Biochemical changes indicate developmental stage in the

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Biochemical Changes Indicate Developmental Stage in the Hippocampal Formation Joanna G. Chwiej,*,† Stanislaw W. Ciesielka,† Agnieszka K. Skoczen,† Krzysztof J. Janeczko,‡ Christophe Sandt,§ Karolina L. Planeta,† and Zuzanna K. Setkowicz‡ †

AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow 30-059, Poland Jagiellonian University, Institute of Zoology and Biomedical Research, Krakow 30-387, Poland § SOLEIL, Gif sur Yvette 91192, France ACS Chem. Neurosci. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 11/12/18. For personal use only.

‡

ABSTRACT: The literature showing how age of humans or animals influences the IR absorption spectra recorded in different brain regions is very poor. A very limited number of studies used FTIR microspectroscopy for analysis of the aging process, however there is lack of data concerning the biomolecular changes occurring in the course of postnatal development of the central nervous system. Therefore, in this paper the topographic and semiquantitative biochemical changes occurring within the rat hippocampus during postnatal development were examined. To achieve the goal of the study, three groups of normal male rats differing in age were investigated. These were 6, 30, and 60 day old animals, and the chosen ages correspond to the neonatal period, childhood, and early adulthood in humans, respectively. Already, preliminary topographic analysis identified a number of significant changes in the accumulation of biomolecules within the hippocampal formation occurring during brain development. Such observation was confirmed by further semiquantitative analysis of intensities of selected absorption bands or ratios of their intensities. The detailed examinations were done for four hippocampal cellular layers (multiform, molecular, pyramidal, and granular layers), and the results showed that the accumulation of most biomolecules, including both saturated and unsaturated lipids as well as compounds containing phosphate and carbonyl groups, was significantly higher in adulthood comparing to the neonatal period. What is more, the increases in their levels were observed mostly between 6th and 30th days of animals’ life. The unsaturation level of lipids did not change during postnatal development, although the differences in unsaturated and saturated lipids contents were noticed between examined animal groups. Significant differences in relative secondary structure of proteins were found between young adult rats and animals in neonatal period for which the relative level of proteins with β-type secondary structure was the highest. KEYWORDS: postnatal brain development, hippocampal formation, topographic and semiquantitative biochemical analysis, Fourier transform infrared microspectroscopy

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INTRODUCTION

normal hippocampal functions and could lead to permanent pathological changes.1 The most important factors that may influence both hippocampal neurogenesis and dendritic architecture are positive and negative stress, physical activity, environmental enrichment, memory challenging tasks, and some changes in cellular nutrients.4−12 Extensive remodeling of hippocampal formation including appearance of new granule cells in ectopic locations within the dentate gyrus, dispersion of the granule cell layer, and reorganization of mossy fibers can also be the result of epileptic seizures.13,14 Our earlier research carried out using Fourier transform infrared microspectroscopy showed, moreover, that seizures may induce structural changes of proteins (increased

The hippocampal formation is the brain region presenting exclusive capacity for structural reorganization.1 The creation of new neurons and accompanying growth of dendrites and axons, as well as generation of new synapses, are not limited there only to prenatal development but occur also during the postnatal period, young adulthood, middle age, and senescence.1 The hippocampal formation consists of heterogeneous populations of neurons. Some of them, including pyramidal neurons, are generated only during embryonic development, while the others such as granule cells in the dentate gyrus (DG) are produced throughout the whole life.2,3 This structural plasticity of the hippocampal formation is sensitive to many different experiences, which may have both positive and negative impact on its function. From one side, its extended restructuring results in adaptive plasticity. From the other side, the sensitivity to environmental perturbations may have adverse effects on © XXXX American Chemical Society

Received: September 5, 2018 Accepted: October 30, 2018 Published: October 30, 2018 A

