Perineuronal Nets Alterations Contribute to Stress-Induced Anxiety-Like Behavior.

Anxiety disorder is one of the most common mental disorders worldwide, affecting nearly 30% of adults. However, its underlying molecular mechanisms are still unclear. Here we subjected mice to chronic restraint stress (CRS), a paradigm known to induce anxiety-like behavior in mice. CRS mice exhibited anxiety-like behavior and reduced synaptic transmission in the medial prefrontal cortex (mPFC). Notably, Wisteria Floribunda agglutinin (WFA) staining showed a reduction of perineuronal nets (PNNs) expression in the mPFC of CRS mice. And the mRNA and protein levels of aggrecan (ACAN), a core component of PNNs, were also reduced. Parallelly, enzymatic digestion of PNNs in the mPFC by injecting Chondroitinase ABC (chABC) resulted in anxiety-like behavior in mice. Fluoxetine (FXT) is a clinically prescribed antidepressant/anxiolytic drug. FXT treatment in CRS mice not only ameliorated their deficits in behavior and synaptic transmissions, but also prevented CRS-induced reduction of PNNs and ACAN expressions. This study demonstrates that proper PNNs level is critical to brain functions, and their decline may serve as a pathological mechanism of anxiety disorders.

Zinc-a2-Glycoprotein Acts as a Component of PNN to Protect Hippocampal Neurons from Apoptosis.

In the adult mouse brain, perineuronal net (PNN), a highly structured extracellular matrix, surrounds subsets of neurons. The AZGP1 gene encodes zinc-2-glycoprotein (ZAG) is a lipid-mobilizing factor. However, its expression and distribution in the adult brain have been controversial. Here, for the first time, we demonstrate that the secreted ZAG is localized to Wisteria floribunda agglutinin (WFA)-positive PNNs around parvalbumin (PV)-expressing interneurons in the hippocampus, cortex, and a number of other PNN-bearing neurons and co-localizes with aggrecan, one of the components of PNNs. Few ZAG-positive nets were seen in the area without WFA staining by chondroitinase ABC (ChABC) which degrades glycosaminoglycans (GAGs) from the chondroitin sulfate proteoglycans (CSPGs) in the PNN. Reanalysis of single-cell sequencing data revealed that ZAG mRNA was mainly expressed in oligodendrocyte lineages, specifically in olfactory sheathing cells. The ZAG receptor ß3 adrenergic receptor (ß3AR) is also selectively co-localized with PV interneurons and CA2 pyramidal neurons in the hippocampus. In addition, molecular docking provides valuable new insights on how GAGs interfere with ZAG and ZAG/ß3AR complex. Finally, our results indicated that human recombinant ZAG could significantly inhibit serum derivation-induced cell apoptosis in HT22 cells. Our combined experimental and theoretical approach raises a unique hypothesis namely that ZAG may be a crucial functional attribute of PNNs in the brain to protect neuronal cell from apoptosis.

PGC-1α regulates critical period onset/closure, mediating cortical plasticity.

Peroxisome proliferator-activated receptor PPARγ coactivator-α (PGC-1α) is concentrated in inhibitory interneurons and plays a vital role in neuropsychiatric diseases. We previously reported some characteristic features of schizophrenia (SZ) in GABAergic neuron-specific Pgc-1alpha knockout (KO) mice (Dlx5/6-Cre: Pgc-1alphaf/f). However, there is a fundamental gap in the molecular mechanism by which the Pgc-1alpha gene is involved in the neurobehavioral abnormalities of SZ. The loss of critical period (CP) triggers-maturations of parvalbumin interneurons (PVIs) and brakes-and the formation of perineuronal nets (PNNs) implicates mistimed trajectories during adult brain development. In this study, using the Pgc-1alpha KO mouse line, we investigated the association of Pgc-1alpha gene deletion with SZ-like behavioral deficits, PVI maturation, PNN integrity and synaptic ultrastructure. These findings suggest that Pgc-1alpha gene deletion resulted in a failure of CP onset and closure, thereby prolonging cortical plasticity timing. To determine whether the manipulation of the PNN structure is a potential method of altering neuronal plasticity, GM6001, a broad-spectrum matrix metalloproteinase (MMP)-inhibitor was applied. Here we confirmed that the treatment could effectively correct the CP plasticity window and ameliorate the synaptic ultrastructure in the Pgc-1alpha KO brain. Moreover, the intervention effect on neuronal plasticity was followed by the rescue of short-term habituation deficits and the mitigation of aberrant salience, which are some characteristic features of SZ. Taken collectively, these findings suggest that the role of PGC-1α in regulating cortical plasticity is mediated, at least partially, through the regulation of CP onset/closure. Strategically introduced reinforcement of molecular brakes may be a novel preventive therapy for psychiatric disorders associated with PGC-1α dysregulation.

