Perineuronal Pockets Prevent Spillage from Synapses

The latticework of extracellular protein that surrounds brain cells forms a particularly dense mesh around fast-spiking interneurons. Why do these cells need such thick coats? In the July 17 Nature Neuroscience, scientists led by Harald Sontheimer at the University of Virginia School of Medicine, Charlottesville, suggest that it acts as a form of insulation.

  • Around inhibitory interneurons, astrocytes contact synapses via holes in the extracellular matrix.
  • Without this perineuronal net, astrocyte processes expand, and glutamate leaks.
  • This extrasynaptic glutamate triggers seizures.

In wild-type mice, tiny pockets in this perineuronal net give astrocytes access to interneuron synapses, the authors report. The pockets also corralled glutamate and other signaling proteins, keeping these molecules from activating extrasynaptic receptors. This containment was crucial for brain health, because when the authors enzymatically degraded the meshwork, glutamate spilled from synapses, and mice developed seizures.

Notably, perineuronal nets broke down spontaneously in mouse models of epilepsy and amyloidosis. “These findings may explain the hyperexcitability associated with Alzheimer’s disease,” the authors speculated.

PNN Pockets. In a single-plane confocal image, tiny holes in the perineuronal net (pink) around inhibitory interneurons are filled with excitatory (white) or inhibitory (blue) synapses, or sometimes both (circles). [Courtesy of Tewari et al., Nature Neuroscience.]

Scientists have long known that perineuronal nets (PNNs) around inhibitory interneurons lock their synapses into place, preventing connectivity changes in adult mice (Pizzorusso et al., 2002; Mar 2013 conference news; Jul 2021 news). Excitatory neurons are sparsely covered by extracellular matrix (ECM). These neurons develop so-called tripartite synapses, meaning that astrocyte processes surround the synaptic cleft, from which they vacuum up stray chemical messengers. Scientists had not previously explored whether astrocytes also penetrate dense PNNs to do the same job for inhibitory neurons.

To investigate this, first author Bhanu Tewari first immunostained inhibitory neurons in the somatosensory cortices of adult mice for PNN and synaptic markers. This revealed small holes riddling the PNN coat. Nearly every hole contained a synapse. Overall, almost a third of the pockets had excitatory synapses, a third inhibitory, and a third had both (image above). A small percentage were empty. Staining for astrocyte markers revealed it was this cell type that filled most synaptic pockets, implying astrocytes might be cleaning up neurotransmitters after the synapse had fired (image below). Overall, about three-quarters of excitatory synapses, and two-thirds of inhibitory ones, were cupped by astrocyte processes known as leaflets.

Armed with Astrocytes. In a confocal microscope image, immunostaining of the dense perineuronal net (pink) around inhibitory interneurons (blue) reveals holes, most of which contain astrocyte processes (green, arrows). [Courtesy of Tewari et al., Nature Neuroscience.]

To learn how changes to the PNN would affect this arrangement, the authors degraded the meshwork in adult mice by injecting the enzyme chondroitinase ABC into the cortex. Six days later, PNNs had become thinner and more porous. As a result, astrocyte leaflets had expanded over the surface of interneurons, but the synapses hadn’t budged. The same thing happened when the authors abolished PNNs entirely by knocking out a key ECM gene in interneurons of adult mice (image below).

To examine what these astrocyte extensions do, the authors made cortical slice cultures and enzymatically degraded PNNs. After synapses had fired, expanded astrocytes mopped up only half as much glutamate and potassium as they did in intact slices. Without a PNN pocket to contain the neurotransmitters, the molecules were free to spill across the neuronal surface and activate extrasynaptic glutamate receptors.

Such spillage caused excitotoxicity when the authors injected a dozen mice with the enzymes chondroitinase ABC and hyaluronidase to rapidly break down PNNs over a broad portion of the rostral and caudal cortex. Over the next 24 hours, half of the mice had seizures.

Breach in Containment. In a three-dimensional reconstruction of confocal microscopy images from immunostainings of healthy mice (left), astrocyte processes (green) occupy small holes in the perineuronal net (pink) around inhibitory interneurons (gray). After genetically abolishing the PNN (right), astrocyte coverage expands. [Courtesy of Tewari et al., Nature Neuroscience.]

Something similar may happen in diseases. In the 5xFAD model of amyloidosis, and also the Theiler’s murine encephalomyelitis virus (TMEV) model of epilepsy, PNNs were but half as dense as in wild-types, and astrocyte processes covered up to twice as much of the interneuron surface. These changes likely promote excitotoxicity in each disease, the authors noted.

PNNs may break down due to neuroinflammation, which hikes the expression of metalloproteinases that degrade ECM. The authors previously reported that blocking these enzymes prevented seizures in mice with brain tumors that release proteolytic enzymes (Tewari et al., 2018). 

Beyond spurring hyperactivity, glutamate that activates extrasynaptic NMDA receptors can trigger cell death pathways (Oct 2020 news; for review, see Parsons and Raymond, 2014). Fast-spiking interneurons may be particularly susceptible to this, because they fire up to hundreds of times per second, releasing massive amounts of neurotransmitters. Thus, interneuron PNNs may have evolved to cage these molecules and protect the cells from themselves, the authors speculated.—Madolyn Bowman Rogers

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References

News Citations

  1. In Pursuit of Toxic Tau
  2. Not Just Alzheimer's: Microglia Sculpt the Brain in Health and Disease
  3. Excitotoxicity: Could a New Class of Drug Stop a Bad TRP?

Research Models Citations

  1. 5xFAD (B6SJL)

Paper Citations

  1. Pizzorusso T, Medini P, Berardi N, Chierzi S, Fawcett JW, Maffei L. Reactivation of ocular dominance plasticity in the adult visual cortex. Science. 2002 Nov 8;298(5596):1248-51. PubMed.
  2. Tewari BP, Chaunsali L, Campbell SL, Patel DC, Goode AE, Sontheimer H. Perineuronal nets decrease membrane capacitance of peritumoral fast spiking interneurons in a model of epilepsy. Nat Commun. 2018 Nov 9;9(1):4724. PubMed.
  3. Parsons MP, Raymond LA. Extrasynaptic NMDA receptor involvement in central nervous system disorders. Neuron. 2014 Apr 16;82(2):279-93. PubMed.

Further Reading

Primary Papers

  1. Tewari BP, Woo AM, Prim CE, Chaunsali L, Patel DC, Kimbrough IF, Engel K, Browning JL, Campbell SL, Sontheimer H. Astrocytes require perineuronal nets to maintain synaptic homeostasis in mice. Nat Neurosci. 2024 Aug;27(8):1475-1488. Epub 2024 Jul 17 PubMed.

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