Ca²âº/cAMP-sensitive covariation of I(A) and I(H) voltage dependences tunes rebound firing in dopaminergic neurons.

The level of expression of ion channels has been demonstrated to vary over a threefold to fourfold range from neuron to neuron, although the expression of distinct channels may be strongly correlated in the same neurons. We demonstrate that variability and covariation also apply to the biophysical properties of ion channels. We show that, in rat substantia nigra pars compacta dopaminergic neurons, the voltage dependences of the A-type (I(A)) and H-type (I(H)) currents exhibit a high degree of cell-to-cell variability, although they are strongly correlated in these cells. Our data also demonstrate that this cell-to-cell covariability of voltage dependences is sensitive to cytosolic cAMP and calcium levels. Finally, using dynamic clamp, we demonstrate that covarying I(A) and I(H) voltage dependences increases the dynamic range of rebound firing while covarying their amplitudes has a homeostatic effect on rebound firing. We propose that the covariation of voltage dependences of ion channels represents a flexible and energy-efficient way of tuning firing in neurons.

Evidence from computer simulations for alterations in the membrane biophysical properties and dendritic processing of synaptic inputs in mutant superoxide dismutase-1 motoneurons.

A critical step in improving our understanding of the development of amyotrophic lateral sclerosis (ALS) is to identify the factors contributing to the alterations in the excitability of motoneurons and assess their individual contributions. Here we investigated the early alterations in the passive electrical and morphological properties of neonatal spinal motoneurons that occur by 10 d after birth, long before disease onset. We identified some of the factors contributing to these alterations, and estimated their individual contributions. To achieve this goal, we undertook a computer simulation analysis using realistic morphologies of reconstructed wild-type (WT) and mutant superoxide dismutase-1 (mSOD1) motoneurons. Ion channel parameters of these models were then tuned to match the experimental data on electrical properties obtained from these same motoneurons. We found that the reduced excitability of mSOD1 models was accompanied with decreased specific membrane resistance by approximately 25% and efficacy of synaptic inputs (slow and fast) by 12-22%. Linearity of summation of synaptic currents was similar to WT. We also assessed the contribution of the alteration in dendritic morphology alone to this decreased excitability and found that it reduced the input resistance by 10% and the efficacy of synaptic inputs by 7-15%. Our results were also confirmed in models with dendritic active conductances. Our simulations indicated that the alteration in passive electrical properties of mSOD1 models resulted from concurrent alterations in their morphology and membrane biophysical properties, and consequently altered the motoneuronal dendritic processing of synaptic inputs. These results clarify new aspects of spinal motoneurons malfunction in ALS.

Early abnormalities in transgenic mouse models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative and fatal human disorder characterized by progressive loss of motor neurons. Transgenic mouse models of ALS are very useful to study the initial mechanisms underlying this neurodegenerative disease. We will focus here on the earlier abnormalities observed in superoxide dismutase 1 (SOD1) mutant mice. Several hypotheses have been advanced to explain the selective loss of motor neurons such as apoptosis, neurofilament disorganisation, oxidative stress, mitochondrial dysfunction, astrogliosis and excitotoxicity. Although disease onset appears at adulthood, recent studies have detected abnormalities during embryonic and postnatal maturation in animal models of ALS. We reported that SOD1(G85R) mutant mice exhibit specific delays in acquiring sensory-motor skills during the first week after birth. In addition, physiological measurements on in vitro spinal cord preparations reveal defects in evoking rhythmic activity with N-methyl-DL-aspartate and serotonin at lumbar, but not sacral roots. This is potentially significant, as functions involving sacral roots are spared at late stages of the disease. Moreover, electrical properties of SOD1 lumbar motoneurons are altered as early as the second postnatal week when mice begin to walk. Alterations concern the input resistance and the gain of SOD1 motoneurons which are lower than in control motoneurons. Whether or not the early changes in discharge firing are responsible for the uncoupling between motor axon terminals and muscles is still an open question. A link between these early electrical abnormalities and the late degeneration of motoneurons is proposed in this short review. Our data suggest that ALS, as other neurodegenerative diseases, could be a consequence of an abnormal development of neurons and network properties. We hypothesize that the SOD1 mutation could induce early changes during the period of maturation of motor systems and that compensatory mechanisms-linked to developmental spinal plasticity-might explain the late onset of the disease.

Optimization of neuronal cultures from rat superior cervical ganglia for dual patch recording.

