Spinal premotor interneurons controlling antagonistic muscles are spatially intermingled.

Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized, we used multiple complementary viral-tracing approaches in mice to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components.

The spinal cord contains circuits of nerve cells that control how the body moves. Within these networks are interneurons that project to motor neurons, which innervate different types of muscle to contract: flexors (such as the biceps), which bend, or 'flex', the body's joints, and extensors (such as the triceps), which lead to joint extension. These motor signals must be carefully coordinated to allow precise and stable control of the body's movements. Previous studies suggest that where interneurons are placed in the spinal cord depends on whether they activate the motor neurons responsible for flexion or extension. To test if these findings were reproducible, Ronzano, Skarlatou, Barriga, Bannatyne, Bhumbra et al. studied interneurons which flex and extend the ankle joint in mice. In collaboration with several laboratories, the team used a combination of techniques to trace how interneurons and motor neurons were connected in the mouse spinal cord. This revealed that regardless of the method used or the laboratory in which the experiments were performed, the distribution of interneurons associated with flexion and extension overlapped one another. This finding contradicts previously published results and suggests that interneurons in the spinal cord are not segregated based on their outputs. Instead, they may be positioned based on the signals they receive, similar to motor neurons. Understanding where interneurons in the spinal cord are placed will provide new insights on how movement is controlled and how it is impacted by injuries and disease. In the future, this knowledge could benefit work on how neural circuits in the spinal cord are formed and how they can be regenerated.

In vitro longitudinal lumbar spinal cord preparations to study sensory and recurrent motor microcircuits of juvenile mice.

In vitro spinal cord preparations have been extensively used to study microcircuits involved in the control of movement. By allowing precise control of experimental conditions coupled with state-of-the-art genetics, imaging, and electrophysiological techniques, isolated spinal cords from mice have been an essential tool in detailing the identity, connectivity, and function of spinal networks. The majority of the research has arisen from in vitro spinal cords of neonatal mice, which are still undergoing important postnatal maturation. Studies from adults have been attempted in transverse slices, however, these have been quite challenging due to the poor motoneuron accessibility and viability, as well as the extensive damage to the motoneuron dendritic trees. In this work, we describe two types of coronal spinal cord preparations with either the ventral or the dorsal horn ablated, obtained from mice of different postnatal ages, spanning from preweaned to 1 mo old. These semi-intact preparations allow recordings of sensory-afferent and motor-efferent responses from lumbar motoneurons using whole cell patch-clamp electrophysiology. We provide details of the slicing procedure and discuss the feasibility of whole cell recordings. The in vitro dorsal and ventral horn-ablated spinal cord preparations described here are a useful tool to study spinal motor circuits in young mice that have reached the adult stages of locomotor development.NEW & NOTEWORTHY In the past 20 years, most of the research into the mammalian spinal circuitry has been limited to in vitro preparations from embryonic and neonatal mice. We describe two in vitro longitudinal lumbar spinal cord preparations from juvenile mice that allow the study of motoneuron properties and respective afferent or efferent spinal circuits through whole cell patch clamp. These preparations will be useful to those interested in the study of microcircuits at mature stages of motor development.

The Effects of Optogenetic Activation of Astrocytes on Spike-and-Wave Discharges in Genetic Absence Epileptic Rats.

