Andrew J. Moorhouse

Resting Microglia Directly Monitor the Functional State of Synapses In Vivo and Determine the Fate of Ischemic Terminals

Recent studies have identified the important contribution of glial cells to the plasticity of neuronal circuits. Resting microglia, the primary immune effector cells in the brain, dynamically extend and retract their processes as if actively surveying the microenvironment. However, just what is being sampled by these resting microglial processes has not been demonstrated in vivo, and the nature and function of any interactions between microglia and neuronal circuits is incompletely understood. Using in vivo two-photon imaging of fluorescent-labeled neurons and microglia, we demonstrate that the resting microglial processes make brief (∼5 min) and direct contacts with neuronal synapses at a frequency of about once per hour. These contacts are activity-dependent, being reduced in frequency by reductions in neuronal activity. After transient cerebral ischemia, the duration of these microglia–synapse contacts are markedly prolonged (∼1 h) and are frequently followed by the disappearance of the presynaptic bouton. Our results demonstrate that at least part of the dynamic motility of resting microglial processes in vivo is directed toward synapses and propose that microglia vigilantly monitor and respond to the functional status of synapses. Furthermore, the striking finding that some synapses in the ischemic areas disappear after prolonged microglial contact suggests microglia contribute to the subsequent increased turnover of synaptic connections. Further understanding of the mechanisms involved in the microglial detection of the functional state of synapses, and of their role in remodeling neuronal circuits disrupted by ischemia, may lead to novel therapies for treating brain injury that target microglia.

Microglia: actively surveying and shaping neuronal circuit structure and function

The traditional role of microglia has been in brain infection and disease, phagocytosing debris and secreting factors to modify disease progression. Recent evidence extends their role to healthy brain homeostasis, including the regulation of cell death, synapse elimination, neurogenesis, and neuronal surveillance. These actions contribute to the maturation and plasticity of neural circuits that ultimately shape behavior. Here we review microglial contributions to the development, plasticity, and maintenance of neural circuits with a focus on interactions with synapses. We introduce this topic by reviewing recent studies on the migration and proliferation of microglia within the brain, and conclude with the proposal that microglia dysfunction may adversely affect brain function, and thereby contribute to the development of psychiatric and neurological disorders.

Techniques: applications of the nerve-bouton preparation in neuropharmacology.

Single mammalian neurons can be isolated with adherent functional synaptic terminals using an enzyme-free, mechanical dissociation procedure. This allows investigations of the effects of presynaptic modulators of synaptic transmission with unprecedented ease and accuracy. Furthermore, single presynaptic terminals and boutons can be visualized using fluorescent markers and can also be focally stimulated with electrical pulses. In this article, the isolated-nerve-adherent-synaptic-bouton preparation and some examples of its general properties and uses are discussed.

Early Changes in KCC2 Phosphorylation in Response to Neuronal Stress Result in Functional Downregulation

The K+ Cl− cotransporter KCC2 plays an important role in chloride homeostasis and in neuronal responses mediated by ionotropic GABA and glycine receptors. The expression levels of KCC2 in neurons determine whether neurotransmitter responses are inhibitory or excitatory. KCC2 expression is decreased in developing neurons, as well as in response to various models of neuronal injury and epilepsy. We investigated whether there is also direct modulation of KCC2 activity by changes in phosphorylation during such neuronal stressors. We examined tyrosine phosphorylation of KCC2 in rat hippocampal neurons under different conditions of in vitro neuronal stress and the functional consequences of changes in tyrosine phosphorylation. Oxidative stress (H2O2) and the induction of seizure activity (BDNF) and hyperexcitability (0 Mg2+) resulted in a rapid dephosphorylation of KCC2 that preceded the decreases in KCC2 protein or mRNA expression. Dephosphorylation of KCC2 is correlated with a reduction of transport activity and a decrease in [Cl−]i, as well as a reduction in KCC2 surface expression. Manipulation of KCC2 tyrosine phosphorylation resulted in altered neuronal viability in response to in vitro oxidative stress. During continued neuronal stress, a second phase of functional KCC2 downregulation occurs that corresponds to decreases in KCC2 protein expression levels. We propose that neuronal stress induces a rapid loss of tyrosine phosphorylation of KCC2 that results in translocation of the protein and functional loss of transport activity. Additional understanding of the mechanisms involved may provide means for manipulating the extent of irreversible injury resulting from different neuronal stressors.

M2 Pore Mutations Convert the Glycine Receptor Channel from Being Anion- to Cation-Selective

Three mutations in the M2 transmembrane domains of the chloride-conducting alpha1 homomeric glycine receptor (P250Delta, A251E, and T265V), which normally mediate fast inhibitory neurotransmission, produced a cation-selective channel with P(Cl)/P(Na), = 0.27 (wild-type P(Cl)/P(Na) = 25), a permeability sequence P(Cs) > P(K) > P(Na) > P(Li), an impermeability to Ca(2+), and a reduced glycine sensitivity. Outside-out patch measurements indicated reversed and accentuated rectification with extremely low mean single channel conductances of 3 pS (inward current) and 11 pS (outward current). The three inverse mutations, to those analyzed in this study, have previously been shown to make the alpha7 acetylcholine receptor channel anion-selective, indicating a common location for determinants of charge selectivity of inhibitory and excitatory ligand-gated ion channels.

