Central Amygdala Projections to Lateral Hypothalamus Mediate Avoidance Behavior in Rats

Abstract

Persistent avoidance of stress-related stimuli following acute stress exposure predicts negative outcomes such as substance abuse and traumatic stress disorders. Previous work using a rat model showed that the central amygdala (CeA) plays an important role in avoidance of a predator odor stress-paired context. Here, we show that CeA projections to the lateral hypothalamus (LH) are preferentially activated in male rats that show avoidance of a predator odor-paired context (termed Avoider rats), that chemogenetic inhibition of CeA-LH projections attenuates avoidance in male Avoider rats, that chemogenetic stimulation of the CeA-LH circuit produces conditioned place avoidance (CPA) in otherwise naive male rats, and that avoidance behavior is associated with intrinsic properties of LH-projecting CeA cells. Collectively, these data show that CeA-LH projections are important for persistent avoidance of stress-related stimuli following acute stress exposure.SIGNIFICANCE STATEMENT This study in rats shows that a specific circuit in the brain [i.e., neurons that project from the central amygdala (CeA) to the lateral hypothalamus (LH)] mediates avoidance of stress-associated stimuli. In addition, this study shows that intrinsic physiological properties of cells in this brain circuit are associated with avoidance of stress-associated stimuli. Further characterization of the CeA-LH circuit may improve our understanding of the neural mechanisms underlying specific aspects of stress-related disorders in humans.

Keywords: HCN channels; avoidance; central amygdala; lateral hypothalamus; stress.

Figure 1.

CeA projections form functional connections with LH neurons. A, Schematic of intra-CeA microinjections of AAV5-hSyn-mCherry. Right, Image showing mCherry expression in a coronal brain section containing the CeA and LH (scale bar: 500 µm). Inset, High-magnification image of mCherry fibers in LH (scale bar: 20 µm). 3V: third ventricle, f: fornix, ZI: zona incerta, LH: lateral hypothalamus, Opt: optic tract, IC: internal capsule, MeA: medial amygdala, CeA: central amygdala, BLA: basolateral amygdala. B, Schematic showing approximate location of mCherry+ terminals (red) in LH along the rostro-caudal axis. C, Representative trace showing optically evoked current of LH neuron in the absence (black) and presence (orange) of the GABA-A receptor blocker PTX. D, In neurons that received PTX, relative synaptic response amplitudes were significantly decreased (*p < 0.001). E, An example recording of postsynaptic current in an LH neuron showing response to optical stimulation of CeA neuron axon terminals before and after bath application of TTX and 4AP. F, Schematic of intra-LH microinjections of Retrobeads. Right, Representative image of intra-LH Retrobeads microinjection site (scale bar: 200 µm). G, left, Low-magnification (2×) image showing medial (CeM) and lateral (CeL) subdivisions of CeA and surrounding landmarks. LA: lateral amygdala, BLA: basolateral amygdala, PIR: piriform cortex. Middle and right, Representative images of Retrobeads+ cells in CeM versus CeL (scale bar: 50 µm). H, Retrobeads+ cell counts in CeM versus CeL of Avoider, Non-Avoider, and Controls rats (*p < 0.001; n = 6 or 7 rats per group). I, Expression of Crhr1 in CeA-LH projection cells (labeled with CTB-555; scale bar: 50 µm). Arrows indicate examples of double-labeled cells.

Figure 2.

Avoiders have more c-Fos+ CeA-LH cells. A, Timeline schematic of experiment 2. B, Predator odor stress produces conditioned avoidance in a subset of rats. C, Representative images of c-Fos+ (white) and Retrobeads+ (red) cells in the CeA of Controls, Non-Avoiders, and Avoiders (scale bar: 20 µm). Total number of c-Fos+ cells in CeA (D), CeM (E), and CeL (F) of Avoiders (red), Non-Avoiders (blue), and Controls (black). Percent of c-Fos+/Retrobeads+ cells over total Retrobeads+ cells in CeA (G), CeM (H), and CeL (I) of Avoiders, Non-Avoiders, and Controls; *p < 0.05 (see text for exact p value), n = 6 or 7 rats per group.

