Francisco, for fostering in her the joy for science and the scientific process, during the gap years after her undergraduate education. We would like to thank the following students in helping us get started with this project and for technical

Rett syndrome is characterized by an early period of typical development and then, regression of learned motor and speech skills in girls. The underlying mechanisms from normalcy to regression are unclear. Due to random X-chromosome inactivation, female patients of Rett syndrome, and female mouse models of Rett syndrome (Mecp2Heterozygous, Het) express a functional copy of wild-type MECP2 protein in half of all mature cells in brain and body. By analyzing wild-type MECP2 expression in the context of whisker tactile sensory perception, here we report compensatory increase in MECP2 protein expression in 6-week-old adolescent Het mice, which display normal levels of perineuronal net expression, mild tactile sensory perception deficit and efficient pup retrieval behavior. Comparatively, 12-week-old adult Het mice display decreased MECP2 expression and significant tactile sensory perception deficits. Thus, we have identified a period of normalcy and regression in this female mouse model, which coincide with variable wild-type expression of MECP2. We speculate that compensatory increases in MECP2 expression in Het brains allows for normal functioning, while the inability to maintain these expression level changes leads to behavioral deficits.

Cortical neuronal circuits along the sensorimotor pathways are shaped by experience during critical periods of heightened plasticity in early postnatal development. After closure of critical periods, measured histologically by the formation and maintenance of extracellular matrix structures called perineuronal nets (PNNs), the adult mouse brain exhibits restricted plasticity and maturity. Mature PNNs are typically considered to be stable structures that restrict synaptic plasticity on cortical parvalbumin+ GABAergic neurons. Changes in environment (i.e. novel behavioral training) or social contexts (i.e. motherhood) are known to elicit synaptic plasticity in relevant neural circuitry. However, little is known about concomitant changes in the PNNs surrounding the cortical parvalbumin+ GABAergic neurons. Here, we show novel changes in PNN density in the primary somatosensory cortex (SS1) of adult female mice after maternal experience, using systematic microscopy analysis of a whole brain region. On average, PNNs were increased in the right barrel field and decreased in the left forelimb regions. Individual mice had left hemisphere dominance in PNN density. Using adult female mice deficient in methyl-CpG-binding protein 2 (MECP2), an epigenetic regulator involved in regulating experience-dependent plasticity, we found that MECP2 is critical for this precise and dynamic expression of PNN. Adult naïve Mecp2-heterozygous females (Het) had increased PNN density in specific subregions in both hemispheres before maternal experience. The laterality in PNN expression seen in naïve Het was lost after maternal experience, suggesting possible intact mechanisms for plasticity. Together, our results identify subregion and hemisphere-specific alterations in PNN expression in adult females, suggesting extracellular matrix plasticity as a possible neurobiological mechanism for adult behaviors in rodents.

Rett syndrome is characterized by an early period of typical development and then, regression of learned motor and speech skills in girls. Loss of MECP2 protein is thought to cause Rett syndrome phenotypes. The specific underlying mechanisms from typical developmental trajectory to regression features throughout life are unclear. Lack of established timelines to study the molecular, cellular, and behavioral features of regression in female mouse models is a major contributing factor. Due to random X-chromosome inactivation, female patients with Rett syndrome and female mouse models for Rett syndrome (Mecp2 Heterozygous , Het) express a functional copy of wild-type MECP2 protein in approximately half of all cells. As MECP2 expression is regulated during early postnatal development and experience, we characterized the expression of wild-type MECP2 in the primary somatosensory cortex of female Het mice. Here, we report increased MECP2 levels in non-parvalbumin-positive neurons of 6-week-old adolescent Het relative to age-matched wild-type controls, while also displaying typical levels of perineuronal net expression in the barrel field subregion of the primary somatosensory cortex, mild tactile sensory perception deficits, and efficient pup retrieval behavior. In contrast, 12-week-old adult Het express MECP2 at levels similar to age-matched wild-type mice, show increased perineuronal net expression in the cortex, and display significant tactile sensory perception deficits. Thus, we have identified a set of behavioral metrics and the cellular substrates to study regression during a specific time in the female Het mouse model, which coincides with changes in wild-type MECP2 expression. We speculate that the precocious increase in MECP2 expression within specific cell types of adolescent Het may provide compensatory benefits at the behavioral level, while the inability to further increase MECP2 levels leads to regressive behavioral phenotypes over time.

The purpose of this protocol is to outline the process by which we count high-intensity perineuronal nets (PNNs) in whole brain analysis. Specifically, high-intensity PNNs within the primary somatosensory cortex of adult mouse brain sections cut in a sagittal orientation. This protocol accompanies the paper "Lateralized expression of cortical perineuronal nets during maternal experience is dependent on MECP2" (https://doi.org/10.1101/787267).First, we align a digital map from the Franklin & Paxinos Mouse Brain Atlasonto individual brain sections. We then explain the process by which the S1 and its subregions areoutlined based on this atlas overlay. Finally, we outline the process of counting high-intensity PNNs within these regions.High-intensity PNNs are thought to be more mature structures due to a larger accumulation of the proteoglycans that make-up the structures 1-3 . We look at PNNs as a marker for changes in experience-dependent plasticity within the brain, where it can inhibit plasticity or solidify the changes that occurred in response to an experience. Video tutorial accompanying this protocol:Different regions of the mouse brain have differing densities of PNNs. Adult somatosensory cortex has the highest density based on what we have observed. This protocol can also be used to count all PNNs, just skip the contrast step (7.2) in PNN analysis. Citations 1 02/12/2020 C ita tio n : C ita tio n : Dana Layo, Billy Y.B. Lau, Keerthi Krishnan (02/12/2020). Mapping and Counting High-Intensity Perineuronal Nets in the Somatosensory Cortex. https://dx.doi.org/10.17504/protocols.io.bcf8itrw This is an open access protocol distributed under the terms of the C rea tiv e C o m m o n s A ttrib u tio n Licen se C rea tiv e C o m m o n s A ttrib u tio n Licen se (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited