Microglial activation results in neuron-type-specific increase in mPFC GABAergic transmission and abnormal behavior in mice
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Abstract
Neuroinflammation and synaptic dysfunction are two early symptoms of most neurological diseases. However, the mechanisms underlying microglia-associated neuroinflammation in the regulation of synaptic activity remain obscure. We report here that acute neuroinflammation induced by a single-dose proinflammatory cytokine inducer, lipopolysaccharide (LPS), results in enhanced inhibitory postsynaptic currents (IPSCs) of glutamatergic neurons, upregulated levels of GABA A R subunits, glutamine synthetase (GS) and vGAT, and downregulated BDNF and pTrkB levels, due to enhanced activation of microglia in the medial prefrontal cortex (mPFC). Blockage of microglial activation by minocycline ameliorated LPS-induced aberrant mIPSCs and associated aberrant protein expression and behavior. Exogenous application of BDNF prior to LPS challenge also ameliorated LPS-induced abnormal mIPSCs. Thus, this study elucidates a critical role for microglia in the neurobiology of GABAergic synaptic dysfunction induced by neuroinflammation, revealing a novel GABAergic signaling pathway that might be targeted therapeutically to treat neuroinflammation-induced abnormal synaptic activity and associated aberrant behavior.
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###Reviewer #3:
The authors have used a number of different experimental approaches to investigate the actions of LPS (as a model for inflammation) on modifying GABAergic inhibition in the medial prefrontal cortex (mPFC). They conclude that the inhibition of pyramidical neurons is selectively enhanced by the subsequent upregulated levels of GABAAR subunits, glutamine synthetase (GS) and vGAT, and downregulated BDNF and pTrkB levels as a result of microglia activation. Unfortunately the authors use a number of different approaches that preclude comparing results because of the different experimental conditions. For example, IP injection of LPS 2 hours before recording from acutely prepared brain slices is not necessarily comparable to a 20 min bath application of LPS directly onto brain slices. The entry of LPS directly into the brain is …
###Reviewer #3:
The authors have used a number of different experimental approaches to investigate the actions of LPS (as a model for inflammation) on modifying GABAergic inhibition in the medial prefrontal cortex (mPFC). They conclude that the inhibition of pyramidical neurons is selectively enhanced by the subsequent upregulated levels of GABAAR subunits, glutamine synthetase (GS) and vGAT, and downregulated BDNF and pTrkB levels as a result of microglia activation. Unfortunately the authors use a number of different approaches that preclude comparing results because of the different experimental conditions. For example, IP injection of LPS 2 hours before recording from acutely prepared brain slices is not necessarily comparable to a 20 min bath application of LPS directly onto brain slices. The entry of LPS directly into the brain is likely to be minimal and is not equivalent to the bath application of LPS. In addition, the attenuation of the "sickness behavior" after LPS injection and the attenuation by minocycline (Fig 7) is a fairly old story well studied by Dantzer's group (e.g. PMCID: PMC2683474) and previously shown to be blocked by minocycline (Henry et al 2008 PMID: 18477398).
There are discrepancies in the methods descriptions and details about the conditions. Technically some of the recordings aren't whole cell patch recordings because the pipettes contain gramicidin indicating that these were perforated patch recordings. However it is uncertain which recordings are obtained using perforated patch approach. The authors don't provide enough information on the evaluations of the perforated patch recordings to ensure there were no access resistance problems. In addition there are two different pipette solutions described in the methods. This has to be clarified. The authors also do not provide information on when the animals were sacrificed after the LPS injections and slices were obtained.
Finally the authors describe the actions of BDNF on LPS application on brain slices not on the LPS injection into the animals. They also mention two different concentrations. I am not certain the effects of LPS injection IP in the awake animal are equivalent to the LPS application for 20 min prior to BDNF. Page 6- I don't think the acute application of LPS onto inhibitory interneurons is equivalent to the effects of LPS injection in the whole animal and the preparation of slices leading to recordings from pyramidal neurons. These experiments are unconvincing and would have to be conducted under similar conditions for comparisons to be made.
The authors puff supernatant extracted from PFCs and compare +- LPS. They find a higher amplitude current from the LPS treated mice and interpret this as indicating a higher GABA content. This is insufficient evidence as there are other components in extracts such as this and the authors have no evidence using GABA antagonists that these currents truly are due to GABA-A Cl- channels.
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###Reviewer #2:
The manuscript by Tang et al 2020 entitled "Microglia activation leads to neuron-type-specific increase in mPFC GABAergic transmission and abnormal behavior in mice" investigates how changes in inflammation acutely modify GABAergic neurotransmission in the medial prefrontal cortex. The authors provide evidence that 2h-post LPS systemic injection (i.p.) leads to enhanced mIPSC amplitude and frequency and upregulation of GABAaR, vGAT, and GS protein levels. In addition, BDNF application or pre-treatment with minocycline prevents aberrant GABAergic transmission following LPS exposure. They conclude that microglia are responsible for these changes in neurotransmission. The experiments are generally well-done and the manuscript was nicely written and easy to follow. However, there are significant concerns related to the …
###Reviewer #2:
The manuscript by Tang et al 2020 entitled "Microglia activation leads to neuron-type-specific increase in mPFC GABAergic transmission and abnormal behavior in mice" investigates how changes in inflammation acutely modify GABAergic neurotransmission in the medial prefrontal cortex. The authors provide evidence that 2h-post LPS systemic injection (i.p.) leads to enhanced mIPSC amplitude and frequency and upregulation of GABAaR, vGAT, and GS protein levels. In addition, BDNF application or pre-treatment with minocycline prevents aberrant GABAergic transmission following LPS exposure. They conclude that microglia are responsible for these changes in neurotransmission. The experiments are generally well-done and the manuscript was nicely written and easy to follow. However, there are significant concerns related to the interpretation that this is a microglial effect. Above all, LPS and minocycline are very blunt and not specific to microglia. Besides their effects on the peripheral immune system, which could also affect the brain, they can also directly affect other cell types in the brain (neurons, glia, vasculature, etc.) in addition to microglia. Therefore, it cannot be concluded, without more cell-specific manipulations, that the effects are attributed to microglia. Other concerns are detailed below:
Are changes in neurotransmission restricted and specific to the mPFC or is this a more global disruption in neurotransmission due to full body systemic inflammation?
