Perturbation of in vivo Neural Activity Following α-Synuclein Seeding in the Olfactory Bulb

Abstract

Background: Parkinson's disease (PD) neuropathology is characterized by intraneuronal protein aggregates composed of misfolded α-Synuclein (α-Syn), as well as degeneration of substantia nigra dopamine neurons. Deficits in olfactory perception and aggregation of α-Syn in the olfactory bulb (OB) are observed during early stages of PD, and have been associated with the PD prodrome, before onset of the classic motor deficits. α-Syn fibrils injected into the OB of mice cause progressive propagation of α-Syn pathology throughout the olfactory system and are coupled to olfactory perceptual deficits.

Objective: We hypothesized that accumulation of pathogenic α-Syn in the OB impairs neural activity in the olfactory system.

Methods: To address this, we monitored spontaneous and odor-evoked local field potential dynamics in awake wild type mice simultaneously in the OB and piriform cortex (PCX) one, two, and three months following injection of pathogenic preformed α-Syn fibrils in the OB.

Results: We detected α-Syn pathology in both the OB and PCX. We also observed that α-Syn fibril injections influenced odor-evoked activity in the OB. In particular, α-Syn fibril-injected mice displayed aberrantly high odor-evoked power in the beta spectral range. A similar change in activity was not detected in the PCX, despite high levels of α-Syn pathology.

Conclusion: Together, this work provides evidence that synucleinopathy impacts in vivo neural activity in the olfactory system at the network-level.

Keywords: Lewy pathology; Parkinson’s disease; dementia; local field potential; olfaction; olfactory bulb; piriform cortex; synucleinopathy.

Fig 1.. Experimental design for α-Syn seeding and subsequent multi-site LFP recordings in awake mice.

A, 2–3 months old C57BL/6J mice received unilateral OB injections of either α-Syn PFFs or PBS and survived for 1, 2, or 3 months post injection. The mice were then surgically implanted ipsilaterally to the initial surgical site in the OB and PCX with twisted bipolar electrodes for LFP recordings. Following this, the mice were allowed 2–3 days to recover. Spontaneous and odor-evoked LFPs were recorded from awake mice while they were head fixed (to allow control for the positioning of the snout relative to the odor port) in the absence and presence of odors respectively, and then were perfused within 3 days for post-mortem histology. Image made in BioRender. B, Example localization of the electrode implants in the OB and PCX of a mouse injected with PBS using 10X magnification. * = former sites of bipolar electrode tips.

Fig 2.. Verification of PFF length prior to OB injections.

A, Transmission electron microscopy images of PFFs before and after sonication. To allow for visualization and quantification of sonicated individual fibrils, the sonicated sample was diluted prior to imaging. Scale bars represent 100 nm. B, Histogram of PFF length post sonication illustrating that >50% of the fibril population are <50 nm. Dashed line represents 50% of total population of PFFs quantified. Data from two separate sonication runs, 6–7 electron micrographs each.

Fig 3.. Paradigm for recording spontaneous and odor-evoked LFPs from head-fixed awake mice.

A, After a variable inter-trial interval (ITI), the odor valves are turned on for 8 secs and the vacuum for 4 secs. This allows the animal to be presented with an odor for 4 secs. B, Schematic showing the recording paradigm. An epoch of spontaneous LFP activity was recorded before and after odor presentation. During odor presentation, 7 odors were presented in a pseudo-random order for 4–5 sessions, and their odor-evoked activity recorded. C, Average photoionization detector trace in response to 12 presentations of 1 Torr isopentyl acetate, depicting the rapid temporal dynamics and stability of odor presentation (10 Hz, low pass filtered). Data are mean +/− SEM.

Fig 4.. PFFs injected in the OB induced an amplification and spread of pathology to interconnected regions, including the PCX.

Pser129 immunofluorescence was used as an assay to detect pathological α-Syn. A, Coronal panel showing the regions (bold boxes) used for quantifying Pser129 expression level in the OB and PCX. B, Representative images of Pser129 immunofluorescence staining of the OB and PCX of mice that survived for 1, 2, or 3 months post PFF seeding. Arrowheads in the OB and PCX panels indicate areas of neuritic pathology. The images were gray-scaled and inverted to show pathology more readily, for illustration purposes of this figure only. C, Quantification of mean % area in the OB and PCX, showing Pser129 immunofluorescence in PFF and a subset of PBS injected mice that survived for 1 (PFF n= 8, PBS n= 4), 2 (PFF n= 10, PBS n= 4), and 3 (PFF n= 12, PBS n= 4) months post injection. Animals injected with PFFs had a significantly greater Pser129 immunopositive signal than the PBS injected animals, including in both the OB and PCX. Significant increase in mean % area Pser129 was observed in the PCX when compared to the OB. ***p ≤ 0.001 ANOVA followed by Tukey’s multiple comparison’s test.

Fig 5.. Example spontaneous LFP activity.

A, Representative spontaneous LFP traces from two separate mice injected with either PBS (left) or PFF (right), 2 months prior to recording. Shown are full band traces from simultaneous OB and PCX recordings (0.1–100Hz) which were also filtered to separately display beta and gamma band activity as defined in the figure. Respiratory theta from the OB is also displayed along with dashed vertical lines indicating the timing of OB respiratory cycles for visual aid. B, 2-dimensional histograms of the same spontaneous full band power spectrograms with power displayed in dB to help illustrate the diversity of the full band data.

Fig 6.. No effect of PFF injection on spontaneous LFP powers in the OB or PCX.

Spontaneous OB (A) and PCX LFP power (B), consisting of theta (1–10 Hz), beta (15–34 Hz), and gamma (40–75 Hz) spectral bands in either PFF seeded mice or PBS injected mice that survived for 1 (PFF: n= 8, PBS: n= 9), 2 (PFF: n= 10 , PBS: n= 9) , or 3 (PFF: n=12, PBS: n=8) months post injection. No treatment or age-post injection effect was observed in either brain region.

Fig 7.. OB PFF seeding entails heightened odor-evoked OB beta-band power.

Representative odor-evoked LFP traces from two separate mice injected with either PBS (left) or PFF (right), 2 months prior to recording. Shown are full band traces from odor-evoked OB and PCX recordings (0.1–100Hz) which were also filtered to separately display beta band activity as defined in the figure. Respiratory theta from the OB is also displayed as is the root mean square of the beta band activity to illustrate elevated power of beta activity in the PFF injected versus PBS injected mouse. Gray shaded boxes indicate the time of odor delivery.

Fig 8.. Analyses of odor-evoked activity uncover PFF seeding induced aberrant OB beta-band power during odor.

A,B, Odor-evoked OB and PCX LFP power, consisting of theta (1–10 Hz), beta (15–34 Hz), and gamma (40–75 Hz) spectral bands in either PFF seeded mice or PBS injected mice that survived 1 (PFF: n= 8, PBS: n= 9), 2 (PFF: n= 10 , PBS: n= 9), or 3 (PFF: n=12, PBS: n=8) months post injection. Across all age groups, a significant increase in the beta band power in the OB of PFF seeded mice was observed when compared to PBS treated. *ANOVA. Solid data point indicates an animal whose elevated OB beta power was associated with high OB Pser129 pathological burden.

Fig 9.. Lack of statistical correlation between OB Pser129 burden and aberrant beta band activity.

Scatterplot illustrating the relationship between the mean % area occupied by Pser129 in the OB (as quantified in Fig. 4) and odor-evoked beta power (gray, 15–34 Hz). Gray dashed line indicates the linear fit bounded by 95% confidence intervals (Pearson r(28) = 0.156, p = 0.411).