Astrocytic modulation of neuronal network oscillations

The synchronization of the neuron’s membrane potential results in the emergence of neuronal oscillations at multiple frequencies that serve distinct physiological functions (e.g. facilitation of synaptic plasticity) and correlate with different behavioural states (e.g. sleep, wakefulness, attention). It has been postulated that at least ten distinct mechanisms are required to cover the large frequency range of neuronal oscillations in the cortex, including variations in the concentration of extracellular neurotransmitters and ions, as well as changes in cellular excitability. However, the mechanism that gears the transition between different oscillatory frequencies is still unknown. Over the past decade, astrocytes have been the focus of much research, mainly due to (1) their close association with synapses forming what is known today as the “tripartite synapse”, which allows them to bidirectionally interact with neurons and modulate synaptic transmission; (2) their syncytium-like activity, as they are electrically coupled via gap junctions and actively communicate through Ca2+ waves; and (3) their ability to regulate neuronal excitability via glutamate uptake and tight control of the extracellular K+ levels via a process termed K+ clearance. In this thesis we hypothesized that astrocytes, in addition to their role as modulators of neuronal excitability, also act as “network managers” that can modulate the overall network oscillatory activity within their spatial domain. To do so, it is proposed that astrocytes fine-tune their K+ clearance capabilities to affect neuronal intrinsic excitability properties and synchronization with other neurons, thus mediating the transitions between neuronal network oscillations at different frequencies. To validate or reject this hypothesis I have investigated the potential role of astrocytes in modulating cortical oscillations at both cellular and network levels, aiming at answering three main research questions: a) what is the impact of alterations in astrocytic K+ clearance mechanisms on cortical networks oscillatory dynamics? b) what specific neuronal properties underlying the generation of neuronal oscillations are affected as a result of impairments in the astrocytic K+ clearance process? and c) what are the bidirectional mechanisms between neurons and astrocytes (i.e. neuromodulators) that specifically affect the K+ clearance process to modulate the network activity output? In the first experimental chapter I used electrophysiological recordings and pharmacological manipulations to dissect the contribution of the different astrocytic K+ clearance mechanisms to the modulation of neuronal network oscillations at multiple frequencies. A key finding was that alterations in membrane properties of layer V pyramidal neurons strongly correlated with the network behaviour following impairments in astrocytic K+ clearance capabilities, depicted as enhanced excitability underlying the amplification of high-frequency oscillations, especially within the beta and gamma range. The second experimental chapter describes a combinatorial approach based on K+-selective microelectrode recordings and optical imaging of K+ ions used to quantitatively determine extracellular K+ changes and to follow the spatiotemporal distribution of K+ ions under both physiological and altered K+ clearance conditions, which affected the K+ clearance rate. The impact of different neuromodulators on astrocytic function is discussed in the third experimental chapter. Using extracellular K+ recordings and Ca2+ imaging I found that some neuromodulators act specifically on astrocytic receptors to affect both K+ clearance mechanisms and Ca2+ signalling, as evidenced by reduced K+ clearance rates and altered evoked Ca2+ signals. Overall, this thesis provides new insights regarding the impact of astrocytic K+ clearance mechanisms on modulating neuronal properties at both cellular and network levels, which in turn imposes alterations on neuronal oscillations that are associated with different behavioural states

Generating brain waves, the power of astrocytes

Synchronization of neuronal activity in the brain underlies the emergence of neuronal oscillations termed “brain waves”, which serve various physiological functions and correlate with different behavioral states. It has been postulated that at least ten distinct mechanisms are involved in the formulation of these brain waves, including variations in the concentration of extracellular neurotransmitters and ions, as well as changes in cellular excitability. In this mini review we highlight the contribution of astrocytes, a subtype of glia, in the formation and modulation of brain waves mainly due to their close association with synapses that allows their bidirectional interaction with neurons, and their syncytium-like activity via gap junctions that facilitate communication to distal brain regions through Ca2+ waves. These capabilities allow astrocytes to regulate neuronal excitability via glutamate uptake, gliotransmission and tight control of the extracellular K+ levels via a process termed K+ clearance. Spatio-temporal synchrony of activity across neuronal and astrocytic networks, both locally and distributed across cortical regions, underpins brain states and thereby behavioral states, and it is becoming apparent that astrocytes play an important role in the development and maintenance of neural activity underlying these complex behavioral states

Neuromodulation of astrocytic K+ clearance

Potassium homeostasis is fundamental for brain function. Therefore, effective removal of excessive K+ from the synaptic cleft during neuronal activity is paramount. Astrocytes play a key role in K+ clearance from the extracellular milieu using various mechanisms, including uptake via Kir channels and the Na+-K+ ATPase, and spatial buffering through the astrocytic gap-junction coupled network. Recently we showed that alterations in the concentrations of extracellular potassium ([K+]o) or impairments of the astrocytic clearance mechanism affect the resonance and oscillatory behavior of both the individual and networks of neurons. These results indicate that astrocytes have the potential to modulate neuronal network activity, however, the cellular effectors that may affect the astrocytic K+ clearance process are still unknown. In this study, we have investigated the impact of neuromodulators, which are known to mediate changes in network oscillatory behavior, on the astrocytic clearance process. Our results suggest that while some neuromodulators (5-HT; NA) might affect astrocytic spatial buffering via gap-junctions, others (DA; Histamine) primarily affect the uptake mechanism via Kir channels. These results suggest that neuromodulators can affect network oscillatory activity through parallel activation of both neurons and astrocytes, establishing a synergistic mechanism to maximize the synchronous network activity

