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1.IntroductionOptogenetics is a powerful tool in neuroscience allowing researchers to use light of specific wavelengths to stimulate select neurons with millisecond temporal and spatial precision. This technique comprises two essential components; the expression of an opsin, or light-sensitive ion channel, in specific neurons forming the optic component, and a controllable light-source required to activate the opsin, the electrical component. Opsins, the transducers at the heart of optogenetics, are a class of ion channels expressed on the cell membrane that open to allow ion flow when they receive light of a specific wavelength.1 There are many opsin subtypes that have been categorized based on their function and their activating wavelength of light but the most commonly used class of opsins is channelrhodopsin-2 (ChR2). When activated, ChR2 opens and allows the conductance of cations such as sodium, potassium, and calcium across the cell membrane to initiate depolarization and generate action potentials.2 The majority of ChR2s are stimulated with the application of blue light of wavelengths ranging from 465 to 473 nm, whereas other variants have been engineered to respond to the red light of higher wavelengths around 620 to 750 nm.3 Other opsins have been discovered, generated, or mutated to allow different kinetics of activation, rates of desensitization, responsivity, and multiphoton activation.4,5 Further genetic engineering in vivo allows cell-type specific expression of the opsins using the cre-recombinase/loxP system where loxP-flanked opsins are introduced by a virus or genetic engineering into specific cells expressing cre-recombinase. This results in the expression of the opsin in only those cells expressing Cre. Further specificity is provided by the location of the fiber-optic when the optoprobe is implanted. Together, this process allows the activation of specific cells and circuits with millisecond precision and is proving to be an invaluable tool in neuroscience research. Optogenetics provides neuroscientists the unprecedented ability to investigate the causal relationships between neural activity and behavior in animal models. The wired systems typically consist of an external light source connected via a long fiber-optic cable and commutator, thus tethering the subject to the light source. This imposes some restrictions on a test animal’s movement and limits the application of the experiment. Wireless optogenetic stimulators are commercially available, but they are often too expensive for a standard research budget. One such system available is a head mounted, inductively powered wireless device for mice models with a high starting price for the control unit and further costs per head mount.6 On the other hand, there are notable open-source designs available but these require specialized manufacturing facilities. Several designs have been published by the Bruchas and Rogers Research Groups. These use specialized micro- and nanofabrication techniques and require a number of days to manufacture.7,8 Another design by the Poon group utilizes radio-frequency (RF) power to wirelessly power and control a subcutaneously implanted light source but requires manufacturing of a complex RF harvesting resonant cavity. Additionally, operation of the device is restricted to the 21-cm diameter of the RF chamber.9 RF has also been used in an earlier design by the Boyden Group, perhaps one of the first wireless devices to be published.10 The field has also advanced by incorporating microfluidics to allow a precise delivery of fluids to the neurons being activated11 and to assess simultaneous electrical activity.12 Another notable design is the infrared (IR)-based stimulator described by Hirase et al.,13 which is the most similar available device. While being easier to implement compared to other devices, the need of proprietary software such as LabView to control the parameters of activation for the light-emitting diode (LED) light raises the startup cost. This device also requires more sophisticated manufacturing techniques than CerebraLux, which was designed and built by under-graduate students. To address these hurdles, we developed CerebraLux: a low-cost, wireless optogenetic probe. The device is designed to be simple to manufacture and assembled without the need for specialized facilities or training. CerebraLux is powered by a light rechargeable lithium polymer (LiPo) battery with sufficient capacity for full behavioral experiments. Most components of the device such as the fiber-optic and electronic surface-mount devices (SMD) are inexpensive and commercially available, whereas custom parts, like the printed circuit board (PCB) and the baseplate, can be manufactured using consumer-grade machines or commissioned from widely available manufacturing