Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

All procedures follow the Guide for the Care and Use of Laboratory Animals (NIH Publications N°. 8023, revised in 1978) implemented by National Health Institutions and local Mexican laws to reduce the number of animals used for animal welfare and prohibition of animal suffering. The Ethics Committee of the Universidad Iberoamericana approved the experimental protocols for the use of animals in this study.

1. General setup

1. Install the Behavioral Tracking Software on a computer according to the manufacturing instructions.
2. Mount the camera directly above the device, so it faces downward. The camera should be connected to the computer.
3. Install the driver's software required by the camera (following the manufacturing instructions).
4. If the camera includes zoom lens, adjust them to fit perfectly in the camera's display.
5. Turn off the camera autofocus (AF) mode following the manufacturing software.
6. Ensure that the camera is working correctly in real-time and test the video capture mode until it is ready to use.

2. Environmental enrichment protocol (see Figure 1)

NOTE: Three-month old male Wistar rats were used for this experiment and were maintained under natural dark-light conditions.

1. Place sawdust bedding in a transparent acrylic square arena (500 x 500 x 500 mm).
2. Put three different kinds of toys on the arena for rodents to interact with (e.g., activity wheels, double deck, stairs, etc.).
3. Add four 2-inches and four curved gray opaque PVC tubes.
4. Provide food and water dispensers with ad libitum access to animals.
5. Place three rodents per cage inside the vivarium room under regular conditions.
6. Leave the animals in this arena for the time required according to the corresponding protocol. In this experiment, animals should stay inside the arena for 20 days.
   NOTE: After the electrode implantation surgery, animals do not go back to environmental enrichment treatment. Instead, they were put in single cages until the novel object recognition test is completed.

3. Electrodes manufacturing process

1. Cut a piece of copper wire at approximately 2 cm and use a sandpaper to rub approximately 0.5 cm from each end.
2. Roll one end of the copper wire to the head of a small-sized screw (electrodes) and make sure it is firmly fixed since this is a crucial step. Correct contact between both materials must be guaranteed to avoid artifacts in the EEG signals.
3. Insert the other end on the connector’s terminal tip and make sure it is properly fixed by reinforcing using fine forceps. This tip should connect with an amplifier cable.
4. Measure the suitable conductivity from the tip to screw using a multimeter. This process guarantees that the electrode connection is properly installed.

4. Epidural (skull screw) electrodes implantation surgery

NOTE: After 20 days of environmental enrichment treatment, the animals will undergo surgery following the procedure described below:

