In the cortex, PNN neurons occur in high density in the motor and sensory cortex, as well as in the prefrontal and the temporal cortex. They are mostly found in layers 2–5 of the cortex (Brückner et al., 1999). However, there is some variation between cortical regions in the layers in which the PNNs can be found: the visual cortex shows the PNNs mostly in layer 4 and the lower part of layer 5, while in the medial entorhinal cortex (mEC) the PNNs are mostly found in layer 2 and 3(Lensjø et al., 2017a). The time at which PNNs mature also varies between cortical regions, in the mEC, PNNs mature earlier than in the visual cortex. Maturation of PNNs in the mEC coincides with the maturation of the grid cell pattern at postnatal day 30 (Lensjø et al., 2017a), while maturation of the PNNs in the visual cortex occurs at postnatal day 42 (Ye and Miao, 2013). The majority of neurons surrounded by PNNs in the cortex are GABAergic interneurons, while a smaller number of neurons surrounded by PNNs are pyramidal cells (Härtig et al., 1999; Beebe et al., 2016). There is a high co-localization of the GABAergic neurons ensheathed by PNNs and parvalbumin (Baig et al., 2005), specifically the myosin binding protein C (mbpC) positive parvalbumin neurons, while the somatostatin parvalbumin positive neurons do not show PNNs (Rossier et al., 2015). Parvalbumin positive neurons are inhibitory interneurons and they form the largest population of PNN positive cells in the brain (Baig et al., 2005). They are fast spiking interneurons which regulate pyramidal neurons, which in turn project out of the cerebral cortex and provide excitatory signals. When PNNs are removed from the visual cortex with ChABC, inhibitory activity is indeed reduced (Lensjø et al., 2017b). Electrophysiological recordings display an altered excitatory-inhibitory balance which resemble a reset to a juvenile state of the cortex, with increased plasticity (Lensjø et al., 2017b).

In the visual cortex, the formation of the PNNs can be delayed by dark rearing (Pizzorusso et al., 2002). Deprivation of stimuli by dark rearing disturbs the formation of the PNNs, delays the closure of critical period (a period of high plasticity during development) and thus the maturation of neural circuits in the visual cortex (Pizzorusso et al., 2002, 2006; Hensch, 2004). Furthermore, plasticity in the visual cortex can be reopened in adults by removing PNNs using ChABC treatment (Pizzorusso et al., 2002). Similar results were found in the auditory cortex when treatment with hyaluronidase reopened the window of plasticity (Happel et al., 2014). This suggests CS and hyaluronan in the PNNs are important for controlling plasticity in the cortex. Moreover, in C6ST-1 knockin mice, in which the ratio of 4S:6S is reduced, both the formation of PNNs and the maturation of parvalbumin neurons are impaired (Miyata et al., 2012). This genetic mutation also delays the closure of the critical period. In HAPLN1 KO mice, which show a reduction in PNNs, long term depression (LTD) is facilitated (Romberg et al., 2013). LTD is a reduction of the efficacy of the neuronal synapse lasting long after the stimulus, a form of plasticity. These changes in plasticity enhance the long term memory of the HAPLN1 KO mice (Romberg et al., 2013). Interestingly, PNNs in the cortex are also necessary for fear learning, which also requires plasticity. However, instead of blocking plasticity, in fear learning PNNs need to be dynamically regulated to make plasticity possible. A recent study shows that 4 h after fear conditioning, mRNA of PNN components were upregulated and more cells were surrounded with PNNs (Banerjee et al., 2017). This finding indicates that cells may respond to episodes of plasticity by increasing their PNNs. Together, these results show that PNNs in the cortex surround parvalbumin positive GABAergic interneurons and pyramidal cells and it regulates the closure of critical periods of plasticity.

Perineuronal nets (PNNs) in the amygdala ensheath parvalbumin and calbindin positive neurons (Härtig et al., 1995). Both parvalbumin and calbindin positive neurons are GABAergic interneurons and both innervate pyramidal neurons. PNNs are also found surrounding excitatory neurons in the amygdala; PNNs in this region co-localize with Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (Morikawa et al., 2017). These excitatory neurons are necessary for fear memory. When cFos was used to mark activity in neurons, a correlation was found between the level of activity in the PNN excitatory neurons and the amount of freezing seen in the behavior of the animal (Hisaoka et al., 2010). Moreover, the appearance of the PNNs in the amygdala coincides with a developmental switch in fear memory resilience (Gogolla et al., 2009). This developmental switch specifies a period when the brain changes from a young brain, able to erase fear memories, to a mature brain in which fear memories can no longer be erased. The removal of the PNNs with ChABC completely blocks the expression of fear memory, suggesting that the memory is erased, which is not normally possible in the adult brain when the neuronal circuit is mature and PNNs have formed (Gogolla et al., 2009). Interestingly, the PNNs in the amygdala are both necessary for the protection and renewal of fear memories (Gogolla et al., 2009). This indicates the PNNs are not simply blocking the plasticity of neurons, but are also a crucial component of the plasticity. Treatment with ChABC abolishes spontaneous recovery and renewal of conditioned fear memory, while it does not interfere with memory consolidation (Gogolla et al., 2009). In the amygdala, the PNNs envelope both inhibitory and excitatory interneurons and the development of PNNs in the amygdala coincides with a switch in fear memory regulation.

