Microglial morphology and dynamic behavior is regulated by ionotropic glutamatergic and GABAergic neurotransmission

Purpose

Microglia represent the primary resident immune cells in the CNS, and have been implicated in the pathology of neurodegenerative diseases. Under basal or “resting” conditions, microglia possess ramified morphologies and exhibit dynamic surveying movements in their processes. Despite the prominence of this phenomenon, the function and regulation of microglial morphology and dynamic behavior are incompletely understood. We investigate here whether and how neurotransmission regulates “resting” microglial morphology and behavior.

Methods

We employed an ex vivo mouse retinal explant system in which endogenous neurotransmission and dynamic microglial behavior are present. We utilized live-cell time-lapse confocal imaging to study the morphology and behavior of GFP-labeled retinal microglia in response to neurotransmitter agonists and antagonists. Patch clamp electrophysiology and immunohistochemical localization of glutamate receptors were also used to investigate direct-versus-indirect effects of neurotransmission by microglia.

Results

Retinal microglial morphology and dynamic behavior were not cell-autonomously regulated but are instead modulated by endogenous neurotransmission. Morphological parameters and process motility were differentially regulated by different modes of neurotransmission and were increased by ionotropic glutamatergic neurotransmission and decreased by ionotropic GABAergic neurotransmission. These neurotransmitter influences on retinal microglia were however unlikely to be directly mediated; local applications of neurotransmitters were unable to elicit electrical responses on microglia patch-clamp recordings and ionotropic glutamatergic receptors were not located on microglial cell bodies or processes by immunofluorescent labeling. Instead, these influences were mediated indirectly via extracellular ATP, released in response to glutamatergic neurotransmission through probenecid-sensitive pannexin hemichannels.

Conclusions

Our results demonstrate that neurotransmission plays an endogenous role in regulating the morphology and behavior of “resting” microglia in the retina. These findings illustrate a mode of constitutive signaling between the neural and immune compartments of the CNS through which immune cells may be regulated in concert with levels of neural activity.     

Role of astrocytes in the maintenance and modulation of glutamatergic and GABAergic neurotransmission

The functional activity in the brain is primarily composed of an interplay between excitation and inhibition. In any given region the output is based upon a complex processing of incoming signals that require both excitatory and inhibitory units. Moreover, these units must be regulated and balanced such that an integrated and finely tuned response is generated. In each of these units or synapses the activity depends on biosynthesis, release, receptor interaction, and inactivation of the neurotransmitter in question; thus, it is easily understood that each of these processes needs to be highly regulated and controlled. It is interesting to note that in case of the most prevailing neurotransmitters, glutamate and GABA, which mediate excitation and inhibition, respectively, the inactivation process is primarily maintained by highly efficient, high-affinity transport systems capable of maintaining transmembrane concentration gradients of these amino acids of 10⁴–10⁵-fold. The demonstration of the presence of transporters for glutamate and GABA in both neuronal and astrocytic elements naturally raises the question of the functional importance of the astrocytes in the regulation of the level of the neurotransmitters in the synaptic cleft and hence for the activity of excitatory and inhibitory neurotransmission. Obviously, this discussion has important implications for the understanding of the role of astrocytes in disease states in which imbalances between excitation and inhibition are a triggering factor, for example, epilepsy and neurodegeneration.     

Regulation of glutamatergic neurotransmission in the striatum by presynaptic adenylyl cyclase-dependent processes

