ISSN 00124966, Doklady Biological Sciences, 2014, Vol. 456, pp. 160–164. © Pleiades Publishing, Ltd., 2014. Original Russian Text © A.A. Kolacheva, E.A. Kozina, E.V. Volina, M.V. Ugryumov, 2014, published in Doklady Akademii Nauk, 2014, Vol. 456, No. 2, pp. 246–250.
PHYSIOLOGY
Time Course of Degeneration of Dopaminergic Neurons and Respective Compensatory Processes in the Nigrostriatal System in Mice A. A. Kolacheva, E. A. Kozina, E. V. Volina, and Academician M. V. Ugryumov Received January 27, 2014
DOI: 10.1134/S0012496614030041
Neurons are known to die continuously during the whole life of a human or animals [1]. However, until advanced age or even death, this neuronal loss does not influence negatively the functioning of the brain, including its involvement in neural or neuroendocrine regulation of the most important body functions. Even an increase in the rate of demise of specific neurons induced by unknown reasons in neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease (PD), and others, does not impair brain func tions, such as cognitive functions in Alzheimer’s dis ease or motor functions in PD, for several decades [1]. This may be explained by compensation of functional insufficiency caused by neuronal loss related to the mechanisms of brain plasticity. However, at a certain threshold level of neuronal degeneration, compensa tory capabilities of the brain become insufficient or exhausted, which results in impairments of specific body functions [2]. From the viewpoint of basic neu rophysiology, it is important to understand the tempo ral and functional relationships between two linked processes, neurodegeneration and compensation of functions of dead neurons, which continuously occur in the brain. This basic knowledge is very important for medicine too, because it may allow us to understand the mechanisms of regulation of both processes and to cope with them using prospective drugs. The aim of the present study was to examine the development of the linked processes of degeneration of dopaminergic (DAergic) neurons after treatment with specific neurotoxin 1methyl4phenyl1, 2, 3, 6tetrahydropyridine (MPTP) and compensation expressed in the features of the functional conditions of surviving DAergic neurons. Using a recently devel oped mouse model of the early clinical stage of PD [3], Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, 117808 Russia Anokhin Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow, 103009 Russia email:
[email protected]
we expected to study the time course and rate of degeneration of DAergic neurons after treatment with the specific neurotoxin and the compensatory changes in the functional activity of surviving DA ergic neurons. Two to threemonthold C57BL/6JSto mice weighting 22–26 g were used for the study. The model of the early clinical stage of PD was obtained by means of four subcutaneous injections of MPTP (Sigma, United States) at a dose of 12 mg/kg at intervals of 2 h between injections. The animals of the control group were injected with 0.9% NaCl according to the same scheme. For immunocytochemical study of tyrosine hydroxylase (TH), a marker of DAergic neurons, mice were anesthetized with chloral hydrate (Sigma, United States) at a dose of 12.5 mg/kg 3, 6, 12, and 24 h after the last injection of MPTP, perfused with a phosphatebuffered saline solution (PBS; pH 7.2–7.4) and 4% paraformaldehyde in 0.2 M phosphate buffer for 15 min, and decapitated. Brain was postfixed in the same fixative for 12 h at 4°C, washed in 0.02 M PBS, placed into 20% sucrose solution for 48 h, frozen in hexane at –40°C, and stored at –70°C until use. Frontal serial 20µm sections through the substan tia nigra (SN) from bregma 2.54 to bregma 4.04 and frontal 12µm sections of the striatum from bregma 1.70 to bregma 0.14 were prepared using a cryostat (Leica, Germany). Each section of the substantia nigra and one of three sections of the striatum were mounted on slides. The sections were incubated with rabbit antiTH antibodies (kindly provided by J. Thibaut, France) and goat antirabbit biotinylated antibodies at dilutions of 1 : 2000 and 1 : 200, respec tively, and peroxidase conjugated avidin–biotin com plex (Vector Laboratories, United States) as described previously [3]. Peroxidase of the avidin–biotin complex was detected by incubating the sections with 0.05% 3,3' diaminodenzidinetetrahydrochloride (Sigma, United States) and 0.02% H2O2 in PBS. Color devel