Chondroitin 4-O-sulfation regulates hippocampal perineuronal nets and social memory.

Glycan alterations are associated with aging, neuropsychiatric, and neurodegenerative diseases, although the contributions of specific glycan structures to emotion and cognitive functions remain largely unknown. Here, we used a combination of chemistry and neurobiology to show that 4-O-sulfated chondroitin sulfate (CS) polysaccharides are critical regulators of perineuronal nets (PNNs) and synapse development in the mouse hippocampus, thereby affecting anxiety and cognitive abilities such as social memory. Brain-specific deletion of CS 4-O-sulfation in mice increased PNN densities in the area CA2 (cornu ammonis 2), leading to imbalanced excitatory-to-inhibitory synaptic ratios, reduced CREB activation, elevated anxiety, and social memory dysfunction. The impairments in PNN densities, CREB activity, and social memory were recapitulated by selective ablation of CS 4-O-sulfation in the CA2 region during adulthood. Notably, enzymatic pruning of the excess PNNs reduced anxiety levels and restored social memory, while chemical manipulation of CS 4-O-sulfation levels reversibly modulated PNN densities surrounding hippocampal neurons and the balance of excitatory and inhibitory synapses. These findings reveal key roles for CS 4-O-sulfation in adult brain plasticity, social memory, and anxiety regulation, and they suggest that targeting CS 4-O-sulfation may represent a strategy to address neuropsychiatric and neurodegenerative diseases associated with social cognitive dysfunction.

Selective Disruption of Perineuronal Nets in Mice Lacking Crtl1 is Sufficient to Make Fear Memories Susceptible to Erasure.

The ability to store, retrieve, and extinguish memories of adverse experiences is an essential skill for animals' survival. The cellular and molecular factors that underlie such processes are only partially known. Using chondroitinase ABC treatment targeting chondroitin sulfate proteoglycans (CSPGs), previous studies showed that the maturation of the extracellular matrix makes fear memory resistant to deletion. Mice lacking the cartilage link protein Crtl1 (Crtl1-KO mice) display normal CSPG levels but impaired CSPG condensation in perineuronal nets (PNNs). Thus, we asked whether the presence of PNNs in the adult brain is responsible for the appearance of persistent fear memories by investigating fear extinction in Crtl1-KO mice. We found that mutant mice displayed fear memory erasure after an extinction protocol as revealed by analysis of freezing and pupil dynamics. Fear memory erasure did not depend on passive loss of retention; moreover, we demonstrated that, after extinction training, conditioned Crtl1-KO mice display no neural activation in the amygdala (Zif268 staining) in comparison to control animals. Taken together, our findings suggest that the aggregation of CSPGs into PNNs regulates the boundaries of the critical period for fear extinction.

Behavioral regulation by perineuronal nets in the prefrontal cortex of the CNTNAP2 mouse model of autism spectrum disorder.

