PBN-PVT projections modulate negative affective states in mice

  1. Ya-Bing Zhu
  2. Yan Wang
  3. Xiao-Xiao Hua
  4. Ling Xu
  5. Ming-Zhe Liu
  6. Rui Zhang
  7. Peng-Fei Liu
  8. Jin-Bao Li
  9. Ling Zhang  Is a corresponding author
  10. Di Mu  Is a corresponding author
  1. Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, China
  2. The First Rehabilitation Hospital of Shanghai, Tongji University School of Medicine, China
  3. Department of Respiratory, The First Affiliated Hospital of Guangzhou Medical University, China
  4. SUSTech Center for Pain Medicine, School of Medicine, Southern University of Science and Technology, China

Abstract

Long-lasting negative affections dampen enthusiasm for life, and dealing with negative affective states is essential for individual survival. The parabrachial nucleus (PBN) and thalamic paraventricular nucleus (PVT) are critical for modulating affective states in mice. However, the functional roles of PBN-PVT projections in modulating affective states remain elusive. Here, we show that PBN neurons send dense projection fibers to the PVT and form direct excitatory synapses with PVT neurons. Activation of the PBN-PVT pathway induces robust behaviors associated with negative affective states without affecting nociceptive behaviors. Inhibition of the PBN-PVT pathway reduces aversion-like and fear-like behaviors. Furthermore, the PVT neurons innervated by the PBN are activated by aversive stimulation, and activation of PBN-PVT projections enhances the neuronal activity of PVT neurons in response to the aversive stimulus. Consistently, activation of PVT neurons that received PBN-PVT projections induces anxiety-like behaviors. Thus, our study indicates that PBN-PVT projections modulate negative affective states in mice.

Editor's evaluation

This study will interest neuroscientists, in particular those interested in the neural circuits that support emotional processing. Using modern neuroscience techniques, the authors demonstrate that anatomical projections from the parabrachial nucleus to the paraventricular nucleus thalamus contribute to aversive states like fear and anxiety. Overall, the study offers important details of a previously uncharacterized brain circuit.

https://doi.org/10.7554/eLife.68372.sa0

Introduction

Threat and injury often induce defensive behaviors, such as flight, freezing, hiding (Ohman and Mineka, 2001), and negative affective states, such as fear and anxiety (Jimenez et al., 2018). Such behavioral adaptations and psychological responses are essential for animal survival, and understanding their mechanisms is of great interest. It is worth noting that the parabrachial nucleus (PBN) in the brainstem plays a critical role in encoding danger signals and promoting affective behavior states to limit harm in response to potential threats (Campos et al., 2018).

The PBN receives the majority of the ascending inputs from the spinal cord (Todd, 2010), and PBN neurons respond robustly to nociception, food neophobia, hypercapnia, and threat to maintain homeostasis in stressful circumstances (Campos et al., 2018; Kaur et al., 2013). The PBN relays this information (visceral malaise, taste, temperature, pain, itch) to brain areas, such as the hypothalamus, central amygdala (CeA), thalamus, insular cortex (IC), and periaqueductal gray (PAG), to participate in diverse physiological processes (Chiang et al., 2019; Palmiter, 2018; Saper, 2016). A recent study found that subpopulations of the PBN have distinct projection patterns and functions (Chiang et al., 2020). Neurons in the dorsal division of the PBN projecting to the ventromedial hypothalamus (VMH) and PAG mediate escape behaviors. In contrast, neurons in the external lateral division of the PBN projecting to the bed nucleus of the stria terminalis (BNST) and the CeA mediate aversion and avoidance memory (Chiang et al., 2020). Optogenetic manipulation of specific outputs from PBN accomplishes distinct functions (Bowen et al., 2020). In the thalamus, the intralaminar thalamus nucleus (ILN) is the downstream target of PBN neurons that receive spinal cord inputs, and this ILN pathway participates in nociception processing (Deng et al., 2020). In addition to the ILN, the thalamic paraventricular nucleus (PVT) is another primary target of projections from the PBN nucleus in the thalamus (Chiang et al., 2020).

