Measurement of startle reflex
Authors: Chikhman V.N. , Solnushkin S.D. , Smirnov V.Yu. , Molodtsov V.O. 
Published: July 2024

Observation and registration of the behavior of laboratory animals in response to a sound stimulus (Acoustic Startle Reflex - ASR) is one of the methods of experimental physiological research. The manifestation of this reflex is a startle - a rapid muscle twitching caused by a sudden and intense acoustic signal. Despite its relative simplicity, the magnitude of the startle response is a kind of behavioral tool that can be used to assess the physiological mechanisms of sensory motor reactions. The startle reflex parameters have been measured for a long time. One paper describes a device for measuring the acoustic startle response in mice that uses a spring-loaded arm, a strain gauge, and a polygraph. Individual startle responses were recorded using an ink recorder. Subsequently, they began to measure the reflex using electromyography. With the automation of scientific research, computerized devices are being developed. Known startle detection devices use accelerometers as sensors. These systems are characterized by excessive complexity and high cost. Purpose of our work was development of a simple device and problem-oriented software for the quantitative measurement of ASR based on the IBM PC with the Windows operating system. The floor of the experimental cage, in which the laboratory animal (white rats) is placed, is made in the form of a plastic plate mounted on four foam supports, which, in turn, are supported by four piezoelectric sensors (MakerHawk Analog Ceramic Piezo Vibration Sensor) placed in the bottom of the cage. An acoustic speaker is attached to the top of the cage to give stimulus signal. The board with electronic components is placed under the bottom of the cage. A relatively simple MD308 device has been developed and a program has been written to quantify ASR. The MD308 is based on the STM32L053C8T6 microcontroller, which provides sampling of analog signals from piezoelectric sensors into a digital code using an ADC, transferring the ADC output codes to a computer via the USB interface protocol. Simultaneously with the sampling of signals from the piezoelectric sensors, the audio signal is digitized for time reference in order to further process the recorded signals. The audio signal is formed by a computer using a standard sound card and connected to one of the ADC inputs through a high-pass filter with a signal shift to the middle of the range to convert a bipolar signal to an unipolar one. MD308 digitizes the voltage at the inputs from the piezo sensors and from the audio input in the sampling range from 0 to 2047, with a frequency of 1 kHz, i.e. on five channels, generates packets of 6 such measurements (30 values of 16 bits) and sends them to USB. This sampling frequency is quite sufficient, since the latent time of the startle reaction is about 50–300 ms. MD308 is a USB Custom HID device, data exchange with which is carried out using reports (HID_Reports). The device opens with the name "MD308". At startup, the microcontroller connects via USB to a computer and transfers the results of sampling signals from sensors and audio signal in portions of 60 bytes (ID_Report - 0x03): 6 samples in 5 channels of 2 bytes (channel 0 is the first sample, channel 1 is the first sample, ..., channel 4 is the first reading, then - channel 0 is the second reading, etc.). Since HID devices have a maximum burst rate of 1 kHz and a single burst size of maximum 64 bytes, grouping sample output values by 6 is done for the convenience of further monitoring data on the screen. The microcode for the microcontroller was developed in C++ in the IAR Embedded Workbench for ARM environment. The installation of SMD components (capacitors, resistors, etc.) on the MD308 circuit with the corresponding nominal values is made according to the approved circuit solutions. Schematic development was performed using Altium Designer CAD software. The software for the ASR reflex registration experiment based on the MD308 module is implemented in the Borland Delphi 7 environment, uses the JEDI VCL component library, which includes the TJvHidDeviceController component, which provides access to Custom HID devices. Software modules provide quantitative recording of discretized signals from four piezoelectric sensors, synthesis of an acoustic signal with specified parameters (amplitude, frequency, duration, number of sound stimuli), fixing the time of the beginning of the stimulus. Implemented dynamic display of signals on the display, their saving in the database. There are two blocks in the software - recording signals in the oscilloscope mode and displaying the preliminary processing of signals, which are stored in the database. The registration block allows you to configure the parameters of the experiment. To adjust the parameters of the sound stimulus, a group of visual components "sound" is provided. The experimenter sets the frequency of sound parcels, amplitude and duration. For testing, the buttons "start sound", "stop sound" are provided. The group of components "experiment" is intended for setting the parameters of the experiment - identification of the experiment, the number of sound bursts, the interval between sound bursts. The “start series” button starts the registration of signals and their display in real time. Upon completion of the series, the registration stops, the data can be saved in the database (button "save in the database"). The number displayed in the right corner of the "experience" group corresponds to the number of the sound message. The group of buttons at the bottom of the interface is designed to test the operation of piezoelectric sensors ("start", "stop"), set the signal display scale, and control the "sound" panel. The upper graphic field displays in real time the integrated signal of four piezoelectric sensors, the mark of the beginning of the sound stimulus in the form of a vertical lines (benchmarks). The bottom four fields display the signals separately from each piezo sensor. Graphical interface of the preprocessing block. To navigate through the database of saved data, the "base" group is used, which allows you to select a specific experiment conducted at a designated time, with the specified parameters of the sound stimulus. In the graphical window "response to sound", the registered integrated signal of the piezoelectric sensors, vertical reference points of the beginning of sound parcels, marks of maximum values are displayed in the full recording time. The "processing" graphic windows present the quantitative parameters of the reaction of the experimental animal to the sound impact, tied to the labels of the maximum value of the amplitude of the integrated response - latency, amplitude and integral characteristic of the total response. Transfer of the registered data for the subsequent processing in Excel is possible. The developed hardware and software tools provide a cost-effective measurement of the ASR parameters. The registration of signals is implemented using available standard sensors and an electronic device made using the STM32F042F4 microcontroller at the modern technical level. The software for conducting experiments implements an information system for real-time registration of signals, dynamic display of processes on the display. The system is used for ASR-tests in the study