services. To demonstrate the feasibility of our system in vivo, we implanted our device in the right striatum of mice genetically engineered to express ChR2 crossed with transgenic mice expressing cre-recombinase in dopamine receptor 1 (D1) neurons. This mouse model was chosen as it provides a visually identifiable response to light activation, counter-clockwise turning behavior, and increased motor activity. For the full realization of the open-source concept, we have included all of our materials, design specifications, computer aided designs (CAD), and computer-aided manufacturing (CAM) files, PCB design, and programming protocols for the microcontroller unit (MCU) and IR communication along with detailed instructions for device assembly and troubleshooting in our compiled CerebraLux manual, included as an Appendix, with this paper. We propose that this information provides a building block to be used by all, either as is or as a building block for further development of the technology. 2.Methods2.1.Design and Manufacturing of CerebraLux2.1.1.Overview of the systemCerebraLux consists of an optic and an electronic component (Fig. 1). The optic component includes a baseplate that holds the fiber-optic and its ferrule. The electronic component includes an MCU, an IR receiver, and an SMD LED soldered on a double-sided PCB with a removable LiPo battery. Only the optic component is permanently housed on the mouse’s head. The two components are aligned and held together using small magnets. This allows the electronic component to be removed when not in use. 2.1.2.Optic componentAn overview of the optic component is shown in Fig. 2. This figure shows the baseplate, made of high-density polyethylene (HDPE), containing the fiber-optic at specific - and -coordinates and two magnets for alignment with the electronic component. The baseplate was designed in Autodesk Fusion 360 https://www.autodesk.com/products/fusion-360/students-teachers-educators, an open-source three-dimensional (3-D) design software.14 We used this software to generate the CAM toolpaths, and exported the resulting G-code to Otherplan to be milled by an Othermill computer numerical control (CNC) mill (Other Machine Company https://www.bantamtools.com/products/hdpe, Berkeley, California). Step-by-step instructions for the baseplate design, milling, and files for download are available in the CerebraLux manual (Appendix: Secs. A1.1–A1.3) and lab website (Walwyn Lab http://www.walwynlab.semel.ucla.edu/new-page), respectively. We inserted a fiber-optic of specified length based on the coordinates for surgery (Doric Lenses, diameter, NA 0.63, plastic optical fiber) into a ferrule (Doric Lenses, Zirconia Ferrule OD 1.25 mm). Finally, we inserted the ferrule with the fiber-optic into its channel in the baseplate and glued the magnets (Magnet4us Inc., D0201N40) into their respective slots in the baseplate (Appendix: CerebraLux manual Sec. A1.4). 2.1.3.Electronic componenti. Software. We used an open-source IR remote library developed by Ken Shirriff for the Arduino platform to send and receive signals, which control the LED. A simple graphical user interface (GUI) was designed to control the stimulator. Figure 3 shows the GUI, which receives the input values for on time, off time, and light intensity, and forwards the information to the device. The GUI communicates serially through a universal serial bus (USB) port to an Arduino Uno (ArduinoBoardUno) to send the corresponding IR LED pulses to the device. We designed the GUI in the Python 2.7 TkInter library, the most widely accessed GUI library. Constructing the GUI in Python instead of proprietary software such as LabVIEW and MATLAB ensures free access and programmability of the interface. By providing our interface as a standalone executable file, researchers can use the interface as is or modify it further to fit their experimental needs. The TkInter library is also well supported, with thorough documentation online in forums and tutorials. The program operates by sending three signals for on time, off time, and light intensity, through the infrared 950-nm LED (SparkFun, Boulder, Colorado) in the form of three IR LED flashes. The IR receiver on the stimulator receives these IR pulses, and the microcontroller processes them to assign the corresponding on–off times and light intensity for the stimulating LED. The order of activation is outlined in Fig. 4. The codes for the sending and receiving protocols in the Arduino interactive development environment (IDE) can be found in the Appendix; CerebraLux manual (Secs. A2.2 and A2.3) while the GUI is downloadable (Walwyn Lab http://www.walwynlab.semel.ucla.edu/new-page). ii. Hardware. We ported the code onto an ATMega328p MCU using the protocol outlined by Arduino. We then soldered the ATMega328p microcontroller (Atmel, San Jose, California) surface mount IR receiver (Vishay Electronics, Malvern, Pennsylvania), and surface-mount the LED (Kingbright USA, City of Industry, California) onto a custom PCB. We designed the PCB in Cadsoft EAGLE and ordered the PCB to be manufactured by OSHpark. The fully assembled PCB is shown in Fig. 5. We connected a 3.7-V, 20-mAh capacity rechargeable LiPo battery (Tenergy, Fremont, California) to the PCB using female headers (SparkFun, Boulder, Colorado). The LiPo battery chosen weighs under 1 g and fits within a footprint on the PCB. Finally, we placed two magnets on the bottom side of the PCB that are aligned with those on the baseplate. The final assembly of the electronic component is shown in Fig. 5. Details of all materials used are in the Appendix: CerebraLux manual (Sec. A2.1) and step-by-step instructions to manufacture the PCB are described in the Appendix: CerebraLux manual (Sec. A2.4). 2.2.In Vivo Testing of CerebraLux2.2.1.Surgery to install the CerebraLux baseplateAll animal experiments were conducted with the approval of the UCLA IACUC (OARO). Surgeries were completed in a sterile surgery suite, using a table that allowed 360 deg access and tools that had been sterilized prior to surgery. A Model 1900 Stereotaxic alignment system (Kopf, Tujunga, California) was used for the surgeries. Before use, the Stereotaxic instrument was calibrated using a centering scope (Model 1915, Kopf, Tujunga, California) by centering over the height gauge, identifying, and setting the zero point of reference for the manipulator readout display. The optogenetic mice used were created from two strains: Ai27[RCL-hChR2(H134R)/tdT]-D (JAX #01256) and B6.FVB(Cg)-Tg(Drd1a-cre)EY262Gsat (Mmucd# 030989-UCD) to obtain ChR2 expression in D1 neurons in this Ai27 x D1 Cre mouse line. The mouse was placed in an induction chamber (Patterson Scientific, Foster City, California) and anesthetized with 2.5% isoflurane (Isoflo, Zoetis Inc., Kalamazoo, Michigan). Once breathing slowed, the mouse was removed from the chamber and depth of anesthesia was verified by no response to a toe pinch (stage 3, plane 2 of general anesthesia). Mice were shaved (Wahl, Sterling, Illinois) from eyes to neck along the midline and placed into the nose cone (Model 1923-B Mouse Gas Anesthesia Head Holder, Kopf, Tujunga, California) on a surgical heating pad (Harvard Apparatus, Holliston, Massachusetts). Anesthesia was maintained by 1.5% to 2% isoflurane. 0.5 ml of saline solution (0.9% sodium chloride USP, Hospira, Lake Forest, Illinois) with 1% carprofen (Rimadyl, Zoetis Inc., Kalamazoo, Michigan) was injected subcutaneously to provide fluids and pain relief. Ear-bars were placed so as to secure the skull. The shaved area was cleaned with alternating alcohol and betadine wipes. A 2-cm long incision was made with a #22 scalpel (Miltex, York, Pennsylvania) along the midline from between the eyes to the back of the skull. Brain planarity was verified with an alignment indicator tool (Model 1905, Kopf Instruments, Tujunga, California) on the bregma. Coordinates of bregma were recorded, and the coordinates of probe insertion into the caudate calculated [striatum; , from bregma, and ]. The skull was softly scored with a scalpel and the hole drilled at the correct -coordinate (Model 1911 Stereotaxic Drilling Unit, Kopf, Tujunga, California). The baseplate with aligned fiber-optic was secured to the stereotaxic frame by the stereotaxic adapter (Appendix: CerebraLux Manual; Sec. A1.5) attached to a cannula holder (Kopf Instruments, model 1766-AP). The baseplate was lowered slowly to the correct coordinate. Isoflurane was lowered to 1% and a two-part dental cement (Bosworth Trim II, Keystone Industries, Gibbstown, New Jersey) was applied around the baseplate to form a “dome” around the baseplate and to the edge of the scalp. After 5 to 10 min of drying time, the stereotaxic arm and adapter were raised slowly to break the magnetic attachment, leaving the baseplate securely attached to the skull. Isoflurane was then reduced to 0% allowing the mouse to recover from the anesthetic. A triple antibiotic ointment (Perrigo Company plc, Allegan, Michigan) was applied externally around the incision using a Q-tip. After heart rate and breathing increased, the mouse was placed into its home cage preheated on a heating pad (Sunbeam Products Inc., Boca Raton, Florida) until sternal and normal behavior observed. The sham surgery to implant an optoprobe lacking a fiber-optic was completed in the same manner but no hole was drilled in the skull, and the fiber-optic was not inserted. 