Background: Absence seizures (petit mal seizures) are characterized by a brief loss of consciousness without loss of postural tone. The disease is diagnosed by an electroencephalogram (EEG) showing spike-wave discharges (SWD) caused by hypersynchronous thalamocortical (TC) oscillations. There has been an explosion of research highlighting the role of astrocytes in supporting and modulating neuronal activity. Despite established in vitro evidence, astrocytes' influence on the TC network remains to be elucidated in vivo in the absence epilepsy (AE). Purpose: In this study, we investigated the role of astrocytes in the generation and modulation of SWDs. We hypothesize that disturbances in astrocytes' function may affect the pathomechanism of AE. Methods: To direct the expression of channelrhodopsin-2 (ChR2) rAAV8-GFAP-ChR2(H134R)-EYFP or to control the effect of surgical intervention, AAV-CaMKIIa-EYFP was injected into the ventrobasal nucleus (VB) of the thalamus of 18 animals. After four weeks following the injection, rats were stimulated using blue light (~473 nm) and, simultaneously, the electrophysiological activity of the frontal cortical neurons was recorded for three consecutive days. The animals were then perfused, and the brain tissue was analyzed by confocal microscopy. Results: A significant increase in the duration of SWD without affecting the number of SWD in genetic absence epileptic rats from Strasbourg (GAERS) compared to control injections was observed. The duration of the SWD was increased from 12.50 ± 4.41 s to 17.44 ± 6.07 following optogenetic stimulation in GAERS. The excitation of the astrocytes in Wistar Albino Glaxo Rijswijk (WAG-Rij) did not change the duration of SWD; however, stimulation resulted in a significant increase in the number of SWD from 18.52 ± 11.46 bursts/30 min to 30.17 ± 18.43 bursts/30 min. Whereas in control injection, the duration and the number of SWDs were similar at pre- and poststimulus. Both the background and poststimulus average firing rates of the SWD in WAG-Rij were significantly higher than the firing recorded in GAERS. Conclusion: These findings suggest that VB astrocytes play a role in modulating the SWD generation in both rat models with distinct mechanisms and can present an essential target for the possible therapeutic approach for AE.

Comparison of the temporal properties of medium latency responses induced by cortical and peripheral stimulation.

Sudden foot dorsiflexion lengthens soleus muscle and activates stretch-based spinal reflexes. Dorsiflexion can be triggered by activating tibialis anterior (TA) muscle through peroneal nerve stimulation or transcranial magnetic stimulation (TMS) which evokes a response in the soleus muscle referred to as Medium Latency Reflex (MLR) or motor-evoked potential-80 (Soleus MEP80), respectively. This study aimed to examine the relationship between these responses in humans. Therefore, latency characteristics and correlation of responses between soleus MEP80 and MLR were investigated. We have also calculated the latencies from the onset of tibialis activity, i.e., subtracting of TA-MEP from MEP80 and TA direct motor response from MLR. We referred to these calculations as Stretch Loop Latency Central (SLLc) for MEP80 and Stretch Loop Latency Peripheral (SLLp) for MLR. The latency of SLLc was found to be 61.4 ± 5.6 ms which was significantly shorter (P = 0.0259) than SLLp (64.0 ± 4.2 ms) and these latencies were correlated (P = 0.0045, r = 0.689). The latency of both responses was also found to be inversely related to the response amplitude (P = 0.0121, r = 0.451) probably due to the activation of large motor units. When amplitude differences were corrected, i.e. investigating the responses with similar amplitudes, SLLp, and SLLc latencies found to be similar (P = 0.1317). Due to the identical features of the soleus MEP80 and MLR, we propose that they may both have spinal origins.

Post-activation depression of primary afferents reevaluated in humans.

Amplitude variation of Hoffmann Reflex (H-reflex) was used as a tool to investigate many neuronal networks. However, H-reflex itself is a subject to intrinsic changes including post-activation depression (P-AD). We aimed to investigate P-AD and its implication on motor control in humans. Upon tibial nerve stimulation in 23 healthy participants, peak-to-peak amplitude change of H-reflex was investigated using surface electromyography (SEMG) of soleus muscle. Variety of stimulus intensities, interstimulus intervals (ISIs), voluntary contraction levels/types and force recording were used to investigate the nature of P-AD. We have shown that P-AD was significantly stronger in the shorter ISIs. The only exception was the ISI of 200 msecs which had a weaker P-AD than some of the longer ISIs. Sudden muscle relaxation, on the other hand, further increased the effectiveness of the ongoing P-AD. Moreover, P-AD displayed its full effect with the first stimulus when there was no muscle contraction and was efficient to reduce the muscle force output by about 30%. These findings provide insight about the variations and mechanism of P-AD and could lead to improvements in diagnostic tools in neurological diseases.

Coculture model of blood-brain barrier on electrospun nanofibers.