In the hippocampus, a high density of PNNs is found in the CA2 (Lensjø et al., 2017a), fewer PNNs are found in the CA1, CA3 (Kochlamazashvili et al., 2010) and the dentate gyrus (Jansen et al., 2017) The PNNs are found around basket cells and bistratified neurons with high parvalbumin levels (Yamada et al., 2015). These inhibitory interneurons control pyramidal cells in the local circuit. In the CA2, the PNNs are also found around the excitatory pyramidal neurons (Carstens et al., 2016). The PNNs on the excitatory neurons suppress plasticity of excitatory synapses. Removal of the PNNs with ChABC leads to synaptic potentiation and the excitatory postsynaptic current (EPSC) amplitude increases (Carstens et al., 2016).

In a mouse model where PNN components brevican, neurocan, TN-R, and Tenascin C were deleted, synaptic depression was found amplified in the dentate gyrus (Jansen et al., 2017). When neurons and astrocytes from this KO model are cultured, a reduction in frequency of both EPSCs and inhibitory postsynaptic currents (IPSCs) was observed (Geissler et al., 2013). These studies indicate that the deletion of the PNN components leads to a loss of synaptic plasticity in hippocampal neurons. Similarly, in a TN-R KO model, reduction of long term potentiation (LTP) was found in the CA1 region (Bukalo et al., 2001). The threshold for the induction for LTP is also increased and the basal excitatory synaptic transmission is elevated (Bukalo et al., 2007). LTP is a persistent strengthening of the synapse and a form of synaptic plasticity. The loss of the potential to strengthen synapses when TN-R, a component of the PNNs, is missing, shows the PNNs do not only block plasticity but regulate plasticity of neurons in various manners. Indeed, removal of the PNNs with hyaluronidase treatment reduces calcium currents, which are crucial for neuronal activity, in the CA3 and CA1 (Kochlamazashvili et al., 2010).

Similar to findings in the amygdala and the cortex, PNN removal with ChABC and hyaluronidase in the hippocampus disrupts fear memory formation (Hylin et al., 2013). The function of the PNNs as a regulator of plasticity is also demonstrated in status epilepticus, a prolonged state of seizure. After status epilepticus, a period of enhanced neuronal activity, PNNs are lost (McRae et al., 2012). This finding indicates the PNNs are regulated by neuronal activity. Status epilepticus causes synaptic reorganization in the hippocampus, which is possible due to PNN removal (McRae et al., 2012). These findings indicate the PNNs in the hippocampus surround both excitatory and inhibitory neurons and in both cases the PNNs are crucial for the control of plasticity.

In the cerebellum, the PNNs can be found in the cerebellar cortex (Mabuchi et al., 2001) as well as in lateral and basolateral nuclei (Lafarga et al., 1984; Blosa et al., 2016) of the cerebellum. The PNNs in the cerebellum envelope the large excitatory Golgi neurons (Carulli et al., 2006), which synapse on the granule cells. Thin and compact PNNs can also be found around the Purkinje cells (Mabuchi et al., 2001), which project to the deep cerebellar nuclei. Both Golgi neurons and Purkinje cells are positive for parvalbumin. When PNN formation is blocked by the removal of one of the link proteins, HAPLN1, the Purkinje cell terminal is enhanced (Foscarin et al., 2011). Furthermore, when animals are reared in an enriched environment, which leads to higher plasticity in the cerebellum, the formation of the PNNs is reduced (Foscarin et al., 2011). The formation of the PNNs is linked to the level of plasticity in the cerebellum.

