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.

Figure 1.. LH neurons: physiology, behavior, and mapping input neurocircuitry.

(a) Left, representative image of Hcrt-LH-AAV-DIO-GCaMP6f expression and fiberoptic placement; inset panels show specific co-expression of viral GCaMP6f labeling (green) with Hcrt immunostaining (red), replicated independently with similar results in six mice. Right, schematic of coronal brain slice showing target area in the LH. (b) Left; mean (± S.E.M.) fluorescent Ca²⁺ activity traces from in vivo fiber photometry recordings of Hcrt-LH neurons during baseline 60s (left of dashed line), and during 60s exposure to salient odorant stimuli (right of dashed line). Right; mean (± S.E.M.) fluorescence levels during the 60s stimuli exposure phase (n=6 mice; 1–2 trials with each stimulus per subject, one-way ANOVA; F_3,32 = 11.38, p < 0.0001, Bonferroni post-hoc comparison ***p < 0.0001 vs. no-scent control). (c) Representative image of the LH of LepRb-Cre x Cre-inducible Ai14 mice (tdTomato, red), with Hcrt immunostaining (green) demonstrating complete lack of LepRb-Cre and Hcrt co-expression, replicated independently with similar results in four mice. (d) Left; mean (± S.E.M.) fluorescent Ca²⁺ activity traces from in vivo fiber photometry recordings of LepRb-LH neurons during baseline 60s (left of dashed line), and during 60s exposure to salient odorant stimuli (right of dashed line). Right; mean (± S.E.M.) fluorescence levels during the 60s stimuli exposure phase (n=4 mice; 1–2 trials with each stimulus per subject, one-way ANOVA; F_3,20 = 5.01, p = 0.0094). (e) Direct comparison of mean (± S.E.M.) fluorescence levels from Hcrt-LH (n=6) and LepRb-LH (n=4) mice during the 60s stimuli exposure phase (two-way ANOVA; stimuli x LH cell type interaction F_3,52 = 8.75, p < 0.0001, Hcrt vs. LepRb Bonferroni post-hoc comparisons *p < 0.05 ***p < 0.0001) (f) Representative image of Hcrt-LH-AAV-DIO-ChR2-eYFP expression; inset panels show specific co-expression (yellow) of viral ChR2-eYFP labeling (green) with Hcrt immunostaining (red), replicated independently with similar results in ten mice. (g) Representative heat maps indicating activity of Hcrt-LH-eYFP and Hcrt-LH-ChR2-eYFP mice in the RTPT apparatus during 10Hz stimulation trial. (h) Hcrt-LH-ChR2 photostimulation is aversive in the RTPT (n=7 eYFP, n=10 ChR2-eYFP), two-way repeated measures ANOVA; ChR2 x stimulation interaction: F_2,30 = 6.72, p = 0.004, Bonferroni post-hoc comparisons ***p < 0.0005 vs. Hcrt-LH-eYFP control group). Centre and error bars are mean ± S.E.M. (i) LepRb-LH-ChR2 photostimulation is rewarding in the RTPT (n=8 LepRb-LH-eYFP mice, n=6 LepRb-LH-ChR2-eYFP mice, two-way repeated measures ANOVA; ChR2 x stimulation interaction: F_2,24 = 10.18, p = 0.0006, Bonferroni post-hoc comparisons **p < 0.005, ***p < 0.0005 vs. LepRb-LH-eYFP control group). (j) Representative image of Hcrt-LH-RVdG-GFP expression; inset panels show starter cells co-expressing RVdG-GFP (green) and AAV5-DIO-TVA-mCherry (red), replicated independently with similar results with four mice (k) Representative images of upstream RVdG-GFP cells that provide direct monosynaptic inputs on Hcrt-LH neurons. (l) Visualization of relative abundance of brainwide inputs onto Hcrt LH neurons vs. LepRb LH neurons (n=4 Hcrt-LH, n=3 LepRb-LH, two-way RM-ANOVA; LH subpopulation x input region interaction: F_40,205 = 4.03, p < 0.0001, Bonferroni post-hoc comparisons **p < 0.005, ***p < 0.0005 vs. opposite LH subpopulation). Centre and error bars are mean ± S.E.M. Abbreviations: AH; anterior hypothalamus, AVPV; anteroventral periventricular nuclei, BLA; basolateral amygdala, BNST; bed nuclei of the stria terminalis, CeA; central nucleus of the amygdala, CoA; cortical amygdala nuclei, DBB; diagonal band of Broca; DMH; dorsomedial hypothalamus, DRN; dorsal raphe nuclei, GP; globus pallidus, Layer 5; fifth and/or sixth-layer pyramidal neurons (sparsely, in various anterior cortical regions), LC; locus coeruleus, LDTg; laterodorsal tegmentum, LH; lateral hypothalamus, LPO; lateral preoptic area, LS; lateral septum, MeA; medial amygdala, MHb; medial habenula, MnPO; median preoptic nucleus, MPoA; medial preoptic area, MRN; median raphe nucleus, MS; medial septum, NAcc; nucleus accumbens, NTS; nucleus tractus solitarus, PBN; parabrachial nucleus, PMv; ventral premammilary nucleus, PVN; paraventricular hypothalamus, PVT; paraventricular thalamus, RMg; raphe magnus, SCN; suprachiasmatic nuclei, SNc; substantia nigra compacta, SON; supraoptic hypothalamus, STN; subthalamic nucleus, SUM; supramammilary nucleus, TMN; tuberomammilary nucleus, VMH; ventromedial hypothalamus, VP; ventral pallidum, VTA; ventral tegmental area, ZI; zona incerta. Scalebars: all 100 μm.