The PVT has been implicated in a range of affective behaviors (Hsu et al., 2014). The functional roles of the PVT include modulation of a diverse array of processes, such as arousal (Ren et al., 2018), drug addiction (Zhu et al., 2016), reward seeking (Do-Monte et al., 2017; Engelke et al., 2021), stress (Beas et al., 2018; Gao et al., 2020), and associative learning and memory retrieval (Penzo et al., 2015; Do-Monte et al., 2015; Zhu et al., 2018; Keyes et al., 2020). The PVT receives a significant amount of input from the brainstem, hypothalamus, and prefrontal cortical areas and projects to the infralimbic cortex, nucleus accumbens (NAc), BNST, and CeA (Kirouac, 2015; Vertes et al., 2015). The convergent signals include arousal from the hypothalamus (Ren et al., 2018), emotional saliency from the prefrontal cortex (Yamamuro et al., 2020), and stress responsivity from the locus coeruleus (LC) (Beas et al., 2018), which might help promote appropriate behavioral responses to environmental challenges. However, despite substantial improvements in our understanding of the neurocircuitry of the PVT, the functional role of PBN-PVT projections remains mostly unknown.

In this study, we used viral tracing and electrophysiology to dissect the anatomical and functional connections between the PBN and the PVT. By using optogenetic and pharmacogenetic approaches, we then demonstrated that PBN-PVT projections modulate negative affective states in mice.

Results

Functional connectivity pattern of PBN-PVT projections

Although it has been shown that the PVT receives input from the PBN (Chiang et al., 2020; Li and Kirouac, 2012), the detailed morphology of PBN-PVT projections and whether these two nuclei form direct functional synapses remain unknown. To examine the synaptic connectivity between the PBN and the PVT, we injected AAV2/8-hSyn-ChR2-mCherry virus into the PBN and employed whole-cell patch-clamp recording. There were dense projection fibers in the PVT (Figure 1A–C) and precisely time-locked action potentials induced by brief laser pulses in PBN ChR2+ neurons (5 Hz, 10 Hz, and 20 Hz; Figure 1G). We found that optogenetic activation of PBN projection fibers evoked excitatory postsynaptic currents (EPSCs) in 34 of 52 PVT neurons. The medial PVT (bregma: –0.94 to –1.82 mm; 18 of 25 cells, 72.0%) and posterior PVT (pPVT; bregma: –1.82 to –2.3 mm; 14 of 21 neurons, 66.7%) showed higher connectivity than the anterior PVT (bregma: –0.22 to –0.94 mm; 2 of 6 cells, 33.3%; Figure 1D–F). The average amplitude of the light-evoked EPSCs was 103.4 ± 11.93 pA (Figure 1H). Moreover, the latency of EPSCs was short with small jitter (Figure 1I and J), indicating monosynaptic connections between the PBN and PVT. Consistently, the EPSCs were sensitive to the Na+ channel blocker tetrodotoxin (TTX, 1 μM) and were rescued by the K+ channel blocker 4-aminopyridine (4-AP, 100 μM). The EPSCs were further blocked by the AMPA receptor antagonist NBQX (10 μM), confirming the monosynaptic glutamatergic innervation of PVT neurons by PBN neurons (Figure 1K and L). In addition, we also observed light-evoked inhibitory postsynaptic currents (IPSCs) in only 4 of 52 PVT neurons (less than 30 pA). Thus, these results indicate that most monosynaptic connections between PBN and PVT are glutamatergic.

Figure 1 with 3 supplements see all
Functional connectivity pattern of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections.

