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An Advanced 3D Printed Design of the Hybrid Infusion-Multielectrode Recording System for Local Field Potential and Single Unit Acquisition and Intrabrain Drug Delivery in Freely Moving Mice
DOI: 10.17691/stm2016.8.4.17 Received February 14, 2016
O. Senkov, PhD, Head of in vivo Electrophysiology Department1;
A. Mironov, PhD, Associate Professor, Department of Neurotechnology, Institute of Biology and Biomedicine2; Senior Researcher, Experimental Modeling Unit, Central Research Laboratory; Associate Professor,
Department of Normal Physiology named after N.Y. Belenkov3;
A. Dityatev, PhD, Professor, Head of Molecular Neuroplasticity Research Group1; Professor, Medical Faculty4
1German Center for Neurodegenerative Diseases (DZNE), 44/64 Leipziger St., Magdeburg, 39120, Germany;
2Lobachevsky State University of Nizhni Novgorod, 23 Prospekt Gagarina, Nizhny Novgorod, 603950,
Russian Federation;
3Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005,
Russian Federation;
4Medical Faculty, Otto-von-Guericke University, 44 Leipziger St., 39120, Magdeburg, Germany
In our recent methodological paper (Senkov et al., 2015, Front Neurosci), we have introduced a highly versatile reusable hybrid infusion-multielectrode recording (HIME) system, which can be utilized in freely moving mice performing cognitive tasks, while their brain activity is being recorded electrophysiologically in terms of local field potential and at single unit resolution, and can be combined with local intrahippocampal or intracortical drug delivery a few hours before behavior experiments. Here, we describe a new more advanced version of the HIME system, where some of its parts can be quickly fabricated using modern 3D printing technology. Our preliminary results show that usage of 3D printers for the construction of small plastic parts of the system facilitates and speeds up the whole fabrication process and improves precision and reliability of HIME implants. It also improves wearability, safety and welfare of mice carrying the system on their heads in the postoperative period and during long-term behavior tests.
Key words: hybrid infusion-recording system; multielectrode system; electroencephalography; local field potentials; hippocampus; drugs delivery.
How to cite: Senkov O., Mironov A., Dityatev A. An advanced 3D printed design of the hybrid infusion-multielectrode recording system for local field potential and single unit acquisition and intrabrain drug delivery in freely moving mice. Sovremennye tehnologii v medicine 2016; 8(4): 129-132, https://doi.org/10.17691/stm2016.8A17
Russian
новая конструкция гибридной инфузионной многоэлектродной системы, отпечатанной на Эй-принтере, для регистрации локальных полевых потенциалов внутримозговой доставки лекарств свободно-передвигающимся мышам
O. Senkov, PhD, Head of in vivo Electrophysiology Department1;
А. Миронов, к.б.н., доцент кафедры нейротехнологий Института биологии и биомедицины2; For contacts: Oleg Senkov, e-mail: oleg.senkov@dzne.de
Hybrid Infusion-Multielectrode Recording System СТМ J 2016 — VQl. 8, No.4 129
старший научный сотрудник отдела экспериментального моделирования ЦНИЛ;
доцент кафедры нормальной физиологии им. Н.Ю. Беленкова3;
A. Dityatev, PhD, Professor, Head of Molecular Neuroplasticity Research Group1;
Professor, Medical Faculty4
1German Center for Neurodegenerative Diseases (DZNE), 44/64 Leipziger St., 39120, Magdeburg, Germany;
Нижегородский государственный университет им. Н.И. Лобачевского, Н. Новгород, 603950, пр. Гагарина, 23;
Нижегородская государственная медицинская академия, Н. Новгород, 603005, пл. Минина и Пожарского, 10/1;
4Medical Faculty, Otto-von-Guericke University, 44 Leipziger St., 39120, Magdeburg, Germany
В нашей последней методологической статье (Senkov и др., 2015 г., журнал "Frontiers in Neuroscience") мы представили универсальную гибридную инфузионно-регистрирующую систему многоразового применения (ГИРС), которую можно использовать для тестирования свободно передвигающихся мышей, выполняющих когнитивные задачи, с одновременной электрофизиологической записью их мозговой активности в терминах локального потенциала поля и с разрешением по каждому электроду. Систему можно комбинировать с доставкой лекарственных средств внутрь коры головного мозга и гиппокампа за несколько часов до поведенческих экспериментов. В данной работе мы приводим описание усовершенствованной версии ГИРС-системы, в которой некоторые детали можно быстро изготовить, используя современную технологию 3D-печати. Наши предварительные результаты показывают, что применение 3D-принтеров для изготовления мелких деталей системы облегчает и ускоряет весь производственный процесс и улучшает точность и надёжность ГИРС-имплантатов. Кроме того, увеличиваются износостойкость, безопасность имплантатов, что способствует нормальному состоянию мышей в послеоперационный период и в течение длительных поведенческих тестов.