2.2.2.Behavioral testingBehavioral testing was completed in a quiet, dark room. A Basler camera model aca1300-60 gc (Ahrensburg, Germany), equipped with a 1/2” Computar IR lens (CBC AMERICAS Corp., Cary, North Carolina) was secured (0.85 m) below the frosted glass panel, in which the subject chambers were placed. The camera was connected to the computer (Windows 10 OS), which ran the behavioral testing software, Ethovision XT 7 (Noldus, Leesburg, Virginia). Tests were conducted in a behavioral arena that was either square ( WxLxH) or circular (17.5 cm in diameter) and data recorded and analyzed by Ethovision (vXT7). Testing protocol. Mice were handled for 3 to 5 days prior to testing. On the day of the test, we first connected the fully charged battery to the PCB, then attached the PCB to the baseplate on the mouse’s head while holding the awake mouse in an open gloved hand. The mouse was then placed into the arena and 5 min of baseline behavior with the light off recorded followed by 5 min of behavior with the light on and the last 5 min with the light off. Statistical analysis. We used Ethovision to measure the number of counter-clockwise and clockwise rotations, total distance traveled, velocity, and time spent mobile in mice implanted with a white-HDPE baseplate (). Video recordings were checked for extraneous tracking and the data exported to and analyzed in Graphpad Prism v7 (Graphpad, La Jolla, California). Ethovision was also used to assess the number of counter-clockwise and clockwise rotations in mice implanted with a black HDPE baseplate () or the sham mouse implanted with the optoprobe lacking the fiber-optic () and the data exported to Microsoft Excel (v10) and graphed in Graphpad Prism v7. 3.Results3.1.Transmission RangeWe first measured the distance that our device was able to consistently send and receive signals and found this to be 1.8 m. This distance was not affected by glass or plastic in the light path. However, we did find that fluorescent lighting interfered with the IR transmission so it is advisable to use low or alternative sources of light. 3.2.Stimulator Light OutputThe ability to adjust the light output from the stimulator is critical. Excessive light power may compromise neuron function, whereas light power below the activation threshold will not activate ChR2. The minimum light power needed to activate ChR2 depends on a number of factors including the light attenuation properties of the target brain region, ChR2 expression levels, and the age of the tissue. Typically, 2 to is considered an acceptable irradiance range to successfully stimulate opsins.15 Power can also be manipulated by incorporating pulse-width modulation (PWM) to apply the light at a specific frequency (Hz). A range of frequencies may be used in vivo and in vitro16–19 but for the tests in this study we used 10 Hz, or 10 ms on and 90 ms off. This is done by changing the “ON” and “OFF” time variables in the GUI (Fig. 3). Power can also be manipulated by changing the forward voltage, shown as the intensity variable in the GUI (Fig. 3). This was assessed in vitro by placing a fully assembled device in an integrating sphere (PMD100, Thorlabs, New Jersey) to assess light power output from the tip of the fiber-optic. Furthermore, the performance of rechargeable batteries may be affected by the number of recharge cycles. We, therefore, tested power output using three different batteries that had been recharged once (battery 1), three times (battery 2), or multiple times (battery 3) beforehand. The data are shown as total power output () or irradiance [, Figs. 6(a) and 6(b)]. We found a linear relationship () between the forward voltage and light power output with a peak average output of or . Interestingly, the power output declined by only 3% when the forward voltage was reduced by 40%. Across all voltages, the newer battery (1) had a higher power output compared to the older batteries (2,3), but all batteries showed the same % reduction with decreasing forward voltage. Details of the assembly and use of these batteries can be found in the Appendix: CerebraLux manual (Sec. A2.5). 3.3.Stimulator Light Output over a Single Discharge Cycle (Battery Runtime)Well-characterized battery life and power output over a discharge cycle provide critical information for a proposed experimental design. We measured the runtime of the 3.7-V 20 mAh battery tested at a 10-Hz frequency and 100% forward voltage while recording light power output from the fiber-optic tip every 5 min until the battery was not able to maintain the threshold light output of . We used the same three batteries as used in the previous test that been recharged once (battery 1), three times (battery 2), or multiple times (battery 3) beforehand. The data show a decline in power output over time (Fig. 7). During the first 30 min, the power output of all three batteries show a steady decrease of for each 10 min of stimulation that is independent of the initial power output. Thereafter, power output declined but remained above 50% of the initial output for 35 min (battery 3) or 50 min (batteries 1 and 2). Together, these data (Figs. 6 and 7) show that power output varies relatively little if the forward voltage is reduced, but it is influenced by the number of prior recharge cycles of the battery and the time in use. These data show that it is important to monitor these two parameters during and between experiments. 