(A) The schematic for virus injection of AAV2/8-hSyn-ChR2-mCherry into the PBN nucleus and the slice recording with 473 nm laser stimulation. (B) The projection fibers in the PVT nucleus. Scale bar: 100 μm. (C) The AAV2/8-hSyn-ChR2-mCherry virus expression in the PBN nucleus. Scale bar: 200 μm. (D−F) The locations of the recorded cells in the anterior PVT (D), the middle PVT (E), and the posterior PVT (F). Red circles indicate neurons with excitatory postsynaptic currents (EPSCs), and blue circles indicate neurons without EPSCs. (G) The 473 nm laser-induced time-locked action potential firing at 5 Hz (top), 10 Hz (middle), and 20 Hz (bottom) in the ChR2-expressing neuron in the PBN. Scale bars: 100 ms, 40 mV. (H−J) The amplitude of light-evoked EPSCs (H), the latency of EPSCs (I), and the latency jitter of EPSCs (J) from all 34 responsive neurons in the PVT. (K) Amplitudes of light-evoked EPSCs recorded from a PVT neuron (right panel). (L) The light-evoked EPSC was completely blocked by 1 μM tetrodotoxin (TTX), rescued by 100 μM 4-aminopyridine (4-AP), and blocked by 10 μM NBQX (AMPA/kainate receptor antagonist). Scale bars: 5 ms, 10 pA. (M) Schematic shows retroAAV2/2-hSyn-Cre injection into the PVT nucleus on Rosa26-tdTomato mice. (N) The injection site in the PVT nucleus. Scale bar: 200 μm. (O, P) The distribution of the tdTomato-positive neurons in the left PBN (O) and the right PBN (P). (Q) The quantification of the tdTomato-positive neurons in the lateral PBN (LPBN) and the media PBN (MPBN). n = 4 mice. Scale bar: 200 μm. (R−T) Double staining of tdTomato with Slc17a6 mRNA by in situ hybridization. Scale bar: 50 μm, the scale bar in the quadrangle was 25 μm. (U) Quantification of the double-positive neurons over the total number of tdTomato-positive neurons, n = 6 sections from three mice. ***p<0.001, data are represented as mean ± SEM. Paired Student’s t-test for (Q).

Next, we asked what are the distribution pattern and molecular identity of PBN-PVT neurons. We injected retroAAV2/2-hSyn-Cre virus into the PVT of Rosa26-tdTomato mice, which could retrogradely label projection neurons in the PBN (Figure 1M–P). We found that tdTomato+ neurons were bilaterally located in the lateral PBN (55 ± 6 neurons, n = 4 mice) and rarely in the medial PBN (8 ± 1 neurons; Figure 1O–Q). These results indicate that most PVT inputs originate in the lateral aspect of the PBN. We then performed tdTomato staining and in situ hybridization with Slc17a6 (Vglut2) probe and found that approximately 94.4% of tdTomato+ neurons expressed Slc17a6 mRNA (Vglut2+, Figure 1R–U). We also examined several markers for subpopulations of PBN neurons, including tachykinin 1 receptor (Tacr1), tachykinin 1 (Tac1), prodynorphin (Pdyn), and calcitonin gene-related peptide (CGRP). We found that tdTomato+ neurons were only partially co-labeled with Tacr1 mRNA (38.0%), Tac1 mRNA (6.4%), or Pdyn mRNA (23.0%) and not with CGRP protein (Figure 1—figure supplement 1). These results indicate that the majority of PBN-PVT neurons are glutamatergic neurons, and most of these neurons are not labeled by Tacr1, Tac1, Pdyn, or CGRP.

To examine the collateral projections from the PVT-projecting PBN glutamatergic neurons, we injected AAV2/8-EF1α-DIO-EGFP virus into the PBN of Slc17a6tm2(cre)Lowl (also called Vglut2-ires-Cre) mice (Figure 1—figure supplement 2A). Robust expression of AAV2/8-EF1α-DIO-EGFP was found in both the lateral and medial PBN (Figure 1—figure supplement 2B–D). It is worth noting that the density of EGFP+ fibers was higher in the middle and pPVT (Figure 1—figure supplement 2E–H), considering the notion that the pPVT is particularly sensitive to aversion (Gao et al., 2020). We also found collateral projections from PBN-PVT neurons in the BNST, lateral hypothalamus (LH), paraventricular nucleus of the hypothalamus (PVN), and PAG but not in the CeA or VMH (Figure 1—figure supplement 3).

Optogenetic activation of PBN-PVT projections induces anxiety-like behaviors and aversion-like behaviors

To examine the functional role of PBN-PVT projections in modulating affective behaviors, we injected AAV2/9-EF1α-DIO-ChR2-mCherry virus or AAV2/9-EF1α-DIO-mCherry virus bilaterally into the PBN of Vglut2-ires-Cre mice and implanted optic fibers above the PVT to selectively activate PBN-PVT projections (Figure 2A). Four weeks after surgery, we found robust expression of ChR2-mCherry (Figure 2B and C, Figure 2—figure supplement 1A) or mCherry (Figure 2—figure supplement 1C) in bilateral PBN neurons and axon terminals in the PVT (Figure 2D, Figure 2—figure supplement 1B and D).

Figure 2 with 4 supplements see all
Optogenetic activation of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections induced negative affective states.