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opment was performed in all slides simultaneously under visual control. The sections were coverslipped in Mowiol 488 (Sigma, United States) and studied using an Olympus BX51 light microscope (Olympus, Japan). THimmunoreactive (THIR) neuronal bod ies with visible nuclei were counted in the serial sec tions of the SN pars compacta in its rostrocaudal extension. Optical density (OD) of neurons, correlat ing with TH content, was calculated as log ( level of grey background ) OD = . log ( level of grey signal ) The count of the THIR axonal terminals was per formed in the dorsal striatum in four randomly chosen optical fields of 1752 × 1318 µm2 using the Striatum Image Analysis software (Russia), which was created by us in collaboration with Dorodnitsyn Computing Center, Russian Academy of Sciences [4]. The OD of axonal terminals was calculated according to the equation presented above. The data of quantitative analysis are presented as percent of changes in the number of bodies and density of distribution of the axonal terminals in experimental animals as compared eith the control values, which were taken to be 100%. The contents of DA and its metabolite 3,4dihy droxyphenylacetyc acid (DOPAC) were measured using high performance liquid chromatography with electrochemical detection. For this purpose, mice were anesthetized with chloral hydrate at a dose of 12.5 mg/kg 3, 6, 12, and 24 h after the last injection of MPTP and decapitated. The brain was taken out and dissected along the midsagittal plane. The striatum and SN were dissected from the left hemisphere according to the atlas [5]. The samples were weighed, frozen in liquid nitrogen, and stored at –70°C. Prior to measurement of DA and DOPAC, the samples of the stiratum and SN were thawed and homogenized in 70 and 150 µL of 0.1 N HClO4, respectively, with addi tion of 250 pmol/mL of 3,4dihydroxybenzylamine as an internal standard. The experimental procedure and data analysis were described previously [3]. The data of measurement of DA and DOPAC are presented as percent of changes in experimental animals as compared to the control values, which were taken to be 100%. First, we studied the time course of degeneration of DAergic neurons in a model of the early preclinical stage of PD and analyzed the rate of degeneration of DAergic neurons on the levels of neuronal bodies and their axonal terminals. To date, direct quantitative methods of estimation of degenerating neurons are absent; therefore, we used a reversed index, i.e., the count of DAergic neurons that “survived” MPTP administration compared to the control value. A marker of DAergic neurons is TH, a ratelimiting enzyme of DA synthesis equally distributed around neuron. Complete coincidence of the number of TH stained and Nisslstained neurons in the control and DOKLADY BIOLOGICAL SCIENCES
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after MPTP administration supports the efficiency of this approach [6]. Degeneration of bodies of DAergic neurons started 3 h after the last MPTP injection and contin ued for 3 h (Fig. 1). These data poorly agree with the previously published study [7], where the time course of degeneration of DAergic neuronal bodies in the SN was estimated in C57BL/6 mice after four admin istrations of a higher dose of MPTP, 20 mg/kg, at 2h intervals. The authors of the study [7] have reported that the degeneration of DAergic neurons started 12 h after the last MPTP injection, i.e., later compared to our experiment, and continued longer, i.e., up to 36 h after the treatment. This discrepancy may be explained by the use of different methods of counting of the total number of neurons in the SN. In our study, we estimated the total number of neurons in the SN as the sum of neurons counted in all serial sections in the rostrocaudal extension of the SN, whereas Jackson Lewis et al. [7] used only selected sections and did not take into account unequal distribution of the bodies of DAergic neurons along the rostrocaudal extension in normal and MPTPtreated animals [8]. In PD, degeneration of DAergic neurons is assumed to start from the axonal terminals in the stri atum and to expand in a retrograde direction to the neuronal bodies [9]. In spite of the key role of the axonal terminals in DAergic neurotransmission in the striatum, there are only a few studies where the axonal terminals were counted in the striatum using experimental models of PD [10]. Moreover, none of these studies reported the time course of the degener ation of the axonal terminals after MPTP treatment. This is an advantage of our study because we per formed this analysis. Here, we found that 3 h after the last MPTP injection, the