All the initial values of the differential equations were taken as zero. All parametric and steady-state values are given in Supplementary Table 3. We developed a comprehensive model of SNc neuron, which exhibits characteristic ionic dynamics Fig. The model also exhibits energy consumption by different cellular processes Fig. Oscillations in intracellular molecular concentrations in relation to the oscillations of the membrane potential.
Then, we studied the effect of electrical Fig. Finally, we showed model responses to energy deficiency conditions Figs. Model response to electrical stimulation.
Frequency of firing A and Energy consumption C by ionic pumps blue trace and all other whole cellular processes orange trace of the model concerning the amplitude of stimulating depolarized current 1 s , Extracellular dopamine concentration B and Energy consumption D by ionic pumps blue trace and all other whole cellular processes orange trace of the model concerning the frequency of stimulating depolarized current 2 secs.
Model response to chemical stimulation glutamate. Frequency of firing A , Apoptosis signal C due to excess stimulation, Energy consumption by ionic pumps B and all other whole cellular processes D of the model concerning the concentration of glutamate application 1 s. Model response to hypoglycemia and hypoxia conditions. Responses of whole A—D and reduced E—H models to hypoglycemia and hypoxia conditions.
Average normal alpha-synuclein asyn concentration A , E , average misfolded alpha-synuclein asyn mis concentration B , F , average aggregated alpha-synuclein asyn agg concentration C , G , and average reactive oxygen species ROS concentration D , H of the fast and slow dynamic models for varying glucose and oxygen concentrations. The bursting type of firing also observed in the proposed model with a different range of synaptic inputs 19 not shown here.
The ionic flux concentrations, which drive membrane potential, were in the range of values used in previous models 13 , In general, the calcium concentration in the MT will be lesser than the cytoplasm, but due to the higher mitochondrial density 4 and higher calcium loading in the SNc cells 38 , 39 , the SNc mitochondrial calcium concentration was much higher than other cells Fig.
Accompanying slow calcium buffering mechanisms, calcium-binding proteins such as calbindin and calmodulin act as rapid calcium buffering mechanisms 40 mobile calcium buffers , which are present near calcium hotspots and bind rapidly to excess cytoplasmic calcium Fig. The link between membrane potential, which was driven by the exchange of ionic concentrations, and extracellular release of DA, which was driven by that membrane potential was described in Tello-Bravo model of DA neuron 9 Fig.
Active pumps and exchangers maintained the ionic equilibrium across the cell membrane, where ATP drives the sodium—potassium and calcium pumps. Utilizing glucose and oxygen, ATP was produced in the cell through stages of processes such as glycolysis and oxidative phosphorylation Fig. Other intermediate metabolites in the energy metabolism were in the range similar to Cloutier et al.
The energy consumption in the SNc neuron by different cellular processes, namely action potential propagation, vesicle recycling, DA packing, ER calcium sequestration, and protein degradation was estimated using the proposed model Fig. In order to study the effect of increased electrical stimulation on firing frequency and DA release, electrical stimulation was carried on the proposed SNc neuronal model. The consumed ATP by ionic pumps, and all other cellular processes increased with increased frequency of stimulation Fig.
In order to study the effect of glutamate application on the different properties such as firing frequency, energy consumption, and apoptotic signal, chemical stimulation was carried on the proposed SNc neuronal model. By introducing energy deficiency in the form of hypoxia and hypoglycemia, we now studied the effect of hypoglycemia and hypoxia on the various critical molecular players in the SNc neuron.
The energy deficiency conditions were implemented by varying glucose and oxygen levels in the proposed comprehensive model of SNc. The firing frequency of the model decreases Fig. The average cytoplasmic calcium concentration was higher, which might be due to the reduced outward flux of calcium by active calcium pump and sodium-calcium exchangers as a result of lesser ATP availability at higher extent of hypoglycemia and hypoxia conditions Fig.
