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. 2024 Apr 24;15(1):3455.
doi: 10.1038/s41467-024-47966-2.

Synergism between two BLA-to-BNST pathways for appropriate expression of anxiety-like behaviors in male mice

Ren-Wen Han  1   2 , Zi-Yi Zhang  3   4 , Chen Jiao  3   4 , Ze-Yu Hu  3   4 , Bing-Xing Pan  5   6
Affiliations

Synergism between two BLA-to-BNST pathways for appropriate expression of anxiety-like behaviors in male mice

Ren-Wen Han et al. Nat Commun. .

Abstract

Understanding how distinct functional circuits are coordinated to fine-tune mood and behavior is of fundamental importance. Here, we observe that within the dense projections from basolateral amygdala (BLA) to bed nucleus of stria terminalis (BNST), there are two functionally opposing pathways orchestrated to enable contextually appropriate expression of anxiety-like behaviors in male mice. Specifically, the anterior BLA neurons predominantly innervate the anterodorsal BNST (adBNST), while their posterior counterparts send massive fibers to oval BNST (ovBNST) with moderate to adBNST. Optogenetic activation of the anterior and posterior BLA inputs oppositely regulated the activity of adBNST neurons and anxiety-like behaviors, via disengaging and engaging the inhibitory ovBNST-to-adBNST microcircuit, respectively. Importantly, the two pathways exhibited synchronized but opposite responses to both anxiolytic and anxiogenic stimuli, partially due to their mutual inhibition within BLA and the different inputs they receive. These findings reveal synergistic interactions between two BLA-to-BNST pathways for appropriate anxiety expression with ongoing environmental demands.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The aBLA and pBLA PNs have distinct projection patterns in dBNST.
a Schematic showing injection of AAV vectors encoding EYFP and mCherry into aBLA and pBLA respectively, to anterogradely label the axonal terminals of their PNs in the two dBNST subregions. b Representative images showing EYFP and mCherry expression in aBLA (left) and pBLA (right) respectively. D dorsal, L left, CeA central amygdala, vHPC ventral hippocampus. This experiment was repeated 4 times with similar results. c Representative images showing the axonal terminals of aBLA (top) and pBLA (middle) PNs in the adBNST and ovBNST subregions of dBNST along the antero-posterior axis. Images were merged on the bottom. ac anterior commissure, icv intracerebroventricular. d Relative fluorescence intensity of the axonal terminals of aBLA and pBLA PNs in adBNST and ovBNST as shown in (c). n = 4 mice, *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test. e Schematic showing injection of the retro-AAV vector encoding mCherry into adBNST to retrogradely label the BLA→adBNST PNs. f Representative image showing the injection site in adBNST. This experiment was repeated 4 times with similar results. g Representative images showing the retrogradely labeled BLA→adBNST PNs along the antero-posterior axis. This experiment was repeated 4 times with similar results. h Quantification of the retrogradely labeled BLA→adBNST PNs in (g). n = 4 mice. i–l Same as (e–h) except that the retro-AAV was injected into the ovBNST. n = 3 mice. All data shown as means ± s.e.m. Source data including statistics are provided as a Source Date file.
Fig. 2
Fig. 2. aBLA and pBLA inputs oppositely regulate the activity of adBNST neurons.
a Experimental procedures for investigating the effect of optogenetic activation of aBLA or pBLA inputs on the dBNST neuronal activity in vitro. b Schematic showing recording of the firing of adBNST neurons to optogenetic activation of the entire aBLA inputs in dBNST. c Representative trace showing the firing of an adBNST neuron before, during and after aBLA inputs activation. d Summary of the spike frequency as shown in (c). Data from individual neurons were shown in gray. Inset showing the percentage of adBNST cells excited or inhibited following aBLA inputs activation. n = 16 neurons/5 mice; ***p < 0.001, one-way ANOVA with Bonferroni’s multiple comparisons test. e Schematic showing recording of the firing of ovBNST neurons upon activation of pBLA inputs in dBNST. f Representative trace showing the firing of an ovBNST neuron before, during and after pBLA inputs activation. g Summary of the spike frequency as shown in (f). Data from individual neurons were shown in gray. Inset showing the percentage of ovBNST cells excited or inhibited following pBLA inputs activation. n = 14 neurons/5 mice, ***p < 0.001, one-way ANOVA with Bonferroni’s multiple comparisons test. h–j Same as in (e–g) except that the recordings were made on the adBNST neurons. n = 16 neurons/5 mice, ***p < 0.001, one-way ANOVA with Bonferroni’s multiple comparisons test. k Experimental procedures for investigating the effect of chemogenetic activation of aBLA→dBNST or pBLA→dBNST PNs on adBNST neuronal activity in vivo. i.p., intraperitoneal injection. l Left: Representative images showing the expression of GCaMP6s (green) in adBNST cells and the projections (red) from aBLA→dBNST PNs. The positioning of optical fiber was marked with the dashed lines. Right: Expression of hM3Dq in aBLA→dBNST PNs. Scale bar: 200 μm. This experiment was repeated 7 times with similar results. m Representative traces showing the calcium signals in adBNST cells before and after vehicle (top) or CNO (bottom) administration. n–p Summary of the calcium signal changes in adBNST cells following vehicle and CNO administration. Data from individual mice were shown in gray. n = 7 mice, *p < 0.05, **p < 0.01, ***p < 0.001, two tailed paired Student’s t-test. q–u Same as in (n–p) except that hM3Dq was expressed in pBLA→dBNST PNs. n = 6 mice, **P < 0.01, two tailed paired Student’s t-test. All data shown as means ± s.e.m. Source data including statistics are provided as a Source Date file.