(A) The illustration shows the injection of AAV2/9-EF1α-DIO-ChR2-mCherry virus into the PBN nucleus and the optic fiber above the PVT on the Vglut2-ires-Cre mice. (B, C) The virus injection sites of the left PBN (B) and the right PBN (C). Scale bar: 200 μm. (D) The projection axons from the PBN and the location of the optic fiber (rectangle) in the PVT. Scale bar: 200 μm. (E) The schematic of the open field test (OFT) with optogenetic activation via a 473 nm laser (20 Hz, 5 mW, 5 ms). (F, G) The example traces of the 15 min optogenetic manipulation OFT from an AAV2/9-EF1α-DIO-mCherry virus-injected mouse (F) or an AAV2/9-EF1α-DIO-ChR2-mCherry virus-injected mouse (G). (H, I) Quantification of the velocity (H) and the center time (I) in the OFT, mCherry group: n = 7 mice; ChR2 group: n = 8 mice. (J) The illustration of the real-time place aversion (RTPA) test with optogenetic activation via a 473 nm laser. The right side was paired with the laser. (K, L) The example traces of the RTPA and post-test from the mice injected with AAV2/9-EF1α-DIO-mCherry (K) or AAV2/9-EF1α-DIO-ChR2-mCherry (L). (M) Quantification of the time spent in the laser-paired chamber in the pre-test (Pre), RTPA, and post-test (Post), mCherry group: n = 7 mice; ChR2 group: n = 8 mice. (N) Schematic timeline of cue-dependent optogenetic conditioning. (O) Conditioned-freezing responses to sound cue paired with optogenetic activation of the PBN-PVT projection during training, mCherry group: n = 4 mice; ChR2 group: n = 5 mice. (P, Q) Optogenetic activation of the projection fibers from the PBN in the PVT did not induce context-dependent fear (P) and cue-dependent fear (Q), mCherry group: n = 4 mice; ChR2 group: n = 5 mice. *p<0.05, **p<0.01, ***p<0.001, all data are represented as mean ± SEM. Two-way ANOVA followed by Bonferroni test for (H), (I), (M), and (O). Unpaired Student’s t-test for (P) and (Q).

We performed a 15 min optogenetic manipulation in the open field test (OFT; 0–5 min laser off, 5–10 min laser on, 10–15 min laser off; Figure 2E). Optogenetic activation (473 nm, 20 Hz, 5 mW, 5 ms) of the efferents from the PBN to the PVT elicited instant running behavior along the chamber wall with a significantly increased velocity (Figure 2F–H, Figure 2—video 1). The activation of PBN-PVT projections also reduced the center time compared with that of the control mice (Figure 2I). It is worth noting that the velocity returned to normal once the laser was off, but the time spent in the center was still lower than that of the control group during the 5 min after stimulation. These results indicate that anxiety could last for at least several minutes after acute activation of PBN-PVT projections. Although the speed increased during the laser on period, the immobility time of the ChR2 mice during the laser on period was also increased (Figure 2—figure supplement 2A). Therefore, the distance during the laser on period and the total distance in 15 min were not changed (Figure 2—figure supplement 2B).

To provide a more detailed profile of behavior in the OFT, we further divided the laser on period (5–10 min) into five 1-min periods and analyzed the velocity, immobility time, center time, distance, and jumping (Figure 2—figure supplement 2C–G). We found that the velocity and immobility time were increased, and the center time was decreased in the ChR2 mice during most periods (Figure 2—figure supplement 2C–E). Furthermore, we observed that the distance and jumping behaviors were increased mainly in the first 1-min period in ChR2 mice (Figure 2—figure supplement 2F and G). This detailed analysis indicates that optogenetic activation of PBN-PVT projections could induce brief and robust running, jumping behaviors, and persistent anxiety-like behaviors, such as less time spent in the center.

In addition to anxiety, another critical component of negative affective states is aversion. Therefore, we used the real-time place aversion (RTPA) test to explore the function of optogenetic activation of PBN-PVT projections in modulating aversion (Figure 2J). We found that the activation of PBN-PVT projections reduced the time spent in the laser-paired chamber, and the aversion disappeared when the laser was off (Figure 2K–M, Figure 2—video 2). We also used a prolonged conditioning protocol that mimics drug-induced conditioned place aversion (CPA; Figure 2—figure supplement 2H). We found that prolonged activation of PBN-PVT projections did not display aversion in the postconditioning test (Figure 2—figure supplement 2I).

