In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

Poznań University of Physical Education

Patterns of afferent connections from receptors of the distal forelimb were investigated in neurones located in C6-C7 segments of the spinal cord with branching axons projecting to the lateral reticular nucleus and the cerebellum. Experiments were made on five adult cats under alpha-chloralose anaesthesia. After antidromic identification, EPSPs and IPSPs were recorded from 22 neurones following stimulation of deep radial, superficial radial, median and ulnar nerves. Both excitatory and inhibitory effects were found in the majority of the cells, however, in 2 cases no synaptic actions were recorded. EPSPs were evoked from group I or II muscle, or cutaneous afferents - mostly monosynaptically. IPSPs from muscle, cutaneous or flexor reflex afferents were mostly polysynaptic. Seven various types of convergence were established in the cells investigated. Significance of parallel transmission of integrated information from various receptors of the distal forelimb to the reticular formation and cerebellum is discussed.

Afferent input from distal forelimb nerves to branching spinoreticular-spinocerebellar neurones in the cat.

Propriospinal neurones located in the cervical enlargement and projecting bilaterally to sacral segments of the spinal cord were investigated electrophysiologically in eleven deeply anaesthetized cats. Excitatory or inhibitory postsynaptic potentials from forelimb afferents were recorded following stimulation of deep radial (DR), superficial radial (SR), median (Med) and ulnar (Uln) nerves. 26 cells were recorded from C7, 22 from C8 and 3 from Th1 segments. The majority of the cells were located in the Rexed's laminae VIII and the medial part of the lamina VII. In 10 cases no afferent input from the forelimb afferents was found. In the remaining neurones effects were evoked mostly from DR (88%) and Med (63%), less often from SR (46%) and Uln (46%). Inhibitory actions were more frequent than excitatory. The highest number of IPSPs was evoked from high threshold flexor reflex afferents (FRA)--all connections were polysynaptic. However, inhibitory actions were often evoked from group I or II muscle afferents (polysynaptic or disynaptic) and, less frequently, from cutaneous afferents (mostly polysynaptic). Di- or polysynaptic IPSPs often accompanied monosynaptic EPSPs from group I or II muscle afferents. Disynaptic or polysynaptic EPSPs from muscle and cutaneous afferents were also recorded in many neurones, while polysynaptic EPSPs from FRA were observed only exceptionally. Various patterns of convergence in individual neuronal subpopulations indicate that they integrate different types of the afferent input from various muscle and cutaneous receptors of the distal forelimb. They transmit this information to motor centers controlling hind limb muscles, forming a part of the system contributing to the process of coordination of movements of fore--and hind--limbs.

Convergence of forelimb afferent actions on C7-Th1 propriospinal neurones bilaterally projecting to sacral segments of the cat spinal cord.

The purpose of the study was to analyze the interspecies differences of motor unit contractile properties in two most frequently studied mammals: cats and rats. A total sample of 166 motor units (79 in cats and 85 in rats) was investigated in the medial gastrocnemius muscle. Considerable differences were found in composition of the studied muscle. In cats, fast fatigable, fast resistant and slow units formed 68, 18 and 14% of the investigated population, whereas in rats 36, 52 and 12%, respectively. The contraction and relaxation times of motor units in the cat muscle were evidently longer than in the rat and the border values for fast/slow motor units division in these species were 44 and 20 ms, respectively. The mean values of twitch and tetanic forces appeared to be 7-8 times lower in rats, for fast, while 2-5 times for slow motor units. Also variability between the strongest and the weakest units within each type revealed differences 10-60 times in cats, whereas only 3.5-14 times in rats. The summation of twitches into tetanus for fast units was comparable in both species, but for S units was evidently more effective in the cat. In fast motor units' tetanic contractions evident interspecies differences concerned sag appearance and profiles of unfused tetani of FF and FR units. Differences in contractile properties described in the study may depend on the size, number and innervation ratio of motor units in the muscle of cat and rat, as well as their biochemical variability. Differences in composition of motor unit types and uneven mechanisms of force development may reflect biological adaptation to variable behaviour of cats and rats.