Autism spectrum disorders (ASDs) arise from altered development of the central nervous system, and manifest behaviorally as social interaction deficits and restricted and repetitive behaviors. Alterations to parvalbumin (PV) expressing interneurons have been implicated in the neuropathological and behavioral deficits in autism. In addition, perineuronal nets (PNNs), specialized extracellular matrix structures that enwrap the PV-expressing neurons, also may be altered, which compromises neuronal function and susceptibility to oxidative stress. In particular, the prefrontal cortex (PFC), which regulates several core autistic traits, relies on the normal organization of PNNs and PV-expressing cells, as well as other neural circuit elements. Consequently, we investigated whether PNNs and PV-expressing cells were altered in the PFC of the CNTNAP2 knockout mouse model of ASD and whether these contributed to core autistic-like behaviors in this model system. We observed an overexpression of PNNs, PV-expressing cells, and PNNs enwrapping PV-expressing cells in adult CNTNAP2 mice. Transient digestion of PNNs from the prefrontal cortex (PFC) by injection of chondroitinase ABC in CNTNAP2 mutant mice rescued some of the social interaction deficits, but not the restricted and repetitive behaviors. These findings suggest that the neurobiological regulation of PNNs and PVs in the PFC contribute to social interaction behaviors in neurological disorders including autism.

Interactions between astrocytes and extracellular matrix structures contribute to neuroinflammation-associated epilepsy pathology.

Often considered the "housekeeping" cells of the brain, astrocytes have of late been rising to the forefront of neurodegenerative disorder research. Identified as crucial components of a healthy brain, it is undeniable that when astrocytes are dysfunctional, the entire brain is thrown into disarray. We offer epilepsy as a well-studied neurological disorder in which there is clear evidence of astrocyte contribution to diseases as evidenced across several different disease models, including mouse models of hippocampal sclerosis, trauma associated epilepsy, glioma-associated epilepsy, and beta-1 integrin knockout astrogliosis. In this review we suggest that astrocyte-driven neuroinflammation, which plays a large role in the pathology of epilepsy, is at least partially modulated by interactions with perineuronal nets (PNNs), highly structured formations of the extracellular matrix (ECM). These matrix structures affect synaptic placement, but also intrinsic neuronal properties such as membrane capacitance, as well as ion buffering in their immediate milieu all of which alters neuronal excitability. We propose that the interactions between PNNs and astrocytes contribute to the disease progression of epilepsy vis a vis neuroinflammation. Further investigation and alteration of these interactions to reduce the resultant neuroinflammation may serve as a potential therapeutic target that provides an alternative to the standard anti-seizure medications from which patients are so frequently unable to benefit.

A glial perspective on the extracellular matrix and perineuronal net remodeling in the central nervous system.

A structural scaffold embedding brain cells and vasculature is known as extracellular matrix (ECM). The physical appearance of ECM in the central nervous system (CNS) ranges from a diffused, homogeneous, amorphous, and nearly omnipresent matrix to highly organized distinct morphologies such as basement membranes and perineuronal nets (PNNs). ECM changes its composition and organization during development, adulthood, aging, and in several CNS pathologies. This spatiotemporal dynamic nature of the ECM and PNNs brings a unique versatility to their functions spanning from neurogenesis, cell migration and differentiation, axonal growth, and pathfinding cues, etc., in the developing brain, to stabilizing synapses, neuromodulation, and being an active partner of tetrapartite synapses in the adult brain. The malleability of ECM and PNNs is governed by both intrinsic and extrinsic factors. Glial cells are among the major extrinsic factors that facilitate the remodeling of ECM and PNN, thereby acting as key regulators of diverse functions of ECM and PNN in health and diseases. In this review, we discuss recent advances in our understanding of PNNs and how glial cells are central to ECM and PNN remodeling in normal and pathological states of the CNS.

Changes in Brain Matrix Glycan Sulfation Associate With Reactive Gliosis and Motor Coordination in Mice With Head Trauma.

