Physiology, Pathophysiology and Clinical Relevance of D-Amino Acids Dynamics: From Neurochemistry to Pharmacotherapy.

Over three decades ago, two independent groups of investigators identified free D-aspartic and later D-serine in specific brain nuclei and endocrine glands. This finding revealed a novel, non-proteinogenic role of these molecules. Moreover, the finding that aged proteins from the human eye crystallin, teeth, bone, blood vessels or the brain incorporate D-aspartic acids to specific primary protein sequences fostered the hypothesis that aging might be related to D-amino acid isomerization of body proteins. The experimental confirmation that schizophrenia and neurodegenerative diseases modify plasma free D-amino acids or tissue levelsnurtured the opportunity of using D-amino acids as therapeutic agents for several disease treatments, a strategy that prompted the successful current application of D-amino acids to human medicine.

Enhanced Astrocyte Activity and Excitatory Synaptic Function in the Hippocampus of Pentylenetetrazole Kindling Model of Epilepsy.

Epilepsy is a chronic condition characterized by recurrent spontaneous seizures. The interaction between astrocytes and neurons has been suggested to play a role in the abnormal neuronal activity observed in epilepsy. However, the exact way astrocytes influence neuronal activity in the epileptogenic brain remains unclear. Here, using the PTZ-induced kindling mouse model, we evaluated the interaction between astrocyte and synaptic function by measuring astrocytic Ca2+ activity, neuronal excitability, and the excitatory/inhibitory balance in the hippocampus. Compared to control mice, hippocampal slices from PTZ-kindled mice displayed an increase in glial fibrillary acidic protein (GFAP) levels and an abnormal pattern of intracellular Ca2+-oscillations, characterized by an increased frequency of prolonged spontaneous transients. PTZ-kindled hippocampal slices also showed an increase in the E/I ratio towards excitation, likely resulting from an augmented release probability of excitatory inputs without affecting inhibitory synapses. Notably, the alterations in the release probability seen in PTZ-kindled slices can be recovered by reducing astrocyte hyperactivity with the reversible toxin fluorocitrate. This suggests that astroglial hyper-reactivity enhances excitatory synaptic transmission, thereby impacting the E/I balance in the hippocampus. Altogether, our findings support the notion that abnormal astrocyte-neuron interactions are pivotal mechanisms in epileptogenesis.

GABAergic Regulation of Astroglial Gliotransmission through Cx43 Hemichannels.

Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA activates the influx of Cl- via the GABAA receptor or efflux or K+ via the GABAB receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABAA and GABAB receptors induces an increase in intracellular Ca2+ and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca2+-dependent cellular mechanisms for the astroglial release of glutamate and ATP. However, no study has evaluated the effect of GABA on astroglial Cx43 hemichannel activity and Cx43 hemichannel-mediated gliotransmission. Here we assessed the effects of GABA on Cx43 hemichannel activity in DI NCT1 rat astrocytes and hippocampal brain slices. We found that GABA induces a Ca2+-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABAA receptor, as it was blunted by the GABAA receptor antagonist bicuculline but unaffected by GABAB receptor antagonist CGP55845. Moreover, GABA induced the Cx43 hemichannel-dependent release of glutamate and ATP, which was also prevented by bicuculline, but unaffected by CGP. Gliotransmission in response to GABA was also unaffected by pannexin 1 channel blockade. These results are discussed in terms of the possible role of astroglial Cx43 hemichannel-mediated glutamate and ATP release in regulating the excitatory/inhibitory balance in the brain and their possible contribution to psychiatric disorders.

Revealing the contribution of astrocytes to glutamatergic neuronal transmission.

Research on glutamatergic neurotransmission has focused mainly on the function of presynaptic and postsynaptic neurons, leaving astrocytes with a secondary role only to ensure successful neurotransmission. However, recent evidence indicates that astrocytes contribute actively and even regulate neuronal transmission at different levels. This review establishes a framework by comparing glutamatergic components between neurons and astrocytes to examine how astrocytes modulate or otherwise influence neuronal transmission. We have included the most recent findings about the role of astrocytes in neurotransmission, allowing us to understand the complex network of neuron-astrocyte interactions. However, despite the knowledge of synaptic modulation by astrocytes, their contribution to specific physiological and pathological conditions remains to be elucidated. A full understanding of the astrocyte's role in neuronal processing could open fruitful new frontiers in the development of therapeutic applications.

P2Y1 receptor inhibition rescues impaired synaptic plasticity and astroglial Ca2+-dependent activity in the epileptic hippocampus.