The aim here was to examine the possible roles of adenylyl cyclase- and protein kinase A (PKA)-dependent processes in ionotropic glutamate receptor (iGluR)-mediated neurotransmission using superfused mouse striatal slices and a non-metabolized L-glutamate analogue, D-[3H]aspartate. The direct and indirect presynaptic modulation of glutamate release and its susceptibility to changes in the intracellular levels of cyclic AMP (cAMP), Ca(2+) and calmodulin (CaM) and in protein phosphorylation was characterized by pharmacological manipulations. The agonists of iGluRs, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate, stimulated the basal release of D-[3H]aspartate, while N-methyl-D-aspartate (NMDA) was without effect. Both the AMPA- and kainate-mediated responses were accentuated by the beta-adrenoceptor agonist isoproterenol. These facilitatory effects were mimicked by the permeable cAMP analogue dibutyryl-cAMP. The beta-adrenoceptor antagonist propranolol, the adenylyl cyclase inhibitor MDL12,330A, the inhibitor of PKA and PKC, H-7, and the PKA inhibitor H-89 abolished the isoproterenol effect on the kainate-evoked release. The dibutyryl-cAMP-induced potentiation was also attenuated by H-7. Isoproterenol, propranolol and MDL12,330A failed to affect the basal release of D-[3H]aspartate, but dibutyryl-cAMP was inhibitory and MDL12,330A activatory. In Ca(2+)-free medium, the kainate-evoked release was enhanced, being further accentuated by the CaM antagonists calmidazolium and trifluoperazine, though these inhibited the basal release. The potentiating effect of calmidazolium on the kainate-stimulated release was counteracted by both MDL12,330A and H-7.We conclude that AMPA- and kainate-evoked glutamate release from striatal glutamatergic terminals is potentiated by beta-adrenergic receptor-mediated adenylyl cyclase activation and cAMP accumulation. Glutamate release is enhanced if the Ca(2+)- and CaM-dependent, kainate-evoked processes do not prevent the excessive accumulation of intracellular cAMP.     

Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain

Climate cooling over the past one hundred thousand years has resulted in seasonal ice cover at northern and southern latitudes that has selected for hypoxia and anoxia tolerance in some species, such as freshwater turtles. At the northern reaches of their range, North American freshwater turtles spend 4 months or more buried in the mud bottom of ice covered lakes and ponds. From a comparative perspective this gives us the opportunity to understand how an extremely oxygen-sensitive organ, such as the vertebrate brain, can function without oxygen for long periods. Brain function is based on complex excitatory (on) and inhibitory (off) circuits involving the major neurotransmitters glutamate and, γ-aminobutyric acid (GABA) respectively. When a mammalian brain becomes anoxic, glutamate levels rise within minutes resulting in excitotoxic cell death which does not occur in anoxic turtle brain. The response in turtle brain has been remodelled – GABA levels rise rapidly resulting in large inhibitory GABA receptor currents and inhibition of glutamate receptor function that together depress neuronal activity.     

Neuromuscular glutamatergic and GABAergic channels

Our laboratory has worked extensively on glutamatergic and GABA-ergic channels, predominantly in crayfish, but also in locust,Drosophila and recentlyAscaris. Channel currents were recorded in the different modes of the patch-clamp technique (Hamillet al., 1981). The opening kinetics of the channels were derived from open and closed time histograms obtained from single channel recordings. From these, channel conductances could also be evaluated. The most relevant data were obtained by very rapidly rising and falling pulses (time of change about 0.1 ms) of agonists applied to outside-out patches containing the respective channels (Frankeet al., 1987). From such recordings we constructed dose-response curves for peak and steady-state currents, for the rise times of the currents and for the time constants of desensitization. In double-pulse experiments we measured recovery from desensitization and predesensitization due to low agonist concentrations. For most of the channel types, we succeeded in constructing a reaction scheme which in computer simulations mimicked channel behaviour to a good approximation.     

Redefining chemical anatomy of glutamatergic neurotransmission

With the recent identification of the two isoforms of vesicular glutamate transporters VGLUT1 and VGLUT2 and of the presumed neuronal glutamine transporter SAT1 novel tools have been made available to unequivocally define the anatomy of glutamatergic pathways on the cellular and synaptic level. Using highly specific antisera and cRNA probes two distinct glutamatergic pathways expressing either VGLUT1 or VGLUT2 could be detected throughout the central nervous system. Areas where VGLUT1 predominated included the cerebral and cerebellar cortex and the hippocampus. VGLUT2 was mainly expressed in the thalamus, hypothalamus and brain stem. VGLUT1 and VGLUT2 synapses exhibited distinct region- and pathway-specific relationships with each other and with other classical transmitter and peptidergic systems. The glutamine transporter SAT1 was expressed in CNS neurons and in ependymal cells. Neuronal SAT1 expression comprised virtually all glutamatergic neurons but also specific subsets of cholinergic, GABAergic and aminergic neurons in the CNS. In addition to widespread expression of VGLUT1 and VGLUT2 in the CNS, peripheral tissues such as sensory neurons and pancreatic islet cells differentially expressed VGLUT isoforms and SAT1.
Our results suggest pathway-specific functional duality in the regulation of vesicular glutamate release at excitatory synapses and provide evidence for glutamine transport and metabolism in excitatory glutamatergic and diverse nonglutamatergic neurons as well.     

