The optogenetic manipulation of light-activated ion-channels/pumps (i.e., opsins) can reversibly activate or suppress neuronal activity with precise temporal control. Therefore, optogenetic techniques hold great potential to establish causal relationships between specific neuronal circuits and their function in freely moving animals. Due to the critical role of the hippocampal CA1 region in memory function, we explored the possibility of targeting an inhibitory opsin, ArchT, to CA1 pyramidal neurons in mice. We established a transgenic mouse line in which tetracycline trans-activator induces ArchT expression. By crossing this line with a CaMKIIα-tTA transgenic line, the delivery of light via an implanted optrode inhibits the activity of excitatory CA1 neurons. We found that light delivery to the hippocampus inhibited the recall of a contextual fear memory. Our results demonstrate that this optogenetic mouse line can be used to investigate the neuronal circuits underlying behavior.

Funding: This work was partially supported by the World Premier International Research Center Initiative and Grants for Excellent Graduate Schools from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan Society for the Promotion of Science KAKENHI grants (numbers 26115502, 25116530, and 24700340), RIKEN Special Postdoctoral Fellowship, the strategic program for R&D (President’s discretionary fund) of RIKEN, Uehara Memorial Foundation, Takeda Science Foundation, Brain Science Foundation, Research Foundation for Optoscience and Technology, and Japan Foundation for Applied Enzymology to MS and by RIKEN, U.S. National Institutes of Health (R01DA17310), Grant-in-Aid for Scientific Research (A), Scientific Research on Innovative Area ‘Foundation of Synapse and Neurocircuit Pathology’ from MEXT, Human Frontier Science Program, Takeda Pharmaceutical Co. Ltd., and Fujitsu Laboratories to Y. Hayashi. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Sakaguchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Introduction

Optogenetics have become a popular technique for demonstrating a causal relationship between the function of a specific neuronal circuit and behavior [1–8]. The use of light-activated ion channels (i.e., opsins) in combination with other genetic tools allows for temporally precise, reversible, and selective manipulation of target neuron activity. This technique has begun to challenge conventional views of brain regions responsible for memory storage and retrieval and the temporal nature of their involvement in memory [9]. The primary advantage of optogenetic techniques is their ability to overcome many limitations inherent in older techniques. For instance, microinjections of reagents do not permit temporally precise control of neuronal activity such as spiking or allow the restriction of reagent effects to defined time windows or particular cell types. Similarly, electrical stimulation affects all neurons and axons within the vicinity of the electrode and cannot be used to inhibit neurons.

The use of inhibitory opsins, in particular, has begun to highlight the importance of temporally precise neuronal activity for memory function. For example, Goshen et al. showed the real-time involvement of hippocampal CA1 excitatory neurons during the acquisition and recall of recent contextual fear memory [1]. Gu et al. clarified the critical time point when adult-born hippocampal neurons most efficiently encode contextual and spatial memory [4]. Denny et al. and Tanaka et al. showed evidence that the deactivation of hippocampal neurons activated during learning is necessary for the retrieval of the memory [10,11]. Furthermore, other studies using inhibitory opsins show the importance of brain regions other than hippocampus in various stages of memory [2,3,5].

Two types of inhibitory opsins—archaerhodpsin (Arch; light-driven outward proton pump) and halorhodopsin (NpHR; inward chloride transporter)—have been widely used in neuroscience research [12–14]. Both opsins produce nA-scale photocurrents upon light stimulation, thereby generating reversible membrane hyperpolarization with step-like kinetic stability. However, after the cessation of an extended period of photo-activation, as is often required for studies of learning and memory, NpHR causes a rebound increase in the probability of synapse-evoked spiking through changes in the reversal potential of GABA A receptors [15]. Therefore, Arch-based optogenetics may be a good alternative to NpHR-based techniques in certain experimental settings [16].

A challenge in utilizing optogenetic approaches in neuroscience research is the need to express high levels of opsin in each neuron due to the relatively small current mediated by each channel or pump [17,18]. Previous studies often used viral vectors to deliver opsins to target brain regions, resulting in incomplete coverage of the target region and variable expression levels between neurons and between animals. The use of transgenic, tetracycline-controlled transcriptional activation systems, however, allows opsins to be reversibly expressed in genetically defined cell populations by turning their expression on and off via the application of tetracycline or its derivatives (e.g., doxycycline) through the animal’s diet. Although some studies have applied similar approaches to express Arch [19] in the brain [10,11,17,20], to our knowledge, ArchT, a more sensitive version of Arch, has not previously been used to generate an inducible transgenic animal to study memory.

Here, we generated a TetO-ArchT mouse line in which ArchT is expressed in a defined cell population through its cross with an appropriate driver line. Using these mice, we confirmed that light delivery reliably inhibits CA1 neuronal activity in vivo. Furthermore, we showed that the recall of a fear memory in TetO-ArchT mice is reversibly inhibited by light delivery to the hippocampus, providing evidence of the utility of this new mouse line in memory research.