Astrocytes have long been regarded as essentially unexcitable cells that do not contribute to active signaling and information processing in the brain. Contrary to this classical view, it is now firmly established that astrocytes can specifically respond to glutamate released from neurons. Astrocyte glutamate signaling is initiated upon binding of glutamate to ionotropic and/or metabotropic receptors, which can result in calcium signaling, a major form of glial excitability. Release of so-called gliotransmitters like glutamate, ATP and D-serine from astrocytes in response to activation of glutamate receptors has been demonstrated to modulate various aspects of neuronal function in the hippocampus. In addition to receptors, glutamate binds to high-affinity, sodium-dependent transporters, which results in rapid buffering of synaptically-released glutamate, followed by its removal from the synaptic cleft through uptake into astrocytes. The degree to which astrocytes modulate and control extracellular glutamate levels through glutamate transporters depends on their expression levels and on the ionic driving forces that decrease with ongoing activity. Another major determinant of astrocytic control of glutamate levels could be the precise morphological arrangement of fine perisynaptic processes close to synapses, defining the diffusional distance for glutamate, and the spatial proximity of transporters in relation to the synaptic cleft. In this review, we will present an overview of the mechanisms and physiological role of glutamate-induced ion signaling in astrocytes in the hippocampus as mediated by receptors and transporters. Moreover, we will discuss the relevance of astroglial glutamate uptake for extracellular glutamate homeostasis, focusing on how activity-induced dynamic changes of perisynaptic processes could shape synaptic transmission at glutamatergic synapses.

In the neonate forebrain, network formation is driven by the spontaneous synchronized activity of pyramidal cells and interneurons, consisting of bursts of electrical activity and intracellular Ca2+ oscillations. By employing ratiometric Na+ imaging in tissue slices obtained from animals at postnatal day 2–4 (P2–4), we found that 20% of pyramidal neurons and 44% of astrocytes in neonatal mouse hippocampus also exhibit transient fluctuations in intracellular Na+. These occurred at very low frequencies (~2/h), were exceptionally long (~8 min), and strongly declined after the first postnatal week. Similar Na+ fluctuations were also observed in the neonate neocortex. In the hippocampus, Na+ elevations in both cell types were diminished when blocking action potential generation with tetrodotoxin. Neuronal Na+ fluctuations were significantly reduced by bicuculline, suggesting the involvement of GABAA-receptors in their generation. Astrocytic signals, by contrast, were neither blocked by inhibition of receptors and/or transporters for different transmitters including GABA and glutamate, nor of various Na+-dependent transporters or Na+-permeable channels. In summary, our results demonstrate for the first time that neonatal astrocytes and neurons display spontaneous ultraslow Na+ fluctuations. While neuronal Na+ signals apparently largely rely on suprathreshold GABAergic excitation, astrocytic Na+ signals, albeit being dependent on neuronal action potentials, appear to have a separate trigger and mechanism, the source of which remains unclear at present.

Astrocytes are the main cell type responsible for the regulation of brain homeostasis, including the maintenance of ion gradients and neurotransmitter clearance. These processes are tightly coupled to changes in the intracellular sodium (Na +) concentration. While activation of the sodium-potassium-ATPase (NKA) in response to an elevation of extracellular K + may decrease intracellular Na + , the cotransport of transmitters, such as glutamate, together with Na + results in an increase in astrocytic Na +. This increase in intracellular Na + can modulate, for instance, metabolic downstream pathways. Thereby, astrocytes are capable to react on a fast time scale to surrounding neuronal activity via intracellular Na + fluctuations and adjust energy production to the demand of their environment. Beside the well-documented conventional roles of Na + signaling mainly mediated through changes in its electrochemical gradient, several recent studies have identified more atypical roles for Na + , including protein interactions leading to changes in their biochemical activity or Na +-dependent regulation of gene expression. In this review, we will address both the conventional as well as the atypical functions of astrocytic Na + signaling, presenting the role of transporters and channels involved and their implications for physiological processes in the central nervous system (CNS). We will also discuss how these important functions are affected under pathological conditions, including stroke and migraine. We postulate that Na + is an essential player not only in the maintenance of homeostatic processes but also as a messenger for the fast communication between neurons and astrocytes, adjusting the functional properties of various cellular interaction partners to the needs of the surrounding network.

In the rodent forebrain, the majority of astrocytes are generated during the early postnatal phase. Following differentiation, astrocytes undergo maturation which accompanies the development of the neuronal network. Neonate astrocytes exhibit a distinct morphology and domain size which differs to their mature counterparts. Moreover, many of the plasma membrane proteins prototypical for fully developed astrocytes are only expressed at low levels at neonatal stages. These include connexins and Kir4.1, which define the low membrane resistance and highly negative membrane potential of mature astrocytes. Newborn astrocytes moreover express only low amounts of GLT-1, a glutamate transporter critical later in development.Furthermore, they show specific differences in the properties and spatio-temporal pattern of intracellular calcium signals, resulting from differences in their repertoire of receptors and signalling pathways. Therefore, roles fulfilled by mature astrocytes, including ion and transmitter homeostasis, are underdeveloped in the young brain.Similarly, astrocytic ion signalling in response to neuronal activity, a process central to neuron-glia interaction, differs between the neonate and mature brain. This review describes the unique functional properties of astrocytes in the first weeks after birth and compares them to later stages of development. We conclude that with an immature neuronal network and wider extracellular space, astrocytic support might not be as demanding and critical compared to the mature brain. The delayed differentiation and maturation of astrocytes in the first postnatal weeks might thus reflect a reduced need for active, energy-consuming regulation of the extracellular space and a less tight control of glial feedback onto synaptic transmission.

