A remote controlled multimode micro-stimulator for freely moving

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					Acta Physiologica Sinica, April 25, 2006, 58 (2): 183-188                                                                              183
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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: htzhen@mail.xjtu.edu.cn; YUAN
Kui: Tel: +86-10-82614510; Fax: +86-10-62613695; E-mail: kui.yuan@mail.ia.ac.cn
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[16], 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[17] and monopolar pulses are chosen with ref-
                                                                   erence to Stephen et al[18]. 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)[19],                     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


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                                                                                129.
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