The Role of Cerebellar Perineuronal Nets in Synaptic Plasticity and Associative Learning

Post by Shireen Parimoo

What's the science?

The cerebellum is a brain structure that contains nearly half of all the neurons in the brain and is involved in a wide variety of functions, ranging from motor control to learning. Cerebellar neurons are surrounded by perineuronal nets (PNNs), which are specialized extracellular matrix structures, made up of carbohydrate and protein molecules like chondroitin sulfate proteoglycans (CSPGs). Eye-blink conditioning is a type of associative learning that is dependent on deep cerebellar neurons. Normally, delivering puffs of air to the eye elicits a reflexive blinking response. In eye-blink conditioning, air puffs are paired with a neutral stimulus, such as light, so that after repeated exposures, simply presenting the light elicits the blinking response. Studies have found that disrupting PNNs can enhance structural plasticity and alter memory formation, but the precise role of cerebellar PNNs in learning and neuroplasticity is not known. This week in PNAS, Carulli, and colleagues investigated the molecular mechanisms underlying PNN-mediated synaptic and structural plasticity during associative learning in mice.

How did they do it?

The authors assigned mice to eye-blink conditioning, pseudo-conditioning, or a control group. Mice in the conditioning group were repeatedly exposed to air puffs paired with light, whereas the pseudo-conditioning group was exposed to air puffs and light separately, but these were never presented together. Learning and memory were assessed by the percentage of eye-blinks after eye-blink conditioning and the fraction of eyelid closure when the air puff was delivered (e.g. 1 = eyelid was fully closed, 0 = eyes were fully open).

First, the authors examined the effect of eye-blink conditioning on PNN expression during learning (after five days of conditioning) and following memory formation (after ten days of conditioning). To do this, they stained the deep cerebellar nuclei for CSPGs and further classified the PNNs based on staining intensity (e.g. low intensity = weak PNNs). They then investigated the effect of PNN disruption on plasticity by overexpressing the enzyme “chondroitinase”, which degrades PNNs, in the deep cerebellar nuclei. Using a combination of staining, immunocytochemistry, and single-unit recordings, the authors assessed how PNN degradation altered learning, as well as the structural (e.g. number and size of axon terminals) and functional plasticity (e.g. spiking activity) of cerebellar neurons. Finally, they explored the long-term effects of PNN digestion on memory and structural plasticity of cerebellar neurons, 21 days after eye-blink conditioning.

What did they find?

After five days of training, mice in the conditioning group learned the association between the air puff and light, showing an increase in eye-blinks and a fraction of eyelid closure in response to the light. This was accompanied by a reduction in the proportion of strong PNNs but an increase in medium and weak PNNs in the deep cerebellar nuclei. In contrast, pseudo-conditioned and control mice did not show changes in PNN expression over time. After training for ten days, there was no difference in PNN expression between conditioned and pseudo-conditioned mice. These findings suggest that associative learning in the cerebellum is related to a reduction in strong PNNs, which then return to normal levels after the associative memories are formed.

Animals with overexpressed chondroitinase showed better learning than the control mice. Long-term memory initially declined for both groups, but while it stabilized in the control group, memory retention continued to decline over time among the chondroitinase mice. Thus, although PNN degradation facilitated initial learning, disrupting PNNs was detrimental for long-term retention of associative memories. Disrupting PNNs also altered the structural plasticity of cerebellar neurons, with an increase in the number of inhibitory, GABAergic axon terminals, but a reduction in the number of excitatory, glutamatergic terminals. These structural changes were further accompanied by reduced baseline spiking activity of cerebellar neurons in the chondroitinase mice.

What's the impact?

This is the first study to demonstrate the importance of cerebellar PNNs in associative learning, particularly the finding that PNNs modulate synaptic and functional plasticity at different phases of memory acquisition (learning vs retention). These findings pave the way for future research to elucidate the role of PNNs in other cerebellum-dependent cognitive processes like emotional and motor learning.

Carulli et al. Cerebellar plasticity and associative memories are controlled by perineuronal nets. Proceedings of the National Academy of Sciences (2020). Access the original scientific publication here.