1. Inject a cocktail of Ketamine/Xylazine (90/10 mg/kg, i.p.) to the animal.
   NOTE: To avoid airway obstruction, wait until the rat stops moving, then take it out of the housing cage and place the animal on a flat surface. Inject a nonsteroidal anti-inflammatory (meloxicam 1 mg/kg, s.c.) and an antibiotic (enrofloxacin 2.5 mg/kg, p.o.) as pre-emptive analgesia.
2. Once the rat is entirely anesthetized, shave the head area of the rat.
   NOTE: Make sure that the animal is completely anesthetized before continuing with the surgery. Carefully pinch one of the legs or the tail. If the animal reacts to the stimulus, wait a few more minutes and pinch it again. If the animal does not react to the pinch, go to the next step. If the required equipment is available, the use of gas anesthesia (such as isoflurane) is highly recommended, as it is more easily titrated for safety.
3. Place the animal on the stereotaxic apparatus by fixating both ears first with the ear bars (be careful not to hurt the animal's internal ear). Finally, place the front teeth over the bite bar and secure the nose bar.
   NOTE: Provide the animal with a heating pad for all the surgery since the anesthesia used in this procedure usually causes hypothermia and breathing problems.
4. Clean the top of the head area using three alternating rounds of Chlorhexidine or iodine-based scrub followed by saline or alcohol rinse.
5. Administer lidocaine subcutaneously (20 mg/mL) under the skin of the head area (0.5 mL).
6. Instill a drop of ophthalmic solution or saline to each animal’s eyes every 5-10 min to help them not dry out.
7. Using a scalpel, make an incision of approximately 2 cm from anterior to posterior direction to properly expose the skull's top region.
8. Retract the skin using bulldog clamps and scrape the tissue overlying the skull.
9. Identify and record the bregma coordinates obtained.
10. Starting from Bregma, using the stereotaxic Paxinos and Watson Atlas³⁰, locate and mark the position of each of the seven points (coordinates) where the electrodes will be fixed in.
    NOTE: In this experiment, F3, F4 screws (+2.0 mm from Bregma, 2.25 mm lateral from midline); C3, C4 screws (−3.0mm from Bregma, 2.75 mm lateral from midline); and P3, P4 screws (−7.0 mm from Bregma, 2.75 mm lateral from midline) were installed. A seventh screw was located posterior of the nasal bone (NZ), as the ground reference (see Figure 2).
11. Using a variable speed drill tool, make a hole with a tip size 2 (length 44.5 mm) on each one of the marks, be careful not to penetrate the skull fully.
12. Insert the electrode into the hole and screw it into the skull carefully.
13. Repeat steps 4.10 and 4.11 until all seven screws are fixed in appropriately.
14. Fix all 7 screws with a first layer of dental cement. Insert each electrode into a connector. Cover the wires entirely with a second layer of dental cement (it will prevent the animal from pulling screws off), and the bottom of the connector. If necessary, cover with a third layer of dental cement, leaving the EEG connector clean for a proper connection, so that the EEG device can be connected appropriately (see Figure 3).
    NOTE: After placing each pair of bilateral screws, these could be fixed in with dental cement (optional step).
15. Leave the rat in post-operative care overnight. Observe the animal and provide the animal with a heating pad for 1-2 h after surgery since the anesthesia used in this procedure usually causes hypothermia and breathing problems.
16. Administer 50mL/kg/24h (maintenance dose) of saline solution subcutaneously to prevent dehydration. Inject a nonsteroidal anti-inflammatory (meloxicam 2 mg/kg, s.c.) and an antibiotic (enrofloxacin 5 mg/kg, p.o.) after surgery and for the next 24 h.
17. After the surgery, keep the rats in single cages for full recovery during seven days before conducting the behavioral tests.
18. Gently manipulate the animal on a periodical basis (at least once a day) to help to reduce the stress in future manipulations. While holding the rat with one hand, finger pressure is gently applied to the back of the animal, sliding the fingers through the fur. Check the head wound, health condition, behavior in general, and body weight for a period of one week after the surgery.
    NOTE: If any abnormality or signs of disease/stress are found in the animal, notify the responsible veterinary physician. After this period, perform the Novel object recognition test and EEG recording technique.

5. Novel object recognition test (NORT)

NOTE: Seven days after surgery, proceed to behavioral tests. All behavioral procedures, in the presented experiment were performed between the 14 h 00 min and 16 h 00 min, which corresponds to the rat's light cycle.