In the spinal cord, the PNNs are mostly found around interneurons, as well as around 30% of the motoneurons (Smith et al., 2015) in the ventral horn and around 20% of the neurons in the dorsal horn (Galtrey et al., 2008). Thickness of PNNs in the spinal cord increases with exercise, which suggests higher synaptic activity can enlarge PNNs (Smith et al., 2015). Treatment with ChABC in the spinal cord reinstates plasticity, which allows for the recovery of forelimb functions after spinal cord injury (Galtrey et al., 2007; Wang et al., 2011). This suggests that PNNs also act as a brake on plasticity in the spinal cord. Interestingly, the removal of the PNNs only leads to recovery when it is accompanied by rehabilitation (Wang et al., 2011). New stimuli to the spinal cord are needed to make use of the reopening of the plasticity by the removal of the PNNs.

A review on the identities on PNN neurons in different regions of the CNS allows us to draw a comparison on the properties on PNN neurons. There are many parallels on the formation of PNNs from the various CNS regions. In the cerebellum, the CSPGs which form the PNNs are produced both by glial cells and by neurons (Carulli et al., 2006). However, only neurons have the mRNA for the hyaluronan synthases (HASs) which produce hyaluronan, the backbone of the PNNs. Similar to the findings in the cerebellum, in the spinal cord HAS, together with aggrecan, HAPLN1 and brain link protein 2 (BRAL2) are only expressed by neurons (Galtrey et al., 2008). In both regions many PNN components are produced by multiple cell types but only neurons produce HASs (Carulli et al., 2007; Galtrey et al., 2008). This similarity indicates there is a shared underlying mechanism for the formation of the PNNs although variation between regions can also be found. Indeed, recombinant expression of HASs in cell culture allows the formation of pericellular matrix similar to PNNs (Kwok et al., 2010; Giamanco and Matthews, 2012).

Divergence between PNNs in different regions is caused by variations in the components of the PNNs, such as aggrecan (Matthews et al., 2002). Aggrecan exists with different glycosylation patterns and subtypes of neurons produce aggrecan with different glycosylation patterns (Lander et al., 1997; Matthews et al., 2002). The components of the PNNs also have distinct spatial patterns in the brain (Dauth et al., 2016). For example, aggrecan, brevican, and Tn-R differ in their spatial patterns, brevican intensity is higher in the caudate-putamen than in the thalamus, whilst aggrecan, and Tenascin-R show lower intensity in the caudate-putamen than in the thalamus. It is likely the heterogeneity of the PNNs reflects the variety of functions carried out by neurons with PNNs.

Overall, across CNS regions, PNNs surround a variety of interneurons and regulates plasticity. Since PNNs are mostly found around inhibitory interneurons in the cortex, an initial proposal for PNN functions is that PNNs act as a regulator on inhibitory neurons to balance the inhibitory and excitatory inputs (Dityatev et al., 2010). However, this proposal has been adjusted due to the identification of PNNs on excitatory neurons. It has been proposed that PNNs surrounding excitatory synapses could lead to an early closure of critical period (Carstens et al., 2016). The fast spiking interneurons have a high capacity for plasticity and the consistent presence of PNNs on the fast spiking interneurons provides an extra “tool” for the regulation of plasticity.

The PNNs act as a physical barrier between the neuron and the extracellular space. It acts as a local buffering reservoir for ions (Morawski et al., 2015), protects the neuron from oxidative stress (Suttkus et al., 2014) and toxic protein species (Miyata et al., 2007). Both hyaluronan and CSs are highly negatively charged molecules, and their presence allows PNNs to act as a local buffer for cations close to the synapse (Härtig et al., 1999). The PNNs mostly envelope fast spiking interneurons, which have high activity at their synapses and therefore need buffering for the cations involved in the neurotransmission. Using Fe³⁺ as a cationic probe, PNNs have been detected as a structure with high local charge density in patches on neuronal surfaces on tissue slices (Morawski et al., 2015). The concentration of negative charge in the PNNs is high enough for the PNNs to perform ion sorting properties on the neuronal surface (Morawski et al., 2015). The PNNs can also buffer ions, such as Fe³⁺, to prevent oxidative damage in the neuronal microenvironment (Suttkus et al., 2014). This was shown in the aggrecan, TN-R and HAPLN1 triple KO model, in which PNN formation is impaired (Suttkus et al., 2014). The study showed that PNNs protect neurons from oxidative stress induced by FeCl₃. After a deletion of aggrecan, TN-R or HAPLN1, protection against the oxidative stress caused by FeCl₃ is lost (Suttkus et al., 2014). Similarly, PNN removal with ChABC makes parvalbumin neurons more vulnerable to oxidative stress (Cabungcal et al., 2013). The PNNs also protect neurons from the toxicity of amyloid beta (Miyata et al., 2007). Amyloid beta has a neurotoxic effect on neurons without PNNs, but not on neurons surrounded with PNNs. When the PNNs are removed with ChABC, neuronal death caused by amyloid beta is increased. Similar observation is made in human brains where PNN neurons are indeed protected from amyloid beta plaques (Morawski et al., 2012). These examples show that PNNs act as a physical barrier to molecules that can damage their underlying neurons.

