Intramuscular Botulinum toxin A injections induce central changes to axon initial segments and cholinergic boutons on spinal motoneurones in rats.

Intramuscular injections of botulinum toxin block pre-synaptic cholinergic release at neuromuscular junctions producing a temporary paralysis of affected motor units. There is increasing evidence, however, that the effects are not restricted to the periphery and can alter the central excitability of the motoneurones at the spinal level. This includes increases in input resistance, decreases in rheobase currents for action potentials and prolongations of the post-spike after-hyperpolarization. The aim of our experiments was to investigate possible anatomical explanations for these changes. Unilateral injections of Botulinum toxin A mixed with a tracer were made into the gastrocnemius muscle of adult rats and contralateral tracer only injections provided controls. Immunohistochemistry for Ankyrin G and the vesicular acetylcholine transporter labelled axon initial segments and cholinergic C-boutons on traced motoneurones at 2 weeks post-injection. Soma size was not affected by the toxin; however, axon initial segments were 5.1% longer and 13.6% further from the soma which could explain reductions in rheobase. Finally, there was a reduction in surface area (18.6%) and volume (12.8%) but not frequency of C-boutons on treated motoneurones potentially explaining prolongations of the after-hyperpolarization. Botulinum Toxin A therefore affects central anatomical structures controlling or modulating motoneurone excitability explaining previously observed excitability changes.

Increased mast cell numbers in a calcaneal tendon overuse model.

Tendinopathy is often discovered late because the initial development of tendon pathology is asymptomatic. The aim of this study was to examine the potential role of mast cell involvement in early tendinopathy using a high-intensity uphill running (HIUR) exercise model. Twenty-four male Wistar rats were divided in two groups: running group (n = 12); sedentary control group (n = 12). The running-group was exposed to the HIUR exercise protocol for 7 weeks. The calcaneal tendons of both hind limbs were dissected. The right tendon was used for histologic analysis using Bonar score, immunohistochemistry, and second harmonic generation microscopy (SHGM). The left tendon was used for quantitative polymerase chain reaction (qPCR) analysis. An increased tendon cell density in the runners were observed compared to the controls (P = 0.05). Further, the intensity of immunostaining of protein kinase B, P = 0.03; 2.75 ± 0.54 vs 1.17 ± 0.53, was increased in the runners. The Bonar score (P = 0.05), and the number of mast cells (P = 0.02) were significantly higher in the runners compared to the controls. Furthermore, SHGM showed focal collagen disorganization in the runners, and reduced collagen density (P = 0.03). IL-3 mRNA levels were correlated with mast cell number in sedentary animals. The qPCR analysis showed no significant differences between the groups in the other analyzed targets. The current study demonstrates that 7-week HIUR causes structural changes in the calcaneal tendon, and further that these changes are associated with an increased mast cell density.

The time course of serotonin 2C receptor expression after spinal transection of rats: an immunohistochemical study.

In the spinal cord serotonin (5-HT) systems modulate the spinal network via various 5-HT receptors. Serotonin 2A receptor and serotonin 2C receptor (5-HT2A and 2C receptors) are likely the most important 5-HT receptors for enhancing the motoneuron excitability by facilitating the persistent inward current (PIC), and thus play an important role for the pathogenesis of spasticity after spinal cord injury. In conjunction with our 5-HT2A receptor study, using a same sacral spinal transection rat model we have in this study examined 5-HT2C receptor immunoreactivity (5-HT2CR-IR) changes at seven different time intervals after spinal injury. We found that 5-HT2CR-IR was widely distributed in different regions of the spinal gray matter and was predominantly located in the neuronal somata and their dendrites although it seemed also present in axonal fibers in the superficial dorsal horn. 5-HT2CR-IR in different regions of the spinal gray matter was seen to be increased at 14days after transection (with an average ∼1.3-fold higher than in sham-operated group) but did not reach a significant level until at 21days (∼1.4-fold). The increase sustained thereafter and a plateau level was reached at 45days (∼1.7-fold higher), a value similar as that at 60days. When 5-HT2CR-IR analysis was confined to the ventral horn motoneuron somata (including a proportion of proximal dendrites) a significant increase was not detected until 45days post-operation. 5-HT2CR upregulation in the spinal gray matter is confirmed with Western blot in the rats 60days post-operation. The time course of 5-HT2CR upregulation in the spinal gray matter and motoneurons was positively correlated with the development of tail spasticity (clinical scores). This indicates that 5-HT2CR is probably an important factor underlying this pathophysiological development by increasing the excitability of both motoneurons and interneurons.

Voltage-dependent amplification of synaptic inputs in respiratory motoneurones.

