Hello! I am Mary Pearce, a rising Junior double majoring in Biology and Music at Gettysburg College. This summer I am doing research on vocal behavior, and the brain circuits the produce this behavior, in fish with Prof. Kittelberger. We are working on this project at the Marine Biological Laboratory in Woods Hole, MA, which is located on Cape Cod, just a ferry ride away from Martha’s Vineyard. When I have free time, I can go to the beach, ride my bike along the bike trail, walk to the lighthouse, go into town, or do many other great touristy activities. We are working on our research project in the Grass Laboratory alongside many other neuroscientists conducting their own experiments. While the lab can get very cramped sometimes due to the number of people working in such a small space, the work environment is always interesting. Everyone in the lab is very enthusiastic about their projects and curious to learn more about the other work taking place in the lab.
The fish we use in our experiments is the plainfin midshipman (Porichthys notatus), a species of teleost fish found along the western coast. For most of the year these fish live in deeper waters, but during their mating season, they migrate into the intertidal zone where the males make nests under rocks. These fish are interesting because they produce two types of vocal sounds, grunts and hums. Both male and female midshipman can grunt, which is a defensive vocalization used to scare off predators. Only the large male midshipman can produce a hum, which is a mating call. Grunts are very short in duration, while hums can last several hours. Nesting males use their hum to attract a female to the nest. The female will then lay eggs and leave, while the male remains to care for the eggs by cleaning and protecting them.
A male midshipman produces a hum by radically inflating his swim bladder, then contracting two muscles located on either side of it at very rapid rates. These two muscles are controlled by sonic motor neurons, which are connected to pacemaker neurons that synchronize their activity. These two types of neurons are located in the portion of the spinal cord that connects to the brain. A circuit in the brain controls the motor neurons involved in vocalization. The structures and neurons that do this are part of the vocal and auditory circuits in the midshipman brain. Many neurons in these vocal structures are thought to be involved in the reward system of the fish. It is known that certain neurons in or near vocal structures are also dopamine-producing neurons. Dopamine is a neurotransmitter that plays a role in the brain’s internal reward system, across many vertebrate species, including humans. When a reward or goal is reached, dopamine is released, a signal that links the preceding behavior with the ensuing reward. It has been shown that when dopamine is injected into the midshipman brain, humming behavior decreases. This suggests that neurons that are producing dopamine are involved in modulating vocal behavior, perhaps related to the “reward” of a female arriving in a male’s nest, a natural stimulus that causes males to stop humming. If dopamine releasing neurons are shown to be active during vocalization, it would strengthen the hypothesis that these neurons are directly involved in vocal behavior in midshipman.
This summer, our goal is to map the vocal circuit in the brain using immunohistochemistry with antibodies targeting active neurons and dopamine-releasing neurons. To do this, we had to get our midshipman naturally behaving in their tanks. This involved building a light-proof tent over the fish tanks. My first week here, I got a nice tour of the town as we went in search of hardware stores that sold PVC piping and thick black plastic sheeting. Once we collected our supplies, we journeyed back to the MBL to build the tent. Several power tools and a large amount of duct tape later, we had created a light-proof tent over our fish tanks. Inside, we placed lights that were attached to a timer. All of this was done so we could day-night reverse the nocturnal midshipman to get them naturally behaving during what they thought was nighttime, but was actually the middle of the day (so we wouldn’t have to stay up all night to do our experiments!). Once our shipment of fish arrived from the west coast, we settled them into tanks equipped with ceramic flowerpot saucers as nests. We began recording the sound in the tanks using a hydrophone. On their third day in the tanks, we heard humming on our recording. Our plan to get the fish behaving naturally was successful!
To run an experiment, I listen to a tank for an hour to ensure that there is humming occurring. Then, Prof. Kittelberger goes into the tank and removes either a humming or non-humming control male from a nest. The brain of the fish will be quickly fixed with formaldehyde to keep it preserved. I then section the brain into paper-thin sections using a freezing microtome. The sections are rinsed and stained with primary antibody for the immediate early gene c-fos, which is expressed in active neurons, followed by a fluorescent secondary antibody. If we are double labeling for dopamine releasing neurons, we also use antibodies for tyrosine hydroxylase, the enzyme that synthesizes dopamine in the brain. Once the staining is complete, the sections are mounted on slides and coverslipped with a medium containing DAPI, a fluorescent DNA stain. We can then view the slides under a microscope to see which neurons are active in the brain during humming. With the combination of three fluorescent stains, we will be able to figure out exactly what neurons are active, dopamine producing, and where they are located. This will help us understand the connections and roles different neurons in the brain play in the vocal behavior of the midshipman fish.