Experimental Cortical Spreading Depression Induces NMDA Receptor Dependent Potassium Currents in Microglia.

UNLABELLED: Cortical spreading depression (CSD) is a propagating event of neuronal depolarization, which is considered as the cellular correlate of the migraine aura. It is characterized by a change in the intrinsic optical signal and by a negative DC potential shift. Microglia are the resident macrophages of the CNS and act as sensors for pathological changes. In the present study, we analyzed whether microglial cells might sense CSD by recording membrane currents from microglia in acutely isolated cortical mouse brain slices during an experimentally induced CSD. Coincident with the change in the intrinsic optical signal and the negative DC potential shift we recorded an increase in potassium conductance predominantly mediated by K(+) inward rectifier (Kir)2.1, which was blocked by the NMDA receptor antagonist D-AP5. Application of NMDA and an increase in extracellular K(+) mimics the CSD-induced Kir activation. Application of D-AP5, but not the purinergic receptor antagonist RB2, blocks the NMDA-induced Kir activation. The K(+) channel blocker Ba(2+) blocks both the CSD- and the NMDA-triggered increase in Kir channel activity. In addition, we could confirm previous findings that microglia in the adult brain do not express functional NMDA receptors by recording from microglia cultured from adult brain. From these observations we conclude that CSD activates neuronal NMDA receptors, which lead to an increase in extracellular [K(+)] resulting in the activation of Kir channel activity in microglia. SIGNIFICANCE STATEMENT: Cortical spreading depression (CSD) is a wave of neuronal depolarization spreading through the cortex and is associated with the aura of migraine. Here we show that microglial cells, which are viewed as pathologic sensors of the brain, can sense this wave. The increase in the extracellular potassium concentration associated with that wave leads to the activation of an inward rectifying potassium conductance in microglia. The involvement of neuronal NMDA receptors is crucial because NMDA mimics that response and microglia do not express functional NMDA receptors. Although it is now evident that CSD leads to a signal in microglia, the consequences of this microglial activation during CSD needs to be explored.

Spontaneous Ca2+ transients in mouse microglia.

Microglia are the resident immune cells in the central nervous system and many of their physiological functions are known to be linked to intracellular calcium (Ca2+) signaling. Here we show that isolated and purified mouse microglia-either freshly or cultured-display spontaneous and transient Ca2+ elevations lasting for around ten to twenty seconds and occurring at frequencies of around five to ten events per hour and cell. The events were absent after depletion of internal Ca2+ stores, by phospholipase C (PLC) inhibition or blockade of inositol-1,4,5-trisphosphate receptors (IP3Rs), but not by removal of extracellular Ca2+, indicating that Ca2+ is released from endoplasmic reticulum intracellular stores. We furthermore provide evidence that autocrine ATP release and subsequent activation of purinergic P2Y receptors is not the trigger for these events. Spontaneous Ca2+ transients did also occur after stimulation with Lipopolysaccharide (LPS) and in glioma-associated microglia, but their kinetics differed from control conditions. We hypothesize that spontaneous Ca2+ transients reflect aspects of cellular homeostasis that are linked to regular and patho-physiological functions of microglia.

A comparative morphological, electrophysiological and functional analysis of axon regeneration through peripheral nerve autografts genetically modified to overexpress BDNF, CNTF, GDNF, NGF, NT3 or VEGF.

The clinical outcome of microsurgical repair of an injured peripheral nerve with an autograft is suboptimal. A key question addressed here is: can axon regeneration through an autograft be further improved? In this article the impact of six neurotrophic factors (BDNF, CNTF, GDNF, NGF, NT3 or VEGF) on axon regeneration was compared after delivery to a 1cm long nerve autograft by gene therapy. To distinguish between early and late effects, regeneration was assessed at 2 and 20weeks post-surgery by histological, electrophysiological and functional analysis. BDNF, GDNF and NGF exhibited a spectrum of effects, including early stimulatory effects on axons entering the autograft and excessive axon growth and Schwann cell proliferation at 20weeks post-surgery. Persistent expression of these factors in autografts interfered with target cell reinnervation and functional recovery in a modality specific way. Autografts overexpressing VEGF displayed hypervascularization, while grafts transduced with CNTF and NT3 were indistinguishable from control grafts. These three factors did not have detectable pro-regenerative effects. In conclusion, autograft-based repair combined with gene therapy for three of the six growth factors investigated (BDNF, GDNF, NGF) showed considerable promise since these factors enhanced modality specific axon outgrowth in autografts. The remarkable and selective effects of BDNF, GDNF and NGF on motor or sensory regeneration will be exploited in future experiments that aim to carefully regulate their temporal and spatial expression since this has the potential to overcome the adverse effects on long-distance regeneration observed after uncontrolled delivery.