Fig. 3
Fig. 3. pBLA inputs activation drives strong feedforward inhibition from ovBNST to adBNST.
a Representative traces showing oEPSCs and oIPSCs in adBNST neurons upon optogenetic activation of the entire aBLA (left) or pBLA (right) inputs with blue light pulses of increasing intensity. b, c Summary of the oEPSCs and oIPSCs amplitudes against the increasing intensity of light stimuli delivered to aBLA (b) and pBLA (c) inputs. n = 15 neurons/5 mice for both groups. *p < 0.05, two-way ANOVA with Bonferroni’s multiple comparisons test. d, e Distribution of the oEPSCs and oIPSCs amplitudes in each recorded adBNST cells when the light stimuli were delivered to aBLA (d) and pBLA (e) inputs at an intensity of 100 µW/mm2. f Summary of the I/E ratio in (d and e). n = 15 neurons/5 mice for both groups. ***p < 0.001, two tailed unpaired Student’s t-test. g Schematic diagrams showing recording of the oIPSCs in adBNST neurons or their firings upon selective activation of the pBLA terminals in either ovBNST or adBNST with focalized light stimuli. Note that the stimuli were sequentially delivered to either of the two subregions at a randomized order. h Representative traces showing oIPSCs recorded from one identical adBNST neuron when the light stimuli were delivered to adBNST (left) and ovBNST (right). i Summary of the oIPSCs amplitudes in adBNST neurons as shown in (h). n = 14 neurons/4 mice. *p < 0.05, **p < 0.01, two-way ANOVA with Bonferroni’s multiple comparisons test. j Representative traces showing the firing of one identical adBNST cell in response to the light activation of pBLA terminals in adBNST (left) and ovBNST (right). k Summary of the spike frequency of adBNST neurons before, during and after selective activation of pBLA inputs in either the adBNST or ovBNST as shown in (j). n = 17 neurons/5 mice; **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni’s multiple comparisons test. All data shown as means ± s.e.m. Source data including statistics are provided as a Source Date file.
Fig. 4
Fig. 4. Optogenetic activation of aBLA→dBNST and pBLA→dBNST pathways oppositely regulates the anxiety-like behaviors in mice.
a, Experimental procedures for investigating the effect of optogenetic activation of aBLA→dBNST pathway on mice’s anxiety-like behaviors. OFT, open field test, EPM, elevated plus maze. b, Schematic showing injection of AAV vectors encoding EYFP or ChR2 into aBLA and implantation of optical cannula onto dBNST for optogenetic manipulations. c, Left: Representative image showing the axonal terminals from aBLA PNs in dBNST. The positioning of optical fiber was marked with the dashed lines. Right: ChR2 expression in aBLA PNs. This experiment was repeated 11 times with similar results. d, Representative activity tracking across epochs in OFT for a mouse expressing ChR2 in aBLA. The designated center zone was marked with dashed lines. e, Summary of the time in center and total distance travelled before, during and after light stimulation of the EYFP- or ChR2-expressing aBLA inputs. Data from individual mice were shown in light colors. n = 10 (EYFP), 11 (ChR2) mice. **p < 0.01, two-way ANOVA with Bonferroni’s multiple comparisons test. f, Representative activity tracking across epochs in EPM for a mouse expressing ChR2 in aBLA. g, Summary of the time in open arms and open-arm entries measured before, during and after light stimulation of the EYFP- or ChR2-expressing aBLA inputs. Data from individual mice were shown in light colors. *p < 0.05, **p < 0.01, two-way ANOVA with Bonferroni’s multiple comparisons test. h–n, same as in (a–g), except that EYFP or ChR2 was expressed in the pBLA PNs. n = 9 (EYFP), 10 (ChR2) mice. *p < 0.05, ***p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test. All data shown as means ± s.e.m. Source data including statistics are provided as a Source Date file.