The average ER and mitochondrial calcium concentrations were low, which might be due to reduced sequestration of calcium into ER and MT, which in turn happens due to lesser ATP availability under more severe hypoglycemia and hypoxia conditions Fig. The average cytoplasmic DA concentration was higher, which might be due to reduced DA packing into the vesicles as a result of lesser ATP availability under more severe hypoglycemia and hypoxia conditions Fig.
The average extracellular and vesicular DA concentrations were low, which might be due to reduced readily releasable vesicle pool as a result of lesser ATP availability, which might affect the DA packing into the vesicles under more severe hypoglycemia and hypoxia conditions Fig. The average F26P accumulation was higher during high glucose and low oxygen, which was an integrator of metabolic stress 17 Fig.
In the whole fast dynamics model simulation, the healthy alpha-synuclein protein asyn was misfolded, and the available healthy alpha-synuclein protein was low at low glucose and low oxygen Fig. Under low glucose and low oxygen conditions, the accumulation of misfolded alpha-synuclein asyn mis and alpha-synuclein aggregates asyn agg was higher due to lesser ATP availability, which leads to reduced proteolysis or protein degradation Fig.
The average ROS concentration was increased at low glucose and low oxygen levels due to misfolded alpha-synuclein, thereby inducing further release of ROS by hindering mitochondrial functioning Fig. For a better representation of molecular markers under pathological conditions, the reduced slow dynamics model was simulated, which was obtained by assuming fast substrates reaching their steady states rapidly, and associated differential equations were transformed into functions that is, at steady-state values.
The average normal alpha-synuclein concentration decreases with a decrease in glucose and oxygen levels due to increased ROS-induced misfolding of alpha-synuclein Fig. Due to higher ROS concentration, alpha-synuclein misfolding and aggregation were prominent, and the concentrations are reaching values similar to high-stress conditions 10 Fig. The central objective of this computational study is to show that metabolic deficiency is the root cause that connects various molecular level pathological manifestations of PD in SNc cells.
More importantly, we want to investigate the hypothesis that metabolic deficit is perhaps the root cause of SNc cell loss in PD. The proposed model is one of its kind, which explains how deficits in supply of energy substrates glucose and oxygen can lead to the pathological molecular changes, including alpha-synuclein aggregation, ROS production, calcium elevation, and DA deficiency.
The proposed model is compared to other models, that at least had more than one cellular process modeled together Table 1. The proposed model with its biophysical framework shows four regimes of ATP dynamics as a function of glucose and oxygen levels: A Unperturbed no change in Basal ATP Concentration BAC , B adaptation initial drop and a subsequent return to initial BAC 52 , C no adaptation initial drop and stabilized at a lower BAC, however, generally astrocytes and other energy sources glycogen, glutamine will restore ATP levels 53 , and D oscillating BAC fluctuates, where anaerobic respiration might occur 54 and other regimes in which neuron undergoes degeneration Fig.
The model also suggests that hypoglycemia plays a more crucial role in leading to ATP deficits than hypoxia Fig. Model responses to hypoglycemia and hypoxia conditions. A Different regimes of the model response to hypoglycemia and hypoxia conditions, B average ATP concentration for different initial glucose concentration concerning oxygen concentration. In PD, energy deficiency occurs in a targeted fashion over a long period of time, which first affects the most vulnerable neurons and spreads to less vulnerable neurons in the brain.
So, when compared to the glutamatergic neurons, SNc neurons are one of the most vulnerable and energy-consuming neuronal clusters, due to their structural and functional properties 57 such as complex axonal arborization 4 , 5 , pacemaking ion channels 58 auto-rhythmicity , presence of reactive neuromodulator 59 dopamine , excitotoxicity 3 , 7 , calcium loading and higher basal metabolic rates associated with chronically elevated ROS production 4.
Taking out all these plausible factors, SNc cells prone to be the most susceptible to energy deficiency. Under energy deficit conditions, SNc neurons undergo apoptosis due to overexcitation with even physiological concentrations of glutamate when compared to normal conditions 52 not shown here.
