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Intracellular Calcium Networ CA3 Region in Rat Postnatal Deve
DOI: 10.17691/stm2016.8.4.21 Received March 15, 2016
Y.I. Mitaeva, PhD, Junior Researcher, Department of Neurotechnology, Institute i A.M. Mozherov, PhD Student, Junior Researcher, Department of Neurotechnology, Institute of Biology and Biomedicine1;
IA Kastalskiy, Researcher, Laboratory for Development of Intellectual Biomechatronic ' Centre for Biotechnology Development, Institute of Biology and Biomedicine1; T.A. Mishchenko, PhD, Senior Researcher, Molecular and Cell Technologies Department, Central Research Laboratory2; Senior Researcher, Laboratory for Neuroprotection Methods Develo Institute of Biology and Biomedicine1; Research Fellow, Laboratory of Laser Chemestry3; I.V. Mukhina, DSc, Professor, Head of the Central Research Laboratory; Head of the Department of Normal Physiology named after N.Y. Belenkov2; Professor, Department of Neurotechnology, Institute of Biology and Biomedicine; Head of the Center of Translational Technology1
1Lobachevsky State University of Nizhni Novgorod, 23 Prospect Gagarina, Nizhny Novgorod, 603950, Russian Federation;
2Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russian Federation;
institute of Photon Technologies, Federal Scientific Research Center "Crystallography and Photonics" of the Russian Academy of Sciences, 2 Pionerskaya St., Moscow, Troitsk, 142190, Russian Federation
Hippocampus — the structure of the central nervous system, which is involved in the mechanisms of memory consolidation. The hippocampus has a certain topology distribution of cellular elements, which provides the many cellular networks. One of them is the network of neurons in the CA3 field. In this article discusses the features of the Ca2+ signaling system of cells of CA3 field of rat hippocampus of early (P5-8, P14—16) and late (P21-25) postnatal development in different physiological conditions: spontaneous activity, in violation of the excitation in the neural network by adding tetrodotoxin (Ca2+ signaling), as well as the effects of excitatory neurotransmitters (ATP, L-glutamate). This work extends the concepts of Ca2+ signaling in rat hippocampus cells of early and late stages of postnatal ontogenesis. The study showed that changes in Ca2+ activity in the cells CA3 field of rat hippocampal taking place during the neonatal period of postnatal ontogenesis directly related to the functioning of neural networks and the metabolic state of the cells.
Key words: neuronal network; hippocampus; CA3 cells; astrocytes; Ca2+ imaging; postnatal development.
How to cite: Mitaeva Y.I., Mozherov A.M., Kastalskiy I.A., Mishchenko T.A., Mukhina I.V. Intracellular calcium network activity in the hippocampus CA3 region in rat postnatal development. Sovremennye tehnologii v medicine 2016; 8(4): 167-177, https://doi.org/10.17691/ stm2016.8.4.21
Russian
сетевая кальциевая активность в 0А3-поле гиппокампа крыс в неонатальном периоде развития
Я.И. Митаева, к.б.н., младший научный сотрудник кафедры нейротехнологий Института биологии и биомедицины1;
А.М. Можеров, аспирант, младший научный сотрудник кафедры нейротехнологий Института биологии и биомедицины1;
И.А. Кастальский, научный сотрудник лаборатории разработки интеллектуальных биомехатронных технологий Центра развития биотехнологий Института биологии и биомедицины1; Т.А. Мищенко, к.б.н., старший научный сотрудник отдела молекулярно-клеточных технологий ЦНИЛ2; старший научный сотрудник лаборатории по разработке методов нейропротекции Института биологии и биомедицины1; старший научный сотрудник лаборатории лазерной химии3;
И.В. Мухина, д.б.н., профессор, зав. ЦНИЛ; зав. кафедрой нормальной физиологии им. Н.Ю. Беленкова2; профессор кафедры нейротехнологий Института биологии и биомедицины; руководитель Центра трансляционных технологий1
For contacts: Artem M. Mozherov, e-mail: artemmozherov@gmail.com
