From sleep spindles of natural sleep to spike and wave discharges of typical absence seizures: is the hypothesis still valid?

The temporal coincidence of sleep spindles and spike-and-wave discharges (SWDs) in patients with idiopathic generalized epilepsies, together with the transformation of spindles into SWDs following intramuscular injection of the weak GABAA receptor (GABAAR) antagonist, penicillin, in an experimental model, brought about the view that SWDs may represent ‘perverted’ sleep spindles. Over the last 20 years, this hypothesis has received considerable support, in particular by in vitro studies of thalamic oscillations following pharmacological/genetic manipulations of GABAARs. However, from a critical appraisal of the evidence in absence epilepsy patients and well-established models of absence epilepsy it emerges that SWDs can occur as frequently during wakefulness as during sleep, with their preferential occurrence in either one of these behavioural states often being patient dependent. Moreover, whereas the EEG expression of both SWDs and sleep spindles requires the integrity of the entire cortico-thalamo-cortical network, SWDs initiates in cortex while sleep spindles in thalamus. Furthermore, the hypothesis of a reduction in GABAAR function across the entire cortico-thalamo-cortical network as the basis for the transformation of sleep spindles into SWDs is no longer tenable. In fact, while a decreased GABAAR function may be present in some cortical layers and in the reticular thalamic nucleus, both phasic and tonic GABAAR inhibitions of thalamo-cortical neurons are either unchanged or increased in this epileptic phenotype. In summary, these differences between SWDs and sleep spindles question the view that the EEG hallmark of absence seizures results from a transformation of this EEG oscillation of natural sleep

The Role of Transmembrane AMPA Receptor Regulatory Proteins (TARPs) in Synapse Remodeling and Homeostatic Plasticity

Tese de doutoramento em Biologia, na especialidade de Biologia Molecular apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.Durante o desenvolvimento do sistema nervoso central o refinamento dos circuitos neuronais é primariamente controlado por mecanismos de plasticidade de Hebb. No entanto, esta plasticidade tende a destabilizar os circuitos neuronais, levando a um descontrolo da excitabilidade neuronal. Existem evidências convincentes na literatura do controlo homeostático da excitabilidade neuronal em diversos sistemas. Conhecem-se algumas das vias de sinalização que controlam a plasticidade homeostática, mas os mecanismos moleculares que regulam este processo permanecem uma área de investigação activa. Neste trabalho testámos o papel da stargazina, uma proteína da família das proteínas transmembranares que interagem com os receptores do glutamato do tipo AMPA (TARPs), na plasticidade sináptica homeostática. Para isso, investigámos de que modo a perda da função da stargazina afecta o scaling sináptico induzido por tetrodotoxina (TTX) em culturas de neurónios corticais. Descobrimos que o bloqueio crónico da actividade com TTX aumenta os níveis de stargazina bem como a sua fosforilação, causando acumulação sináptica da stargazina que coincide com aumento da expressão superficial de receptores do glutamato do tipo AMPA (AMPAR), uma característica do mecanismo de plasticidade homeostática em resposta a inibição crónica da actividade neuronal. A fosforilação da stargazina está aumentada após bloqueio da actividade e regula o scaling sináptico já que a expressão de mutantes para a fosforilação da stargazina, S9A e S9D, impede a acumulação sináptica da subunidade GluA1 dos receptores AMPA. Demonstrámos também o envolvimento das proteínas cinase PKC e CaMKIIβ no scaling sináptico sendo a stargazina um substrato destas cinases. O estudo do papel da stargazina na plasticidade dependente de actividade da sinapse retinogenicular envolveu a caracterização do desenvolvimento desta xiv sinapse nos ratinhos stargazer. Descobrimos que a stargazina é essencial para a manutenção sináptica e plasticidade dependente de actividade, já que os ratinhos stargazer apresentam um defeito na maturação da sinapse retinogenicular no período sensível à visão. Analisámos ainda os níveis de TARPs e das subunidades dos AMPAR no núcleo geniculado lateral (dLGN) após diferentes manipulações visuais. É importante ressaltar que os níveis de stargazina são regulados pela visão, e estão aumentados no tálamo visual quando os ratinhos são privados de visão entre p20-27 (LDR-late dark rearing), sem se registarem alterações noutro membro das TARPs, a TARPγ4. Finalmente, descobrimos que, de acordo com o que foi descrito anteriormente (Gainey et al. 2009), o bloqueio crónico da actividade de neurónios corticais aumenta a expressão da subunidade GluA2. Esta subunidade está também aumentada no dLGN após LDR, demostrando que haverá semelhantes efectores moleculares da plasticidade homeostático in vitro e in vivo. De acordo com o nosso conhecimento, esta é a primeira evidência de indução de plasticidade homeostática em regiões sub-corticais em resposta à privação de visão binocular. Com base nos nossos resultados propomos um modelo em que a fosforilação da stargazina é o interruptor principal para a acumulação sináptica de AMPAR durante a plasticidade homeostática, uma forma de plasticidade essencial para o desenvolvimento de circuitos e adaptação ao ambiente. O nosso trabalho mostra a stargazina como um novo regulador da homeostase dos circuitos.Activity-dependent synaptic refinement during development occurs primarily through Hebbian mechanisms of plasticity. However, this plasticity tends to destabilize the neuronal circuits leading to runway excitation or inhibition. There is now compelling evidence from a number of systems for homeostatic control of firing rates in central neurons. Molecular control of homeostatic processes is an active area of research. We tested whether stargazin participates in homeostatic synaptic plasticity by investigating how loss of stargazin function affects TTX-induced synaptic scaling in cultured cortical neurons. We found that chronic activity blockade with TTX increased stargazin levels and stargazin phosphorylation state, leading to increased synaptic stargazin accumulation coincident with increased surface expression of AMPA-type glutamate receptors, a hallmark of homeostatic plasticity induced by chronic inhibition of neuronal activity. Stargazin phosphorylation was increased upon chronic activity blockade and regulates synaptic scaling as expression of stargazin phospho-mutants, S9A and S9D, disrupted increased surface expression of GluA1, an AMPA receptor subunit. We were also able to show the involvement of PKC and CaMKIIβ in synaptic scaling. The role of stargazin in experience-dependent plasticity was studied by characterizing the development of the retinogeniculate synapse of stargazer mice. We found that stargazin is essential for synaptic maintenance and experiencedependent plasticity, as stargazer mice showed disrupted retinogeniculate synapse maturation at the vision sensitive period. Furthermore, we biochemically analyzed TARPs and AMPA receptor subunits in the dorsal lateral geniculate nucleus (dLGN) after different visual manipulations. Importantly, stargazin levels are regulated by vision, and stargazin was up-regulated in the visual thalamus when experiencexvi dependent plasticity was induced by late-dark rearing, without significant changes in the thalamus-expressed family member TARPγ4. Finally, we found that, similarly to what was previously described (Gainey et al. 2009) and confirmed in our in vitro system, GluA2 subunit is increased in dLGN after late dark-rearing mice, showing similar molecular effectors of homeostatic plasticity in vitro and in vivo. To our knowledge this is the first evidence for homeostatic plasticity mechanisms being induced in sub-cortical regions in response to binocular visual deprivation. Herein we propose a model for stargazin phosphorylation as the main switch controlling AMPAR synaptic accumulation and trapping during homeostatic plasticity. This form of plasticity is essential for circuit development and adaptation to the ever-changing environment, making stargazin a new regulator of circuit homeostasis