The blood-brain barrier (BBB) is a control mechanism that limits the diffusion of many substances to the central nervous system (CNS). In this study, we designed an in-vitro 3-dimensional BBB system to obtain a fast and reliable model to mimic drug delivery characteristics of the CNS. A support membrane of polycaprolactone nanofiber surfaces was prepared using electrospinning. After confirming the fiber morphology and size, endothelial cells (HUVEC) and glial cells were cultured on either side of this membrane. The model's similarity to in vivo physiology was tested with a home-designed transmembrane resistance (TR) device, with positive and negative control molecules. Finally, 2 doses of methotrexate (MTX), a chemotherapy agent, were applied to the model, and its permeability through the model was determined indirectly by a vitality test on the MCF-7 cell line. Nicotine, the positive control, completed its penetration through the model almost instantly, while albumin, the negative control, was blocked significantly even after 2 days. MTX reached a deadly threshold 24 h after application. The TR value of the model was promising, being around 260 ohm.cm2. The provided model proposes a disposable and reliable tool for investigating drug permeability through the BBB and has the potential to reduce the number of animal experiments.

Effect of aging on H-reflex response to fatigue.

Injury as a result of tripping is relatively common among older people. The risk of falling increases with fatigue and of importance is the ability to dorsiflex the foot through timely activation of the tibialis anterior (TA) muscle to ensure the foot clears the ground, or an obstacle, during the swing phase of walking. We, therefore, questioned whether the muscle spindle input to the motoneurons alters with ongoing fatigue in older people. We electrically stimulated the common peroneal nerve to assess the TA primary afferent efficacy using H-reflex before, immediately following and after a fatiguing maximal isometric contraction. M-response was kept unchanged throughout the experiment to ensure a similar stimulus intensity was delivered across time points. H-reflex increased significantly while the TA muscle was in a state of fatigue for the younger participants but tended to decrease with increasing age. The main contributor to the tonicity of TA muscle, i.e., excitatory synapses of spindle primary endings of motoneurons that innervate TA muscle, tend to lose their efficacy during fatigue in the older individuals but increased efficiency in the majority of the younger people. Since TA muscle is the main dorsiflexor of the foot and it needs to be active during the swing phase of stepping to prevent tripping, older individuals become more susceptible to falling when their muscles are fatigued. This finding may help improve devices/treatments to overcome the problem of tripping among older individuals.

Transcranial magnetic stimulation induced early silent period and rebound activity re-examined.

Despite being widely studied, the underlying mechanisms of transcranial magnetic brain stimulation (TMS) induced motor evoked potential (MEP), early cortical silent period (CSP) and rebound activity are not fully understood. Our aim is to better characterize these phenomena by combining various analysis tools on firing motor units. Responses of 29 tibialis anterior (TA) and 8 abductor pollicis brevis (APB) motor units to TMS pulses were studied using discharge rate and probability-based tools to illustrate the profile of the synaptic potentials as they develop on motoneurons in 24 healthy volunteers. According to probability-based methods, TMS pulse produces a short-latency MEP which is immediately followed by CSP that terminates at rebound activity. Discharge rate analysis, however, revealed not three, but just two events with distinct time courses; a long-lasting excitatory period (71.2 ± 9.0 ms for TA and 42.1 ± 11.2 ms for APB) and a long-latency inhibitory period with duration of 57.9 ± 9.5 ms for TA and 67.3 ± 13.8 ms for APB. We propose that part of the CSP may relate to the falling phase of net excitatory postsynaptic potential induced by TMS. Rebound activity, on the other hand, may represent tendon organ inhibition induced by MEP activated soleus contraction and/or long-latency intracortical inhibition. Due to generation of field potentials when high intensity TMS is used, this study is limited to investigate the events evoked by low intensity TMS only and does not provide information about later parts of much longer CSPs induced by high intensity TMS. Adding discharge rate analysis contributes to obtain a more accurate picture about the characteristics of TMS-induced events. These results have implications for interpreting motor responses following TMS for diagnosis and overseeing recovery from various neurological conditions.

