Somatostatin Neurons of the Bed Nucleus of Stria Terminalis Enhance Associative Fear Memory Consolidation in Mice

Abstract

Excessive fear learning and generalized, extinction-resistant fear memories are core symptoms of anxiety and trauma-related disorders. Despite significant evidence from clinical studies reporting hyperactivity of the bed nucleus of stria terminalis (BNST) under these conditions, the role of BNST in fear learning and expression is still not clarified. Here, we tested how BNST modulates fear learning in male mice using a chemogenetic approach. Activation of GABAergic neurons of BNST during fear conditioning or memory consolidation resulted in enhanced cue-related fear recall. Importantly, BNST activation had no acute impact on fear expression during conditioning or recalls, but it enhanced cue-related fear recall subsequently, potentially via altered activity of downstream regions. Enhanced fear memory consolidation could be replicated by selectively activating somatostatin (SOM), but not corticotropin-releasing factor (CRF), neurons of the BNST, which was accompanied by increased fear generalization. Our findings suggest the significant modulation of fear memory strength by specific circuits of the BNST.SIGNIFICANCE STATEMENT The bed nucleus of stria terminalis (BNST) mediates different defensive behaviors, and its connections implicate its integrative modulatory role in fear memory formation; however, the involvement of BNST in fear learning has yet to be elucidated in detail. Our data highlight that BNST stimulation enhances fear memory formation without direct effects on fear expression. Our study identified somatostatin (SOM) cells within the extended amygdala as specific neurons promoting fear memory formation. These data underline the importance of anxiety circuits in maladaptive fear memory formation, indicating elevated BNST activity as a potential vulnerability factor to anxiety and trauma-related disorders.

Keywords: BNST; DREADD; extended amygdala; fear; somatostatin..

Figure 1.

BNST neurons are activated during fear conditioning but not during CS-dependent fear recall. A, B, Freezing behavior during auditory fear conditioning and CS recall. Control mice were exposed to the auditory conditioned stimuli (CSs) without footshocks, whereas conditioned animals received seven pairings of shock-cue (CS+US) pairings. During auditory fear recall, mice were exposed to 15 CS+ in an altered context (illustrated in panel B). C, D, Representative fluorescent photomicrographs showing c-Fos immunostaining of BNST subregions during fear conditioning and auditory fear recall, respectively. E, Illustration of investigated BNST subregions for c-Fos quantification. F, Fear conditioning significantly increased c-Fos expression of all BNST subregions (n = 10/groups). G, In contrast, cued fear recall induced no changes in c-Fos expression (n = 10–11/groups). On freezing time curves, each major tick depicts two to three footshock (FS) blocks in case of conditioning, and a 180-s block, starting with a 150-s pretone baseline period (BL) in case of recall test. All data are represented as mean ± SEM. Asterisks represent main effect of ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001. aBNST, anterior BNST; ac, anterior commissure; amBNST, anteromedial BNST; avBNST, anteroventral BNST; fx, fornix; ic, internal capsule; ovBNST, oval nucleus of the BNST; pBNST, posterior BNST; sm, stria medullaris.

Figure 2.