Interspecies differences of motor units properties in the medial gastrocnemius muscle of cat and rat.

Properties of motor unit action potentials (MUAPs) were compared for medial gastrocnemius (MG) motor units (MUs) in cats and rats. The experiments on functionally isolated MUs were performed under general anaesthesia, under comparable conditions (surgery, stimulating protocol and recording methods) for both species investigated. The proportions of motor units and contractile properties of the sample used in the study were consistent with previous studies performed on the MG muscle in both animal species, so comparisons of action potentials of individual types of MUs were acknowledged as fully reliable. The most prominent differences concerning MUAPs were observed in total duration and peak-to-peak times which for all MU types were about twice longer in cat MUs, in comparison to the rat MUs. The considerable disproportions were observed between the MUAP amplitudes of FF (fast fatigable), FR (fast resistant to fatigue) and S (slow) MUs in each species (the highest amplitudes were measured for FF and the lowest for S MUs), but there were no significant differences between cat and rat when respective types of MUs were compared. The shapes of MUAPs were commonly characterized by biphasic waveforms composed of two or three turns in all types of units, and no interspecies differences were revealed. Several factors influencing MUAP parameters were discussed indicating most of all importance of variable length of cat and rat muscle fibres and ambiguous influence of motor unit size, thickness of muscle fibres and their density around the recording electrode in the MG muscle of both species.

Comparative analysis of motor unit action potentials of the medial gastrocnemius muscle in cats and rats.

During natural contractions of a muscle, motor units (MUs) are activated by irregular discharge patterns of motoneurones. The aim of this study was to analyze changes in contractile forces of MUs following patterns of stimulation at variable frequency. Experiments were performed on 33 functionally isolated MUs of the fast-type in the medial gastrocnemius (MG) muscle of adult Wistar rats, under pentobarbital anaesthesia. The MUs forces evoked at five different regular stimulation patterns of constant frequencies were compared to forces generated during five random patterns of irregular stimulation with the same mean values of interpulse intervals, between 10 and 75 ms, and variability of these intervals of +/- 50% in each case. These values cover the natural range of the preferred firing rates of the MG motoneurones from unfused to nearly fused tetanic contractions. Analysis of changes in tetanic forces indicated a linear relationship between the interpulse interval as well as the initial level of the force and the amplitude of the force increase of the next contraction. It was demonstrated that variability of the instantaneous tetanic force during the irregular discharge pattern depends on the level of tetanic fusion. Moreover, it was demonstrated that for low and moderately-fused tetani, effectiveness of a MU contraction (expressed as the force-time area) is considerably higher for contractions evoked by irregular stimulation patterns. On the basis of the results of this study it was supposed that during voluntary contractions, the influence of changes in the motoneuronal firing rate on the motor unit force depends on the initial level of force.

Force generated by fast motor units of the rat medial gastrocnemius muscle during stimulation with pulses at variable intervals.

In the medial gastrocnemius muscle of intact rats, division of motor units (MUs) into slow (S) or fast (F) types is typically based on presence of a sag phenomenon in 40 Hz unfused tetanic contraction. MUs with sag are classified as F, while those without sag as S. However, in rats one month after spinal cord injury this phenomenon almost completely disappears and cannot be used as a basis for MUs differentiation, whereas the twitch contraction time increased significantly. Analysis of myosin heavy chain (MHC) isoform composition confirmed transformational changes of muscle fibres after spinal cord transection and indicated unchanged proportion of type I MHC isoforms, disappearance of type IIa MHC isoforms, and increase of type IIb MHC isoforms. We proposed an additional method for division of MUs into types when standard criteria are not applicable. It was observed that relative effectiveness of force summation during 20 Hz tetanus, described as a ratio of the force of the last contraction of this tetanus to the force of the first contraction, did not change after spinal cord injury. This ratio for S MUs both in intact and spinal rats exceeded 2.0, whereas for F units was lower than 2.0. Calculations of this ratio made for better fused tetani, evoked by 30 Hz or 40 Hz stimulation, showed overlapping values. We conclude that this 20 Hz tetanus index appears to be an alternative method useful for division of motor units into S and F types.

