This article is licensed under CC-BY 4.0 pubs.acs.org/chemneuro Research Article Roadmap for Postnatal Brain Maturation: Changes in Gray and White Matter Composition during Development Measured by Fourier Transformed Infrared Microspectroscopy Marta Peris,# Núria Benseny-Cases,# Gemma Manich,* Oriana Zerpa, Beatriz Almolda, À lex Perálvarez-Marín, Berta González, and Bernardo Castellano Cite This: ACS Chem. Neurosci. 2023, 14, 3088−3102 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Key events in postnatal brain development, such as neuronal migration, synaptogenesis, and myelination, shape the adult brain. These events are reflected in changes in gray and white matter (GM and WM) occurring during this period. Therefore, precise knowledge of GM and WM composition in perinatal brain development is crucial to characterizing brain formation as well as the neurodevelopmental disruption observed in diseases such as autism and schizophrenia. In this study, we combined histochemical and immunohistochemical staining with biochemical and biophysical analyses using Fourier transform infrared (IR) microspectroscopy (μFTIR) to better understand the chemical changes during postnatal developmental myelination. For this purpose, we analyzed the GM and WM in the mouse brain and cerebellum (strain C57BL/6) from postnatal day 0 (P0) to day P28 and established presumed correlations between staining and IR data. IR spectra allowed the (i) quantification of lipid and protein content through the CH2/amide I ratio, (ii) determination of chemical characteristics of lipids, such as the presence of unsaturated bonds in the carbonate chain or carbonyls from ester groups in the polar head, and (iii) determination of the protein secondary structure (α-helix and intramolecular β-sheets). The results indicate that the increase in the CH2/amide I ratio calculated from the μFTIR data correlates well with lipid histochemical staining. IR data indicated a change in the lipid composition in WM since carbonyl and unsaturated olefinic groups do not increase when lipids accumulate during myelination. Our correlation analysis between IR data and immunohistochemical staining of myelin-associated proteins revealed that myelin oligodendrocyte protein correlated well with lipid accumulation, while myelin basic protein appeared before lipid modifications, which indicated that myelin- associated proteins and lipid deposition were not synchronic. These events were related to a decrease in the intramolecular β/α protein ratio. Our results indicate that lipids and proteins in WM substantially change their composition due to primary myelination, and according to results obtained from staining, these modifications are better described by lipid histochemical staining than by immunohistochemistry against myelin-related proteins. In conclusion, μFTIR can be a useful technique to study WM during perinatal development and provide detailed information about alterations in the chemical composition related to neuro- developmental diseases. KEYWORDS: myelination, development, white matter, gray matter, μFTIR 1. INTRODUCTION Determination of the lipid and protein composition of GM Key processes of central nervous system (CNS) maturation and WM in experimental animal models, such as mice and occur during perinatal development, including oligodendro- rabbits, has been obtained by chromatography, mass genesis, primary myelination, and neural network formation spectrometry, or imaging techniques in whole-brain samples through synaptogenesis and synaptic pruning.1,2 Alterations in and isolated myelin,5−9 techniques that lack area specificity and these processes can lead to several neurodevelopmental diseases, such as autism and hypomyelination.3,4 Postnatal Received: April 11, 2023 brain maturation under physiological conditions is reflected in Accepted: July 24, 2023 modifications of the gray matter (GM) and white matter Published: August 4, 2023 (WM) composition, and their alteration can be related to pathophysiological mechanisms involved in neurodevelopmen- tal diseases. © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acschemneuro.3c00237 3088 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 1. PCA in the second derivative spectra of μFTIR from brain and cerebellum WM and GM during postnatal development. Score plots (A,E) and loading plots (B,F) of PCA in the fingerprint region of the brain and cerebellum GM and WM spectra are shown. Clear differences are observed in GM (full circles, G) and WM (empty circles, W) of the brain, and GM in the granular (GM-G) and molecular (GM-M) layers (full circles (G) and crosses (M), respectively) and WM (empty circles, W). A normalized average of the second derivative spectra in WM and GM areas of the mouse brain (C,D) and the cerebellum (G,H) at P0 (blue lines), P14 (red lines), and P28 (green lines) is represented. Peaks corresponding to functional groups are illustrated in groups according to their absorbance (d2A, in arbitrary units) of the following bands (wavenumber in cm−1): 3012 cm−1 (C�CH, unsaturated olefinic group), 2921 cm−1 (CH2 asymmetric stretching vibrations), 1743 cm−1 (C�O, carbonyl group), 1656 cm−1 (α-helix secondary protein structure), 1637 cm−1 (β-sheet secondary protein structure). have few time points of study. In humans, these changes have Primary myelination in WM has been extensively studied been described using imaging and chromatography.10−15 using histochemical techniques, such as Luxol Fast Blue (LFB), 3089 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Sudan Black (SB), and Oil Red O (ORO) staining, and region (1800−1300 cm−1) was carried out. The score plot and immunohistochemistry against myelin sheath-specific proteins, loadings of the analysis of all brain and cerebellar spectra (GM such as myelin basic protein (MBP), proteolipid protein and WM) are presented in Figure 1A,E and B,F, respectively. A (PLP), and myelin oligodendrocyte glycoprotein (MOG), in separate analysis of GM and WM was also performed with animal brains.16 However, histochemical techniques provide similar loadings and score plots (data not shown). only partial molecular information and do not describe detailed For the analysis of the brain spectra, the score plot mainly biochemical modifications in the GM and WM. separates the different areas and ages on PC1 (78% of Current knowledge of postnatal brain composition is limited discrimination). The PC1 loading plot shows the bands that to the description of general changes. In mice, during the first distinguish the spectra of WM and GM during development in early phase of 10 days, changes are related to brain growth the fingerprint region. The bands in the PC1 correspond to since the total protein content, phospholipids, cholesterol, and carbonyl groups (band at 1740 cm−1), amide I (1656 cm−1 for nucleic acids increase globally. In the second phase, from day α-helix structures and 1637 cm−1 for β-sheet structures), and 14 onward, the brain composition is characterized by abrupt CH groups (bands at 1470 and 1380 cm−1). Since principal increases of cerebrosides and sulfatides due to myelin component