We suggest that the excitotoxic loss of SNc neurons in PD might be precipitated by energy deficiency 3. Any therapeutic interventions that can reduce ionic flux through these glutamatergic receptors or enhance energy production can be neuroprotective in nature 62 , 63 , PD can be caused due to damage to glutaminergic neurons as a result of energy deficiency which is caused by ischemic stroke. However, PD is a slowly evolving disease unlike sudden ischemic stroke which leads to a sudden drop in energy substrates.
In PD, energy deficiency occurs in a targeted fashion over a long period of time which first affects the most vulnerable neurons and spreads to less vulnerable neurons in the brain. So, when compared to the glutamatergic neurons, SNc neurons are one of the most vulnerable and energy consuming neuronal clusters, due to their structural and functional properties. We list out some of the plausible factors which make SNc cells to be most susceptible.
Complex axonal arbors Large axonal arborisation that requires large amounts of energy to drive currents along these axons 5 , 65 ,. Reactive neurotransmitter When a reactive neurotransmitter like DA is present in excess, it would readily oxidize with proteins, nucleic acids and lipids 38 eventually leading to neurodegeneration. NMDA synaptic activation Due to pacemaker type of firing, magnesium blockage of NMDA receptors is ineffective, resulting in substantial NMDA receptor currents even with weak glutamatergic inputs resulting in additional burden to maintain calcium homeostasis; the resulting energy deficiency leads to excitotoxicity 39 , 69 ,.
Prone to neuroinflammation Astrocytes play a modulatory role in microglial activation 70 , 71 , 72 and any miscommunication between them results in neuroinflammation which eventually leads to neurodegeneration 73 , The risk of inflammation in SNc neurons is high due to the small proportion of astrocytes regulating the huge population of microglia in this region 75 , It has been reported that neuromelanin can induce microglial activation 77 , SNc neurons are more susceptible to neuro-melanin induced inflammation compared to VTA neurons due to their high neuro-melanin biosynthesis as a result of underexpression of VMAT-2 Weak microvasculature SNc neurons are more prone to environmental toxins due to weak surrounding cerebral microvasculature Since the metabolic demands of SNc neurons are particularly high when compared to any other neuronal types 38 including neurons of other dopaminergic systems 4 , 5 , 80 , any sustained insufficiency in the supply of energy can result in cellular degeneration, characteristic of PD The effect of glutamate released from glutaminergic neurons onto SNc neurons can be considered as toxic, in addition to its regular action of neurotransmission during energy deficit conditions 82 , As a result, a slightly increased glutamate stimulation can create a calcium storm in SNc neurons However, under energy deficit conditions, even physiological levels of glutamate are toxic as a result of increased intracellular calcium concentration, which leads to oxidative stress through a mechanism known as indirect excitotoxicity or weak excitotoxicity It was reported that the glutamatergic excitation of SNc neurons by STN neurons 85 under the conditions of bioenergetic deficiency might lead to aggravation of degeneration processes 3 , 52 , In genetics, the phenotype of an organism depends on the underlying genotype Similarly, the occurrence of different phenotypes of a disease can be driven by underlying dysfunctions occurring at different levels in the hierarchy, such as molecular, cellular, and systems levels 87 , In PD, the loss of dopaminergic neurons in SNc results in the manifestation of PD symptoms, and the cause of the SNc cell loss is still not clearly elucidated.
The PD phenotypes are distinct, and this specificity might be arising out of a combination of interactions between key determinants at the same or different levels.
The dysfunction causing interactions among different molecular determinants 91 , 92 , 93 was elaborated in Fig. Interactions among the determinants at different levels of hierarchy. See Box-1 for description of the Figure.