The role of persistent inward currents (PICs) in cat respiratory motoneurones (phrenic inspiratory and thoracic expiratory) was investigated by studying the voltage-dependent amplification of central respiratory drive potentials (CRDPs), recorded intracellularly, with action potentials blocked with the local anaesthetic derivative, QX-314. Decerebrate unanaesthetized or barbiturate-anaesthetized preparations were used. In expiratory motoneurones, plateau potentials were observed in the decerebrates, but not under anaesthesia. For phrenic motoneurones, no plateau potentials were observed in either state (except in one motoneurone after the abolition of the respiratory drive by means of a medullary lesion), but all motoneurones showed voltage-dependent amplification of the CRDPs, over a wide range of membrane potentials, too wide to result mainly from PIC activation. The measurements of the amplification were restricted to the phase of excitation, thus excluding the inhibitory phase. Amplification was found to be greatest for the smallest CRDPs in the lowest resistance motoneurones and was reduced or abolished following intracellular injection of the NMDA channel blocker, MK-801. Plateau potentials were readily evoked in non-phrenic cervical motoneurones in the same (decerebrate) preparations. We conclude that the voltage-dependent amplification of synaptic excitation in phrenic motoneurones is mainly the result of NMDA channel modulation rather than the activation of Ca2+ channel mediated PICs, despite phrenic motoneurones being strongly immunohistochemically labelled for CaV1.3 channels. The differential PIC activation in different motoneurones, all of which are CaV1.3 positive, leads us to postulate that the descending modulation of PICs is more selective than has hitherto been believed.

Pro-inflammatory effector Th cells transmigrate through anti-inflammatory environments into the murine fetus.

The presence of maternal DNA or even maternal cells within the offspring (microchimerism) has been reported for many fetal tissues, including the liver, heart, and spleen. Microchimerism is believed to be involved in the pathogenesis of autoimmune diseases; however, the cellular origin of this phenomenon remains unknown. Here, we determined whether differentiated T lymphocytes could transmigrate through the immunosuppressive environment of the placenta to reach the fetus. In vitro-differentiated effector/memory Th1 and Th17 cells from OVA323₋339-specific TCR(tg) T cells of OT-II mice were adoptively transferred (i.v.) into the tail veins of pregnant Ly5.1 mice at d15 and d19 of gestation. Mice were then sacrificed 40 h after adoptive cell transfer. Using radioactive labeling of T cells with sodium chromate [Cr5¹] prior to adoptive transfer, we observed that homing of pro-inflammatory Th cells was equally efficient in both pregnant and non-pregnant mice. Transmigration of Th1- and Th17-like cells through the highly immunosuppressive environment of the placenta into the fetus was significantly enhanced in experimental mice compared to control mice (P < 0.0001). In addition, a substantial amount of effector Th cells accumulated in the placenta. Finally, we found that treatment with Pertussis Toxin resulted in a 3-fold increase in the transmigration of effector Th17 cells into the fetus (P < 0.0001). When pro-inflammatory Th1-or Th17-like cells were injected into syngeneic mothers, almost all of the fetuses analyzed exhibited radioactivity, suggesting that transmigration of effector T cells occurs frequently. Our results suggest the possibility of novel roles for these maternal effector cells in the pathogenesis or reduction of disease.

The time course of serotonin 2A receptor expression after spinal transection of rats: an immunohistochemical study.

Hyperexcitability of motoneurons is one of the key mechanism that has been demonstrated to underlie the pathogenesis of spasticity after spinal injury. Serotonin (5-HT) denervation supersensitivity is one of the mechanisms underlying this increased motoneuron excitability. In this study, to examine whether the supersensitivity is caused by 5-HT receptor upregulation we investigated changes in levels of 5-HT2A receptor immunoreactivity (5-HT2AR-IR) following a spinal transection in the sacral spinal cord of rats at seven different time points post injury: 2, 8, 16 h, and 1, 2, 7 and 28 days, respectively. 5-HT2AR-IR density was analyzed in motoneurons (regions containing their somata and dendrites) in the spinal segments below the lesion. The results showed no significant changes in 5-HT2AR-IR in the motoneurons up to 16 h following the transection. After 1-day, however the levels of 5-HT2AR-IR increased in the motoneurons and their dendrites, with the density level being 3.4-fold higher in spinalized rats than in sham-operated rats. The upregulation increased progressively until a maximal level was reached at 28 days post-injury. We also investigated 5-HT and 5-HT transporter expressions at five different post injury time points: 1, 2, 7, 21 and 60 days and they showed concurrent down-regulation changes after 2 days. These results suggest that the upregulation of 5-HT2ARs may at least partly underlie the development of 5-HT denervation supersensitivity in spinal motoneurons following spinal injury and thereby implicates their involvement in the pathogenesis of the subsequent development of spasticity.