analysis (PCA) was performed on the second formation, which lasts for 8 days and decreases onward.17 derivative of the spectra, the spectra on the positive axis of the Specific investigation of myelin-enriched fractions reveals a score plot contained more proteins (negative band in the predominance of phosphatidylcholine at postnatal day (P) 15, loading plot) and fewer lipids (positive band in the loading followed by an increase in monounsaturated fatty acids and plot) than the spectra on the negative part of the score plot. phosphatidylserine, phosphatidylinositol, and phosphatidyle- The score plot shows clear differences between GM and thanolamine because of myelin formation, as similarly observed WM, especially at more advanced stages, showing a higher in rats.5,9 Therefore, a general characterization of GM and WM lipid content in WM than in GM. The analysis of WM and GM changes during postnatal development with complete and showed that mature GM composition was achieved at 7 days specific biochemical and molecular features, spatial resolution, since only the P0 spectra differed from the rest of the GM data. and representative points of study of postnatal development is However, for WM, P0, P7, and P14 showed similar spectra needed. (the spectra colocalized in the score plot), whereas P21 and One of the most sensitive techniques for analyzing the P28 differed from the early stages, showing that the WM biochemical composition of biological samples, including fixed composition was still changing until 21 and 28 days. and nonfixed tissue slices, is Fourier transform infrared PCA for the cerebellum spectra for each area analyzed (GM- microspectroscopy (μFTIR).18 μFTIR provides complete G, GM-M, and WM) is shown in panels E and F. The PC1 information on the functional groups of lipids and protein loading plot (43% of discrimination) shows very similar bands secondary structures, while sample handling prior to analysis is to those for the brain spectra. For GM-G, all spectra for all ages minimized. μFTIR has been used in neuroscience as an colocalized on the right part of the score plot, indicating that interesting tool for studying tissues under homeostatic no changes could be detected in the fingerprint region using conditions, such as WM and GM in adult mice and humans μFTIR. However, GM-M P0 and P7 spectra colocalized with during aging,19−21 or to study neurodegenerative diseases.22−26 GM-G at the positive part of the graph, while P14, P21, and In the case of postnatal development, only one study P28 spectra were situated more in the center of the score plot, determined changes in the hippocampal formation by μFTIR indicating a higher content of lipids at those stages. For in 6, 30, and 60 day-old rats.27 cerebellar WM, as observed for brain data, a higher lipid The technical advantages of μFTIR and its proven efficacy in content was detected because the spectra were found in the CNS tissues make it a suitable option for studying changes in negative part of the score plot. In this case, the increase in lipid brain composition, especially lipids, during perinatal develop- (shift of the spectra to the negative part) starts at P14, at an ment. Owing to their key role in proper and abnormal brain earlier stage than in the brain. maturation, data on specific biochemical changes in GM and The changes detected in the PCA could also be observed in WM can provide a deeper insight into relevant molecular the second derivative (d2) of the average spectra for each information, especially when comparing μFTIR results with condition, normalized by amide I, as shown in Figure standard myelin histochemical and immunohistochemical 1C,D,G,H. Changes in CH could be observed more accurately methods. Therefore, this study sets the basis for understanding in the 3050−2800 cm−1 region. The most pronounced changes GM and WM composition during mammal brain maturation, in the IR spectra occurred in the CH2 band (d2A2921 and as well as for studying its alterations in animal models of d2A2852). Changes in the C�O band (d2A1743) showed a neurodevelopmental diseases and in the developing human marked gradual increase with development, especially in the brain. cerebellar WM. In the cerebellar GM and brain, the modifications were almost imperceptible. This was also the 2. RESULTS case for the amide I band (d2A1656), corresponding to the α- 2.1. μFTIR Data Show Important Modifications in WM helix secondary protein structure. However, bands correspond- Composition during Postnatal Development. To study ing to the β-sheet structure (d2A1637) and C�CH (d2A3012) the changes in the general composition of GM and WM during showed no apparent differences between the groups as animals postnatal development, we analyzed the spectra of the grew. The average spectra calculated from the spectra of each following CNS areas: for GM, we studied the cerebral cortex condition are also shown in Figure S1. and cerebellar molecular (GM-M) and granular layers (GM- To statistically analyze the changes observed in PCA and in G); for WM, we investigated the corpus callosum (CC) and the average spectra, we first analyzed the changes in the lipid/ cerebellar arbor vitae. protein ratio during development. For this, we used the To determine the specific bands that change during intensity of the second derivative of the CH2 (d2A2952) band, development, a principal component analysis of the fingerprint representing the total lipid content, and amide I (d2A1656+1637) 3090 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 2. Changes in the CH2/amide I ratio in the second derivative spectra of μFTIR obtained from brain and cerebellum WM and GM during postnatal development. (A,B) Quantification of the CH2/amide I ratio (CH2/α + β) after second derivatives of the respective absorbances (d2A2852/d2A1656+1637) in the brain (A) and cerebellum (B) GM and WM areas during postnatal development. Data are represented as the mean ± standard error mean (SEM) Figure 6; Figure 6; Figure 6. Statistical treatment was performed with the two-way ANOVA test (time effect p < 0.0001; region effect p < 0.0001), and posthoc comparisons of differences between time points or areas were detected with Tukey’s posthoc test and represented as follows: **p < 0.01 and ****p < 0.0001 when WM compared to GM; “a” p < 0.0001 when compared to the previous time point. (C−V) Tissue heat maps represent the CH2/amide I (d2A2852/d2A1656+1637; CH2/α + β) ratio in the CC (C−F), cerebral cortex (G−J), arbor vitae (K−N), molecular layer (O−R), and granular layer (S−V) of the cerebellum. Scale bar = 10 μm. The bottom left is a representative drawing, where the studied areas are indicated: 1-cortex, 2-CC, 3-molecular layer, 4-granular layer, and 5-arbor vitae. Created with Biorender.com. representing the protein absorption.8,25 Although the band raw data for lipid (CH2) and amide I absorption, as shown in intensity in the absorption spectrum would be proportional to Supporting Information Figures S2 and S3, confirmed that the amount of lipid or protein, the efficiency in the IR changes observed in the CH2/amide I ratio corresponded to absorption varies depending on the bond and the vibration modifications in the total lipid content. Little change was mode; therefore, the CH2/amide I ratio (d2A2952/d2A1656+1637; observed in amide I absorption, while a significant increase was CH2/(α + β)) used in this study cannot be directly interpreted observed for the lipid band, corroborating the increase in lipids as the actual lipid/protein ratio but as an estimation of the during development in WM. variation in the lipid/protein ratio. 