Modulation of GABAergic neurotransmission in the brain by dipeptides

The effects of endogenous and synthetic peptides containing GABA or its analogues on the GABA/benzodiazepine/chloride ionophore, complex, GABA_B receptor, Cl fluxes, GABA release and GABA uptake were studied using synaptic membranes, crude synaptoneurosomal preparations and slices prepared from the rat and mouse brain. The sodium-independent binding of GABA was strongly inhibited by GABA-histidine, followed by -glutamyl-homotaurine, GABA-glycine and -glutamyl-GABA. The binding of diazepam was slightly enhanced by the same peptides. The peptides alone had no effect on the chloride fluxes, but GABA-histidine, -glutamyl-GABA and GABA-glycine enhanced while -glutamyl-homotaurine and GABA-taurine inhibited GABA-stimulated chloride uptake. GABA-histidine was the most effective displacer of baclofen binding, but -glutamyl-homotaurine was entirely ineffective. The uptake of GABA was markedly inhibited in synaptosomal preparations by GABA-histidine, while all other peptides were less effective. -Glutamyl-taurine attenuated but -glutamyl-homotaurine and GABA-glycine enhanced the potassium-stimulated release of GABA. The present actions of GABA-histidine in vitro may be of significance for GABAergic neurotransmission in vivo.     

GABAergic neurotransmission and retinal ganglion cell function

Ganglion cells are the output retinal neurons that convey visual information to the brain. There are ~20 different types of ganglion cells, each encoding a specific aspect of the visual scene as spatial and temporal contrast, orientation, direction of movement, presence of looming stimuli; etc. Ganglion cell functioning depends on the intrinsic properties of ganglion cell’s membrane as well as on the excitatory and inhibitory inputs that these cells receive from other retinal neurons. GABA is one of the most abundant inhibitory neurotransmitters in the retina. How it modulates the activity of different types of ganglion cells and what is its significance in extracting the basic features from visual scene are questions with fundamental importance in visual neuroscience. The present review summarizes current data concerning the types of membrane receptors that mediate GABA action in proximal retina; the effects of GABA and its antagonists on the ganglion cell light-evoked postsynaptic potentials and spike discharges; the action of GABAergic agents on centre-surround organization of the receptive fields and feature related ganglion cell activity. Special emphasis is put on the GABA action regarding the ON–OFF and sustained–transient ganglion cell dichotomy in both nonmammalian and mammalian retina.     

Role of glutamatergic and GABAergic systems in alcoholism

The pharmacological effects of ethanol are complex and widespread without a well-defined target. Since glutamatergic and GABAergic innervation are both dense and diffuse and account for more than 80% of the neuronal circuitry in the human brain, alterations in glutamatergic and GABAergic function could affect the function of all neurotransmitter systems. Here, we review recent progress in glutamatergic and GABAergic systems with a special focus on their roles in alcohol dependence and alcohol withdrawal-induced seizures. In particular, NMDA-receptors appear to play a central role in alcohol dependence and alcohol-induced neurological disorders. Hence, NMDA receptor antagonists may have multiple functions in treating alcoholism and other addictions and they may become important therapeutics for numerous disorders including epilepsy, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's chorea, anxiety, neurotoxicity, ischemic stroke, and chronic pain. One of the new family of NMDA receptor antagonists, such as DETC-MESO, which regulate the redox site of NMDA receptors, may prove to be the drug of choice for treating alcoholism as well as many neurological diseases.     