Network Mechanisms in the Hippocampus CA3 Region in Rat Postnatal Development СТМ J 2016 — vol. 8, No.4 167
Нижегородский государственный университет им. Н.И. Лобачевского, Н. Новгород, 603950, пр. Гагарина, 23;
Нижегородская государственная медицинская академия, Н. Новгород, 603005, пл. Минина и Пожарского, 10/1;
3Институт фотонных технологий, Федеральный научно-исследовательский центр «Кристаллография и фотоника» РАН, Москва, Троицк, 142190, ул. Пионерская, 2
Гиппокамп — это структура центральной нервной системы, вовлеченная в механизмы консолидации памяти. Гиппокамп имеет определенную топологию распределения клеточных элементов, которая обеспечивает работу множества клеточных сетей. Одной из них является нейронная сеть поля СА3. Рассмотрены функции Са2+-сигнальной системы клеток САЗ-поля гиппокампа крыс раннего (Р5-8, Р14—16) и позднего (Р21-25) постнатального развития при различных физиологических состояниях: спонтанная активность при нарушении возбудимости в нейронной сети путем добавления тетродотоксина (Са2+-сигнализация) и под воздействием возбуждающих нейротрансмиттеров (АТФ, L-глутамат). Работа расширяет представление о Са2+-сигнализации в клетках гиппокампа крыс на ранней и поздней стадиях постнатального онтогенеза. Выявлено, что изменения в Са2+-активности клеток в поле САЗ гиппокампа крыс, происходящие в течение раннего неонатального периода, непосредственно связанны с началом функционирования нейронных сетей и метаболическим состоянием клеток.
Ключевые слова: нейронная сеть; гиппокамп; САЗ-клетки; астроциты; Са2+-изображения; постнатальное развитие.
Introduction. Meaning of calcium signaling system in the brain is very large, because it is directly involved in the regulation of fundamental processes of neural integration, regulates various localized functional responses, and maintains the set of intracellular signaling pathways. Calcium signaling is the subject of many scientific research areas related to the study of the role of neurons and astrocytes in the functioning of the central nervous system (CNS) in health and disease [1-7]. Metabolism is impossible at millimolar levels of intracellular Ca2+, so all living cells retain the Ca2+ concentration at the low nanomolar level, thus creating a difference in gradients between cellular compartments and intercellular space [8, 9]. Increase of intracellular Ca2+ concentration in the cell is a signaling process that triggers many biochemical reactions. Ca2+ activity of cells is the ability to quickly modify the intracellular concentration of Ca2+ in the form of oscillations. Spontaneous Ca2+ activity in the cells formed during the activation of ionotropic and metabotropic receptors, channels, regulates by the transporter proteins and probably depends on the stage of ontogenesis [3, 4].
Hippocampus — the structure of the central nervous system, which is involved in the mechanisms of emotion and memory consolidation. The hippocampus has a certain topology distribution of cellular elements, which provides work of many cellular networks [10-12]. One of these is the network of neurons in the CA3 field. This network receives inputs from cells of the entorhinal cortex and the dentate gyrus, moreover CA3 pyramidal neurons form the connection between themselves and interneurons, forming a closed network that operates in conditions of acute slice and generates spontaneous Ca2+ activity [4, 11, 13, 14]. Therefore, to estimate the age dependence of Ca2+ activity in the cells were investigated Ca2+ oscillations in neuronal and glial networks and the interactions between them.
In this study, we investigated changes in the characteristics of Ca2+ oscillations of cells of rat hippocampal CA3 field of early (P5-8, P14-16) and late (P21-25) postnatal development. Besides in the study
was valued role of network activity in the formation of spontaneous Ca2+ oscillations of cells of rat hippocampal CA3 field in early and late stages of postnatal development.
