Maternal immune activation induces sustained changes in fetal microglia motility.

Maternal infection or inflammation causes abnormalities in brain development associated with subsequent cognitive impairment and in an increased susceptibility to schizophrenia and autism spectrum disorders. Maternal immune activation (MIA) and increases in serum cytokine levels mediates this association via effects on the fetal brain, and microglia can respond to maternal immune status, but consensus on how microglia may respond is lacking and no-one has yet examined if microglial process motility is impaired. In this study we investigated how MIA induced at two different gestational ages affected microglial properties at different developmental stages. Immune activation in mid-pregnancy increased IL-6 expression in embryonic microglia, but failed to cause any marked changes in morphology either at E18 or postnatally. In contrast MIA, particularly when induced earlier (at E12), caused sustained alterations in the patterns of microglial process motility and behavioral deficits. Our research has identified an important microglial property that is altered by MIA and which may contribute to the underlying pathophysiological mechanisms linking maternal immune status to subsequent risks for cognitive disease.

Microglial Contact Prevents Excess Depolarization and Rescues Neurons from Excitotoxicity.

Microglia survey and directly contact neurons in both healthy and damaged brain, but the mechanisms and functional consequences of these contacts are not yet fully elucidated. Combining two-photon imaging and patch clamping, we have developed an acute experimental model for studying the role of microglia in CNS excitotoxicity induced by neuronal hyperactivity. Our model allows us to simultaneously examine the effects of repetitive supramaximal stimulation on axonal morphology, neuronal membrane potential, and microglial migration, using cortical brain slices from Iba-1 eGFP mice. We demonstrate that microglia exert an acute and highly localized neuroprotective action under conditions of neuronal hyperactivity. Evoking repetitive action potentials in individual layer 2/3 pyramidal neurons elicited swelling of axons, but not dendrites, which was accompanied by a large, sustained depolarization of soma membrane potential. Microglial processes migrated to these swollen axons in a mechanism involving both ATP and glutamate release via volume-activated anion channels. This migration was followed by intensive microglial wrapping of affected axons and, in some cases, the removal of axonal debris that induced a rapid soma membrane repolarization back to resting potentials. When the microglial migration was pharmacologically blocked, the activity-induced depolarization continued until cell death ensued, demonstrating that the microglia-axon contact served to prevent pathological depolarization of the soma and maintain neuronal viability. This is a novel aspect of microglia surveillance: detecting, wrapping, and rescuing neuronal soma from damage due to excessive activity.

Mutation of the pore glutamate affects both cytoplasmic and external dequalinium block in the rat olfactory CNGA2 channel.

Dequalinium has recently been reported to block CNGA1 and CNGA2 channels expressed in Xenopus laevis. Using the inside-out configuration of the patch-clamp technique, we examined the effects of dequalinium on rat olfactory CNGA2 channels expressed in human embryonic kidney (HEK293) cells and studied aspects of its molecular mechanism of action. We found that cytoplasmic dequalinium blocked wild-type (WT) CNGA2 channels in a voltage-dependent manner with an IC(50) of approximately 1.3 muM at a V(m) of + 60 mV, and an effective fractional charge, zdelta, of +0.8 (z=2, delta=+0.4), suggesting that cytoplasmic dequalinium interacts with a binding site that is about two fifths of the way along the membrane electric field (from the intracellular side). Neutralizing the negatively charged pore lining glutamate acid residue (E342Q) still allows effective channel block by cytoplasmic dequalinium with an IC(50) of approximately 2.2 muM at a V(m) of +60 mV but now having a zdelta of +0.1 (delta=+0.05), indicating a profoundly decreased level of voltage-dependence. In addition, by comparing the extent of block under different levels of channel activation, we show that the block by cytoplasmic dequalinium displayed clear state-dependence in WT channels by interacting predominantly with the closed channel, whereas the block in E342Q channels was state-independent. Application of dequalinium to the external membrane surface also blocked currents through WT channels and the E342Q mutation significantly increased the IC(50) for external block approximately fivefold. These results confirm dequalinium as a potent, voltage-dependent and state-dependent blocker of cyclic-nucleotide-gated channels, and show that neutralization of the E342 residue profoundly affects the block by both cytoplasmic and external application of dequalinium.

Differential modulation of evoked and spontaneous glycine release from rat spinal cord glycinergic terminals by the cyclic AMP/protein kinase A transduction cascade.

The mechanisms underlying cyclic AMP modulation of action potential-dependent and -independent (spontaneous) release of glycine from terminals synapsing onto sacral dorsal commissural nucleus neurons of lamina X were studied in spinal cord slices using conventional patch-clamp recordings. 3-Isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and forskolin increased the amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in a sensitive manner to protein kinase A (PKA) inhibition (with KT-5720). Direct activation (with adenosine 3',5'-cyclic-monophosphothioate, Sp-isomer) and inhibition (with adenosine 3',5'-cyclic-monophosphothioate, Rp-isomer) of PKA increased and decreased the eIPSC amplitude, respectively. Paired pulse experiments and direct injection of PKA inhibitor fragment 6-22 amide (PKI(6-22)) into the recording neuron revealed that these effects on eIPSC amplitude occurred presynaptically, indicating that evoked glycine release is regulated by presynaptic cAMP via changes in PKA activity. Increasing cAMP also increased spontaneous release of glycine, causing an increased frequency of miniature IPSCs (mIPSCs). In contrast to the effects on evoked release, this response was not solely mediated via PKA, as it was not occluded by PKA inhibition, and both direct inhibition and direct activation of PKA actually enhanced mIPSC frequency. Direct inhibition of cAMP (with SQ 22536) did, however, reduce mIPSC frequency. These results suggest cAMP modulation of evoked and spontaneous release involves different presynaptic mechanisms and proteins.

GABAB receptor transduction mechanisms, and cross-talk between protein kinases A and C, in GABAergic terminals synapsing onto neurons of the rat nucleus basalis of Meynert.

The transduction mechanisms underlying presynaptic GABAB receptor-mediated inhibition of transmitter release have been characterized for a variety of synapses in the central nervous system (CNS). These studies have suggested a range of transduction mechanisms, including a role for second messengers such as protein kinases A (PKA) and C (PKC). In the present study, we have examined the intracellular signalling pathways underlying baclofen-induced inhibition of GABA release from terminals synapsing onto rat basalis of Meynert neurons using patch-clamp recordings. Baclofen, a selective GABAB receptor agonist, reversibly decreased both evoked and spontaneous, miniature, GABAergic inhibitory postsynaptic currents (eIPSCs and mIPSCs, respectively). Such baclofen actions were completely abolished by CGP55845A, a selective GABAB receptor antagonist, and by staurosporine, a non-selective PKA and PKC inhibitor. The mIPSC frequency was still decreased by baclofen even in the presence of 4 AP, a K+ channel blocker, and Cd2+, a voltage-dependent calcium channel blocker. Pharmacological activation or inhibition of PKC activity affected basal GABA release and mildly affected the response to baclofen. Inhibition of the cAMP/PKA cascade also affected basal GABA release and, in a subset of neurons, occluded the effects of baclofen, suggesting that the GABAB receptor-mediated inhibitory action on GABA release was mediated via decreases in PKA activity. In addition, PKA inhibition occluded the effects of PKC modulation on both basal GABA release and on the response to baclofen. Our results characterize the transduction pathway of baclofen at these nucleus basalis of Maynert (nBM) synapses and show, for the first time, some cross-talk between the cAMP/PKA and PKC pathways in mammalian presynaptic nerve terminals.