Kalan Gardiner

Frequency–current relationships of rat hindlimb α‐motoneurones

The purpose of this study was to describe the frequency–current (f–I) relationships of hindlimb α‐motoneurones (MNs) in both anaesthetized and decerebrate rats in situ. Sprague–Dawley rats (250–350 g) were anaesthetized with ketamine and xylazine (KX) or subjected to a precollicular decerebration prior to recording electrophysiological properties from sciatic nerve MNs. Motoneurones from KX‐anaesthetized rats had a significantly (P < 0.01) hyperpolarized resting membrane potential and voltage threshold (Vth), increased rheobase current, and a trend (P= 0.06) for a smaller after‐hyperpolarization (AHP) amplitude compared to MNs from decerebrate rats. In response to 5 s ramp current injections, MNs could be categorized into four f–I relationship types: (1) linear; (2) adapting; (3) linear + sustained; and (4) late acceleration. Types 3 and 4 demonstrated self‐sustained firing owing to activation of persistent inward current (PIC). We estimated the PIC amplitude by subtracting the current at spike derecruitment from the current at spike recruitment. Neither estimated PIC nor f–I slopes differed between fast and slow MNs (slow MNs exhibited AHP half‐decay times > 20 ms) or between MNs from KX‐anaesthetized and decerebrate rats. Motoneurones from KX‐anaesthetized rats had significantly (P < 0.02) hyperpolarized ramp Vth values and smaller and shorter AHP amplitudes and decay times compared to MNs from decerebrate rats. Pentobarbitone decreased the estimated PIC amplitude and almost converted the f–I relationship from type 3 to type 1. In summary, MNs of animals subjected to KX anaesthesia required more current for spike initiation and rhythmic discharge but retained large PICs and self‐sustained firing. The KX‐anaesthestized preparation enables direct recording of PICs in MNs from intact animals.

Does elimination of afferent input modify the changes in rat motoneurone properties that occur following chronic spinal cord transection

The purpose of this study was to determine the effects of 6–8 weeks of chronic spinal cord isolation (SI, removal of descending, ascending and afferent inputs), compared with the same duration of spinal cord transection (ST, removal of descending input only) on hindlimb motoneurone biophysical properties. Adult female Sprague–Dawley rats were placed into three groups: (1) control (no removal of inputs), (2) ST and (3) SI. The electrophysiological properties from sciatic nerve motoneurones were recorded from deeply anaesthetized rats. Motoneurones in SI rats had significantly (P < 0.01) lower rheobase currents and higher spike afterhyperpolarization amplitudes and input resistances compared with motoneurones in control rats. A higher percentage (χ2, P= 0.01) of motoneurones in SI than control rats demonstrated frequency‐current (f–I) relationships consistent with activation of persistent inward currents. Motoneurone steady state f–I slopes determined by increasing steps of 500 ms current pulses were significantly lower (P < 0.02) in SI than control rats. Motoneurone spike frequency adaptation measured using 30 s square‐wave current injections (1.5–3.0 nA above the estimated rhythmic firing threshold), was similar for control and SI motoneurones. Changes in motoneurone properties following SI did not differ from ST. These findings indicate that the removal of afferent and ascending inputs along with descending inputs has little additional affect on motoneurone properties than removal of descending inputs alone. This study is the first to demonstrate that intact ascending and afferent input does not modify the effects of spinal transection on basic and rhythmic firing properties of rat hindlimb motoneurones.

Extensor motoneurone properties are altered immediately before and during fictive locomotion in the adult decerebrate rat

This is the first report, in adult decerebrate rats, to examine intracellular hindlimb motoneurone properties during quiescence, fictive locomotion and a tonic period immediately before fictive locomotion that is characterized by increased peripheral nerve activity. It is shown for the first time during fictive locomotion that motoneurones become more responsive in the tonic period, suggesting that the motoneurone pool becomes primed before patterned motor output commences. Spike frequency adaptation exists in quiescence and during fictive locomotion during constant excitation with injected current but not during centrally driven fictive locomotion. Motoneurones within the extensor motor pool show changes in excitability even when they are not directly involved in locomotion. The data show increased responsiveness of motoneurones during locomotion via a lowered threshold for spike initiation and decreased rheobase.

Caloric restriction does not offset age-associated changes in the biophysical properties of motoneurons.

