$G_i$-dependent microglial dynamics prevent neural network hyperexcitability

• They generated $Mg^{PTX}$mice to genetically inhibit $G_i$ in microglia

  

  Left: scheme of MgPTX mice generation. Right: confocal microscopy images of the cortex of PTX (red) and microglia (green) in MgPTX and MgWT mice. Arrows indicate PTX+ microglia. Scale bar, 50 μm. A total of 60.5 ± 2.7% (mean ± s.e.m.) of cortical GFP+ cells express PTX. n = 3 mice.

  • We generated Tg(Cx3cr1-Cre):R osaPTX/+ (MgPTX) mice for microglia-specific Gi inhibition. In the presence of Cre recombinase, RosaPTX/+ mice express the pertussis toxin (PTX) S1 catalytic subunit, which ADP ribosylates the α subunits of Gi/o proteins, thereby uncoupling them from upstream GPCRs for cell-autonomous Gi/o inhibition in vivo.

• They showed that sustained reduction of microglia brain surveillance and directed process mobility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice.

Microglia process dynamics are perturbed in neuroinflammation and neurodegenration

• but poorly understood on the role of microglial surveillance and directed process motility

Hypothesis

1. Base surveillance (not directed process motility) is regulated by the TWIK-related halothane-inhibited $K^+$channel (THIK-1), which is gated by the $P2Y_{12}$ receptor ($P2Y_{12}R$, P2ry12).
2. Genetic depletion of $P2Y_{12}R$ transiently delays microglial-directed motility to laser ablation without affecting baseline surveillance, which is potentially due to compensation by other G-protein-coupled receptors (GPCRs).
3. Hypothesis: Inhibiting the $G_i$ gateway in microglia would lead to a sustained inhibition of microglial dynamics.

Findings

1. Using in vivo 2P (two-photon) time-lapse imaging, authors found dramatically reduced microglia surveillance in $Mg^{PTX}$ mice, compared with littermate control $Rosa^{PTX/+}$($Mg^{WT}$) mice.

   

   Time-lapse in vivo 2P imaging of cumulative volume sampling of microglia surveillance in MgPTX and MgWT mice. A 20-μm z-stack was acquired in the cortex every 2 min for 1 h. Each frame is an overlay of the maximum-intensity projection acquired at ti and all previous frames. Scale bar, 20 μm.

   Time-lapse in vivo 2P imaging of cumulative volume sampling of microglia surveillance in MgPTX and MgWT mice. A 20-μm z-stack was acquired in the cortex every 2 min for 1 h. Each frame is an overlay of the maximum-intensity projection acquired at ti and all previous frames. Scale bar, 20 μm.

2. Lack of $P2Y_{12}R$ delays, but does not abolish, the microglia response to laser ablation. In contrast, in $M_g^{PTX}$ mice, directed process motility was abolished even 2h after laser ablation

   

   Time-lapse in vivo 2P imaging of microglia-directed process motility toward a tissue laser ablation in MgPTX and MgWT mice. Maximum-intensity projection of a 40-μm z-stack acquired every 4 min for 116 min. Scale bar, 20 μm.

   Time-lapse in vivo 2P imaging of microglia-directed process motility toward a tissue laser ablation in MgPTX and MgWT mice. Maximum-intensity projection of a 40-μm z-stack acquired every 4 min for 116 min. Scale bar, 20 μm.

3. $M_g^{PTX}$ mice had decreased microglial process length and branch points and fewer microglia contacts onto neuronal somata

   

   Animation of 3D reconstruction of cortical microglia in MgPTX and MgWT mice. Scale bar, 20 μm.

   Animation of 3D reconstruction of cortical microglia in MgPTX and MgWT mice. Scale bar, 20 μm.

4. Expression levels of P2ry12 and Kcnk13 were similar between the genotypes

   • which suggests that the decreased microglial motility in $Mg^{PTX}$ mice occurred downstream of Gi. Filamentous actin (F-actin), an indicator of cyclic AMP regulation downstream of Gi in microglia, was highly decompartmentalized and spread throughout the cytoplasm in $Mg^{PTX}$ mice (Extended Data Fig. 2f), which indicates an increase in cAMP after Gi inhibition.

5. Inhibition of Gi signaling in microglia impairs both microglia surveillance and lesion-induced directed motility

   • $Mg^{PTX}$ mice exhibited normal densities of dendritic spines, synapses and neurons, as shown by in vivo 2P imaging, by serial block-face scanning electron microscopy (SBEM) and by immunostaining for NeuN and the inhibitory and excitatory presynaptic puncta markers GAD65/67 and vGLUT1, respectively (Extended Data Fig. 3).

6. $Mg^{PTX}$ mice developed spontaneous seizures that decreased survival.

   

   Spontaneous seizure in a MgPTX mouse. A spontaneous seizure was observed in a MgPTX mouse during routine cage inspection.

   Spontaneous seizure in a MgPTX mouse. A spontaneous seizure was observed in a MgPTX mouse during routine cage inspection.

7. Microglia numbers were decreased in $M_g^{PTX}$mice, potentially due to effects of $G_i$ signaling during development.

   

   Confocal images of GFP-positive microglia in MgPTX and MgWT cortex.