Fluorescent dyes and genetically encoded fluorescence indicators (GEFI) are common tools for visualizing concentration changes of specific ions and messenger molecules during intra- as well as intercellular communication. Using advanced imaging technologies, fluorescence indicators are a prerequisite for the analysis of physiological molecular signaling. Automated detection and analysis of fluorescence signals requires to overcome several challenges, including correct estimation of fluorescence fluctuations at basal concentrations of messenger molecules, detection and extraction of events themselves as well as proper segmentation of neighboring events. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. Here, we present two algorithms (PBasE and CoRoDe) for accurate baseline estimation of fluorescent detection of messenger molecules and automated detection of fluorescence fluctuations.

21Spontaneous neuronal and astrocytic activity in the neonate forebrain is believed to drive 22 Author Summary 46Spontaneous neuronal and astrocytic activity during the early postnatal period is crucial to 47 the development and physiology of the neonate forebrain. Elucidating the origin of this activity is 48 key to our understanding of the cell maturation and formation of brain-region-specific networks. 49This study reports spontaneous, ultraslow, large-amplitude, long-lasting fluctuations in the 50 intracellular Na + concentration of neurons and astrocytes in the hippocampus of mice at postnatal 51 days 2-4 that mostly disappear after the first postnatal week. We combine ratiometric Na + imaging 52 and pharmacological manipulations with a detailed computational model of neuronal networks in 53 the neonatal and adult brain to provide key insights into the origin of these Na + fluctuations. 54 Furthermore, our model predicts that these periods of spontaneous Na + influx leave neonatal 55 neuronal networks more vulnerable to hyperactivity when compared to mature brain. 56 57 Spontaneous neuronal activity is a hallmark of the developing central nervous system [1], 58 and has been described in terms of intracellular Ca 2+ oscillations both in neurons and astrocytes 59 [2][3][4][5] and bursts of neuronal action potentials [6][7][8]. This activity is believed to promote the 60 maturation of individual cells and their integration into complex brain-region-specific networks 61 [1,[9][10][11]. In the rodent hippocampus, early network activity and Ca 2+ oscillations are mainly 62 attributed to the excitatory role of GABAergic transmission originating from inhibitory neurons 63 [7,[12][13][14]. 64The excitatory action of GABAergic neurotransmission is one of the most notable 65 characteristics that distinguish neonate brain from the mature brain, where GABA typically 66 inhibits neuronal networks [1, 7, 8,[10][11][12][15][16][17]. While recent work has also called the inhibitory 67 action of GABA on cortical networks into question [18], there are many other pathways that could 68 play a significant role in the observed spontaneous activity in neonate brain (discussed below). 69Additional key features of the early network oscillations in the hippocampus include their 70 synchronous behavior across most of the neuronal network, modulation by glutamate, recurrence 71 with regular frequency, and a limitation to early post-natal development [2, 7, 12]. 72More recently, Felix and co-workers [5] reported a new form of seemingly spontaneous 73 activity in acutely isolated tissue slices of hippocampus and cortex of neonatal mice. It consists of 74 spontaneous fluctuations in intracellular Na + both in astrocytes and neurons, which occur in ~25% 75 of pyramidal neurons and ~40% of astrocytes tested. Na + fluctuations are ultraslow in nature, 76 averaging ~2 fluctuations/hour, are not synchronized between cells, and are not significantly 77 affected by an array of pharmacological blockers for various channels, receptors, and transpo...

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Spontaneous neuronal and astrocytic activity in the neonate forebrain is believed to drive the maturation of individual cells and their integration into complex brain-region-specific networks. The previously reported forms include bursts of electrical activity and oscillations in intracellular Ca2+ concentration. Here, we use ratiometric Na+ imaging to demonstrate spontaneous fluctuations in the intracellular Na+ concentration of CA1 pyramidal neurons and astrocytes in tissue slices obtained from the hippocampus of mice at postnatal days 2-4 (P2-4). These occur at very low frequency (~2/h), can last minutes with amplitudes up to several mM, and mostly disappear after the first postnatal week. To further investigate their mechanisms, we model a network consisting of pyramidal neurons and interneurons. Experimentally observed Na+ fluctuations are mimicked when GABAergic inhibition in the simulated network is made depolarizing. Our experiments and computational model show that blocking voltage-gated Na+ channels or GABAergic signaling significantly diminish the neuronal Na+ fluctuations. On the other hand, blocking a variety of other ion channels, receptors, or transporters including glutamatergic pathways, does not have significant effects. Our model also shows that the amplitude and duration of Na+ fluctuations decrease as we increase the strength of glial K+ uptake. Furthermore, neurons with smaller somatic volumes exhibit fluctuations with higher frequency and amplitude. As opposed to this, larger extracellular to intracellular volume ratio observed in neonatal brain exerts a dampening effect. Finally, our model predicts that these periods of spontaneous Na+ influx leave neonatal neuronal networks more vulnerable to seizure-like states when compared to mature brain.