2.2. Modifications in the Biochemical Composition of In the brain GM and granular cerebellum layers, no Lipids and Proteins Are Related to Primary Myelination. significant changes were observed in the CH2/amide I ratio To study the changes in the biochemical composition of lipids throughout development, while in the molecular layer, the in different brain and cerebellar areas during development, we CH2/amide I ratio was significantly increased at postnatal day measured the amounts of unsaturated olefinic (d2A3012; C� 14 (P14) (*p < 0.05, P0 GM-M vs P14 GM-M) and then CH) and carbonyl (d2A1743; C�O) normalized to the total remained stable (Figure 2B). In contrast, significant changes lipid content (d2A2852, CH2) (Figure 3). Because the were observed in the WM during development. In the early absorption of the different groups is highly dependent on the stages, the CH2/amide I ratio in both the cerebellum and brain efficiency of the IR absorption, changes in these ratios was slightly higher than that in the GM and remained stable represent changes in composition but do not represent the until P7 and P14, respectively (Figure 2). After these time exact ratio of these groups in the sample. Also, we studied points, this ratio significantly increased, and lipids progressively changes in the ratio CH2/CH3 (d2A2852/d2A2959), which could accumulated in the WM at later time points. In general, WM be highly dependent on changes in the lipid/protein ratio.25 areas showed changes that appeared later and were larger than In the brain GM, carbonyl groups remained constant across those observed in GM areas (two-way ANOVA, region effect all time points (Figure 3A−C), while CH2 and the unsaturated ****p < 0.0001 in the brain and cerebellum). olefinic functional group showed lower levels compared to To eliminate the contribution of possible changes in sample WM that were lost at P7. In brain WM, CH2 increased thickness, we used CH2/amide I ratios; however, analysis of following a pattern similar to that described for the CH2/amide 3091 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 3. Biochemical composition of lipids in the brain and cerebellar WM and GM of WT mice during postnatal development. (A−C,G−I) Representation of the ratio of the second derivative of the absorbances (d2A) of CH2 (A,G), unsaturated olefinic (B,H)m and carbonyl (C,I) functional groups compared to the CH3 or the CH2/amide I ratio (an estimation of the total lipid content of the brain GM and WM (A,B) and cerebellar GM-formed by the molecular layer (GM-M) and granular layer (GM-G)- and WM from postnatal day 0 (P0) to P28. Data are represented as median ± SEM. Statistical treatment for brain areas was performed with a two-way ANOVA test (time effect ****p < 0.0001; region effect for the CH2 and unsaturated olefinic groups ****p < 0.0001 and for the carbonyl group p = 0.0003) and Tukey’s posthoc comparisons (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 WM compared to GM); “a” p < 0.05, “b” p < 0.01, “c” p < 0.001, and “d” p < 0.0001 when compared to the previous time point). Statistical treatment for cerebellar areas was performed with a two-way ANOVA (time effect n.s. for olefinic groups; p < 0.0001 for the CH2 and carbonyl groups; p < 0.0001 regional effect for all three groups) and Tukey’s posthoc comparisons (indicated as in the brain). (D−F,J−L) Correlation between the normalized CH2/amide I ratio (between 0 and 1) and the normalized ratio of CH2/CH3 ratio (D,J) unsaturated olefinic group (E,K) or carbonyl group (F,L) in WM. See Materials and Methods for further details about the obtention of normalized values. Drawings indicate areas of study: 1-CC, 2-cortex; 1-arbor vitae, 2-granular layer, and 3-molecular layer. Created with Biorender.com. 3092 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 4. Secondary structure of proteins in the cerebellum and brain WM and GM of WT mice during postnatal development. (A,B) Representation of the ratio of the second derivative of the absorbances (d2A) of amide I (β-sheet)/amide I (α-helix) of proteins (d2A1637/d2A1656) in the (A) cerebral cortex (brain GM) and CC (WM), CC in the (B) cerebellar WM, molecular layer, and granular layer of GM from postnatal day 0 (P0) to P28. Data are represented as median ± SEM. Statistical treatment was performed with the two-way ANOVA test (for the brain: time effect p < 0.0001, region effect p = 0.003; for the cerebellum: time effect p < 0.0001; region effect n.s.), and Tukey’s posthoc comparisons (*p < 0.05 and ***p < 0.001, WM compared to GM; “a” p < 0.05 and “b” p < 0.01 compared to a previous time point). (C,D) α-helix (blue) and β-sheet (green) secondary structure proportions out of total protein (measured as amide I) in brain and cerebellar WM during postnatal development. S.S. = secondary structure (α-helix, or β-sheet) and T.P. = total protein (amide I). I ratio. Since lipids have a larger amount of CH2 than CH3 and WM at later developmental stages (Figure 3H,I). As compared to proteins, which have a more similar amount of similarly described for the brain, in WM, unsaturated olefinic these two groups in their lateral chains, an increase in the and carbonyl functional groups showed an expression dynamic CH2/amide I ratio will translate into an increase in the CH2/ pattern opposite that of the CH2/amide I ratio. CH3 ratio. This is demonstrated in the correlation graph, To better analyze the composition of lipids accumulating in which shows a strong correlation between these two groups WM during maturation, the correlation between the different (Figure 3D,J). Besides, in WM, carbonyl and olefinic ratios and the CH2/amide I ratio normalized between 0 and 1 unsaturated functional groups normalized by total lipid content was calculated using linear regression. A moderate-to-poor decreased from P21 onward, thus presenting lower amounts linear correlation was observed for the unsaturated olefinic than in GM (Figure 3A,B). Considering that, as we previously groups (Figure 3E,K) while lipids accumulate. However, a described, the total lipid content of WM increases during strong linear correlation of carbonyl groups with the CH2/ development, carbonyl and olefinic functional groups express a amide I ratio is shown, which demonstrates a dependent dynamic pattern opposite that of the CH2/amide I ratio. decrease in the ratio of carbonyl groups (Figure 3F). In the In the case of cerebellar GM, the CH2 group levels remained WM of the cerebellum, the correlation is poor for olefinic constant (Figure 3G), but we found a significant increase in unsaturation (Figure 3L), as observed in the brain, while the unsaturated olefinic