Figure 2.. Neurochemical identification of genetically-defined BNST→LH circuitry

(a) Schematic showing upstream RVdG-GFP BNST cells that directly synapse onto downstream Hcrt-LH cells. (b) Representative images of DAPI-labeled coronal anterior (left) and posterior (right) BNST slices overlaid with subnuclei distinctions and anatomical landmarks, showing preferential expression of Hcrt-LH-RVdG-GFP input cells in lateral vs. medial BNST neurons, replicated independently with similar results in four mice. (c) Quantification of BNST→LH input cells (n=4 Hcrt-LH, n=3 LepRb-LH mice; two-way ANOVA; LH subpopulation x BNST division interaction for Anterior vs. Posterior: F_1,10 = 0.43, p = 0.53 n.s.; LH subpopulation x BNST division interaction for Lateral vs. Medial: F_1,10 = 23.50, p = 0.0007, Bonferroni post-hoc comparisons *p = 0.0129 vs. opposite LH subpopulation). Centre and error bars are mean ± S.E.M. (d) Representative images of CRF (top) and CCK (bottom) neuropeptide immunostaining (red) in coronal BNST slices. (e) Representative images of Hcrt-LH-RVdG-GFP dorsal BNST stained for CRF (top) and CCK (bottom). White arrowheads indicate double-labeled cells. Note the abundance of Hcrt-LH-RVdG-GFP BNST input cells co-expressing CRF, but not CCK. (f) Top left: equal numbers of CRF and CCK neuropeptide neurons (mean per hemisphere, per slice, n=4 mice, two-tailed paired t-test; t₃ = 0.09, p = 0.93). Top right: significantly greater number Hcrt-LH-RVdG-GFP+ neurons co-expressing CRF vs. CCK (mean per slice, per hemisphere, n=4 mice, two-tailed paired t-test; t₃ = 7.73, **p = 0.0045). Bottom left: significantly greater percentage of CRF vs. CCK BNST neurons co-expressing Hcrt-LH-RVdG (n=4 mice, two-tailed paired t-test; t₃ = 5.04, *p = 0.0151). Bottom right: significantly greater percentage of Hcrt-LH-RVdG-GFP+ neurons co-expressing CRF vs. CCK (n=4 mice, two-tailed paired t-test; t₃ = 3.81, *p = 0.0319). Centre and error bars are mean ± S.E.M. Abbreviations: ac; anterior commissure, ad; anterodorsal BNST, adl; anterodorsolateral BNST, adm; anterodorsomedial BNST, av; anteroventral BNST, jc; juxtacapsular nucleus, lv; lateral ventricle, ov; oval nucleus, p; posterior BNST, pl; posterolateral BNST, pm; posteromedial BNST, vLS; ventral lateral septum. Scalebars: all 100 μm, except panel d (500 μm).