Epilepsy is characterized by a progressive predisposition to suffer seizures due to neuronal hyperexcitability, and one of its most common co-morbidities is cognitive decline. In animal models of chronic epilepsy, such as kindling, electrically induced seizures impair long-term potentiation (LTP), deteriorating learning and memory performance. Astrocytes are known to actively modulate synaptic plasticity and neuronal excitability through Ca2+-dependent gliotransmitter release. It is unclear, however, if astroglial Ca2+ signaling could contribute to the development of synaptic plasticity alterations in the epileptic hippocampus. By employing electrophysiological tools and Ca2+ imaging, we found that glutamatergic CA3-CA1 synapses from kindled rats exhibit an impairment in theta burst (TBS) and high frequency stimulation (HFS)-induced LTP, which is accompanied by an increased probability of neurotransmitter release (Pr) and an abnormal pattern of astroglial Ca2+-dependent transients. Both the impairment in LTP and the Pr were reversed by inhibiting purinergic P2Y1 receptors (P2Y1R) with the specific antagonist MRS2179, which also restored the spontaneous and TBS-induced pattern of astroglial Ca2+-dependent signals. Two consecutive, spaced TBS protocols also failed to induce LTP in the kindled group, however, this impairment was reversed and a strong LTP was induced when the second TBS was applied in the presence of MRS2179, suggesting that the mechanisms underlying the alterations in TBS-induced LTP are likely associated with an aberrant modulation of the induction threshold for LTP. Altogether, these results indicate that P2Y1R inhibition rescues both the pattern of astroglial Ca2+-activity and the plastic properties of CA3-CA1 synapses in the epileptic hippocampus, suggesting that astrocytes might take part in the mechanisms that deteriorate synaptic plasticity and thus cause cognitive decline in epileptic patients.

Gliotransmission: A Novel Target for the Development of Antiseizure Drugs.

For more than a century, epilepsy has remained an incapacitating neurological disorder with a high incidence worldwide. Mesial temporal lobe epilepsy (TLE) is a common type of epilepsy without an effective pharmacological treatment. An increase in excitability and hypersynchrony of electrical neuronal activity during development are typically associated with an excitatory/inhibitory imbalance in the neuronal network. Astrocytes release gliotransmitters, which can regulate neuronal excitability and synaptic transmission; therefore, the classical neurocentric vision of the cellular basis of epileptogenesis has begun to change. Growing evidence suggests that the key contribution of astrocyte-to-neuron signaling in the mechanisms underlies the initiation, propagation, and recurrence of seizure activity. The aim of this review was to summarize current evidence obtained from experimental models that suggest how alterations in astroglial modulation of synaptic transmission and neuronal activity contribute to the development of this brain disease. In this article, we will summarize the main pharmacological, Ca2+-imaging, and electrophysiological findings in the gliotransmitter-mediated modulation of neuronal activity and their possible regulation as a novel cellular target for the development of pharmacological strategies for treating refractory epilepsies.

Astroglial Ca2+-Dependent Hyperexcitability Requires P2Y1 Purinergic Receptors and Pannexin-1 Channel Activation in a Chronic Model of Epilepsy.

Astrocytes from the hippocampus of chronic epileptic rats exhibit an abnormal pattern of intracellular calcium oscillations, characterized by an augmented frequency of long lasting spontaneous Ca2+ transients, which are sensitive to purinergic receptor antagonists but resistant to tetrodotoxin. The above suggests that alterations in astroglial Ca2+-dependent excitability observed in the epileptic tissue could arise from changes in astrocyte-to-astrocyte signaling, which is mainly mediated by purines in physiological and pathological conditions. In spite of that, how purinergic signaling contributes to astrocyte dysfunction in epilepsy remains unclear. Here, we assessed the possible contribution of P2Y1R as well as pannexin1 and connexin43 hemichannels-both candidates for non-vesicular ATP-release-by performing astroglial Ca2+ imaging and dye uptake experiments in hippocampal slices from control and fully kindled rats. P2Y1R blockade with MRS2179 decreased the mean duration of astroglial Ca2+ oscillations by reducing the frequency of slow Ca2+ transients, and thereby restoring the balance between slow (ST) and fast transients (FT) in the kindled group. The potential contribution of astroglial pannexin1 and connexin43 hemichannels as pathways for purine release (e.g., ATP) was assessed through dye uptake experiments. Astrocytes from kindled hippocampi exhibit three-fold more EtBr uptake than controls, whereby pannexin1 hemichannels (Panx1 HCs) accounts for almost all dye uptake with only a slight contribution from connexin43 hemichannels (Cx43 HCs). Confirming its functional involvement, Panx1 HCs inhibition decreased the mean duration of astroglial Ca2+ transients and the frequency of slow oscillations in kindled slices, but had no noticeable effects on the control group. As expected, Cx43 HCs blockade did not have any effects over the mean duration of astroglial Ca2+ oscillations. These findings suggest that P2Y1R and Panx1 HCs play a pivotal role in astroglial pathophysiology, which would explain the upregulation of glutamatergic neurotransmission in the epileptic brain and thus represents a new potential pharmacological target for the treatment of drug-refractory epilepsy.