3.4.Testing the Stimulator In VivoActivating dopamine 1 (D1) receptors in direct pathway striatal projection neurons increases turning behavior.20 Optogenetic activation of these neurons also increases locomotion and velocity.21,22 We proposed that CerebraLux activation of ChR2 in neurons expressing D1 receptors in the right side of the striatum would increase both counter-clockwise rotations and total mobility. This was tested by connecting the electronic component to the in situ baseplate, made of white HDPE [Fig. 8(A)], and placing the mouse, from the genetically engineered Ai27 × D1 cre line, in the recording chamber. Details of the operating instructions of the probe and software can be found in the Appendix: CerebraLux manual (Sec. A3). Three mice were used for these tests. After 5 min of basal recording, the optoprobe was turned on (10 Hz, 100% intensity) for 5 min and then off for the final 5 min of the test. Figure 8(Aa) shows the mouse with the optoprobe turned on in the arena used. During this period, counter-clockwise rotations increased [, ] when compared with the basal off period [Fig. 8(Ab)] without changing clockwise rotations [, Fig. 8(Ac)]. Activation of the optoprobe also increased distance traveled [, , Fig. 8(Ad)], velocity [, , Fig. 8(Ae)], and mobility [, , Fig. 8(Af)] versus % basal levels. An example of this behavior is seen in the video (Fig. 9). Together these data show that CerebraLux produces the predicted behavioral outcome, an increase in turning behavior, in mice expressing Ch2R in direct pathway striatonigral neurons. So as to reduce light scatter, we repeated these tests using a baseplate made of black HDPE in two additional mice of the same genotype [Fig. 8(B)]. This reduced light scatter [shown by the frame taken when the oproptobe was turned on in Figs. 8(Bg) versus 8(Aa)] but did not change the % increase in counter-clockwise rotations during the on versus basal off period [350% versus 348% in mice with black versus white HDPE, respectively, Fig. 8(Bh)]. Turning the optoprobe on also induced a similar % change in clockwise rotations compared with the basal off period; 138% versus 157% in mice with black versus white HDPE, respectively [Fig. 8(Bi)]. In addition, the sham mouse, from the Ai27 × D1 cre line, in which an optoprobe lacking the fiber-optic was implanted, showed no change in counter-clockwise rotations [Fig. 8(Bh)] and a decrease in clockwise rotations [Fig. 8(Bi)] when the probe was turned on. These data show that the use of the black HDPE baseplate did reduce light scatter but did not affect turning behavior resulting from activation of striatal D1 neurons. 4.DiscussionThis paper provides all the information and methodology required to build an inexpensive wireless optoprobe, CerebraLux. Using these guidelines and specifications, any laboratory interested in optogenetic studies, regardless of budget, will be able to build this lightweight and inexpensive wireless probe. The highlights of CerebraLux are: 4.1.Ease of UseThere are several features of the CerebraLux design that make it easy for a researcher to use. The low-profile lightweight baseplate is stable, so it is unlikely to come loose once secured to the skull. The magnets on both the baseplate and PCB allow easy application of the PCB to the mouse. This plug-and-play feature contrasts with wired system optoprobes where anesthesia is often required to connect the mouse. Once in place, the IR-based controller with a transmission range of 1.8 m facilitates complex experimental designs that are not possible using wired or some wireless devices. Once the device has been activated, the mouse can move through mazes or doors allowing further flexibility in experimental designs. Not only is the total light power output adjustable using PWM, the rechargeable battery is also able to sustain 30 min of activation, considerably longer than the typical activation period. Finally, the ability to control CerebraLux through downloadable software on any computer is both easy and inexpensive. The GUI is written in open-source Python to ensure access and programmability by researchers, whereas the IR communication protocol is written in the widely documented Arduino IDE. With all of the implemented features, the total weight of CerebraLux is 2.8 g, of which the mouse permanently carries 0.5 g of the optic component, with the remaining 2.3 g electronic component being detachable. This allows the mouse to move unencumbered during and after experiments. A summary of the features of our device in comparison with similar devices is shown in Table 1. Table 1Comparison of CerebraLux with other available devices.