Medium latency excitatory reflex of soleus re-examined.

We aimed to study the receptor origin and postsynaptic potential profile of the medium latency reflex (MLR) response that develops in the soleus muscle when common peroneal nerve of antagonist tibialis anterior (TA) muscle is electrically stimulated. To achieve this aim, we electrically stimulated common peroneal nerve and recorded surface electromyography (SEMG) responses of soleus and TA muscles of informed volunteers. Additionally, we recorded intramuscular EMG from the soleus muscle. Stimulation of common peroneal nerve induced a direct motor response (M-response) in the TA and MLR in SEMG of the soleus. Using voluntarily-activated single motor units (SMUs) from the soleus muscle we noted that there were two distinct responses following the stimulus. The first response was a reciprocal inhibitory reflex probably originating from the antagonist muscle spindle primary (Ia) afferents. This was followed by an indirect reflex response activated by the contraction of the TA muscle during the M-response. This contraction generated a rapid acceleration in the direction of dorsiflexion hence inducing a stretch stimulus on soleus muscle. The response of soleus to this stimulus was a stretch reflex. We suggest that this stretch reflex is the main contributor to the so-called soleus MLR in the literature. This study illustrated the importance of using SMUs and also using discharge-rate based analysis for closely examining previously 'established' reflexes.

Motor units as tools to evaluate profile of human Renshaw inhibition.

KEY POINTS: To uncover the synaptic profile of Renshaw inhibition on motoneurons, we stimulated thick motor axons and recorded from voluntarily-activated motor units. Stimuli generated a direct motor response on the whole muscle and an inhibitory response in active motor units. We have estimated the profile of Renshaw inhibition indirectly using the response of motor unit discharge rates to the stimulus. We have put forward a method of extrapolation that may be used to determine genuine synaptic potentials as they develop on motoneurons. These optimized techniques can be used in research and in clinics to fully appreciate Renshaw cell function in various neurological disorders. ABSTRACT: Although Renshaw inhibition (RI) has been extensively studied for decades, its precise role in motor control is yet to be discovered. One of the main handicaps is a lack of reliable methods for studying RI in conscious human subjects. We stimulated the lowest electrical threshold motor axons (thickest axons) in the tibial nerve and analysed the stimulus-correlated changes in discharge of voluntarily recruited low-threshold single motor units (SMUs) from the soleus muscle. In total, 54 distinct SMUs from 12 subjects were analysed. Stimuli that generated only the direct motor response (M-only) on surface electromyography induced an inhibitory response in the low-threshold SMUs. Because the properties of RI had to be estimated indirectly using the background discharge rate of SMUs, its profile varied with the discharge rate of the SMU. The duration of RI was found to be inversely proportional to the discharge rate of SMUs. Using this important finding, we have developed a method of extrapolation for estimating RI as it develops on motoneurons in the spinal cord. The frequency methods indicated that the duration of RI was between 30 and 40 ms depending on the background firing rate of the units, and the extrapolation indicated that RI on silent motoneurons was ∼55 ms. The present study establishes a novel methodology for studying RI in human subjects and hence may serve as a tool for improving our understanding of the involvement of RI in human motor control.

Optimal location for eliciting the tibial H-reflex and motor response.

INTRODUCTION: Although there are numerous protocols to adjust the amplitude of the Hoffmann reflex (H-reflex) relative to the size of the direct motor response (M-response), the optimal stimulating location has not been described. We sought to determine the optimal positioning of the stimulating cathode when evoking the tibial nerve H-reflex and M-response. METHODS: A small cathode was placed on defined points in the popliteal fossa while an anode was fixed on the patella. The tibial nerve was stimulated electrically, and the response of the soleus muscle was recorded using intramuscular and surface electromyography. RESULTS: We found that more-lateral points along a line drawn across the popliteal fossa were the best locations to obtain only the M-response, whereas stimulating the midpoint was optimal for obtaining only the H-reflex. DISCUSSION: By using specified locations for electrical stimulation to evoke H-reflex and M-response, the functionality of the tibial nerve can be assessed. Muscle Nerve 58:828-833, 2018.

