1. Investigating the role of the evolutionary conserved limbic circuit within vertebrates.
Limbic system is believed to control emotions and emotional behaviors. This neural circuit contains the hippocampus, amygdala, stria terminalis, or formix. The function of the limbic system assists us not only in expressing emotions such as joy or angry, but also in selecting behavior using emotional information memorized within this circuit.
The neural circuit specialized in the behavioral selection is called the cortico-basal ganglia circuit, which includes the cortex, basal ganglia (which consists of the striatum and globus pallidus) and thalamus. The external environmental information and associated emotional information enter to this neural circuit through the hippocampus and amygdala, respectively, and are believed to influence on the process of establishing a neural ensemble which guides an optimal behavioral output (Grace et al., 2000, Brain Res Rev. 31, Figures 1, 2). However, how a particular neural ensemble becomes selected via the cortico-basal ganglia circuit remains unclear.
In order to understand the role of this neural circuit, it is necessary to study the activity of groups of neurons in the whole circuit. However, it is difficult to observe the neural activity of such a big number of neurons by the electrophysiological recording, a classical way of investigate the neural activities in the mammals. On the contrary, the recently developed calcium imaging technique may permit to observe the activity of neurons in much broader region. Nevertheless, the mammalian brain is too big to observe the whole neural circuit by this method.
In mammals, the cortico-basal ganglia circuit is located in the part of brain called telencephalon, which is the most anterior part of the brain. It has been long thought that there was almost no similarity in the brain structure between the zebrafish, a small tropical fish, and the mammals. It has been even considered that the fish did not have the cortico-basal ganglia circuit itself. However, it has been recently demonstrated that the apparent structural difference of the brain between the fish and the mammals is the result of the difference of the way of developing the brain during fetus stage, and if we consider this fact, the telencephalon of the fish and the mammals is actually much more similar than it has been thought before (Wulliman MF and Mueller T, 2004, J Comp Neurol, 475).
The mammalian telencephalon is formed as a tube becomes broaden although the fish telencephalon is formed as a flower opens (Figure 3). Thus, the region corresponding to the cortex becomes located on the dorsal most position in the zebrafish telencephalon. This dorsal position of the cortex and the very small brain size (whole brain measures less than 10 mm) in zebrafish permits us to observe the neural activity of the whole circuit by the calcium imaging methods.
Recently, we have succeeded in visualizing how information stored as long-term memory in the cerebral cortex is processed to guide behavioral choices (Aoki et al., 2013, Neuron).
First, the transgenic fish in which the calcium sensitive protein has been introduced in all neurons learned the avoidance behavior (Figure 4A). In this learning paradigm, fish is put into the chamber that is separated into two compartments. If the fish did not go to the opposite compartment within 15 seconds during which the red LED lamp is presented, an aversive mild electric shock is given. By repeating this, fish learns to avoid to the opposite compartment when the red LED lamp turns on.
Once the fish learned the avoidance behavior, we put the fish under a fluorescent microscope which can visualize the changes in fluorescence intensity of each part of the brain, and showed a red LED lamp, a predictive sign for the electric shock (Figure 4B). As a result, we observed bilateral spot-like activities in the brain region corresponding to the cortex when the fish remember the learned avoidance behavior 24 hours after the training (Figure 4C-d), although no such activity has been observed 30 hours after the training (Figure 4C-c). This means that we succeeded in visualizing the process of reading out the behavioral program, written and stored for a long period (long-term memory) within a cortical region.
By measuring a large numbers of neurons in a whole cortical region of the fish, we succeeded in visualizing the process of reading out the behavioral programs, written as a memory, during the decision-making. In a future, we are planning to measure the neural activities of the basal ganglia that receive the information from the cortex, located in a deeper region of the brain by using the laser microscope. The measurement of the neural activity of the basal ganglia, in combination with the recently developped optogenetic tecniques, allows us to understand in details the mechanisms in which the behavioral programs become written, stored and read out through the cortico-basal ganglia circuit.
Our brain operates the decision-making process by choosing correctly the behavioral programs depending on the environmental changes. This ability is indispensible for our daily social life. It is now pointed out the possibility that the dysfunction in the behavioral choice and decision-making can lead psychiatric symptoms such as manic behavior and repetitive behavior often seen in the obsessive-compulsive disorders, schizophrenia or autisms. Our results using the zebrafish, a prototype of the vertebrates, opened the way to investigate and understand how these symptoms appear in human psychiatric disorders.