Publications by authors named "Masahiko Takada"

116 Publications

Effects of Optogenetic Suppression of Cortical Input on Primate Thalamic Neuronal Activity during Goal-Directed Behavior.

eNeuro 2021 Mar-Apr;8(2). Epub 2021 Mar 23.

Department of Physiology, Hokkaido University School of Medicine, Sapporo 060-8638, Japan.

The motor thalamus relays signals from subcortical structures to the motor cortical areas. Previous studies in songbirds and rodents suggest that cortical feedback inputs crucially contribute to the generation of movement-related activity in the motor thalamus. In primates, however, it remains uncertain whether the corticothalamic projections may play a role in shaping neuronal activity in the motor thalamus. Here, using an optogenetic inactivation technique with the viral vector system expressing halorhodopsin, we investigated the role of cortical input in modulating thalamic neuronal activity during goal-directed behavior. In particular, we assessed whether the suppression of signals originating from the supplementary eye field at the corticothalamic terminals could change the task-related neuronal modulation in the oculomotor thalamus in monkeys performing a self-initiated saccade task. We found that many thalamic neurons exhibited changes in their firing rates depending on saccade direction or task event, indicating that optical stimulation exerted task-specific effects on neuronal activity beyond the global changes in baseline activity. These results suggest that the corticothalamic projections might be actively involved in the signal processing necessary for goal-directed behavior. However, we also found that some thalamic neurons exhibited overall, non-task-specific changes in the firing rate during optical stimulation, even in control animals without vector injections. The stimulation effects in these animals started with longer latency, implying a possible thermal effect on neuronal activity. Thus, our results not only reveal the importance of direct cortical input in neuronal activity in the primate motor thalamus, but also provide useful information for future optogenetic studies.
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http://dx.doi.org/10.1523/ENEURO.0511-20.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009665PMC
March 2021

Morphological features of large layer V pyramidal neurons in cortical motor-related areas of macaque monkeys: analysis of basal dendrites.

Sci Rep 2021 Feb 18;11(1):4171. Epub 2021 Feb 18.

Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Aichi, 484-8506, Japan.

In primates, large layer V pyramidal neurons located in the frontal motor-related areas send a variety of motor commands to the spinal cord, giving rise to the corticospinal tract, for execution of skilled motor behavior. However, little is known about the morphological diversity of such pyramidal neurons among the areas. Here we show that the structure of basal dendrites of the large layer V pyramidal neurons in the dorsal premotor cortex (PMd) is different from those in the other areas, including the primary motor cortex, the supplementary motor area, and the ventral premotor cortex. In the PMd, not only the complexity (arborization) of basal dendrites, i.e., total dendritic length and branching number, was poorly developed, but also the density of dendritic spines was so low, as compared to the other motor-related areas. Regarding the distribution of the three dendritic spine types identified, we found that thin-type (more immature) spines were prominent in the PMd in comparison with stubby- and mushroom-type (more mature) spines, while both thin- and stubby-type spines were in the other areas. The differential morphological features of basal dendrites might reflect distinct patterns of motor information processing within the large layer V pyramidal neurons in individual motor-related areas.
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http://dx.doi.org/10.1038/s41598-021-83680-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893167PMC
February 2021

MacaquePose: A Novel "In the Wild" Macaque Monkey Pose Dataset for Markerless Motion Capture.

Front Behav Neurosci 2020 18;14:581154. Epub 2021 Jan 18.

Department of Human Intelligence Systems, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu, Japan.

Video-based markerless motion capture permits quantification of an animal's pose and motion, with a high spatiotemporal resolution in a naturalistic context, and is a powerful tool for analyzing the relationship between the animal's behaviors and its brain functions. Macaque monkeys are excellent non-human primate models, especially for studying neuroscience. Due to the lack of a dataset allowing training of a deep neural network for the macaque's markerless motion capture in the naturalistic context, it has been challenging to apply this technology for macaques-based studies. In this study, we created MacaquePose, a novel open dataset with manually labeled body part positions (keypoints) for macaques in naturalistic scenes, consisting of >13,000 images. We also validated the application of the dataset by training and evaluating an artificial neural network with the dataset. The results indicated that the keypoint estimation performance of the trained network was close to that of a human-level. The dataset will be instrumental to train/test the neural networks for markerless motion capture of the macaques and developments of the algorithms for the networks, contributing establishment of an innovative platform for behavior analysis for non-human primates for neuroscience and medicine, as well as other fields using macaques as a model organism.
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http://dx.doi.org/10.3389/fnbeh.2020.581154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874091PMC
January 2021

Nonhuman Primate Optogenetics: Current Status and Future Prospects.

Adv Exp Med Biol 2021 ;1293:345-358

Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan.

Nonhuman primates (NHPs) have widely and crucially been utilized as model animals for understanding various higher brain functions and neurological disorders since their behavioral actions mimic both normal and disease states in humans. To know about how such behaviors emerge from the functions and dysfunctions of complex neural networks, it is essential to define the role of a particular pathway or neuron-type constituting these networks. Optogenetics is a potential technique that enables analyses of network functions. However, because of the large size of the NHP brain and the difficulty in creating genetically modified animal models, this technique is currently still hard to apply effectively and efficiently to NHP neuroscience. In this article, we focus on the issues that should be overcome for the development of NHP optogenetics, with special reference to the gene introduction strategy. We review the recent breakthroughs that have been made in NHP optogenetics to address these issues and discuss future prospects regarding more effective and efficient approaches to successful optogenetic manipulation in NHPs.
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http://dx.doi.org/10.1007/978-981-15-8763-4_22DOI Listing
February 2021

An Open Resource for Non-human Primate Optogenetics.