To further confirm this instant aversion phenomenon, we subjected mice to the cue-dependent optogenetic conditioning test (Figure 2N). A 30 s auditory conditioning stimulus (CS) co-terminated with 30 s of synchronous optogenetic activation of PBN-PVT projections (laser stimulus [LS]) in this test. Activation of the PBN-PVT projections induced significant freezing behavior during six CS-LS pairings (Figure 2O). However, the freezing behavior to the same context or to the auditory cue in a novel context disappeared on the second day (Figure 2P and Q). These results indicate that optogenetic activation of PBN-PVT projections induces instant aversion and freezing but does not drive associative fear memory formation.

Pharmacogenetic activation of PBN-PVT neurons induces anxiety-like behaviors and freezing behaviors

To further confirm the effects of activating the PBN-PVT pathway, we also used retrograde viral tracing and pharmacogenetic manipulation. We first injected retroAAV2/2-hSyn-Cre virus into the PVT and AAV2/9-hSyn-DIO-hM3Dq-mCherry virus or control virus bilaterally into PBN to specifically transduce PBN-PVT neurons with the hM3Dq, a designer receptor exclusively activated by designer drugs (Figure 3A; Armbruster et al., 2007). PBN-PVT neurons could be activated by intraperitoneal (i.p.) injection of clozapine N-oxide dihydrochloride (CNO; Figure 3B–D). The region of virus expression in the PBN is shown in Figure 3—figure supplement 1. Consistent with the optogenetic activation results, pharmacogenetic activation of PBN-PVT neurons reduced the center time, increased immobility time, and reduced the travel distance in the OFT (Figure 3E–I). At the same time, the velocities were not significantly different (Figure 3J). We also found that activation of PBN-PVT neurons did not affect motor ability in the rotarod test (Figure 3—figure supplement 2G). In addition, pharmacogenetic activation of PBN-PVT neurons decreased exploration time of open quadrants in the elevated zero maze (EZM; Figure 3K and L), further suggesting that activation of PBN-PVT neurons induces anxiety-like behaviors.

Figure 3 with 2 supplements see all
Pharmacogenetic activation of parabrachial nucleus-paraventricular nucleus (PBN-PVT) neurons induced anxiety-like behaviors and fear-like behaviors.

(A) The illustration shows virus injection of retroAAV2/2-hSyn-Cre into the PVT nucleus and bilateral injection of AAV2/9-hSyn-DIO-hM3Dq-mCherry into the PBN nucleus. (B, C) Clozapine N-oxide dihydrochloride (CNO) administration evokes Fos expression in AAV2/9-hSyn-DIO-hM3Dq-mCherry-injected mice (B) but not in AAV2/9-EF1α-DIO-mCherry-injected mice (C). Scale bar: 200 μm. (D) Percentage of co-labeled neurons in the PBN, mCherry group: n = 3 mice; hM3Dq group: n = 4 mice. (E) The illustration of the open field test (OFT) with pharmacogenetic activation. (F) Example of the OFT traces from the mice infected with AAV2/9-EF1α-DIO-mCherry or AAV2/9-hSyn-DIO-hM3Dq-mCherry. (G−I) Quantification of the center time (G), the immobility time (H), and the total distance (I) in the OFT, mCherry group: n = 7 mice; hM3Dq group: n = 8 mice. (J) Quantification of the velocity in the OFT, mCherry group: n = 7 mice; hM3Dq group: n = 5 mice. (K) Example elevated zero maze (EZM) traces from the mice infected with AAV2/9-EF1α-DIO-mCherry and AAV2/9-hSyn-DIO-hM3Dq-mCherry. (L) Quantification of the time spent in open quadrants in the EZM test, n = 8 mice per group. (M) The illustration of pharmacogenetic activation-induced fear-like freezing behavior. (N) Pharmacogenetic activation of PBN-PVT neurons induced fear-like freezing behaviors, mCherry group: n = 8 mice; hM3Dq group: n = 7 mice. *p<0.05, ***p<0.001, all data are represented as mean ± SEM. Unpaired Student’s t-test for (D), (G), (H), (I), and (J). Two-way ANOVA followed by Bonferroni test for (L) and (N).