Figure 3.

Conditioned avoidance of a predator odor-paired context is attenuated by CeA-LH cell inhibition and mimicked by CeA-LH cell activation. A, Schematic of intersectional viral expression strategy for targeting hM4D(Gi)-mCherry, hM3D(Gq)-mCherry, or mCherry control to CeA-LH cells. B, Image of mCherry expression in CeA cell bodies (left panel) and fibers in LH (right panel). BLA: basolateral amygdala, CeA: central amygdala, f: fornix, IC: internal capsule, LH: lateral hypothalamus, MeA: medial amygdala, ZI: zona incerta. C, Timeline schematic for testing the effect of CeA-LH circuit inhibition on avoidance behavior. D, Avoidance scores in Avoider and Non-Avoider rats that were given intra-CeA active Gi-DREADD or control virus and CNO or vehicle pretreatment during test 1 and test 2. E, Timeline schematic of CNO/vehicle place conditioning procedure. F, Avoidance scores of rats that received intra-CeA active Gq-DREADD or control virus, split by CNO treatment group and across three posttest sessions; *p < 0.05 (see text for exact p values), n = 6–9 rats per group.

Figure 4.

CeA-LH cells in Avoiders have altered intrinsic properties after stress. A, Representative image showing spatial distribution of fluorescent Retrobeads-labeled cells in CeA (scale bar: 200 µm). RMP (B) and input resistance (C) of CeA-LH cells in Avoiders, Non-Avoiders, and Controls. D, Example traces of postsynaptic currents from a CeA-LH cell recorded with membrane voltage clamped at −70 and −50 mV. sEPSC event amplitude (E) and frequency (F) of CeA-LH cells in Avoiders, Non-Avoiders, and Controls. sIPSC event amplitude (G) and frequency (H) in CeA-LH cells of Avoiders, Non-Avoiders, and Controls. I, A set of membrane voltage responses in step current injections of varying amplitudes in an example CeA cell. Scale bars: 100 mV and 500 ms. J, Input-output current-firing rate function of CeA-LH cells in Avoiders, Non-Avoiders, and Controls constructed using spike counts from current step responses. K, Firing rate in response to 200-pA step current injection. L, An example voltage trace (baseline −70 mV) in response to a hyperpolarizing current. The SAG fraction is characterized as the ratio of: (1) the voltage difference between the minimum hyperpolarization and the new steady state voltage, and (2) the difference between new steady state and baseline resting voltage (−70 mV). These quantities are denoted A and B, respectively, here. M, The SAG voltage fraction of CeA→LH cells from Avoiders, Non-Avoiders, and Controls; *p < 0.05, **p < 0.01; n = 74 cells from 17 rats.

Figure 5.

HCN subunit-specific function in CeA-LH cells after stress. A, SAG fraction magnitude pre-ZD7288 and post-ZD7288 (20 μm) application. B, Voltage SAG decay fitted with a single exponential decay function to estimate relative timescales of the voltage response from HCN currents. C, Decay timescale of the SAG in Avoiders, Non-Avoiders, and Controls. D, Example membrane voltage traces in response to hyperpolarizing step current injections before and after bath application of capsazepine (20 μm) in Control, Non-Avoider, and Avoider rats. E, Relative SAG magnitude in CeA-LH cells of Avoiders, Non-Avoiders, and Controls following capsazepine application. F, The change in decay time constant of the voltage SAG after capsazepine bath application. G, Correlation between relative SAG amplitude and change in timescale (from E, F) when data from Avoiders, Non-Avoiders, and Controls are pooled; *p < 0.05, **p < 0.01; n = 26 cells from 15 rats.