Acta Physiologica Sinica, April 25, 2006, 58 (2): 183-188 183 http://www.actaps.com.cn Experimental Technique A remote controlled multimode micro-stimulator for freely moving animals SONG Wei-Guo1, CHAI Jie2, HAN Tai-Zhen2,*, YUAN Kui1,* 1 Hi-Tech Innovation Center, Institute of Automation, Chinese Academy of Sciences, Beijing 100080, China; 2Department of Physiology, Xi’an Jiaotong University, Xi’an 710061, China Abstract: This paper presents a remote controlled multimode micro-stimulator based on the chip nRF24E1, which consists mainly of a micro-control unit (MCU) and a radio frequency (RF) transceiver. This micro-stimulator is very compact (18 mm×28 mm two layer printed circuit board) and light (5 g without battery), and can be carried on the back of a small animal to generate electrical stimuli according to the commands sent from a PC 10 meters away. The performance and effectiveness of the micro-stimulator were validated by in vitro experiments on the sciatic nerve (SN) of the frog, where action potentials (APs) as well as artifacts were observed when the SN was stimulated by the micro-stimulator. It was also shown by in vivo behavioral experiments on operant conditioned reflexes in rats which can be trained to obey auditory instruction cues by turning right or left to receive electrical stimulation (‘virtual’ reward) of the medial forebrain bundle (MFB) in a maze. The correct response for the rats to obey the instructions increased by three times and reached 93.5% in an average of 5 d. This micro-stimulator can not only be used for training small animals to become an ‘animal robots’, but also provide a new platform for behavioral and neurophysiological experiments. Key words: electrical stimulation; electrophysiology; nRF24E1; bio-robotic; multimode micro-stimulator 一种用于自由活动动物的微型多模式遥控刺激器 宋卫国 1，柴 洁 2，韩太真 2,*，原 魁 1,* 1 中国科学院自动化研究所高技术创新中心，北京 100080；2 西安交通大学医学院生理教研室，西安 710061 摘 要：本文采用集成有射频功能的片上系统(nRF24E1)，研制了一种用于自由活动小动物的微型多模式遥控刺激器。刺激 参数的设置及结果分析均由 10 m 外的个人计算机进行。通过离体实验，即刺激蛙坐骨神经干时产生动作电位并可观察到伪 迹；及在体大鼠的训练操作性条件反射的行为学实验，即在 Y 臂迷宫中训练大鼠听到不同声音完成左右运动，如果完成正确 给予前脑内侧束以电刺激，我们观察到 5 d 内大鼠的正确率增加 3 倍，达到 93.5%。上述实验都验证了该刺激器的有效性与 稳定性。本系统具有体积小(18 mm×28 mm 双层线路板)、重量轻(不含电池 5 g)、简单、实用、可靠等特点，能有效地用 于自由活动小动物的实验研究。为基于电刺激的小动物行为训练(动物机器人)及相关神经生理学实验研究，提供了新的实验 条件和实验手段。 关键 词：电刺激；电生理；n R F 2 4 E 1 ；动物机器人；多模式微型刺激器 中图分类号：Q 4 2 4 ；R 3 3 -3 3 Electrical stimulation has been widely used in electrophysi- and rat[6,7], is also arousing refreshed interests from ological research and for the treatment of a variety of neu- bioengineers and artificial intelligence (AI) researchers. rologic and psychiatric disorders for a long time[1-5]. Re- These achievements not only provide insights into how cently the combination of electrophysiology with behav- animals learn, which may eventually lead to better pros- ioral science, such as the locomotion control of cockroach thetics[8-10], but also open new possibilities for AI study. Received 2005-08-25 Accepted 2005-12-08 This work was supported by the National Natural Science Foundation of China (No. 60375026). * Corresponding author. HAN Tai-Zhen: Tel: +86-29-82655274; Fax: +86-29-82655274; E-mail: firstname.lastname@example.org; YUAN Kui: Tel: +86-10-82614510; Fax: +86-10-62613695; E-mail: email@example.com 184 Acta Physiologica Sinica, April 25, 2006, 58 (2): 183-188 The commonly used electrical stimulators need high op- then transmitted to the micro-stimulator. The micro-simu- erating voltage, isolated stimulating channels, or cable lator will generate a specified stimulus sequence to the brain connections, which make them very complex and heavy, of the animal or drive the speaker to give a sound accord- and thus limit their usage in the experiments. With the ad- ing the needs of the experiment. The behavior patterns of vance of microelectronic technology and the validity of the animal can be tracked and recorded automatically by direct brain stimulation by digital signal, different types of an image processing system developed in our lab at the stimulators have emerged for different purposes. But most same time. of them are customized with special technology such as Micro-stimulator application-specific integrated circuit (ASIC)[8,10-13], and the Experiments showed that the intensity needed for direct stimulating parameters are preset or can only be set within neural stimulation is about 100 times smaller than what is a few centimeters via radio frequency (RF) link. Instead needed for muscle stimulation, which means that the stimu- of being used for small animal experiments, these stimula- lators can be more compact with the advances of system- tors can only be used for the experiments with large ani- on-chip technology. The nRF24E1 chip, which has a mi- mals or human beings. Since these stimulators, as well as cro-control unit (MCU) (8051) core and integrates a RF the multi-array electrodes, usually need very complex sur- transceiver, is chosen as the main processor of the micro- gery operation be implemented into animals to carry out an stimulator. The nRF24E1 has the properties of compact experiment, the experiments are usually quite costly and package size, low power consumption, high transmission inflexible. rate, etc. It also provides a pulse width modulation (PWM) Apart from electrical stimuli, physical stimuli, such as analog output to drive the speaker. In order to minimize sound stimuli, may also play an important role in behav- the weight and size of the micro-stimulator, all compo- ioral research, which is lacking in the present stimulators. nents are chosen as surface mounted devices (SMD 0603 Therefore, the aim of our work is to develop a remote package), thus the total weight of the micro-stimulator is controlled micro-stimulator, which is integrated with both less than 5 g. The outlook of the stimulator is shown in audio and electrical stimuli, to explore the behavioral and Fig.2A and Fig.2B. electrophysiological characteristics of small animals in nor- Stimulating pulses can be set either monopolar, as in the mal living conditions. This work is meaningful for the re- traditional stimulator, or bipolar depending according to search on animal’s locomotor behavior under direct brain the needs of the experiments and the characteristics of the stimulation, and then for biomedical, AI and cognitive func- electrodes. Electrodes used for both recording and stimu- tion researches[14,15]. It also has a potential possibility in lating have a sharp tip plated with gold, and bipolar stimu- training an animal into “a remote controlled robot”, as well lating pulses are preferred, in order to minimize the po- as in designing high-efficient brain-machine interfaces larization of the electrode as well as the permanent lesion (BMI)[9,10,12,13]. to the tissue, which is caused by the charge accumulation at the electrode. Bipolar pulses can be achieved by provid- 1 SYSTEM DESIGN ing another negative power supply, which means addi- tional weight and size, so it is impractical for small animal The system is composed of two parts: the remote con- use. By arranging P0 port (P0.3 P0.4), (P0.5 P0.6) of the trolled multimode micro-stimulator, which is fixed on the nRF24E1 as two pairs and by setting high and low voltage back of an animal, and the remote control station, which is alternatively and simultaneously for each pair, we can a commonly used PC. The configuration of the system is achieve bipolar stimulating pulses effectively without the shown in Fig.1. need of negative power. The commanding parameters are set from the PC, and The stimulating electrodes used in this study are self- made concentric needle electrodes, which have been proved to be quite reliable under monopolar stimulation in our pre- vious studies and monopolar pulses are chosen with ref- erence to Stephen et al. The PWM port of the nRF24E1 is used to drive the speaker, and all the idle ports of the nRF24E1 are set to low level in order to minimize the power consumption and to minimize the electromagnetic Fig. 1. The schematic representation of the system. interference. The remote controlled micro-stimulator de- SONG Wei-Guo et al: Remote Controlled Multimode Micro-stimulator for Freely Moving Animals 185 Fig. 2. A rat with the stimulator (A), the main circuit board (B) and program flow chart of the stimulator (C). codes commands from the RF data package and delivers a Table 1. Serial communication