Authors:
Sébastien Tremblay Leah Acker Arash Afraz Daniel L Albaugh Hidetoshi Amita Ariana R Andrei Alessandra Angelucci Amir Aschner Puiu F Balan Michele A Basso Giacomo Benvenuti Martin O Bohlen Michael J Caiola Roberto Calcedo James Cavanaugh Yuzhi Chen Spencer Chen Mykyta M Chernov Andrew M Clark Ji Dai Samantha R Debes Karl Deisseroth Robert Desimone Valentin Dragoi Seth W Egger Mark A G Eldridge Hala G El-Nahal Francesco Fabbrini Frederick Federer Christopher R Fetsch Michal G Fortuna Robert M Friedman Naotaka Fujii Alexander Gail Adriana Galvan Supriya Ghosh Marc Alwin Gieselmann Roberto A Gulli Okihide Hikosaka Eghbal A Hosseini Xing Hu Janina Hüer Ken-Ichi Inoue Roger Janz Mehrdad Jazayeri Rundong Jiang Niansheng Ju Kohitij Kar Carsten Klein Adam Kohn Misako Komatsu Kazutaka Maeda Julio C Martinez-Trujillo Masayuki Matsumoto John H R Maunsell Diego Mendoza-Halliday Ilya E Monosov Ross S Muers Lauri Nurminen Michael Ortiz-Rios Daniel J O'Shea Stéphane Palfi Christopher I Petkov Sorin Pojoga Rishi Rajalingham Charu Ramakrishnan Evan D Remington Cambria Revsine Anna W Roe Philip N Sabes Richard C Saunders Hansjörg Scherberger Michael C Schmid Wolfram Schultz Eyal Seidemann Yann-Suhan Senova Michael N Shadlen David L Sheinberg Caitlin Siu Yoland Smith Selina S Solomon Marc A Sommer John L Spudich William R Stauffer Masahiko Takada Shiming Tang Alexander Thiele Stefan Treue Wim Vanduffel Rufin Vogels Matthew P Whitmire Thomas Wichmann Robert H Wurtz Haoran Xu Azadeh Yazdan-Shahmorad Krishna V Shenoy James J DiCarlo Michael L Platt

Neuron 2020 12 19;108(6):1075-1090.e6. Epub 2020 Oct 19.

Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Marketing, Wharton School, University of Pennsylvania, Philadelphia, PA 19104, USA.

Optogenetics has revolutionized neuroscience in small laboratory animals, but its effect on animal models more closely related to humans, such as non-human primates (NHPs), has been mixed. To make evidence-based decisions in primate optogenetics, the scientific community would benefit from a centralized database listing all attempts, successful and unsuccessful, of using optogenetics in the primate brain. We contacted members of the community to ask for their contributions to an open science initiative. As of this writing, 45 laboratories around the world contributed more than 1,000 injection experiments, including precise details regarding their methods and outcomes. Of those entries, more than half had not been published. The resource is free for everyone to consult and contribute to on the Open Science Framework website. Here we review some of the insights from this initial release of the database and discuss methodological considerations to improve the success of optogenetic experiments in NHPs.
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http://dx.doi.org/10.1016/j.neuron.2020.09.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962465PMC
December 2020

Deschloroclozapine, a potent and selective chemogenetic actuator enables rapid neuronal and behavioral modulations in mice and monkeys.

Nat Neurosci 2020 09 6;23(9):1157-1167. Epub 2020 Jul 6.

Department of Functional Brain Imaging, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan.

The chemogenetic technology designer receptors exclusively activated by designer drugs (DREADDs) afford remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities and potential off-target effects of CNO represent areas for improvement. Here, we provide a new high-affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 μg per kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 μg per kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents a potent, selective, metabolically stable and fast-acting DREADD agonist with utility in both mice and nonhuman primates for a variety of applications.
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http://dx.doi.org/10.1038/s41593-020-0661-3DOI Listing
September 2020

Olig2-Induced Semaphorin Expression Drives Corticospinal Axon Retraction After Spinal Cord Injury.

Cereb Cortex 2020 Oct;30(11):5702-5716

Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

Axon regeneration is limited in the central nervous system, which hinders the reconstruction of functional circuits following spinal cord injury (SCI). Although various extrinsic molecules to repel axons following SCI have been identified, the role of semaphorins, a major class of axon guidance molecules, has not been thoroughly explored. Here we show that expression of semaphorins, including Sema5a and Sema6d, is elevated after SCI, and genetic deletion of either molecule or their receptors (neuropilin1 and plexinA1, respectively) suppresses axon retraction or dieback in injured corticospinal neurons. We further show that Olig2+ cells are essential for SCI-induced semaphorin expression, and that Olig2 binds to putative enhancer regions of the semaphorin genes. Finally, conditional deletion of Olig2 in the spinal cord reduces the expression of semaphorins, alleviating the axon retraction. These results demonstrate that semaphorins function as axon repellents following SCI, and reveal a novel transcriptional mechanism for controlling semaphorin levels around injured neurons to create zones hostile to axon regrowth.
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http://dx.doi.org/10.1093/cercor/bhaa142DOI Listing
October 2020

Primate Amygdalo-Nigral Pathway for Boosting Oculomotor Action in Motivating Situations.

iScience 2020 Jun 23;23(6):101194. Epub 2020 May 23.

Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

A primary function of the primate amygdala is to modulate behavior based on emotional cues. To study the underlying neural mechanism, we first inactivated the amygdala locally and temporarily by injecting a GABA agonist. Then, saccadic eye movements and gaze were suppressed only on the contralateral side. Next, we performed optogenetic activation after injecting a viral vector into the amygdala. Optical stimulation in the amygdala excited amygdala neurons, whereas optical stimulation of axon terminals in the substantia nigra pars reticulata inhibited nigra neurons. Optical stimulation in either structure facilitated saccades to the contralateral side. These data suggest that the amygdala controls saccades and gaze through the basal ganglia output to the superior colliculus. Importantly, this amygdala-derived circuit mediates emotional context information, whereas the internal basal ganglia circuit mediates object value information. This finding demonstrates a basic mechanism whereby basal ganglia output can be modulated by other areas conveying distinct information.
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http://dx.doi.org/10.1016/j.isci.2020.101194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281789PMC
June 2020

Optogenetic manipulation of a value-coding pathway from the primate caudate tail facilitates saccadic gaze shift.

Nat Commun 2020 04 20;11(1):1876. Epub 2020 Apr 20.

Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.

In the primate basal ganglia, the caudate tail (CDt) encodes the historical values (good or bad) of visual objects (i.e., stable values), and electrical stimulation of CDt evokes saccadic eye movements. However, it is still unknown how output from CDt conveys stable value signals to govern behavior. Here, we apply a pathway-selective optogenetic manipulation to elucidate how such value information modulates saccades. We express channelrhodopsin-2 in CDt delivered by viral vector injections. Selective optical activation of CDt-derived terminals in the substantia nigra pars reticulata (SNr) inhibits SNr neurons. Notably, these SNr neurons show inhibitory responses to good objects. Furthermore, the optical stimulation causes prolonged excitation of visual-saccadic neurons in the superior colliculus (SC), and induces contralateral saccades. These SC neurons respond more strongly to good than to bad objects in the contralateral hemifield. The present results demonstrate that CDt facilitates saccades toward good objects by serial inhibitory pathways through SNr.
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http://dx.doi.org/10.1038/s41467-020-15802-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171130PMC
April 2020

Store-Operated Calcium Channels Are Involved in Spontaneous Slow Calcium Oscillations in Striatal Neurons.

Front Cell Neurosci 2019 17;13:547. Epub 2019 Dec 17.

Department of Radiological Imaging and Informatics, Tohoku University Graduate School of Medicine, Sendai, Japan.

The striatum plays an important role in linking cortical activity to basal ganglia output. Striatal neurons exhibit spontaneous slow Ca oscillations that result from Ca release from the endoplasmic reticulum (ER) induced by the mGluR5-IP3R signaling cascade. The maximum duration of a single oscillatory event is about 300 s. A major question arises as to how such a long-duration Ca elevation is maintained. Store-operated calcium channels (SOCCs) are one of the calcium (Ca)-permeable ion channels. SOCCs are opened by activating the metabotropic glutamate receptor type 5 and inositol 1,4,5-trisphosphate receptor (mGluR5-IP3R) signal transduction cascade and are related to the pathophysiology of several neurological disorders. However, the functions of SOCCs in striatal neurons remain unclear. Here, we show that SOCCs exert a functional role in striatal GABAergic neurons. Depletion of calcium stores from the ER induced large, sustained calcium entry that was blocked by SKF96365, an inhibitor of SOCCs. Moreover, the application of SKF96365 greatly reduced the frequency of slow Ca oscillations. The present results indicate that SOCCs contribute to Ca signaling in striatal GABAergic neurons, including medium spiny projection neurons (MSNs) and GABAergic interneurons, through elevated Ca due to spontaneous slow Ca oscillations.
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http://dx.doi.org/10.3389/fncel.2019.00547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927941PMC
December 2019

Layer specificity of inputs from supplementary motor area and dorsal premotor cortex to primary motor cortex in macaque monkeys.

Sci Rep 2019 12 3;9(1):18230. Epub 2019 Dec 3.

Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Aichi, 484-8506, Japan.

The primate frontal lobe processes diverse motor information in parallel through multiple motor-related areas. For example, the supplementary motor area (SMA) is mainly involved in internally-triggered movements, whereas the premotor cortex (PM) is highly responsible for externally-guided movements. The primary motor cortex (M1) deals with both aspects of movements to execute a single motor behavior. To elucidate how the cortical motor system is structured to process a variety of information, the laminar distribution patterns of signals were examined between SMA and M1, or PM and M1 in macaque monkeys by using dual anterograde tract-tracing. Dense terminal labeling was observed in layers 1 and upper 2/3 of M1 after one tracer injection into SMA, another tracer injection into the dorsal division of PM resulted in prominent labeling in the deeper portion of layer 2/3. Weaker labeling was also visible in layer 5 in both cases. On the other hand, inputs from M1 terminated in both the superficial and the deep layers of SMA and PM. The present data indicate that distinct types of motor information are arranged in M1 in a layer-specific fashion to be orchestrated through a microcircuit within M1.
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http://dx.doi.org/10.1038/s41598-019-54220-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890803PMC
December 2019

Oral splint ameliorates tic symptoms in patients with tourette syndrome.

Mov Disord 2019 10 23;34(10):1577-1578. Epub 2019 Aug 23.

Department of Oral Anatomy and Neurobiology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.

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http://dx.doi.org/10.1002/mds.27819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6852427PMC
October 2019

Optogenetic recruitment of spinal reflex pathways from large-diameter primary afferents in non-transgenic rats transduced with AAV9/Channelrhodopsin 2.

