ノルアドレナリンによる恐怖学習・記憶調節の神経回路および分子メカニズム

2013 Fiscal Year Annual Research Report

• Project/Area Number: 13F03209
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: JOHANSEN JOSHUA 独立行政法人理化学研究所, 脳科学総合研究センター, チームリーダー
• Co-Investigator(Kenkyū-buntansha): TAN Bao Zhen 独立行政法人理化学研究所, 脳科学総合研究センター, 外国人特別研究員
• Keywords: optogenetics / animal behavioral testing / fear learning & memory / synaptic plasticity / locus coeruleus / lateral amygdala / noradrenaline

Research Abstract

Background : Palvovian auditory fear conditioning is the standard paradigm for exploring the circuit and molecular mechanisms of learning and memory, and emotion. An emotionally neutral auditory conditioned stimulus (CS), such as a tone, is paired with an emotionally potent, innately aversive unconditioned stimulus (US), such as an electric shock, during the acquisition phase. The assessment of conditioning then involves measuring conditioned responses (behavioral freezing) elicited by the auditory CS, independent of the aversive US during a memory test phase. Pathways transmitting the CS and US converge in the lateral amygdala (LA), and synaptic plasticity within the LA may enhance the response of LA neurons to the auditory CS.
Project 1 : To determine whether the locus coeruleus (LC) is the source of noradrenaline (NA) to the LA and to define the specific temporal epochs during fear conditioning in which these inputs are important. Results : We have virally infected the NA neurons in the LC with archaerhodopsin (Arch, a green light-activated outward proton pump used to inhibit neural activity) and use laser light to inactivate LC neuron terminals in the LA during the unconditioned stimulus. Different adenoviral serotypes were injected to check for viral expression in the LC, and various incubation times were tested to verify sufficient terminal expression in the LA for light manipulation. In our hands, only the AAVJCAG-FLEX-GFP vectors in serotype AAV9 infected LC neurons of TH-Cre rats robustly and at least 8 weeks of incubation was required for terminal expression. Preliminary data unexpectedly showed higher behavioral freezing in ArchT-GFP group, compared to GFP controls. However, the n number is small (ArchT-GFP, n =2 ; GFP, n = 3) and we need to test more animals.
Project 2 : To determine if NA from LC facilitates fear learning by activation of CRTC1 and CREB-dependent gene expression.
Results : Preliminary data showed significant increase in nuclear translocation of CRTC1 in LA neurons (immunohistochemical sections) 1 hour after fear conditioning (0.704, n=4) versus box control (0.323, n=4). Immediate shock control experiments were carried out, together with additional box control and fear conditioned groups, and immunohistochemical sections are being examined to determine if increase in nuclear translocation in LA neurons is specific to CS-US pairing, and not just US exposure alone.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Experiments are being conducted as initially planned for Projects 1 and 2.

Strategy for Future Research Activity

Project 1 : Increase the n number of ArchT GFP animals for behavioral test, as well as use laser to inactivate LC neuron terminals in the LA at other time points outside of US periods (e.g. CS periods or tonically) to define when these inputs are important during fear conditioning. β-adrenergic receptors (ARs, G-protein coupled receptors) in the LA are known to be required for fear conditioning, and we will determine if changes in strength of fear conditioning via stimulation of LC terminals in LA is dependent on β-AR activation in the LA via pharmacological manipulations with β-AR agonist or viral injection of DREADDs (engineered GPCRs that permit spatial and temporal control of G-protein signaling in vivo).
Project 2 : Test if CRTC1 nuclear translocation is necessary for behavioral fear conditioning, via viral expression of dominant-negative CRTC1 (prevents activation of CREB in the nucleus) or shRNAs targeting CRTC1 (down-regulate endogenous CRTC1 expression) in the LA. To determine if NA from LC is necessary for CRTC1 nuclear translocation, immunohistochemical sections of LA from optogenetically-manipulated or β-AR-activated animals will be examined as well. Both CREB and transcription of some CREB-dependent genes are necessary for plasticity in the LA and fear-conditioning. Real-time quantitative RT-PCR will be performed to determine changes in CREB-dependent gene transcription after viral manipulations.