Gender-specific perturbations in modulatory inputs to motoneurons in a mouse model of amyotrophic lateral sclerosis.

The fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) is characterised by loss of motoneurons of the brainstem and spinal cord, and corticospinal neurons of the motor cortex. There is also increasing evidence of involvement of glial cells and interneurons, with non-cell autonomous disease mechanisms now thought to contribute to motoneuron degeneration in ALS. Given the apparent involvement of altered motoneuron excitability in ALS and the recent demonstration that motoneuron excitability is controlled by C-boutons, a specific class of synaptic input recently shown to originate from a small cluster of spinal interneurons, we hypothesised that perturbations in C-bouton inputs to motoneurons may contribute to altered excitability and the eventual degeneration of motoneurons in ALS. To begin to assess this we performed a detailed, developmental study of the anatomy of C-boutons in a mouse model of ALS (G93A SOD1 mutant). We found that C-bouton number is unchanged in ALS mice compared to wildtype littermates at any age. In contrast, we found that the size of C-boutons increases in ALS mice between postnatal day (P)8 and P30, with boutons remaining larger throughout symptomatic stages (P120-P140). Interestingly, we found that C-boutons are only enlarged in male mice. We found no evidence of concomitant changes in clusters of postsynaptic proteins known to align with C-boutons (Kv2.1 K(+) channels and m(2)-type muscarinic receptors). In conclusion, these data support the involvement of pre-symptomatic changes in C-bouton anatomy in ALS pathogenesis and in particular mechanisms underlying the male bias of this disease.

A multitarget basal ganglia dopaminergic and GABAergic transplantation strategy enhances behavioural recovery in parkinsonian rats.

The current transplantation paradigm for Parkinson's disease that places foetal dopaminergic cells in the striatum neither normalizes neuronal activity in basal ganglia structures such as the substantia nigra (SN) and subthalamic nucleus (STN) nor leads to complete functional recovery. It was hypothesized that restoration of parkinsonian deficits requires inhibition of the pathological overactivity of the STN and SN in addition to restoration of dopaminergic activity in the striatum. To achieve inhibition, a multitargeted basal ganglia transplantation strategy using GABAergic cells derived from either foetal striatal primordia (FSP) cells or human neural precursor cells (hNPCs) expanded in suspension bioreactors was investigated. In hemiparkinsonian rats, transplantation of foetal rat dopaminergic cells in the striatum in conjunction with GABAergic grafts in the STN and/or SN promoted significant improvement in forelimb akinesia and motor function compared to transplantation of intrastriatal dopaminergic grafts alone or in conjunction with undifferentiated hNPCs. In culture, FSP cells exhibited neuronal electrophysiological properties. However, recordings from GABAergic hNPCs revealed limited ionic conductances and an inability to fire action potentials. Despite this, they were almost as efficacious as FSP cells in inducing functional recovery following transplantation, suggesting that such recovery may have been mediated by secretion of GABA rather than by functional integration into the host. Thus, restoration of dopaminergic activity to the striatum in concert with inhibition of the STN and SN by GABAergic grafts may be beneficial for improving clinical outcomes in patients with Parkinson's disease and potential clinical application of this strategy may be enhanced by the use of differentiated hNPCs.

Mechanisms underlying the early phase of spike frequency adaptation in mouse spinal motoneurones.

Spike frequency adaptation (SFA) is a fundamental property of repetitive firing in motoneurones (MNs). Early SFA (occurring over several hundred milliseconds) is thought to be important in the initiation of muscular contraction. To date the mechanisms underlying SFA in spinal MNs remain unclear. In the present study, we used both whole-cell patch-clamp recordings of MNs in lumbar spinal cord slices prepared from motor functionally mature mice and computer modelling of spinal MNs to investigate the mechanisms underlying SFA. Pharmacological blocking agents applied during whole-cell recordings in current-clamp mode demonstrated that the medium AHP conductance (apamin), BK-type Ca2+ -dependent K+ channels (iberiotoxin), voltage-activated Ca2+ channels (CdCl2), M-current (linopirdine) and persistent Na+ currents (riluzole) are all unnecessary for SFA. Measurements of Na+ channel availability including action potential amplitude, action potential threshold and maximum depolarization rate of the action potential were found to correlate with instantaneous firing frequency suggesting that the availability of fast, inactivating Na+ channels is involved in SFA. Characterization of this Na+ conductance in voltage-clamp mode demonstrated that it undergoes slow inactivation with a time course similar to that of SFA. When experimentally measured parameters for the fast, inactivating Na+ conductance (including slow inactivation) were incorporated into a MN model, SFA could be faithfully reproduced. The removal of slow inactivation from this model was sufficient to remove SFA. These data indicate that slow inactivation of the fast, inactivating Na+ conductance is likely to be the key mechanism underlying early SFA in spinal MNs.