Fig. 5
Fig. 5. Optogenetic activation of mpBLA→adBNST and lpBLA→ovBNST pathways oppositely regulates the anxiety-like behaviors in mice.
a Experimental procedures for investigating the effect of optogenetic activation of mpBLA→adBNST pathway in regulating the mice’s anxiety-like behaviors. OFT, open field test, EPM, elevated plus maze. b Schematic showing injection of AAV vectors encoding EYFP or ChR2 into mpBLA and implantation of cannula onto adBNST for optogenetic manipulations. c Left: Representative image showing the axonal terminals from mpBLA PNs. The position of optical fiber was marked with the dashed lines. Right: ChR2 expression in mpBLA PNs. This experiment was repeated 10 times with similar results. d Summary of the time in center region and total distance travelled in OFT before, during and after light stimulation of the EYFP- or ChR2-expressing mpBLA projections. n = 12 (EYFP), 10 (ChR2) mice. Data from individual mice were shown in gray. ***p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test. e Summary of the time in open arms and open-arm entries in EPM before, during and after light stimulation of EYFP- or ChR2-expressing mpBLA projections. n = 12 (EYFP), 10 (ChR2) mice. *p < 0.05, two-way ANOVA with Bonferroni’s multiple comparisons test. f–j same as in (a–e), except that EYFP or ChR2 was expressed in lpBLA PNs and the cannula was implanted onto ovBNST. n = 10 mice for both. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test. All data shown as means ± s.e.m. Source data including statistics are provided as a Source Date file.
Fig. 6
Fig. 6. The aBLA→adBNST and lpBLA→ovBNST pathways oppositely regulate the adBNST neuronal activity and anxiety-like behaviors in OFT and EPM.
a Experimental procedures for investigating the effect of optogenetic activation of aBLA→dBNST or pBLA→dBNST PNs on adBNST neuronal activity and anxiety-like behaviors in OFT and EPM. OFT, open field test, EPM, elevated plus maze. b Left: Representative images showing the expression of jRGECO1a (red) in adBNST cells and the projections (green) from aBLA→dBNST PNs. Right: Expression of ChR2 in aBLA→dBNST PNs. The positions of optical fiber were marked with the dashed lines. This experiment was repeated 7 times with similar results. c, d Heat maps showing the calcium signal in the adBNST neurons of individual mice before, during and after light stimulation of the EYFP- (top) or ChR2-expressing (bottom) aBLA→dBNST PNs in OFT (c) and EPM (d). n = 7 mice for both groups. e, f Summary of the calcium signal, time in center and total distance travelled in OFT before and during light stimulation of the EYFP- (e) or ChR2-expressing (f) aBLA→dBNST PNs. Data from individual mice were shown in gray. **P < 0.01, two tailed paired Student’s t-test. g, h Summary of the calcium signal, time in open arms and open arm entries in EPM before and during light stimulation of the EYFP- (g) or ChR2-expressing (h) aBLA→dBNST PNs. Data from individual mice were shown in gray. **P < 0.01 and ***P < 0.001, two tailed paired Student’s t-test. i–o Same as in (b–h) except that ChR2 was expressed in lpBLA→dBNST PNs. n = 7 (EYFP), 6 (ChR2) mice. *P < 0.05 and **P < 0.01, two tailed paired Student’s t-test. All data shown as means ± s.e.m. Source data including statistics are provided as a Source Date file.
Fig. 7
Fig. 7. The aBLA→adBNST and lpBLA→ovBNST pathways synergistically encode the anxiety-relevant cues in the environment.
a Experimental procedures for real-time monitoring of the calcium signal changes in the aBLA→adBNST axonal terminals when the mice explored in the elevated plus maze (EPM). b Schematic showing injection of AAV vectors encoding GCaMP6s into aBLA and implantation of the recorded optical cannula onto adBNST. c Left: Representative image showing the axonal terminals expressing GCaMP6s from aBLA PNs. The positioning of optical cannula was marked with the dashed lines. Right: GCaMP6s expression in aBLA PNs. This experiment was repeated 9 times with similar results. d Changes of average calcium transients in aBLA→adBNST fibers when the mice moved from the closed arm to the open arm. n = 9 mice. e Heat maps showing the calcium fluorescence in the aBLA→adBNST terminals of individual mice (2–7 trails for each) in (d). f, g Same as in (d–e) except that the calcium signals were measured when the mice transitioned from open to the closed arm. h summary of the changes of the average calcium signals in aBLA→adBNST fibers when mice explored between the closed and open arms of EPM. ***p < 0.001, two tailed one sample t test. i–p Same as in (a–h), except that GCaMP6s was expressed in lpBLA PNs and the cannula was implanted onto ovBNST. n = 9 mice. ***p < 0.001, two tailed one sample t-test. All data shown as means ± s.e.m. Source data including statistics are provided as a Source Date file.