Cation-selective Mutations in the M2 Domain of the Inhibitory Glycine Receptor Channel Reveal Determinants of Ion-Charge Selectivity

Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective α1 homomeric glycine receptor (α1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1′E GlyR) was sufficient to convert the channels to select cations over anions with PCl/PNa = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2′Δ mutant GlyR retained its anion selectivity (PCl/PNa = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (PCl/PNa = 27.9). When the A-1′E and the P-2′Δ mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (PCl/PNa = 0.13). The SDM GlyR was also Ca2+ permeable (PCa/PNa = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19′A GlyR) produced a more Ca2+-permeable channel (PCa/PNa = 0.73), without drastically altering monovalent charge selectivity (PCl/PNa = 0.23). The SDM+R19′E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca2+ permeability (PCa/PNa = 0.92), with little effect on monovalent selectivity (PCl/PNa = 0.19). Estimates of the minimum pore diameter of the A-1′E, SDM, SDM+R19′A, and SDM+R19′E GlyRs revealed that these pores are larger than the α1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity.

Microglia contact induces synapse formation in developing somatosensory cortex

Microglia are the immune cells of the central nervous system that play important roles in brain pathologies. Microglia also help shape neuronal circuits during development, via phagocytosing weak synapses and regulating neurogenesis. Using in vivo multiphoton imaging of layer 2/3 pyramidal neurons in the developing somatosensory cortex, we demonstrate here that microglial contact with dendrites directly induces filopodia formation. This filopodia formation occurs only around postnatal day 8–10, a period of intense synaptogenesis and when microglia have an activated phenotype. Filopodia formation is preceded by contact-induced Ca2+ transients and actin accumulation. Inhibition of microglia by genetic ablation decreases subsequent spine density, functional excitatory synapses and reduces the relative connectivity from layer 4 neurons. Our data provide the direct demonstration of microglial-induced spine formation and provide further insights into immune system regulation of neuronal circuit development, with potential implications for developmental disorders of immune and brain dysfunction.

Microglia and synapse interactions: fine tuning neural circuits and candidate molecules

Brain function depends critically on the interactions among the underlying components that comprise neural circuits. This includes coordinated activity in pre-synaptic and postsynaptic neuronal elements, but also in the non-neuronal elements such as glial cells. Microglia are glial cells in the central nervous system (CNS) that have well-known roles in neuronal immune function, responding to infections and brain injury and influencing the progress of neurodegenerative disorders. However, microglia are also surveyors of the healthy brain, continuously extending and retracting their processes and making contacts with pre- and postsynaptic elements of neural circuits, a process that clearly consumes considerable energy. Pruning of synapses during development and in response to injury has also been documented, and we propose that this extensive surveillance of the brain parenchyma in adult healthy brain results in similar “fine-tuning” of neural circuits. A reasonable extension is that a dysfunction of such a homeostatic role of microglia could be a primary cause of neuronal disease. Indeed, neuronal functions including cognition, personality, and information processing are affected by immune status. In this review we focus on the interactions between microglia and synapses, the possible cellular and molecular mechanisms that mediate such contacts, and the possible implications these interactions may have in the fine tuning of neural circuits that is so important for physiological brain function.

Clustering of Neuronal K+-Cl− Cotransporters in Lipid Rafts by Tyrosine Phosphorylation

The neuronal K+-Cl− cotransporter (KCC2) is a membrane transport protein that extrudes Cl− from neurons and helps maintain low intracellular [Cl−] and hyperpolarizing GABAergic synaptic potentials. Depolarizing γ-aminobutyric acid (GABA) responses in neonatal neurons and following various forms of neuronal injury are associated with reduced levels of KCC2 expression. Despite the importance for plasticity of inhibitory transmission, less is known about cellular mechanisms involved in more dynamic changes in KCC2 function. In this study, we investigated the role of tyrosine phosphorylation in KCC2 localization and function in hippocampal neurons and in cultured GT1-7 cells. Mutation to the putative tyrosine phosphorylation site within the long intracellular carboxyl terminus of KCC2(Y1087D) or application of the tyrosine kinase inhibitor genistein shifted the GABA reversal potential (EGABA) to more depolarized values, indicating reduced KCC2 function. This was associated with a change in the expression pattern of KCC2 from a punctate distribution to a more uniform distribution, suggesting that functional tyrosine-phosphorylated KCC2 forms clusters in restricted membrane domains. Sodium vanadate, a tyrosine phosphatase inhibitor, increased the proportion of KCC2 associated with lipid rafts membrane domains. Loss of tyrosine phosphorylation also reduced oligomerization of KCC2. A loss of the punctuate distribution and oligomerization of KCC2 and a more depolarized EGABA were seen when the 28-amino-acid carboxyl terminus of KCC2 was deleted. These results indicate that direct tyrosine phosphorylation of KCC2 results in membrane clusters and functional transport activity, suggesting a mechanism by which intracellular Cl− concentrations and GABA responses can be rapidly modulated.

Functions of microglia in the central nervous system – beyond the immune response

Microglia cells are the immune cells of the central nervous system and consequently play important roles in brain infections and inflammation. Recent in vivo imaging studies have revealed that in the resting healthy brain, microglia are highly dynamic, moving constantly to actively survey the brain parenchyma. These active microglia can rapidly respond to pathological insults, becoming activated to induce a range of effects that may contribute to both pathogenesis, or to confer neuronal protection. However, interactions between microglia and neurons are being recognized as important in shaping neural circuit activity under more normal, physiological conditions. During development and neurogenesis, microglia interactions with neurons help to shape the final patterns of neural circuits important for behavior and with implications for diseases. In the mature brain, microglia can respond to changes in sensory activity and can influence neuronal activity acutely and over the long term. Microglia seem to be particularly involved in monitoring the integrity of synaptic function. In this review, we discuss some of these new insights into the involvement of microglia in neural circuits.