Cerebral edema affects millions of people worldwide and is associated with a plethora of diseases, disorders, and conditions such as traumatic brain injury, stroke, cardiac arrest, autism and epilepsy. Cellular edema has been known to increase epileptiform activity and seizure susceptibility in vitro and in vivo. However, the identity of the cell types undergoing volume increases and the types of excitability changes that occur in neurons remain unclear. Electrophysiological whole-cell patch clamp techniques were used to record currents and potentials from CA1 pyramidal neurons during the application of hypoosmolar ACSF (hACSF) in acutely isolated hippocampal slices from mice. Hypoosmolar ACSF evoked slow inward currents (SICs) in neurons, which initiated after ~1 minute of hACSF application. Neuronal excitability increased as osmolarity decreased in a dose-dependent manner. Even 5% reductions in osmolarity were sufficient to significantly increase neuronal excitability. In addition, hACSF induced neuronal firing of action potentials (APs), independent of AMPA receptor activation. Neuronal excitability was also increased during application of hACSF while blocking both APs and AMPA receptors. Increased sub-threshold EPSPs, neuronal APs, and bursting activity were also evoked in the presence of Mg2+, suggesting that hypoosmolar insults increase neuronal excitability under more physiological conditions. Hypoosmolar insults increased neuronal excitability in both juvenile (P15-P21) and adult 2- to 5-month-old) mice. Bursting activity in adult mice during osmotic insult was elevated compared to juvenile mice. During hypoosmolar insults the frequency of SICs recorded at physiological temperature were significantly elevated from SICs recorded at room temperature. SICs were potentiated by D-serine, and blocked by both DL-AP5 and the NR2B specific compound Ro25-6981. Together, these results indicate that osmotic insults produce cellular edema in both neurons and astrocytes, and increase neuronal excitability within minutes through a combination of synaptic and non-synaptic activation of glutamate receptors.

Abstract:

Neurotransmitter and ion influx into astrocytes generates osmotic gradients coupled to water movement into the cell, resulting in transient or prolonged fluctuations in cell volume. Increases in cell volume reduce the size of the extracellular space (ECS) and are associated with elevated brain tissue excitability. However, the precise mechanisms at play in coupling astrocyte volume changes to ion movements remain controversial, as does the effect of acute astrocyte volume fluctuations on neuronal function. Here we set out to determine the effects of raised extracellular potassium concentrations ( [K+]o) on volume responses of CA1 pyramidal neurons and stratum radiatum astrocytes in the hippocampus. First, we found that elevated [K+]o within a physiological range (6.5 and 10.5 mM from a baseline of 2.5 mM) and up to 26 mM produces dose-dependent increases in astrocyte volume, with no effect on neuronal volume. Astrocyte volume increases in elevated [K+]o were not dependent on AQP4, Kir4.1, the sodium-bicarbonate cotransporter NBCe1, or the electroneutral cotransporter NKCC1, but were significantly attenuated in 1 mM BaCl2 and by the Na+/K+ pump inhibitor ouabain, suggesting that astrocyte volume increases are due to K+ influx from nonspecific K+ channels and/or the Na+/K+ ATPase. High [K+]o-induced astrocyte swelling resulted in significant increases in neuronal excitability in the form of NMDA receptor-dependent slow inward currents (SICs) and mixed AMPA/NMDA mEPSCs. Direct depolarizing effects of high [K+]o on neuronal spiking were prevented by application of TTX, and the amount of depolarization was insufficient to activate voltage-gated Ca2+ channels, suggesting that changes in neuronal excitability were not due to elevated [K+]o-related increases in synaptic transmission. Finally, we show that astrocyte-specific swelling in elevated [K+]o and effects on neuronal excitability can be completely negated by addition of mannitol, which we found selectively shrinks astrocytes. Overall, our findings suggest that astrocyte-selective volume increases in elevated [K+]o conditions are due to activity of the Na+/K+ ATPase, which result in astrocyte-specific increases in neuronal excitability independent of direct depolarizing effects of high [K+]o on neurons.