protocol specified stimulating pulse sequence or drives the speaker Byte No. 1 2 3~7 This to give sound. The flow chart of the main program is given Data 0xFF 0xAA DATA CHECKSUM in Fig.2C, and the program is written in C51 language and stored in a memory chip (25AA320). The software run- ning on the PC for commanding parameter setting and data which meets the experimental requirement. The protocol analyzing is written in VC++ and, part of the interface is is shown in Table 1, where two bytes of synchronization shown in Fig.3. head (0xFF, 0xAA) is followed by five bytes of command package (DATA), in which each byte represents stimulat- ing polarity (monopolar or bipolar), pulse duration (50 ms ~10 ms), pulse number (1~255), time interval (1 s~ 255 s), data frequency (0.1 Hz ~255 Hz), respectively. The last byte (CHECKSUM) is the check sum of the whole data package. An interruption mode is adopted for the transmitter’s serial communication; receiving data from the PC and transmitting them to the stimulator are accom- plished in the interruption service routine, while the main program will carry out the tasks of initialization and RF configuration. 2.2 Wireless communication The active mode of nRF24E1 is operated at ShockBurst, Fig. 3. The control and monitoring interface of the micro-stimulator. which can clock in data at a low data rate (depending on the speed of the MCU) and transmit data at a high rate (up to 1 Mbps), thus enabling extreme power reduction (60 2 COMMUNICATION MODES AND PROTO- mW) and high sensitivity. While utilizing 250 kbps instead COLS of 1 Mbps would improve the receiver sensitivity by 10 2.1 Serial communication dB, we set a lower rate to achieve high sensitivity. As The communication between the PC and the transmitter is the RF transceiver can only run in simplex mode, in in serial mode and the baud rate is set to 19.6 kbps. Then, order to establish the bi-direction wireless data link, the the time delay of the command package is less than 5 ms, nRF24E1s in both ends need to be configured properly. 186 Acta Physiologica Sinica, April 25, 2006, 58 (2): 183-188 The configuration words consist of 144 bits, which one end of the SN was placed on a recording electrode, complete the ShockBurst configuration and the general while the other end was stimulated by the micro-stimulator, configuration. The data package of the RF is listed in and a reference electrode was laid between them. Ringer Table 2. solution was added to the SN in the experimental session to keep a stable excitability of the sample. After being set Table 2. Radio frequency communication protocol from the PC, the command package was transmitted to Byte No. 1 2~6 7~11 12~13 the micro-stimulator 10 meters away via RF. Then, the Data PREAMBLE ADDRESS DATA CRC action potentials (APs) aroused from the nerve trunk were sent to a data acquisition and analysis system (SMUP, Fudan University) and to an oscilloscope simultaneously. Figure 4 The PREAMBLE is the package head, which is added shows the recorded APs from the SN and the correspond- and removed automatically at the transmitter and receiver ing stimulating pulse (intensity: 3.3 V; monopolar; pulse respectively. The ADDRESS is a receiving address, which needs to be set at the transmitter and would be removed duration: 0.4 ms; frequency: 0.1 Hz; train number: 1 pulse). automatically at the receiver. The DATA is the payload, the The Stimu and Spike in Fig.4 are corresponding to the maximum width (bits) of which is 256 minus the address stimulating pulses and the APs, and the inside box is a width (bits) and the check width (bits). The CRC repre- close-up of a single pair. It shows clearly that the stimulat- sents an 8 or 16 bit checksum. The first byte of the receiv- ing pulses of the micro-stimulator could arouse APs from ing address should not start with 0x55 or 0xAA, which the SN, and the waveforms of the APs and the shapes of might be interpreted as part of preamble and cause address the stimulating artifact shows that it can be used for elec- mismatch for the rest of the address. Also, the addresses trophysiology experiments effectively. made by (5, 4, 3, or 2) equal bytes should be avoided