Perineuronal nets (PNNs) are extracellular matrix (ECM) structures that enmesh and regulate neurocircuits involved in motor and sensory function. Maladaptive changes to the composition and/or abundance of PNNs have been implicated in preclinical models of neuroinflammation and neurocircuit destabilization. The central nervous system (CNS) is limited in its capacity to repair and reorganize neural networks following traumatic brain injury (TBI) and little is known about mechanisms of ECM repair in the adult brain after TBI. In this study, adult male C57BL/6 mice were subjected to a TBI via a controlled cortical impact (CCI) to the right motor and somatosensory cortices. At 7 days following CCI, histological analysis revealed a loss of Wisteria floribunda agglutinin (WFA) positive PNN matrices in the ipsilateral cortex. PNNs are comprised of chondroitin sulfate (CS) and dermatan sulfate (DS)-glycosaminoglycans (GAGs), the composition of which are known to influence neuronal integrity and repair. Using an innovative liquid chromatography tandem mass spectrometry (LC-MS/MS) method, we analyzed the relative abundance of six specific CS/DS-GAG isomers (Δ4S-, Δ6S-, Δ4S6S-, Δ2S6S-, Δ0S-CS, and Δ2S4S-DS) from fixed-brain sections after CCI injury. We report a significant shift in CS/DS-GAG sulfation patterns within the rostro-caudal extent of the injury site from mice exposed to CCI at 7 days, but not at 1 day, post-CCI. In the ipsilateral thalamus, the appearance of WFA+ puncta occurred in tandem with gliosis at 7 days post-CCI, but weakly colocalized with markers of gliosis. Thalamic WFA+ puncta showed moderate colocalization with neuronal ubiquitin C-terminal hydrolase L1 (UCHL1), a clinical biomarker for TBI injury. A shift in CS/DS-GAG sulfation was also present in the thalamus including an increase of 6S-CS, which is a specific isomer that associates with the presence of glial scarring. Upregulation of the 6S-CS-specific sulfotransferase (CHST3) gene expression was accompanied by reactive gliosis in both the ipsilateral cortex and thalamus. Moreover, changes in 6S-CS extracted from the thalamus positively correlated with deficits in motor coordination after CCI. Collectively, these data argue that CCI alters CS/DS-GAG sulfation in association with the spatiotemporal progression of neurorepair. Therapeutic interventions targeting restoration of CS/DS-GAG sulfation patterns may improve outcomes from TBI.

Impact of Perineuronal Nets on Electrophysiology of Parvalbumin Interneurons, Principal Neurons, and Brain Oscillations: A Review.

Perineuronal nets (PNNs) are specialized extracellular matrix structures that surround specific neurons in the brain and spinal cord, appear during critical periods of development, and restrict plasticity during adulthood. Removal of PNNs can reinstate juvenile-like plasticity or, in cases of PNN removal during early developmental stages, PNN removal extends the critical plasticity period. PNNs surround mainly parvalbumin (PV)-containing, fast-spiking GABAergic interneurons in several brain regions. These inhibitory interneurons profoundly inhibit the network of surrounding neurons via their elaborate contacts with local pyramidal neurons, and they are key contributors to gamma oscillations generated across several brain regions. Among other functions, these gamma oscillations regulate plasticity associated with learning, decision making, attention, cognitive flexibility, and working memory. The detailed mechanisms by which PNN removal increases plasticity are only beginning to be understood. Here, we review the impact of PNN removal on several electrophysiological features of their underlying PV interneurons and nearby pyramidal neurons, including changes in intrinsic and synaptic membrane properties, brain oscillations, and how these changes may alter the integration of memory-related information. Additionally, we review how PNN removal affects plasticity-associated phenomena such as long-term potentiation (LTP), long-term depression (LTD), and paired-pulse ratio (PPR). The results are discussed in the context of the role of PV interneurons in circuit function and how PNN removal alters this function.

PGC-1α regulate critical period plasticity via gene × environment interaction in the developmental trajectory to schizophrenia.