J Physiol 2019 10 28;597(19):5025-5040. Epub 2019 Aug 28.

Department of Neurophysiology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.

Key Points: We demonstrated optical activation of primary somatosensory afferents with high selectivity to fast-conducting fibres by means of adeno-associated virus 9 (AAV9)-mediated gene transduction in dorsal root ganglion (DRG) neurons. AVV9 expressing green fluorescent protein showed high selectivity and transduction efficiency for fast-conducting, large-sized DRG neurons. Compared with conventional electrical stimulation, optically elicited volleys in primary afferents had higher sensitivity with stimulus amplitude, but lower sensitivity with stimulus frequency. Optically elicited dorsal root volleys activated postsynaptic neurons in the segmental spinal pathway. This proposed technique will help establish the causal relationships between somatosensory afferent inputs and neural responses in the CNS as well as behavioural outcomes in higher mammals where transgenic animals are not available.

Abstract: Previously, fundamental structures and their mode of action in the spinal reflex circuit were determined by confirming their input-output relationship using electrophysiological techniques. In those experiments, the electrical stimulation of afferent fibres was used as a core element to identify different types of reflex pathways; however, a major disadvantage of this technique is its non-selectivity. In this study, we investigated the selective activation of large-diameter afferents by optogenetics combined with a virus vector transduction technique (injection via the sciatic nerve) in non-transgenic male Jcl:Wistar rats. We found that green fluorescent protein gene transduction of rat dorsal root ganglion (DRG) neurons with a preference for medium-to-large-sized cells was achieved using the adeno-associated virus 9 (AAV9) vector compared with the AAV6 vector (P = 0.021). Furthermore, the optical stimulation of Channelrhodopsin 2 (ChR2)-expressing DRG neurons (transduced by AAV9) produced compound action potentials in afferent nerves originating from fast-conducting nerve fibres. We also confirmed that physiological responses to different stimulus amplitudes were comparable between optogenetic and electrophysiological activation. However, compared with electrically elicited responses, the optically elicited responses had lower sensitivity with stimulus frequency. Finally, we showed that afferent volleys evoked by optical stimulation were sufficient to activate postsynaptic neurons in the spinal reflex arc. These results provide new ways for understanding the role of sensory afferent input to the central nervous system regarding behavioural control, especially when genetically manipulated animals are not available, such as higher mammals including non-human primates.
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http://dx.doi.org/10.1113/JP278292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851594PMC
October 2019

A note on retrograde gene transfer efficiency and inflammatory response of lentiviral vectors pseudotyped with FuG-E vs. FuG-B2 glycoproteins.

Sci Rep 2019 03 5;9(1):3567. Epub 2019 Mar 5.

Systems Neuroscience Section, Department of Neuroscience, Primate Research Institute, Kyoto University, Inuyama, Aichi, 484-8506, Japan.

Pseudotyped lentiviral vectors give access to pathway-selective gene manipulation via retrograde transfer. Two types of such lentiviral vectors have been developed. One is the so-called NeuRet vector pseudotyped with fusion glycoprotein type E, which preferentially transduces neurons. The other is the so-called HiRet vector pseudotyped with fusion glycoprotein type B2, which permits gene transfer into both neurons and glial cells at the injection site. Although these vectors have been applied in many studies investigating neural network functions, it remains unclear which vector is more appropriate for retrograde gene delivery in the brain. To compare the gene transfer efficiency and inflammatory response of the NeuRet vs. HiRet vectors, each vector was injected into the striatum in macaque monkeys, common marmosets, and rats. It was revealed that retrograde gene delivery of the NeuRet vector was equal to or greater than that of the HiRet vector. Furthermore, inflammation characterized by microglial and lymphocytic infiltration occurred when the HiRet vector, but not the NeuRet vector, was injected into the primate brain. The present results indicate that the NeuRet vector is more suitable than the HiRet vector for retrograde gene transfer in the primate and rodent brains.
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http://dx.doi.org/10.1038/s41598-019-39535-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400974PMC
March 2019

Primate Nigrostriatal Dopamine System Regulates Saccadic Response Inhibition.

Neuron 2018 12 8;100(6):1513-1526.e4. Epub 2018 Nov 8.

Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan; Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan; Transborder Medical Research Center, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. Electronic address:

Animals need to inhibit inappropriate actions that would lead to unwanted outcomes. Although this ability, called response inhibition, is impaired in neurological/psychiatric disorders with dopaminergic dysfunctions, how dopamine regulates response inhibition remains unclear. Here we investigated neuronal signals of the nigrostriatal dopamine system in monkeys performing a saccadic countermanding task. Subsets of dopamine neurons in the substantia nigra and striatal neurons receiving the dopaminergic input were activated when the monkey was required to cancel a planned saccadic eye movement. These activations were stronger when canceling the eye movements was successful compared with failed and were enhanced in demanding trials. The activated dopamine neurons were distributed mainly in the dorsolateral, but not in the ventromedial, part of the nigra. Furthermore, pharmacological blockade of dopaminergic neurotransmission in the striatum dampened the performance of canceling saccadic eye movements. The present findings indicate that disruption of nigrostriatal dopamine signaling causes impairments in response inhibition.
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http://dx.doi.org/10.1016/j.neuron.2018.10.025DOI Listing
December 2018

Enhancement of the transduction efficiency of a lentiviral vector for neuron-specific retrograde gene delivery through the point mutation of fusion glycoprotein type E.

