In 1898, Camillo Golgi, an eminent Italian physician and pathologist, published a landmark paper on the structure of “nervous cells.” In addition to the organelle that still bears his name, the Golgi apparatus, he described “a delicate covering” surrounding neurons’ cell bodies and extending along their dendrites. That same year, another Italian researcher, Arturo Donaggio, observed that these coverings, now known as perineuronal nets (PNNs), had openings in them, through which, he correctly surmised, axon terminals from neighboring neurons make synapses.
Since then, however, PNNs have been largely neglected by the scientific community—especially after Santiago Ramón y Cajal, a fierce rival of Golgi (who would later share the Nobel Prize with him), dismissed them as a histological artifact. It wasn’t until the 1970s, thanks to the improvement of histological techniques and the development of immunohistochemistry, that researchers confirmed the existence of PNNs around some types of neurons in the brain and spinal cord of many vertebrate species, including humans.
Composed of extracellular matrix (ECM) molecules, PNNs form during postnatal development, marking the end of what’s known as the “critical period” of heightened brain plasticity. For a while after birth, the external environment has a profound effect on the wiring of neuronal circuits and, in turn, on the development of an organism’s skills and behaviors, such as language, sensory processing, and emotional traits. But during childhood and adolescence, neuronal networks become more fixed, allowing the individual to retain the acquired functions. Evidence gathered over the past 15 years suggests that PNNs contribute to this fixation in many brain areas, by stabilizing the existing contacts between neurons and repelling incoming axons.
Because limited neuronal plasticity underlies the irreversibility of many afflictions of the central nervous system (CNS), from stroke to spinal cord injury to neurodegenerative diseases, PNNs have been considered promising targets to enhance CNS repair. Moreover, they are increasingly recognized as important players in the regulation of memory processes.
PNNs may also play a supportive role in the normal functioning of the CNS. These coatings have been repeatedly observed around highly active neurons, and researchers have proposed that the structures provide a buffered, negatively charged environment that controls the diffusion of ions such as sodium, potassium, and calcium, thus serving as a rapid cation exchanger to support neuronal activity.1 PNNs have also been shown to protect neurons from oxidative stress, as they limit the detrimental effect of excessive reactive oxygen species on neuronal function or survival. Indeed, enzymatic degradation of the PNNs renders neurons more susceptible to oxidative stress.2
A lot of progress has been made in the last two decades toward illuminating the structural and functional properties of PNNs, defining their roles in CNS plasticity, and developing methods to manipulate them to increase plasticity, memory, and CNS repair. Still, how exactly PNNs work in the brain, and which precise mechanisms underlie their remodeling in physiological or pathological conditions, are still open questions.
PNNs and the plastic brain
PNNs’ role in closing the critical period of brain plasticity is now well established. In 2010, for instance, my colleagues and I showed that knockout mice lacking a PNN component called link protein display a reduced formation of PNNs, and they maintain juvenile levels of plasticity throughout adulthood.3
Another example comes from rats with amblyopia, a neurodevelopmental disorder resulting from an imbalance between the neural signals coming from the two eyes during the critical period for visual development. Inputs from the right and left eyes compete when they first converge on neurons in the primary visual cortex, leading to a physiological and anatomical cortical representation of the relative inputs contributed by either eye. When one eye is deprived of visual input—for instance, due to a congenital cataract—individuals suffer a loss of cortical response to that eye and an overrepresentation of the input from the healthy eye, resulting in visual impairment. In adulthood, because the critical period is closed, vision will remain defective even if the cause of amblyopia is treated. The removal of PNNs in the visual cortex of adult rats, however, has proven effective in treating amblyopia.4
Under particular circumstances, such as enriched environmental stimulation, the adult brain regains certain levels of plasticity, and here too, PNNs appear to be important mediators. Adult amblyopic rats reared in a cage enriched with toys, ladders, and running wheels show a reduction of PNNs in the visual cortex and recover normal visual acuity after two to three weeks in this environment with no other treatment.5
In 2016, my colleagues and I documented similar links between PNNs and plasticity in the vestibular system, which detects head position and acceleration, stabilizes gaze and body posture, and contributes to self-motion perception. Mice that have suffered permanent damage to the inner ear vestibular receptors generally show severe deficits in their posture and balance, but will improve over time. This improvement comes along with an initial decrease of PNNs in the areas of the brain stem that regulate vestibular functions, followed by a complete restoration of the PNNs after posture and balance strengthen.6
Beyond these examples of recovering from sensory deficiencies, the adult brain exhibits plastic tendencies during normal learning, and recent evidence points to the role of PNNs in memory formation and retention. In rodents, explicit memory—information that can be consciously recollected—can be assessed by a novel object recognition test: when animals are exposed to familiar and novel objects, they spend more time exploring the novel one. This type of memory requires synaptic plasticity in a specific area of the cerebral cortex. Mutant mice lacking link proteins, and thus having reduced PNNs, exhibit a prolonged memory for familiar objects, and degradation of the PNN via the application of the bacterial enzyme chondroitinase ABC gives similar results. In both cases, the removal of PNNs facilitates the induction of synaptic plasticity.7
In addition to resisting memory formation, PNNs may also be to blame for blocking memory destruction. Whereas young individuals can permanently erase a fear memory by extinction training—a form of learning involving associating the fear-inducing stimulus with neutral scenarios—adults exhibit fear behaviors that are resistant to erasure. These behaviors depend on the amygdala, where PNNs are present in adult, but not young, animals. Interestingly, in adult mice, PNN degradation in the amygdala by chondroitinase ABC reopens a critical period during which fear memories can be fully erased by extinction training.8 In addition, PNNs in various cortical areas have recently been shown to be important for storage of fear memories, as their removal disrupts such memories.9,10
Currently, chondroitinase ABC is widely applied for removing PNNs in experimental animals, but it lacks specificity, causing a degradation of ECM molecules not only in PNNs but throughout CNS tissue. Researchers are looking for more subtle ways to manipulate the PNN in animal models in order to further understand their functions as well as to fine-tune neuronal plasticity. Additionally, while behavioral studies have clearly demonstrated the role of PNNs in mediating the plasticity of the brain, researchers still don’t have a good grasp on the molecular details of these processes. Recently, revelations about the composition of PNNs have begun to yield clues.
PNN structure and the control of plasticity
A number of ECM molecules are present at higher concentrations within the PNN than in the rest of the extracellular space. The sugar hyaluronan serves as the backbone of PNN structure. Bound to hyaluronan are chondroitin sulfate proteoglycans (CSPGs). This binding is stabilized by the link proteins. CSPGs are composed of a core protein and attached sugar chains. Different sulfation patterns in the CSPG sugar chains create specific binding sites for a wide variety of molecules and receptors, affecting CSPG function. (See illustration below.)
One mechanism by which PNNs control neuronal plasticity is the interaction between CSPGs and the homeoprotein Otx2. Homeoproteins are transcription factors that play major roles during embryonic development, controlling the organization of the vertebrate brain into distinct regions. Many homeoproteins also serve as paracrine signaling factors that shuttle between cells. In mice, experimentally reducing the capture of Otx2 by visual cortex neurons, which happens through binding to CSPGs, reduced PNN assembly, increased plasticity, and prompted the recovery of visual acuity in adult animals with amblyopia.11 And research last year demonstrated Otx2’s role in regulating PNNs’ influence on the experience-dependent formation of tonotopic maps, the spatial arrangement of neurons according to their sound frequency responses, in the primary auditory cortex and the acquisition of acoustic preferences (which is mediated by the medial prefrontal cortex)……