Figure 3.. Crf and Cck BNST neurons for opposing emotional states

(a) Fluorescent Ca²⁺ activity traces from in vivo fiber photometry recordings of Crf-BNST neurons before and after (dashed line) exposure to salient odorant stimuli. Inset; mean fluorescence levels from Crf-BNST neurons during the 60s stimuli exposure phase (n=6 mice; 1–3 trials with each stimulus per subject, one-way ANOVA; F_3,24 = 13.33, p < 0.0001, Bonferroni post-hoc comparison ***p < 0.0001 vs. no-scent control). (b) Fluorescent Ca²⁺ activity traces from in vivo fiber photometry recordings of Cck-BNST neurons before and after (dashed line) exposure to salient odorant stimuli. Inset; mean fluorescence levels from Cck-BNST neurons during the 60s stimuli exposure phase (n=6 mice; 1–3 trials with each stimulus per subject, one-way ANOVA; F_3,27= 11.93, p < 0.0001, Bonferroni post-hoc comparison ***p = 0.0001 vs. no-scent control). (c) Direct comparison of mean Crf-BNST and Cck-BNST fluorescence levels during the 60s stimuli exposure phase (n=6 Crf and n=6 Cck; two-way ANOVA; stimuli x BNST cell type interaction F_3,51 = 12.49, p < 0.0001, Crf vs. Cck Bonferroni post-hoc comparisons **p < 0.005 ***p < 0.0001). (d) Left; ex vivo validation of ChR2-evoked spiking at 5Hz and 10Hz in Crf-BNST cell bodies. Right; Crf-BNST-ChR2 photostimulation is aversive in the RTPT (n=9 eYFP, n=15 ChR2-eYFP mice, two-way RM-ANOVA; ChR2 x stimulation interaction: F_2,44 = 22.63, p < 0.0001, Bonferroni post-hoc comparisons ***p < 0.0001 vs. Crf-BNST-eYFP control group). (e) Left; ex vivo validation of ChR2-evoked spiking at 5Hz and 10Hz in Cck-BNST cell bodies. Right; Cck-BNST-ChR2 photostimulation is rewarding in the RTPT (n=4 eYFP, n=6 ChR2-eYFP mice, two-way RM-ANOVA; ChR2 x stimulation interaction: F_2,16 = 8.60, p = 0.003, Bonferroni post-hoc comparisons ***p < 0.0005 vs. Cck-BNST-eYFP control group). (f) Representative images of BNST starter cells co-expressing RVdG-GFP (green) and AAV5-DIO-TVA-mCherry (red) in Crf (left) and Cck (right) mice, replicated independently with similar results in four mice per group. (g) Visualization of relative abundance of BNST inputs onto Crf vs. Cck BNST neurons. (h) Quantification of BNST inputs onto Crf vs. Cck BNST neurons. (n=4 per group, two-way RM-ANOVA; BNST genetic subpopulation x BNST subdivision interaction: F_4,24 = 12.01, p < 0.0001, Bonferroni post-hoc comparisons **p < 0.005, ***p < 0.0005 vs. opposite genetic BNST subpopulation). (i) Representative images of upstream RVdG-GFP cells providing direct inputs onto Crf (left) or Cck (right) BNST neurons, replicated independently with similar results in four mice per group. (j) Quantification of relative abundance of brainwide inputs onto Crf vs. Cck BNST neurons (n=4 per group, two-way RM-ANOVA; BNST genetic subpopulation x input region interaction: F_23,138 = 10.76, p < 0.0001, Bonferroni post-hoc comparisons *p < 0.05, ***p < 0.0001 vs. opposite genetic BNST subpopulation). In panels a–e, h and j, centre and error bars are mean ± S.E.M. Scalebars: all 100 μm.