Chemogenetic activation of the BNST facilitates fear learning, but not fear recall. A, Schematics of virus injections in vgat-ires-cre mice and representative photomicrograph of mCherry expression. Right panels show minimum (filled areas) and maximum (areas with colored outlines) extensions of mCherry expression in the BNST. B, Patch clamp recordings from BNST slices: representative photomicrograph depicting a vGAT+ neuron filled with biocytin. Right panel shows representative trace of a hM3Dq expressing neuron indicating CNO-mediated depolarization in the presence of TTX, which was absent in BNST^vGAT neurons expressing control fluorophore. C, CNO administration also elevated the frequency of APs evoked by depolarizing current steps and decreased the rheobase. D, Intraperitoneal injection of CNO under homecage condition induced significant c-Fos expression in hM3Dq-mCherry-expressing neurons (control: n = 9, hM3Dq: n = 9). E, Experimental design for auditory fear conditioning. F, I, Schematics of chemogenetic modulation of the BNST during fear conditioning or memory consolidation, respectively, and freezing behavior exhibited during conditioning. BNST stimulation did not affect acute fear response (freezing) during conditioning. G, BNST stimulation during conditioning did not affect contextual fear recall, but enhanced cued fear recall (H; control: n = 8, hM3Dq: n = 9). Similarly, chemogenetic activation of BNST^vGAT neurons during fear memory consolidation resulted in enhanced cued fear recall with additional fear generalization on day 2 (freezing during baseline; K), without altering contextual fear recall in context A (J). L, Experimental design for chemogenetic activation of the BNST during cued fear recall with conditioned (CS+) and safety cue (CS–) presentations. Freezing response of hM3Dq and control groups were similar during all testing phase, i.e., conditioning (M), short CS+ and CS– recall (N), CS+ and CS– induced fear recall (O; control: n = 8, hM3Dq: n = 13). On freezing time curves, each major tick depicts two to three footshock (FS) blocks in case of conditioning, and a 180-s block, starting with a 150-s pretone baseline period (BL) in case of recall test. All data are represented as mean ± SEM. Asterisks represent main effect of one-way, two-way, or repeated-measure ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; # represents significant difference in BL period p < 0.05.

Figure 3.

Postsynaptic activity following chemogenetic stimulation of the BNST during memory consolidation. A, Schematics of virus injections and representative photomicrograph of mCherry expression in the BNST. Right panel shows experimental design: c-Fos expression was assesses 6 h after fear conditioning combined with subsequent chemogenetic activation (i.e., in the consolidation phase). B, Representative wide-field fluorescence photomicrographs depicting major projection areas of BNST^vGAT neurons. C, Representative single-plane confocal photomicrographs showing altered c-Fos expression during consolidation in the BNST and downstream regions, where white arrows indicate activated hM3Dq-expressing BNST^vGAT neurons (mCherry+c-Fos). D, C-Fos activity was significantly increased in the BNST, and DMT, vlPAG, ifVTA downstream regions (F), with additional decrease in PVN (E). ac, anterior commissure; amBNST, anteromedial BNST; Aq, cerebral aqueduct; BLA, basolateral amygdala; CeL/CeC, central amygdala, lateral/capsular part; CeM, central amygdala, medial part; cpd, cerebral peduncle; D3V, dorsal part of the third ventricle; DMT, dorsal midline thalamus; DR, dorsal raphe; fx, fornix; ic, internal capsule; LHA, lateral hypothalamic area; MD, mediodorsal thalamus; MHb, medial habenula; MM, medial mammillary nucleus; NAc, nucleus accumbens; opt, optic tract; PVN, paraventricular hypothalamic nucleus; SNc, substantia nigra, pars compacta; st, stria terminalis; vlPAG/lPAG, periaqueductal gray, ventrolateral/lateral part; ifVTA, ventral tegmental area, interfascicular nucleus. Asterisks represent main effect of one-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Chemogenetic activation of BNST^SOM neurons during fear memory consolidation enhances CS-induced fear recall. A, Distribution of BNST^SOM neurons illustrated by representative single-plane confocal photomicrographs from reporter ZsGreen fluorescent protein-expressing mouse lines, and their proportional quantification (% of all neurons, NeuN+) across subregions. B, Experimental design for chemogenetic modulation of BNST^SOM neurons during fear memory consolidation. C, Photomicrograph showing mCherry expression in BNST^SOM neurons and illustration of minimum (filled areas) and maximum (areas with colored outlines) extensions of mCherry expression. D, Intraperitoneal injection of CNO (1 mg/kg) under baseline (homecage) condition significantly increased c-Fos expression in hM3Dq-mCherry-expressing BNST^SOM neurons, while hM4Di-expressing BNST^SOM neurons showed reduced c-Fos expression (control: n = 3, hM3Dq: n = 5, hM4Di: n = 5). E, Freezing behavior during fear conditioning of som-ires-cre mice. Chemogenetic activation of BNST^SOM neurons during fear memory consolidation resulted in enhanced cued fear recall (G, left panel) with additional contextual fear generalization during baseline period 1 d later (G, right panel) and disrupted discrimination between CS and ITI (H). Contextual fear recall in context A was similar between groups (F; control: n = 8, hM3Dq: n = 7). In contrast to stimulation, chemogenetic inhibition of BNST^SOM neurons had no impact on fear recalls (F–H, hM4Di: n = 9). I, Experimental design to test CS-independent contextual fear generalization following chemogenetic stimulation of BNST^SOM neurons. J–L, Chemogenetic stimulation of BNST^SOM neurons did not affect contextual fear recall, either in context A or B (control: n = 9, hM3Dq: n = 11). On freezing time curves, each major tick depicts two to three footshock (FS) blocks in case of conditioning, and a 180-s block, starting with a 150-s pretone baseline period (BL) in case of recall test. All data are represented as mean ± SEM. Asterisks represent main effect of repeated-measure ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001; Hash sign represents significant difference during BL period: #p < 0.05. ac, anterior commissure; aBNST, anterior BNST; amBNST, anteromedial BNST; avBNST, anteroventral BNST; ovBNST, oval nucleus of the BNST; pBNST, posterior BNST.