DOI: 10.1021/acschemneuro.8b00471 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience relative content of proteins with β-type secondary structure) and lipids, decrease unsaturation level of lipids, and lead to anomalies in the accumulation of creatine within the hippocampal formation.15−18 From the other side, some neuroplastic changes triggered in the hippocampus in response to seizure-induced neuronal damage may lead to the establishment of temporal lobe epilepsy, which is the most common type of focal human epilepsy.19 Therefore, there is a close relation between epileptogenesis and neuroplasticity, which are the two processes that may positively intensify one another.19 Our previous study carried out using X-ray fluorescence microscopy showed that structural reorganization of rat hippocampal formation during postnatal development is accompanied by significant changes in the elemental composition of this brain area. The found variations concerned Fe and Zn levels, which were higher in young adults compared to animals in neonatal period. Although, both elements increased together with the animals age, the progress of these changes differed significantly. While the increase of Zn areal density was found only between the 6th and 30th day of animal postnatal life, the level of Fe increased slightly until the early adulthood.20 This paper is the continuation of the mentioned study, and its main goal is the analysis of biochemical changes accompanying structural reorganization of hippocampal formation and occurring between early postnatal period and adulthood in rats. To achieve it, the brain of animals at the age of 6, 30, and 60 days (further indicated as N06, N30, and N60, respectively) were examined. It is necessary to mention that the chosen age of animals corresponds to the neonatal period, childhood, and early adulthood in humans.3,21,22 As the neurogenesis and dendritic restructuring is limited only to some of cellular populations and areas of hippocampal formation, the use of the technique enabling highly spatially resolved biomolecular analysis was necessary.2 Fourier transform infrared (FTIR) microspectroscopy meets such requirements and what is more enables simultaneous topographic and semiquantitative analysis of the distribution of main biomolecules as well as changes in their structure, and therefore it was used for this study.

were calculated. This was done separately for each examined slice based on the spectra recorded for 25 pixels chosen from particular layer. 5. Evaluation of median values of biochemical parameters for all the three examined age groups. 6. Determination of the statistically significant differences between biochemical parameters recorded for the three examined animal groups. This was done using nonparametric Mann−Whitney U test. Table 1. List of Examined Biochemical Parameters absorption band (ratio of absorption bands) 1658 cm−1 1634 cm−1/1658 cm−1 2955 cm−1 3012 cm−1 3012 cm−1/2800−3000 cm−1 1740 cm−1 1080 cm−1 1240 cm−1

1360−1480 cm−1

remarks amide I band, distribution of proteins structural changes of proteinsa distribution of saturated lipids distribution of unsaturated fatty acids unsaturation level of lipidsb distribution of compounds containing carbonyl group(s) including phospholipids, cholesterol esters, ketone bodiesc distribution of compounds containing phosphate groups(s) including nucleic acids, phospholipids, phosphorylated carbohydrates, differences in the degree of phosphorylation of carbohydrates, and glycoproteinsd distribution of lipids, cholesterol esters, and cholesterol

a

From Kneipp et al.;23 Miller et al.;24 Kretlow et al.;25 SzczerbowskaBoruchowska et al.;26 Chwiej et al.15,16 bFrom Petibois and Déleris.27,28 cFrom Kretlow et al.;29 Chwiej et al.16 dFrom Kneipp et al.;30 Diem et al.;31 Liquier and Taillandier;32 SzczerbowskaBoruchowska et al.26

As one can see from Figure 1, presenting the comparison of mean spectra recorded for four examined cellular layers of hippocampal formation, the GR and PY layers presented higher level of proteins and lower level of lipids compared to MO and MU layers. These relations were confirmed by chemical mapping of main absorption bands specific for proteins and lipids, the result of which is shown in Figure 2. What is more, already preliminary topographic analysis of chemical maps presented in the Figure 2 pointed at the occurrence of differences in the accumulation of some biomolecules between the examined groups of rats. Together with the brain postnatal development, the levels of saturated and unsaturated lipids, as well as compounds containing phosphate and carbonyl groups, tended to increase within the hippocampal formation. What is more, the biomolecular changes occurred mainly between the 6th and 30th day of animal postnatal life. Four cellular layers of hippocampal formation, namely, molecular, multiform, pyramidal, and granular (abbreviations, MO, MU, PY, and GR, respectively), were subjected to detailed quantitative comparisons. Their typical localization within the scanned tissue area is presented in Figure 3. For each hippocampal slice (one per each of the examined animal), the mean values of biochemical parameters were calculated from 25 spectra chosen from individual hippocampal layers. Afterward, in order to evaluate the differences existing between the animals in different age groups, the median, minimal, and maximal values of biochemical parameters were evaluated for N06, N30, and N60 groups and presented in the form of box-and-whiskers plots in Figure 4.