Materials and Methods
Slice preparations. Experiments were performed on acute hippocampal slices prepared from the brains of male Wistar rats early (P5-8, P14-16) and late (P21-25) stages of postnatal development. Male Wistar rats killed by cervical vertebra dislocation, according to the protocols approved by the National Ministry of Public Health for the care and use of laboratory animals and by the Bioethics Committee of the Nizhny Novgorod State Medical Academy. The work was performed in accordance with ethical principles established by European Convention for the Protection of Vertebrata used for Experimental and other Scientific Purposes (the Convention was passed in Strasburg, March 18, 1986, adopted in Strasburg, June 15, 2006). The brain was mounted on a vibratome (Microm HM 650V, Germany) and submerged in ice-cold Ringer solution that contained (in mM) 87 NaCl, 2.5 KCl, 26.2 NaHCO3, 1.24 NH2PO4, 0.5 CaCl2, 8.48 MgSO4, 11 D-glucose and gassed with carbogen (95% O2/5% CO2); pH 7.4; 295 mOsm. Three hundred fifty — micrometer — thick slices of the hippocampus were prepared and incubated for 1 h in the same solution at 35.0±0.5°C before recording. Bath solutions were bubbled with carbogen (95% O2/5% CO2) and had a pH 7.4.
Ca2+ imaging
Dye loading. Oregon Green 488 BAPTA-1 AM (OGB-1) (0.4 |jM) (Invitrogen, USA) dissolved in dimethylsulfoxide with 4% pluronic acid (pH 7.4) and gassed with 95% O2 and 5% CO2 at 35.5°C. After 40-min incubation for near-full absorption of the OGB-1 molecules by the hippocampal slices. Additionally, the exposed hippocampal slices were also loaded with the astrocyte-specific indicator sulforhodamine SR101 (10 jM) (Sigma, USA) [15, 16]. Then slices were transferred to a recording chamber perfused with Ringer solution that contained (in mM) 119 NaCl, 2.5 KCl, 1.3 MgSO4, 1 NaH2PO4,
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168 СТМ J 2016 — v0l. 8, N0.4 Y.I. Mitaeva, A.M. Mozherov, IA Kastalskiy, T.A. Mishchenko, I.V. Mukhina
1 1 1 I 1 1
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Figure 1. Visualization of the method for cross-correlation calculating for i and j discrete traces of the same duration within the non-zero delay time t. For this example, t=2 s, 8=1 s. Ratio of pulses calculated by the formula Ci/=nsyncAr/min(n,; n) is: Cij=3/min(6; 5)=0.6
26.2 NaHCO3, 2 CaCl2, 11 D-glucose and gassed with carbogen (95% O2/5% CO2); pH 7.4; 295 mOsm, at a rate of 1-1.5 ml/min. Calcium activity was imaged from the CA3 field of hippocampus.
Optical techniques. A confocal laser-scanning microscope, Zeiss LSM 510 (Germany), with a W Plan-Apochromat 20*/1.0 objective used to investigate the spontaneous activity of the neuronal and astrocytic network. Cytosolic Ca2+ was visualized via OGB-1 excitation with the 488 nm line of Argon laser radiation and emission detection with a 500-530 nm filter, while astrocytes were visualized with SR101, which was excited by 543 nm radiation from a He-Ne laser and detected with the use of a 650-710 nm filter for emission. Time series of 512*512-pixel images with a 400*400-|jm field of view recorded at a rate of 1 Hz. A confocal pinhole of 1 Airy unit ensured an axial optical slice resolution of 1.6 |m [17].
Image analysis. Quantitative evaluation of Ca2+ transients was performed off-line using custom-made software in C++ Builder. Cell regions from fluorescent images manually selected. The Ca2+ fluorescence for each cell in each frame calculated as the average fluorescence intensity (relative units from 0 to 255) of the pixels within the defined cell region. Single Ca2+ signals found using the following algorithm. First, averaging two neighboring points in the sample set filtered each trace from all of the cells. Next, we calculated a simple derivative of the signal by calculating a difference between each pair of consequent points. The pulses found from the derivative of the trace using a threshold detection algorithm. The threshold estimated as the detection accuracy coefficient Kdf (Knockdown factor) multiplied by the standard deviation of the derivative of the trace. Suprathreshold points on the derivative of the trace were taken as the beginnings and endings of the pulses. The detection accuracy coefficient Kdf was empirically set to 0.45.