Figure 5.

Chemogenetic modulation of BNST^CRF neurons during fear memory consolidation does not affect fear recalls. A, Distribution of BNST^CRF neurons illustrated by representative single-plane confocal photomicrographs from reporter ZsGreen fluorescent protein-expressing mouse lines and their proportional quantification (% of all neurons, NeuN+) across subregions. B, Representative photomicrographs of mCherry expression in BNST^CRF neurons and illustration of minimum (filled areas) and maximum (areas with colored outlines) extensions of mCherry expression. C, Intraperitoneal injection of CNO (1 mg/kg) under baseline (homecage) condition significantly increased c-Fos expression in hM3Dq-mCherry-expressing BNST^CRF neurons (control: n = 7, hM3Dq: n = 7, hM4Di n = 6). D and H, Illustrations depicting high (0.7-mA footshocks) and low-intensity (0.4-mA footshocks) fear conditioning in crh-ires-cre mice with CNO administration after conditioning. Fear recalls were independent of BNST manipulation following both high-intensity (F–G) and low-intensity (J–K) trainings. E and I, Freezing during high- and low-intensity trainings, respectively (control: n = 15 and n = 10, hM3Dq: n = 12 and n = 14 for low-intensity and high-intensity trainings, respectively; hM4Di: n = 13). Asterisks represent main effect of one-way ANOVA: **n < 0.01.

Figure 6.

AAV based mapping of major projection areas of BNST^CRF (A) and BNST^SOM (B) neurons. Representative wide-field fluorescence photomicrographs show a highly similar distribution of BNST^CRF and BNST^SOM projections. 3V, third ventricle; 4V, fourth ventricle; ac, anterior commissure; amBNST, anteromedial BNST; avBNST, anteroventral BNST; BLAa, basolateral amygdala, anterior part; CeL, central amygdala, lateral part; CeM, central amygdala, medial part; CLi, central linear nucleus raphe; cpd, cerebral peduncle; D3V, dorsal part of the third ventricle; DRD, dorsal raphe, dorsal part; DRL, dorsal raphe, lateral part; DRV, dorsal raphe, ventral part; fx, fornix; GP, globus pallidus; ic, internal capsule; IPN, interpeduncular nucleus; LC, locus coeruleus; LHa, lateral hypothalamic area; LHb, lateral habenula; MeA, medial amygdala; MeAad, medial amygdala nucleus, anterodorsal part; MHb, medial habenula; MM, medial mammillary nucleus; opt, optic tract; ov, oval nucleus of the BNST; pBNST, posterior BNST; PMv, ventral premammillary nucleus; PSTh, parasubthalamic nucleus; PVN, paraventricular hypothalamic nucleus; PVT, paraventricular thalamic nucleus; RRF, retrorubral field; scp, superior cerebellar peduncles; SI, substantia innominate; sm, stria medullaris; SNc, substantia nigra, pars compacta; st, stria terminalis; STh, subthalamic nucleus; SUM, supramammillary nucleus; TMV, tuberomammillary nucleus, ventral part; vlPAG, ventrolateral periaqueductal gray; VTA, ventral tegmental area.