracic rodent contusion SCI model of neuropathic pain that has been shown to associate with increased spontaneous activity in primary afferents and hindlimb mechanosensory stimulus hypersensitivity. Here we paired capture of these variables with chronic capture of three state sleep and respiration to more broadly understand SCI-induced physiological dysfunction and to assess possible interrelations. Noncontact electric field sensors were embedded into home cages to non-invasively capture the temporal evolution of sleep and respiration changes for six weeks after SCI in naturally behaving mice. Hindlimb mechanosensitivity was assessed weekly, and terminal experiments measured primary afferent spontaneous activity in situ from intact lumbar dorsal root ganglia (DRG). We observed that SCI led to increased spontaneous primary afferent activity (both firing rate and the number of spontaneously active DRGs) that correlated with increased respiratory rate variability and measures of sleep fragmentation. This is the first study to measure and link sleep dysfunction and variability in respiratory rate in a SCI model of neuropathic pain, and thereby provide broader insight into the magnitude of overall stress burden initiated by neural circuit dysfunction after SCI.

Unobtrusive Heartbeat Detection from Mice Using Sensors Embedded in the Nest.

Unobtrusive monitoring of physio-behavioral variables from animals can minimize variability in preclinical research and thereby maximize the potential for clinical translation. In this paper, we present the design, implementation, and validation of an instrumented nest providing continuous recordings of seismocardiogram (SCG) signals and skin temperature. SCG represents the chest-wall vibrations associated with the heartbeat, and can potentially provide a measure by which individual heartbeats can be detected without the need for electrodes or implantable devices. A non-contact electric field sensor placed in proximity to the animal in the nest was also used to detect respiratory dynamics. The setup was tested with a total of six anesthetized mice. To understand the effects of mouse positioning within the nest on signal quality, the error in heartbeat detection at different positions of the sensor on the body was quantified, with a simultaneously-obtained electrocardiogram (ECG) as the reference standard. At the optimal placement determined with this approach, multiple perturbations were performed such as pinching, changing ambient temperature, and norepinephrine injection to modulate physiology and assess measurement capability. Heartbeat intervals obtained from the ECG and SCG during the perturbations were correlated (R2=0.82) and were in agreement according to Bland-Altman methods (bias: 0.006ms, 95% confidence interval: [-3.79, 3.78]ms) suggesting that SCG can be reliably used for unobtrusive heartbeat detection. Accordingly, the setup can provide a means by which individual heartbeats - and thereby heart rate and heart rate variability indices - can be quantified without the need for any sensors to be attached to the body of the animal.

Automated Gait Analysis Through Hues and Areas (AGATHA): A Method to Characterize the Spatiotemporal Pattern of Rat Gait.

While rodent gait analysis can quantify the behavioral consequences of disease, significant methodological differences exist between analysis platforms and little validation has been performed to understand or mitigate these sources of variance. By providing the algorithms used to quantify gait, open-source gait analysis software can be validated and used to explore methodological differences. Our group is introducing, for the first time, a fully-automated, open-source method for the characterization of rodent spatiotemporal gait patterns, termed Automated Gait Analysis Through Hues and Areas (AGATHA). This study describes how AGATHA identifies gait events, validates AGATHA relative to manual digitization methods, and utilizes AGATHA to detect gait compensations in orthopaedic and spinal cord injury models. To validate AGATHA against manual digitization, results from videos of rodent gait, recorded at 1000 frames per second (fps), were compared. To assess one common source of variance (the effects of video frame rate), these 1000 fps videos were re-sampled to mimic several lower fps and compared again. While spatial variables were indistinguishable between AGATHA and manual digitization, low video frame rates resulted in temporal errors for both methods. At frame rates over 125 fps, AGATHA achieved a comparable accuracy and precision to manual digitization for all gait variables. Moreover, AGATHA detected unique gait changes in each injury model. These data demonstrate AGATHA is an accurate and precise platform for the analysis of rodent spatiotemporal gait patterns.

Quantitative histological grading methods to assess subchondral bone and synovium changes subsequent to medial meniscus transection in the rat.

AIM OF THE STUDY: The importance of the medial meniscus to knee health is demonstrated by studies which show meniscus injuries significantly increase the likelihood of developing osteoarthritis (OA), and knee OA can be modeled in rodents using simulated meniscus injuries. Traditionally, histological assessments of OA in these models have focused on damage to the articular cartilage; however, OA is now viewed as a disease of the entire joint as an organ system. The aim of this study was to develop quantitative histological measures of bone and synovial changes in a rat medial meniscus injury model of knee OA. MATERIALS AND METHODS: To initiate OA, a medial meniscus transection (MMT) and a medial collateral ligament transection (MCLT) were performed in 32 male Lewis rats (MMT group). MCLT alone served as the sham procedure in 32 additional rats (MCLT sham group). At weeks 1, 2, 4, and 6 post-surgery, histological assessment of subchondral bone and synovium was performed (n = 8 per group per time point). RESULTS: Trabecular bone area and the ossification width at the osteochondral interface increased in both the MMT and MCLT groups. Subintimal synovial cell morphology also changed in MMT and MCLT groups relative to naïve animals. CONCLUSIONS: OA affects the joint as an organ system, and quantifying changes throughout an entire joint can improve our understanding of the relationship between joint destruction and painful OA symptoms following meniscus injury.

Spatiotemporal gait compensations following medial collateral ligament and medial meniscus injury in the rat: correlating gait patterns to joint damage.