Ключевые слова: гибридная инфузионно-регистрирующая система; мультиэлектродная система; электроэнцефалография; локальные полевые потенциалы; гиппокамп; доставка лекарств.
Introduction. Recently, Frontiers in Neuroscience has published our methodological paper in the "Neural Technology" section [1]. There we described a design of a new hybrid infusion-multielectrode recording (HIME) system for local field potential and provided detailed information how to build it from scratch by using inexpensive commonly available small parts. In short, we have used for electrode construction: tungsten wires (0 50/112 |jm) insulated with teflon (originally from A-M Systems, USA, distributed in Europe by Science Products, Germany) bound together in a stainless steel tubing (0 0.7, 0.5, or 0.4 mm), depending on number of wires inside. Each of such electrodes contains from 1 to 5 single tungsten wires, for example, to record from different cortical and/or hippocampal layers, and can be implanted simultaneously up to 6 different brain structures as we show in the paper, but if required, the number of electrodes can be easily increased, if implantation of guide cannulas is omitted. Instead of tungsten wires, other insulated metal wires can be used, such as stainless steel, or even non-movable tetrodes made of 4* formvar coated nichrome wires twisted and melted by heat together (0 18/25 jm). For interfacing electrodes with a 16-32 channels preamplifier, we used soldering-free "pin" technology, when each electrode wire is inserted into its own hole on the printed circuit electrode interface board (EIB-16 or EIB-32 by Neuralynx, USA) and electrical connection is realized through mechanical pinning the electrode wires by small golden pins. Instead of Neuralynx ElBs with Omnetics connectors, one can easily adapt to our HIME system any other boards available from www.open-ephys.org, www.arnoaarts.wix.com,www.adinstruments.com, www.
trianglebiosystems.com and others, or even design one's own customized EIB and order it from a local printed circuitry boards fabrication company. For protection of fragile electrodes, cannulas and headstages on the mouse head from dust and damage during mice housing in animal facilities and during behavior experiments, we proposed to use an easy-to-make protection hat, which additionally serves as a Faraday cage, to reduce electromagnetic/electrostatic artifacts on EEG/units recording in freely moving mice.
Previously, we suggested building it from 5/10 ml plastic syringes properly cut and covered with a grounded cupper or aluminum adhesive tape around the hat to function as a Faraday cage (See Figure (i)). Such design is very practical and can be made in any lab within a few minutes. However, it has some disadvantages, namely: 1) it has two ear-like plastic parts from the syringe on both the hat and the acrylic-cemented pedestal, which might be less ergonomic and comfortable for mice; 2) through these ear-like plastic parts two screws are inserted to hold the hat on the implanted pedestal, insertion of these screws requires some force to be applied, which may damage or displace the whole implant, if done improperly; 3) the protective hat is yet bulky and not customizable for different implants. To overcome all these drawbacks of the previous design, we decided to try to print all plastic parts by using a 3D printer.
Methods. All treatments and behavioral procedures were conducted in accordance with ethical animal research standards defined by German law and approved by the Ethical Committee on Animal Health and Care of the State of Saxony-Anhalt, Germany; the license number: 42502-2-1159DZNE. Special care has been
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130 СТМ J 2016 - vol. 8, No.4 O. Senkov, A. Mironov, A. Dityatev
Hat
Pedestal
O'O
Design of 3D printed plastic parts for the HIME system: (a)-(g) 3D models made in Fusion 360 CAD software for printing with the 3D printer Ultimaker 2; (a)-(c) the protecting hat with a small hole (0 2 mm) for the 10 mm M1 screw to be used to hold the hat on the bottom pedestal; (d)-(g) the bottom pedestal which is supposed to be fixed with dental cement on the mouse head; (e) magnified view of the pedestal bottom part depicting a bigger hole (0 3 mm) for insertion a female screw for the M1 male screw, which is fixed there by melting plastic around with a hot soldering tip; (h) an example of implanted mouse bearing electrodes, 3D printed plastic parts and connected with a 16 channels Neuralynx preamplifier with a long tether/cable; (i) an awake mouse with the removed protecting hat exposing Neuralynx EIB-18, golden pins, electrodes, a ground wire and the dental acrylic fixed to the mouse cranium bone bottom pedestal
taken to minimize suffering, discomfort and number of animals used in the experiments.
