**1. Introduction**

In the brains of higher vertebrates, both neurons and glial cells produce and secrete the molecules that accumulate and form the extracellular matrix (ECM). In the nineteenth century, pioneers

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of brain cell biology including Camillo Golgi and Santiago Ramon y Cajal have discovered a mesh-like structure surrounding the most neurons and synapses in the adult brain [1]. This extracellular scaffold has been seen initially as a key component for ensuring the structural stabilityoftherespectivetissue[2].LaterresearchevidencedthattheECMhasmultiplefunctions including regulation of cell adhesion, cell-to-cell communication, cell differentiation, cellular compartmentalization, and several forms of neuronal plasticity [3]. During brain maturation, the ECM undergoes profound changes. In late embryonic phases, the ECM is composed of particular proteoglycans and glycoproteins like neurocan and tenascin-C, which are downre‐ gulated during adulthood. Other components like the glycoprotein tenascin-R and chondroi‐ tin sulfate (CS) proteoglycans (CSPGs), such as brevican and aggrecan, are the main molecular components of the adult brain ECM. One of the most important structural components in the adult ECM is the unbranched polysaccharide hyaluronic acid (HA). HA forms the backbone that structurally orchestrates the enmeshment of all other ECM components. Interestingly, it hasbeenshownthatthisdevelopmentalshiftofthejuvenileandadultformsoftheECMcoincides with the closure of the so-called critical periods during brain maturation of respective brain regions. This led to the hypothesis that the brain's ECM is involved in regulating the switch from juvenile to adult plasticity by structural tenacity restricting the potential for neuronal reorganization[4].Thereby,thebrainhasevolvedmechanismsthatguaranteestructuralstability oftheneuronalnetworksestablishedduringexperience-dependentlearning.Thisbrainfunction is fundamental for strengthening neuronal connections and their respective forms of process‐ ing impacting on long-term memory storage and recall. Nevertheless, current research has shown that dynamic adaptations of the ECM can alter several forms of synaptic plasticity and thereby regulate flexible learning and memory organization in the adult brain.

#### **2. The structural foundation of the ECM in the vertebrate's brain**

Components of the ECM in the mature brain are interlinked in a complex netlike architecture [5]. The linear HA backbone binds and coordinates proteoglycans especially of the lectican family that are cross-linked by glycoproteins such as the tenascins. Thus, this form of ECM is referred to as HA-based ECM. It is rich in the glycosaminoglycan CS attached to CSPGs of the lectican family, as for instance brevican and aggrecan [6]. The main carrier of CS within the ECM is aggrecan with its multiple attachment sites, while Brevican is a part-time proteoglycan existing glycoprotein and proteoglycan [7,8]. Brevican and aggrecan both bind to the ECM glycoprotein tenascin-R (**Figure 1A**). Other so-called cartilage link proteins form a complex with N-terminal domains of the lecticans and HA and thereby contribute to the ECM stability [6]. A large variety of other components including reelin, laminins, thrombospondins, heparinsulfate proteoglycans, guidance molecules, and even transcription factors are incorporated in the complex ECM structure (**Figure 1A**). This form of the homogenous HA-based ECM loosely enwraps cell bodies, dendritic, and synaptic structures of most neurons. Further, the mature brain contains a specialized glycosaminoglycan-rich ECM structure around synapses and somata of a small proportion of neurons. This more rigid form is called the perineuronal nets (PNNs), which are especially rich in aggrecan and CS. Such PNNs are most abundant on GABAergic interneurons expressing the calcium buffer protein parvalbumin (PV; **Figure 1B**). Recent evidence shows that PNNs are highly heterogeneous and can be found on various types of neurons throughout the CNS. For the formation of WFA-positive PNNs, the cartilage link protein Crtl1/Hapln1 has been identified as a key regulator [9].

of brain cell biology including Camillo Golgi and Santiago Ramon y Cajal have discovered a mesh-like structure surrounding the most neurons and synapses in the adult brain [1]. This extracellular scaffold has been seen initially as a key component for ensuring the structural stabilityoftherespectivetissue[2].LaterresearchevidencedthattheECMhasmultiplefunctions including regulation of cell adhesion, cell-to-cell communication, cell differentiation, cellular compartmentalization, and several forms of neuronal plasticity [3]. During brain maturation, the ECM undergoes profound changes. In late embryonic phases, the ECM is composed of particular proteoglycans and glycoproteins like neurocan and tenascin-C, which are downre‐ gulated during adulthood. Other components like the glycoprotein tenascin-R and chondroi‐ tin sulfate (CS) proteoglycans (CSPGs), such as brevican and aggrecan, are the main molecular components of the adult brain ECM. One of the most important structural components in the adult ECM is the unbranched polysaccharide hyaluronic acid (HA). HA forms the backbone that structurally orchestrates the enmeshment of all other ECM components. Interestingly, it hasbeenshownthatthisdevelopmentalshiftofthejuvenileandadultformsoftheECMcoincides with the closure of the so-called critical periods during brain maturation of respective brain regions. This led to the hypothesis that the brain's ECM is involved in regulating the switch from juvenile to adult plasticity by structural tenacity restricting the potential for neuronal reorganization[4].Thereby,thebrainhasevolvedmechanismsthatguaranteestructuralstability oftheneuronalnetworksestablishedduringexperience-dependentlearning.Thisbrainfunction is fundamental for strengthening neuronal connections and their respective forms of process‐ ing impacting on long-term memory storage and recall. Nevertheless, current research has shown that dynamic adaptations of the ECM can alter several forms of synaptic plasticity and