Chiropractic spinal manipulation alters TMS induced I-wave excitability and shortens the cortical silent period.

The objective of this study was to construct peristimulus time histogram (PSTH) and peristimulus frequencygram (PSF) using single motor unit recordings to further characterize the previously documented immediate sensorimotor effects of spinal manipulation. Single pulse transcranial magnetic stimulation (TMS) via a double cone coil over the tibialis anterior (TA) motor area during weak isometric dorsiflexion of the foot was used on two different days in random order; pre/post spinal manipulation (in eighteen subjects) and pre/post a control (in twelve subjects) condition. TA electromyography (EMG) was recorded with surface and intramuscular fine wire electrodes. Three subjects also received sham double cone coil TMS pre and post a spinal manipulation intervention. From the averaged surface EMG data cortical silent periods (CSP) were constructed and analysed. Twenty-one single motor units were identified for the spinal manipulation intervention and twelve single motor units were identified for the control intervention. Following spinal manipulations there was a shortening of the silent period and an increase in the single unit I-wave amplitude. No changes were observed following the control condition. The results provide evidence that spinal manipulation reduces the TMS-induced cortical silent period and increases low threshold motoneurone excitability in the lower limb muscle. These finding may have important clinical implications as they provide support that spinal manipulation can be used to strengthen muscles. This could be followed up on populations that have reduced muscle strength, such as stroke victims.

Assessment of the corticospinal fiber integrity in mirror movement disorder.

Mirror movements are unintended movements occurring on one side of the body that mirror the contralateral voluntary ones. It has been proposed that mirror movements occur due to abnormal decussation of the corticospinal pathways. Using detailed multidisciplinary approach, we aimed to enlighten the detailed mechanism underlying the mirror movements in a case subject who is diagnosed with mirror movements of the hands and we compared the findings with the unaffected control subjects. To evaluate the characteristics of mirror movements, we used several techniques including whole exome sequencing, computed tomography, diffusion tensor imaging and transcranial magnetic stimulation. Computed tomography showed the absence of a spinous process of C5, fusion of the body of C5-C6 vertebrae, hypoplastic dens and platybasia of the posterior cranial fossa. A syrinx cavity was present between levels C3-C4 of the spinal cord. Diffusion tensor imaging of the corticospinal fibers showed disorganization and minimal decussations at the lower medulla oblongata. Transcranial magnetic stimulation showed that motor commands were distributed to the motor neuron pools on the left and right sides of the spinal cord via fast-conducting corticospinal tract fibers. Moreover, a heterozygous missense variation in the deleted in colorectal carcinoma gene has been observed. Developmental absence of the axonal guidance molecules or their receptors may result in abnormalities in the leading of the corticospinal fibers. Clinical evaluations and basic neuroscience techniques, in this case, provide information for this rare disease and contribute to our understanding of the normal physiology of bimanual coordination.

Chiropractic Manipulation Increases Maximal Bite Force in Healthy Individuals.

Recent research has shown that chiropractic spinal manipulation can alter central sensorimotor integration and motor cortical drive to human voluntary muscles of the upper and lower limb. The aim of this paper was to explore whether spinal manipulation could also influence maximal bite force. Twenty-eight people were divided into two groups of 14, one that received chiropractic care and one that received sham chiropractic care. All subjects were naive to chiropractic. Maximum bite force was assessed pre- and post-intervention and at 1-week follow up. Bite force in the chiropractic group increased compared to the control group (p = 0.02) post-intervention and this between-group difference was also present at the 1-week follow-up (p < 0.01). Bite force in the chiropractic group increased significantly by 11.0% (±18.6%) post-intervention (p = 0.04) and remained increased by 13.0% (±12.9%, p = 0.04) at the 1 week follow up. Bite force did not change significantly in the control group immediately after the intervention (−2.3 ± 9.0%, p = 0.20), and decreased by 6.3% (±3.4%, p = 0.01) at the 1-week follow-up. These results indicate that chiropractic spinal manipulation can increase maximal bite force.