To detect super-oscillations, we filtered the signal with a low-pass elliptic filter (0.2 Hz) that removed any regular short calcium pulses. Then, we calculated the derivative of the filtered signal. Each point of the derivative estimated as an average of the differences of the 20 subsequent pairs of points. This definition allowed us to clearly visualize the super-oscillation beginnings and endings. Next, we applied a threshold detection algorithm to
detect these super-oscillations. The threshold estimated as the super-oscillation detection accuracy coefficient KSdf (Super-oscillation Knockdown factor) multiplied by the standard deviation of the derivative of the trace. All of the time points that crossed the threshold defined as the beginnings and endings of the super-oscillation. The super-oscillation detection accuracy coefficient KSdf was empirically set to 0.8.
Pharmacological agents. Tetrodotoxin (TTX) (1 pM), L-glutamate (5 pM), adenosine 5'-triphosphate (ATP) disodium salt hydrate (5 pM) were purchased from Sigma-Aldrich (USA). The drug solution was prepared immediately before use and perfused into the recording chamber.
Statistical analysis. All data quantification is presented as the mean ± standard error of the mean (SEM), where n — the number of cells. Was performed statistical analysis using a Student's t-test in the program Origin 7.0. The experimental data were normally distributed, and then was conducted a two-sided paired Student's t-test for samples with unequal variances. The difference between groups was considered significant if the p value was less than 0.05.
Cross-correlation analysis. The method for calculation of the cross-correlations used to estimate the degree of synchronization of all pairs of cells. According to this method, two traces removed by the delay time t. Further, the number of synchronous pulses located on both channels in the ranges of tolerance of 25 is calculated. In our case t=0 s, and the interval 5 was taken equal to 1 s. Since the active astrocytes oscillation frequency varies in a small, range from cell to cell, the minimum number of pulses in the compared pair of channels used for normalization (Figure 1). Thus, the formula for calculating the cross-correlation matrix looks like:
Cij=nSynchr/min(n; n)
Calculation and visualization of cross-correlation matrices was performed by using standard Matlab algorithms.
Results
Dynamics of spontaneous Ca2+ oscillations cells in the hippocampal area CA3 in early (P5-8, P14-16) and late (P21-25) postnatal development. Calcium activity of pyramidal neurons, interneurons and astrocytes in the
Network Mechanisms in the Hippocampus CA3 Region in Rat Postnatal Development CTM J 2016 — vol. 8, No.4 169
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Figure 2. Change (a) the amount of Ca2+ oscillations and (b) the duration in cells of the CA3 field of rat hippocampus of early (P5-8) and late (P14-16, P21-25) stages of postnatal development. Student's t-test; * p<0.001, ** p<0.05; n=180
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hippocampal area CA3 in early (P5-8, P14-16) and late (P21-25) postnatal development was monitored using functional Ca2+ imaging. In this study, we investigated such parameters of calcium cells activity as a number of Ca2+ oscillations per minute and duration of Ca2+ oscillations. We have demonstrated that spontaneous Ca2+ activity varied depending on the ages of the rat (Figure 2).
Revealed an increase in the duration and decrease in the number of Ca2+ oscillations of pyramidal neurons and interneurons in the transition from early stage to late postnatal development. Duration of Ca2+ oscillations in astrocytes also increased during postnatal ontogenesis, and frequency change was complex. Thus, when comparing animal's younger age group P5-8 with animals P14-16 there was an increase number of astrocytic Ca2+ oscillations, when compared with a group P21-25 showed a significant decrease to values below the level in animal's younger age groups. It is shown that the number of Ca2+ oscillations and its duration depends on the temperature of the perfusion.