Apart from acting as a barrier between the extracellular matrix molecules and the neurons, the PNNs also inhibit the growth of neurites and thus deters synapse formation. CSPGs, one of the main components of the PNNs, are strong inhibitory molecules highly up-regulated in the glial scar after CNS injury, they collapse growth cones and inhibit neural regeneration (Cheah et al., 2016; Shinozaki et al., 2016). Breaking down CSPGs with ChABC treatment improves the regeneration of axons (Shinozaki et al., 2016). The role as a plasticity brake of PNNs is partially attributed to the presence of CSPGs in the structure.

The different components of the PNNs have specific binding capacities for proteins. Several growth factors, such as midkine and fibroblast growth factor 2, have been shown to bind specifically to chondroitin sulfate E (CS-E) which is enriched in PNNs compared to the soluble extracellular matrix (ECM) (Deepa et al., 2002). Similarly, the chemorepulsive molecule Semaphorin3a (Sema3A) binds to the PNNs via CS-E (Dick et al., 2013). Removal of the PNNs with ChABC removes Sema3A from the neuronal surface (Vo et al., 2013). The finding suggests that the PNNs present Sema3A to approaching axons from other neurons to allow Sema3A to function as a repulsive signaling molecule (de Winter et al., 2016).

TN-R carries a HNK-1 epitope, which allows it to bind GABA receptors (Bukalo et al., 2007). GABA receptors are key regulators of synaptic activity, so this binding capacity allows the PNNs to regulate the synapse. In TN-R KO animals, a higher calcium influx is observed (Bukalo et al., 2007), which shows a relation to plasticity of the synapse. The hyaluronan backbone of the PNNs has the capacity to modulate postsynaptic L-type calcium channels (Kochlamazashvili et al., 2010). Treatment with hyaluronidase indeed reduced calcium currents in the hippocampus (Kochlamazashvili et al., 2010). Another regulator of synaptic proteins is brevican, which regulates potassium channels and AMPA receptors (Favuzzi et al., 2017). This function allows brevican to regulate the activity dependent gating of parvalbumin interneuron function (Favuzzi et al., 2017). Brevican deficient mice indeed have impaired LTP in the CA1 of the hippocampus (Brakebusch et al., 2002).

The transcription factor orthodenticle homeobox 2 (Otx2), needs to be captured by PNNs to be internalized by the neuron (Beurdeley et al., 2012). This internalization is crucial for the maturation of parvalbumin positive neurons in the cortex and regulate plasticity. Treatment with ChABC removes the PNNs and reduces the amount of Otx2 inside the neuron. A recent study also demonstrates that the PNNs regulate the level of PV expression and thus the plasticity capacity of a neuron (Donato et al., 2013). In an animal model with a point mutation on the Otx2 gene, maturation of parvalbumin interneurons is delayed (Lee et al., 2017). Similar to the previous literature using OTX2 blocking peptide, the closure of critical period plasticity is also delayed in this model, confirming the importance of the binding of Otx2 to the function of PNNs in critical period plasticity. This suggests that PNNs affect neurons through its capacity to selectively bind growth factors, transcription factors and signaling molecules, which ultimately affect the neuronal functions.

In addition to the binding partners discussed above, there are other proteins which may play a similar role as Sema3A or OTX2 to the PNNs. Recently, the tumor necrosis factor-stimulated gene-6 (TSG-6) was identified in the glial scar (Coulson-Thomas et al., 2016). The expression of TSG-6 is up-regulated after spinal cord injury (Coulson-Thomas et al., 2016). TSG-6 binds to hyaluronan and stabilizes the hyaluronan matrices (Baranova et al., 2011). Since the backbone of PNNs consists of hyaluronan it is possible TSG-6 binds to the PNNs and has a stabilizing role comparable to its role in the glial scar. Other than TSG-6, another extracellular protein neuronal pentraxin 2 (Nptx2) has also been proposed as a PNN binding partner. ChABC treatment, which removes PNNs, removes Nptx2 from the surface of neurons (Chang et al., 2010). Since Nptx2 is involved in the regulation of critical periods, just like the PNNs, and ChABC removes Nptx2, it is likely the protein is a binding partner of the PNNs via the CS component (Gu et al., 2013). Nptx2 controls plasticity through clustering of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Pelkey et al., 2015). It has been previously shown that the PNNs limit AMPA receptor movement (see the next section) (Frischknecht et al., 2009). PNNs may regulate AMPA receptors through the regulation of NPTX2. Both proteins are potential candidates for specific binding partners of the PNNs.

