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Parallel circuits from the bed nuclei of stria terminalis to the lateral hypothalamus drive opposing emotional states

Abstract

Lateral hypothalamus (LH) neurons containing the neuropeptide hypocretin (HCRT; orexin) modulate affective components of arousal, but their relevant synaptic inputs remain poorly defined. Here we identified inputs onto LH neurons that originate from neuronal populations in the bed nuclei of stria terminalis (BNST; a heterogeneous region of extended amygdala). We characterized two non-overlapping LH-projecting GABAergic BNST subpopulations that express distinct neuropeptides (corticotropin-releasing factor, CRF, and cholecystokinin, CCK). To functionally interrogate BNST→LH circuitry, we used tools for monitoring and manipulating neural activity with cell-type-specific resolution in freely behaving mice. We found that Crf-BNST and Cck-BNST neurons respectively provide abundant and sparse inputs onto Hcrt-LH neurons, display discrete physiological responses to salient stimuli, drive opposite emotionally valenced behaviors, and receive different proportions of inputs from upstream networks. Together, our data provide an advanced model for how parallel BNST→LH pathways promote divergent emotional states via connectivity patterns of genetically defined, circuit-specific neuronal subpopulations.

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Fig. 1: LH neurons: physiology, behavior, and mapping input neurocircuitry.
Fig. 2: Neurochemical identification of genetically defined BNST→LH circuitry
Fig. 3: Crf- and Cck-BNST neurons for opposing emotional states
Fig. 4: Crf- and Cck-BNST neurons: sufficiency and necessity for opposing motivated states
Fig. 5: Crf- and Cck-BNST neurons: parallel LH pathways for opposing emotional states

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Acknowledgements

We acknowledge all de Lecea lab members, A.D. Abraham, and P.F. Apostolides for critical feedback. We thank A. Khan, T.A. Lucas, K. Malacon, M. Silvestre, D. Hoang, C. Liang, K. Choudhury, K. Cruz, and A. Yao for excellent technical assistance. This work was supported by National Institutes of Health grants F32 AA022832 (W.J.G.), R01 MH087592 (L.d.L.), R01 MH102638 (L.d.L.), and F32 MH106206 (D.J.C.). We also recognize support from A. Olson and the Stanford Neuroscience Microscopy Service, NIH NS069375.

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W.J.G. and L.d.L. conceived and designed the studies. W.J.G., A.E.-R., D.J.C., and S.-B. L. performed experiments. W.J.G. and D.J.C. analyzed data. R.C.M. provided equipment and resources, W.J.G. wrote the manuscript with contributions from A.E.-R., D.J.C., and L.d.L.

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Correspondence to Luis de Lecea.

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Integrated supplementary information

Supplementary Figure 1 Hcrt-IRES-Cre validation and Hcrt-ChR2-LH RTPT behavioral and pharmacological analyses.

(a) Schematic of targeting vector for Hcrt-IRES-Cre knockin mouse line. (b) Knockin strategy for Hcrt-IRES-Cre. (c) Representative images showing co-expression of AAV-DIO-eYFP viral labeling (green) and Hcrt (top) or melanin-concentrating hormone (MCH; bottom) immunostaining (red) in LH of Hcrt-Cre mice, replicated independently with similar results in four mice. (d) Quantification of specificity and efficiency of Hcrt-LH infection with Cre-inducible viruses in Hcrt-Cre mice, n=4. (e) Raw Ca2+ activity traces from in vivo fiber photometry recordings of Hcrt-LH neurons in Hcrt-Cre mice injected with AAV-DIO-GCaMP6f virus. Traces show 60s baseline (left of dashed line), and 60s of Ca2+ activity data following exposure to salient odorant stimuli (right of dashed line) (f) Mean (± S.E.M.) fluorescent Ca2+ activity traces from in vivo fiber photometry recordings of Hcrt-LH neurons in control mice injected with AAV-DIO-GFP virus during baseline (left of dashed line), and following exposure to salient odorant stimuli (right of dashed line). Inset; mean (± S.E.M.) fluorescence levels during the 60s stimuli exposure phase (n=2 mice; 1-2 stimulus trials per subject). (g) Number of entries into the stimulation side for Hcrt-LH-ChR2 and control mice tested in the RTPT (n=7 eYFP, n=10 ChR2-eYFP, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,30 = 1.37, p = 0.27). Centre and error bars are mean ± S.E.M. (h) Mean time spent per entry to the stimulation side for Hcrt-LH-ChR2 and eYFP control mice tested in the RTPT (n=7 eYFP, n=10 ChR2-eYFP mice, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,30 = 2.28, p = 0.12, Bonferroni post-hoc comparison **p < 0.005 vs. Hcrt-LH-eYFP). Centre and error bars are mean ± S.E.M. (i) Hcrt-LH-ChR2 real-time place avoidance is significantly blocked by pharmacological treatment with double Hcrt-R antagonist MK-6096 (5mg/kg i.p., n=10 mice, two-tailed paired t-test; t9= 1.11, p = 0.297, n.s.). Centre and error bars are mean ± S.E.M. (j) Significant Hcrt-LH-ChR2 real-time place avoidance following pharmacological treatment with KOR antagonist norBNI (10 mg/kg i.p., n=6 mice, two-tailed paired t-test; t5 = 4.33, p = 0.0075, **p < 0.01 vs. No Light control). Centre and error bars are mean ± S.E.M. Scalebars: all 100 μm.