number of the terminals of DAergic axons in the striatum decreased down to 66% of their control value. In contrast to the neuronal bodies, the axonal terminals are more sensitive to the MPTP effect because their membrane contains more the DA transporters providing the entrance of the toxin into neurons compared to the membrane of the bodies of DAergic neurons. Degradation of the axonal terminals continues for another 3 h, and their number additionally decreases by 10% at this time (Fig. 1). If we suppose that the degeneration rate is sta ble, then the degradation of the axonal terminals should start immediately after the first MPTP injec tion, which closely corresponds to our previous study where we have demonstrated a decreased number of the axonal terminals immediately after a single MPTP administration at a dose of 12 mg/kg [3]. We did not observe any changes in the numbers of neuronal bod ies in the SN or axonal terminals in the striatum 6, 12, or 24 h after the last MPTP injection. In addition to the estimation of the numbers of cell bodies and axonal terminals of nigrostriatal DAergic
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Fig. 1. Tyrosine hydroxylase immunoreactivity (THIR) in the substantia nigra (SN) and dorsal striatum 3, 6, 12, and 24 h after the last injection of MPTP (1methyl1, 2, 3, 6tetrahydropyridine). Data are presented as percent of the control level (0.9% NaCl) taken to be 100%. 1, number of THIR neurons in the SN; 2, density of THIR axonal terminals, calculated as number of terminals per unit of section area, in the dorsal striatum; 3, * p < 0.05, significant differences compared to the control level; 4, # p < 0.05, significant differences between experimental groups.
Fig. 2. Tyrosine hydroxylase immunoreactivity (THIR) in the substantia nigra (SN) and dorsal striatum 3, 6, 12, and 24 h after the last injection of MPTP (1methyl1, 2, 3, 6tetrahydropyridine). Data are presented as percent of the control level (0.9% NaCl) taken to be 100%. 1, optical density (OD) of THIR neuronal bodies in the SN; 2, OD of THIR axonal terminals, cal culated as number of terminals per unit of section area, in the dorsal striatum. The other indications see in Fig. 1.
neurons, we studied the functional conditions of “sur viving” DAergic neurons. We used the intracellular contents of TH, DA, and the product of DA degrada tion, DOPAC, as indices of the functional activity of neurons. The intracellular TH content was measured using OD of the THIR material, whereas the intrac ellular contents of DA and DOPAC were indirectly assayed by dividing the DA and DOPAC contents in the SN and striatum by the number of cell bodies and axonal terminals in these structures, respectively (Figs. 2–4). The DA contents in neuronal bodies decreased by 70%, whereas the TH level remained to be stable 3 h after the last MPTP injection. This was probably due to the lower TH activity after MPTP treatment [11], which was followed by a decreased rate of DA synthe sis. On the other hand, this decrease in the DA content may be related to the development of one of compen satory processes of their combination. As a rule, the compensatory processes include an increased rate of
DA release and inhibition of DA reuptake [2]. The ele vated enzymatic DA cleavage, an additional method of protection from the toxic effect of cytosolic DA, could also have decreased the DA content in neuronal bod ies. However, this was not the case, because the DA and DOPAC contents were similar 3 h after the last MPTP injection. For estimation of the functional conditions of DA ergic neurons, the assay of the functional conditions of the axonal terminals forming synaptic contacts with target neurons is very important. We observed an approximately 80% decrease in the DA content in the axonal terminals 3 h after the last MPTP injection. In contrast to neuronal bodies, the TH content in the axonal terminals was decreased approximately by 25%, probably, due to the impairment of axoplasmic transport. In the present and our previous studies, indirect evidence for impaired axonal transport induced by MPTP was presented; however, what is even more important, impaired axonal transport is DOKLADY BIOLOGICAL SCIENCES
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Fig. 3. Contents of dopamine (DA) in the substantia nigra (SN) and striatum 3, 6, 12, and 24 h after the last injection of MPTP. Data are presented as percent of the control level (0.9% NaCl) taken to be 100%. 1, body of DAergic neuron; 2, axonal terminal of the dorsal striatum. The other indications see in Fig. 1.