bonds throughout postnatal development, carbonyl functional group showed a strong and linear decrease first in the molecular layer at P14 (two-way ANOVA, posthoc that correlated with an increase in the lipid content (Figure Tukey’s P0−P14 ****p < 0.0001) and later in the granular 3K). Therefore, in both cases, the increase in lipids was related layer at P28 (two-way ANOVA, posthoc Tukey’s P14−P28 to a decrease in both functional groups normalized by the total ****p < 0.0001) (Figure 3G). Finally, carbonyl levels lipid (Figure 3E,F,K,L), and the correlation was especially decreased only in the GM-G (Figure 3I; two-way ANOVA, strong for carbonyl groups in both the brain and cerebellum posthoc Tukey’s P14−P28 ***p < 0.001). In the cerebellar (Figure 3F,L), indicating that myelin lipids synthesized during WM (arbor vitae), CH2 followed a similar pattern to the one primary myelination are poor in carbonyl groups and olefinic observed for the CH2/amide I ratio. Unsaturated and carbonyl unsaturation. groups progressively and significantly decreased (Figure 3H,I, The amide I is sensitive to the protein’s secondary structure, two-way ANOVA, posthoc Tukey’s P0−P14 ****p < 0.0001 so the ratio d2A1637/d2A1656 (β-sheet/α-helix) was used to and P7−P28 *p < 0.05, respectively). Owing to the gradual study changes in the secondary structure of proteins during growth of unsaturated olefinic groups in GM and the decrease development. In the brain GM, the β-sheet/α-helix ratio of both functional groups in WM, significant differences in the slightly increased at P7 and then remained stable across all content of both functional groups were found between GM time points (Figure 4A). In the GM of the cerebellum, no 3093 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 5. Histochemical lipid staining of brain WM in WT mice during postnatal development. (A−I) Representative microphotographs of (A−C) LFB, (D−F) SB, and (G−I) ORO staining of the CC of WT mice from postnatal day (P14) to P28. (J−L) Quantification of the stained area (%) of (J) LFB, (K) SB, and (L) ORO staining in the CC of WT mice. Statistical treatment was performed with a one-way ANOVA test (time effect p < 0.0001) and Tukey’s posthoc comparison (****p < 0.0001, **p < 0.01, and *p < 0.05 compared to the previous time point). Data are represented as median ± SEM (M−O) Correlation between the normalized CH2/amide I ratio of the CC and (M) LFB, (N) SB, and (O) ORO staining. See Materials and Methods for further details about the obtention of normalized values. Scale bar: 20 μm. changes were observed in the granular layer, whereas in the P21 (two-way ANOVA, posthoc Tukey P7−P21 GM-M **p < molecular layer, the β-sheet/α-helix ratio decreased from P7 to 0.01) and then returned to previous levels at P28 (Figure 4B). 3094 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 6. Histochemical lipid staining of cerebellum WM in WT mice during postnatal development. (A−I) Representative microphotographs of (A−C) LFB, (D−F) SB, and (G−I) ORO staining of the arbor vitae of WT mice from postnatal day 7 (P7) to P21. (J−L) Quantification of (J) LFB, (K) SB, and (L) ORO staining in WT. Statistical treatment was performed with the one-way ANOVA test (time effect p < 0.0001) and Tukey’s posthoc comparison (****p < 0.0001, ***p < 0.001, and *p < 0.05 compared to the previous time point). Data are represented as median ± SEM (M−O) Correlation between the CH2/amide I ratio and (M) LFB, (N) SB, and (O) Oil Red, O staining. See Materials and Methods for further detail about the obtention of normalized values. Scale bar = 20 μm. WM = white matter, GM-M = molecular layer of gray matter, and GM- G = granular layer of gray matter. A progressive decrease in the β-sheet/α-helix ratio was brain and cerebellar WM, the β-sheet/α-helix ratio decreased observed in the WM of both the brain and cerebellum. In the from P7 onward (two-way ANOVA, posthoc Tukey P7−P21 3095 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 7. Immunostaining of myelin-associated proteins MBP and MOG in the brain and cerebellar WM of WT mice during postnatal development. (A−H,K−R) Representative microphotographs of the immunohistochemical staining of MBP and MOG in the CC (A−H) and arbor vitae (M−T) of WT mice. (I,J,S,T) Quantification of the percentage of area immunostained by MBP and MOG in CC and arbor vitae. Statistical treatment of a one-way ANOVA test (time effect p < 0.0001 for the brain and cerebellum) and Tukey’s posthoc comparison (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 respect to the previous time point). Data are represented as median ± SEM. Scale bar = 20 μm. cerebellar WM ***p = 0.0001; Figure 4A,B); at P28, in the 2.3. Changes in the Brain Composition Associated brain and cerebellar WM, the β-sheet/α-helix ratio was with Primary Myelination Are Well Reflected by LFB, SB significantly decreased compared to GM (Figure 4A,B). and ORO Histochemical Staining. Our data indicate that Analysis of the proportion of α-helix or β-sheet compared to the most important changes in brain composition during total protein (amide I) in WM of both the brain and postnatal development are related to WM and primary cerebellum (Figure 4C,D) suggests that the α-helix secondary myelination. Myelination has traditionally been studied using structure is by far the most dominant during all time points other available experimental histological techniques. Therefore, and, coinciding with myelination, it continues to increase to study how changes in postnatal brain composition are throughout development. reflected by these techniques, we compared and correlated the 3096 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article μFTIR results with those obtained using histochemistry or work, we fixed our samples for preservation purposes and to be immunohistochemistry. able to directly compare our samples in the histochemical First, we compared the WM brain lipid composition with study. The effect of fixation in the IR spectra has shown different lipid stains, such as LFB, SB, and ORO (Figures 5 and contradictory results in several works.28−31 Our results in the 6). For all lipid stains, no specific staining was detected in the GM and WM areas of P28, the age closer to adult mice, are WM at P7. Staining was detected at P14 and increased rapidly consistent with those shown in mice, rats, and humans and significantly at P21 and P28 (Figure 5A−L). To determine independently of tissue processing.24,32−34 In our study, the whether staining reflected trustworthy changes in lipid brain main limitations of sample fixation were the inability to analyze composition, μFTIR values were correlated to the area of phosphate and carbohydrate regions due to the use of positive staining for LFB, SB, and ORO. The CC results phosphate buffer (PB) and sugar-based cryoprotectants. showed a high linear correlation between lipid content During postnatal CNS development, the main differences in detected by μFTIR and the intensity of lipid staining (Figure composition were detected in the WM. Changes in brain 5M,O). All staining results presented high linear correlations composition