The dorsal striatum constitutes the basal ganglia circuit and is a brain region important in the selection and initiation of rewarding and aversive behaviours. The cells that make up the dorsal striatum are known as medium spiny neurons (MSNs), which receive input from dopamine and are broadly divided into dMSNs (direct MSNs), which express dopamine D1 receptors and form a direct pathway in the basal ganglia circuit, and iMSNs (indirect MSNs), which express dopamine D2 receptors and form an indirect pathway. The iMSN (indirect MSN) expresses dopamine D2 receptors and constitutes the indirect pathway. Since dopamine receptors regulate PKA activity, it is expected that PKA activity in the MSN is dynamically regulated by dopamine fluctuations during behavioural selection, but this has not been measured. Therefore, we established a technique to monitor PKA activity and its downstream ERKs in the dMSN and iMSN of the striatum of freely moving mice in real time.

First, we generated transgenic mice that express the FRET biosensor in a dMSN- and iMSN-specific manner. To do so, we generated transgenic mice that express the FRET biosensor of PKA or ERK in a Cre-dependent manner and crossed them with mice that express Cre specifically for dMSN and iMSN (D1-Cre, D2-Cre) (Figure 5B).

To observe the molecular activity of PKA and ERK in the MSN of freely moving mice in real time, we introduced a fiber endomicroscope in collaboration with the Shigetada Nakanishi Laboratory, Osaka Bioscience Institute. In this microscope, one end of a fiber bundle consisting of thousands of 2-micron diameter optical fibers is inserted into each region of the brain, and the other end is scanned with a confocal microscope to enable in vivo FRET imaging under free-moving conditions (Figure 5A).

Figure 5. a. Photograph and schematic of fiber endomicroscopy. b. Transgenic mice expressing FRET biosensor specifically in direct and indirect tracts of striatum. c. Real-time measurement of PKA activity in direct and indirect tracts of free-moving mice by in vivo FRET. PKA activity of electrical stimulation shows opposite responses in direct and indirect tracts, and even more so during cocaine and male sexual behavior.

To examine the activity of PKA and ERK during reward behavior, we monitored PKA and ERK activity in real time after cocaine administration. Cocaine administration increased PKA and ERK activity in the direct pathway, but decreased PKA and ERK activity in the indirect pathway (Figure 5C).

To examine the activity of PKA and ERK during aversive behavior, we observed the activity of PKA and ERK in real time after electrical stimulation. Electrical stimulation decreased PKA and ERK activity in the direct pathway, but increased PKA and ERK activity in the indirect pathway (Figure 5C). This was the opposite pattern of the response to cocaine administration, a mirror image relationship.

Next, we observed real-time PKA and ERK activity during sexual behavior. Males showed the same activity when accepted by the female as when cocaine was administered (Figure 5C), but when rejected by the female, the activity was similar to that of electrical stimulation. Interestingly, the activity changed depending on the interaction with the female at that time. In other words, it became clear for the first time that PKA and ERK are dynamically regulated in opposite directions, with the activity of the direct and indirect pathways depending on the rewarding and aversive nature of the behavior at that time.