Regulation of presynaptic neurotransmission by macroautophagy

mTOR is a regulator of cell growth and survival, protein synthesis-dependent synaptic plasticity, and autophagic degradation of cellular components. When triggered by mTOR inactivation, macroautophagy degrades long-lived proteins and organelles via?sequestration into autophagic vacuoles. mTOR further regulates synaptic plasticity, and neurodegeneration occurs when macroautophagy is deficient. It is nevertheless unknown whether macroautophagy modulates presynaptic function. We find that the mTOR inhibitor rapamycin induces formation of autophagic vacuoles in prejunctional dopaminergic axons with associated decreased axonal profile volumes, synaptic vesicle numbers, and evoked dopamine release. Evoked dopamine secretion was enhanced and recovery was accelerated in?transgenic mice in which macroautophagy deficiency was restricted to dopaminergic neurons; rapamycin failed to decrease evoked dopamine release in the striatum of these mice. Macroautophagy that follows mTOR inhibition in presynaptic terminals, therefore, rapidly alters presynaptic structure and neurotransmission.     

The undertow of sulfur metabolism on glutamatergic neurotransmission

Metabolic interdependence between specialized cells in an organ represents a strategy for energy economy by requiring expression of only a subset of pathway genes in a given cell type. In brain, sulfur metabolism exemplifies this principle of metabolic cooperation between glial and neuronal cells and furnishes three key reagents: S-adenosylmethionine, glutathione and taurine. The pathways for glutathione and taurine syntheses depend on metabolic integration between astrocytes and neurons and intersect with the glutamine-glutamate cycle, which underlies glutamatergic synaptic transmission and requires cooperation between these cell types. We propose that underlying waves of glutamate clearance by astrocytes are activation of cystine import and taurine efflux that result, respectively, from a shared transporter and an increase in solute concentration that triggers osmoregulatory responses.     

Function of GABAergic and glutamatergic neurons in the stomach

-Aminobutyric acid (GABA) and L-glutamic acid (L-Glu) are transmitters of GABAergic and glutamatergic neurons in the enteric interneurons, targeting excitatory or inhibitory GABA receptors or glutamate receptors that modulate gastric motility and mucosal function. GABAergic and glutamatergic neuron immunoreactivity have been found in cholinergic enteric neurons in the stomach. GABA and L-Glu may also subserve hormonal and paracrine signaling. Disruption in gastrointestinal function following perturbation of enteric GABA receptors and glutamate receptors presents potential new target sites for drug development.     

Differential roles of alanine in GABAergic and glutamatergic neurons

Studies in different preparations of neurons and astrocytes of alanine transport and activities of its metabolizing enzyme alanine aminotransferase have led to the proposal that this amino acid is preferentially synthesized in astrocytes and transferred from the astrocytic to the neuronal compartment. From a functional point of view this may well be the case in a GABAergic synapse since theoretically alanine can be utilized as a metabolic fuel in GABAergic neurons where the GABA shunt is operating. Thus, a metabolic scheme is proposed, according to which alanine catabolism is coupled to the TCA cycle where the GABA shunt replaces the alpha-ketoglutarate dehydrogenase/succinyl CoA synthetase reactions. In a glutamatergic synapse in which the large demand for synthesis of neurotransmitter glutamate leads to a large production of ammonia, it is possible that alanine could play a completely different role. Hence, experimental evidence is reviewed suggesting that alanine may serve as a carrier of ammonia nitrogen from the neuronal compartment to the astrocytic compartment using a flux of lactate in the opposite direction to account for transfer of the C-3 carbon skeleton.     