J Neurosci Methods 2019 01 19;311:147-155. Epub 2018 Oct 19.

Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan. Electronic address:

Background: Pseudotyping of a lentiviral vector with fusion glycoproteins composed of rabies virus glycoprotein (RVG) and vesicular stomatitis virus glycoprotein (VSVG) segments achieves high gene transfer efficiency through retrograde transport in the nervous system. In our previous study, we determined the junction of RVG/VSVG segments of glycoproteins that enhances the transduction efficiency of the neuron-specific retrograde gene transfer (NeuRet) vector (termed fusion glycoprotein type E or FuG-E).

New Method: We aimed to optimize the amino acid residue at position 440 in the membrane-proximal region of FuG-E to improve the efficiency of retrograde gene transfer in the brain.

Results: We constructed variants of FuG-E with 18 kinds of single amino acid substitutions at residue 440 to generate lentiviral vectors pseudotyped with these variants, and tested in vivo gene transfer of the vectors in the mouse brain. The FuG-E (P440E) variant, in which proline was substituted by glutamate at residue 440 in FuG-E, showed the greatest retrograde gene transfer efficiency in the brain, bearing the property of the NeuRet vector. The FuG-E (P440E) pseudotype also displayed efficient retrograde gene transfer in the common marmoset brain.

Comparison With Existing Methods: The NeuRet vector with the FuG-E (P440E) variant demonstrated higher retrograde gene transfer efficiency into different neural pathways compared with the parental FuG-E vector.

Conclusions: The FuG-E (P440E) pseudotype provides a powerful tool to investigate neural circuit mechanisms underlying various brain functions and for gene therapy trials of neurological and neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.jneumeth.2018.10.023DOI Listing
January 2019

Causal Role of Neural Signals Transmitted From the Frontal Eye Field to the Superior Colliculus in Saccade Generation.

Front Neural Circuits 2018 28;12:69. Epub 2018 Aug 28.

Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Japan.

The frontal eye field (FEF) and superior colliculus (SC) are major and well-studied components of the oculomotor system. The FEF sends strong projections to the SC directly, and neurons in these brain regions transmit a variety of signals related to saccadic eye movements. Electrical microstimulation and pharmacological manipulation targeting the FEF or SC affect saccadic eye movements. These data suggest the causal contribution of each region to saccade generation. To understand how the brain generates behavior, however, it is critical not only to identify the structures and functions of individual regions, but also to elucidate how they interact with each other. In this review article, we first survey previous works that aimed at investigating whether and how the FEF and SC interact to regulate saccadic eye movements using electrophysiological and pharmacological techniques. These works have reported what signals FEF neurons transmit to the SC and what roles such signals play in regulating oculomotor behavior. We then highlight a recent attempt of our own that has applied an optogenetic approach to stimulate the neural pathway from the FEF to the SC in nonhuman primates. This study has shown that optogenetic stimulation of the FEF-SC pathway is sufficiently effective not only to modulate SC neuron activity, but also to evoke saccadic eye movements. Although the oculomotor system is a complex neural network composed of numbers of cortical and subcortical regions, the optogenetic approach will provide a powerful strategy for elucidating the role of each neural pathway constituting this network.
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http://dx.doi.org/10.3389/fncir.2018.00069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120992PMC
March 2019

Recruitment of calbindin into nigral dopamine neurons protects against MPTP-Induced parkinsonism.

Mov Disord 2019 02 30;34(2):200-209. Epub 2018 Aug 30.

Systems Neuroscience Section, Department of Neuroscience, Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan.

Background: Parkinson's disease is caused by dopamine deficiency in the striatum, which is a result of loss of dopamine neurons from the substantia nigra pars compacta. There is a consensus that a subpopulation of nigral dopamine neurons that expresses the calcium-binding protein calbindin is selectively invulnerable to parkinsonian insults. The objective of the present study was to test the hypothesis that dopamine neuron degeneration might be prevented by viral vector-mediated gene delivery of calbindin into the dopamine neurons that do not normally contain it.

Methods: A calbindin-expressing adenoviral vector was injected into the striatum of macaque monkeys to be conveyed to cell bodies of nigral dopamine neurons through retrograde axonal transport, or the calbindin-expressing lentiviral vector was injected into the nigra directly because of its predominant uptake from cell bodies and dendrites. The animals in which calbindin was successfully recruited into nigral dopamine neurons were administered systemically with MPTP.

Results: In the monkeys that had received unilateral vector injections, parkinsonian motor deficits, such as muscular rigidity and akinesia/bradykinesia, appeared predominantly in the limbs corresponding to the non-calbindin-recruited hemisphere after MPTP administration. Data obtained from tyrosine hydroxylase immunostaining and PET imaging for the dopamine transporter revealed that the nigrostriatal dopamine system was preserved better on the calbindin-recruited side. Conversely, on the non-calbindin-recruited control side, many more dopamine neurons expressed α-synuclein.

Conclusions: The present results indicate that calbindin recruitment into nigral dopamine neurons protects against the onset of parkinsonian insults, thus providing a novel approach to PD prevention. © 2018 International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.107DOI Listing
February 2019

Promoting functional recovery by inhibition of repulsive guidance molecule-a after spinal cord injury.

Neural Regen Res 2018 Jun;13(6):981-982

Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Japan.

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http://dx.doi.org/10.4103/1673-5374.233437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022473PMC
June 2018

Preferential Representation of Past Outcome Information and Future Choice Behavior by Putative Inhibitory Interneurons Rather Than Putative Pyramidal Neurons in the Primate Dorsal Anterior Cingulate Cortex.