We further evaluated freezing behaviors in the fear conditioning chamber and found that pharmacogenetic activation of PBN-PVT neurons induced more freezing behaviors (Figure 3M and N). Although activation of PBN-PVT neurons induced significant anxiety-like behavior, it did not affect the depressive-like behaviors evaluated by the tail suspension test (TST; Figure 3—figure supplement 2A) or the forced swimming test (FST; Figure 3—figure supplement 2B). Previous studies have revealed that the PBN receives direct projections from the spinal cord and plays a vital role in pain processing (Deng et al., 2020; Sun et al., 2020). We then assessed whether pharmacogenetic activation of PBN-PVT neurons affected nociceptive behaviors. By performing the von Frey test and Hargreaves test, we found that the basal nociceptive thresholds were not affected after pharmacogenetic activation of PBN-PVT neurons (Figure 3—figure supplement 2C and D). Given the distinct mechanisms between the reflexive and coping responses induced by nociceptive stimulation (Huang et al., 2019), we injected formalin into the paw to induce inflammatory pain. We found that activation of PBN-PVT neurons did not affect formalin-evoked licking behaviors (Figure 3—figure supplement 2E and F). These results indicate that activating the PBN-PVT pathway induces anxiety-like and freezing behaviors but not nociceptive behaviors.

Inhibition of PBN-PVT projections reduces aversion-like behaviors and freezing behaviors

We next asked whether inhibition of PBN-PVT projections could modulate negative affective states. We first injected AAV2/9-EF1α-DIO-NpHR3.0-EYFP virus or AAV2/8-EF1α-DIO-EGFP virus bilaterally into the PBN and implanted optic fibers into the PVT of Vglut2-ires-Cre mice (Figure 4A–C, Figure 4—figure supplement 1). We used 2-methyl-2-thiazoline (2-MT), a widely used odorant molecule that can generate innate fear-like freezing responses in rodents (Isosaka et al., 2015), to induce a fear-like state. We found that 589 nm laser-induced inhibition of PBN-PVT projections reduced the aversion caused by 2-MT exposure (Figure 4D–F) and increased the moving duration (Figure 4G). We also observed that inhibition of PBN-PVT projections increased the time spent in the 2-MT zone in the OFT (Figure 4H–I). In addition to 2-MT, footshock is another paradigm that induces robust freezing behaviors. We found that constant inhibition of PBN-PVT projections reduced footshock-induced freezing behaviors (Figure 4J and K).

Figure 4 with 3 supplements see all
Optogenetic inhibition of parabrachial nucleus-paraventricular nucleus (PBN-PVT) projections reduced aversion-like behavior and fear-like behaviors.

(A) The illustration shows the bilateral injection of AAV2/9-EF1α-DIO-NpHR3.0-EYFP virus into the PBN and placement of optic fiber above the PVT on Vglut2-ires-Cre mice. (B, C) Examples of AAV2/9-EF1α-DIO-NpHR3.0-EYFP expression in the PVT (B) and PBN (C). The cyan rectangle represents the position of the optic fiber. Scale bar: 200 μm. (D) Schematic of 2-methyl-2-thiazoline (2-MT)-induced aversion test with optogenetic inhibition via the 589 nm laser. (E) Representative traces of the mice infected with AAV2/8-EF1α-DIO-EGFP or AAV2/9-EF1α-DIO-NpHR3.0-EYFP in two chambers. (F, G) Quantification of the time spent in the 2-MT paired chamber (F) and the total moving duration (G), n = 7 mice per group. *p<0.05, **p<0.01, (H) Representative traces of the mice infected with AAV2/8-EF1α-DIO-EGFP or AAV2/9-EF1α-DIO-NpHR3.0-EYFP in the open field test (OFT) chamber. (I) Quantification of the time spent in the 2-MT zone, n = 7 mice per group. (J) Illustration of footshock-induced freezing behavior with optogenetic inhibition via a 589 nm laser. (K) Quantification of the freezing behavior, n = 7 mice per group. *p<0.05, all data are represented as mean ± SEM. Unpaired Student’s t-test for (F), (G), (I), and (K).