Perineuronal nets (PNNs) limit the mobility of membrane bound proteins on the neuronal surface. When the PNNs are removed from the neuronal surface using hyaluronidase in neuronal cultures, lateral diffusion of AMPA receptor subunits increases (Frischknecht et al., 2009). Whole-cell patch-clamp recordings showed that the induced increase in diffusion allows a fast exchange of desensitized receptors under high stimulation, which then leads to an increase in paired-pulse ratio, a form of short term synaptic plasticity (Frischknecht et al., 2009). The limitation to the lateral mobility of membrane bound proteins caused by the PNNs allow the PNNs to inhibit synaptic plasticity.

As discussed earlier in this review, many of the components of the PNNs have binding capacities for specific proteins. The PNNs can apply this function to attract proteins to the neuron. This process has been investigated with Otx2. Otx2 relies on the PNNs to enter the neuron and influence plasticity (Beurdeley et al., 2012). Otx2 is expressed in the choroid plexus and spreads to other brain regions through the cerebrospinal fluid (Spatazza et al., 2013). When Otx2 reaches the visual cortex, it binds the PNNs and enters the neuron to regulate maturation and thus affect plasticity (Sugiyama et al., 2008). The Otx2 internalization is mediated through its binding to CS-D and CS-E in the PNNs. Consequently, when the PNN is removed with ChABC, the amount of Otx2 in the neurons is reduced (Beurdeley et al., 2012). This indicates that the communication between choroid plexus and the visual cortex relies on the PNNs to bind Otx2. The PNNs enable long distance communication between neurons by its binding specificity.

The PNNs also regulate the signals which a neuron presents to its surrounding ECM and potentially other cells. Semaphorin 3A (Sema3A) binds the PNNs and potentiates the inhibition of PNNs to the growth of axons (Dick et al., 2013) and potentially to the formation of synapses. Inhibition of neurite outgrowth of dorsal root ganglion cultures on a surface of PNN glycans, is stronger when the surface of PNN glycans is mixed with Sema3A (Dick et al., 2013). This finding indicates the binding of Sema3A to the PNNs allows the PNNs to inhibit the approach of axons from other neurons toward the neuron it surrounds. In contrast, the PNNs block integrin activation and signaling to other neurons (Orlando et al., 2012). Removal of the PNN with ChABC allows activation of integrins which leads to enhanced spine motility (Orlando et al., 2012). In conclusion, the PNNs control communication of the neurons they surround toward other neurons through the altered presentation of protein signals.

Neurons communicate through signaling at the synapse and the PNNs regulate the synapse with several methods. As described earlier in this review, PNNs acts as a physical barrier to limit AMPA receptor mobility on the neuronal membrane (Frischknecht et al., 2009). The limitation of the AMPA receptor mobility blocks them from leaving the synaptic cleft, which keeps the amount of AMPA receptors at the synapse constant. The physical barrier function of the PNNs thus reduces the capacity for plasticity.

The PNNs also regulate the synapse through parvalbumin. Many of the neurons surrounded by PNNs are parvalbumin expressing interneurons (Härtig et al., 1995; Baig et al., 2005; Yamada et al., 2015). Parvalbumin is a calcium binding protein which regulates short term synaptic plasticity, which was shown with electrophysiology experiments on a parvalbumin knockout mouse model (Caillard et al., 2000). It is likely parvalbumin regulates the short term plasticity by binding the calcium ions. In the mouse hippocampus, digestion of PNNs with ChABC leads to a decrease of parvalbumin levels inside neurons (Yamada et al., 2015). Both the amount of parvalbumin mRNA as well as the amount of the protein itself are reduced by ChABC injection into the brain. These results show that PNNs help in maintaining the amount of parvalbumin in the neuron which allows it to regulate plasticity.

The PNNs also block the formation of new synapses between neurons (Geissler et al., 2013). When the formation of the PNNs is decreased in cultured neurons by knockout of brevican, neurocan, TN-R, and Tenascin-C, the neurons show an increase in the amount of inhibitory synapses they form. Similarly, when the PNNs are removed with ChABC, synaptogenesis is increased between transplant and host neurons (Suzuki et al., 2007). The formation of new synapses allows neurons to form a new route to communicate. In conclusion, the PNNs regulate cell signaling at the synapse.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.