At the cellular level, the determinants that might contribute to differential PD phenotypes are complex morphology 4 , 5 , 65 due to large axonal arborization and numerous synaptic connectivity , lesser mitochondrial mass 94 , 95 due to higher level of mitochondrial DNA deletions , high levels of reactive cytosolic DA 66 , 96 , 97 due to underexpression of vesicular monoamine transporter 2 and overexpression of DA transporter , distinctive electrophysiology 98 , 99 , due to broad spikes and pacemaking activity , calcium loading 97 , , due to presence of Ca v 1.
These cellular determinants individually or collectively would result in higher basal metabolic rate and increased oxidative stress 4 , which in turn converges to common pathologies Fig. At the systems level, the determinants that might contribute to differential PD phenotypes are excitotoxicity 3 , due to overexcitation by STN or pedunculopontine nucleus , aging , due to proteostatic dysfunction, mitochondrial dysfunction, genetic mutations or telomere shortening , genetic instability , , due to changes in nucleic acid sequences, chromosomal rearrangements or aneuploidy , environmental toxins , due to exposure to insecticides, commercial solvents, metal exposure or traumatic head injury , neuroinflammation , due to traumatic head injury, exotoxins or immune dysfunctions , prion-like infection , bacteria or viruses , telomere shortening , due to aging or oxidative stress , glial dysfunction , , due to phagocytic or inflammatory impairments, enteric glial dysfunction and vascular dysfunction , due to endothelial dysfunction or cardiovascular autonomic dysfunction.
These systems-level determinants interact among themselves and also across different levels in the hierarchy resulting in different PD phenotypes Fig. Dysfunctions at any level of hierarchy would make SNc cells move from normal state to pathological state directly or indirectly via an intermediate vulnerable state Fig. Any therapeutics that can bring back SNc neurons from a pathological or vulnerable state to normal state can be beneficiary for the survival of SNc neurons.
In Pavlin et al. Firstly, the insoluble amyloid plaques prevent the vesicular transport functioning which leads to progressive neurodegeneration. These amyloid plaques are formed as a result of enhanced interaction between alpha-synuclein and oxidized heavy atom ions increased ROS oxidizes heavy atom ions.
Secondly, the dyshomeostasis occurring due to loss of lipid bilayer membrane permeability of the mitochondrial wall or cellular membrane leads loss of electronic gradients in turn resulting in loss of resting potential and neurodegeneration.
The membrane permeability is disturbed due to increased interaction between ROS and methylene groups of lipid bilayer. In the present study, the ROS formation is contributed by respiratory chain complexes Eq.
The ROS is scavenged by catalase Eq. Apart from these factors, the ROS formation is contributed by DA metabolized by MAO B enzyme, heavy metal ions and inflammatory responses late stages of the disease also need to be considered. However, it should be mentioned that there are several factors that aggravate or mitigate the effect of ROS, incorporating all these factors will increase the complexity of the model whereas our main focus was to study the effect of energy deficiency on the major molecular players such as calcium, DA, ATP and the membrane voltage Supplementary Fig.
The interaction among the various important players such as calcium, DA, spike frequency and ATP was illustrated and along with both positive and negative feedback loops in the Supplementary Fig. In normal conditions, ATP maintains low levels of calcium in the cytoplasm by efflux of excess calcium into the extracellular space and sequestrating the excess calcium into the endoplasmic reticulum.
As cytoplasmic calcium increases, the extracellular DA release also increases. However, on continued release of extracellular DA, the cytoplasmic calcium subsequently decreases by the feedback, regulatory action of DA via DA autoreceptors The deficiency in the supply of energy substrates results in reduced levels of ATP which in turn affects the homeostasis of cytoplasmic calcium and the amount of extracellular DA released. As ATP decreases, extracellular DA released also decreases as a result of reduced packing of DA into the vesicles in turn leading to excess DA build up in cytoplasm which eventually results in oxidative stress.