during postnatal development are mainly (R2 > 0.850). concentrated in lipids, as observed and measured in the IR In the cerebellar WM (arbor vitae), we found positive spectra (Figures 1, 2, S2, and S3), and these modifications are staining at P7 in the SB but not in the LFB and ORO (Figure stronger in the WM than in the GM, as expected. The study of 6A,D,G). All stains showed a steep increase at P14 and GM showed that at P0, the cerebral cortex contained lower progressed to a slower path until P28 (Figure 6A−L). amounts of lipids and fewer unsaturated olefinic functional Quantification of the percentage of stained area revealed groups than the brain WM (Figure 3). These differences progressive significant increases in all staining from P14 to P21, between GM and WM were reduced or lost at later time points and later to P28, except for LFB, which achieved maximal (P7, P14) due to the total increase in lipids containing both staining at P21 (Figure 6J−L). Results in the arbor vitae functional groups (Figure 2). In our study, the stable reflected that the myelination process starts earlier in the composition of GM from P7 onward represented a reliable cerebellum than in the brain and also showed high linear internal control of myelination. Changes in brain GM lipid correlations between μFTIR data and lipid staining in SB and content and composition at early postnatal stages, from P0 to the ORO and moderate linear correlations with the LFB P7, had been similarly observed when comparing the (Figure 6M,O). hippocampus of P6 to P30 rats27 and could be explained by 2.4. Modifications in Protein Structure Composition the peak of dendrite and axon extension as well as Are Related to the Increase of Myelination Proteins in synaptogenesis in cortical areas.35,36 Newly formed synaptic WM. The general decrease found in the β-sheet/α-helix connections increase the number of cell membranes, which protein ratio of the WM of both the brain and cerebellum explains the increase in the CH2/amide I ratio and carbonyl (Figure 4) is coincident with the time points of myelination. and unsaturated olefinic functional groups. In addition, the β- Then, we assessed whether changes in the proportion of α- sheet/α-helix ratio raised at P7, as similarly observed in the rat helix and β-sheet proteins in cerebral and cerebellar WM hippocampus,27 indicating that β-sheet proteins could increase reflect the new synthesis of myelin-associated proteins during proportionally during this process. In the cerebellum, an primary myelination. As a reference, we performed two increase in the number of unsaturated olefinic groups was first immunohistochemical staining against two proteins abundant detected in the molecular layer and then in the granular layer. in myelinated areas: the MBP and the MOG (Figure 6). These These specific changes could be related to the migration of two proteins were chosen for their different compositions in granular neurons from the outer to inner cortical areas at these secondary structures since MBP is a protein-rich in α-helices stages.37 Detection of those modifications opens the door to [Uniprotkb: P04370], whereas MOG is rich in β-sheets the study of changes in GM composition and their relation to [Uniprotkb: Q61885]. hallmarks of CNS formation, such as neuronal migration and/ In the CC, MBP began its expression at P7, where or synaptogenesis. oligodendrocytes were marked with extensions that began to In WM, primary myelination is represented by an abrupt myelinate (Figure 6A). At P14, myelinated fibers were lipid increase, a process that occurs during postnatal CNS observed throughout the CC, and at P21 and P28, the entire development in mice, rats, and rabbits and in perinatal human CC was stained, showing complete myelination (Figure 7B− stages.7,16,38 Accordingly, our data showed a significant D). Surprisingly, MOG showed a later expression onset than increase in lipids of the cerebellar arbor vitae at P14 and MBP since it started to be expressed at P14 (Figure 7E,F,J). later at P21 in the brain CC, which progressed at later time From P14 onward, MOG expression increased gradually until points in both areas (Figures 2 and 3). These results reflect the P28 (Figure 7F−H,J). In the cerebellum, we observed a very cauda-rostral directionality of myelination in the CNS, as similar dynamic for MBP and MOG expression in the WM observed in mice, rats, and humans.38 Our results also (Figure 6L−U). For MBP and MOG proteins, expression reinforce a threshold previously proposed to distinguish started at either P7 or P14, respectively, as was also detected in WM-GM of 1.5 or 1.05 for the CH2/amide I ratio, based on CC (Figure 7L,Q). data from GM and WM areas in adult mice and the human brain.19,23 The WM-GM myelination threshold of 1.05, 3. DISCUSSION proposed by Bonda et al.,24 specifically distinguishes between The present work describes in detail changes in lipids and lack of or emergent myelin synthesis and partially myelinated proteins during CNS development in mice from birth through WM. We and others have reported myelin formation at P7 in postnatal stages at the very beginning of life. In addition, we the cerebellum and P14 in the brain, according to the correlated our IR data on biochemical changes in WM with the histological detection of lipids, MBP, and MOG proteins,16,39 results of the histochemical and immunohistochemical although the increase in lipids at P14 and P21 detected by techniques commonly used to study myelination. In this μFTIR did not reach full myelination. The threshold of 1.5, 3097 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article proposed by Sanchez-Molina et al.,19 was achieved in the changes in WM in physiological and pathological contexts, cerebellar arbor vitae at P14 and in the CC at P21, time points such as demyelination/remyelination.16,47−49 Lipid staining that coincided with significant increases in the CH2/amide I used in this study is one of the most popular methods for ratio, and that were only surpassed at later stages. Based on characterizing myelin49,50 and shows different properties. ORO these observations and our μFTIR data, we propose that the stains hydrophobic or neutral lipids, such as fats and CH2/amide I threshold of 1.5 distinguishes well between cholesterol esters of unsaturated fatty acids, and SB shows a incomplete and completely myelinated WM since partially similar pattern to ORO, in which less hydrophobic lipids, such myelinated WM is found close to 1.5 but fully myelinated WM as phospholipids and sphingolipids, are also stained.47,51 LFB is is above this threshold. The reliability of this threshold is also usually referred to as phospholipid staining, although this reinforced by the fact that the lipid−protein ratio in myelin is association is unclear.47,52 The CH2/amide I ratio was highly maintained throughout life, as reported by others, meaning correlated with the intensity of three different lipid histological that its lipid−protein ratio is constant;5 therefore, the increase staining results in both the CC and cerebellar arbor vitae in lipids observed in the quantification of the CH2/amide