INTRODUCTION: After transection of the medial collateral ligament and medial meniscus (MCLT + MMT) in the rat, focal cartilage lesions develop over 4-6 weeks; however, sham surgery (MCLT alone) does not result in cartilage damage over a similar period. Thus, comparison of MCLT + MMT with the MCLT sham group offers an opportunity to investigate behavioral modifications related to focal cartilage and meniscus damage in the rat. METHODS: MCLT or MCLT + MMT surgery was performed in the right knees of male Lewis rats, with spatiotemporal gait patterns and hind limb sensitivity assessed at 1, 2, 4, and 6 weeks postsurgery (n = 8 rats per group per time point, n = 64 total). After the animals were euthanized, Histology was performed to assess joint damage. RESULTS: MCLT + MMT animals had unilateral gait compensations at early time points, but by week 6 bilateral gait compensations had developed in both the MCLT sham and MCLT + MMT groups. Conversely, heightened tactile sensitivity was detected in both MCLT sham and MCLT + MMT animals at week 1, but only the MCLT + MMT animals maintained heightened sensitivity to week 6. Cartilage lesions were found in the MCLT + MMT group but not in the MCLT sham group. Correlations could be identified between joint damage and gait changes in MCLT + MMT animals; however, the same gait changes were found with MCLT sham animals despite a lack of joint damage. CONCLUSIONS: Combined, our data highlight a common conundrum in osteoarthritis (OA) research: Some behavioral changes correlate to cartilage damage in the OA group, but the same changes can be identified in non-OA controls. Of the behavioral changes detected, allodynia was maintained in MCLT + MMT animals but not in the MCLT sham group. However, the correlation between cartilage damage and hind limb sensitivity is relatively weak (R = -0.4498), and the range of sensitivity measures overlaps between groups. The factors driving gait abnormalities in MCLT and MCLT + MMT animals also remain uncertain. The gait modifications are similar between groups and do not appear until weeks after surgery, despite cartilage damage being focused in the MCLT + MMT group. Combined, our data highlight the need to evaluate the links between noncartilage changes and behavioral changes following joint injury in the rat.

A novel operant-based behavioral assay of mechanical allodynia in the orofacial region of rats.

BACKGROUND: Detecting behaviors related to orofacial pain in rodent models often relies on subjective investigator grades or methods that place the animal in a stressful environment. In this study, an operant-based behavioral assay is presented for the assessment of orofacial tactile sensitivity in the rat. NEW METHODS: In the testing chamber, rats are provided access to a sweetened condensed milk bottle; however, a 360° array of stainless steel wire loops impedes access. To receive the reward, an animal must engage the wires across the orofacial region. Contact with the bottle triggers a motor, requiring the animal to accept increasing pressure on the face during the test. To evaluate this approach, tolerated bottle distance was measured for 10 hairless Sprague Dawley rats at baseline and 30 min after application of capsaicin cream (0.1%) to the face. The experiment was repeated to evaluate the ability of morphine to reverse this effect. RESULTS: The application of capsaicin cream reduced tolerated bottle distance measures relative to baseline (p<0.05). As long as morphine did not cause reduced participation due to sedation, subcutaneous morphine dosing reduced the effects of capsaicin (p<0.001). Comparison with existing method: For behavioral tests, experimenters often make subjective decisions of an animal's response. Operant methods can reduce these effects by measuring an animal's selection in a reward-conflict decision. Herein, a method to measure orofacial sensitivity is presented using an operant system. CONCLUSIONS: This operant device allows for consistent measurement of heightened tactile sensitivity in the orofacial regions of the rat.

An in vitro adult mouse muscle-nerve preparation for studying the firing properties of muscle afferents.

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

Gait analysis methods for rodent models of osteoarthritis.

Patients with osteoarthritis (OA) primarily seek treatment due to pain and disability, yet the primary endpoints for rodent OA models tend to be histological measures of joint destruction. The discrepancy between clinical and preclinical evaluations is problematic, given that radiographic evidence of OA in humans does not always correlate to the severity of patient-reported symptoms. Recent advances in behavioral analyses have provided new methods to evaluate disease sequelae in rodents. Of particular relevance to rodent OA models are methods to assess rodent gait. While obvious differences exist between quadrupedal and bipedal gait sequences, the gait abnormalities seen in humans and in rodent OA models reflect similar compensatory behaviors that protect an injured limb from loading. The purpose of this review is to describe these compensations and current methods used to assess rodent gait characteristics, while detailing important considerations for the selection of gait analysis methods in rodent OA models.

Characterization of muscle spindle afferents in the adult mouse using an in vitro muscle-nerve preparation.

We utilized an in vitro adult mouse extensor digitorum longus (EDL) nerve-attached preparation to characterize the responses of muscle spindle afferents to ramp-and-hold stretch and sinusoidal vibratory stimuli. Responses were measured at both room (24°C) and muscle body temperature (34°C). Muscle spindle afferent static firing frequencies increased linearly in response to increasing stretch lengths to accurately encode the magnitude of muscle stretch (tested at 2.5%, 5% and 7.5% of resting length [Lo]). Peak firing frequency increased with ramp speeds (20% Lo/sec, 40% Lo/sec, and 60% Lo/sec). As a population, muscle spindle afferents could entrain 1:1 to sinusoidal vibrations throughout the frequency (10-100 Hz) and amplitude ranges tested (5-100 µm). Most units preferentially entrained to vibration frequencies close to their baseline steady-state firing frequencies. Cooling the muscle to 24°C decreased baseline firing frequency and units correspondingly entrained to slower frequency vibrations. The ramp component of stretch generated dynamic firing responses. These responses and related measures of dynamic sensitivity were not able to categorize units as primary (group Ia) or secondary (group II) even when tested with more extreme length changes (10% Lo). We conclude that the population of spindle afferents combines to encode stretch in a smoothly graded manner over the physiological range of lengths and speeds tested. Overall, spindle afferent response properties were comparable to those seen in other species, supporting subsequent use of the mouse genetic model system for studies on spindle function and dysfunction in an isolated muscle-nerve preparation.