In animals, of older age groups are reduced in amount of Ca2+ oscillations in pyramidal neurons by 75 and 96%, interneurons in 41 and 90% in the age groups P14-16 and P21-25, respectively, when compared with a group of P5-8. The amount of Ca2+ oscillations in astrocytes increased by 84% in the group P14-16 and decreased by 54% in the group P21-25, in comparison with a group of animals of 5-8-day postnatal ontogenesis. In the analysis of another parameter of Ca2+ activity of cells — "duration of Ca2+ oscillations", the opposite found age-related dynamics. Since the duration of Ca2+ oscillations pyramidal neurons and astrocytes significantly increased (47 and 71%, respectively) only in the transition from the early neonatal period of ontogenesis (P5-8, P14-16) to
late (P21-25). A duration Ca2+ oscillations in interneurons increased by 12 and 66% in group P14-16 and P21-25, respectively, in comparing with the group P5-8.
Dynamics of Ca2+ oscillations cells in the hippocampal area CA3 of early (P5-8, P14-16) and late (P21-25) postnatal development at infringement conduction of excitation in the neural network. In order to investigate the role of network factors conduction of excitation by outgrowth of neurons from cell to cell in the formation of spontaneous Ca2+ oscillations in neurons and astrocytes of the CA3 field experiments were carrying out with the blockade of voltage-dependent sodium channel by tetrodotoxin (TTX). In Figure 3 is a histogram showing the change in number of Ca2+ oscillations when added to the perfusion solution of TTX (1 |jM).
When we added to the perfusion solution TTX (1 jM) there was observed reduction in the number of Ca2+ oscillations in pyramidal neurons, astrocytes and interneurons in the hippocampal area CA3 of early (P5-8) postnatal development by the 95, 83 and 38%, respectively. In the age group P14—16 also observed reduction of the number of Ca2+ oscillations, but at 62, 69 and 61% of the pyramidal neurons, interneurons and astrocytes, respectively. In animals, the older age group P21-25, the adding TTX to a perfusion solution not caused significantly changes in Ca2+ activity of the cells. It shown that in the early period of postnatal ontogenesis Ca2+ neuronal activity was completely dependent on the activity of the neural network and dramatically decreased with violations of conduct signal on the network, where neurons connected by electrical synapses. In the study, the structure of neural networks shown that in the early postnatal period there is a strict synchronization of activity of pyramidal neurons and interneurons, due to
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170 CTM J 2016 — v0l. 8, N0.4 Y.I. Mitaeva, A.M. Mozherov, I.A. Kastalskiy, T.A. Mishchenko, I.V. Mukhina
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formation of a stable neural network, subject to excitation by electrical synapses. Astrocytes are not involved in general functional network at this stage of development (Figure 4).
Spontaneous Ca2+ activity of hippocampal cells of animals of middle age group also depended on network activity, which exists due to partial preservation of electrical synapses at this stage of development, so the addition of TTX also caused reduce the amount of Ca2+ oscillations in neurons. Reduced astrocytic Ca2+ activity in the group P14 is a response of astrocytes on the activity of neuronal networks, which proved the neuron-glial interactions with 14 days of postnatal development. 14-16 postnatal day the number of working synchronously neurons decreases. Likely, those electrical synapses remained only on pyramidal neurons while on interneurons they were almost absent (Figure 5).
Further studies carrying out on the effect of t TTX on calcium activity of the cells of the hippocampus of
rats 21-25 days of ontogenesis. Therefore, absence of effect of TTX on spontaneous Ca2+ cell activity in group P21-25, due to the lack of spontaneous activity in the neural network CA3 field of hippocampal slices, in which neurons are connected by chemical synapses. We did not observe changes in Ca2+ activity of astrocytes accordingly (Figure 6).
At the age of 21-25 days of postnatal period is observed fully formed neural network with significant predominance of chemical synapses, the presence of a molecular network of the extracellular matrix of the brain active metabolic system synthesis and degradation of neurotransmitters, neuromodulators, a set of proteins of the transport system, both on the outer cell membrane and internal organelles.