GluR2 AMPA receptor subunit expression in motoneurons at low and high risk for degeneration in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder that results in selective degeneration of most, but not all, groups of motoneurons. The greater susceptibility of vulnerable motoneurons to glutamate excitotoxicity and neurodegeneration has been hypothesized to result from their lower expression of the GluR2 AMPA receptor subunit under control conditions, which renders these receptors permeable to calcium. To address the question of whether there is differential expression of the GluR2 subunit in motoneurons, we compared in normal adult rats expression of GluR2 mRNA and protein within two cranial motor nuclei that are either resistant (III; oculomotor nucleus) or vulnerable (XII; hypoglossal nucleus) to degeneration in ALS. RT-PCR analysis of tissue punched from III and XII motor nuclei detected mRNA for all AMPA subunits (GluR1-R4). In situ hybridization demonstrated no significant difference in GluR2 mRNA expression between III and XII nuclei. Immunohistochemical examination of GluR2 (and GluR4) protein levels demonstrated a similar pattern of the subunit expression in both motor nuclei. This equivalent expression of GluR2 mRNA and protein in motoneurons that differ in their vulnerability to degeneration in ALS suggests that reduced expression of GluR2 is not a factor predisposing motoneurons to degeneration.

Calcium binding proteins in motoneurons at low and high risk for degeneration in ALS.

Recent reports challenge the hypothesis that expression of calcium binding proteins contributes to the greater resistance of some motoneurons to degeneration in amyotrophic lateral sclerosis (ALS). We therefore re-examined, using immunohistochemistry, the expression of calbindin, calretinin and parvalbumin in vulnerable (hypoglossal, XII; and cervical spinal) and resistant (oculomotor, III) motoneurons of adult rats. Calbindin immunoreactivity was lacking in motor nuclei but strong in the dorsal horn. Calretinin was expressed in spinal, but not III or XII, motoneurons. Parvalbumin immunoreactivity, tested with a polyclonal antibody, was intense in spinal and III, but not XII, motoneurons; however, no staining in the ventral horn was observed with a monoclonal antibody. Differential expression of calretinin and parvalbumin within vulnerable motoneurons suggests that immunoreactivity for these proteins is not a reliable marker for resistance to degeneration in ALS.

Synaptic control of motoneuron excitability in rodents: from months to milliseconds.

1. Motoneurons (MN) shape motor patterns by transforming inputs into action potential output. This transformation, excitability, is determined by an interaction between synaptic inputs and intrinsic membrane properties. Excitability is not static, but changes over multiple time scales. The purpose of the present paper is to review our recent data on synaptic factors important in the dynamic control of MN excitability over time scales ranging from weeks to milliseconds. 2. Developmental changes in modulation of MN excitability are well established. Noradrenergic potentiation of hypoglossal (XII) MN inspiratory activity in rhythmically active medullary slice preparations from rodents increases during the first two postnatal weeks. This is due to increasing alpha 1- and beta-adrenoceptor excitatory mechanisms and to a decreasing inhibitory mechanism mediated by alpha 2-adrenoceptors. Over a similar period, ATP potentiation of XII inspiratory activity does not change. 3. Motoneuron excitability may also change on a faster time scale, such as between different behaviours or different phases of a behaviour. Examination of this has been confounded by the fact that excitatory synaptic drives underlying behaviour can obscure smaller concurrent changes in excitability. Using the rhythmically active neonatal rat brain-stem-spinal cord preparation, we blocked excitatory inspiratory drive to phrenic MN (PMN) to reveal a reduction in PMN excitability specific to the inspiratory phase that: (i) arises from an inhibitory GABAergic input; (ii) is not mediated by recurrent pathways; and (iii) is proportional to and synchronous with the excitatory inspiratory input. We propose that the proportionality of the concurrent inhibitory and excitatory drives provides a means for phase-specific modulation of PMN gain. 4. Modulation across such diverse time scales emphasizes the active role that synaptic factors play in controlling MN excitability and shaping behaviour.