Cereb Cortex 2019 06;29(6):2339-2352

Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.

The dorsal anterior cingulate cortex (dACC) plays crucial roles in monitoring the outcome of a choice and adjusting a subsequent choice behavior based on the outcome information. In the present study, we investigated how different types of dACC neurons, that is, putative pyramidal neurons and putative inhibitory interneurons, contribute to these processes. We analyzed single-unit database obtained from the dACC in monkeys performing a reversal learning task. The monkey was required to adjust choice behavior from past outcome experiences. Depending on their action potential waveforms, the recorded neurons were classified into putative pyramidal neurons and putative inhibitory interneurons. We found that these neurons do not equally contribute to outcome monitoring and behavioral adjustment. Although both neuron types evenly responded to the current outcome, a larger proportion of putative inhibitory interneurons than putative pyramidal neurons stored the information about the past outcome. The putative inhibitory interneurons further represented choice-related signals more frequently, such as whether the monkey would shift the last choice to an alternative at the next choice opportunity. Our findings suggest that putative inhibitory interneurons, which are thought not to project to brain areas outside the dACC, preferentially transmit signals that would adjust choice behavior based on past outcome experiences.
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http://dx.doi.org/10.1093/cercor/bhy103DOI Listing
June 2019

Multisynaptic Projections from the Amygdala to the Ventral Premotor Cortex in Macaque Monkeys: Anatomical Substrate for Feeding Behavior.

Front Neuroanat 2018 19;12. Epub 2018 Jan 19.

Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Japan.

The amygdala codes the visual-gustatory/somatosensory valence for feeding behavior. On the other hand, the ventral premotor cortex (PMv) plays a central role in reaching and grasping movements prerequisite for feeding behavior. This implies that object valence signals derived from the amygdala may be crucial for feeding-related motor actions exerted by PMv. However, since no direct connectivity between the amygdala and PMv has been reported, the structural basis of their functional interactions still remains elusive. In the present study, we employed retrograde transneuronal labeling with rabies virus to identify the amygdalar origin and possible route of multisynaptic projections to PMv in macaque monkeys. Histological analysis of the distribution pattern of labeled neurons has found that PMv receives disynaptic input primarily from the basal nucleus, especially from its intermediate subdivision. It has also been revealed that the medial (e.g., the cingulate motor areas, CMA) and lateral (e.g., the insular cortices) cortical areas, and the cholinergic cell group 4 in the basal forebrain probably mediate the projections from the amygdala to PMv. Such multisynaptic pathways might represent amygdalar influences on PMv functions for feeding behavior.
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http://dx.doi.org/10.3389/fnana.2018.00003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780351PMC
January 2018

Treatment With the Neutralizing Antibody Against Repulsive Guidance Molecule-a Promotes Recovery From Impaired Manual Dexterity in a Primate Model of Spinal Cord Injury.

Cereb Cortex 2019 02;29(2):561-572

Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Japan.

Axons in the mature mammalian central nervous system have only a limited capacity to grow/regenerate after injury, and spontaneous recovery of motor functions is therefore not greatly expected in spinal cord injury (SCI). To promote functional recovery after SCI, it is critical that corticospinal tract (CST) fibers reconnect properly with target spinal neurons through enhanced axonal growth/regeneration. Here, we applied antibody treatment against repulsive guidance molecule-a (RGMa) to a monkey model of SCI. We found that inhibition of upregulated RGMa around the lesioned site in the cervical cord resulted in recovery from impaired manual dexterity by accentuated penetration of CST fibers into laminae VII and IX, where spinal interneurons and motoneurons are located, respectively. Furthermore, pharmacological inactivation following intracortical microstimulation revealed that the contralesional, but not the ipsilesional, primary motor cortex was crucially involved in functional recovery at a late stage in our SCI model. The present data indicate that treatment with the neutralizing antibody against RGMa after SCI is a potential target for achieving restored manual dexterity in primates.
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http://dx.doi.org/10.1093/cercor/bhx338DOI Listing
February 2019

Alterations in the reduced pteridine contents in the cerebrospinal fluids of LRRK2 mutation carriers and patients with Parkinson's disease.

J Neural Transm (Vienna) 2018 01 1;125(1):45-52. Epub 2017 Sep 1.

Department of Neurology, St. Olavs Hospital, Norwegian University of Science and Technology (NTNU), 7030, Trondheim, Norway.

Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase that is essential for the biosynthesis of dopamine. Parkinson's disease (PD) is characterized by a progressive degeneration of nigrostriatal dopaminergic neurons, and biomarkers reflecting the degree of neurodegeneration are important not only for basic research but also for clinical diagnosis and the treatment of the disease. Although the total neopterin and biopterin levels in the cerebrospinal fluids (CSF) of the patients with PD were reported, alterations in the composition of reduced and oxidized forms of pteridine compounds have not been examined. In this study, we first examined the time-dependent alterations in BH4 and other reduced pteridine compounds in the CSF of an MPTP-treated monkey as a primate PD model. We found that the CSF levels of BH4 and dihydroneopterin, an intermittent metabolite of BH4-biosynthesis, altered inversely with progression of neurodegeneration, whereas those of dihydrobiopterin and neopterin were relatively low and constant. Next, we assayed the amounts of reduced pteridine compounds in the CSF of 36 pre-symptomatic LRRK2-mutation (N1437H or G2019S) carriers (LRRK2-carrier), 13 patients with PD symptoms (LRRK2-PD), 46 patients with sporadic PD (sPD), and 26 non-PD individuals. The BH4 levels were significantly lower in both the LRRK2-PD and sPD patients, and the LRRK2-carriers exhibited higher BH4 levels compared with the sPD patients. The total neopterin levels in the CSF of the LRRK2-PD were significantly higher than those in the sPD and non-PD individuals, which indicated greater inflammatory responses in the brains of LRRK2-PD patients. The present results suggest that detailed analyses of pteridine levels in the CSF might be useful for understanding the pathophysiology of familial PD and for monitoring PD progression.
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http://dx.doi.org/10.1007/s00702-017-1784-xDOI Listing
January 2018

Pseudotyped Lentiviral Vectors for Retrograde Gene Delivery into Target Brain Regions.