Astrocytic modulation of neuronal excitability through K+ spatial buffering

The human brain contains two major cell populations, neurons and glia. While neurons are electrically excitable and capable of discharging short voltage pulses known as action potentials, glial cells are not. However, astrocytes, the prevailing subtype of glia in the cortex, are highly connected and can modulate the excitability of neurons by changing the concentration of potassium ions in the extracellular environment, a process called K+ clearance. During the past decade, astrocytes have been the focus of much research, mainly due to their close association with synapses and their modulatory impact on neuronal activity. It has been shown that astrocytes play an essential role in normal brain function including: nitrosative regulation of synaptic release in the neocortex, synaptogenesis, synaptic transmission and plasticity. Here, we discuss the role of astrocytes in network modulation through their K+ clearance capabilities, a theory that was first raised 50 years ago by Orkand and Kuffler. We will discuss the functional alterations in astrocytic activity that leads to aberrant modulation of network oscillations and synchronous activity

Dynamic interplay between H-current and M-current controls motoneuron hyperexcitability in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a type of motor neuron disease (MND) in which humans lose motor functions due to progressive loss of motoneurons in the cortex, brainstem, and spinal cord. In patients and in animal models of MND it has been observed that there is a change in the properties of motoneurons, termed neuronal hyperexcitability, which is an exaggerated response of the neurons to a stimulus. Previous studies suggested neuronal excitability is one of the leading causes for neuronal loss, however the factors that instigate excitability in neurons over the course of disease onset and progression are not well understood, as these studies have looked mainly at embryonic or early postnatal stages (pre-symptomatic). As hyperexcitability is not a static phenomenon, the aim of this study was to assess the overall excitability of upper motoneurons during disease progression, specifically focusing on their oscillatory behavior and capabilities to fire repetitively. Our results suggest that increases in the intrinsic excitability of motoneurons are a global phenomenon of aging, however the cellular mechanisms that underlie this hyperexcitability are distinct in SOD1 G93A ALS mice compared with wild-type controls. The ionic mechanism driving increased excitability involves alterations of the expression levels of HCN and KCNQ channel genes leading to a complex dynamic of H-current and M-current activation. Moreover, we show a negative correlation between the disease onset and disease progression, which correlates with a decrease in the expression level of HCN and KCNQ channels. These findings provide a potential explanation for the increased vulnerability of motoneurons to ALS with aging

A small-molecule TrkB ligand restores hippocampal synaptic plasticity and object location memory in Rett syndrome mice

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in methyl-CpG-binding protein-2 (MECP2), a transcriptional regulator of many genes, including brain-derived neurotrophic factor (BDNF). BDNF levels are reduced in RTT autopsy brains and in multiple brain areas of Mecp2-deficient mice. Furthermore, experimental interventions that increase BDNF levels improve RTT-like phenotypes in Mecp2 mutant mice. Here, we characterized the actions of a small-molecule ligand of the BDNF receptor TrkB in hippocampal function in Mecp2 mutant mice. Systemic treatment of female Mecp2 heterozygous (HET) mice with LM22A-4 for 4 weeks improved hippocampal-dependent object location memory and restored hippocampal long-term potentiation (LTP). Mechanistically, LM22A-4 acts to dampen hyperactive hippocampal network activity, reduce the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs), and reduce the frequency of spontaneous tetrodotoxin-resistant Ca2+ signals in Mecp2 mutant hippocampal neurons, making them comparable to those features observed in wild-type neurons. Together, these observations indicate that LM22A-4 is a promising therapeutic candidate for the treatment of hippocampal dysfunction in RTT

Extending the life span of acute neuronal tissue for imaging and electrophysiological studies

Slice preparations of neuronal tissue are among the most commonly used experimental approaches in the field of neuroscience. They are employed for a variety of techniques addressing questions across the entire neuroscience spectrum, including immunohistochemical, anatomical, and electrophysiological methods to study the properties of individual, and networks of neurons. In the past decades, slice preparations have provided information that has allowed us to develop our understanding of the central nervous system. Unlike cultures, slice preparations leave the topography of neurons and glia intact and therefore retain a considerable degree of functionality that allows molecular, cellular, and network investigations. However, a major limitation of using acute brain slices is their life span which is limited to 6–8 h due to intrinsic and extrinsic factors. Recently, new technological and methodological modifications have proved efficient in extending the life span of acute neuronal tissue. In this chapter, we will review the mechanisms leading to tissue deterioration and describe in detail the steps required to achieve a significant enhancement in neuronal viability and longevity

Astrocytic modulation of cortical oscillations

Brain waves are rhythmic voltage oscillations emerging from the synchronization of individual neurons into a neuronal network. These oscillations range from slow to fast fluctuations, and are classified by power and frequency band, with different frequency bands being associated with specific behaviours. It has been postulated that at least ten distinct mechanisms are required to cover the frequency range of neural oscillations, however the mechanisms that gear the transition between distinct oscillatory frequencies are unknown. In this study, we have used electrophysiological recordings to explore the involvement of astrocytic K+ clearance processes in modulating neural oscillations at both network and cellular levels. Our results indicate that impairment of astrocytic K+ clearance capabilities, either through blockade of K+ uptake or astrocytic connectivity, enhance network excitability and form high power network oscillations over a wide range of frequencies. At the cellular level, local increases in extracellular K+ results in modulation of the oscillatory behaviour of individual neurons, which underlies the network behaviour. Since astrocytes are central for maintaining K+ homeostasis, our study suggests that modulation of their inherent capabilities to clear K+ from the extracellular milieu is a potential mechanism to optimise neural resonance behaviour and thus tune neural oscillations