As we started to develop the proposed model, we looked into several factors that contribute to neurodegeneration and we tried to incorporate the primary factors that affect the neuronal survivability. We agree that the missing factors which contribute towards ROS formation should be incorporated in the future studies which we believe will enhance the scope of the model. Several researchers suggested that L-DOPA might be harmful to SNc cells by a mechanism that probably involves oxidative stress , , However, several others proposed that L-DOPA might not accentuate neurodegeneration of SNc neurons , , and sometimes acts a neuroprotective agent , , or promote recovery of dopaminergic markers in the striatum , After several studies, it is still not clear whether L-DOPA is toxic , , , , , Therefore, in order to have the same symptom-relieving effect, the dosage of L-DOPA needs to be increased.
It has been suggest that adjunct therapies such as antioxidants , , , , and other potential therapies such as D2 agonists , glycogen synthase kinase 3 inhibitors , calcium-binding protein drugs , etc.
Thus, the beneficial or toxic effects of L-DOPA needs to be investigated with more thorough experiments performed at preclinical and clinical levels. Synaptic transmission requires the presynaptic release of neurotransmitter from synaptic vesicles SVs onto the postsynaptic neuron.
Vesicular neurotransmitter transporter proteins, which use a V-ATPase-generated proton gradient, play a crucial role in packaging neurotransmitter into SVs. Acidification of SVs represents a critical point during the SV cycle: without acidification, neurotransmitters cannot be loaded into SVs , Excess cytoplasmic DA undergoes non-enzymatic autoxidative reaction as pH value in the cytoplasm is about 7 giving rise to a superoxide anion that further decomposes to reactive oxygen species result in oxidative stress , The pacemaking type of behavior is necessary to maintain a constant DA level to their innervating regions, such as striatum in case of SNc.
Tonic and phasic signaling are both required for the execution of motivated behaviors and work together to reinforce advantageous outcomes while reducing disadvantageous behaviors. DA levels in the synaptic gap, cytoplasm and extracellular space increases as a result of VMAT-2 inhibition by amphetamine or reserpine and DAT inhibition by amphetamine or cocaine Both cocaine and amphetamine acutely elevate tonic DA levels, but result in reduced basal extracellular DA levels as measured by microdialysis 18 h following extended access self-administration, possibly as a compensatory response to chronic drug-induced DA elevations One possible mechanism for reductions in basal DA levels following cocaine or amphetamine self-administration is that increased synaptic DA levels, due to drug-induced inhibition of uptake, are subject to enzymatic degradation rather than repacking into vesicles, thus, release may be reduced, and be made more dependent on DA synthesis.
In support of this hypothesis, it has been observed that reductions in electrically evoked DA release following extended access cocaine self-administration, suggesting that intracellular DA levels are reduced In contrast, following extended access amphetamine self-administration, intracellular and extracellular DA levels appear to be differently affected, whereby extracellular levels are decreased and electrically evoked DA release is increased.
Both cocaine and amphetamine acutely augment the amplitude and frequency of phasic DA signals which likely results in enhanced phasic DA responses to environmental stimuli when cocaine or amphetamine are on board MAO is an enzyme found everywhere in the body inside the cells.
Rasagiline is about 10 times more potent in the inhibition of MAO-B than selegiline as demonstrated , This higher potency of rasagiline is corrected in the clinic with dose adjustments approved daily dose 1 and 5—10 mg for rasagiline and selegiline, respectively In the proposed model, DA metabolism by MAO-B in synaptic bouton and extracellular space was simplified where the excess cytoplasmic DA after packing into vesicles is metabolized see Eq.
In the proposed model, the inhibitory effect of selegiline and rasagiline can be implemented by regulating the kinetic rate constants of Eqs. However, to understand the potency of rasagiline over selegiline, we should be formulating the metabolism of DA in greater detail which can be incorporated in the future studies so the differential effect of MAO inhibitors can be elucidated.
The dopaminergic synapse which was proposed by Best et al. However, there are some factors which gives the dopaminergic synapse in the proposed model an edge over the dopaminergic synapse proposed by Best et al. The following aspects of the dopaminergic synapse in the proposed model are listed below:. When an action potential arrives at the nerve terminal, it induces membrane depolarization, causing the opening of voltage-gated ion channels.