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RESULTS AND DISCUSSION Each slice of hippocampal formation was subjected to raster scanning using the infrared (IR) beam. As a result of this process, FTIR spectra were recorded for all examined tissue points. The analysis of the experimental data included the following steps: 1. Identification of absorption bands present in the spectra recorded for hippocampal formation (Figure 1). 2. Chemical mapping of selected absorption bands or their intensity ratios. The detailed list of biochemical parameters examined in frame of the study is presented in the Table 1, while in Figure 2 the chemical maps obtained for selected animals representing N06, N30, and N60 groups are shown. 3. Identification of spectra recorded for particular cellular layers based on the correlation of chemical maps and microscopic views of the scanned tissue areas. 4. Four cellular layers of hippocampal formation, namely, molecular (MO), pyramidal (PY), multiform (MU), and granular (GR) layers were subjected separately to detailed quantitative analysis. For them, the mean intensities or mean ratios of intensities of selected absorption bands B


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was found only in the pyramidal layer for which the content of proteins was lower in 60 day old rats compared to 6 day old animals (“−” sign for pyramidal layer in case of N60 group in the Figure 5). On the other hand, for all the examined hippocampal layers, significant differences in the relative secondary structure of proteins were noticed for 30 and 60 day old rats in comparison to animals in the neonatal period. For both these animal groups, the ratio of the absorbance at the wavenumbers of 1635 and 1658 cm−1 was lower (“−” signs for all the examined cellular layers in case of N30 and N60 groups), which means that they demonstrated reduced relative level of proteins with β-sheet secondary structure. The relative content of proteins with β-type secondary structure decreased significantly between the 6th and 30th day of life. Although afterward, in MU and MO cells the ratio of absorbance at the wavenumbers of 1635 and 1658 cm−1 increased, 60 day old rats still presented lower levels of this parameter for all the examined hippocampal layers. For MU, MO, and PY layers, the levels of both saturated and unsaturated lipids presented the same pattern of changes. In the first observation period, the content of both types of lipids increased, while between the 30th and 60th day of animal life, it decreased (MO and MU cells) or remained stable (PY layer). In GR layer, the content of saturated lipids increased and that of unsaturated lipids remained stable in both observation periods. Despite this, the unsaturation level also in GR layer did not alter in any of the examined time intervals. Moreover, the relative content of unsaturated lipids did not differ among the examined animal groups. The chemical mapping of 1080 and 1240 cm−1 absorption bands followed by further statistical analysis showed that the content of compounds containing phosphate groups increased significantly between the first week and first month of rat life and afterward remained stable. Such course of changes in the accumulation of the compounds was found for most cellular layers. The only exception from this rule was noticed for granular layer for which significant changes in phosphate group accumulation were found neither in the first nor in the second observation period. Nonetheless, 60 day old animals presented higher intensity of the 1080 cm−1 band in the granular layer, compared to rats in neonatal period. A great body of literature confirms that biomolecular analysis of brain samples using FTIR microspectroscopy may be very helpful in the investigation of mechanisms involved in different pathologies of the central nervous system.15−18,33−38 Most of these investigations have been carried out based on animal models of diseases;15−18,36−39 however one can find also the examples of studies using human biopsy or autopsy tissues.36,40,41 One of the factors that may influence biochemical composition of brain tissue is the age of patients or animals in case of studies using animal models. Surowka et al. in 201439 and Fimognari et al. in 201834 used FTIR microspectroscopy to study the changes in lipids and proteins occurring in selected brain areas as a result of aging. The first study showed that the total content of lipids decreased and lipid saturation increased within the human substantia nigra tissue with age.39 In turn, the second investigation demonstrated diminished lipid unsaturation and elevated lactate within the corpus callosum white matter of the mouse model of accelerated aging.34 However, according to our best knowledge, there is still a lack of evidence showing how biochemical composition of brain tissue changes during postnatal development. Therefore, in the frame of this paper, we evaluated the biomolecular changes of

Figure 1. Comparison of mean baseline corrected IR absorption spectra calculated for examined layers of hippocampal formation in the case of selected animals representing N06, N30, and N60 groups.