Note that only CerebraLux can be manufactured without microscale fabrication techniques. All parts of CerebraLux are available either off the shelf or are easily reproducible using standard manufacturing tools accessible to most laboratories. 4.2.Ease of AssemblyThe CerebraLux device is designed to be assembled without prior electronic and manufacturing experience. Step-by-step instructions detailing how to manufacture every component of the device are included in the CerebraLux manual. It is also possible to outsource the manufacturing of certain components (e.g., the baseplate) to high-resolution 3-D printing or laser sintering services such as Protolabs. Most of the components, except those that are milled, are commercially available. The baseplate can be machined in 1 h using a low-cost, consumer-grade CNC mill, and the stereotaxic adapter printed in 15 min using a 3-D printer (uPrint SE, Stratasys, Eden Prairie, Minnesota). The PCB board can also be manufactured on the Othermill on a copper board, which is less expensive than OSH Park. Once the PCB is manufactured, researchers can follow the instructions in the CerebraLux manual on soldering all the surface mount components (Sec. A2.4). Manufacturing and assembly of CerebraLux requires a total of 3 h once all the components are attained. In contrast, the cellular-scale stimulator created by the Bruchas research group requires specialized microfabrication facilities and a lengthy production time of two weeks to complete.7 CerebraLux’s rapid turnaround time of a day in manufacturing means that design iterations can be developed and tested in a shorter span of time. Additionally, multiple devices can be assembled and implanted at the same time, thus increasing the number of optogenetic studies that a laboratory can conduct within a particular time frame. 4.3.Cost AnalysisCerebraLux alleviates the financial barriers to optogenetics technology with its open-source design and low cost. Table 2 shows a price breakdown of all components used in our device and a cost analysis comparing our device with the current standards in the field of optogenetics. Our starting price of $108.10 is considerably less expensive than the Cambridge Neurotech device6 or the RF resonant cavity proposed by the Poon group9 and other suppliers. It is important to note that this cost analysis does not include the cost of purchasing a CNC mill. However, these are often available on university campuses and, even if outsourced, CerebraLux still remains less expensive than similar commercially available devices. Table 2Price breakdown of all components used in CerebraLux.
