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Perineuronal net expression in the brain of a hibernating mammal

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Abstract

During hibernation, mammals like the 13-lined ground squirrel cycle between physiological extremes. Most of the hibernation season is spent in bouts of torpor, where body temperature, heart rate, and cerebral blood flow are all very low. However, the ground squirrels periodically enter into interbout arousals (IBAs), where physiological parameters return to non-hibernating levels. During torpor, neurons in many brain regions shrink and become electrically quiescent, but reconnect and regain activity during IBA. Previous work showed evidence of extracellular matrix (ECM) changes occurring in the hypothalamus during hibernation that could be associated with this plasticity. Here, we examined expression of a specialized ECM structure, the perineuronal net (PNN), in the forebrain of ground squirrels in torpor, IBA, and summer (non-hibernating). PNNs are known to restrict plasticity, and could be important for retaining essential connections in the brain during hibernation. We found PNNs in three regions of the hypothalamus: ventrolateral hypothalamus, paraventricular nucleus (PVN), and anterior hypothalamic area. We also found PNNs throughout the cerebral cortex, amygdala, and lateral septum. The total area covered by PNNs within the PVN was significantly higher during IBA compared to non-hibernating and torpor (P < 0.01). Additionally, the amount of PNN coverage area per Nissl-stained neuron in the PVN was significantly higher in hibernation compared to non-hibernating (P < 0.05). No other significant differences were found across seasons. The PVN is involved in food intake and homeostasis, and PNNs found here could be essential for retaining vital life functions during hibernation.

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Funding

This study was funded by a University of Wisconsin-La Crosse Faculty Research Grant to CS and a University of Wisconsin-La Crosse Undergraduate Research and Creativity Grant to AM.

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  1. Department of Biology, University of Wisconsin-La Crosse, 1725 State St., La Crosse, WI, 54601, USA

    Anna Marchand & Christine Schwartz

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  1. Anna Marchand
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  2. Christine Schwartz
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Correspondence to Christine Schwartz.

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The authors declare that they have no conflict of interest.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the University of Wisconsin-La Crosse Institutional Animal Care and Use Committee (protocol #14–15). This article does not contain any studies with human participants performed by any of the authors.

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429_2019_1983_MOESM1_ESM.tif

Fig. S1 Representative analysis of PNNs shown in cingulate cortex (a,b) and paraventricular nucleus (c,d). Raw grayscale images (a and c) were converted to binary images (b and d) by adjusting image threshold to account for background staining using the auto threshold function in ImageJ. Depending on the brain region, total pixels were counted in a specified box size (cingulate cortex as an example in b) or using a polygon to encompass the entire structure (PVN as an example in d). Total pixels in the analysis area were then quantified as the area covered by PNNs (TIFF 9798 kb)

429_2019_1983_MOESM2_ESM.tif

Fig. S2 Lectin histochemistry control without lectin results in no PNN staining. The lectin histochemistry protocol was performed in the same manner without Wisteria lectin during the 2-h incubation, resulting in no staining in any region. Shown are representative images from cingulate cortex (a), dorsolateral cerebral cortex (b), lateral septum (c), ventrolateral hypothalamus (d), and paraventricular nucleus/anterior hypothalamic area (e). Scale bar applies to all images. AHA: anterior hypothalamic area; Cg: cingulate cortex; DLc: dorsolateral cerebral cortex; LS: lateral septum; PVN: paraventricular nucleus; VLH: ventrolateral hypothalamus (TIFF 5270 kb)

429_2019_1983_MOESM3_ESM.tif

Fig. S3 Perineuronal nets surround neurons in all brain regions analyzed. The images shown are representative images with lectin histochemistry (green) merged with Nissl staining for neurons (red). a. Dorsal cerebral cortex with prominent staining in layer III (a’) and V (a’’). b and b’. Cingulate cortex. c. Dorsolateral cerebral cortex with prominent staining in layer III (c’) and V (c’’). d and d’. Anterior hypothalamic area. e. Lateral septum with representative cells from LSD (e’) and LSI (e’’). f and f’. Paraventricular nucleus. g. Amygdala with representative cells from AA (g’) and BMA (g’’). h. Ventrolateral hypothalamus with representative staining from mid-structure (h’) and dorsal (h’’). All images shown are from torpor animals. Scale bar in a applies to images a-h. Scale bar in a’ applies to all ‘and ‘’ images. 3 V: Third ventricle; AA: Anterior amygdaloid area; BMA: Basomedial amygaloid nucleus; III: Cerebral cortex layer III; V: Cerebral cortex, layer V; LSD: Dorsal lateral septum; LSI: Intermediate lateral septum; LV: lateral ventricle, oc: optic chiasm; PVN: paraventricular nucleus (TIFF 54522 kb)

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Marchand, A., Schwartz, C. Perineuronal net expression in the brain of a hibernating mammal. Brain Struct Funct 225, 45–56 (2020). https://doi.org/10.1007/s00429-019-01983-w

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