1. Place a vest made of soft fabric (to which the EEG device would be placed during the behavioral test) on the rat. Allow habituation for 2-3 days before conducting the behavioral test.
2. Place a black acrylic square arena (500 x 500 x 500 mm) in a dim-light illuminated recording room.
3. Fix two identical novel objects to the floor center of the arena using double-sided tape (to prevent its displacement by the animals). Objects must be equidistant from each other and the arena walls.
4. Clean each object thoroughly beforehand with 50% ethanol, as well the arena's floor after each trial (to avoid olfactory cues).
   NOTE: Always transfer the animals to housing rooms (from the vivarium room to the experimental room) at least half an hour before starting each session. After completing the recording session, leave the animals in the experimental room for an additional hour. This is to avoid the stress that could affect the performance of this test.
5. Connect the EEG device, before starting each test. Gently restrain the animal and firmly insert the cable to the connector on the animal's head with the EEG kit attached to the animal's back (see Figure 4). Only one position is allowed.
   NOTE: Gentle previous manipulation of the animal might help to reduce the stress in animals during the connection procedure. Otherwise, the risk of damage to the device or the animals increases. Fully pre-charge the device battery using a USB-Port.
6. Novel object recognition tests phases
   1. Habituation: Handle the animal at 5-min intervals for two consecutive days, and immediately after, place the animal on the arena (without any objects) and allow them to explore for 10 min freely.
      NOTE: Before executing any acquisition and memory test sessions, rats were carefully handled and connected to the corresponding EEG device, which was properly fixed before starting the test.
   2. Acquisition Session: Place the animal on the arena facing one of the walls opposite to the objects. Allow animals to explore freely for 10 min. Go to step 6.13 for test recording using Behavioral Tracking Software.
      NOTE: Make sure the EEG device properly holds the vest attached to the back of the rat (to ensure proper tracking of the animal). For additional reinforcement, use masking tape.
   3. Short-term memory test (SMT): Replace one of the objects with any other completely different in shape, color, and texture. Place the animal, 2 h after the acquisition session, in the arena facing one of the walls opposite to the objects. Allow the animal to explore freely for 10 min. Go to step 6.13 for test recording using the Behavioral Tracking Software.
   4. Long-term memory test (LMT): Replace the object used with any other completely different in shape, color, and texture from the short-term memory test. Place the animal 24 h after the acquisition session, in the arena facing one of the walls opposite to the objects. Allow the animal to explore freely for 10 min Go to step 6.13 for test recording using the Behavioral Tracking Software.

6. Behavioral tracking software setup

1. Open the Behavioral Tracking Software.
2. Log in to the account using the institution’s user and password.
3. Open the tap “New empty experiment” and choose a name for the protocol (e.g., “NORT”).
4. Select “Video tracking mode.”
   NOTE: In this experiment, the camera is set up to stream the video tracking live. However, there is an additional option to select pre-recorded videos.
5. Go to “Apparatus.” Define the arena area, by adjusting the orange rectangle to the limits of the projected arena. Determine the object's region, fitting the orange circles at the objects border inside the arena projected from the camera on the screen.
6. Setup the scale moving ruler line to a position along the known length of the image (the arena). Enter the length of the object in millimeters in the option “The length of the ruler line is” on the Settings panel. In this case, the arena measures 500 x 500 mm.
7. Go to “Tracking and behavior.” Continue to “Zones.” Click on the “Add item” menu and select “New Zone.” Select the arena area and name the new zone (e.g., “Field”).
8. Repeat the previous step with the objects' area and name the new zone (e.g., “Objects”).
9. Go to “Animal color” and select “The animals are lighter than the apparatus background” option.
   NOTE: White (Wistar) rats were used for this experiment. However, the software has additional options for researchers who use black and spotted rats. Both breeds of animals can be used in the same experiment.
10. Go to “Tracking the animal’s head & tail” and select “Yes, I want the animal’s head and tail to be tracked.”
11. Go to “Testing” | “Stages,” and from the menu “Add item,” select “New stage.” Name the new stage, “Acquisition.” Define the duration of the stage (e.g., 600 s).
12. Repeat the previous step of “Short-term memory test” and “Long-term memory test” stages.
    NOTE: In this protocol, all stages have the same duration (10 min).
13. Go to “Procedures.” Define the events to be tracked for each stage (acquisition, short-term memory test, and long-term memory test).
14. Start the test (with each animal). Go to “Tests” (in the top menu bar) and select “Add a test (+).” Assign a number for the animal to be tested (e.g., “1”).
15. Select “Record” and name the animals and session (e.g., “M1 Acq”).
16. Before placing the animal in the arena, click one time on the “Play” button. A message “waiting to start” will be displayed.
17. After placing the animal in the arena, click a second time on the “Play” button. The test will start and end automatically.
18. Repeat steps 6.13-6.16 for the short-term memory test (2 h after the acquisition session) and the long-term memory test (24 h after the acquisition session).

