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In the brain the extracellular concentration of glutamate is controlled by glial transporters that restrict the neurotransmitter action to synaptic sites and avoid excitotoxicity. Impaired transport of glutamate occurs in many cases of amyotrophic lateral sclerosis, a devastating motoneuron disease. Motoneurons of the brainstem nucleus hypoglossus are among the most vulnerable, giving early symptoms like slurred speech and dysphagia. However, the direct consequences of extracellular glutamate build-up, due to uptake block, on synaptic transmission and survival of hypoglossal motoneurons remain unclear and have been studied using the neonatal rat brainstem slice preparation as a model. Patch clamp recording from hypoglossal motoneurons showed that, in about one-third of these cells, inhibition of glutamate transport with the selective blocker dl-threo-β-benzyloxyaspartate (TBOA; 50 μm) unexpectedly led to the emergence of rhythmic bursting consisting of inward currents of long duration with superimposed fast oscillations and synaptic events. Synaptic inhibition block facilitated bursting. Bursts had a reversal potential near 0 mV, and were blocked by tetrodotoxin, the gap junction blocker carbenoxolone, or antagonists of AMPA, NMDA or mGluR1 glutamate receptors. Intracellular Ca2+ imaging showed bursts as synchronous discharges among motoneurons. Synergy of activation of distinct classes of glutamate receptor plus gap junctions were therefore essential for bursting. Ablating the lateral reticular formation preserved bursting, suggesting independence from propagated network activity within the brainstem. TBOA significantly increased the number of dead motoneurons, an effect prevented by the same agents that suppressed bursting. Bursting thus represents a novel hallmark of motoneuron dysfunction triggered by glutamate uptake block.
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease primarily affecting motoneurons (Bruijn et al. 2004). A large number of ALS patients show a deficit in the transport process of the excitatory transmitter glutamate that builds up extracellularly to produce excitotoxic damage to motor cells (Rothstein et al. 1992; Cleveland & Rothstein, 2001; Rao & Weiss, 2004). Because in the vast majority of cases the disease is sporadic and associated with normal synthesis of the transporter proteins (Rao & Weiss, 2004), various environmental factors are suspected to generate this condition by inhibiting glutamate transport in vulnerable brain regions (Bruijn et al. 2004).
One important form of ALS (termed bulbar) is clinically manifested as severe degeneration of brainstem motoneurons, although some motor nuclei are more vulnerable than others (Rowland & Shneider, 2001). In particular, the nucleus hypoglossus, that exclusively innervates tongue muscles, is among the most strongly involved in ALS (Krieger et al. 1994; Lips & Keller, 1999; Laslo et al. 2001), producing slurred speech, difficulty in mastication, swallowing and breathing. While the early damage of hypoglossal motoneurons (HMs) may be related to their characteristic intracellular Ca2+ homeostasis (Ladewig et al. 2003) and expression of Ca2+-permeable AMPA receptors (Del Cano et al. 1999; Laslo et al. 2001; Essin et al. 2002), it is also suggested that vulnerable motor nuclei normally possess distinctive properties of glutamate uptake to protect them against the risk factor of excitoxocity (Medina et al. 1996).
In the present study, based on electrophysiological recording, and intracellular Ca2+ imaging from HMs of the rat brainstem slice preparation, we used the very selective glutamate transport inhibitor dl-threo-β-benzyloxyaspartate (TBOA; Shigeri et al. 2004) to explore how it may change synaptic transmission, and its consequences on HM survival estimated with histochemical methods. Even after a short period of uptake block, we discovered the emergence of a novel type of bursting with significant neurotoxic damage to HMs.