because it would make the packet-error-rate increase. In general, more bits in the address and the checksum will give less false detection, which in the end might give lower data packet loss, but it would result in low transmission efficiency. In order to send commands (6 bytes) reliably and with short delay, the application protocol is configured as single receive channel, 40 bits receive address width, 16 bits check width and 6 bytes of payload for the commands. The nRF24E1 in the PC side is configured as transmitter and in the stimulator side is configured as receiver, which establishes the wireless link. 2.3 Power supply The power supply of the whole system is composed of Fig. 4. The stimulating pulses (lower) and the action potentials (upper) two separate parts: the power supply of the transmitter recorded from SN. The box shows an enlarged single stimulating at the PC end is derived from the USB port of the host pulse and an action potential. PC after voltage regulation (LM2576) from 12 V to 3.3 V, which is very convenient and practical especially in 3.2 In vivo the open field. The power supply for the micro-stimula- Three adult male Sprague-Dawley rats, weighing 290 ~ tor was a lithium cell (3.6 V, 650 mAh, 9 g), which can 320 g, were used (provided by Xi'an Jiaotong University sustain for about 20 h when the micro-stimulator works Animal Center). The surgery operation was performed continuously. under anesthesia, induced by administration of 2% sodium pentobarbital (50 mg/kg). Supplemental anesthesia was 3 METHODS AND RESULTS given when necessary. The animals were stereotaxically 3.1 In vitro planted with several self-made microelectrodes (a teflon At first, a piece of sciatic nerve (SN) trunk was separated coated stainless steel with diameter of 50 mm was put in a from a frog and put in a custom-made shield box. Then, cannula with a diameter of 80 mm) in the medical fore- SONG Wei-Guo et al: Remote Controlled Multimode Micro-stimulator for Freely Moving Animals 187 brain bundle (MFB) (L: –7.5 mm; A: 2.6 mm; P: 2 mm), about 100 kΩ at 100 Hz, thus the effective stimulating cur- and four stainless steel screws were driven in the skull rent is around 30 μA. The parameters of the conditional around the cannula evenly. After the surface of the skull stimulation sound, used as instruction cues, were as follows: was cleaned of all fascias and thoroughly dried, it was one 1-minute of 1 kHz for left turn and three 0.5-minute of treated with erythromycin ointment. The screws and the 1 kHz for right turn. At the end of the behavioral tests, cannula were fixed to the skull firmly with dental cement. each rat was deeply anaesthetized with sodium pentobar- And then, the animals were injected with antibiotics for bital and the stimulating site was marked by the equipment recovery in the following three days. Training was per- (SS-202J, Nihon Kohden, Japan). Then brain sections (40 formed one week after surgery, when the animals had fully mm in thickness) were prepared for identifying the elec- recovered and were behaving normally. A Y-maze para- trode tip in the MFB (Fig.5). Statistical results showed that digm was used to start the following task: for five days the in an average of five days rats reached 93.5% correct cri- rats were trained to run down the maze and respond to terion for acquisition and the averaged correct turns in- auditory instruction cues by turning right or left to receive creased by three times (Fig.6). Behavioral experiments an electrically stimulating reward (MFB). It has been found showed the preference of the animal for the MFB stimula- that stimulating the MFB in the hypothalamus is a good tion as well as the effectiveness of the micro-stimulator enough reward so that the animal will work to obtain the and the method for ‘intelligent-rat’ controlling. stimulation just as it will for food or water[20,21]. Therefore, this method can be used as a ‘virtual’ reward for training. A daily training session consisted of two 5-minute ses- sions with 10 min interval in between (each tone presented randomly). Whenever the maze presented the rats a choice of turning left or right, the micro-stimulator generated a specific