Note: Excluding the Arduino, the fiber-optic and its ferrule holder, all other components are individually priced at under $10 each. 4.4.In Vivo TestingOur behavioral experiments demonstrate proof-of-concept that we are able to target and stimulate opsin-expressing cells in the brain with our low-cost device. As previously mentioned, the appropriate light power needed for an experiment often falls within a narrow range. Our device is able to produce of power, which, for a diameter fiber, corresponds to irradiance values of . Our behavioral testing shows that this is adequate to stimulate ChR2. It must be noted that this is affected by the battery, in particular, the number of recharge cycles. Our data show that a greater number of recharge cycles reduces the maximum light power output and runtime. This reduction can be partially offset by careful soldering of the connections and observing the manufacturer guidelines for care and recharging of the battery. In addition, as these batteries are relatively inexpensive and available, a supply of backup batteries would be advisable. 4.5.Future DirectionsIn providing the complete specifications of CerebraLux, we are providing the foundation, in which this and future iterations of CerebraLux may be built. We have shown that, as is, CerebraLux works in vivo. As with any engineering project, there are also future directions and improvements that would enhance and improve this probe. One of these is the modality used to activate the probe. We successfully utilized IR signaling as a medium of communication between the researcher and the stimulator. IR signaling is easy to implement using commercial off-the-shelf components such as IR LEDs and Arduino boards. The ease of IR programmability was also demonstrated by sending signals with an Arduino Uno utilizing popular open-source IR libraries. However, IR requires line-of-sight communication and is affected by ambient light sources, which may restrict the type of behavioral studies that can be done. In future iterations, an approach similar to Hirase et al.13 can be adopted using an array of LEDs to transmit the signals from multiple angles, ultimately reducing interference and signal loss. Alternatively, the fragility of IR transmission can be bypassed through use of Bluetooth Low Energy 5.0 technology embedded in microcontrollers; however, this method also presents challenges, such as implementing Bluetooth communication protocols. Future iterations of our device should also aim to maximize efficiency of the optic assembly. Currently, the LED we used produces 8.1 mW of light power at maximum forward voltage, but 0.824 mW of total power or is transmitted through the fiber-optic at a 10-Hz frequency, a transmission efficiency of 10.6%. It must be noted that our device utilizes direct butt-coupling between the diode and the waveguide with no optical focusing mechanism, explaining the low transmission efficiency. An optical focusing mechanism could be established by lenses and condensers that would collimate the light from the LED before transmission through the fiber-optic and allow the device to be operated in experimental setups with greater light power needs. However, incorporating such focusing elements would also increase the weight and height profile of the device. The baseplate could also be manufactured with higher precision and tolerance to improve alignment between the diode and fiber-optic and to further improve light power transmission. These iterations would increase power output and would also allow a lower voltage to be used, prolonging overall battery life. Another iteration would be to use black HDPE [Fig. 8(B)] for the baseplate so as to minimize light leakage and prevent the mouse from visually reacting to the light. Our baseplate design can also be altered to target different classes of opsins. By simply changing the location of the ferrule hole in the baseplate during milling, we can align the fiber-optic with a different color diode on the LED. The LED used here has three color settings (i.e., red, green, blue), which can be programmed to allow for stimulation of a wider range of opsin subtypes. A new baseplate design would also be needed to include bilateral probes. This would require multiple LEDs or a single LED with a light splitter being used to stimulate different targets in the brain. A change in the baseplate design may also be needed for different -coordinates for different brain regions or for use in other animal models. One more consideration for a future design is the addition of electrophysiological recording capabilities. To acquire measurements such as temperature and electric signals, our device will require extensive modifications to the device programming, microcontroller selection, and chassis design. However, the addition of this function would require specialized fabrication techniques, which may make the device harder to manufacture and less accessible for all to use. 5.ConclusionWe have presented a complete system for optogenetic stimulation in rodents. Our primary objective was to make our device inexpensive and fully open source. We have successfully kept the cost of manufacturing a fully functional and lightweight optogenetic stimulator under $200. We have included our designs and assembly instructions for the neuroscience community to use and/or change as necessary. Thus, our optoprobe