Fig. 8
Fig. 8. The aBLA→adBNST and lpBLA→ovBNST PNs are reciprocally inhibited through recruiting the local interneurons.
a Experimental procedures for investigating the effect of optogenetic activation of aBLA→adBNST PNs on the activity of lpBLA→ovBNST PNs. b Left: Representative image showing unbiased mCherry expression in both aBLA→dBNST and pBLA→dBNST PNs and selective ChR2 expression in aBLA→adBNST PNs in a horizontal slice. Right: The magnified image of BLA. icv intracerebroventricular; HPC hippocampus. This experiment was repeated 4 times with similar results. c Schematic showing recording of the firing of lpBLA→ovBNST PNs upon optogenetic activation of aBLA→adBNST PNs. d Representative trace showing the firing of lpBLA→ovBNST PN before, during and after activation of aBLA→adBNST PNs. e Summary of the spike frequency as shown in (d). Data from individual neurons were shown in gray. Inset showing the percentage of lpBLA→ovBNST PNs excited or inhibited following aBLA→adBNST neuronal activation. n = 11 neurons/4 mice; ***p < 0.001, one-way ANOVA with Bonferroni’s multiple comparisons test. f–h Same as in (c–e), except that the GABAergic interneurons in lpBLA (lpBLAGABA) were recorded. n = 10 neurons/4 mice; **p < 0.01, one-way ANOVA with Bonferroni’s multiple comparisons test. i Summary of the spike frequency in lpBLA→ovBNST PNs before, during and after aBLA→adBNST neuronal activation in the presence of PTX (100 µM). Data from individual neurons were shown in gray. Inset showing the percentage of lpBLA→ovBNST PNs excited or inhibited following aBLA→adBNST neuronal activation. n = 6 neurons/3 mice. j–r Same as in (a–i) except that ChR2 was selectively expressed in lpBLA→ovBNST PNs and the recordings were made on the aBLA neurons. k This experiment was repeated 4 times with similar results. n n = 10 neurons/4 mice; ***p < 0.001, one-way ANOVA with Bonferroni’s multiple comparisons test. q n = 7 neurons/3 mice; *p < 0.05, one-way ANOVA with Bonferroni’s multiple comparisons test. r n = 7 neurons/3 mice. All data shown as means ± s.e.m. Source data including statistics are provided as a Source Date file.
Fig. 9
Fig. 9. Whole-brain mapping of the monosynaptic inputs to aBLA→adBNST and lpBLA→ovBNST PNs.
a Experimental procedures for whole-brain tracing of the monosynaptic inputs to aBLA→adBNST and lpBLA→ovBNST PNs using pseudotyped rabies. b Representative images showing the aBLA→adBNST (green, left) and lpBLA→ovBNST (green, right) PNs. The arrowed showing the start cells expressing RVG, TVA-EYFP and ENVA-N2C(dG)-tdtomato. This experiment was repeated 4 times with similar results. c Representative images showing the retrogradely labeled neurons in brain regions which exhibit distinct projections to aBLA→adBNST and lpBLA→ovBNST PNs. AI agranular insular area, AUD auditory areas, CA1v field CA1 ventral region, ENT entorhinal area. d Percentages of the retrogradely labeled cells in different upstream regions in ipsilateral sides of the injected sites. n = 4 mice. *p < 0.05, **p < 0.01, ***p < 0.001, multiple t-test. Data shown as means ± s.e.m. Source data including statistics are provided as a Source Date file.
Fig. 10
Fig. 10. A proposed model for the coordination between the two functionally opposing BLA → dBNST circuits.
In this model, we propose that when the anxiolytic information is transmitted to the BLA, it triggers the activation of aBLA→adBNST PNs. Apart from eliciting anxiolytic behaviors by directly stimulating the adBNST neurons, aBLA→adBNST PN activation also enhance the activity of the inhibitory interneurons in the lpBLA. The enhanced activation of these interneurons not only dampens the activity of anxiogenic lpBLA→ovBNST PNs, but also relieves the inhibition imposed by the aBLA interneurons onto aBLA→adBNST PNs, thereby further augmenting the activity of the latter. On the other hand, when the anxiogenic information is passed onto BLA, it activates lpBLA→ovBNST PNs and elicits anxiogenic behaviors through engaging the inhibitory ovBNST→adBNST microcircuit. In addition, it also activates the interneurons in aBLA, which further suppress the activity of anxiolytic aBLA→adBNST PNs with subsequent removal of their indirect inhibition onto lpBLA→ovBNST PNs via disengaging the local interneurons.
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