Astrocytic glutamate transporters, GLAST and GLT-1 (rodent analogs of EAAT1 and EAAT2, respectively) constitute the major pathway of glutamate uptake in the central nervous system. Uptake of synaptically released glutamate by astrocytes is essential for maintaining a healthy level of excitatory activity in the brain. It is also known that astrocytes in situ possess a myriad of G-protein coupled receptors (GPCRs) of the Gi, Gs, and Gq families. Stimulation of glutamatergic afferents not only leads to astrocytic glutamate uptake but also has been shown to stimulate group I (Gq type) metabotropic glutamate receptor (mGluR) mediated intracellular Ca2+ elevations in astrocytes both in vivo and in situ. Activation of mGluRs has been associated with short and long-term plastic changes of glutamate transport in cultured astroglia and in Purkinje neurons in cerebellar slices. This led us to hypothesize that astrocytic Gq GPCRs modulate glutamate transport. This hypothesis was tested in acute mouse hippocampal slices using two different approaches: (1) activation of astrocytic mGluRs in slices from wild-type mice using a tetanic high frequency stimulus (200Hz, 1s) applied to the Schaffer collaterals (SCs); (2) activation of a transgenic Gq GPCR (the MrgA1R) which is selectively expressed by astrocytes. Synaptically evoked glutamate transporter currents (STCs) were isolated from the total evoked astrocytic currents and analyzed for changes in amplitude, rise time, rise slope and decay time constant. High frequency stimulation (HFS) of SCs led to potentiation of the amplitude of the STCs without changes in kinetics. Similar potentiation was not observed in the presence of group I mGluRs or the PKC inhibitor, PKC 19-36, suggesting that HFS induced potentiation of glutamate uptake is group I mGluR - PKC dependent. Activation of the MrgA1Rs selectively expressed by astrocytes also potentiated the STC amplitude indicating the sufficiency of astrocytic Gq GPCR activation for potentiation of glutamate uptake. Surprisingly, the amplitude of the slow inward astrocytic current due to potassium uptake is also enhanced following activation of either the endogenous mGluRs or the astrocyte-specific MrgA1Rs. These findings collectively suggest that astrocytic Gq GPCR activation has a synergistic modulatory effect on the uptake of both glutamate and potassium.

In addition to synaptic communication between neurons, there is now strong evidence for neuron-to-astrocyte receptor signaling in the brain. During trains of action potentials or repetitive stimulation, neurotransmitter spills out of the synapse to activate astrocytic Gq protein-coupled receptors (Gq GPCRs). To date, very little is known about the ability of astrocytic receptors to exhibit plasticity as a result of long-term changes in neuronal firing rates. Here we describe for the first time bidirectional scaling of astrocytic group 1 metabotropic glutamate receptor (mGluR) signaling in acute mouse hippocampal slices on a rapid timescale following either long-term blockade or increase in neuronal synaptic transmission. Plasticity of astrocytic mGluRs was measured by recording changes in spontaneous and evoked astrocyte Ca2+ elevations in both astrocytic soma as well as fine processes. In response to 4 to 6 hour blockade of CA3-CA1 neurotransmission, the following changes in astrocyte Ca2+ signaling were observed: 1) a significant increase in the percentage of astrocytes in the slice population exhibiting spontaneous Ca2+ elevations; 2) significantly faster rise times of the spontaneous Ca2+ transients; 3) a significant increase in response probability to the group I mGluR agonist; 4) significantly faster rise times of evoked Ca2+ responses; 5) significantly shorter response latencies of evoked Ca2+ responses in astrocyte microdomains; and 6) a dose-dependent shift in astrocytic responses to DHPG in TTX vs. control incubated slices. In response to 4 to 6 hour elevation of CA3-CA1 neurotransmission, the opposite effects on the previous parameters were observed. Further study using transgenic mice expressing a novel Gq GPCR suggested that the changes observed in astrocytic group I mGluR Ca2+ signaling were due to changes in expression of the group I mGluRs in astrocytes, while the intracellular signaling pathway activated by the Gq GPCRs remained unchanged. This study introduces a sensitive assay for recording changes in astrocytic Gq GPCR expression levels, and the results demonstrate active astrocytic detection of basal and elevated frequencies of neuronal action potentials that lie within a physiological range.