strikes a balance between cost, ease of manufacture, and functionality and hope that this will enhance accessibility to the optogenetics toolkit. AppendicesAppendix:Design and Operations ManualA1.BaseplateA1.1.DesignThe baseplate is the “landing pad” that aligns the LED and its controllers with the fiber-optic. It is permanently attached to the mouse’s head. The top of the baseplate has a rectangular slot for the LED to fit into. Adjacent to the LED’s slot are recesses to accommodate the LED leads and solder joints. The top face of the baseplate also has two cylindrical slots for magnets to be glued into. These two magnets are how the electronic components of CerebraLux are secured onto the baseplate. The hole for the fiber-optic ferrule is positioned so that when the LED is inserted into the baseplate face-down, the blue diode aligns with the fiber-optic. The proper position for the fiber-optic ferrule channel was determined with digital calipers. The ferrule of the fiber-optic also functions as a surface for the dental cement to adhere to. A1.2.MaterialsThe baseplate used in CerebraLux was manufactured by CNC milling a block of HDPE. Any soft plastic or metal may be used to create the baseplate, but a lightweight material should be chosen to reduce burden on the test animal. A diameter fiber-optic was used in CerebraLux to maximize the amount of light that the fiber can receive from the LED. Because CerebraLux does not incorporate any light focusing mechanism, a large diameter fiber is recommended to maximize total light power even though light intensity will decrease as a result of the larger surface area. A 1.27-mm diameter ceramic ferrule was used to house the bare fiber-optic, increasing our ability to polish and handle the fiber-optic. A ferrule is not strictly necessary if the baseplate is modified to accommodate the smaller diameter of the bare fiber-optic instead of a ferrule. 1/16” diameter magnets are used to secure the electronic components to the baseplate. The magnets are glued into their respective slots on the baseplate and on the PCB in alternating polarity to ensure only one way of alignment. A1.3.MillingOur team used an Othermill tabletop CNC machine to mill the baseplate out of white/black HDPE (HDPE https://othermachine.co/store/materials/hdpe/). Below are instructions for milling the baseplate using the Othermill. A1.3.1.Downloads
A1.3.2.Steps to mill
A1.4.Assembly
A1.5.Stereotaxic adapterTo assist in implanting the device, a stereotaxic adapter can be designed and 3-D printed (Fig. 13). The stereotaxic adapter attaches to the baseplate through magnets (shown below) and fits into a cannula holder available from Kopf (Kopf, #1766). Our adapter CAD design is available for download at the Walwyn Lab http://www.walwynlab.semel.ucla.edu/new-page website. A2.ElectronicsA2.1.MaterialsCustom PCB
IR transmitter and receiver modules
Battery and Charger
Microcontroller programming
Soldering
A2.2.DownloadsArduino IDE 1.6+: https://www.arduino.cc/en/Main/Software To use IRremote library:
Python IDE 2.7+ (NOT Python 3): https://www.python.org/downloads/ Pyserial: http://pyserial.readthedocs.org/en/latest/pyserial.html
Python Imaging Library (PIL): http://pillow.readthedocs.org/en/3.0.x/installation.html
Source code
Table 3IR Sending Protocol.
Table 4IR Receiving Protocol.
A2.3.Programming the MCU
A2.4.Building the PCB
A2.5.Battery
AcknowledgmentsThanks are due to Dr. Stephanie Seidlits and Dr. Dino Di Carlo from the Department of Bioengineering at UCLA for advice and guidance. Funding from the Hatos Foundation and NIDA (DA 005010) is gratefully acknowledged. R.D., Y.Y.L., J.A., M.C., A.J., V.Q., J.C., W.W. designed and built the probe. M.M., T.K. assisted and advised. A.M. and J.H. implanted the probe. R.D., Y.Y.L., A.J., A.M., J.H., G.S., and J.C. tested the probe in vivo. R.D., Y.Y.L., G.S., and W.W. wrote this paper. Data Availability: All data generated or analyzed during this study are included in this published paper and supporting files. ReferencesK. Deisseroth,
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BiographyRobel Dagnew is originally from Addis Ababa, Ethiopia, where he finished his secondary education. He moved to Los Angeles in 2012 to pursue higher education and received his BSc degree in bioengineering from UCLA in 2016. From 2016 to 2017, he was in charge of manufacturing and maintaining an IR-based optogenetic device for the Psychiatry, Biobehavioral Sciences and Neuropharmacology Department at UCLA. He has been currently enrolled in the computational biology PhD program at USC, California. Jesse Cushman received his PhD from UCLA in the lab of Dr. Michael Fanselow and postdoctoral training in the lab of Dr. Mayank Mehta. He ran the behavioral testing core at UCLA for 3 years before recently moving to start up an intramural neurobehavioral core at the National Institute of Environmental Health Sciences. He received initial training in optogenetics at the Stanford Optogenetics Innovation Laboratory. Wendy Walwyn is a professor of the Semel Institute, David Geffen School of Medicine at UCLA. She has spent many years working on the addictive properties of prescription opioids. Her research focuses on why this type of addiction is so difficult to treat. She has developed this optoprobe so as to define the pathways and circuits in the brain that play important roles in opioid abuse. |