For studying mechanisms of spontaneous Ca2+ oscillations in neurons and astrocytes of mature network, we have carried out experiments with the addition of excitatory neurotransmitters — L-glutamate and ATP.
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In order to prove the role of neural networks in the formation of spontaneous Ca2+ oscillations in the cells of the CA3 field of hippocampal slices of different age groups, we performed experiments with the blockade of excitation in the neural networks with the help of a blocker of voltage-dependent sodium channel TTX (1 |M). We have shown that in the early neonatal period, of postnatal ontogenesis Ca2+ activity of the neurons was completely dependent on the activity of neural networks and abruptly decreased with violations of the signal of a neural network in which the neurons connected by electrical synapses. Spontaneous Ca2+ activity of the cells of the hippocampus of animals the middle age group (P14-16) also dependent on network activity, the current due to partial preservation of electrical synapses at this stage of development, so the addition of TTX also reduces the amount of Ca2+ oscillations in neurons. Decrease of astrocytic Ca2+ activity in the group P14-16 is a response of astrocytes on the activity of neuronal networks that proves the presence of neuron-glia interactions with 14 days of postnatal development. Lack of effect of TTX on spontaneous Ca2+ activity of the cells of P21-25, due to the lack of spontaneous activity of the neural network in the CA3 field of hippocampal slices in which neurons connected by chemical synapses. Consequently, we did not observe changes in astrocytic Ca2+ activity. Also has been studied the structure of neural networks, whereby it was shown that in the early neonatal period of postnatal ontogenesis there is a strict synchronization of activity of pyramidal neurons and interneurons by forming a stable neural network subject to excitation through electrical synapses. Astrocytes at this stage of development of common functional network are not involved. In a mature network, violation of excitation of axons almost had no effect on Ca2+ activity of the cells, as a spontaneous network activity in a slice missing.
Further research has focused on the Ca2+ activity of the cells of the CA3 field of hippocampal slices of rats late (P21-25) neonatal period of postnatal ontogenesis. To study the mechanism of spontaneous Ca2+ oscillations in neurons and astrocytes mature network experiments carried out with the addition of excitatory neurotransmitters — ATP and L-glutamate. In rest in the animal hippocampus extracellular glutamate levels ranging from 1 to 2 |M, and the ATP level of 100 nM. A concentration of these substances in 20 |M considered half maximal effective concentration, and can be observed with increased activity of the brain, for example during a research of animal behavior. This study allowed us to estimate the dependence of Ca2+ activity in the of cell of CA3 field of rat hippocampus of the late (P21-25) postnatal period from metabolic state of cells associated with increased release of neurotransmitters into the synaptic cleft. Furthermore, it was shown that under the conditions of a mature network when spontaneous Ca2+ cell activity is low while maintaining the conduction of excitation by adding the neural network of excitatory neurotransmitters cause strict synchronization of cell
activity. In the papers [4, 10, 11, 13] aimed at studying the neural networks in the hippocampus was also shown that about 60% of the neurons responded to sensory irritation, due to feature in the structure of the hippocampus.
The study showed that changes in Ca2+ activity in the cells CA3 field of rat hippocampal taking place during the neonatal period of postnatal ontogenesis directly related to the functioning of neural networks and the metabolic state of the cells. Ca2+ signaling in the developing brain is a complex multi-component process involving various receptor systems capable of mutual substitution in case of violation the normal functioning of one or more of them.
Study of Ca2+ signaling brain cells refers to a number of key problems of fundamental neuroscience, as the processes of cell signaling and neuron-glial interactions play a decisive role in the functioning of the nervous system.
Acknowledgments. The research was supported by the Federal Target Program "Research and development in priority areas of the development of the scientific and technological complex of Russia for 2014-2020" of the Ministry of Education and Science of Russia (Project ID RFMEFI60715X0117).
Conflicts of Interest. The authors have no conflicts of interest to disclose.
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176 CTM J 2016 — vol. 8, No.4 Y.I. Mitaeva, A.M. Mozherov, IA Kastalskiy, T.A. Mishchenko, I.V. Mukhina
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