Figure 4.. Crf and Cck BNST neurons: sufficiency and necessity for opposing motivated states

(a) Crf- and Cck-Cre mice received bilateral BNST infusion of either control (mCherry) or excitatory DREADD (hM3Dq) virus, replicated independently with similar results in six mice per group. (b) Relative to aCSF, CNO application to BNST neurons elicited robust spiking activity and increased membrane potential (n=7 neurons from two mice; two-tailed paired t-test; t₆ = 2.49, *p = 0.0472). (c) Timeline for CNO drinking hM3Dq self-stimulation studies. (d) CNO intake (left) and CNO preference (right) in Crf-BNST-hM3Dq and control mice (n=6 Crf-mCherry, n=12 Crf-hM3Dq-mCherry mice, two-way RM-ANOVA; intake: DREADD x CNO interaction: F_2,32 = 5.32, p = 0.010, Bonferroni post-hoc comparisons **p < 0.005, ***p < 0.0005 vs. Crf-BNST-mCherry). (e) CNO intake (left) and CNO preference (right) in Cck-BNST-hM3Dq and control mice (n=15 Cck-mCherry, n=11 Cck-hM3Dq-mCherry mice, two-way RM-ANOVA; intake: DREADD x CNO interaction: F_2,48 = 5.41, p = 0.008, Bonferroni post-hoc comparisons **p < 0.005 vs. Cck-BNST-mCherry). (f) Body weight, water intake, total fluid intake, and food intake for Crf BNST mice in CNO drinking studies (n=6 Crf-mCherry, n=12 Crf-hM3Dq-mCherry mice, no significant group x CNO interactions two-way RM-ANOVA DREADD x CNO interaction; p = 0.54, 0.52, 0.81, 0.96). (g) Body weight, water intake, total fluid intake, and food intake for Cck BNST mice in CNO drinking studies (n=15 Cck-mCherry, n=11 Cck-hM3Dq-mCherry mice, no significant group x CNO interactions two-way RM-ANOVA DREADD x CNO interaction; p = 0.62, 0.13, 0.69, 0.14). (h) For chemogenetic inhibition experiments, Crf- and Cck-Cre mice received bilateral BNST infusion of either control (mCherry) or inhibitory DREADD (hM4Di) virus. For physiological inhibition experiments, Crf- and Cck-Cre mice received bilateral BNST infusion of either control (eYFP) or inhibitory K+ channel (Kir2.1) virus. (i) Relative to aCSF, CNO application to hM4Di-expressing BNST neurons reduced spiking activity and significantly decreased resting membrane potential (n=7 neurons from two mice; two-tailed paired t-test; t₆ = 2.59, *p = 0.0412). (j) Relative to eYFP-expressing control BNST neurons, Kir2.1-infected BNST neurons showed reduced spiking activity in response to increasing current injection (n=5 neurons from two mice per group; Current x Virus interaction: F_4,32 = 3.1, *p = 0.029, Bonferroni post-hoc comparisons *p < 0.05). (k) Behavioral approach to female mouse odorant stimuli in male Crf-BNST mice following hM4Di DREADD inhibition (n=5 Crf-mCherry, n=6 Crf-hM4Di-mCherry, two-way RM-ANOVA; DREADD x CNO interaction: F_1,9 = 0.12, p = 0.737). (l) Behavioral approach to female mouse odorant stimuli vs. control no-scent stimuli in male Crf-BNST mice following Kir2.1 manipulation (n=8 Crf-GFP, n=6 Crf-Kir2.1-GFP mice, two-way RM-ANOVA; Kir2.1 x stimuli interaction: F_1,12 = 0.01, p = 0.912, Bonferroni post-hoc comparisons **p < 0.01 vs. no-scent control). (m) Behavioral approach to female mouse odorant stimuli in male Cck-BNST mice following hM4Di DREADD inhibition (n=7 per group, two-way RM-ANOVA; DREADD x CNO interaction: F_1,12 = 4.98, p = 0.046, Bonferroni post-hoc comparisons *p < 0.05 vs. saline control. (n) Behavioral approach to female mouse odorant stimuli vs. control no-scent stimuli in male Cck-BNST mice following Kir2.1 manipulation (n=8 per group, two-way RM-ANOVA; Kir2.1 x stimuli interaction: F_1,14 = 3.73, p = 0.074, Bonferroni post-hoc comparisons ***p < 0.0005 vs. no-scent control). In panels b, d–g, and i–n, centre and error bars are mean ± S.E.M. Scalebars: all 100 μm.