Fig. 4. Contents of 3,4dihydroxyphenylacetic acid (DOPAC) in the substantia nigra (SN) and striatum 3, 6, 12, and 24 h after the last injection of MPTP. Data are presented as percent of the control level (0.9% NaCl) taken to be 100%. 1, body of DAergic neuron; 2, axonal terminal of the dorsal striatum. The other indications see in Fig. 1.
specific for PD patients [12]. On the other hand, the decrease in the DA content in axonal terminals was more expressed compared to the decrease in the TH content. These data show a possible increase in the rate of DA release, a decrease in its reuptake or lower activity of TH after MPTP treatment [2]. Similar to neuronal bodies, elevated enzymatic cleavage of DA in axonal terminals can be excluded, because the con tents of DA and DOPAC are equally declined in axonal terminals. The degeneration of the bodies of DAergic neu rons in the SN 3–6 h after the last MPTP injection was associated with an increased DA content in surviving neurons, whereas the contents of TH and DOPAC remained unchanged. The increased DA level was probably due to recovery of TH activity compared to the control value and/or the progressive development of the compensatory mechanisms. Similar processes were observed in the striatum, where the TH content decreased in axonal terminals, whereas the DA level remained unchanged 6 h after the treatment, which indicates an elevated activity of the enzyme. On the other hand, the decreased TH content in axonal ter DOKLADY BIOLOGICAL SCIENCES
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minals 6 after MPTP administration compared to that 3 h after it demonstrates development of MPTP induced impairment in axoplasmic transport. Twelve hours after the last MPTP injection, we observed an uneven increase in the DA content in neu ronal bodies of the SN compared to that found 6 h after the injection and an insignificant trend towards DOPAC accumulation in some neuronal bodies. These changes were accompanied by a stable TH level, which was similar to the control value. These data indicate an additional compensatory growth of the TH activity and elevated DA synthesis in the bodies of sur viving DAergic neurons. In the axonal terminals, we did not observe any changes in these indices 6–12 h after the last MPTP injection. The content of DOPAC in some bodies of DA ergic neurons of the SN increased almost by half 12– 24 h after the last MPTP injection, whereas the TH and DA contents did not change. The increased DOPAC level associated with a stable DA content demonstrates a higher activity of the enzymes of DA degradation, first of all, monoamine oxidase A, the key enzyme of DA cleavage in neurons of rodents [13].
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In this time period, in the axonal terminals, we observed an 80% increase in the TH content only. This suggests a recovery of axoplasmic transport in DA ergic neurons. On the other hand, the increased TH level in the axonal terminals did not result in elevation of the DA and DOPAC contents, probably, due to a decrease in TH activity and/or the increased rate of DA release and the lower rate of DA reuptake into axonal terminals in the striatum. We plan to clarify this problem in a future study. Thus, in a mouse model of the early clinical stage of PD, the degeneration of DAergic nifrostriatal neu rons stops 6 h after the last injection of the specific neurotoxin. This is accompanied by initiation of com pensatory processes aimed at improving DAergic neurotransmission in the striatum.
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ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research (project nos. 110412121ofi m2011 and 130040375K KOMFI) and the Pro gram of the Presidium of the Russian Academy of Sci ences “Fundamental Science for Medicine.”
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Translated by M. Stepanichev
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