Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

The overall goal of the following procedure is to train rats to swim and voluntarily dive underwater through a five meter long plexiglass maze. This is accomplished by first gradually training newly weaned rats to swim to a raised finished platform at one end of a five channeled maze with progressive extension of the distance to the platform. After the rat has learned to swim the entire maze, the animal is conditioned to dip its head under the vertical edge of the start area.

That is to dive, to enter the maze and swim to the finish platform. The distance the rat has to dive underwater is then extended until a five meter long dive tunnel has been created. Finally, once the rat has been fully trained, pulsatile arterial blood pressure readings can be collected from the animal during the swimming and diving sessions.

Ultimately, the central aspects of the mammalian diving response. For instance, investigating which areas of the brainstem are activated during diving can be analyzed in control, non-driving and diving trained animals. Because the physiology and neuroanatomy of rats are already well characterized, training these animals to voluntarily dive underwater can help answer key questions in the field of diving physiology, such as what are the central integrative pathways responsible for the mammalian diving response.

Before starting the swim training, fill the tank with fresh 32 degree Celsius, tap water to a depth of about 10 centimeters. Stir the water to minimize the presence of any warm or cool pockets, and then insert the finish area and channel dividers to create five swimming channels. To prepare the rat for the first training session, lower the rat by hand until it is directly across from the finished platform, and then let the animal walk to the platform without getting wet.

Next, lower the rat until its feet are just touching the water and let it walk to the platform again. Then once the rat has acclimated to the situation, gently lower the animal into the water about three to five centimeters from the finish area, taking care to support the feet and let the rat swim away rather than drop it into the water. Upon introduction to the water, the rat will appear unsteady as it first encounters the sensation of floating and will paddle about in an uncoordinated fashion while trying to locate a way to exit the water.

Eventually reaching the nearby finish area due to its innate ability to swim. In subsequent trials, the rat will swim in a much more coordinated fashion toward the finish area between trials. Let the rat remain in the finish area for at least one minute to allow it to groom and explore so that the finish area will be regarded as a safe place.

After this wait period, hold the rat for one minute before initiating the next trial, gently drying the animal with a towel to prevent the rat from developing hypothermia during the training. After completing a daily training of three to five trials, remove all the channel dividers and the finish area from the tank and empty out the water. Begin the second day of trials from the distance successfully negotiated on the previous day.

Then for the next few subsequent daily training sessions, increase the distance between where the rats are placed in the water and the finish area by five to 10 centimeters. After the animals appear more comfortable with the swimming, increase the distances by 30 to 50 centimeters per day until a final distance of five meters is reached. When learning to swim, the hairpin turns between the channels.

Smaller increases are preferred, and the animal may require two to three training sessions. Use larger increases in the swimming distances while the rats are learning to swim a straight portion of a swim channel. So here's a little hint.

Place pieces of plexiglass along the corners of the tank. This prevents the animals from climbing out as they swim through the tank and encourages proper training of the rats. Repeat this daily training protocol over a period of three weeks to ensure successful swimming of the entire five channels and a successful completion of the maze, especially as the maze includes alternating left and right hairpin turns.

After the rats have learned to successfully negotiate swimming through the maze, fill the tank until the water is one centimeter below the opening to the start area. Insert the finish area and channel dividers to create five swimming channels, and then to start the diving training. Lower the rat into the water with the elevator at the top of the start chamber.

During this first session, the water level is low enough that the rat can easily swim from the start chamber into the maze. On the second day of diving training, fill the tank until the water level is slightly above the opening to the start area. This will require the rat to dip its head under the edge of the start chamber To enter the maze, consider this the rat's first dive and allow the animal to continue swimming through the maze to the finish area.

As usual, on the third day, fill the tank until the water is above the opening to the start area. The rat will now have to dip its head under the edge of the start chamber and swim five centimeters underwater to reach the open water of the swim channel. For the first few daily training sessions, increase the length of the dive tunnel by adding additional horizontal dividers.

To extend the distance, the rat has to swim underwater by five to 10 centimeters. After the rats appear more comfortable with the diving, increase the distances by 30 to 50 centimeters per day until a final distance of five meters is reached. Repeat this daily training protocol over a period of three weeks to ensure a successful diving of the entire five channels and a successful completion of the maze.

Here's some hints and tricks, and here's the first one. Don't overextend your diving distances. You don't want the animal to go from here all the way to here.

They just might not make it. You just want to gradually extend the underwater diving distance. And here's the second hand.

You can tip up the channel cover. So in this case, the water level is to here, have the animal dive to here. The next training session, lower it just a bit further now has to dive to here, and the next session lower a bit further.

The animal has to die further. The most important part is never let your animals turn around underwater. Keep them moving forward.

Always keep them moving forward. If you let the animals turn around underwater, you'll de train them. They'll get stressed out and you'll never be able to complete the training procedure After completion of the training.

Follow the surgical implantation procedures suggested by the manufacturer for implanting a commercially available telemetric transmitter into the trained animals. Ensure complete recovery from the surgery before returning the rat to the water, and then once in the water, confirm that the antenna receiving the radio signal is nearby. Using a handheld antenna wand rather than a rat cage sized antenna.

Follow the rat as it progresses through the maze, keeping the wand antenna within 30 centimeters of the rat to ensure that the signal is not lost while the rat is underwater. Finally, collect pulsitile arterial blood pressure readings from the animal with the transmitter during the swimming and diving sessions. Successful completion of the described training procedures can decrease the stress experienced by the rats when diving underwater.

For example, blood corticosterone levels indicate that repetitive daily training decreases the stressfulness associated with voluntary diving and that trained rats find diving no more stressful than being handled daily by a human. Additionally, both trained and untrained rats find forced diving to be the most stressful immediately upon voluntary submersion and within a single beat, the heart rate decreases by 78%and the mean arterial blood pressure decreases by 25%in both trained and untrained rats demonstrating that voluntarily diving rats exhibit the same. Cardio respiratory changes typically observed in other diving animals, neurons within the ventral medullary, dorsal horn express phos during voluntary diving, potentially constituting the initial brainstem afferent relay of the diving response.

Thus, in this control non-driving rat, there was no FOSS detected in this swimming rat. There was very little FOSS in the medullary dorsal horn or in the para trigeminal nucleus within the spinal trigeminal tract. In a diving rat, however, FOSS is detected ventrally in both the medullary dorsal horn and the para trigeminal nucleus compared to the swimming and control rats indicating that neurons in these regions of the brainstem are activated during diving but not swimming.

Though this method can provide insight into the field of diving physiology. It can also be used to investigate the central control mechanisms of the cardiovascular, respiratory, and autonomic nervous systems. It can also be used to investigate the mechanisms involved in learning and memory.