Figure 5.. Crf and Cck BNST neurons: parallel LH pathways for opposing emotional states

(a) Representative images of AAV-DIO-eYFP expression in BNST cell bodies and downstream axonal fibers of Crf (top) and Cck (bottom) Cre mice, replicated independently with similar results in four mice per group. (b) Crf-BNST→LH photostimulation is aversive in the RTPT (n=3 Crf-BNST→LH-eYFP, n=6 Crf-BNST→LH-ChR2-eYFP mice, two-way RM-ANOVA; ChR2 x stimulation interaction: F_2,14 = 14.65, p = 0.0004, Bonferroni post-hoc comparisons ***p < 0.0001 vs. Crf-BNST→LH-eYFP). (c) Cck-BNST→LH photostimulation is rewarding in the RTPT (n=5 Cck-BNST→LH-eYFP, n=4 Cck-BNST→LH-ChR2-eYFP mice, two-way RM-ANOVA; ChR2 x stimulation interaction: F_2,14 = 6.04, p = 0.013, Bonferroni post-hoc comparisons ***p < 0.0005 vs. Cck-BNST→LH-eYFP). (d) Strategy for crossing Hcrt-eGFP reporter mice with Crf and Cck Cre mice, in order to inject AAV-DIO-ChR2-mCherry into BNST and record from Hcrt-eGFP+ and non-Hcrt-eGFP+ neurons in slice preparation. (e) Crf-BNST→LH slice recordings. 6/10 Hcrt-eGFP+ neurons and 7/10 non-Hcrt-eGFP+ neurons received direct synaptic input from Crf-BNST-ChR2 neurons. (f) Cck-BNST→LH slice recordings. 1/8 Hcrt-eGFP+ neurons and 3/4 non-Hcrt-eGFP+ neurons received direct synaptic input from Cck-BNST-ChR2 neurons. (g) Example traces showing 473nm blue light-evoked picrotoxin (Ptx)-sensitive inhibitory postsynaptic currents, indicating direct Crf-BNST-ChR2 connectivity onto Hcrt+ and non-Hcrt+ LH neurons, with no significant differences in IPSC amplitude, latency, or decay between responsive LH cell types (n=4 eGFP+, n=7 eGFP- cells). (h) Example traces showing 473nm blue light-evoked Ptx-sensitive inhibitory postsynaptic currents, indicating direct Cck-BNST-ChR2 connectivity in Hcrt+ and non-Hcrt+ LH neurons, with no significant differences in IPSC amplitude, latency, or decay between responsive LH cell types (n=1 eGFP+, n=3 eGFP- cells). In panels b, c, g and h, centre and error bars are mean ± S.E.M. Scalebars: all 100 μm.