Mark C. Bellingham

A review of the neural mechanisms of action and clinical efficiency of riluzole in treating amyotrophic lateral sclerosis: what have we learned in the last decade?

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disease of adults which preferentially attacks the neuromotor system. Riluzole has been used as the only approved treatment for amyotrophic lateral sclerosis since 1995, but its mechanism(s) of action in slowing the progression of this disease remain obscure. Searching PubMed for “riluzole” found 705 articles published between January 1996 and June 2009. A systematic review of this literature found that riluzole had a wide range of effects on factors influencing neural activity in general, and the neuromotor system in particular. These effects occurred over a large dose range (<1 μM to >1 mM). Reported neural effects of riluzole included (in approximate ascending order of dose range): inhibition of persistent Na+ current = inhibition of repetitive firing < potentiation of calcium‐dependent K+ current < inhibition of neurotransmitter release < inhibition of fast Na+ current < inhibition of voltage‐gated Ca2+ current = promotion of neuronal survival or growth factors < inhibition of voltage‐gated K+ current = modulation of two‐pore K+ current = modulation of ligand‐gated neurotransmitter receptors = potentiation of glutamate transporters. Only the first four of these effects commonly occurred at clinically relevant concentrations of riluzole (plasma levels of 1–2 μM with three‐ to four‐fold higher concentrations in brain tissue). Treatment of human ALS patients or transgenic rodent models of ALS with riluzole most commonly produced a modest but significant extension of lifespan. Riluzole treatment was well tolerated in humans and animals. In animals, despite in vitro evidence that riluzole may inhibit rhythmic motor behaviors, in vivo administration of riluzole produced relatively minor effects on normal respiration parameters, but inhibited hypoxia‐induced gasping. This effect may have implications for the management of hypoventilation and sleep‐disordered breathing during end‐stage ALS in humans.

A Novel Presynaptic Inhibitory Mechanism Underlies Paired Pulse Depression at a Fast Central Synapse

Several distinct mechanisms may cause synaptic depression, a common form of short-term synaptic plasticity. These include postsynaptic receptor desensitization, presynaptic depletion of releasable vesicles, or other presynaptic mechanisms depressing vesicle release. At the endbulb of Held, a fast central calyceal synapse in the auditory pathway, cyclothiazide (CTZ) abolished marked paired pulse depression (PPD) by acting presynaptically to enhance transmitter release, rather than by blocking postsynaptic receptor desensitization. PPD and its response to CTZ were not altered by prior depletion of the releasable vesicle pool but were blocked by lowering external calcium concentration, while raising external calcium enhanced PPD. We conclude that a major component of PPD at the endbulb is due to a novel, transient depression of release, which is dependent on the level of presynaptic calcium entry and is CTZ sensitive.

Response of the medullary respiratory network of the cat to hypoxia.

1. The effect of systemic hypoxia was tested in anaesthetized, immobilized, thoracotomized and artificially ventilated cats with peripheral chemoreceptor afferents either intact or cut. Extracellular recordings from different types of medullary respiratory neurones and intracellular recordings from stage 2 expiratory neurones were made to determine the hypoxia‐induced changes in neuronal discharge patterns and postsynaptic activity as an index for the disturbances of synaptic interaction within the network. 2. The general effect of systemic hypoxia was an initial augmentation of respiratory activity followed by a secondary depression. In chemoreceptor‐denervated animals, secondary depression led to central apnoea. 3. The effects of systemic hypoxia were comparable with those of cerebral ischaemia following occlusion of carotid and vertebral arteries. 4. In chemoreceptor‐denervated animals, all types of medullary respiratory neurones ceased spontaneous action potential discharge during hypoxia. 5. Reversal of inhibitory postsynaptic potentials (IPSPs) and/or blockade of IPSPs was seen after 2‐3 min of hypoxia. 6. During hypoxia, the membrane potential of stage 2 expiratory neurones showed a slight depolarization to ‐45 to ‐55 mV and then remained stable. 7. The neurone input resistance increased initially and then decreased significantly during central apnoea. 8. Rhythmogenesis of respiration was greatly disturbed. This was due to blockade of IPSPs and, in some animals, to more complex disturbances of phase switching from inspiration to expiration. 9. Central apnoea occurred while respiratory neurones were still excitable as shown by stimulus‐evoked orthodromic and antidromic action potentials. 10. The results indicate that the medullary respiratory network is directly affected by energy depletion. There is indication for a neurohumoral mechanism which blocks synaptic interaction between respiratory neurones in chemoreceptor‐intact animals.

