Fear conditioning potentiates synaptic transmission onto long-range projection neurons in the lateral subdivision of central amygdala

Abstract

Recent studies indicate that the lateral subdivision of the central amygdala (CeL) is essential for fear learning. Specifically, fear conditioning induces cell-type-specific synaptic plasticity in CeL neurons that is required for the storage of fear memories. The CeL also controls fear expression by gating the activity of the medial subdivision of the central amygdala (CeM), the canonical amygdala output to areas that mediate defensive responses. In addition to the connection with CeM, the CeL sends long-range projections to innervate extra-amygdala areas. However, the long-range projection CeL neurons have not been well characterized, and their role in fear regulation is unknown. Here we show in mice that a subset of CeL neurons directly project to the midbrain periaqueductal gray (PAG) and the paraventricular nucleus of the thalamus, two brain areas implicated in defensive behavior. These long-range projection CeL neurons are predominantly somatostatin-positive (SOM(+)) neurons, which can directly inhibit PAG neurons, and some of which innervate both the PAG and paraventricular nucleus of the thalamus. Notably, fear conditioning potentiates excitatory synaptic transmission onto these long-range projection CeL neurons. Thus, our study identifies a subpopulation of SOM(+) CeL neurons that may contribute to fear learning and regulate fear expression independent of CeM.

Keywords: central amygdala; connectivity; fear conditioning; synaptic plasticity.

Figure 1.

SOM⁺ CeL neurons project to PAG and PVT. A, Schematics of CTB injection. B, Representative coronal brain sections showing CTB injection into PAG (left) and PVT (right). In this mouse, the PAG and PVT, respectively, were injected with the CTB conjugated to AlexaFluor-488 (CTB-488, green) and CTB conjugated to AlexaFluor-555 (CTB-555, red) (arrows indicate the injection locations). DG, dentate gyrus. C, A representative coronal brain section containing CeL, in which the PAG- and PVT-projecting neurons were labeled with CTB-555 and CTB-488, respectively. SOM⁺ neurons were recognized by an antibody. Bottom, Higher-magnification images of the boxed area in the corresponding top. Arrowheads indicate the colocalization of CTB-555, CTB-488, and SOM in the same neurons. Arrow indicates the colocalization of CTB555 with SOM, but not with CTB488, in the same neuron. D, Quantification of the percentage of long-range projection CeL neurons that are SOM⁺ (n = 3 mice for each group; mean ± SEM). E, A coronal brain section from a SOM-Cre;Ai14 mouse that contains CeL, in which the PAG-projecting neurons were labeled with CTB-488 (left). SOM⁺ neurons were recognized by the red fluorescence from tdTomato (middle). Bottom, Higher-magnification images of the boxed area in the corresponding top. Arrowheads indicate the colocalization of CTB-488 and tdTomato in the same neurons. F, Same as E, except that the PVT-projecting CeL neurons were examined.

Figure 2.

PKC-δ⁺ neurons do not appreciably project to PAG or PVT. A, A representative coronal brain section containing CeL, in which the PAG-projecting neurons were labeled with CTB-488 (left). PKC-δ⁺ neurons were recognized by an antibody (middle). Bottom, Higher-magnification images of the boxed area in the corresponding top. B, Same as A, except that the PVT-projecting CeL neurons were examined. C, Quantification of the percentage of long-range projection CeL neurons that are PKC-δ⁺ (n = 4 mice for each group; mean ± SEM).

Figure 3.

Direct synaptic connectivity between CeL and PAG. A, Schematic of viral injection. B, Left, Representative image of a coronal section obtained from a SOM-Cre mouse, in which the CeL was injected with the AAV-DIO-ChR2(H134R)-YFP. This section was recovered after electrophysiological recording. Right, Higher magnification of the CeL region, showing that ChR2-YFP is expressed in the SOM⁺ CeL neurons. C, Left, Schematic recording configuration. Right, Light-evoked excitatory currents (at −70 mV holding potential) recorded from a SOM⁺ CeL neuron expressing ChR2-YFP. An LED (λ = 470 nm) was used to deliver a train of blue light pulses (2 ms at 5 Hz, denoted by blue bars). D, Left, Schematic recording configuration. Right, IPSCs (at 0 mV holding potential) recorded from a PAG neuron, in response to the photo-stimulation (2 ms at 5 Hz, denoted by blue bars) of axon terminals originating from SOM⁺ CeL neurons. LA, Lateral amygdala; BLA, basolateral amygdala.

Figure 4.

Fear conditioning potentiates the excitatory synapses onto long-range projection CeL neurons. A, Schematic experimental procedure. B, The behavioral performance of animals used in the electrophysiology experiments, which is measured as the percentage of time animal spent in freezing, during habituation, conditioning, and recall (n = 6 mice, mean ± SEM). C, Schematic recording configuration. Acute slices for electrophysiological recording were prepared 1.5 h after fear memory recall test (see A). D, Representative mEPSC traces recorded from PAG-projection CeL neurons in a fear-conditioned (left) and a control mouse (right). Control mice were treated with the same procedure as fear-conditioned mice, except for foot shocks. E, Quantification of the frequency (left) and amplitude (right) of mEPSCs recorded from the fear-conditioned and control mice (PAG-projecting, fear, n = 28 cells in 6 mice, control, n = 31 cells in 6 mice; PVT-projecting, fear, n = 32 cells in 6 mice, control, n = 21 cells in 6 mice; dual-projecting, fear, n = 18 in 5 mice, control, n = 16 cells in 4 mice; mean ± SEM). *p < 0.05 (t test). ***p < 0.001 (t test). LA, Lateral amygdala; BLA, basolateral amygdala.