The statistical evaluation of biochemical differences between rats in different age groups was performed using Statistica software, version 7.1 (StatSoft). The U Mann−Whitney statistical test was applied for this purpose, and the observed changes were treated as significant when their p-values were lower than the assumed significance level of 5%. All the detected statistically significant differences were marked in the box-andwhisker plots presenting the distributions of biochemical parameters for examined animal groups and in Figure 5 presenting the statistically relevant biochemical differences between the subsequent points of time. As one can see from Figure 4A,B and 5, the levels of most biomolecules changed within hippocampal formation during postnatal development. During the first observation period (between 6th and 30th day of life), the level of proteins increased (GR layer) or remained stable (MU, MO, and PY layers), while between 30th and 60th day of animal life, in most cellular layers (excluding MU layer where the level remained unchanged), it decreased. Although in almost all cellular layers, the accumulation of proteins fluctuated between the 6th and 60th day of life, statistically significant difference between adult rats and animals in the neonatal period C


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Figure 2. Comparison of chemical maps obtained for selected animals representing N06, N30, and N60 groups. The distribution of saturated and unsaturated lipids was obtained through the chemical mapping of the absorption bands occurring at 2955 and 3012 cm−1, respectively. The unsaturation level of lipids was calculated as the ratio of intensity of 3012 cm−1 band and lipid massif (2800−3000 cm−1).

mechanisms involved in the pathogenesis and progress of epilepsy in the pilocarpine model of seizures.15−18 The subject of this research was also the hippocampal formation, which is clearly identified as an epileptogenic brain region, highly susceptible to both functional and structural damages, which have been associated with the pathophysiology of temporal lobe epilepsy being the most severe type of pharmacoresistant, focally acquired epilepsy with most heavy seizure symptoms.18,39 The assessment of the course of biomolecular changes in the hippocampal formation in the rat pilocarpine model of seizures allowed us to determine a group of processes that may lead to neurodegenerative changes and recurrent spontaneous seizures. Therefore, we expected that analysis of biochemical changes occurring in the hippocampal formation during its postnatal development might also shed some new light on the increased vulnerability of the immature brain to seizures and epilepsy, which is confirmed by many epidemiological studies showing that the incidence rate of this disease is the highest in the first year of life and declines to the adult levels by the end of the first decade.40−45 In addition, getting to know the biochemical composition of the hippocampal formation during developmental changes can provide valuable information on the molecular basis relevant to possible subsequent pathological changes. FTIR microspectroscopy allowed us to analyze within the hippocampal formation the changes in the distribution and accumulation of lipids, proteins, and compounds containing phosphate or carbonyl groups. Moreover, it made possible the examination of modifications of relative secondary structure of

Figure 3. Localization of main cellular layers within hippocampal formation. MU, MO, PY, and GR mean multiform, molecular, pyramidal and granular cell layers, respectively.

hippocampal formation resulting from normal postnatal brain development. The choice of this brain area for the study was dictated by the fact that hippocampal formation is characterized by an extraordinary plasticity.1 The neurogenesis and changes in the structure of connections between existing and newly emerging cells take place there not only during the development of the nervous system, but throughout the whole life.1 This study is also closely related to our earlier investigations, the aim of which was to gain the knowledge about the D