I (Figures 5 and 6). As mentioned above, the three-lipid staining ratio WM reflects myelination. Beyond the myelination began to stain some early fibers at a time point before the rise thresholds, primary myelination progresses further at P14 in in the CH2/amide I ratio in both the brain and cerebellum. the cerebellum and P21 in the CC. Because the CH2/amide I ORO, SB, and LFB staining showed a strong linear correlation ratio at P28 approaches that described for CC WM in the adult with lipid increase in the CC, and only LFB showed a brain, approximately 2.0,19 we could say that in this study, we moderate linear correlation in the cerebellum. characterized the full primary myelination process. Never- Immunohistochemical staining of myelin proteins is widely theless, it should be kept in mind that myelination of WM and used to track the degree of myelination. The most abundant other brain areas progresses further in the postnatal brain,38,40 proteins in myelin are PLP (38%), MBP (30%), 2′,3′-cyclic and according to some studies, whole-brain myelination is not nucleotide 3′-phosphodiesterase (5%), and MOG (1%).52 completed at most until 3 months in mice/rats8 or until Thus, MBP is one of the most commonly used proteins to adolescence in humans.41 determine myelination and demyelination in CNS tissues. In Analysis of functional groups using μFTIR revealed the our study, MBP was detected in a few mature oligodendrocytes biochemical tissue composition in postnatal WM areas more in the CC and whole fibers in the cerebellar WM before ORO, specifically (Figure 3). Unsaturated olefinic functional groups LFB, or SB (Figure 7). MBP is found in the outer lamellar are found in higher amounts in polyunsaturated long-chain sheet of myelin and supports myelin compacting and fatty acids, while carbonyl functional groups are found in lipids adhesion.38,53 Its appearance has been described before myelin containing ester groups, and they can be quantified through formation in the extracellular membranes of mature oligoden- μFTIR spectra.34,42,43 In our work, both olefinic unsaturated drocytes.54 Because MBP onset is related to lipid staining and and carbonyl contents proportionally decreased compared to the CH2/amide I ratio increases, the use of MBP to determine the total lipid content, while concomitantly, the global WM the degree of myelination should be carefully evaluated, lipid amount increased. Our results suggest that the WM especially at early stages, as MBP expression does not reflect a composition is poorer in lipids containing unsaturated and full and compact myelin lipid envelope around axons. In carbonyl functional groups during myelin maturation. This contrast, in our study, MOG appeared later than MBP because interpretation is supported by the strong negative linear its expression has been described in myelinating rather than all correlations obtained for carbonyls in relation to the CH2/ mature oligodendrocytes.55,56 amide I ratio in the brain CC and cerebellar WM. Our results Myelin compaction is supported by two major myelin- agree with a recent report describing a shift from phospholipids associated proteins: MBP and PLP. As mentioned above, MBP to monounsaturated fatty acids when comparing P15 to P40 expression does not accurately reflect myelination; however, myelin-enriched mouse brain tissues.9 Similarly, previous myelin bundle formation is associated with protein post- studies found an increased content of polyunsaturated fatty transduction modifications and changes in their spatial acids in the forebrain of the human brain during development, organization.56 Examination of the secondary structure by whereas myelin-specific lipids contained more polysaturated our μFTIR data showed a decrease in the intramolecular β- long-chain fatty acids and monounsaturated fatty acids, sheet/α-helix ratio (Figure 4), which could be explained by the specifically oleic acid.14 In agreement with the tendency increase in the proportion of α-helix protein domains rather described, in both adult human and mouse brain tissue, WM than intramolecular β-sheets in proteins found in WM during contained a lower content of unsaturated olefinic and carbonyl primary myelination. These data agree with those of previous bonds compared to GM.19,20 These results can be explained by studies reporting a lower intramolecular β-sheet/α-helix ratio the elevated content of monounsaturated fatty acids in WM in WM compared to GM in both human and mouse brain and polyunsaturated fatty acids in GM.20 The changes tissue.19 Furthermore, a shift from the α-helix to the observed in the spectra during WM maturation could be intramolecular β-sheet has been detected by μFTIR in the because of myelin accumulation, which has a different molar peri-infarct zone of rat brain ischemic lesions or in the CC after ratio of cholesterol, phospholipids, and glycosphingolipids traumatic brain injury, coinciding, in the first study, with (40:40:20) than cell membranes (25:65:10).42,43 Lipids found myelin loss detected by electron microscopy.57,58 Changes in most abundantly in myelin are cholesterol, galactosylcer- protein structure have been related to different functions, and amides, ethanolamine plasmalogen, and sphingomyelin.9,42−45 indeed, its alteration has been associated with demyelina- Carbonyl and unsaturated olefinic functional groups are absent tion. 57−59 This early evidence suggests that properly in cholesterol, sphingomyelin, and long chains of monounsa- compacted and functional myelin favored the α-helix turated fatty acids but are present in some phospholipids.46 conformation of the main compacting proteins MBP and Histochemical and immunohistochemical techniques, in- PLP. In support of this data, PLP, the most abundant protein cluding lipid staining, have been extensively used to describe in CNS myelin, contains several domains that adopt an α- 3098 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article helical structure in lipid bilayers.60 MBP has specific domains calcium fluoride (CaF2) optical windows (CAFP20-1; Crystran, UK). with α-helix and β-sheet conformational states, but the α-helix To minimize the water contribution, sections on the CaF2 slides were domain increases with lipid interactions, especially more with air-dried at room temperature (RT) and stored in a vacuum protected saturated than unsaturated fatty acids.54,61−64 Therefore, in this from light until use. Cryostat sections for histology were mounted on Flex IHC slides (Dako, Glostrup, Denmark). Three consecutive study, we suggest that myelin bundle formation and its coronal brain slices (P0 #26,65 3.51 mm bregma66) and medial sagittal alterations could be detected by measuring intramolecular β- cerebellum slices were obtained for each animal. Once collected, the sheet/α-helix ratios in WM. In some contexts, using this slides were stored at −20 °C until use. approach to evaluate myelination could be advantageous over 5.3. Tissue Sample Obtention of Paraffin Sections for other expensive and time-consuming techniques, such as Histological Analysis. Mouse pups from P7 to P28 were transmission electron microscopy. intracardially perfused, as described above. Brains were dissected and postfixed by immersion in the same fixative solution for 24 h at 4 4. CONCLUSIONS °C. After three washes in PB for 30 min each, the brain and cerebellum were separated, and the samples were progressively In this work, we described the changes in lipid and protein dehydrated in 50, 70, and 95° and absolute ethanol, cleared in xylene, compositions during postnatal CNS development in the brain and embedded in paraffin blocks. 