Neonatal Neuronal Circuitry Shows Hyperexcitable Disturbance in a Mouse Model of the Adult-Onset Neurodegenerative Disease Amyotrophic Lateral Sclerosis

Distinguishing the primary from secondary effects and compensatory mechanisms is of crucial importance in understanding adult-onset neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Transgenic mice that overexpress the G93A mutation of the human Cu-Zn superoxide dismutase 1 gene (hSOD1G93A mice) are a commonly used animal model of ALS. Whole-cell patch-clamp recordings from neurons in acute slice preparations from neonatal wild-type and hSOD1G93A mice were made to characterize functional changes in neuronal activity. Hypoglossal motoneurons (HMs) in postnatal day 4 (P4)–P10 hSOD1G93A mice displayed hyperexcitability, increased persistent Na+ current (PCNa), and enhanced frequency of spontaneous excitatory and inhibitory transmission, compared with wild-type mice. These functional changes in neuronal activity are the earliest yet reported for the hSOD1G93A mouse, and are present 2–3 months before motoneuron degeneration and clinical symptoms appear in these mice. Changes in neuronal activity were not restricted to motoneurons: superior colliculus interneurons also displayed hyperexcitability and synaptic changes (P10–P12). Furthermore, in vivo viral-mediated GFP (green fluorescent protein) overexpression in hSOD1G93A HMs revealed precocious dendritic remodeling, and behavioral assays revealed transient neonatal neuromotor deficits compared with controls. These findings underscore the widespread and early onset of abnormal neural activity in this mouse model of the adult neurodegenerative disease ALS, and suggest that suppression of PCNa and hyperexcitability early in life might be one way to mitigate or prevent cell death in the adult CNS.

ADENOSINERGIC MODULATION OF RESPIRATORY NEURONES AND HYPOXIC RESPONSES IN THE ANAESTHETIZED CAT

1. The modulatory effects of intracellularly injected adenosine on membrane potential, input resistance and spontaneous or evoked synaptic activity were determined in respiratory neurones of the ventral respiratory group. 2. The membrane potential hyperpolarized and sometimes reached values which were beyond the equilibrium potential of Cl(‐)‐dependent IPSPs. At the same time, neuronal input resistance decreased. 3. Spontaneous and stimulus‐evoked postsynaptic activities were decreased, as were mean respiratory drive potentials. 4. Systemic injection of the A1 adenosine receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 0.01‐0.05 mg kg‐1) resulted in an increase in mean peak phrenic nerve activity when arterial chemoreceptors were denervated. In contrast, phrenic nerve activity decreased when arterial chemoreceptors were left intact. 5. The depressant effect of adenosine on synaptic activity was abolished after systemic DPCPX administration. DPCPX caused an increase in respiratory drive potentials, increased the amplitude of stimulus‐evoked IPSPs, and hyperpolarized membrane potential. 6. Administration of DPCPX blocked the early hypoxic depression of stimulus‐evoked IPSPs, doubled the delay of onset of hypoxic apnoea and shortened the time necessary for recovery of the respiratory rhythm. 7. The data indicate that adenosine acts on pre‐ and postsynaptic A1 receptors resulting in postsynaptic membrane hyperpolarization and depression of synaptic transmission. Blockade of A1 receptors increases respiratory activity, indicating that adenosine A1 receptors are tonically activated under control conditions. Further activation contributes to the hypoxic depression of synaptic transmission in the respiratory network.

Developmental changes in EPSC quantal size and quantal content at a central glutamatergic synapse in rat

1 Developmental changes in amplitude and time course of single‐fibre‐evoked and spontaneous EPSCs mediated by AMPA and NMDA receptors at the endbulb‐bushy cell synapse of rats from 4 to 22 days of age were recorded using whole‐cell patch‐clamp methods in in vitro slices of cochlear nucleus. 2 The mean conductance of the AMPA component of evoked EPSCs increased by 66 %, while that of the NMDA component decreased by 61 %, for 12‐ to 18‐day‐old rats cf. 4‐ to 11‐day‐old rats. 3 The mean AMPA spontaneous EPSC conductance increased by 54 %, while mean NMDA spontaneous EPSC conductance decreased by 83 %, for 12‐ to 22‐day‐old rats cf. 4‐ to 11‐day‐old rats. The mean number of quanta contributing to peak evoked AMPA conductance also increased by 78 % in the older age group, after correction for the asynchrony of evoked quantal release. 4 The decay time constant of spontaneous AMPA EPSCs showed a small decrease in older animals, while the decay time constant of spontaneous NMDA EPSCs was markedly decreased in older animals. The decay time constants of evoked NMDA EPSCs showed a quantitatively similar decrease to that of spontaneous NMDA EPSCs. This suggests that AMPA receptor subunit composition is unlikely to undergo developmental change, while NMDA receptor subunit composition may be substantially altered during synaptic maturation. 5 These data are consistent with a developmentally increased efficacy of AMPA receptor‐mediated synaptic transmission at the endbulb‐bushy cell synapse, due to an increase in underlying AMPA‐mediated quantal size and content during the same period as a transient co‐localization of NMDA receptors.

