Extracellular matrix supports excitation-inhibition balance in neuronal networks by stabilizing inhibitory synapses

  1. Egor Dzyubenko
  2. Michael Fleischer
  3. Daniel Manrique-Castano
  4. Mina Borbor
  5. Christoph Kleinschnitz
  6. Andreas Faissner
  7. Dirk M Hermann

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Abstract

Maintaining the balance between excitation and inhibition is essential for the appropriate control of neuronal network activity. Sustained excitation-inhibition (E-I) balance relies on the orchestrated adjustment of synaptic strength, neuronal activity and network circuitry. While growing evidence indicates that extracellular matrix (ECM) of the brain is a crucial regulator of neuronal excitability and synaptic plasticity, it remains unclear whether and how ECM contributes to neuronal circuit stability. Here we demonstrate that the integrity of ECM supports the maintenance of E-I balance by retaining inhibitory connectivity. Depletion of ECM in mature neuronal networks preferentially decreases the density of inhibitory synapses and the size of individual inhibitory postsynaptic scaffolds. After ECM depletion, inhibitory synapse strength homeostatically increases via the reduction of presynaptic GABA B receptors. However, the inhibitory connectivity reduces to an extent that inhibitory synapse scaling is no longer efficient in controlling neuronal network activity. Our results indicate that the brain ECM preserves the balanced network state by stabilizing inhibitory synapses.

Significance statement

The question how the brain’s extracellular matrix (ECM) controls neuronal plasticity and network activity is key for an appropriate understanding of brain functioning. In this study, we demonstrate that ECM depletion much more strongly affects the integrity of inhibitory than excitatory synapses in vitro and in vivo. We revealed that by retaining inhibitory connectivity, ECM ensures the efficiency of inhibitory control over neuronal network activity. Our work significantly expands our current state of knowledge about the mechanisms of neuronal network activity regulation. Our findings are similarly relevant for researchers working on the physiological regulation of neuronal plasticity in vitro and in vivo and for researchers studying the remodeling of neuronal networks upon brain injury, where prominent ECM alterations occur.

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  1. eLife

    ###Reviewer #2:

    Dzyubenko et al. have addressed the role of ECM in the control of inhibition and excitation in primary neuronal cultures. Their impact statement reads: "this study revealed the essential role of brain extracellular matrix in controlling synaptic inhibition and neuronal network activity", which makes it erroneously appear that no other past studies have addressed exactly this topic. There is a vast amount of literature on the link between ECM, particularly on PV-INs and development of inhibition, critical period and regulation by the orthodenticle homeobox 2 (Otx2) by the Hensch group. None of this literature is cited in the text. Moreover, there are numerous references indicating clear functional changes following depletion of ECM in vivo (e.g., PMID: 32457072, just to mention one of the most recent studies). In addition to failing to cite previous evidence obtained in vivo for the role of ECM in the regulation of E/I balance and development, with the exception of an anatomical study in the cortex, the authors limit themselves to studying the effects of ECM depletion in immature neuronal cultures. The following list of major concerns with the study is far from complete:

    1. It is unclear how the ratio of excitatory to inhibitory cells of 2:1 was established in the primary cultures. This seems purely coincidental based on Fig.S2, but it surely does not reflect the 4:1 or 5:1 ratio found in vivo. With such an abundance of I-cells vs E-cells in the culture, one can immediately question the physiological relevance of the findings.

    2. One of the physiological consequences of the deletion of ECM in culture is the increased amplitude and frequency of mIPSCs. However, the bimodal distribution of these mIPSC parameters begs the question of how the authors made sure that they recorded from the same neuronal types in their cultures. Moreover, the use of TTX may not ensure that the mIPSCs are Ca2+-entry independent events. Depolarized terminals, and spontaneous closures of K channels within may lead to the opening of voltage-gated Ca channels that could increase both amplitude and frequency of the "mIPSCs".

    3. A similar concern as above surrounds the MFR and MBR of the cultures as measured with the MAE. In these recordings there is no distinguishing between the firings and bursting of E- or I-neurons.

    4. The modeling part of the study cannot be but biased by the results obtained in cultures. Does it also accurately predict the effects of BMI and CGP46381? How was the effect of CGP46381 distinguished between excitatory and inhibitory terminals, as the antagonist affects GABA-B receptors on both?