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Figure 4. (A,B) Box-and-whisker plots presenting the distributions of biochemical parameters for examined animal groups: median (point in the box), first and third quartiles (box), and minimal and maximal values (whiskers) of selected biochemical parameters in the examined cellular layers obtained for N06, N30, and N60 groups. Statistically significant differences (Mann−Whitney U test, 95% confidence level) between 30- and 6-day-old rats are marked with *, those between 60- and 6-day-old rats are marked with **, and those between 30- and 60-day-old animals are marked with #. MU, MO, PY, and GR mean multiform, molecular, pyramidal, and granular cell layers, respectively.

conclusion seems to be in agreement with the research of Cauley et al., who examined brain tissue changes as a function of age.46 The study was based on advanced analysis of CT scans of patients between neonatal period and two years of age with no known neurologic, neurocognitive, and developmental deficits. Its results showed that the mean density of brain tissue increases in the neonatal period, and most likely this is the effect of an overall decrease in the percentage of water in the tissue and a relative increase in gray matter volume.49,47,48 The level of proteins, in most of cellular layers, did not differ for adult rats compared to animals in the neonatal period. However, significant differences were observed with respect to the secondary structure of proteins. The animals in the neonatal period demonstrated the highest relative content of proteins with β-sheet compared to α-helix secondary structure. As is commonly known, β-sheet aggregates and amyloid fibrils appearing as a result of conformational changes of proteins are the common hallmark of several pathological conditions of central nervous tissue including neurodegerative disorders such as Alzheimer’s, Parkinson’s, Creutzfeld-Jacob’s, and Hunting-

proteins as well as of unsaturation level of lipids in tissue occurring during brain development. The levels of both saturated and unsaturated lipids were generally higher in young adult rats compared to animals in the neonatal period. However, the course of their biochemical changes differed among the examined cellular layers of hippocampal formation. In molecular and multiform layers, the levels of lipids increased between the 6th and 30th days of animal life. Afterward, they decreased slightly, but nonetheless, the median intensities of lipid bands were around two times higher in N60 compared to N06 group. For the pyramidal layer, the changes in the intensity of absorption bands characteristic for lipids were found only during the first observation period. In turn, for the granular layer, the increases, although observed during both observation periods, not always were statistically significant. Also the content of compounds containing phosphate and carbonyl groups was usually elevated in adult animals. The simultaneous increases in the levels of most of biomolecules may reflect the general changes in the density of brain tissue occurring during the postnatal development. Such a E


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Figure 5. Dynamics of biochemical changes occurring in multiform (MU), molecular (MO), pyramidal (PY), and granular (GR) layers of hippocampal formation during postnatal brain development (statistically significant differences in the elemental accumulation between the subsequent points of time). Statistically relevant increases found for the examined periods were marked as the sloping up arrows while the decreases as sloping down arrows. The lack of statistically significant differences between the subsequent time points was marked as black horizontal arrow. Moreover, the values significantly higher (lower) compared to 6-day old animals were indicated with “+” (“−”).

ton’s diseases, and amyotrophic lateral sclerosis.49 Since the βsheet protein structure can be considered as a predictor of pathological changes,50 it is rather surprising that just this conformation is elevated in the early developmental stage comparing to adult animals. What is more, this interesting phenomenon has never been described before. We assume that the here presented significant changes in the accumulation of main biomolecules are hallmarks of normal brain development. According to experimental and clinical data, brain development and aging are continuous processes overlapping each other without a defined borderline between them. These two processes may show different patterns of interregional similarities or differences in their dynamics, which subsequently may determine variations in individual susceptibility to diseases. Consequently, different brain regions should be explored for comparative purposes using similar parameters. The obtained results suggest, therefore, not only further research on animal models but also wide-range clinical trials based on a similar methodological approach and finally aimed at developmental determinants of neurodegenerative diseases in humans. Also, studies on different brain regions at various developmental stages, including aging, offer good opportunities to optimize the methodology.