The indicators of microglial activation by immunostaining for Iba-1 and measuring soma size are fairly superficial. More in-depth molecular analyses with more microglia-specific markers would be more informative.
GFAP does not label all reactive astrocytes and is therefore not the best indicator of changes in reactive astrogliosis. The authors should include additional markers in their analysis outlined in Liddelow et al. Nature 2017.
Behavioral changes, which are largely locomotor, within 2 h post-LPS are more likely a sickness behavior rather than a specific effect of changes in neurotransmission in the mPFC.
It is unclear what specific pyramidal neuron population are being recorded in the mPFC. Specifying the layer would be informative.
The authors attempt to link the results with BDNF application with a microglial affect. This link is not particularly strong. While there are studies demonstrating microglial BDNF can affect circuits, the majority of BDNF is made by other cell types in the brain, not microglia. Without cell-specific manipulations, the authors should tone down this link.
Experiments displayed in Figure 4 should include a minocycline-only condition.
It would be informative to perform electrophysiological recordings on organotypic slices treated with minocycline followed by +/- acute LPS treatment.
The authors use an interesting method whereby they puff lysate from control and LPS brains to assess the impact on e-phys recordings. Due to the increased inhibitory transmission, the authors conclude that there is increased GABA content. However, it seems there could be other explanations such as other neuroactive factors, including cytokines, that could potentiate GABA transmission. Measuring GABA by, for example, immunohistochemistry could help to address this concern.
In several western blot panels the bands are saturated and are, thus, not ideal for use in quantifications.
The increase in GABAaR, vGAT, and GS at the protein level within 2 h-post LPS treatment is quite rapid and more typical of immediate early genes (e.g. c-FOS, Arc, etc.). Could the authors comment on this in the manuscript?
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###Reviewer #1:
In this manuscript, Drs Tang and colleagues study how inhibitory synapses are modulated upon intraperitoneal injection of LPS or upon direct application of LPS onto acute slices. The manuscript could certainly be strengthened by addressing the following points, which are all related:
The authors seem to consider that microglial "activation" identified by a morphological modification and enhanced Iba1 signal is an homogeneous all-or-none state that can be reached or blocked by different stimuli. Therefore, they compare the result of an "activation" by a 2h intraperitoneal (ip) injection of LPS with a direct 10 min application of LPS onto acute brain slices. However, it is now acknowledged that different stimuli induce different microglial phenotypes (Perry et al. Nat Rev Neurol 2010, 6:193) that may not be comparable. LPS …
###Reviewer #1:
In this manuscript, Drs Tang and colleagues study how inhibitory synapses are modulated upon intraperitoneal injection of LPS or upon direct application of LPS onto acute slices. The manuscript could certainly be strengthened by addressing the following points, which are all related:
The authors seem to consider that microglial "activation" identified by a morphological modification and enhanced Iba1 signal is an homogeneous all-or-none state that can be reached or blocked by different stimuli. Therefore, they compare the result of an "activation" by a 2h intraperitoneal (ip) injection of LPS with a direct 10 min application of LPS onto acute brain slices. However, it is now acknowledged that different stimuli induce different microglial phenotypes (Perry et al. Nat Rev Neurol 2010, 6:193) that may not be comparable. LPS binds to TLR4 protein which is expressed by microglia in the brain, but also by peripheral immune cells such as macrophages. The effects of ip injection of LPS might thus be due to microglia (if LPS pass the blood brain barrier), and / or to an indirect effect of peripheral immune cells activation. The effect of LPS application on acute slices is directly due to the binding to microglial TLR4. At this stage, it seems not possible to rule out the possibility that a signaling molecule coming from the periphery could both activate microglia and modulate inhibitory synapses (see point 2). It is therefore not possible to claim (as in the title) that activation of microglia results in the increase of GABAergic transmission.
The authors propose a role for BDNF based on the decrease of BDNF in 2h LPS mice observed by WB (figure 4D). However, they have focussed their WB analysis on this protein and have not examined any other signaling molecules. In figure S3, they showed that LPS increases the mRNAs encoding TNFα, IL1b and IL6. How can they exclude that these proteins are involved in the activation of microglia of microglia and upregulation of GABAR?
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript. Gary L Westbrook (Oregon Health and Science University) served as the Reviewing Editor.
###Summary:
The impact of neuroinflammation on brain circuits is an important topic. However, all reviewers had significant and overlapping concerns and were not convinced that the data adequately supported the authors’ conclusions.
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