Effects of 5-aminolevulinic acid on the glutamatergic neurotransmission

The haem precursor 5-aminolevulinic acid (ALA) has been proposed to be involved in the neurological dysfunctions presented by patients with acute porphyrias. The effects of ALA on the [3H]glutamate and [3H]MK-801 (dizocilpine) binding to rat cortical membranes and on [3H]glutamate uptake by rat astrocyte cultures were evaluated in the present study in order to elucidate the interaction of ALA with the glutamatergic system and its possible contribution to the in vivo excitatory properties of ALA. ALA (0-1mM) did not affect the binding of 100 nM [3H]glutamate, nor the equilibrium binding constants (K(d) and B(max)) of this neurotransmitter in rat or human cortical membranes. The binding of the NMDA-channel blocker, [3H]MK-801, was not affected by ALA (0-10mM) either. ALA (0-3mM) dose-dependently inhibited glutamate uptake by astrocyte cultures. ALA significantly reduced both the K(m) and V(max) of glutamate uptake indicating an uncompetitive inhibition. The inhibitory effect was irreversible and apparently related to the selective inhibition of the GLT-1 (EAAT2) subtype of glutamate transporter. The finding that ALA significantly increased astrocyte lipoperoxidation in astrocytes incubated under these conditions suggests that the inhibitory effect of ALA might be related to an oxidative damage of the transporter. We propose that the inhibition of glutamate uptake may underlie ALA-induced convulsions.     

Balanced GABAergic and glutamatergic synapse development in hippocampal neurons

Coordinated development of excitatory and inhibitory synapses is crucial for normal function of neuronal circuits. Using homo- and heterochronic cultures of hippocampal neurons, we compared the formation of glutamatergic and GABAergic synapses at different stages and asked whether the age of dendrites affects their ability to accept new glutamatergic and GABAergic synapses. Neurons were transfected with either CFP-actin as a dendritic marker or GFP-synaptophysin as a presynaptic marker. We found that GFP-synaptophysin clusters formed on CFP-actin-labeled dendrites at similar density regardless of pre- and postsynaptic cell type or the age of dendrites (0-2 weeks) upon co-culturing. Therefore, the age of mature dendrites does not affect their ability to accept new synapses. Because GABAergic transmission switches from depolarizing to hyperpolarizing during 1-2 weeks in these cultures, our observations also suggest that this developmental switch does not alter the formation of GABAergic synapses.     

Functional significance of brain glycogen in sustaining glutamatergic neurotransmission

The involvement of brain glycogen in sustaining neuronal activity has previously been demonstrated. However, to what extent energy derived from glycogen is consumed by astrocytes themselves or is transferred to the neurons in the form of lactate for oxidative metabolism to proceed is at present unclear. The significance of glycogen in fueling glutamate uptake into astrocytes was specifically addressed in cultured astrocytes. Moreover, the objective was to elucidate whether glycogen derived energy is important for maintaining glutamatergic neurotransmission, induced by repetitive exposure to NMDA in co-cultures of cerebellar neurons and astrocytes. In the astrocytes it was shown that uptake of the glutamate analogue d -[³H]aspartate was impaired when glycogen degradation was inhibited irrespective of the presence of glucose, signifying that energy derived from glycogen degradation is important for the astrocytic compartment. By inhibiting glycogen degradation in co-cultures it was evident that glycogen provides energy to sustain glutamatergic neurotransmission, i.e. release and uptake of glutamate. The relocation of glycogen derived lactate to the neuronal compartment was investigated by employing d -lactate, a competitive substrate for the monocarboxylate transporters. Neurotransmitter release was affected by the presence of d -lactate indicating that glycogen derived energy is important not only in the astrocytic but also in the neuronal compartment.     

Altered GABAergic and glutamatergic transmission in audiogenic seizure-susceptible mice

The C57BL/10 SPS/sps mouse mutant are audiogenic seizure-susceptible. The enzymatic activities of glutamate decarboxylase (GAD), GABA aminotransferase (GABA-T), alanine aminotransferase (ALA-T), aspartate aminotransferase (ASP-T), and glutamate dehydrogenase (GDH) of whole brain supernatant are significantly reduced in these epileptic mice. GABA uptake is decreased in cortex, midbrain, and pons medulla. Previous studies showed the presence of two sodium-dependent GLU uptake systems in normal (SPS/SPS) mice. Glutamate U_max by System 1 is significantly decreased in these mice, whereas the U_max value for System 2 is significantly increased in the epileptic mice.