PeriNeuronal Net RNA expression in olfactory perceptual learning
In one project, we are mapping the endogenous activity of PNNs in the acquisition of highly overlapping olfactory LTMs. Anthony, Kayla, Naomi, Swati, and Arish trained mice on an olfactory perceptual learning task (Mandairon et al., 2006, Behav Neuro). They began by testing mice’s initial abilities to discriminate between the two enantiomers of limonene (+ and −) using a spontaneous discrimination task. Then, for the next 14 days, the mice were divided into two groups. The experimental group received one hour per day of exposure to the two enantiomers. The control group received exposure to mineral oil (the vehicle we use to mix odours). After this “enrichment” period, the students again tested the mice’s ability to discriminate between the two enantiomers. Throughout the study period, we harvested the olfactory bulb, hippocampus, and cerebellum at various time points: first immediately after the initial pre-test, and then after the first, third, seventh, eighth, and last day of enrichment.
Our behavioural results show that only mice who were exposed to both of the enantiomers during the enrichment period were able to discriminate between them during the post-test. In other words, enrichment decreased the overlap between the representations of the two limonene enantiomers. We hypothesize that this decreased overlap (or interference reduction) will correspond to higher levels of mRNA expression for the three proteoglycan components of PNNs (aggrecan, neurocan, and brevican) in the experimental group as compared to the control group. We further expect mRNA expression levels to be highest early in the enrichment period (Day 1 or 2). This semester, Aishat is working in the lab to run qPCRs on our brain samples.
Necessity of PNNs in olfactory perceptual learning
In the first project, we investigated the activity of PNNs during the acquisition of a high-interference mem- ory. To test whether PNN activity is indeed necessary for the acquisition of this memory, we are again training mice on the perceptual learning paradigm detailed above (i.e. pre-test, 14-day enrichment with (+)- and (−)-limonene or mineral oil, post-test). In this experiment, a group of mice received an infusion of Chrondroitinase ABC (ChABC) in their olfactory bulb immediately after pre-testing. (The control group received a vehicle infusion.) ChABC is an enzyme that degrades the sugar chains of PNNs and that has been shown to impede full PNN expression (as measured by Wisteria floribunda agglutinin reactivity) for up to four weeks (Bruckner et al., 1998, Exp Brain Res). This means that PNNs would not be present during our enrichment period. If PNNs are needed for the acquisition of high-interference memories, then ChABC infusions would prevent or decrease enrichment-induced discrimination of the two limonene enantiomers.
This past summer, Earlham was an NSF REU (No. 1559992, “A distributed network of neuroscience scholars”) site, and I participated as a mentor. The REU students collected pilot data for this project. Currently, three students are continuing this project during the semester. The work is funded by an Indiana Academy of Sciences Senior Research Grant.
Visualization of PNN-neuron interactions
To more directly examine how PNNs are involved in stabilizing neuronal ensembles, I need methods that allow me to examine the location of PNNs and their expression relative to learning-activated neurons. The most recent project in my lab is a first step toward imaging PNNs. Currently, Khyrul and I are optimizing a protocol to perform fluorescence immunohistochemistry (IHC) on PNNs. In contrast to more common PNN-staining methods that rely on unconjugated Wisteria floribunda agglutinin labeling, a fluorescence-based protocol will pair better with methods that allow us to target specific neuron types. Our protocol is based on work by Enwright et al., 2016, Neuropsychpharm, and we are in contact with the first author for guidance as we work.