Connexin-Mediated Functional and Metabolic Coupling Between Astrocytes and Neurons.

The central nervous system (CNS) requires sophisticated regulation of neuronal activity. This modulation is partly accomplished by non-neuronal cells, characterized by the presence of transmembrane gap junctions (GJs) and hemichannels (HCs). This allows small molecule diffusion to guarantee neuronal synaptic activity and plasticity. Astrocytes are metabolically and functionally coupled to neurons by the uptake, binding and recycling of neurotransmitters. In addition, astrocytes release metabolites, such as glutamate, glutamine, D-serine, adenosine triphosphate (ATP) and lactate, regulating synaptic activity and plasticity by pre- and postsynaptic mechanisms. Uncoupling neuroglial communication leads to alterations in synaptic transmission that can be detrimental to neuronal circuit function and behavior. Therefore, understanding the pathways and mechanisms involved in this intercellular communication is fundamental for the search of new targets that can be used for several neurological disease treatments. This review will focus on molecular mechanisms mediating physiological and pathological coupling between astrocytes and neurons through GJs and HCs.

Enhanced astroglial Ca2+ signaling increases excitatory synaptic strength in the epileptic brain.

The fine-tuning of synaptic transmission by astrocyte signaling is crucial to CNS physiology. However, how exactly astroglial excitability and gliotransmission are affected in several neuropathologies, including epilepsy, remains unclear. Here, using a chronic model of temporal lobe epilepsy (TLE) in rats, we found that astrocytes from astrogliotic hippocampal slices displayed an augmented incidence of TTX-insensitive spontaneous slow Ca(2+) transients (STs), suggesting a hyperexcitable pattern of astroglial activity. As a consequence, elevated glutamate-mediated gliotransmission, observed as increased slow inward current (SICs) frequency, up-regulates the probability of neurotransmitter release in CA3-CA1 synapses. Selective blockade of spontaneous astroglial Ca(2+) elevations as well as the inhibition of purinergic P2Y1 or mGluR5 receptors relieves the abnormal enhancement of synaptic strength. Moreover, mGluR5 blockade eliminates any synaptic effects induced by P2Y1R inhibition alone, suggesting that the Pr modulation via mGluR occurs downstream of P2Y1R-mediated Ca(2+)-dependent glutamate release from astrocyte. Our findings show that elevated Ca(2+)-dependent glutamate gliotransmission from hyperexcitable astrocytes up-regulates excitatory neurotransmission in epileptic hippocampus, suggesting that gliotransmission should be considered as a novel functional key in a broad spectrum of neuropathological conditions.

Channel-mediated lactate release by K⁺-stimulated astrocytes.

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

Glial cells modulate the synaptic transmission of NTS neurons sending projections to ventral medulla of Wistar rats.

There is evidence that sympathoexcitatory and respiratory responses to chemoreflex activation involve ventrolateral medulla-projecting nucleus tractus solitarius (NTS) neurons (NTS-VLM neurons) and also that ATP modulates this neurotransmission. Here, we evaluated whether or not astrocytes is the source of endogenous ATP modulating the synaptic transmission in NTS-VLM neurons. Synaptic activities of putative astrocytes or NTS-VLM neurons were recorded using whole cell patch clamp. Tractus solitarius (TS) stimulation induced TS-evoked excitatory postsynaptic currents (TS-eEPSCs) in NTS-VLM neurons as well in NTS putative astrocytes, which were also identified by previous labeling. Fluoracetate (FAC), an inhibitor of glial metabolism, reduced TS-eEPSCs amplitude (-85.6 ± 16 vs. -39 ± 7.1 pA, n = 12) and sEPSCs frequency (2.8 ± 0.5 vs. 1.8 ± 0.46 Hz, n = 10) in recorded NTS-VLM neurons, indicating a gliomodulation of glutamatergic currents. To verify the involvement of endogenous ATP a purinergic antagonist was used, which reduced the TS-eEPSCs amplitude (-207 ± 50 vs. -149 ± 50 pA, n = 6), the sEPSCs frequency (1.19 ± 0.2 vs. 0.62 ± 0.11 Hz, n = 6), and increased the paired-pulse ratio (PPR) values (∼20%) in NTS-VLM neurons. Simultaneous perfusion of Pyridoxalphosphate-6-azophenyl-2',5'-disulfonic acid (iso-PPADS) and FAC produced reduction in TS-eEPSCs similar to that observed with iso-PPADS or FAC alone, indicating that glial cells are the source of ATP released after TS stimulation. Extracellular ATP measurement showed that FAC reduced evoked and spontaneous ATP release. All together these data show that putative astrocytes are the source of endogenous ATP, which via activation of presynaptic P2X receptors, facilitates the evoked glutamate release and increases the synaptic transmission efficacy in the NTS-VLM neurons probably involved with the peripheral chemoreflex pathways.