We also examined whether inhibition of the PBN-PVT projection affects aversive memory acquisition or retrieval (Figure 4—figure supplement 2A). We briefly suppressed the activity of PBN-PVT projections during footshock stimulation and found that freezing levels during the condition were not changed (Figure 4—figure supplement 2B). We further compared freezing levels in contextual and cue tests without or with laser and found that aversive memory retrieval was not affected either (Figure 4—figure supplement 2C and D). In addition, we performed optogenetic inhibition of PBN-PVT neurons and observed similar phenomena (Figure 4—figure supplement 3). Thus, these results indicate that inhibition of the PBN-PVT pathway reduces aversion-like behaviors and footshock-induced freezing behaviors without affecting aversive memory acquisition or retrieval.

PBN input shapes PVT neuronal responses to aversive stimulation

To further examine the activity of the PVT in response to aversive stimulation, we performed the in vivo fiber photometry and found that calcium signals of PVT neurons were increased after the footshock and air puff (Figure 5—figure supplement 1). In addition, we injected AAV2/1-hSyn-Cre virus, which could anterogradely label downstream neurons (Zingg et al., 2017), into the PBN of Rosa26-tdTomato mice (Figure 5A). The distribution pattern of PVT neurons that received PBN-PVT projection fibers (hereafter referred to as PVTPBN neurons) is shown in Figure 5B–D. We used Fos as a marker to assess the activity change in 2-MT-treated mice and footshock-treated mice. The percentage of Fos+tdTomato+ neurons/tdTomato+ neurons in the PVT was significantly increased in the mice treated with aversive stimuli compared with that of control mice (Figure 5E–H), confirming that the PVTPBN neurons could be activated by aversive stimuli.

Figure 5 with 1 supplement see all
Activation of PVTPBN neurons by diverse aversive stimuli.

(A) The illustration shows the injection of AAV2/1-hSyn-Cre into the parabrachial nucleus (PBN) of Rosa26-tdTomato mice. (B−D) The distribution of the neurons in the paraventricular nucleus (PVT) at bregma –1.06 mm (B), bregma –1.46 mm (C), and bregma –1.70 mm (D). Scale bar: 200 μm. (E−G) Fos induced by habituation control (E), footshock (F), or 2-methyl-2-thiazoline (2-MT) (G) co-labeled with the tdTomato-positive neurons in the PVT. Scale bar: 200 μm. (H) Quantification of the co-labeled neurons, n = 3 mice per group. *p<0.05, **p<0.01, ***p<0.001, all data are represented as mean ± SEM, one-way ANOVA followed by Bonferroni test for (H).

The next question is whether PBN-PVT projections modulate the neuronal activity of PVT neurons in response to aversive stimuli. We first injected AAV2/9-EF1α-DIO-ChR2-mCherry virus into the PBN and performed dual Fos staining (Nakahara et al., 2020), detecting fos mRNA and Fos protein induced by two episodes of stimulation (Figure 6—figure supplement 1A). We found that there was a broad overlap between optogenetic stimulation-activated neurons (expressing the Fos protein) and footshock-activated neurons (expressing the fos mRNA) (Figure 6—figure supplement 1B–E).

Then, we injected AAV2/9-EF1α-DIO-ChR2-mCherry virus into the PBN and implanted the optoelectrode into the PVT of Vglut2-ires-Cre mice (Figure 6A). We first recorded the spiking signals in response to 10 sweeps of 2 s laser pulse trains (20 Hz, 5 mW, 5 ms). Then, we recorded the spiking signals in response to 20 sweeps of 2 s footshock (0.5 mA) without laser in the odd number sweeps or with laser in the even number sweeps (Figure 6A). We found that laser or footshock (without laser) increased firing rates in 22 or 28, respectively, of 40 neurons (Figure 6B and C). There was also a broad overlap between laser-activated and footshock-activated neurons (Figure 6D). This result was consistent with the dual Fos staining result, suggesting that PVTPBN neurons were activated by aversive stimulation. We also analyzed the response latency of laser-activated neurons and footshock-activated neurons (Figure 6—figure supplement 2A–D). The median response latency of 22 laser-activated neurons was 10 ms, suggesting that monosynaptic inputs from PBN could increase the PVT activity.

Figure 6 with 2 supplements see all
Neuronal activity of paraventricular nucleus (PVT) neurons in response to the footshock was modulated by parabrachial nucleus (PBN)-PVT projections.