The physiological role of astrocytic Gq-protein coupled receptors (Gq GPCRs) has now drawn more attention in the field of neuroscience, as it is now clear that astrocytes sense neuronal signals through activation of their Gq GPCRs. Astrocytes are thus considered excitable and their role in synaptic transmission is under intense investigation. Interestingly, in basal conditions without any user-evoked stimulation, astrocytes exhibit spontaneous Gq GPCR activity driven by mechanisms that still remain mysterious. Understanding the mechanisms underlying these astrocytic Gq GPCR signaling domains in physiological conditions will certainly benefit our knowledge regarding neuron-to-astrocyte communication. Therefore, the main goal of this dissertation is to study the underlying mechanisms of astrocytic Gq GPCR signaling domains in two parts: 1) To identify the factors governing spontaneous astrocytic Gq GPCR activity, and 2) To identify the lowest threshold of neuronal action potential-mediated synaptic transmission capable of evoking an astrocytic Gq GPCR response, and then to determine if the response occurs as a microdomain or a whole-cell event. In Chapter 2, we demonstrate that spontaneous astrocytic Gq GPCR signaling domains are driven by mechanisms unrelated to action potential-triggered neurotransmitter release, but are dependent on spontaneous miniature neurotransmitter release. It also appears that multiple types of astrocytic Gq GPCRs play essential roles in the generation of spontaneous Gq GPCR activity. In Chapter 3, our results suggest that astrocytes respond to neuronal afferent stimulation at intensities much lower than previously described. Moreover, the evoked Gq GPCR domains are qualitatively different from the spontaneous ones. Consistent with the findings from Chapter 2, the evoked events appear to involve multiple types of astrocytic Gq GPCRs. These data suggest that astrocytes respond to neuronal activity in a manner much more sensitive than previously thought.

We also explored the role of beta-Arrestin2 in regulating two forms of synaptic plasticity-long-term potentiation (LTP) and long-term depression (LTD). In Chapter 4, we report normal LTP, but a markedly impaired LTD in beta-Arrestin2 knockout mice, suggesting a novel role of beta-Arrestin2 in cellular mechanisms of learning and memory. This finding could potentially provide a base for developing treatments for dementia-related disorders such as Alzheimer's disease.

Epilepsy, a spectrum of over 40 different disorders, is estimated to affect 1 in 26 people worldwide. It is generally characterized by the appearance of spontaneous, recurrent and unpredictable seizures. Approximately one-third of epileptic patients cannot control their seizures with current medications, while the remaining two-thirds of patients often experience negative cognitive side effects, highlighting the need to better understand epilepsy mechanisms. A common theme in multiple seizure models is that cellular swelling is necessary for seizure initiation and recurrence. In experiments described in this dissertation, I set out to determine: 1) The extent to which neurons and astrocytes swell in two experimental conditions that lead to neuronal bursting and epileptiform activity; 2) The mechanisms governing neuronal vs. astrocyte swelling; and 3) The contribution of astrocytes, specifically, to increases in neuronal excitability. These experiments were carried out in acute hippocampal slices from wild type and transgenic mice using a combination of electrophysiology and imaging approaches. The main findings from these studies were as follows: 1) Both reduced extracellular osmolarity and elevations of extracellular potassium ions ([K+]o) elevated neuronal excitability within minutes in an NMDA receptor-dependent manner; 2) Contrary to published reports, neurons are not osmoresistant and swell just as readily as astrocytes in hypoosmolar conditions, while astrocytes swell selectively in elevated [K+]o conditions; 3) Neuronal swelling is not an artifact of our experimental approaches, nor is it a result of NMDA receptor-driven excitotoxicity; and 4) Astrocytic volume-regulated anion channels (VRAC) may contribute to increased excitability of neurons through release of glutamate. Taken together, these findings have important implications for seizure disorders and for understanding the scope of neuron-glial interactions in the brain.

Astrocytes are a type of glial cell found in the CNS and play important physiological roles in maintaining homeostasis of the extracellular space. They are implicated in maintaining extracellular concentrations of certain ions and molecules, including the excitatory neurotransmitter glutamate. Astrocyte volume is strongly effected by changes in the extracellular environment, a phenomenon that is widely recognized and undisputed. Under both physiological and pathological circumstances, astrocyte volume changes can occur and have noticeable effects on the activity of adjacent neurons. This dissertation aimed to (1) characterize astrocyte volume increases that occur in the presence of elevated extracellular potassium, (2) isolate astrocyte-swelling-specific effects on neuronal excitability in a model of elevated extracellular potassium, and (3) ascertain the role of volume-regulated anion channel function in astrocyte volume change and alterations in neuronal excitability.