Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage link protein Crtl1 (Hapln1), which is up-regulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. In the sensory pathway, axons in knockout animals but not controls sprout into the party denervated cuneate nucleus. The organization of chondroitin sulphate proteoglycan into perineuronal nets is therefore the key event in the control of central nervous system plasticity by the extracellular matrix.

Perineuronal nets (PNNs) are extracellular matrix structures surrounding cortical neuronal cell bodies and proximal dendrites and are involved in the control of brain plasticity and the closure of critical periods. Expression of the link protein Crtl1/Hapln1 in neurons has recently been identified as the key event triggering the formation of PNNs. Here we show that the genetic attenuation of PNNs in adult brain Crtl1 knock-out mice enhances long-term object recognition memory and facilitates long-term depression in the perirhinal cortex, a neural correlate of object recognition memory. Identical prolongation of memory follows localized digestion of PNNs with chondroitinase ABC, an enzyme that degrades the chondroitin sulfate proteoglycan components of PNNs. The memory-enhancing effect of chondroitinase ABC treatment attenuated over time, suggesting that the regeneration of PNNs gradually restored control plasticity levels. Our findings indicate that PNNs regulate both memory and experience-driven synaptic plasticity in adulthood.

In the adult brain, the extracellular matrix (ECM) influences recovery after injury, susceptibility to mental disorders, and is in general a strong regulator of neuronal plasticity. The proteoglycan aggrecan is a core component of the condensed ECM structures termed perineuronal nets (PNNs), and the specific role of PNNs on neural plasticity remains elusive. Here, we genetically targeted theAcangene encoding for aggrecan using a novel animal model. This allowed for conditional and targeted loss of aggrecanin vivo, which ablated the PNN structure and caused a shift in the population of parvalbumin-expressing inhibitory interneurons toward a high plasticity state. Selective deletion of theAcangene in the visual cortex of male adult mice reinstated juvenile ocular dominance plasticity, which was mechanistically identical to critical period plasticity. Brain-wide targeting improved object recognition memory.SIGNIFICANCE STATEMENTThe study provides the first direct evidence of aggrecan as the main functional constituent and orchestrator of perineuronal nets (PNNs), and that loss of PNNs by aggrecan removal induces a permanent state of critical period-like plasticity. Loss of aggrecan ablates the PNN structure, resulting in invoked juvenile plasticity in the visual cortex and enhanced object recognition memory.

Chondroitinase ABC (ChABC) in combination with rehabilitation has been shown to promote functional recovery in acute spinal cord injury. For clinical use, the optimal treatment window is concurrent with the beginning of rehabilitation, usually 2–4 weeks after injury. We show that ChABC is effective when given 4 weeks after injury combined with rehabilitation. After C4 dorsal spinal cord injury, rats received no treatment for 4 weeks. They then received either ChABC or penicillinase control treatment followed by hour-long daily rehabilitation specific for skilled paw reaching. Animals that received both ChABC and task-specific rehabilitation showed the greatest recovery in skilled paw reaching, approaching similar levels to animals that were treated at the time of injury. There was also a modest increase in skilled paw reaching ability in animals receiving task-specific rehabilitation alone. Animals treated with ChABC and task-specific rehabilitation also showed improvement in ladder and beam walking. ChABC increased sprouting of the corticospinal tract, and these sprouts had more vGlut1^+ve presynaptic boutons than controls. Animals that received rehabilitation showed an increase in perineuronal net number and staining intensity. Our results indicate that ChABC treatment opens a window of opportunity in chronic spinal cord lesions, allowing rehabilitation to improve functional recovery.

Damaged CNS axons are prevented from regenerating by an environment containing many inhibitory factors. They also lack an integrin that interacts with tenascin-C, the main extracellular matrix glycoprotein of the CNS, which is upregulated after injury. The α9β1 integrin heterodimer is a receptor for the nonalternatively spliced region of tenascin-C, but the α9 subunit is absent in adult neurons. In this study, we show that PC12 cells and adult rat dorsal root ganglion (DRG) neurons do not extend neurites on tenascin-C. However, after forced expression of α9 integrin, extensive neurite outgrowth from PC12 cells and adult rat DRG neurons occurs. Moreover, both DRG neurons and PC12 cells secrete tenascin-C, enabling α9-transfected cells to grow axons on tissue culture plastic. Using adeno-associated viruses to express α9 integrinin vivoin DRGs, we examined axonal regeneration after cervical dorsal rhizotomy or dorsal column crush in the adult rat. After rhizotomy, significantly more dorsal root axons regrew into the dorsal root entry zone at 6 weeks after injury in α9 integrin-expressing animals than in green fluorescent protein (GFP) controls. Similarly, after a dorsal column crush injury, there was significantly more axonal growth into the lesion site compared with GFP controls at 6 weeks after injury. Behavioral analysis after spinal cord injury revealed that both experimental and control groups had an increased withdrawal latency in response to mechanical stimulation when compared with sham controls; however, in response to heat stimulation, normal withdrawal latencies returned after α9 integrin treatment but remained elevated in control groups.

The Hargreaves test is specifically designed to assess thermal pain sensation in rodents such as rats and mice. This test has been used in experiments involving pain sensitization or recovery of thermal pain response following neural injury and regeneration. We present here a step-by-step protocol highlighted with important notes to guide first-time users through the learning process. Additionally, we have also included representative data from a rat model of sensory denervation showing how the data can be analysed to obtain meaningful results. We hope that this protocol can also assist potential users in deciding whether the Hargreaves test is a suitable test for their experiment. Bio-protocol is an online peer-reviewed protocol journal. Its mission is to make life science research more efficient and reproducible by curating and hosting high quality, free access protocols.