To produce improved parts of the HIME system, we used the leading in EU Ultimaker 2 3D printer (www. ultimaker.com/en, Netherlands), which currently offers one of the best quality and precision of prints worldwide, up to 20 ^m layer resolution and speed of 24 mm3/s. However, other modern 3D printers, e.g. with layer resolution 50 or 100 ^m, can be used as well. For designing 3D models, we have utilized Fusion 360 3D CAD/CAM software www.autodesk.com, which is free for non-commercial application and academia. For slicing the 3D models before printing we used Cura, free software available at the Ultimaker website. In short (See Figure (a)-(g)), our EIB protecting hat 3D model has 18 mm in diameter, thickness of the walls just 1 mm and height of the hat 20 mm. In overall, it takes about 20 min to print it with transparent PLA plastic filament (Verbatim,
2.85 mm thickness) with the following printing settings: layer height 100 ^m, fill density 10%, print speed 100 mm/s, nozzle size 0.4 mm. After printing the weight of the hat was ~1 g, its surface was very smooth, and the whole construction was very robust, which allowed reusing the hat several times. The another part of the EIB hat is the implanted on the mouse head pedestal (See Figure (d)-(g)), which has the following parameters: outer diameter is also 18 mm, inner diameter 15 mm, height 5 mm, printing time 10 min, weight 0.5 g. The whole implant including 3D printed parts, EIB, electrodes, pins, screws and Cupper adhesive tape around the hat weighs only ~3 g; together with dental cement it weighs 4-4.5 g (See Figure (h), (i)). Adult wild-type mice with weight ^30 g can easy tolerate up to 6-7 g on their heads, due to very strong neck muscles, so the designed implant can be used in behavior experiments. Depending on the requirements, the protecting hat and the pedestal can
d
a
^mmmmmmmmmmms^^
Hybrid Infusion-Multielectrode Recording System CTM f 2016 — Vol. 8, No.4 131
be printed at least twice smaller, e.g. when only guide cannulas or an optical fiber for optogenetic stimulation are implanted, or less number of electrodes and thus smaller 8-channel-EIB is used.
Results. Testing the HIME system with 3D printed components, we found the following improvements, as compared with the previously published version: 1) it fits to mice more ergonomically and thus wearability is improved; 2) it is easier to restrain mice for 1-2 min by holding on the hat, e.g. while connecting them to the electrophysiological equipment; 3) it also became easier and risk-free for braking the implant to unscrew and remove the hat to expose electrodes/cannulas for manipulations, even on an awake mouse; 4) plastic parts can be printed in different colors, facilitating video tracking of mice in behavior experiments with special software, which can recognize multiple colors and efficiently trace them separately.
Thus, the advanced HIME system exemplifies how 3D printing technology brings more flexibility and potential in design and fabrication of state-of-art neuroscience tools, with a good "quality/costs" ratio and improved characteristics. The improved HIME system can be used for experiments involving injection of extracellular proteases, such as matrix metalloproteinase MMP-9 [2] or enzymes that digest particular glycans, such as endoneuraminidase (EndoNF) for polysialic acid [3], chondroitinase ABC for chondroitin sulfates [4] and heparinase for heparan sulfates [5]. The functions of these and other carbohydrates carried by cell adhesion and extracellular matrix molecules have been mostly studied in vitro [6, 7], and we anticipate that our HIME system will facilitate translation of in vitro insights into mechanistic in vivo studies.
Acknowledgements. The research was supported by the Federal Target Program "Research and development in priority areas of the development of the scientific and technological complex of Russia for 2014-2020" of the
Ministry of Education and Science of Russia, contract 14.581.21.0016 (Project ID RFMEFI58115X0016).
Conflicts of Interest. The authors declare no conflicts of interest.
References
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