Age-associated changes in neuromuscular function may be due to a loss of motor neurons as well as changes in their biophysical properties. Neuronal damage imposed by reactive oxygen species may contribute to age-related deficits in CNS function. Thus we hypothesized that aging would alter the functional properties of motoneurons and that caloric-restriction would offset these changes. Intracellular recordings were made from lumbar motoneurons of old Fisher Brown Norway (FBN) fed ad libitum (oldAL, 30.8+/-1.3 mo) or on a fortified calorie-restricted diet from 14 wk of age (oldCR, 31.0+/-1.8 mo). Basic and rhythmic firing properties recorded from these aged motoneurons (MNs) were compared with properties recorded from young FBN controls (young, 8.4+/-4.6 mo). Compared with young MNs, old MNs had a 104% greater (P<0.001) afterhyperpolarization potential (AHP), a 21.1% longer AHP half-decay time (P<0.05), 28.7% lower rheobase (P<0.001), 49.7% greater (P<0.001) input resistance, 21.1% (P<0.0001) less spike frequency adaptation, lower minimal (30.2%, P<0.0001) and maximal (16.7%, P<0.0001) steady-state firing frequencies, a lower (35.5%, P<0.0001) frequency-current slope, and an increased incidence of persistent inward current. Because basic properties became more diverse in old MNs and the slope of the frequency-current relationship, which is normally similar for high- and low-threshold MNs, was lower in the old group, we conclude that aging alters the biophysical properties of MNs in a fashion that cannot be simply attributed to a loss of high-threshold MNs. Surprisingly, caloric restriction, which is known to attenuate aging-associated changes in hindlimb muscles, had no effect on the progress of aging in the innervating MNs.

Removal of supraspinal input reveals a difference in the flexor and extensor monosynaptic reflex response to quipazine independent of motoneuron excitation

The purpose of this study was to determine if quipazine, a serotonergic agonist, differentially modulates flexor and extensor motor output. This was achieved by examining the monosynaptic reflex (MSR) of the tibial (extensor) and peroneal (flexor) nerves, by determining the basic and rhythmic properties of extensor and flexor motoneurons, and by recording extracellular Ia field potentials of the tibial and peroneal nerves in the in vivo adult decerebrate rat in both spinal intact and acute spinalized preparations. In the spinal intact preparation, the tibial and peroneal MSR amplitude significantly increased compared with baseline in response to quipazine, with no difference between nerves (P < 0.05). In the spinalized preparation, the MSR was significantly increased in both the tibial and peroneal nerves with the latter increasing more than the former (5.7 vs. 3.6 times; P < 0.05). Intracellular motoneuron experiments demonstrated that rheobase decreased, while input resistance, afterhyperpolarization amplitude, and the firing rate at a given current injection increased in motoneurons following quipazine administration with no differences between extensor and flexor motoneurons. Both the tibial and peroneal nerve extracellular Ia field potentials increased with the peroneal demonstrating a significantly greater increase (7 vs. 38%; P < 0.05) following quipazine. It is concluded that in the spinal intact preparation quipazine does not have a differential effect on flexor or extensor motor output. However, in the acute spinalized preparation, quipazine preferentially affects the flexor MSR compared with the extensor MSR, likely due to the removal of a descending tonic inhibition on flexor Ia afferents.

Daily Passive Cycling Attenuates the Hyperexcitability and Restores the Responsiveness of the Extensor Monosynaptic Reflex to Quipazine in the Chronic Spinally Transected Rat

Activity-based interventions such as locomotor training or passive cycling have a positive influence on the spinal circuitry and recovery following a spinal cord injury (SCI). The use of quipazine in combination with exercise training has demonstrated a greater functional recovery than has exercise training alone. However, the influence of exercise or training on the responsiveness of the spinal cord to quipazine has not been examined following a chronic spinal transection. The purpose of this study was to characterize the flexor and extensor monosynaptic reflex (MSR) response pre- and post-quipazine in chronic complete spinally transected rats that either underwent daily passive cycling for 3 months or did not receive passive cycling. Following a chronic spinal transection, the extensor MSR demonstrated a hyperreflexive response (fivefold increase) to afferent stimuli, and did not respond to quipazine injection. With daily passive cycling, the extensor MSR hyperexcitability was attenuated, and the MSR amplitude increased 72% following quipazine injection (p<0.004), which was comparable to the extensor MSR response (94%) in the control group. For both chronic spinal transection groups, the flexor MSR amplitudes were not altered following quipazine injection, whereas in the control group the flexor MSR amplitude increased 86% in response to quipazine (p<0.004). These results demonstrate that passive cycling attenuates the hyperreflexive response of the extensor MSR following a chronic SCI, and restores the MSR response to quipazine.