Respiratory interneurons in the C5 segment of the spinal cord of the cat

Extracellular recordings were made in the C5 segment of the spinal cord of anaesthetised cats from 129 units which showed respiratory phased discharge. The majority of recordings (88%) were thought to arise from the somata of respiratory spinal interneurons. Inspiratory units and expiratory units comprised 42% and 52% of all recorded units. A small number of postinspiratory units were also found (n = 5). Most units did not respond to electrical stimulation of the ipsilateral superior laryngeal (SLN) and phrenic nerves (PN), but a few expiratory (n = 2) and postinspiratory units (n = 1) were excited by SLN stimulation, while 6 inspiratory units had their discharge suppressed by the same stimulus. PN stimulation evoked a long latency (2-7 ms) burst of firing in 2 inspiratory and 1 expiratory interneurons. It is concluded that these respiratory interneurons may provide a segmental input to phrenic motoneurons, in addition to synaptic drives mediated by bulbospinal pathways.

Contribution of cholinergic systems to state-dependent modulation of respiratory control.

Respiration is altered during different stages of the sleep-wake cycle. We review the contribution of cholinergic systems to this alteration, with particular reference to the role of muscarinic acetylcholine receptors (MAchRs) during rapid eye movement (REM) sleep. Available evidence demonstrates that MAchRs have potent excitatory effects on medullary respiratory neurones and respiratory motoneurones, and are likely to contribute to changes in central chemosensitive drive to the respiratory control system. These effects are likely to be most prominent during REM sleep, when cholinergic brainstem neurones show peak activity levels. It is possible that MAchR dysfunction is involved in sleep-disordered breathing, such as obstructive sleep apnea.

Adenosine suppresses excitatory glutamatergic inputs to rat hypoglossal motoneurons in vitro

Short-latency excitatory postsynaptic potentials (EPSPs), evoked by electrical stimulation lateral to the hypoglossal motor nucleus, were recorded from rat hypoglossal motoneurons (HMs) in brainstem slices. EPSPs were markedly suppressed or abolished by kynurenic acid (1 mM), showing that they were glutamatergic. The adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 100 nM) reduced EPSP amplitude to 42% of control, while the agonist 2-chloroadenosine (2-CA, 0.5-50 microM) caused a dose-dependent reduction of the EPSP. The adenosine receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 0.1-1 microM) increased the EPSP amplitude to 124% of control, and blocked EPSP reduction by CCPA or 2-CA. CCPA, 2-CA and DPCPX did not significantly alter HM input resistance or membrane potential. These data indicate that excitatory glutamatergic inputs to rat HMs are modulated by adenosine A1 receptors, most probably at a presynaptic site. This modulation may be especially significant in hypoxic responses of HMs.

Glycinergic and GABAergic Synaptic Activity Differentially Regulate Motoneuron Survival and Skeletal Muscle Innervation

GABAergic and glycinergic synaptic transmission is proposed to promote the maturation and refinement of the developing CNS. Here we provide morphological and functional evidence that glycinergic and GABAergic synapses control motoneuron development in a region-specific manner during programmed cell death. In gephyrin-deficient mice that lack all postsynaptic glycine receptor and some GABAA receptor clusters, there was increased spontaneous respiratory motor activity, reduced respiratory motoneuron survival, and decreased innervation of the diaphragm. In contrast, limb-innervating motoneurons showed decreased spontaneous activity, increased survival, and increased innervation of their target muscles. Both GABA and glycine increased limb-innervating motoneuron activity and decreased respiratory motoneuron activity in wild-type mice, but only glycine responses were abolished in gephyrin-deficient mice. Our results provide genetic evidence that the development of glycinergic and GABAergic synaptic inputs onto motoneurons plays an important role in the survival, axonal branching, and spontaneous activity of motoneurons in developing mammalian embryos.