sound. When the rat made the corresponding turn, it was immediately given reward stimulation in the MFB and recorded as ‘correct’. If the rat made an incorrect choice, it was deprived of reward stimulation and recorded as a ‘fault’. The animal’s behavior was recorded by a CCD video tracking system. Parameters used for MFB stimulating were as follows: intensity 3.3 V, monopolar pulse width 0.5 ms, pulse inter- Fig. 5. Representative histological section showing an electrode track val 10 ms, pulse number 10, train number 1. The mea- (red line), marking lesion (black arrow) at the bottom of the track and sured resistance of the brain tissue and the electrodes, by the reference marks. A and B are the third ventricle and the using an oscilloscope (VC-10, Nihon Kohden, Japan), is periamygdaloid cortex, respectively. Scale bar, 100 μm. Fig. 6. The training process for three rats. In an average of five days, the rats reached 93.5% correct criterion for acquisition (A) and the averaged correct turns increased by three times (B). 188 Acta Physiologica Sinica, April 25, 2006, 58 (2): 183-188 4 DISCUSSION the ventral tegmental area in rats. Behav Brain Res 1998; 93: 119- 129. This paper presents a remote controlled multimode micro- 6 Holzer R, Shimoyama I. Locomotion control of a bio-robotic stimulator, which can generate physical (audio) and elec- system via electric stimulation. Proc IEEE/RSJ Inter Conf 1997; trical stimulations. The electrical stimulating pulses can be 3: 1514-1519. set to monopolar or bipolar via each pair of isolated channels, 7 Talwar SK, Xu SH, Hawley ES, Weiss SA, Chapin JK, Moxon and the audio stimuli can generate different tones. The elec- KA. Behavioural neuroscience: Rat navigation guided by remote trical stimulating intensity is 3.3 V, and the maximum stimu- control. Nature 2002; 417: 37-38. lating current can reach 15 mA for monopolar or bipolar 8 Heiduschka P, Thanos S. Implantable bioelectronic interfaces square pulses. The effectiveness and performance of this for lost nerve functions. Prog Neurobiol 1998; 55: 433-461. system were validated from both in vitro and in vivo 9 Mussa-lvaldi FA, Miller LE. Brain-machine interfaces: compu- experiments. The results showed that the system can not tational demands and clinical needs meet basic neuroscience. TINS only used as behavioral and neuroscience research plat- 2003; 26(6): 329-334. form for freely moving small animals, it can also be used 10 Nicolelis MAL. Actions from thoughts. Nature 2001; 409: 403- as a tool for small animal’s locomotion training based on 407. electrical stimulation, although more improvements are still 11 Arabi K, Sawan MA. Electronic design of a multichannel pro- needed. grammable implant for neuromuscular electrical stimulation. IEEE As an ongoing work, we have chronically implemented Trans Rehabil Eng 1999; 7(2): 204-214. 12 deCharms RC, Blake DT, Merzenich MM. A multielectrode electrodes in the brain of the rats, and observed some be- implant device for the cerebral cortex. J Neurosci Methods 1999; havioral effects (including learning and memory) by using 93: 27-35. the system to stimulate different nuclei of the brain. At the 13 Chapin JK, Moxon KA, Markowitz RS, Nicolelis MA. Real- same time, we are improving our system by increasing its time control of a robot arm using simultaneously recorded neurons ability to record neuronal activity of the brain and making it in the motor cortex. Nature Neurosci 1999; 2(7): 664- a remote controlled stimulator and telemetric device for 670. freely behaving small animals. We hope to explore the rela- 14 Kohler E, Keysers C, Umilta MA, Fogassi L, Gallese V, Rizzolatti tionships among electrical stimulation, spiking character- G. Hearing sounds, understanding actions: action representation istics and behavioral responses[14,15]. Still further, we hope in mirror neurons. Science 2002; 297: 846-848. it can be used to form a closed loop between the brain and 15 Romo R, Hernandez A, Zainos A, Lemus L, Brody CD. Neu- machine as a BMI [10,12,13]. ronal correlates of decision-making in secondary somatosensory cortex. Nature Neurosci 2002; 5(11): 1217-1225. 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