Serotonin receptor and KCC2 gene expression in lumbar flexor and extensor motoneurons posttransection with and without passive cycling

Sacrocaudal motoneuron gene expression is altered following a spinal transection. Of interest here is the regulation of serotonin (5-HT) receptors (R), glutamate receptor, metabotropic 1 (mGluR1), and potassium-chloride cotransporter (KCC2), which mediate motoneuron excitability, locomotor recovery, and spasticity posttransection. The examination of these genes in lumbar motoneurons posttransection has not been studied, which is necessary for developing potential pharmacological interventions aimed at restoring locomotion and/or reducing spasticity. Also, if activity is to be used to promote recovery or reduce spasticity postinjury, a further examination of neuromuscular activity on gene expression posttransection is warranted. The purpose of this study was to examine motoneuronal gene expression of 5-HT receptors, KCC2, and mGluR1 at 3 mo following a complete thoracic spinal cord transection, with and without the inclusion of daily passive cycling. Physiological hindlimb extensor and flexor motoneurons were differentially identified with two retrograde fluorescent tracers, allowing for the identification and separate harvesting of extensor and flexor motoneurons with laser capture microdissection and the subsequent examination of mRNA content using quantitative RT-PCR analysis. We demonstrate that posttransection 5-HT1AR, 5-HT2CR, and mGluR1 expression was downregulated, whereas the 5-HT2AR was upregulated. These alterations in gene expression were observed in both flexor and extensor motoneurons, whereas passive cycling influenced gene expression in extensor but not flexor motoneurons. Passive cycling in extensor motoneurons further enhanced 5-HT2AR expression and increased 5-HT7R and KCC2 expression. Our results demonstrate that passive cycling influences serotonin receptor and KCC2 gene expression and that extensor motoneurons compared with flexor motoneurons may be more plastic to activity-based interventions posttransection.

Altered transcription of glutamatergic and glycinergic receptors in spinal cord dorsal horn following spinal cord transection is minimally affected by passive exercise of the hindlimbs

Gene expression is altered following a spinal transection (STx) in both motor and sensory systems. Exercise has been shown to influence gene expression in both systems post‐STx. Gene expression alterations have also been shown in the dorsal root ganglia and nociceptive laminae of the spinal cord following either an incomplete spinal cord injury (SCI) or a contusive SCI. However, the effect of STx and exercise on gene expression in spinal cord laminae I‐III has not fully been examined. Therefore, the purpose of this study was to determine whether gene expression in laminae I‐III is altered following STx and determine whether superimposed passive exercise of the hindlimbs would influence gene expression post‐STx in laminae I‐III. Laser capture microdissection was used to selectively harvest laminae I‐III of lumbar spinal cord sections, and quantitative RT‐PCR was used to examine relative expression of 23 selected genes in samples collected from control, STx and STx plus exercise rats. We demonstrate that post‐STx, gene expression for metabotropic glutamate receptors 1, 5 and 8 were up‐regulated, whereas ionotropic glutamatergic receptor (Glur2) and glycinergic subunit GLRA1 expression was down‐regulated. Daily exercise attenuated the down‐regulation of Glur2 gene expression in laminae I‐III. Our results demonstrate that in a STx model, gene expression is altered in laminae I‐III and that although passive exercise influences gene expression in both the motor and sensory systems, it had a minimal effect on gene expression in laminae I‐III post‐STx.

α-Motoneurons maintain biophysical heterogeneity in obesity and diabetes in Zucker rats

Small-diameter sensory dysfunction resulting from diabetes has received much attention in the literature, whereas the impact of diabetes on α-motoneurons (MN) has not. In addition, the chance of developing insulin resistance and diabetes is increased in obesity. No study has examined the impact of obesity or diabetes on the biophysical properties of MN. Lean Zucker rats and Zucker diabetic fatty (ZDF) rats were separated into lean, obese (ZDF fed standard chow), and diabetic (ZDF fed high-fat diet that led to diabetes) groups. Glass micropipettes recorded hindlimb MN properties from identified flexor and extensor MN. MN were separated within their groups on the basis of input conductance, which created high- and low-input conductance subpopulations for each. A significant shorter (20%) afterhyperpolarization half-decay (AHP1/2) was found in low-conductance MN for the diabetic group only, whereas AHP½ tended to be shorter in the obese group (19%). Significant positive correlations were found among rheobase and input conductance for both lean and obese animals. No differences were found between the groups for afterhyperpolarization amplitude (AHPamp), input conductance, rheobase, or any of the rhythmic firing properties (frequency-current slope and spike-frequency adaptation index). MN properties continue to be heterogeneous in obese and diabetic animals. Obesity does not seem to influence lumbar MN. Despite the resistance of MN to the impact of diabetes, the reduced AHP1/2 decay and the tendency for a reduction in AHPamp may be the first sign of change to MN function.NEW & NOTEWORTHY Knowledge about the impact of obesity and diabetes on the biophysical properties of motoneurons is lacking. We found that diabetes reduces the duration of the afterhyperpolarization and that motoneuron function is unchanged by obesity. A reduced afterhyperpolarization may impact discharge characteristics and may be the first sign of change to motoneuron function.