Superior cervical ganglion neurons (SCGN) are often used to investigate neurotransmitter release mechanisms. In this study, we optimized the dissociation and culture conditions of rat SCGN cultures for dual patch clamp recordings. Two weeks in vitro are sufficient to achieve a significant CNTF-induced cholinergic switch and to develop mature and healthy neuronal profiles suited for detailed patch clamp analysis. One single pup provides sufficient material to prepare what was formerly obtained from 12 to 15 animals. The suitability of these cultures to study neurotransmitter release mechanisms was validated by presynaptically perturbing the interaction of the v-SNARE VAMP2 with the vesicular V-ATPase V0c subunit.

Developing electrical properties of postnatal mouse lumbar motoneurons.

We studied the rapid changes in electrical properties of lumbar motoneurons between postnatal days 3 and 9 just before mice weight-bear and walk. The input conductance and rheobase significantly increased up to P8. A negative correlation exists between the input resistance (Rin) and rheobase. Both parameters are significantly correlated with the total dendritic surface area of motoneurons, the largest motoneurons having the lowest Rin and the highest rheobase. We classified the motoneurons into three groups according to their discharge firing patterns during current pulse injection (transient, delayed onset, sustained). The delayed onset firing type has the highest rheobase and the fastest action potential (AP) whereas the transient firing group has the lowest rheobase and the less mature AP. We found 32 and 10% of motoneurons with a transient firing at P3-P5 and P8, respectively. About 20% of motoneurons with delayed onset firing were detected at P8. At P9, all motoneurons exhibit a sustained firing. We defined five groups of motoneurons according to their discharge firing patterns in response to ascending and descending current ramps. In addition to the four classical types, we defined a fifth type called transient for the quasi-absence of discharge during the descending phase of the ramp. This transient type represents about 40% between P3-P5 and tends to disappear with age. Types 1 and 2 (linear and clockwise hysteresis) are the most preponderant at P6-P7. Types 3 and 4 (prolonged sustained and counter clockwise hysteresis) emerge at P8-P9. The emergence of types 3 and 4 probably depends on the maturation of L type calcium channels in the dendrites of motoneurons. No correlation was found between groups defined by step or triangular ramp of currents with the exception of transient firing patterns. Our data support the idea that a switch in the electrical properties of lumbar motoneurons might exist in the second postnatal week of life in mice.

Non-linear developmental trajectory of electrical phenotype in rat substantia nigra pars compacta dopaminergic neurons.

Neurons have complex electrophysiological properties, however, it is often difficult to determine which properties are the most relevant to neuronal function. By combining current-clamp measurements of electrophysiological properties with multi-variate analysis (hierarchical clustering, principal component analysis), we were able to characterize the postnatal development of substantia nigra dopaminergic neurons' electrical phenotype in an unbiased manner, such that subtle changes in phenotype could be analyzed. We show that the intrinsic electrical phenotype of these neurons follows a non-linear trajectory reaching maturity by postnatal day 14, with two developmental transitions occurring between postnatal days 3-5 and 9-11. This approach also predicted which parameters play a critical role in phenotypic variation, enabling us to determine (using pharmacology, dynamic-clamp) that changes in the leak, sodium and calcium-activated potassium currents are central to these two developmental transitions. This analysis enables an unbiased definition of neuronal type/phenotype that is applicable to a range of research questions.

The use of poly(N-[2-hydroxypropyl]-methacrylamide) hydrogel to repair a T10 spinal cord hemisection in rat: a behavioural, electrophysiological and anatomical examination.

There have been considerable interests in attempting to reverse the deficit because of an SCI (spinal cord injury) by restoring neural pathways through the lesion and by rebuilding the tissue network. In order to provide an appropriate micro-environment for regrowing axotomized neurons and proliferating and migrating cells, we have implanted a small block of pHPMA [poly N-(2-hydroxypropyl)-methacrylamide] hydrogel into the hemisected T10 rat spinal cord. Locomotor activity was evaluated once a week during 14 weeks with the BBB rating scale in an open field. At the 14th week after SCI, the reflexivity of the sub-lesional region was measured. We also monitored the ventilatory frequency during an electrically induced muscle fatigue known to elicit the muscle metaboreflex and increase the respiratory rate. Spinal cords were then collected, fixed and stained with anti-ED-1 and anti-NF-H antibodies and FluoroMyelin. We show in this study that hydrogel-implanted animals exhibit: (i) an improved locomotor BBB score, (ii) an improved breathing adjustment to electrically evoked isometric contractions and (iii) an H-reflex recovery close to control animals. Qualitative histological results put in evidence higher accumulation of ED-1 positive cells (macrophages/monocytes) at the lesion border, a large number of NF-H positive axons penetrating the applied matrix, and myelin preservation both rostrally and caudally to the lesion. Our data confirm that pHPMA hydrogel is a potent biomaterial that can be used for improving neuromuscular adaptive mechanisms and H-reflex responses after SCI.