  2. eLife

    ###Reviewer #1:

    The authors of the manuscript entitled "Extracellular matrix supports excitation-inhibition balance in neuronal networks by stabilizing inhibitory synapses" undertook a study to understand the mechanism(s) by which the extracellular matrix (ECM) of the brain may stabilize neuronal excitability and synaptic plasticity. The study heavily utilized in vitro networks consisting of mature, cultured, hippocampal neurons (with a 2:1 ratio of excitatory to inhibitory neurons) where the ECM was disrupted via enzymatic treatment with chondroitinase ABC or hyaluronidase for 16 hours. Control cells were treated with vehicle (0.1 M PBS).

    The study made several interesting observations. Using their in vitro network, the authors were able to show a reduction in both excitatory and inhibitory synapse density after ECM depletion (Figure 1C). In vivo, they observed a specific decrease only in the inhibitory synapse density after ECM depletion (Figure 2D). To understand how ECM depletion-induced reductions in inhibitory synapse density affect synaptic transmission, the authors recorded miniature inhibitory postsynaptic currents (mIPSCs) in control and ECM depleted cultures. These measurements showed an increase rather than a decrease in the amplitude and frequency of mIPSCs (Figure 3C-D). In contrast, spontaneous network activity measured via multielectrode arrays revealed a significant increase in both firing rate and bursting rate after ECM depletion. Ultrastructural microscopic analysis of scaffolds within structurally complete GABAergic and glutamatergic synapses showed that ECM depletion reduced the size of gephyrin, but not PSD95 scaffolds (Figure 4C). Although the size of the gephyrin scaffolds were reduced, the immunoreactivity of GABAA receptors inside gephyrin containing postsynapses was not altered (Figure 4B, D) nor was the total expression of GABAA receptors affected (Figure S3). A significant reduction in GABABR in VGAT+ terminals was however noted.

    The current manuscript provides ample evidence for both an ECM depletion mediated reduction in inhibitory synapse density and an increase of spontaneous network activity. However, essential functional data is needed (see the list of concerns below) to support the conclusion of a homeostatic increase in inhibitory synapse strength via the reduction of presynaptic GABAB receptors. Functional evidence should also be supplied to show an ECM depletion mediated alteration in the excitation-inhibition (E-I) balance.

    Concerns:

    1. To ensure that ECM depletion did not affect cell survival in neuronal cultures, the authors examined DAPI stained neurons for fragmented nuclei, but more specific assays for cell death such as TUNEL, Fluoro-Jade or activated caspase-3 staining should be incorporated into their study.

    2. It is unclear whether enzymatic ECM digestion/disruption is equally efficient at inhibitory and excitatory synapses. Data in Figure 4C shows no magnitude reductions in the PSD95 scaffolds after ECM depletion, is this reflective of specificity or rather a less efficient enzymatic disruption at excitatory synapses?

    3. Although the PBS vehicle and ECM digestion were delivered ipsilaterally, it was unclear whether there was an accompanying effect contralaterally. This was largely because neither quantification of synapse densities nor the magnified images of the yellow contralaterally positioned squares were shown.

    4. Additional functional tests are needed to show that ECM depletion strengthens inhibitory input to single neurons. These functional tests could include measurements of the paired-pulse ratio and uIPSCs, with analysis of both the CV for uIPSCs and the failure rate. Functional tests should also be added to show that in this in vitro cell culture preparation, ECM depletion results in a functional reduction in presynaptic GABABR activation and a subsequent increase in presynaptic release of neurotransmitter.

    5. Given that excitatory synapse densities were also reduced in the cultured neuronal preparations (Figure 1C), measurement of miniature excitatory postsynaptic currents (mEPSCs) should be included in the study. In some cases, reductions in inhibition and excitation can be balanced leading to no net change in E-I balance in the neural circuit, so it's important to consider both parameters.

    6. It is unclear whether the increased firing and bursting are due to the presynaptic blockade of GABABRs or GABABRs localized elsewhere. The equally increased firing rate in the control and ECM depleted condition after bicuculine methiodide application could be interpreted to show that (in the absence of all GABAA-mediated inhibition) the maximum neuronal firing rate is largely unaffected by ECM depletion, and remains similar to the controls.

  4. Version published to 10.1101/2020.07.13.200113 on bioRxiv