Global gene expression analysis of rodent motor neurons following spinal cord injury associates molecular mechanisms with development of postinjury spasticity.

Spinal cord injury leads to severe problems involving impaired motor, sensory, and autonomic functions. After spinal injury there is an initial phase of hyporeflexia followed by hyperreflexia, often referred to as spasticity. Previous studies have suggested a relationship between the reappearance of endogenous plateau potentials in motor neurons and the development of spasticity after spinalization. To unravel the molecular mechanisms underlying the increased excitability of motor neurons and the return of plateau potentials below a spinal cord injury we investigated changes in gene expression in this cell population. We adopted a rat tail-spasticity model with a caudal spinal transection that causes a progressive development of spasticity from its onset after 2 to 3 wk until 2 mo postinjury. Gene expression changes of fluorescently identified tail motor neurons were studied 21 and 60 days postinjury. The motor neurons undergo substantial transcriptional regulation in response to injury. The patterns of differential expression show similarities at both time points, although there are 20% more differentially expressed genes 60 days compared with 21 days postinjury. The study identifies targets of regulation relating to both ion channels and receptors implicated in the endogenous expression of plateaux. The regulation of excitatory and inhibitory signal transduction indicates a shift in the balance toward increased excitability, where the glutamatergic N-methyl-d-aspartate receptor complex together with cholinergic system is up-regulated and the gamma-aminobutyric acid type A receptor system is down-regulated. The genes of the pore-forming proteins Cav1.3 and Nav1.6 were not up-regulated, whereas genes of proteins such as nonpore-forming subunits and intracellular pathways known to modulate receptor and channel trafficking, kinetics, and conductivity showed marked regulation. On the basis of the identified changes in global gene expression in motor neurons, the present investigation opens up for new potential targets for treatment of motor dysfunction following spinal cord injury.

A prolongation of the postspike afterhyperpolarization following spike trains can partly explain the lower firing rates at derecruitment than those at recruitment.

The original motivation for this study was the observation in previous human experiments that the motor neuron firing frequency (recorded from the motor units in the EMG) was lower at derecruitment than that at recruitment, with slow linearly varying voluntary contractions. Are the lower firing rates at derecruitment correlated with a change in the postspike afterhyperpolarization (AHP) after preceding spike trains? This question was investigated by intracellular recordings from cat motor neurons in both unanesthetized and anesthetized preparations. The firing frequencies at recruitment and derecruitment were compared during slow triangular current injections mimicking the slow linearly varying voluntary contractions in humans. There was a lower frequency at derecruitment in almost all motor neurons (83 of 86 motor neurons; mean = 3.35 Hz). Thus intrinsic mechanisms play an important role for the lower frequencies at derecruitment. This was independent of whether the current injection had activated persistent inward current (PIC; plateau potentials, secondary range firing). It was found that a preceding spike train could prolong the AHP duration following a subsequent spike. The lower rate at derecruitment matches the prolongation of the AHP. However, a quantitative comparison between the lowest firing frequency and AHP duration for individual motor neurons reveal that the predicted lowest firing frequency does not match the absolute AHP duration; the lowest frequency is lower than that predicted from AHP duration in fast motoneurons and higher than expected in slow motoneurons. It is suggested that these deviations are explained by the presence of synaptic noise as well as recruitment of PICs below firing threshold. Thus synaptic noise may allow spike discharge even after the end of the AHP in "fast" motor neurons, whereas synaptic noise and PICs below spike threshold tend to give higher minimum firing frequencies in "slow" motor neurons than predicted from AHP duration.

Variable amplification of synaptic input to cat spinal motoneurones by dendritic persistent inward current.

Electrophysiological and computational evidence indicate that the excitatory current from the synapses on the somato-dendritic membrane is not large enough to drive the motoneurones to the firing frequencies actually attained under normal motor activity. It has been proposed that this paradox could be explained if the voltage-dependent persistent inward currents (PICs) present in the dendrites of motoneurones served to amplify synaptic excitation. We report here that dendritic PICs cause a large amplification of synaptic excitation, and that this amplification is enhanced when the background firing by current injection is increased. Moreover the frequency reduction by synaptic inhibition is greatly enhanced at higher firing frequencies, when the current through the recording electrode has activated the dendritic PICs, as is the case when the current-to-frequency slope suddenly becomes steeper. We also demonstrate that synaptic inhibition is several times more effective in reducing the firing caused by synaptic excitation than firing evoked by current injection through the recording microelectrode. That would be explained if motoneuronal discharge by synaptic excitation--but not by current injection in the soma--is always supported by dendritic PICs. We conclude that dendritic PICs contribute dynamically to the transformation of synaptic input into a motoneuronal frequency code.