From Physiology to Pathology of Cortico-Thalamo-Cortical Oscillations: Astroglia as a Target for Further Research

The electrographic hallmark of childhood absence epilepsy (CAE) and other idiopathic forms of epilepsy are 2.5–4 Hz spike and wave discharges (SWDs) originating from abnormal electrical oscillations of the cortico-thalamo-cortical network. SWDs are generally associated with sudden and brief non-convulsive epileptic events mostly generating impairment of consciousness and correlating with attention and learning as well as cognitive deficits. To date, SWDs are known to arise from locally restricted imbalances of excitation and inhibition in the deep layers of the primary somatosensory cortex. SWDs propagate to the mostly GABAergic nucleus reticularis thalami (NRT) and the somatosensory thalamic nuclei that project back to the cortex, leading to the typical generalized spike and wave oscillations. Given their shared anatomical basis, SWDs have been originally considered the pathological transition of 11–16 Hz bursts of neural oscillatory activity (the so-called sleep spindles) occurring during Non-Rapid Eye Movement (NREM) sleep, but more recent research revealed fundamental functional differences between sleep spindles and SWDs, suggesting the latter could be more closely related to the slow (<1 Hz) oscillations alternating active (Up) and silent (Down) cortical activity and concomitantly occurring during NREM. Indeed, several lines of evidence support the fact that SWDs impair sleep architecture as well as sleep/wake cycles and sleep pressure, which, in turn, affect seizure circadian frequency and distribution. Given the accumulating evidence on the role of astroglia in the field of epilepsy in the modulation of excitation and inhibition in the brain as well as on the development of aberrant synchronous network activity, we aim at pointing at putative contributions of astrocytes to the physiology of slow-wave sleep and to the pathology of SWDs. Particularly, we will address the astroglial functions known to be involved in the control of network excitability and synchronicity and so far mainly addressed in the context of convulsive seizures, namely (i) interstitial fluid homeostasis, (ii) K+ clearance and neurotransmitter uptake from the extracellular space and the synaptic cleft, (iii) gap junction mechanical and functional coupling as well as hemichannel function, (iv) gliotransmission, (v) astroglial Ca2+ signaling and downstream effectors, (vi) reactive astrogliosis and cytokine release