Decoding Natural Behavior from Neuroethological Embedding

The scope of my research is to understand how neurodynamics encode natural behavior and how the brain controls complex actions that supports survival in natural environments. Traditional head-fixed paradigms limit our understanding of natural behavior. Our protocol updates this paradigm by embodying precise neural behavior decoding in freely moving animals toward natural brain intelligence.

We will focus on collecting rich uncontrolled data to build digital life models using holistic approaches to understand intelligence in complex living systems. To begin, connect the universal serial bus cable of the synchronization module of the three-dimensional behavior device to the workstation of the same device. Then, connect the synchronization module of the mTPM device to its controller using one SMA cable.

Connect the TTL output port of the synchronization module of the three-dimensional behavior device to the TTL input port of the synchronization module of the mTPM device using one SMA BNC conversion cable. To begin calibration, adjust the shooting angle of all four cameras so that they cover the entire base of the open field and extend their field of view at least 20 centimeters above the farthest boundary to capture mouse rearing behavior. Then, place the calibration module at the center of the shooting area.

Switch off all the lights and run the camera calibration software. Now, fix the mouse restrainer to the micro-manipulator of the mTPM. Using the metal plate, secure the head of the mouse to the restrainer.

Switch off all the lights. Then, fix the mTPM to its holder and switch on the imaging system to locate the fluorescent signal. Add one drop of Carbomer eye gel to the top of the cranial window.

Move the mouse using the motion platform so that the cranial window is aligned directly beneath the objective of the mTPM. Move the micro-manipulator vertically to locate the imaging plane. Then, move the micro-manipulator in plane to center the imaging plane.

Then, fix the upper base to the mTPM. Apply adhesive to glue the lower base to the upper base and secure it to the cranial window. To ensure structural stability, fill the gap between the two bases and the metal plate bracket attached to the mouse's head using a high-performance acrylic structural adhesive.

Then, assess the bond stability by gently probing the base with tweezers. After that, add one drop of Carbomer eye gel into the base chamber. Observe the neuronal fluorescence through the mTPM.

If the fluorescence is not clearly visible, remove the adhesive using a cranial drill to detach the base. Then, repeat the procedure until clear fluorescence is achieved. Then, secure aluminum foil with tape between the fiber of the mTPM and the cranial window.

Switch on the room light and test the clarity of the frames captured by the mTPM. To put the mouse in an open field, inflate at least 10 helium balloons and tie each one separately with cotton twine. Then, detach the metal plate from the mouse restrainer.

Hold the mouse gently by its tail using one hand. With the other hand, support the optical fiber of the mTPM. Carefully place the mouse into the open field.

Suspend the helium balloons by attaching the cotton twine to the fiber. Adjust the number of balloons so that the mouse can move and explore the open field without restriction. Close the door of the mTPM enclosure to reduce external disturbances.

Start the mTPM recording software and the synchronization software. Set the file paths and recording parameters according to the platform establishment procedure. Start the recording of the mTPM through the recording software.

Check the synchronization software to verify that time markers are accurately recorded for each two-photon frame. Evaluate whether the contrast of the two-photon images remain stable during recording. Also confirm that the mouse's movements do not disrupt the stability of the imaging frames.

Now, start the customized camera synchronization script to initiate behavior recording. Set the file path and parameters according to the platform establishment procedure. Then, start the behavior recording using the customized synchronization script.

Confirm the presence of a time marker in the synchronization software for every 30 frames of behavior video. Check that all four video streams from the cameras are synchronized correctly. Verify that the video capture parameters of the three-dimensional behavioral tracking system are set properly.

Once the behavioral recording stops automatically, manually switch off both the mTPM recording and synchronization software to conclude the trial. Correlation coefficient matrices showed no distinct neuron-specific patterns for subject poses, object poses, or body distances, indicating weak correspondence between neural signals and behavioral metrics. All neuron behavior correlation coefficients fell between 0.3 and 0.3, confirming weak associations under naturalistic conditions.

Zebra-derived neural embeddings form intricate patterns, incorporating components from multiple joint embeddings. Zebra embeddings demonstrated consistent alignment of behavioral and neural variables across three mouse pairs, particularly for body distance and social motifs. The decoding error for body distance embeddings was significantly higher than subject and object poses, but remained within expected tracking error limits.

Joint embeddings of neural activity with various behavioral variables revealed high decoding accuracy across subject poses, object poses, and motifs. Cosine similarity analysis using the S1-subject pose embedding as a reference showed lower alignment for object-related motifs, suggesting primary encoding of self and social behavior.