9 μm-thick coronal brain sections and cerebellar GM and WM using μFTIR in detail. Our data and sagittal cerebellar sections were cut using a Leitz 1512 rotatory provide new insights into the most significant changes related microtome and collected on gelatinized slides. Coronal brain slices to primary myelination in WM, consisting of an increase in ranged from 2.79 to 5.07 mm in P7 samples (P6 #12 to #31)65 and lipids containing low olefinic unsaturation and carbonyls. For from bregma 1.10 to −2.64 mm in P14, P21, and P28 samples.66 The the first time, we describe how a proper degree of myelination parasagittal cerebellar slices ranged from central to lateral 0.04 mm. is related to a lower intramolecular β-sheet/α-helix ratio in 5.4. LFB Staining. Myelin staining was performed, as described by WM than in GM. Our data showed that primary myelination Petkovic et al.67 Coronal (in brain samples) or sagittal (in cerebellum may be better studied with routine lipid histochemical staining samples) paraffin sections were deparaffined, hydrated, and incubated in LFB solution (0.1 g of LFB/100 mL of 95% ethanol with 10.5 mL than with immunohistochemical staining against some myelin- of 10% CH3COOH) for 3 h at 60 °C. The excess was washed with associated proteins, such as MBP. These data provide a 95% ethanol and distilled water, and the sections were differentiated reference for further studies determining alterations in using 0.05% Li2CO3 solution, followed by 70% ethanol. Finally, the experimental animals with neurodevelopmental diseases and sections were dehydrated in alcohol, cleared in xylene, and covered constitute a basis for studying brain composition during human with a DPX mounting medium. perinatal development using μFTIR. In this study, we also 5.5. SB Staining. SB staining was performed according to the showed how the use of IR microscopy can easily provide method described by Ineichen et al.68 Coronal brain and sagittal complete data on the lipid and protein content of specific GM cerebellum cryostat sections were air-dried and washed with 0.05 M and WM areas and the degree of myelination with little Tris buffer. The slices were then incubated in a freshly made and biological material. filtered SB solution (1% SB in ethylene glycol) for 20 min at RT. After washing off excess SB solution with 0.05 M Tris buffer, slides were 5. MATERIALS AND METHODS cover-slipped with PermaFluor aqueous mounting medium (TA-030- FM, Thermo Fisher Scientific). 5.1. Animals. C57BL/6 mouse pups P0 to P28 of both sexes were 5.6. ORO Staining. For ORO staining, coronal brain and sagittal used in this study. Pups from P0 to P21 were bred and maintained cerebellum cryostat sections were incubated in freshly made and with their mothers until they were euthanized. After weaning at P21, filtered ORO solution (ORO stock solution: 2.5 g/400 mL 99% P28 animals were housed and bred in the Animal House of the isopropyl alcohol; ORO working solution: 1.5 stock solution/1 Institute of Neurosciences of the Universitat Autònoma de Barcelona distilled water) for 2 h at RT.43 Excess was washed with tap water, and under a 12 h light/dark cycle and maintained at a constant the slides were cover-slipped with PermaFluor aqueous mounting temperature (24 ± 2 °C) with food and water ad libitum. All medium (TA-030-FM, Thermo Fisher Scientific). experimental animal studies were conducted according to Spanish 5.7. Single Immunohistochemistry. Paraffin-embedded coronal regulations (Ley 32/2007, Real Decreto 1201/2005, Ley 9/2003, and brain and sagittal cerebellar sections were immunostained for the Real Decreto 178/2004) in agreement with European Union visualization of myelin (MBP and MOG). Sections were washed twice directives (86/609/CEE, 91/628/CEE, and 92/65/CEE) and were in 0.05 M Tris-buffered saline (TBS, pH 7.4) for 5 min, blocked for approved by the Ethical Committee of the Autonomous University of endogenous peroxidase by incubating 2% H2O2 in 70% methanol for Barcelona. Every effort was made to minimize the number of animals 5 min, and washed again in 0.05 M TBS containing 0.1% Triton X- used to produce reliable scientific data, as well as animal suffering and 100 (TBS-0.1%T). Sections were then blocked for nonspecific pain. Animals were grouped according to age as follows: postnatal day 0 (P0), P7, P14, P21, and P28. Each experimental group consisted of staining by incubating in a blocking buffer solution (BB, containing 4−5 animals. 10% fetal bovine serum and 0.3% bovine serum albumin in TBS-0.1% 5.2. Tissue Sample Obtention of Cryostat Sections for T) for 1 h at RT. Next, the sections were incubated with the primary Histological and μFTIR Analysis. Mouse pups were anesthetized antibody (rat anti-MBP -ab7349 Abcam-, 1:500; rabbit anti-MOG intraperitoneally (ip) with a solution of ketamine (80 mg/kg) and -859901 Biolegend-, 1:50) overnight at 4 followed by 1 h at 37 °C. xylazine (20 mg/kg) at a dose of 0.015 mL/g and intracardially Sections were rinsed three times in TBS-0.1%T and incubated with an perfused with 4% paraformaldehyde in 0.1 M PB (pH 7.4) for 5 or 10 anti-rat (BA-4001 Vector Laboratories) or anti-rabbit (BA-1100 min (P0 to P7 or P14 to P28, respectively). The brain was Vector Laboratories) secondary antibody diluted in BB (1:500) for 1 immediately removed and postfixed by immersion in the same fixative h at RT. After washing with TBS-T, the sections were incubated with solution for 4 h at 4 °C. Samples were then washed with PB and streptavidin-peroxidase (1:500 in BB, SA-5004 Vector Laboratories) cryoprotected for 48 h with a 30% sucrose solution in PB. Then, the for 1 h at RT and then washed twice in TBS and subsequently with cerebellum was separated from the brain before being frozen using TB (0.05 M Trizma base, pH 7.4). The reaction was visualized using a methylbutane (320404, Sigma-Aldrich) and chilled in dry ice. The DAB kit (SK-4100; Vector Laboratories, Inc., Burlingame, CA, USA), samples were stored at −80 °C until use. following the manufacturer’s instructions. Finally, the sections were For either μFTIR analysis or histology, 9 μm-thick frozen coronal dehydrated in alcohol, cleared in xylene, and cover-slipped in the DPX brain and sagittal cerebellar sections were cut using a Leica CM3050 mounting medium. The sections were observed using a bright-field cryostat. Sections for μFTIR analysis were mounted onto polished Nikon Eclipse 80i microscope. 