Genes and environmental conditions are thought to interact in the development of postnatal brain in schizophrenia (SZ). Genome wide association studies have identified that PPARGC1A being one of the top candidate genes for SZ. We previously reported GABAergic neuron-specific PGC-1α knockout mice (Dlx5/6-Cre:PGC-1αfl/fl) presented some characteristic features of SZ. However, there is a fundamental gap of the molecular mechanism by which PGC-1α gene involved in the developmental trajectory to SZ. To explore whether PGC-1α regulates environmental factors interacting with genetic susceptibility to trigger symptom onset and disease progression, PGC-1α deficient mice were utilized to model genetic effect and an additional oxidative stress was induced by GBR injection. We confirm that PGC-1α gene deletion prolongs critical period (CP) timing, as revealed by delaying maturation of PV interneurons (PVIs), including their perineuronal nets (PNNs). Further, we confirm that gene × environment (G × E) influences CP plasticity synergistically and the interaction varies as a function of age, with the most sensitive period being at preweaning stage, and the least sensitive one at early adult age in PGC-1α deficient mice. Along this line, we find that the synergic action of G × E is available in ChABC-infusion PGC-1α KO mice, even though during the adulthood, and the neuroplasticity seems to remain open to fluctuate. Altogether, these results refine the observations made in the PGC-1α deficient mice, a potential mouse model of SZ, and illustrate how PGC-1α regulates CP plasticity via G × E interaction in the developmental trajectory to SZ.

Environmental Enrichment From Birth Impacts Parvalbumin Expressing Cells and Wisteria Floribunda Agglutinin Labelled Peri-Neuronal Nets Within the Developing Murine Striatum.

Environmental enrichment can dramatically affect both the development and function of neural circuits. This is accomplished, at least in part, by the regulation of inhibitory cellular networks and related extracellular matrix glycoprotein structures known as perineuronal nets. The degree to which enhanced housing can influence brain areas involved in the planning and execution of actions is not well known. We examined the effect of enriching mice from birth on parvalbumin expression and perineuronal net formation in developing and adult striatum. This input nucleus of the basal ganglia consists of topographically discernible regions that serve different functions, providing a means of simultaneously examining the influence of environmental factors on discrete, but related networks. Greater densities of striatal parvalbumin positive cells and wisteria floribunda agglutinin labelled perineuronal nets were present in enriched pups during the second postnatal week, primarily within the lateral portion of the nucleus. Housing conditions continued to have an impact into adulthood, with enriched mice exhibiting higher parvalbumin positive cell densities in both medial and lateral striatum. Curiously, no differences due to housing conditions were detected in striatal perineuronal net densities of mature animals. The degree of overlap between striatal parvalbumin expression and perineuronal net formation was also increased, suggesting that heightened neural activity associated with enrichment may have contributed to greater engagement of networks affiliated with cells that express the calcium binding protein. Brain derived neurotrophic factor, an important regulator of inhibitory network maturation, is also subtly, but significantly affected within the striatum of enriched cohorts. Together, these findings suggest that environmental enrichment can exert cell specific effects within different divisions of an area vital for the regulation of action.

Perineuronal Nets and Their Role in Synaptic Homeostasis.

Extracellular matrix (ECM) molecules that are released by neurons and glial cells form perineuronal nets (PNNs) and modulate many neuronal and glial functions. PNNs, whose structure is still not known in detail, surround cell bodies and dendrites, which leaves free space for synapses to come into contact. A reduction in the expression of many neuronal ECM components adversely affects processes that are associated with synaptic plasticity, learning, and memory. At the same time, increased ECM activity, e.g., as a result of astrogliosis following brain damage or in neuroinflammation, can also have harmful consequences. The therapeutic use of enzymes to attenuate elevated neuronal ECM expression after injury or in Alzheimer's disease has proven to be beneficial by promoting axon growth and increasing synaptic plasticity. Yet, severe impairment of ECM function can also lead to neurodegeneration. Thus, it appears that to ensure healthy neuronal function a delicate balance of ECM components must be maintained. In this paper we review the structure of PNNs and their components, such as hyaluronan, proteoglycans, core proteins, chondroitin sulphate proteoglycans, tenascins, and Hapln proteins. We also characterize the role of ECM in the functioning of the blood-brain barrier, neuronal communication, as well as the participation of PNNs in synaptic plasticity and some clinical aspects of perineuronal net impairment. Furthermore, we discuss the participation of PNNs in brain signaling. Understanding the molecular foundations of the ways that PNNs participate in brain signaling and synaptic plasticity, as well as how they change in physiological and pathological conditions, may help in the development of new therapies for many degenerative and inflammatory diseases of the brain.

