Amygdala-Dependent Molecular Mechanisms of the Tac2 Pathway in Fear Learning

Abstract

Recently we determined that activation of the tachykinin 2 (Tac2) pathway in the central amygdala (CeA) is necessary and sufficient for the modulation of fear memories. The Tac2 pathway includes the Tac2 gene, which encodes the neuropeptide neurokinin B and its corresponding receptor neurokinin 3 receptor (NK3R). In this study, using Tac2-cre and Tac2-GFP mice, we applied a combination of in vivo (optogenetics) and multiple in vitro techniques to further explore the mechanisms of action within the Tac2 pathway. In transgenic mice that express ChR2 solely in Tac2 neurons, in vivo optogenetic stimulation of CeA Tac2-expressing neurons during fear acquisition enhanced fear memory consolidation and drove action potential firing in vitro. In addition, Tac2-CeA neurons were shown to co-express striatal-enriched protein tyrosine phosphatase, which may have an important role in regulating Nk3R signaling during fear conditioning. These data extend our current understanding for the underlying mechanism(s) for the role of the Tac2 pathway in the regulation of fear memory, which may serve as a new therapeutic target in the treatment of fear-related disorders.

Figure 1

Optogenetic stimulation of central amygdala tachykinin 2 (Tac2)-expressing neurons drives action potential firing, in vitro, and enhances fear memory consolidation, in vivo. (a) The central amygdala (CeA) of Tac2–cre mice was unilaterally infected with DIO-ChR2 AAV. Top image represents the cannula used for delivering the AAV in the CeA. Bottom image shows the expression of the YFP reporter in Tac2 neurons within the central nucleus (CeM). (b) Light pulse trains at different frequencies. (c) Biocytin colocalizes with recorded CeM–Tac2–ChR2 neurons. Scale bar 20 μm. (d) Electrophysiological properties of Tac2–GFP-positive and Tac2–GFP-negative neurons in the CeM. (e and f) Optical stimulation of CeA–Tac2–ChR2 neurons during fear conditioning does not affect freezing levels or locomotor activity per se. *p<0.05.

Figure 2

Immunohistochemistry studies of the tachykinin 2 (Tac2) neurons in the centromedial amygdala and association with the neurokinin 3 receptor in fear learning. (a) PKCδ neurons detected by immunohistochemistry. (b) Tac2–cre mice were crossed with a GFP fluorescent reporter mouse line, BGeo–GFP. (c) Some Tac2 neurons are colocalized with the PKCδ protein. (d) Nk3R neurons detected by immunohistochemistry. (e) Tac2–GFP neurons from Tac2–cre–BGeo–GFP mice (Tac2–GFP). (f) Some Tac2 neurons are colocalized with Nk3R. (g) Nk3R mRNA levels are upregulated 30 min after auditory fear conditioning (FC) in C57 mice. (h) Wild-type mice received Osanetant (Nk3R antagonist) or vehicle. Two hours after FC, Nk3R mRNA levels were evaluated. *p<0.05; **p<0.01. Scale bar for (a)–(c) is 20 μm, 40 × images. Scale bar for (d)–(f) is 20 μm, 63 × images.

Figure 3

Immunohistochemistry studies of the tachykinin 2 (Tac2) neurons in the central medial amygdala and association with striatal-enriched protein tyrosine phosphatase in fear learning. (a) Striatal-enriched protein tyrosine phosphatase (STEP) neurons detected by immunohistochemistry. (b) Tac2–cre mice were crossed with a GFP fluorescent reporter mouse line and BGeo–GFP. (c) Some Tac2 neurons are colocalized with the STEP protein. (d) STEP neurons detected by immunohistochemistry. (e) Detection of Nk3R neurons with immunohistochemistry. (f) Some STEP neurons are colocalized with Nk3R. (g) STEP mRNA levels are downregulated 30 min after auditory fear conditioning (FC) in wild-type (WT) mice. (h) WT mice received Osanetant (Nk3R antagonist) or vehicle. Two hours after FC STEP mRNA levels were evaluated. *p<0.05. Scale bar for (a)–(b) is 20 μm, 40 × images. Scale bar for (d)–(f) is 20 μm, 63 × images.