The probability of release of DA storage vesicle in response to the nerve impulse depends on the conductance of calcium through N-type channels into the active zone Assuming that intracellular calcium concentration transients are identical at all DA release sites, we model intracellular calcium in the proposed as described in Eq. Following the ideas in Lee et al. Hence, the dopaminergic synapse in the proposed model was effortlessly integrated to dopaminergic soma model.
In this integrated model, calcium oscillations in the soma are driven by ion channel activity, that modulates the DA release from the synapse which is not possible with dopaminergic synapse model proposed by Best et al. DA synthesis originates from the concentration of TYR located in the terminal bouton and is divided into two steps. Each of the steps depends on a specific enzyme that acts as a catalyst for that step. The activity of TH is regulated by a balance among cytosolic DA that acts as an end product inhibitor by competing with its cofactor, by extracellular DA that acts as an inhibitor via the binding with synthesis modulating autoreceptors located on the nerve terminals, and by neuronal activity as a stimulator , As stated earlier, the activity of TH is regulated by neuronal activity.
In the proposed model of dopaminergic synapse, neuronal stimulation is linked to the synthesis of DA as described in Eq. There are four types of DA autoreceptors on the SNc neurons, where they regulate neuronal activity and control DA synthesis, release, and uptake When these autoreceptors get activated, they result in reduced neuronal activity, DA synthesis, release, and uptake.
In the present SNc model, we have considered autoreceptors that regulate DA synthesis and release and excluded one regulating DA uptake unable to find specific data regarding DA-mediated activation of autoreceptors which regulates DA reuptake and neuronal activity.
However, DA regulating neuronal activity can be incorporated in network model of SNc neurons where DA regulates the lateral connections collaterals. We suggest some experimental approaches to evaluate the behavior of dopaminergic neurons at single-cell or network level by capturing the dynamics of critical molecular players in various conditions. During energy-deficient conditions, monitoring important intracellular players such as ATP, glucose, AMP-activated protein kinase AMPK , and lactate using single-cell imaging studies gives an insight into the progressive adaptation of dopaminergic neurons to the energy crisis by activating various compensatory mechanisms 52 , Also, we can determine all the cellular processes that are compromised during energy crisis.
Mitochondria play a major role in maintaining cellular energy levels , and monitoring its functioning capacity provides insights into cellular energy production. Using cellular models , monitoring the mitochondrial calcium, ATP, NADPH, pH, membrane potential, oxygen consumption rate, ROS production, and morphology gives a better understanding of mitochondrial bioenergetic function in the neuron under energy deficits, oxidative stress, and excitotoxicity 4 , , , During progressive energy deficiency, DA and its metabolites can be measured to check for production of ROS leading to oxidative stress in the neuron using toxin-induced animal pathological models , In the proposed model, ketone metabolism can be incorporated to make the model more robust to utilize different substrates as an energy source and understand the role of ketone bodies in PD pathogenesis , Apart from ketone bodies, astrocytes also play an important role in maintaining neuronal energy demands Therefore, combining the SNc neuronal model with astrocyte model will provide a better understanding of compensation due to astrocyte involvement in energy deficit conditions The ischemic condition was implemented by modulating glucose and oxygen levels, which can be extended by introducing the vascular module , where ischemia condition can be simulated more realistically by varying cerebral blood flow.
Cancer cells survive in low oxygen and acidic conditions , where pH plays a vital role in the functioning of cellular processes ; thus, considering potentiometric properties in formulating cellular processes could be more biologically realistic pH plays an essential role in mitochondrial functioning.
In conclusion, we believe that the proposed model provides an integrated modelling framework to understand the neurodegenerative processes underlying PD From the simulation results, it was observed that under conditions of energy starvation, intracellular calcium, DA cytoplasmic , alpha-synuclein, and ROS concentrations significantly deviated from normal values equilibrium.