Division of motor units into fast and slow of the basis of profile of 20 Hz unfused tetanus.

The purpose of this study was to investigate whether age-related changes in motor unit (MU) contractile properties are reflected in parameters of motor unit action potentials (MUAPs). MUs of the medial gastrocnemius muscle were functionally isolated in anaesthetized Wistar rats. A control group of young animals (5-10mo) was compared to two groups of old rats (24-25mo and 28-30mo). The basic contractile properties of MUs as well as the amplitude, total duration, peak-to-peak time, and number of turns within MUAPs were measured. Effects of aging were mainly observed for fast fatigable MUs (a prolongation of MUAPs and increased number of turns). The MUAP amplitude did not change significantly with aging in either MU type, but it correlated to the twitch or tetanic forces, which tended to increase with age, especially for slow MUs. We concluded that the prolongation of MUAPs and the greater incidence of signal turns was probably a result of a decrease in muscle fiber conduction velocity and/or an increase in their dispersion, and enlargement of MU territories - presumably caused by axonal sprouting of surviving motoneurons. The latter might also be responsible for the observed age-related tendency for a increase in MUAP amplitudes in slow MUs. 

Effect of aging on properties of motor unit action potentials in the rat medial gastrocnemius muscle.

The aim of the study was to determine whether chronic muscle overload has measurable effect on electrophysiological properties of motoneurons (MNs), and whether duration of this overload influences intensity of adaptations. The compensatory overload was induced in the rat medial gastrocnemius (MG) by bilateral tenotomy of its synergists (lateral gastrocnemius, soleus, and plantaris); as a result, only the MG was able to evoke the foot plantar flexion. To assure regular activation of the MG muscle, rats were placed in wheel-equipped cages and subjected to a low-level treadmill exercise. The intracellular recordings from MG motoneurons were made after 5 or 12 wk of the overload, and in a control group of intact rats. Some of the passive and threshold membrane properties as well as rhythmic firing properties were considerably modified in fast-type MNs, while remaining unaltered in slow-type MNs. The significant changes included a shortening of the spike duration and the spike rise time, an increase of the afterhyperpolarization amplitude, an increase of the input resistance, a decrease of the rheobase, and a decrease of the minimum current necessary to evoke steady-state firing. The data suggest higher excitability of fast-type MNs innervating the overloaded muscle, and a shift towards electrophysiological properties of slow-type MNs. All of the adaptations could be observed after 5 wk of the compensatory overload with no further changes occurring after 12 wk. This indicates that the response to an increased level of chronic activation of MNs is relatively quick and stable. 

Adaptations of motoneuron properties to chronic compensatory muscle overload.

Resistance training, with repeated short-term and high-intensity exercises, is responsible for an increase in muscle mass and force. The aim of this study was to determine whether such training induces adaptations in the electrophysiological properties of motoneurons innervating the trained muscles and to relate these adaptive changes to previous observations made on motor unit contractile properties. The study was performed on adult male Wistar rats. Animals from the training group were subjected to a 5-wk voluntary progressive weight-lifting program, whereas control rats were restricted to standard cage activity. Intracellular recordings from lumbar spinal motoneurons were made under pentobarbital anesthesia. Membrane properties were measured, and rhythmic firing of motoneurons was analyzed. Strength training evoked adaptive changes in both slow- and fast-type motoneurons, indicating their increased excitability. A shorter spike duration, a higher input resistance, a lower rheobase, a decrease in the minimum current required to evoke rhythmic firing, an increase in the maximum frequencies of the early-state firing (ESF) and the steady-state firing (SSF), and an increase in the respective slopes of the frequency-current (/) relationship were observed in fast motoneurons of the trained group. The increase in the maximum ESF and SSF frequencies and an increase in the SSF / slope were also present in slow motoneurons. Higher maximum firing rates of motoneurons as well as higher discharge frequencies evoked at the same level of intracellular depolarization current imply higher levels of tetanic forces developed by motor units over the operating range of force production after strength training. Neuronal responses to weight-lifting training can be observed in altered properties of both slow and fast motoneurons. Motoneurons of trained animals are more excitable, require lower intracellular currents to evoke rhythmic firing, and have the ability to evoke higher maximum discharge frequencies during repetitive firing.

