Tyrosine phosphorylation of GABAA receptor γ2-subunit regulates tonic and phasic inhibition in the thalamus.

GABA-mediated tonic and phasic inhibition of thalamic relay neurons of the dorsal lateral geniculate nucleus (dLGN) was studied after ablating tyrosine (Y) phosphorylation of receptor γ2-subunits. As phosphorylation of γ2 Y365 and Y367 reduces receptor internalization, to understand their importance for inhibition we created a knock-in mouse in which these residues are replaced by phenylalanines. On comparing wild-type (WT) and γ2(Y365/367F)+/- (HT) animals (homozygotes are not viable in utero), the expression levels of GABAA receptor α4-subunits were increased in the thalamus of female, but not male mice. Raised δ-subunit expression levels were also observed in female γ2(Y365/367F) +/- thalamus. Electrophysiological analyses revealed no difference in the level of inhibition in male WT and HT dLGN, while both the spontaneous inhibitory postsynaptic activity and the tonic current were significantly augmented in female HT relay cells. The sensitivity of tonic currents to the δ-subunit superagonist THIP, and the blocker Zn(2+), were higher in female HT relay cells. This is consistent with upregulation of extrasynaptic GABAA receptors containing α4- and δ-subunits to enhance tonic inhibition. In contrast, the sensitivity of GABAA receptors mediating inhibition in the female γ2(Y356/367F) +/- to neurosteroids was markedly reduced compared with WT. We conclude that disrupting tyrosine phosphorylation of the γ2-subunit activates a sex-specific increase in tonic inhibition, and this most likely reflects a genomic-based compensation mechanism for the reduced neurosteroid sensitivity of inhibition measured in female HT relay neurons.

Riluzole is a potent drug to protect neonatal rat hypoglossal motoneurons in vitro from excitotoxicity due to glutamate uptake block.

Excitotoxic damage to motoneurons is thought to be an important contribution to the pathogenesis of amyotrophic lateral sclerosis (ALS), a slowly developing degeneration of motoneurons that, in most cases of sporadic occurrence, is associated with impaired glial glutamate uptake. Riluzole is the only drug licensed for symptomatic ALS treatment and is proposed to delay disease progression. As riluzole is administered only after full ALS manifestation, it is unclear if its early use might actually prevent motoneuron damage. We explored this issue by using, as a simple in vitro model, hypoglossal motoneurons (a primary target of ALS) of the neonatal rat brainstem slice preparation exposed to excitotoxic stress due to glutamate uptake block by DL-threo-ß-benzyloxyaspartate (TBOA). TBOA evoked sustained network bursting, early (1 h) enhancement of the S100B immunostaining of gray matter astrocytes, and activated the motoneuronal stress ATF-3 transcription factor; 4 h later, loss (30%) of motoneuron staining ensued and pyknosis appeared. Riluzole (5 µM; applied 15 min after TBOA) inhibited bursting, decreased the frequency of spontaneous glutamatergic events, reversed changes in S100B immunostaining and prevented late loss of motoneuron staining. These results show that excitotoxicity induced by glutamate uptake block developed slowly, and was sensed by glia and motoneurons with delayed cell death. Our data provide novel evidence for the neuroprotective action of riluzole on motoneurons and glia when applied early after an excitotoxic stimulus.

Transient oxidative stress evokes early changes in the functional properties of neonatal rat hypoglossal motoneurons in vitro.

Oxidative stress of motoneurons is believed to be an important contributor to neurodegeneration underlying the familial (and perhaps even the sporadic) form of amyotrophic lateral sclerosis (ALS). This concept has generated numerous rodent genetic models with inborn oxidative stress to mimic the clinical condition. ALS is, however, a predominantly sporadic disorder probably triggered by environmental causes. Thus, it is interesting to understand how wild-type motoneurons react to strong oxidative stress as this response might cast light on the presymptomatic disease stage. The present study used, as a model, hypoglossal motoneurons from the rat brainstem slice to investigate how hydrogen peroxide could affect synaptic transmission and intrinsic motoneuron excitability in relation to their survival. Hydrogen peroxide (1 mm; 30 min) induced inward current or membrane depolarization accompanied by an increase in input resistance, enhanced firing and depressed spontaneous synaptic events. Despite enhanced intracellular oxidative processes, there was no death of motoneurons, although most cells were immunopositive for activating transcription factor 3, a stress-related transcription factor. Voltage-clamp experiments indicated increased frequency of excitatory or inhibitory miniature events, and reduced voltage-gated persistent currents of motoneurons. The global effect of this transient oxidative challenge was to depress the input flow from the premotor interneurons to motoneurons that became more excitable due to a combination of enhanced input resistance and impaired spike afterhyperpolarization. Our data show previously unreported changes in motoneuron activity associated with cell distress caused by a transient oxidative insult.

