Implementation of deep neural networks to count dopamine neurons in substantia nigra

Unbiased estimates of neuron numbers within substantia nigra are crucial for experimental Parkinson's disease models and gene‐function studies. Unbiased stereological counting techniques with optical fractionation are successfully implemented, but are extremely laborious and time‐consuming. The development of neural networks and deep learning has opened a new way to teach computers to count neurons. Implementation of a programming paradigm enables a computer to learn from the data and development of an automated cell counting method. The advantages of computerized counting are reproducibility, elimination of human error and fast high‐capacity analysis. We implemented whole‐slide digital imaging and deep convolutional neural networks (CNN) to count substantia nigra dopamine neurons. We compared the results of the developed method against independent manual counting by human observers and validated the CNN algorithm against previously published data in rats and mice, where tyrosine hydroxylase (TH)‐immunoreactive neurons were counted using unbiased stereology. The developed CNN algorithm and fully cloud‐embedded Aiforia™ platform provide robust and fast analysis of dopamine neurons in rat and mouse substantia nigra.

[1]  I. Strömberg,et al.  GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function , 2015, PLoS genetics.

[2]  J. Elson,et al.  Pedunculopontine cell loss and protein aggregation direct microglia activation in parkinsonian rats , 2015, Brain Structure and Function.

[3]  Kunihiko Fukushima,et al.  Neocognitron: A self-organizing neural network model for a mechanism of pattern recognition unaffected by shift in position , 1980, Biological Cybernetics.

[4]  F ROSENBLATT,et al.  The perceptron: a probabilistic model for information storage and organization in the brain. , 1958, Psychological review.

[5]  Jigneshkumar L Patel,et al.  Applications of artificial neural networks in medical science. , 2007, Current clinical pharmacology.

[6]  Anne E Carpenter,et al.  CellProfiler: image analysis software for identifying and quantifying cell phenotypes , 2006, Genome Biology.

[7]  S. Herculano‐Houzel,et al.  The search for true numbers of neurons and glial cells in the human brain: A review of 150 years of cell counting , 2016, The Journal of comparative neurology.

[8]  P. R. Hof,et al.  Design-based stereology in neuroscience , 2005, Neuroscience.

[9]  Richard J Smeyne,et al.  A comparison of model-based (2D) and design-based (3D) stereological methods for estimating cell number in the substantia nigra pars compacta (SNpc) of the C57BL/6J mouse , 2009, Neuroscience.

[10]  W S McCulloch,et al.  A logical calculus of the ideas immanent in nervous activity , 1990, The Philosophy of Artificial Intelligence.

[11]  Lawrence O. Hall,et al.  Unbiased estimation of cell number using the automatic optical fractionator , 2017, Journal of Chemical Neuroanatomy.

[12]  H. Johnson,et al.  A comparison of 'traditional' and multimedia information systems development practices , 2003, Inf. Softw. Technol..

[13]  Victor Tapias,et al.  A highly reproducible rotenone model of Parkinson's disease , 2009, Neurobiology of Disease.

[14]  Anna-Maija Penttinen,et al.  Characterization of a new low‐dose 6‐hydroxydopamine model of Parkinson's disease in rat , 2016, Journal of neuroscience research.

[15]  Ji-Hyuk Park,et al.  Reduced numbers of dopamine neurons in the substantia nigra pars compacta and ventral tegmental area of rats fed an n-3 polyunsaturated fatty acid-deficient diet: A stereological study , 2008, Neuroscience Letters.

[16]  Alex Zhavoronkov,et al.  Applications of Deep Learning in Biomedicine. , 2016, Molecular pharmaceutics.

[17]  Yoshua Bengio,et al.  Gradient-based learning applied to document recognition , 1998, Proc. IEEE.

[18]  D. German,et al.  Midbrain dopaminergic neurons (nuclei A8, A9, and A10): Three‐dimensional reconstruction in the rat , 1993, The Journal of comparative neurology.

[19]  J. Dalley,et al.  Endogenous alpha-synuclein influences the number of dopaminergic neurons in mouse substantia nigra , 2013, Experimental Neurology.

[20]  J. Bolam,et al.  Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat , 2008, Neuroscience.

[21]  P. Auvinen,et al.  Constitutive Ret Activity in Knock-In Multiple Endocrine Neoplasia Type B Mice Induces Profound Elevation of Brain Dopamine Concentration via Enhanced Synthesis and Increases the Number of TH-Positive Cells in the Substantia Nigra , 2007, The Journal of Neuroscience.

[22]  M. Saarma,et al.  Prospects of Neurotrophic Factors for Parkinson's Disease: Comparison of Protein and Gene Therapy. , 2015, Human gene therapy.

[23]  M. Abercrombie Estimation of nuclear population from microtome sections , 1946, The Anatomical record.

[24]  R. Penn,et al.  Developing therapeutically more efficient Neurturin variants for treatment of Parkinson's disease , 2016, Neurobiology of Disease.

[25]  Philip A. Botham,et al.  Assessment of the Effects of MPTP and Paraquat on Dopaminergic Neurons and Microglia in the Substantia Nigra Pars Compacta of C57BL/6 Mice , 2016, PloS one.

[26]  D. Oorschot Total number of neurons in the neostriatal, pallidal, subthalamic, and substantia nigral nuclei of the rat basal ganglia: A stereological study using the cavalieri and optical disector methods , 1996, The Journal of comparative neurology.

[27]  Rogely Waite Boyce,et al.  Design-based Stereology , 2010, Toxicologic pathology.

[28]  H. Haug History of neuromorphometry , 1986, Journal of Neuroscience Methods.

[29]  Luca Maria Gambardella,et al.  Mitosis Detection in Breast Cancer Histology Images with Deep Neural Networks , 2013, MICCAI.

[30]  D. Dexter,et al.  Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6‐hydroxydopamine model of Parkinson’s disease , 2009, Journal of neurochemistry.

[31]  Simon C Watkins,et al.  Automated imaging system for fast quantitation of neurons, cell morphology and neurite morphometry in vivo and in vitro , 2013, Neurobiology of Disease.

[32]  Jitendra Malik,et al.  Region-Based Convolutional Networks for Accurate Object Detection and Segmentation , 2016, IEEE Transactions on Pattern Analysis and Machine Intelligence.

[33]  H. Gundersen,et al.  Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator , 1991, The Anatomical record.

[34]  J R Nyengaard,et al.  Improving efficiency in stereology: a study applying the proportionator and the autodisector on virtual slides , 2013, Journal of microscopy.

[35]  J. Volkmann,et al.  Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment. , 2017, Journal of visualized experiments : JoVE.

[36]  Yolanda T. Chong,et al.  Automated analysis of high‐content microscopy data with deep learning , 2017, Molecular systems biology.

[37]  N. Linder,et al.  Antibody-supervised deep learning for quantification of tumor-infiltrating immune cells in hematoxylin and eosin stained breast cancer samples , 2016, Journal of pathology informatics.

[38]  Guigang Zhang,et al.  Deep Learning , 2016, Int. J. Semantic Comput..