There is a positive feedback loop formed with increased intracellular calcium, or DA levels lead to oligomerization of alpha-synuclein, while alpha-synuclein oligomers increased intracellular calcium and DA levels Any therapeutics that can reduce these key toxicity mediators can be beneficial for the survival of SNc neurons 59 , 91 , To this end, it is desirable to develop a therapeutic computational testbench for PD, wherein the proposed model of SNc will be the center of a larger framework, which will also be integrated to behavioral model This type of framework will help in providing personalized medicine for PD patients rather than the currently employed trial and error approaches.
McDonald, C. Age Ageing 47 , — Goldman, J. Muddapu, V. Neural Circuits 13 , 11 Pacelli, C. Elevated mitochondrial bioenergetics and axonal arborization size are key contributors to the vulnerability of dopamine neurons. Bolam, J. Living on the edge with too many mouths to feed: Why dopamine neurons die. Wellstead, P. Wiley Interdiscip. Programmed cell death in substantia nigra due to subthalamic nucleus-mediated excitotoxicity: A computational model of Parkinsonian neurodegeneration.
BMC Neurosci. Tello-Bravo, D. A Mathematical Model of Dopamine Neurotransmission. Cloutier, M. IET Syst. Cullen, M. Integrated dopaminergic neuronal model with reduced intracellular processes and inhibitory autoreceptors.
Reed, M. Mathematical insights into the effects of levodopa. Francis, F. A single compartment model of pacemaking in dissasociated substantia nigra neurons: Stability and energy analysis. Lloret-Villas, A. The impact of mathematical modeling in understanding the mechanisms underlying neurodegeneration: Evolving dimensions and future directions. CPT Pharmacometrics Syst. Bakshi, S. Marhl, M. Complex calcium oscillations and the role of mitochondria and cytosolic proteins.
BioSystems 57 , 75—86 The control systems structures of energy metabolism. Interface 7 , — Hong, J. Computational modeling of apoptotic signaling pathways induced by cisplatin.
BMC Syst. Marinelli, M. Heterogeneity of dopamine neuron activity across traits and states. Neuroscience , — Destexhe, A. Kinetic models of synaptic transmission. Methods Neuronal. Alzheimer, C. Article Google Scholar. Ford, C. Up to the moment there is no universally accepted scheme of spatial organization of the groups of neurons of substantia nigra pars compacta of the human midbrain.
A detailed study of the architectonics of this structure is necessary for pathomorphological analysis of agerelated changes in the nervous tissue and the associated neurodegenerative diseases with selective death of dopamine neurons.
To clarify the peculiarities of the morphochemical organization of the substantia nigra SN of a human brain and to create a threedimensional model of pars compacta.
Materials and Methods. Sections of the midbrain were stained by Nissl method and by an immunohistochemical method for localization of tyrosine hydroxylase — a marker of dopamine. In the SN pars compacta accumulations of neurons were identified in the form of 9 bands oriented in the rostrocaudal direction and including four areas: medial, lateral, dorsal and ventral.
Morphometric analysis detected significant differences in the density of neurons and in expression of tyrosine hydroxylase between the areas of SN. A model of cellular organization of SN pars compacta proposed by us on the basis of threedimensional reconstruction is characterized by a high degree of detalization as compared to similar works, and shows expressed spatial differentiation of the groups of neurons of SN which should be taken into consideration in pathomorphological examinations.
It is known that physiological ageing and Parkinson disease PD are manifested by morphological alterations of the substantia nigra SN of the brain. Here, age-related involution of SN is characterized by natural reduction in the amount of neurons [ 1 ], while Parkinson disease is associated with a selective death of dopamine neurons in pars compacta [ 2 ].
Intensity and uniformity of quantitative changes of neurons in the structures of SN both in physiological ageing and in pathology — PD — can be evaluated by a morphometric examination.
