In this study, we propose the methods that overcomes the limitation of existing techniques to measure neuronal activity in-vivo. By utilizing them, the role of thalamic reticular nucleus in fear extinction is addressed.
1. The progress of measurement technique in neuroscience allows us to embody the experiments cannot be approached before. For example, the two-photon calcium imaging and the tetrode recording with microdrive are the techniques to record the single neuronal activity with large spatial resolution or high temporal resolution, which enables to get close to the answers for fundamental questions in neuroscience. However, despite the strong advantages, there are practical limitations to apply those techniques.
Although the two-photon microscopy is an excellent physiological technique that enables simultaneous record-ing of hundreds of brain cells in-vivo at a single-cell resolution, a precise targeting of cortical location is not easy for bulk-loading of calcium indicator due to its fine dimensionality. Here, we propose the new method, based on local field potential (LFP) recording, for precise targeting. The heart of this method lies in the use of the same glass pipette for recording LFP and for ejecting calcium dye. After confirming the target area by LFP using a glass pipette, calcium dye is ejected from the same glass pipette without time delay or spatial readjustment. As a result, calcium dye can be loaded to the exact same ensemble of brain cells from which the LFP was obtained. To demonstrate the validity and usefulness of our LFP-based method, we targeted the layer 2/3 of specific bar-rel column in the mouse somatosensory cortex. The result showed that the calcium dye was successfully loaded into the target barrel column, which indicates the proposed method provided precise targeting of small function-al structures such as a cortical functional column for two-photon calcium imaging in-vivo.
Recently, various studies using optogenetics have been performed in neuroscience. Although the optogenetic system allows us to modulate the neurons with a high temporal resolution, simultaneous recording with optical stimulation is not easy due to the mechanical limitations of recording device. We developed a simple way to make a microdrive which can also deliver the optical stimulation simultaneously with the electrophysiological recording, which is called “optical microdrive”. The microdrive originated from commercial manufacturer was modified to be equipped with the thin optical fiber of 125 $\mu$m diameter. The proposed method was validated by the electrophysiological recording accompanied with the optical stimulation in freely moving mice. The result showed that the time-locked single unit response to the optical stimulation, which indicates the proposed method provided successful in-vivo single unit recording with optical stimulation in freely moving mice.
2. We studied the function of the thalamic reticular nucleus (TRN) in fear extinction by utilizing one of the ad-vanced techniques, i.e., optical microdrive, to measure in-vivo neuronal activity.
First, we revealed the specific TRN sector which is related to the fear extinction. The retrograde tracer was in-jected into the area which is known to be a center for fear extinction, and the position of the labeled neurons in TRN was accessed. The result showed that the rostral sector of TRN projects to the center of fear extinction, which indicates that this specific sector might play an important role in fear extinction.
Second, we modulated the rostral sector of TRN with optogenetic methods during fear extinction learning. The channelrhodopsin-2 (ChR2) was specifically expressed in the parvalbumin-positive neurons in the rostral sector of TRN by injecting cre-dependent ChR2 virus into Pvalb-IRES-Cre mouse and the optical fibers were implant-ed to the rostral sector of TRN. The blue light stimulation during fear extinction learning resulted in significantly enhanced fear extinction learning and the lower freezing level in retrieval test of extinction.
Third, using optical microdrive, we recorded the single unit activity in the TRN in freely moving mice during fear extinction learning while the rostral sector of TRN was simultaneously modulated by optogenetic stimula-tion. Compared to the control group, which the optogenetic stimulation was not given to, the stimulated group showed the increased firing rate in TRN during the presentation of conditioned stimulus.
Combining anatomical, behavioral and electrophysiological assessment, our data showed that the rostral sec-tor of TRN is involved in fear extinction and the optogenetic modulation of TRN results in dramatic enhance-ment of fear extinction learning.