Supplementary Figure 2 LepRb-LH: GCaMP6 expression, RTPT behavioral analyses, and rabies mapping.

(a) Representative image of LH-AAV-DIO-GCaMP6 expression and fiberoptic placement in LepRb-Cre x LSL-Ai14 Cre-inducible tdTomato mouse. Merged image shows specific co-expression of viral DIO-GCaMP6f labeling (green) with LepRb-tdTomato reporter expression (red), replicated independently with similar results in three mice. (b) Number of entries into the stimulation side for LepRb-LH-ChR2 and eYFP control mice tested in the RTPT (n=8 eYFP, n=6 ChR2-eYFP, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,24 = 0.55, p = 0.58). Centre and error bars are mean ± S.E.M.). (c) Mean time spent per entry to the stimulation side for LepRb-LH-ChR2 and eYFP control mice tested in the RTPT (n=8 eYFP, n=6 ChR2-eYFP, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,24 = 4.47, p = 0.0224, Bonferroni post-hoc comparison *p < 0.05 ***p < 0.0005 vs. LepRb-LH-eYFP). Centre and error bars are mean ± S.E.M. (d) Representative image showing site of viral injection for Cre-defined rabies input mapping onto LH neurons of LepRb-Cre mice, replicated independently with similar results in three mice. (e) Representative images showing upstream inputs cells throughout the brain in LepRb-LH-RVdG-GFP mice, replicated independently with similar results in three mice. Scalebars: all 100 μm.

Supplementary Figure 3 Crf and Cck: non-overlapping BNST subpopulations, differential inputs onto LH neurons.

(a) Genetic screen of Cre-inducible Ai14 (tdTomato) reporter expression in adBNST neurons of wild-type mice, and mice from seven different BNST marker Cre driver lines, replicated independently with similar results in three mice. (b-c) Representative images of CRF and CCK immunostaining (red) in adBNST and pBNST and slices from LepRb-LH-RVdG-GFP mice, replicated independently with similar results in three mice. White arrowheads indicate neuropeptide co-expressing input cells. (d) Representative co-expression between Crf-Cre, Cck-Cre, Vgat-Cre, or Vglut2-Cre Ai14 (tdTomato, red) reporter expression and CRF or CCK (green) immunostaining in adBNST neurons, replicated independently with similar results in three mice. (e) Representative co-expression between CRF immunostaining & Drd1-Ai14 (top), and Tac1-Ai14 (bottom) in adBNST neurons, replicated independently with similar results in three mice. Scalebars: all 100 μm.

Supplementary Figure 4 Optogenetic stimulation of additional BNST and hypothalamus subpopulations.