Morphological differences between wild-type and transgenic superoxide dismutase 1 lumbar motoneurons in postnatal mice.

Quantitative analysis of the dendritic arborizations of wild-type (WT) and superoxide dismutase 1 (SOD1) postnatal mouse motoneurons was performed following intracellular staining and 3D reconstructions with Neurolucida system. The population of lumbar motoneurons was targeted in the caudal part of the L5 segment, and all labeled motoneurons were located within the same ventrolateral pool. Despite the similar size of the soma and the mean diameter of primary dendrites, the dendritic arborizations of the WT and SOD1 motoneurons showed significant differences in terms of their morphometric parameters. The metric and topological parameters of dendrites show that the total dendritic length and surface area and total number of segments, branching nodes, and tips per motoneuron were significantly higher in SOD1 motoneurons. Our main finding concerns a proliferation of dendritic branches starting at about 100 microm from the soma in the SOD1 motoneurons. However, the longest and mean dendritic paths from soma to terminations were similar, giving a comparable envelope of the dendritic fields. Indeed, the SOD1 motoneurons were larger as a result of abnormal branching. The results suggest that a defect in pruning mechanisms occurs during this developmental period. The abnormal growth of the dendritic arborizations and the reduced excitability of postnatal SOD1 motoneurons could be a neuroprotective response and would represent an early compensatory mechanism against the activity-induced toxicity.

Early electrophysiological abnormalities in lumbar motoneurons in a transgenic mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a lethal, adult-onset disease characterized by progressive degeneration of motoneurons. Recent data have suggested that the disease could be linked to abnormal development of the motor nervous system. Therefore, we investigated the electrical properties of lumbar motoneurons in an in-vitro neonatal spinal cord preparation isolated from SOD1(G85R) mice, which is a transgenic model of amyotrophic lateral sclerosis. The study was performed on young animals at the beginning of their second week, between postnatal days 6 and 10. Measurements of resting membrane potential and action potential characteristics of motoneurons were similar in wild-type and SOD1(G85R) mice. However, the input resistance of motoneurons from transgenic mice was significantly lower than that of wild-type animals, whereas their membrane capacitance was increased, strongly suggesting larger SOD1(G85R) motoneurons. Furthermore, the slope of the frequency-intensity curve was steeper in motoneurons from wild-type pups. Interestingly, the input resistance as well as the slope of the frequency-intensity curves of other spinal neurons did not show such differences. Finally, the amplitude of dorsal root-evoked potentials following high-intensity stimulation was significantly smaller in SOD1(G85R) motoneurons. The superoxide dismutase 1 mutation thus induces specific alterations of the functional properties of motoneurons early in development.

Altered sensorimotor development in a transgenic mouse model of amyotrophic lateral sclerosis.

Most neurodegenerative diseases become manifest at an adult age but abnormalities or pathological symptoms appear earlier. It is important to identify the initial mechanisms underlying such progressive neurodegenerative disease in both humans and animals. Transgenic mice expressing the familial amyotrophic lateral sclerosis (ALS)-linked mutation (G85R) in the enzyme superoxide dismutase 1 (SOD1) develop motor neuron disease at 8-10 months of age. We address the question of whether the mutation has an early impact on spinal motor networks in postnatal mutant mice. Behavioural tests showed a significant delay in righting and hind-paw grasping responses in mutant SOD1G85R mice during the first postnatal week, suggesting a transient motor deficit compared to wild-type mice. In addition, extracellular recordings from spinal ventral roots in an in vitro brainstem-spinal cord preparation demonstrated different pharmacologically induced motor activities between the two strains. Rhythmic motor activity was difficult to evoke with N-methyl-DL-aspartate and serotonin at the lumbar levels in SOD1G85R mice. In contrast to lumbar segments, rhythmic activity was similar in the sacral roots from the two strains. These results strongly support the fact that the G85R mutation may have altered lumbar spinal motor systems much earlier than previously recognized.