Adult vitamin D deficiency disrupts hippocampal-dependent learning and structural brain connectivity in BALB/c mice.

Converging evidence from human and animal studies support an association between vitamin D deficiency and cognitive impairment. Previous studies have shown that hippocampal volume is reduced in adults with vitamin D deficiency as well as in a range of disorders, such as schizophrenia. The aim of the current study was to examine the effect of adult vitamin D (AVD) deficiency on hippocampal-dependent spatial learning, and hippocampal volume and connectivity in healthy adult mice. Ten-week-old male BALB/c mice were fed a control (vitamin D 1500 IU/kg) or vitamin D-depleted (vitamin D 0 IU/kg) diet for a minimum of 10 weeks. The mice were then tested for hippocampal-dependent spatial learning using active place avoidance (APA) and on tests of muscle and motor coordination (rotarod and grip strength). The mice were perfused and brains collected to acquire ex vivo structural and diffusion-weighted images using a 16.4 T MRI scanner. We also performed immunohistochemistry to quantify perineuronal nets (PNNs) and parvalbumin (PV) interneurons in various brain regions. AVD-deficient mice had a lower latency to enter the shock zone on APA, compared to control mice, suggesting impaired hippocampal-dependent spatial learning. There were no differences in rotarod or grip strength, indicating that AVD deficiency did not have an impact on muscle or motor coordination. AVD deficiency did not have an impact on hippocampal volume. However, AVD-deficient mice displayed a disrupted network centred on the right hippocampus with abnormal connectomes among 29 nodes. We found a reduction in PNN positive cells, but no change in PV, centred on the hippocampus. Our results provide compelling evidence to show that AVD deficiency in otherwise healthy adult mice may play a key role in hippocampal-dependent learning and memory formation. We suggest that the spatial learning deficits could be due to the disruption of right hippocampal structural connectivity.

Nicotine self-administration remodels perineuronal nets in ventral tegmental area and orbitofrontal cortex in adult male rats.

Nicotine, a major psychoactive component of tobacco smoke, alters gamma-aminobutyric acid (GABA) modulation of dopamine neurons in the ventral tegmental area (VTA). Changes in structural neuroplasticity can occur in GABAergic parvalbumin (PRV) positive neurons, which are enveloped by structures of the extracellular matrix called perineuronal nets (PNNs). In the current study, rats were trained to self-administer intravenous nicotine (0.03 mg/kg/infusion) for 21 days in 1-hour daily sessions with an incrementing fixed ratio requirement; a control group received saline infusions. At either 45 minutes or 72 hours after the last session, immunofluorescence measurements for PNNs, PRV and c-Fos were conducted. In VTA, nicotine self-administration reduced the number of PRV+ cells surrounded by PNNs at 45 minutes, as well as reducing the intensity of PNNs, suggesting a remodeling of GABA interneurons in this region; the number of PRV+ cells surrounded by PNNs was also reduced at 72 hours. A similar reduction of PNNs occurred in orbitofrontal cortex (OFC) but not in medial prefrontal cortex (prelimbic or infralimbic), 45 minutes after the last session; PNNs were not detected in nucleus accumbens (shell or core). The reduction of PNNs in VTA and OFC was unrelated to c-Fos + cells, as the percent of wisteria floribunda agglutinin + cells co-expressing c-Fos was decreased in OFC but not in VTA. Thus, nicotine self-administration remodeled PNNs surrounding GABA interneurons in VTA and its indirect connections to OFC, suggesting a new possible molecular target where nicotine-induced neuroplasticity takes place. PNN manipulations may prevent or reverse the different stages of tobacco addiction.