Front Neuroanat 2017 2;11:65. Epub 2017 Aug 2.

Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University School of MedicineFukushima, Japan.

Gene transfer through retrograde axonal transport of viral vectors offers a substantial advantage for analyzing roles of specific neuronal pathways or cell types forming complex neural networks. This genetic approach may also be useful in gene therapy trials by enabling delivery of transgenes into a target brain region distant from the injection site of the vectors. Pseudotyping of a lentiviral vector based on human immunodeficiency virus type 1 (HIV-1) with various fusion envelope glycoproteins composed of different combinations of rabies virus glycoprotein (RV-G) and vesicular stomatitis virus glycoprotein (VSV-G) enhances the efficiency of retrograde gene transfer in both rodent and nonhuman primate brains. The most recently developed lentiviral vector is a pseudotype with fusion glycoprotein type E (FuG-E), which demonstrates highly efficient retrograde gene transfer in the brain. The FuG-E-pseudotyped vector permits powerful experimental strategies for more precisely investigating the mechanisms underlying various brain functions. It also contributes to the development of new gene therapy approaches for neurodegenerative disorders, such as Parkinson's disease, by delivering genes required for survival and protection into specific neuronal populations. In this review article, we report the properties of the FuG-E-pseudotyped vector, and we describe the application of the vector to neural circuit analysis and the potential use of the FuG-E vector in gene therapy for Parkinson's disease.
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http://dx.doi.org/10.3389/fnana.2017.00065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5539090PMC
August 2017

High-Speed and Scalable Whole-Brain Imaging in Rodents and Primates.

Neuron 2017 Jun;94(6):1085-1100.e6

Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan; Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, Suita, Osaka 565-0871, Japan; Division of Bioscience, Institute for Datability Science, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address:

Subcellular resolution imaging of the whole brain and subsequent image analysis are prerequisites for understanding anatomical and functional brain networks. Here, we have developed a very high-speed serial-sectioning imaging system named FAST (block-face serial microscopy tomography), which acquires high-resolution images of a whole mouse brain in a speed range comparable to that of light-sheet fluorescence microscopy. FAST enables complete visualization of the brain at a resolution sufficient to resolve all cells and their subcellular structures. FAST renders unbiased quantitative group comparisons of normal and disease model brain cells for the whole brain at a high spatial resolution. Furthermore, FAST is highly scalable to non-human primate brains and human postmortem brain tissues, and can visualize neuronal projections in a whole adult marmoset brain. Thus, FAST provides new opportunities for global approaches that will allow for a better understanding of brain systems in multiple animal models and in human diseases.
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http://dx.doi.org/10.1016/j.neuron.2017.05.017DOI Listing
June 2017

The use of an optimized chimeric envelope glycoprotein enhances the efficiency of retrograde gene transfer of a pseudotyped lentiviral vector in the primate brain.

Neurosci Res 2017 Jul 28;120:45-52. Epub 2017 Feb 28.

Systems Neuroscience Section, Department of Neuroscience, Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan. Electronic address:

Lentiviral vectors have been used not only for various basic research experiments, but also for a wide range of gene therapy trials in animal models. The development of a pseudotyped lentiviral vector with the property of retrograde infection allows us to introduce foreign genes into neurons that are localized in regions innervating the site of vector injection. Here, we report the efficiency of retrograde gene transfer of a recently developed FuG-E pseudotyped lentiviral vector in the primate brain by comparing its transduction pattern with that of the parental FuG-C pseudotyped vector. After injection of the FuG-E vector encoding green fluorescent protein (GFP) into the striatum of macaque monkeys, many GFP-immunoreactive neurons were found in regions projecting to the striatum, such as the cerebral cortex, thalamus, and substantia nigra. Quantitative analysis revealed that in all regions, the number of neurons retrogradely transduced with the FuG-E vector was larger than in the FuG-C vector injection case. It was also confirmed that the FuG-E vector displayed explicit neuronal specificity to the same extent as the FuG-C vector. This vector might promote approaches to pathway-selective gene manipulation and provide a powerful tool for effective gene therapy trials against neurological disorders through enhanced retrograde delivery.
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http://dx.doi.org/10.1016/j.neures.2017.02.007DOI Listing
July 2017

Using a novel PV-Cre rat model to characterize pallidonigral cells and their terminations.

Brain Struct Funct 2017 Jul 19;222(5):2359-2378. Epub 2016 Dec 19.

Laboratory of Neural Circuitry, Graduate School of Brain Science, Doshisha University, Kyotanabe, 610-0394, Japan.