Adaptations of motoneuron properties after weight-lifting training in rats.

This study aimed at investigating the effects of 2, 4 and 8 weeks of endurance training on the contractile properties of slow (S), fast fatigue resistant (FR) and fast fatigable (FF) motor units (MUs) in rat medial gastrocnemius (MG) in relation to the changes in muscle mitochondrial biogenesis. The properties of functionally isolated MUs were examined in vivo. Mitochondrial biogenesis was judged based on the changes in mitochondrial DNA copy number (mtDNA), the content of the electron transport chain (ETC) proteins and PGC-1α in the MG. Moreover, the markers of mitochondria remodeling mitofusins (Mfn1, Mfn2) and dynamin-like protein (Opa1) were studied using qPCR. A proportion of FR MUs increased from 37.9% to 50.8% and a proportion of FF units decreased from 44.7% to 26.6% after 8 weeks of training. The increased fatigue resistance, shortened twitch duration, and increased ability to potentiate force were found as early as after 2 weeks of endurance training, predominantly in FR MUs. Moreover, just after 2 weeks of the training an enhancement of the mitochondrial network remodeling was present as judged by an increase in expression of Mfn1, Opa1 and an increase in PGC-1α in the slow part of MG. Interestingly, no signs of intensification of mitochondrial biogenesis assessed by ETC proteins content and mtDNA in slow and fast parts of gastrocnemius were found at this stage of the training. Nevertheless, after 8 weeks of training an increase in the ETC protein content was observed, but mainly in the slow part of gastrocnemius. Concluding, the functional changes in MUs' contractile properties leading to the enhancement of muscle performance accompanied by an activation of signalling that controls the muscle mitochondrial network reorganisation and mitochondrial biogenesis belong to an early muscle adaptive responses that precede an increase in mitochondrial ETC protein content.

Adaptation of motor unit contractile properties in rat medial gastrocnemius to treadmill endurance training: Relationship to muscle mitochondrial biogenesis.

Trans-spinal direct current stimulation (tsDCS) is a novel neuromodulatory technique that has been used during neurological rehabilitation and sports to modulate muscle activation. However, the physiological mechanisms that underly the long-lasting functional effects of polarization are not yet fully understood, nor are their relationships with specific neuronal populations. The acute facilitatory and depressive effects of anodal and cathodal polarization on motoneurons have been recently demonstrated, and the aim of this study was to determine whether tsDCS-evoked modulations of motoneuron properties are able to persist over several hours. Intracellular recordings from multiple antidromically identified rat motoneurons were performed both before and after the application of tsDCS (0.1 mA for 15 min), at various time points up to 180 min after the offset of anodal or cathodal tsDCS. The examined effects of anodal polarization included decreased rheobase, voltage threshold, the minimum and maximum currents necessary for rhythmic firing, increased rhythmic firing frequencies and the slope of the f-I relationship. The majority of these facilitatory changes to threshold and firing properties were sustained for 30-60 min after polarization. In contrast, the significant effects of cathodal polarization were absent, except the short-lasting decreased ability for motoneurons to induce rhythmic activity. This study provides direct evidence that a single polarization session can alter the electrophysiological properties of motoneurons for at least one hour and provides a basis for the further use of tsDCS techniques under conditions where the sustained modification of motoneuron firing is desired.

Piotr Krutki

Department of Neurobiology

Poznań University of Physical Education

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Piotr Krutki

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neurobiology

Poznań University of Physical Education

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Department of Neurobiology