Neuregulin-1-dependent control of amygdala microcircuits is critical for fear extinction

Highlights

• NRG1-ErbB4 signaling pathway in the ITC is required for fear extinction.

• The NRG1-ErbB4 pathway in the ITC increases feed-forward inhibition of CeM neurons.

• ITC NRG1 might regulate GABAergic transmission through P/Q-type VACCs.

Abstract

The posttraumatic stress disorder is marked by an impaired ability to extinct fear memory acquired in trauma. Although previous studies suggest that fear extinction depends on the function of the amygdala, the underlying mechanisms are unclear. We found that NRG1 receptors (ErbB4) were abundantly expressed in the intercalated cells mass of amygdala (ITC). The NRG1-ErbB4 pathway in the ITC promotes fear extinction. The NRG1-ErbB4 pathway in the ITC did not affect excitatory input to ITC neurons from BLA neurons but increased feed-forward inhibition of (the central medial nucleus of the amygdala) CeM neurons through increased GABAergic neurotransmission of ITC neurons. We also found that the NRG1-ErbB4 signaling pathway in ITC might regulate fear extinction through P/Q-type voltage-activated Ca²⁺ channels (VACCs) but not through L- or N-type VACCs. Overall, our results suggest that the NRG1-ErbB4 signaling pathway in the ITC might represent a potential target for the treatment of anxiety disorders.

Introduction

Healthy individuals can process fearful and other emotional stimuli and extinguish fear in situations that are no longer threatening. However, patients with posttraumatic stress disorder (PTSD) or other anxiety disorders have difficulty extinguishing fearful memories. Pavlovian fear conditioning is a valid model for research on anxiety disorders in the laboratory (Knapska et al., 2012) (Myers and Davis, 2002; Sullivan et al., 2003). PTSD is related to insufficient inhibitory control over fear memory (Hamner et al., 1999). To extinguish learned fear, an animal is given the conditioned stimulus (CS) repeatedly without the unconditioned stimulus (US) (Ramanathan et al., 2018). Therefore, understanding the mechanisms underlying fear memory extinction is important for understanding the mechanism of fear inhibition and may help to improve the treatment of anxiety disorders.

The amygdala is a key brain region for fear learning (Duvarci and Pare, 2014; Jo et al., 2020; Kim and Cho, 2020; LeDoux, 2000; McCullough et al., 2020; Ozawa et al., 2020).

As the main entry for sensory inputs into the amygdala, the lateral amygdala (LA) delivers CS information to the amygdala (Yu, K. et al., 2017). The central medial nucleus of the amygdala (CeM) is the primary output for fear, which projects to brainstem fear effector structures and controls fear behaviors (Duvarci and Pare, 2014). The LA can influence the activity of the CeM indirectly by regulating the basolateral nucleus of the amygdala (BLA) (Pare et al., 2004). GABAergic activity of the amygdala has been implicated in the processes of fear conditioning and extinction regulated by local inhibition (Ehrlich et al., 2009). Meanwhile, glutamatergic neurons of the BLA can project to GABAergic neurons of the intercalated amygdala (ITC), leading to increased feed-forward inhibition of CeM neurons (Pare et al., 2004; Amano et al., 2010). Previous studies also demonstrated that the fear extinction process is associated with increased inhibition of CeM neurons (Amano et al., 2010). Despite these advances, we still know very little about the mechanisms underlying how BLA inputs regulate ITC neurons and increase the inhibition of CeM neurons.

Epidermal growth factor neuregulin 1 (NRG-1) is a trophic factor and its DNA methylation is associated with PTSD (Uddin et al., 2018). NRG1 and ErbB are expressed prefrontal cortex, hippocampus, amygdala, cerebellum, and substantia nigra (Law et al., 2004). Deficits in the Neuronal migration, Synapse formation, axon projection, axon myelination, and axon ensheathment can be caused by lack of function of NRG1 or ErbB4 or perturbation of NRG1 signaling (Mei and Xiong, 2008). As NRG1 receptors, ErbB receptors are transmembrane receptor tyrosine kinases and act in a dimerization paradigm (Mei and Xiong, 2008). And ErbB4 is the only ErbB receptor autonomously activated by NRG1 (Mei and Xiong, 2008). We found that ErbB4 is primarily expressed in GABAergic neurons, and NRG1 binds to ErbB4 and promotes GABA release in BLA slices (Bi et al., 2015). Thus, we focused our research on NRG1-ErbB4 signaling. In past research, there was some evidence showing that NRG1 and ErbB4 are important for innate fear and conditioned fear memory expression (Lu et al., 2014; Mei and Xiong, 2008). However, the role of NRG1-ErbB4 signaling in fear extinction and the underlying mechanism have not yet been investigated.

Previous studies have found that inhibitory synaptic transmission occurs through Ca²⁺-dependent modulation (Lenz et al., 1998). Previous studies also indicate that the spontaneous release of GABA requires the function of multiple coupled voltage-activated Ca²⁺ channels (VACCs) of variable subtypes (Williams et al., 2012). Pharmacological studies have found that at least six different subtypes of Ca²⁺ channels, L, N, T, P, Q, and R, exist (Tsien et al., 1995). At cortical synapses, P/Q channels have the strongest function in GABA release compared to N and R channels (Cao and Tsien, 2005a). However, to date, the mechanism underlying Ca²⁺-dependent modulation of GABAergic neurotransmission is not well understood. Since NRG1-ErbB4 is important for the regulation of GABAergic neurotransmission, whether VACC-dependent GABAergic neurotransmission could be regulated by NRG1-ErbB4 has not yet been determined; therefore, we attempted to use specific channel-type blockers to test this hypothesis.

We performed neuronal anterograde tracing, electrophysiological recording, optogenetic experiment, fear conditioning test and so on in this research. We examined the function of NRG1-ErbB4 signaling in the ITC and explored the function of NRG1-ErbB4 signaling in fear extinction and the underlying mechanism. We also tested if the GABAergic neurotransmission was regulated by NRG1 through Ca²⁺-dependent synaptic transmission. Our observations revealed an important function for NRG1-ErbB4 signaling in controlling GABAergic neurotransmission of the amygdala and might provide potential targets for the treatment of anxiety disorders.