3099 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article 5.8. Densitometric Analysis of Histochemical and Immuno- (R2 correlation <0.65, poor correlation), and Student’s t-test (p < histochemical Staining. For immunostaining analysis, images of a 0.05). A good linear correlation was considered when the slope was minimum of three slices per animal were captured at 20× close to 1 (good correlation >0.8500 or < −0.8500). magnification with a DXM 1200F Nikon digital camera mounted on a bright-field Nikon Eclipse 80i microscope and using ACT-1 2.20 software (Nikon Corporation). For each brain slice, three photo- graphs were taken corresponding to the central and lateral regions of ■ ASSOCIATED CONTENT * Supporting Information sı the CC and three photographs corresponding to the cerebral cortex. The Supporting Information is available free of charge at For cerebellum slices, three photographs were taken corresponding to https://pubs.acs.org/doi/10.1021/acschemneuro.3c00237. regions of the arbor vitae and the two layers of its GM: the molecular layer and the granular layer. μFTIR average absorbance spectra; total lipid and Densitometric analysis for each photograph was performed using protein content in brain GM and WM; and total lipid Analysis software. The percentage of the area occupied by and protein content in the cerebellum GM and WM immunolabeling (% area) was recorded for each photograph. (PDF) 5.9. μFTIR Data Acquisition. μFTIR based on synchrotron radiation was performed at the MIRAS beamline of the ALBA Synchrotron light source (Catalonia, Spain).69 The measurements were performed as described by Sánchez-Molina et al.19 In brief, a Hyperion 3000 microscope with a 36× objective coupled to a Vertex ■ AUTHOR INFORMATION Corresponding Author 70 spectrometer (Bruker, Billerica, MA, USA) was used. Spectra were Gemma Manich − Department of Morphological Sciences, collected in transmission mode at 4 cm−1 resolution, 10 × 10 μm Universitat Autònoma de Barcelona, 08193 Barcelona, aperture, 128 scans in brain samples, and 64 scans in cerebellum Spain; orcid.org/0000-0002-9866-271X; samples using the Opus 7.5 software (Bruker, Billerica, MA, USA). Phone: +34935811825; Email: Gemma.Manich@uab.cat Measurements ranged from 4000 to 600 cm−1 wavenumbers, and zero filling was performed with a fast Fourier transform, obtaining one Authors point every 2 cm−1. Background spectra were collected every 10 min in each CaF2 window from a clean area. For each region of interest, Marta Peris − Department of Cell Biology, Physiology and approximately 50 spectra with a step size of 30 μm × 30 μm were Immunology, Institute of Neuroscience, Universitat Autònoma acquired. To represent regional differences with high spatial de Barcelona, 08193 Barcelona, Spain resolution in the tissue, maps of 100 spectra with a step size of 6 × Núria Benseny-Cases − Biophysics Unit. Department of 6 μm were generated, producing a final map of 60 × 60 μm in one Biochemistry and Molecular Biology, Universitat Autònoma representative sample of each species containing GM (cortex in the de Barcelona, 08193 Barcelona, Spain brain and molecular and granular layers in the cerebellum) and WM Oriana Zerpa − Department of Cell Biology, Physiology and areas (CC in the brain and arbor vitae in the cerebellum). Immunology, Institute of Neuroscience, Universitat Autònoma 5.10. μFTIR Spectrum Analysis. Unscrambler X software de Barcelona, 08193 Barcelona, Spain (CAMO Software, Oslo, Norway) was used for data processing. The second derivative of the spectra was calculated using the Beatriz Almolda − Department of Cell Biology, Physiology and Savitzky−Golay algorithm with an 11-point smoothing filter and a Immunology, Institute of Neuroscience, Universitat Autònoma polynomial order of 3 to enhance the narrow bands and eliminate the de Barcelona, 08193 Barcelona, Spain baseline contribution. With the data already processed, we analyzed À lex Perálvarez-Marín − Biophysics Unit. Department of the absorbances of the following bands:70 3012 cm−1 (C�CH, Biochemistry and Molecular Biology, Universitat Autònoma unsaturated olefinic group), 2852 cm−1 (CH2 symmetric stretching de Barcelona, 08193 Barcelona, Spain; orcid.org/0000- vibrations), 2921 cm−1 (CH2 asymmetric stretching vibrations), 1743 0002-3457-0875 cm−1 (C�O, carbonyl group), 1656 cm−1 (α-helix secondary protein Berta González − Department of Cell Biology, Physiology and structure), and 1637 cm−1 (β-sheet secondary protein structure). The Immunology, Institute of Neuroscience, Universitat Autònoma values mentioned above were obtained by calculating the average of de Barcelona, 08193 Barcelona, Spain the band at 2 cm−1 before and 2 cm−1 after from the secondary derivative of each spectrum. CH2 is a functional group, especially Bernardo Castellano − Department of Cell Biology, present in lipids; therefore, it was used to normalize the values using it Physiology and Immunology, Institute of Neuroscience, as the total lipid value. To study the secondary structure of the Universitat Autònoma de Barcelona, 08193 Barcelona, Spain proteins, the α/β ratio was calculated by taking the absorption values Complete contact information is available at: of the α structures at 1656 cm−1 and the intramolecular β structures at https://pubs.acs.org/10.1021/acschemneuro.3c00237 1637 cm−1 of the second derivative spectra (d2A1637/d2A1656). The sum of both bands was used as the total protein value. 5.11. Statistical Analysis. Statistical analyses were performed Author Contributions # using the GraphPad Prism software (version 8.0; GraphPad Software, M.P. and N.B.-C. contributed equally to the study. M.P., N.B.- San Diego, California, USA). All experimental results are expressed as C., and G.M. designed and performed the experiments, the mean value ± SEM. To study differences in age and region, a two- including acquiring and analyzing data, and wrote and revised way ANOVA with Tukey’s post-hoc test was used to compare the manuscript. B.A. and O.Z. designed experiments, acquired postnatal days and studied areas. A one-way ANOVA was performed data, and revised the manuscript. A.P.-M., B.C., and B.G. to analyze lipid staining and immunohistochemistry. Statistical designed the research and revised the manuscript. All authors significance was set at p < 0.05. contributed to the article and approved the submitted version. To study whether the increase or decrease in lipids was related to changes observed in functional olefinic unsaturated or carbonyl Funding groups or increased histochemical lipid staining, either absorbance This work was supported by the Ministerio de Ciencia e ratios or the percentage of stained area were normalized between 0 Innovación of the Spanish Government (BFU2017-87843 R) and 1 by considering the maximal value equivalent to 1, as performed to B.C. and A.P.-M. in Benseny-Cases et al.25 Correlations were determined using Pearson’s coefficient (R > 0.80 or < −0.80, strong correlation; R > Notes 0.60 or < −0.60, moderate correlation), coefficient of determination The authors declare no competing financial interest. 3100 https://doi.org/10.1021/acschemneuro.3c00237 ACS Chem. Neurosci. 2023, 14, 3088−3102 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article ■ ACKNOWLEDGMENTS We would like to thank Miguel A. 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