(a) Representative image of AAV-DIO-eYFP expression in adlBNST and admBNST of Vgat-Cre mouse. (b) Vgat-BNST-ChR2 optogenetic stimulation is rewarding in the RTPT (n=4 eYFP, n=5 ChR2-eYFP, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,14 = 4.16, p = 0.038, Bonferroni post-hoc comparisons *p < 0.05, ***p < 0.0001 vs. Vgat-BNST-eYFP control group). Centre and error bars are mean ± S.E.M. (c) Representative image of AAV-DIO-eYFP expression in ovBNST of Drd1-Cre mouse. (d) Drd1-BNST-ChR2 optogenetic stimulation is aversive in the RTPT (n=4–8 mice per group, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,20 = 12.38, p = 0.0003, Bonferroni post-hoc comparisons ***p < 0.005 vs. Drd1-BNST-eYFP control group). Centre and error bars are mean ± S.E.M. (e) Representative image of AAV-DIO-ChR2-eYFP expression in admBNST of Six3-Cre mouse. (f) Six3-BNST-ChR2 optogenetic stimulation is rewarding in the RTPT (n=4 per group, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,12 = 8.79, p = 0.0045, Bonferroni post-hoc comparisons ***p < 0.001 vs. Six3-BNST-eYFP control group). Centre and error bars are mean ± S.E.M. (g) Representative image of AAV-DIO-eYFP expression in PVN of Crf-Cre mice. (h) Crf-PVN-ChR2 optogenetic stimulation is aversive in the RTPT (n=4 eYFP, n=6 ChR2-eYFP mice, two-way RM-ANOVA; ChR2 x stimulation interaction: F2,16 = 2.33, p = 0.13, Bonferroni post-hoc comparisons *p < 0.05 vs. Crf-PVN-eYFP control group). Centre and error bars are mean ± S.E.M. Scalebars: all 100 μm.

Supplementary Figure 5 Crf- and Cck-BNST neurons: anxiety-like behavior.

(a) Velocity and (b) percent time spent in the center in the open field test following Crf-BNST DREADD manipulation (n=19 mCherry, n=9 hM3Dq, n=7 hM4Di, two-way RM-ANOVA; DREADD x CNO interaction for velocity: F2,32 = 1.86, p = 0.172, DREADD x CNO interaction for center time: F2,32 = 6.03, p = 0.006, Bonferroni post-hoc comparisons *p < 0.05 vs. saline control). Centre and error bars are mean ± S.E.M. (c) Velocity and (d) percent time spent in the center in the open field test following Cck-BNST DREADD manipulation (n=15 mCherry, n=11 hM3Dq, n=4 hM4Di, two-way RM-ANOVA; DREADD x CNO interaction for velocity: F2,28 = 5.52, p = 0.010, Bonferroni post-hoc comparisons **p < 0.005 vs. saline control, DREADD x CNO interaction for center time: F2,28 = 4.99, p = 0.014, Bonferroni post-hoc comparisons *p < 0.05 vs. saline control). Centre and error bars are mean ± S.E.M. (e) Percent open arm time in the elevated plus maze (EPM) following Crf-BNST DREADD manipulation (n=21 mCherry, n=21 hM3Dq, n=13 hM4Di, two-way RM-ANOVA; DREADD x CNO interaction: F2,52 = 14.51, p < 0.0001, Bonferroni post-hoc comparisons ***p < 0.0001 vs. saline control). Centre and error bars are mean ± S.E.M. (f) Crf-BNST-ChR2 10Hz optogenetic stimulation increased anxiety-like behavior (n=11 mice, two-tailed paired t-test; t10 = 4.16, **p = 0.0020 vs. no-stimulation control). Centre and error bars are mean ± S.E.M. (g) Percent open arm time in the EPM following Cck-BNST DREADD manipulation (n=16 mCherry, n=11 hM3Dq, n=10 hM4Di, two-way RM-ANOVA; DREADD x CNO interaction: F2,34 = 9.431, p = 0.0006, Bonferroni post-hoc comparisons ***p < 0.0001 vs. saline control. Centre and error bars are mean ± S.E.M. (h) Cck-BNST-ChR2 10Hz optogenetic stimulation increased anxiety-like behavior (n=12 mice, two-tailed paired t-test; t11 = 5.53, ***p < 0.0005 vs. no-stimulation control). Centre and error bars are mean ± S.E.M.

Supplementary Figure 6 Crf- and Cck-BNST innervation of Hcrt-eGFP+ LH neurons.

(a) Representative images of BNST-AAV-EF1a-DIO-ChR2-mCherry axonal fibers innervating the Hcrt-LH field in Crf-Cre (left) and Cck-Cre (right) mice crossed with Hcrt-eGFP reporter mice, replicated independently with similar results in six mice per group. Scalebars: 100 μm.

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Giardino, W.J., Eban-Rothschild, A., Christoffel, D.J. et al. Parallel circuits from the bed nuclei of stria terminalis to the lateral hypothalamus drive opposing emotional states. Nat Neurosci 21, 1084–1095 (2018). https://doi.org/10.1038/s41593-018-0198-x

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