In the present study, we generated a novel parvalbumin (PV)-Cre rat model and conducted detailed morphological and electrophysiological investigations of axons from PV neurons in globus pallidus (GP). The GP is considered as a relay nucleus in the indirect pathway of the basal ganglia (BG). Previous studies have used molecular profiling and projection patterns to demonstrate cellular heterogeneity in the GP; for example, PV-expressing neurons are known to comprise approximately 50% of GP neurons and represent majority of prototypic neurons that project to the subthalamic nucleus and/or output nuclei of BG, entopeduncular nucleus and substantia nigra (SN). The present study aimed to identify the characteristic projection patterns of PV neurons in the GP (PV-GP neurons) and determine whether these neurons target dopaminergic or GABAergic neurons in SN pars compacta (SNc) or reticulata (SNr), respectively. We initially found that (1) 57% of PV neurons co-expressed Lim-homeobox 6, (2) the PV-GP terminals were preferentially distributed in the ventral part of dorsal tier of SNc, (3) PV-GP neurons formed basket-like appositions with the somata of tyrosine hydroxylase, PV, calretinin and cholecystokinin immunoreactive neurons in the SN, and (4) in vitro whole-cell recording during optogenetic photo-stimulation of PV-GP terminals in SNc demonstrated that PV-GP neurons strongly inhibited dopamine neurons via GABA receptors. These results suggest that dopamine neurons receive direct focal inputs from PV-GP prototypic neurons. The identification of high-contrast inhibitory systems on dopamine neurons might represent a key step toward understanding the BG function.
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http://dx.doi.org/10.1007/s00429-016-1346-2DOI Listing
July 2017

PET imaging-guided chemogenetic silencing reveals a critical role of primate rostromedial caudate in reward evaluation.

Nat Commun 2016 12 6;7:13605. Epub 2016 Dec 6.

Department of Functional Brain Imaging, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.

The rostromedial caudate (rmCD) of primates is thought to contribute to reward value processing, but a causal relationship has not been established. Here we use an inhibitory DREADD (Designer Receptor Exclusively Activated by Designer Drug) to repeatedly and non-invasively inactivate rmCD of macaque monkeys. We inject an adeno-associated viral vector expressing the inhibitory DREADD, hM4Di, into the rmCD bilaterally. To visualize DREADD expression in vivo, we develop a non-invasive imaging method using positron emission tomography (PET). PET imaging provides information critical for successful chemogenetic silencing during experiments, in this case the location and level of hM4Di expression, and the relationship between agonist dose and hM4Di receptor occupancy. Here we demonstrate that inactivating bilateral rmCD through activation of hM4Di produces a significant and reproducible loss of sensitivity to reward value in monkeys. Thus, the rmCD is involved in making normal judgments about the value of reward.
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http://dx.doi.org/10.1038/ncomms13605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5150653PMC
December 2016

Propagated but Topologically Distributed Forebrain Neurons Expressing Alpha-Synuclein in Aged Macaques.

PLoS One 2016 18;11(11):e0166861. Epub 2016 Nov 18.

Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan.

In neurodegenerative disorders, such as Parkinson's disease (PD), alpha-synuclein (α-syn) accumulates to induce cell death and/or form a cytoplasmic inclusion called Lewy body (LB). This α-syn-related pathology is termed synucleinopathy. It remains unclear how α-syn accumulation expands during the progress of synucleinopathy in the human brain. In our study, we investigated the patterns of distribution and propagation of forebrain neurons expressing α-syn in aged macaques. It was found that the occurrence of α-syn-positive neurons proceeded topologically based on the midbrain dopamine pathways arising from the substantia nigra and the ventral tegmental area where they were primarily observed. In the nigrostriatal or mesolimbic dopamine pathway, the age-dependent increase in α-syn-positive neurons was evident in the striatum or the nucleus accumbens, respectively. Concerning the nigrostriatal pathway, a mediolateral or rostrocaudal gradient was seen in the substantia nigra or the striatum, respectively, and a compensatory increase in dopamine transporter occurred in the striatum regardless of the decreased dopamine level. In the mesocortical dopamine pathway, α-syn-positive neurons appeared in the prefrontal and then motor areas of the frontal lobe. Given that neither LB formation nor clinical phenotype manifestation was detected in any of the monkeys examined in the present study, aged macaques may be useful as a potential presymptomatic model for PD and LB-related neuropsychiatric disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166861PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5115821PMC
June 2017

[Neuroanatomy of Frontal Association Cortex].

Authors:
Masahiko Takada

Brain Nerve 2016 Nov;68(11):1253-1261

Systems Neuroscience Section, Department of Neuroscience, Primate Research Institute, Kyoto University.

The frontal association cortex is composed of the prefrontal cortex and the motor-related areas except the primary motor cortex (i.e., the so-called higher motor areas), and is well-developed in primates, including humans. The prefrontal cortex receives and integrates large bits of diverse information from the parietal, temporal, and occipital association cortical areas (termed the posterior association cortex), and paralimbic association cortical areas. This information is then transmitted to the primary motor cortex via multiple motor-related areas. Given these facts, it is likely that the prefrontal cortex exerts executive functions for behavioral control. The functional input pathways from the posterior and paralimbic association cortical areas to the prefrontal cortex are classified primarily into six groups. Cognitive signals derived from the prefrontal cortex are conveyed to the rostral motor-related areas to transform them into motor signals, which finally enter the primary motor cortex via the caudal motor-related areas. Furthermore, it has been shown that, similar to the primary motor cortex, areas of the frontal association cortex form individual networks (known as "loop circuits") with the basal ganglia and cerebellum via the thalamus, and hence are extensively involved in the expression and control of behavioral actions.
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http://dx.doi.org/10.11477/mf.1416200588DOI Listing
November 2016