144 Composition and Function of the Extracellular Matrix in the Human Body

thereby regulate flexible learning and memory organization in the adult brain.

**2. The structural foundation of the ECM in the vertebrate's brain**

Components of the ECM in the mature brain are interlinked in a complex netlike architecture [5]. The linear HA backbone binds and coordinates proteoglycans especially of the lectican family that are cross-linked by glycoproteins such as the tenascins. Thus, this form of ECM is referred to as HA-based ECM. It is rich in the glycosaminoglycan CS attached to CSPGs of the lectican family, as for instance brevican and aggrecan [6]. The main carrier of CS within the ECM is aggrecan with its multiple attachment sites, while Brevican is a part-time proteoglycan existing glycoprotein and proteoglycan [7,8]. Brevican and aggrecan both bind to the ECM glycoprotein tenascin-R (**Figure 1A**). Other so-called cartilage link proteins form a complex with N-terminal domains of the lecticans and HA and thereby contribute to the ECM stability [6]. A large variety of other components including reelin, laminins, thrombospondins, heparinsulfate proteoglycans, guidance molecules, and even transcription factors are incorporated in the complex ECM structure (**Figure 1A**). This form of the homogenous HA-based ECM loosely enwraps cell bodies, dendritic, and synaptic structures of most neurons. Further, the mature brain contains a specialized glycosaminoglycan-rich ECM structure around synapses and somata of a small proportion of neurons. This more rigid form is called the perineuronal nets (PNNs), which are especially rich in aggrecan and CS. Such PNNs are most abundant on

**Figure 1. Cell types and their specific forms of the hyaluronan-based ECM**. (**A**) The mature ECM is based on the backbone of hyaluronic acid (orange). Schematized are the specialized form of the PNN (*left*) and the loose ECM (*right*) around synaptic contacts. Densely packed PNNs are rich in CS and aggrecan. Small signaling molecules such as sema‐ phorin 3a or Oxt2 are bound to CS within the PNN and mediate several functions or regulate gene expression. The loose ECM around excitatory, spiny synapses is rich in brevican and contains only little aggrecan and is thus less rich in CS. ECM proteins (e.g., reelin) signal through their receptors (e.g., integrins) and regulate several cellular processes including trafficking of glutamate receptors (see C). ECM function is modulated by proteases (scissors) that may free synapses by removing ECM or unmask signaling molecules. (**B**) *Left*, Parvalbumine positive interneuron (PV, red) with typical PNN stained for aggrecan (AGG, green). *Middle*, Dendritic spines and synapses of excitatory neurons (red) are also surrounded by brevican (BC, green), although it is less specific for PNN's. *Right*, Dissociated cortical neuron stained with the dendritic marker Map2 (blue) and Homer (red) to stain excitatory synapses and hyaluronic acid bind‐ ing protein (HABP, green) to label extracellular matrix. Note the loose appearance around dendrites, spines, and syn‐ apses. (**C**) Enzymatic weakening of the ECM by glycosidases (e.g., hyaluronidase, see scissors) changes the micromillieu around synapses by for instance increased lateral diffusion and synaptic exchange of AMPA receptors (blue arrows). Further, removal of stabilizing cues facilitates structural plasticity such as *de-novo* formation of new syn‐ apses and synaptic scaling. Modified from Ref. [22].

The structural foundation of the mature ECM allows regulating several functions beyond mechanical stability of neuronal networks. In the juvenile brain, the ECM regulates neuro- and gliogenesis, cell migration, axonal pathfinding, and synaptogenesis. Adult forms of the HAbased ECM are in the service of multiple functions including regulation of synaptogenesis and synaptic plasticity, compartmentalization of the neuronal surface, neuroprotection, regulation of ion homeostasis, and neuron–glia interactions. The remainder of this review will consider several ECM-based mechanisms for regulating synaptic plasticity and its effects on brain development and adult learning behavior.