Postnatal developmental profile of neurons and glia in motor nuclei of the brainstem and spinal cord, and its comparison with organotypic slice cultures.

In vitro preparations of the neonatal rat spinal cord or brainstem are useful to investigate the organization of motor networks and their dysfunction in neurological disease models. Long-term spinal cord organotypic cultures can extend our understanding of such pathophysiological processes over longer times. It is, however, surprising that detailed descriptions of the type (and number) of neurons and glia in such preparations are currently unavailable to evaluate cell-selectivity of experimental damage. The focus of the present immunohistochemical study is the novel characterization of the cell population in the lumbar locomotor region of the rat spinal cord and in the brainstem motor nucleus hypoglossus at 0-4 postnatal days, and its comparison with spinal organotypic cultures at 2-22 days in vitro. In the nucleus hypoglossus, neurons were 40% of all cells and 80% of these were motoneurons. Astrocytes (35% of total cells) were the main glial cells, while microglia was <10%. In the spinal gray matter, the highest neuronal density was in the dorsal horn (>80%) and the lowest in the ventral horn (≤57%) with inverse astroglia numbers and few microglia. The number of neurons (including motoneurons) and astrocytes was stable after birth. Like in the spinal cord, motoneurons in organotypic spinal culture were <10% of ventral horn cells, with neurons <40%, and the rest made up by glia. The present report indicates a comparable degree of neuronal and glial maturation in brainstem and spinal motor nuclei, and that this condition is also observed in 3-week-old organotypic cultures.

Respiratory motoneurons and pathological conditions: lessons from hypoglossal motoneurons challenged by excitotoxic or oxidative stress.

Hypoglossal motoneurons (HMs) are respiration-related brainstem neurons that command rhythmic contraction of the tongue muscles in concert with the respiratory drive. In experimental conditions, HMs can exhibit a range of rhythmic patterns that may subserve different motor outputs and functions. Neurodegenerative diseases like amyotrophic lateral sclerosis (ALS; Lou-Gehrig disease) often damage HMs with distressing symptoms like dysarthria, dysphagia and breathing difficulty related to degeneration of respiratory motoneurons. While the cause of ALS remains unclear, early diagnosis remains an important goal for potential treatment because fully blown clinical symptoms appear with degeneration of about 30% motoneurons. Using a simple in vitro model of the rat brainstem to study the consequences of excitotoxicity or oxidative stress (believed to occur during the onset of ALS) on HMs, it is possible to observe distinct electrophysiological effects associated with HM experimental pathology. In fact, excitotoxicity caused by glutamate uptake block triggers sustained bursting and enhanced synaptic transmission, whereas oxidative stress generates slow depolarization, augmented repeated firing, and decreased synaptic transmission. In either case, only a subpopulation of HMs shows abnormal functional changes. Although these two insults induce separate functional signatures, the consequences on HMs after a few hours are similar and are preceded by activation of the stress transcription factor ATF-3. The deleterious action of excitotoxicity is inhibited by early administration of riluzole, a drug currently employed for the symptomatic treatment of ALS, demonstrating that this in vitro model can be useful for testing potential neuroprotective agents.

A repertoire of rhythmic bursting produced by hypoglossal motoneurons in physiological and pathological conditions.

The brainstem nucleus hypoglossus contains motoneurons that provide the exclusive motor nerve supply to the tongue. In addition to voluntary tongue movements, tongue muscles rhythmically contract during a wide range of physiological activities, such as respiration, swallowing, chewing and sucking. Hypoglossal motoneurons are destroyed early in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease often associated with a deficit in the transport system of the neurotransmitter glutamate. The present study shows how periodic electrical discharges of motoneurons are mainly produced by a neuronal network that drives them into bursting mode via glutamatergic excitatory synapses. Burst activity is, however, modulated by the intrinsic properties of motoneurons that collectively synchronize their discharges via gap junctions to create 'group bursters'. When glial uptake of glutamate is blocked, a distinct form of pathological bursting spontaneously emerges and leads to motoneuron death. Conversely, H(2)O(2)-induced oxidative stress strongly increases motoneuron excitability without eliciting bursting. Riluzole (the only drug currently licensed for the treatment of ALS) suppresses bursting of hypoglossal motoneurons caused by blockage of glutamate uptake and limits motoneuron death. These findings highlight how different patterns of electrical oscillations of brainstem motoneurons underpin not only certain physiological activities, but also motoneuron death induced by glutamate transporter impairment.