Optogenetic silencing of a corticotropin-releasing factor pathway from the central amygdala to the bed nucleus of the stria terminalis disrupts sustained fear

Abstract

The lateral central nucleus of the amygdala (CeA_L) and the dorsolateral bed nucleus of the stria terminalis (BNST_DL) coordinate the expression of shorter- and longer-lasting fears, respectively. Less is known about how these structures communicate with each other during fear acquisition. One pathway, from the CeA_L to the BNST_DL, is thought to communicate via corticotropin-releasing factor (CRF), but studies have yet to examine its function in fear learning and memory. Thus, we developed an adeno-associated viral-based strategy to selectively target CRF neurons with the optogenetic silencer archaerhodopsin tp009 (CRF-ArchT) to examine the role of CeA_L CRF neurons and projections to the BNST_DL during the acquisition of contextual fear. Expression of our CRF-ArchT vector injected into the amygdala was restricted to CeA_L CRF neurons. Furthermore, CRF axonal projections from the CeA_L clustered around BNST_DL CRF cells. Optogenetic silencing of CeA_L CRF neurons during contextual fear acquisition disrupted retention test freezing 24 h later, but only at later time points (>6 min) during testing. Silencing CeA_L CRF projections in the BNST_DL during contextual fear acquisition produced a similar effect. Baseline contextual freezing, the rate of fear acquisition, freezing in an alternate context after conditioning and responsivity to foot shock were unaffected by optogenetic silencing. Our results highlight how CeA_L CRF neurons and projections to the BNST_DL consolidate longer-lasting components of a fear memory. Our findings have implications for understanding how discrete amygdalar CRF pathways modulate longer-lasting fear in anxiety- and trauma-related disorders.

Figure 1

AAV2/2 CRF promoter driven ArchT vector selectively targets CRF cells. (a) CRF-ArchT and CRF-EGFP control vectors. (b) Schematic of CeA_L viral injection site. (c) Immunohistochemical labeling shows CRF-ArchT-EGFP+ protein is restricted to the CeA. Note the fluorescence extending into the basolateral amygdala (BLA; white arrow) and towards the stria terminalis (asterisk). (d) In situ hybridization, DAPI staining, and intrinsic EGFP+ fluorescence across three neurons in the CeA_L (two aqua arrows and one white arrow). Of the three cells with DAPI stained nuclei (blue stain), only one (white arrow) displays triple expression of cytoplasmic CRF-ArchT-EGFP+ protein (green) and mRNA (yellow-white) with CRF mRNA (red). (e) Magnification of panel d showing the CRF-ArchT-EGFP+ labeled CRF neuron (white arrow). Note the two adjacent cells (aqua arrows) which do not synthesize CRF mRNA also do not exhibit EGFP mRNA or protein. The green-labeled long process appears to be an EGFP+ axonal projection (asterisk).

Figure 2

Confirmation of ArchT function in whole-cell recordings from CeA_L Neurons in an in vitro slice preparation. (a) Schematic showing site of CRF-ArchT injection into CeA_L. (b) Epifluorescent image demonstrating EGFP+ expression in a single CeA_L neuron. (c) Voltage clamp recording (holding potential −60mV) of a neuron demonstrating outward currents elicited by photostimulation (indicated by green bar); the size of the current increased with increasing laser output. (d) Current clamp recording of a neuron demonstrating ArchT-mediated inhibition of firing induced by depolarizing current injection (+100 pA, 3 s) through the patch pipette. The photostimulation period is indicated by the green box. (e) Summary of recordings from CeA_L neurons (n = 4, from 3 rats). Action potentials elicited during a 1-s photostimulation were compared to those preceding and following the stimulation. Neuronal firing was significantly inhibited by laser light. * denotes firing during laser-on was significantly different from pre and post illumination recording.

Figure 3

Silencing of CeA_L CRF+ cells during acquisition disrupts later components of fear retention. (a) Schematic of CeA_L viral injection site and fiber placement. (b) Parameters for contextual fear conditioning. (c) Laser silencing did not affect baseline activity/freezing or fear acquisition. (d) At fear retention, CRF-ArchT rats (n=13) exhibited reduced freezing at time bins 3, 4, and 5 relative to CRF-EGFP (n=11) controls. Each bin represents a three-minute period. *p<.05, error bars are ± S.E.M.

Figure 4

Effects of silencing CRF+ CeA_L cells on retention of auditory fear conditioning. (a) Schematic of CeA_L viral injection site and fiber placement. (b) Schematic of experimental timeline. Animals were trained with five 30-s CSs co-terminating with a 1-s 0.6 mA foot-shock. (c) Silencing of CRF+ CeA_L neurons did not statistically affect auditory fear acquisition, but freezing tended to increase over the last few CS-shock pairings (d) At fear retention, CRF-ArchT (n=11) rats tended to have reduced freezing during the last two presentations of the CS relative to CRF-EGFP (n=10) controls (e) A difference score removing outliers > 2 S.D. and examining the change from training to testing (i.e., CS1 testing – CS1 training, etc.) revealed CRF-ArchT (n=8) animals exhibited a greater reduction in freezing relative to CRF-EGFP (n=7) controls during the last few CSs. A lower difference score indicates a greater reduction in freezing. *p<.05, error bars are ± S.E.M.

Figure 5

Silencing of the CeA_L → BNST_DL CRF pathway during acquisition only disrupts later components of fear retention. (a) Schematic of CeA_L viral injection site and BNST_DL fiber placement. (b) Rat atlas overlay showing EGFP expression in BNST_DL. (c) Axonal projections in BNST_DL (arrows). (d) Laser silencing with same parameters as shown in Fig.2b did not affect baseline activity/freezing or fear acquisition. (e) At fear retention CRF-ArchT (n=8) animals exhibited reduced freezing at time bins 4 and 6 relative to CRF-EGFP (n=10) controls. Each bin represents a three-minute period. *p<.05, error bars are ± S.E.M.