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Sample Preparation. To follow the changes occurring in the hippocampal formation during postnatal brain development, the tissues were taken from rats on the 6th, 30th, and 60th day of life (N06, N30, and N60 groups, respectively). The number of rats in each group was six. After perfusion with physiological saline solution of high analytical purity, the brains were quickly excised, frozen in liquid nitrogen, and cut using a cryomicrotome into 12 μm thick sections. The slices of the dorsal part of the hippocampus were mounted on the MirrIR low-e microscope slides (Kevley Technologies) and freeze-dried at −70 °C. The samples were kept at the same temperature until the measurements, which, in turn, were carried out in air atmosphere at a temperature of 22 ± 1 °C. It is necessary to mention that the usefulness of such samples for studies using the FTIR microspectroscopy was confirmed during our earlier research.15−18,51,52 Measurements and Spectral Analysis. FTIR microscopy was used for qualitative and semiquantitative biochemical analysis of hippocampal formation. The measurements were carried out at the laboratory belonging to the SMIS beamline of SOLEIL synchrotron, but synchrotron source of IR was not used in the study. The investigation was done using Thermo Scientific Nicolet iN10 infrared microscope integrated with dynamically adjusted Michelson interferometer. The setup uses a ceramic source for IR generation and MCT-A detector allowing chemical mapping in the spectral range of at least 650−7800 cm−1 for recording of absorption spectra. Moreover, it is equipped with a motorized sample stage, which was used for raster scanning of the tissue slices deposited on reflective MirrIR slides. The tissues were examined in transflection mode using IR beam with the size of 50 μm × 50 μm. Also the step size applied during scanning was equal to 50 μm. The spectra were collected in the wavenumber range 800−4000 cm−1 with the resolution of 8 cm−1, and each spectrum was the average of 32 scans. The data acquisition was performed using OMNIC Picta, while for the spectral analysis OMNIC software (version 7.3) was applied. The chemical maps were obtained by displaying the area of one peak or the area ratio for the two peaks. Trapezoidal baseline correction was

METHODS

Animals. Male Wistar rats used in the study came from the husbandry of the Department of Neuroanatomy at the Institute of Zoology and Biomedical Sciences (Jagiellonian University, Krakow). All procedures using living animals were carried out there and were approved by the Bioethical Commission of the Jagiellonian University. During experiment, all animals were maintained under the same conditions of controlled temperature (20 ± 2 °C) and illumination (12h light/12-h dark cycle). They had unlimited access to water and food in the form of Labofeed. F


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ACS Chemical Neuroscience used in calculations of integrated peak areas and the background was typically taken at the two extreme frequency values of the peaks. Chemical mapping was carried out on unprocessed spectra. However, before the quantitative analysis the quality of the spectra were verified with respect to the sample thickness and the presence of Mie scattering induced artifacts. The thickness tests were based on criteria proposed by Lasch et al.53 The layered character of the hippocampal formation may result in differences of optical densities between the examined cellular layers and the presence of artifacts connected with resonant and nonresonant Mie scattering.54 All the spectra affected by Mie scattering artifacts were excluded from further analysis.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: joanna.chwiej@fis.agh.edu.pl. ORCID

Joanna G. Chwiej: 0000-0002-3792-7876 Agnieszka K. Skoczen: 0000-0002-4604-4557 Author Contributions

Joanna. G. Chwiej was project leader of the SOLEIL experiment and participated in data collection and analysis, interpretation of experimental results, preparation of manuscript, and supervision of Stanislaw W. Ciesielka (M.Sc. student), Agnieszka K. Skoczen, and Karolina L. Planeta (Ph.D. students). Stanislaw W. Ciesielka participated in data analysis. Agnieszka K. Skoczen participated in data collection and analysis. Krzysztof J. Janeczko provided critical review of the obtained results and participated in manuscript preparation. Christophe Sandt optimized measurement conditions and participated in data collection. Karolina L. Planeta participated in data analysis. Zuzanna K. Setkowicz conducted animal experiments and tissue preparation and participated in data collection and manuscript preparation. Funding

This work was partially supported by the Faculty of Physics and Applied Computer Science AGH UST statutory tasks no. 11.11.220.01/3 within subsidy of the Polish Ministry of Science and Higher Education and the statutory research of the Institute of Zoology and Biomedical Research (Jagiellonian University) K/ZDS/008073. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Synchrotron SOLEIL for the possibility of the use of Nicolet iN10 infrared microscope. REFERENCES

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