7. Wireless electrophysiology device setup

1. Connect the modem to a PC host and turn it on. Turn off any other network device on the PC. Preferably, silence any other wireless communication in the registration room like Bluetooth, cell phones, other modems, or even wireless handsets.
2. Attach the amplifier to the rat’s back, as mentioned in step 5.5.
3. Turn on the EEG device by plugging in the battery.
   NOTE: 2 s after connecting the device, a red led on the EEG amplifier will blink, indicating that the communication with the modem is active, and then green led will be turned on. If the communication is successful, the LEDs on the modem will start to flash continuously. The amplifier is now ready to send information to the modem.
4. Launch the EEG software and set it up according to manufacturer instructions to integrate into the wireless EEG acquisition device
5. Press the "Start Display" button. The EEG software will display the actual signal acquisition.
   NOTE: Use “Windows Task Manager” to assign the “Real-time” priority mode to avoid missing information during experimentation.

8. Electroencephalography (EEG) signal recording

1. After verifying that the EEG software is acquiring data, launch the Behavioral Tracking software, and set experimental protocol to verify that the animal is in the observation zone and the set-up is working correctly.
2. At this point, start the EEG software recording by pressing the "Start Record" button. After checking that the acquisition signal is running, start experimentation in the BTS.
3. After the experiment ends, return to the EEG software and stop the recording process. The recording will be saved by using a default name consisting of the date of recording using the following format: "yyyy-mmdd-hhmm_SubjectID_Ephys.plx". By default, all recordings are saved in the EEG software (NeurophysData) folder.
4. Check that both data files were created. Record the experiment log or change the name to avoid confusion.

9. Behavioral task and EEG signal synchronization

1. Open MATLAB and execute the command: convert_plx2mat. Such function will open a browser box. The converting functions are provided by the manufacturer and must be added to MATLAB’s path.
2. Select the *.plx to convert and press “Enter” on MATLAB’s command line to convert it to default parameters.
3. Open the BTS experimentation file and go to “Protocol.” Click on the option “Results, reports and data” select all the events of both objects and click on “Choose the time format for the report,” select the third option: “Show events times as real-time in HH:MM:SS.sss – for example 13:20:14.791.”
4. Now go to “File” and click on “Export” and “Export experiment as XML,” check “Date and time of the test,” finally click on “Create XML.”
5. Go to “Export test data” and click on “Save Data.” A .csv file with events times will be created.
6. Repeat steps 9.1 to 9.5 for each file. In our case, the three experiments were: ACQ, STM, and LTM.
7. Once the EEG and behavior files are converted, collect them on a single folder. The folder must have six files, the three .mat files, and three .csv, respectively. In our case, files were called: PID_01_ACQ_N.mat, PID_02_STM_N.mat, PID_03_LTM_N.mat, PID_01_ACQ_M.csv, PID_02_STM_M.csv and PID_03_LTM_M.csv. ID refers to an animal’s identification number.
8. Open “procesa_sujeto.m” function using MATLAB, and adjust the second line to the animal’s ID.
9. Now move MATLAB to such folder and execute: “procesa_sujeto” to create figures of alpha and beta relative band to power associated with objects recognition on ACQ, STM and LTM stages.
   NOTE: “procesa_sujeto” is a function that executes/runs several signal processing analyses. These analyses are summarized as follows in steps 9.10 to 9.15.
10. Filter Each EEG signal with a 4th order Butterworth bandpass filter at [5-40] Hz, using phase correction.
11. Visually inspect Signals before to the following analysis, and those channels with artifacts derived from defective electrodes placement or misadjustment by animal movements were excluded from further analysis.
12. Reference signals to common average to alleviate motion artifacts.
13. Segment EEG signals to form epochs of 4 s length synced by timestamps derived from BTS. The target events were the exploration of the object marked by the distance of the animal to objects border. These events are marked on the BTS timestamps and were used as identifiers fix the windows' positions. So, EEG epochs are delimited by 1 s before exploration begins to 3 s after. At this point, no validation about exploration length was used, but it will be considered for future researches.
14. Estimate spectral power density on those epochs by using Welch’s periodogram method using 1 s window length, an overlap of 90%, Hanning window prior to Fourier transform estimation, with these parameters a resolution of 1 Hz was achieved.
15. Assess power spectral on each band by evaluating area under periodogram, and the values presented corresponds to relative energy, it means that the energy of each EEG band was divided by the epoch’s total energy. This procedure also reduces erroneous estimations due to artifacts on EEG signals.