Here, in-depth evaluation of quantitative changes of cell structures of SN is based on examination of the respective parameters of separate aggregations of neurons that make up this structure of the brain. In this context, different variants of division of SN to separate cellular structures were proposed. In one variant SN was divided to two zones — black pars compacta represented by 21 groups of pigmented neurons, and red pars reticulata , consisting of poorly pigmented cells [ 3 ].
Other authors combined dopamine neurons located in the region of the midbrain into 6 separate groups [ 4 ], and arranged dopamine neurons of substantia nigra into groups according to nigrosomes — zones of immunoreactivity of calbindin calcium-binding protein present in nigrostriatal afferent fibers and in cell neuropil [ 5 ]. Besides, there were proposed schemes for division of SN pars compacta of a human brain into regions accessible for studying by morphometric methods [ 6 ].
On the basis of similar schemes and classifications, distribution of cholinergic [ 7 ] and dopaminergic [ 8 ] neurons in SN of the brain was analyzed in laboratory animals, and models of spatial structural organization of SN were described. In the meanwhile, we did not find any information concerning similar three-dimensional models of organization of neuronal structures of SN pars compacta in a human brain in the available literature. The aim of work was to clarify peculiarities of morphochemical organization of SN of a human brain and to obtain a spatial reconstruction of the structures of its pars compacta.
The average age was 71 years old. Binding of antibodies was determined using Thermo Fisher Ultra Vision set on the basis of the polymer system of detection with alkaline phosphatase.
Staining was performed according to the protocols of antibody manufacturers. For three-dimensional reconstruction and morphometry, series of sections were selected from the caudal two thirds of SN with 0. Stained brain sections were scanned using Plustek Opticfilm i slide-scanner China with dpi resolution. For three-dimensional reconstruction, the series of sections were aligned according to their anatomical landmarks and processed using Image J program free software, National Institutes of Health, USA.
The obtained images were used for localization of groups of cells in the examined structures. From the images of sections stained with cresyl violet, maps of distribution of neurons were obtained, with manual marking of neuron somas using Wacom graphic pad.
Images of sections stained for tyrosine hydroxylase, were segmented by brightness for identification of boundaries of dopaminergic structures. Using Free-D program ex-commercial software, Institut Jean-Pierre Bourgin, France [ 9 ], the areas with the highest local density of neurons in SN and boundaries of other structures of the midbrain were marked in the images, after which three-dimensional reconstruction was conducted on the basis of the obtained contours of cell aggregations.
The obtained three-dimensional model of SN was smoothed and its final image was created in Blender program free software, Blender Foundation, the Netherlands. The applied methods were described by us in more detail earlier [ 10 ]. Morphometric examination was carried out using Leica QWin Standart v.
For this, neurons were counted in the field of microscope through the entire depth of the section, with recalculation of their number was per unit volume 0. Besides, in the same structures the intensity of immunostaining for TH was evaluated in standard units from 0 to The work was carried out on 8-bit images obtained with x4 magnification of the objective, taking into account the background staining, with calculation of median values in the group at different levels along the rostro-caudal axis.
The density of arrangement of neurons in the structures of pars compacta of SN was compared using Student test for comparison of groups with pairwise independent variants.
In unstained macropreparations — frontal sections of the midbrain — structures of SN were identified as a separate band of dark brown color Fig. With Nissl method of staining two zones of aggregations of neurons were distinguished Fig. The boundaries of SN were determined by localization of dopamine neurons and their processes Fig. In the obtained three-dimensional model of spatial organization of SN constructed on the basis of sections of the midbrain stained by Nissl method, pars compacta of SN Fig.
In projection of this model on a plane, four areas in SN pars compacta were identified: medial, lateral, dorsal and ventral Fig. Template:Infobox Brain The pars compacta is a portion of the substantia nigra.
Journal of Comparative Neurology. PMID Progress in Brain Research. Brain Research. European Journal of Neuroscience. Seminar in Neuroscience. Categories : Pages with citations using unsupported parameters CS1 maint: Multiple names: authors list Midbrain.
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