(A) Top: schematic shows injection of AAV2/9-EF1α-DIO-ChR2-mCherry into the PBN and placement of the optoelectrode above the PVT of Vglut2-ires-Cre mice. Bottom: the protocol of 10 sweeps of laser stimuli (Trial 1) and 20 sweeps of footshock stimuli without or with laser (Trial 2). (B) Firing rates (z-score) of 40 neurons during laser stimuli (20 Hz, 5 mW, 5 ms, 2 s). Inset: percentages of different groups of neurons according to z-score. (C) Firing rates (z-score) of 40 neurons during footshock (0.5 mA, 2 s) without laser stimuli. (D) Percentage of laser-activated, footshock-activated, and both stimuli-activated neurons. (E–G) Rastergrams and firing rates show the spiking activity of one PVT neuron during laser stimulus (E), footshock without laser stimulus (F), and footshock with laser stimulus (G). (H) Firing rates (z-score) of 40 neurons during footshock (0.5 mA, 2 s) with laser stimuli (20 Hz, 5 mW, 5 ms, 2 s). (I) Quantification of the firing rates of 40 neurons before and during footshock without and with laser, n = 40 neurons. **p<0.01, ***p<0.001, all data are represented as mean ± SEM, two-way ANOVA followed by Bonferroni test for (I).

Next, we analyzed the firing rates of PVT neurons during footshock with laser sweeps and footshock without laser sweeps (Figure 6E–G). We found that the footshock stimulus laser-activated 30 of 40 neurons (Figure 6H) and increased the overall firing rates of 40 neurons compared with the footshock without laser result (Figure 6I). These results indicate that activation of PBN-PVT projections could enhance PVT neuronal responses to aversive stimulation.

Pharmacogenetic activation of PVTPBN neurons induces anxiety-like behaviors

We next investigate the functional role of PVTPBN neurons in modulating negative affective states. We injected AAV2/1-hSyn-Cre virus bilaterally into the PBN and injected AAV2/9-hSyn-DIO-hM3Dq-mCherry virus or control virus into the PVT to activate PVTPBN neurons (Figure 7A). The majority of the PVTPBN neurons could be activated by CNO in the hM3Dq-expressing mice but not the control mice, as demonstrated by the Fos staining (Figure 7B–D). We found that pharmacogenetic activation of PVTPBN neurons reduced the center time (Figure 7E and F). Similarly, the time spent in open quadrants was decreased and overall immobility time was increased in the EZM after activation of PVTPBN neurons (Figure 7G–I). We did not observe obvious nociception-related behaviors, such as forelimb wiping, hindlimb flinching, licking, or biting, after activation of PVTPBN neurons. These results indicate that pharmacogenetic activation of PVTPBN neurons induces anxiety-like behaviors.

Figure 7 with 1 supplement see all
Activation of PVTPBN neurons induced anxiety-like behaviors.

(A) The illustration shows injection of AAV2/1-hSyn-Cre into the parabrachial nucleus (PBN) and AAV2/9-hSyn-DIO-hM3Dq-mCherry into the paraventricular nucleus (PVT). (B, C) Clozapine N-oxide dihydrochloride (CNO) administration evoked Fos expression in AAV2/9-hSyn-DIO-hM3Dq-mCherry-injected mice (B) but not in AAV2/9-EF1α-DIO-mCherry-injected mice (C). Scale bar: 200 μm. (D) Percentage of co-labeled neurons in the PVT, n = 4 mice per group. (E) The illustration of the open field test (OFT) with pharmacogenetic activation. (F) Quantification of center time in the OFT, n = 7 mice per group. (G) Example of elevated zero maze (EZM) traces from the mice injected with AAV2/9-EF1α-DIO-mCherry or AAV2/9-hSyn-DIO-hM3Dq-mCherry. (H, I) Quantification of the time spent in open quadrants (H) and the immobility time in the EZM test (I), n = 7 mice per group. *p<0.05, ***p<0.001, all data are presented as mean ± SEM. Unpaired Student’s t-test for (D), (F), (H), and (I).

Furthermore, we examined the anatomic distribution of terminals of PVTPBN neurons. We labeled PVTPBN neurons in WT mice by injecting AAV2/1-hSyn-Cre virus into the PBN and AAV2/8-EF1α-DIO-EGFP virus into the PVT (Figure 7—figure supplement 1A and B). We found that PVTPBN neurons sent projections to several brain areas, in particular the NAc, BNST, and CeA (Figure 7—figure supplement 1B–H), which was similar to early tracing research on PVT efferent projections (Li and Kirouac, 2008).

Discussion

In this study, we employed viral tracing and electrophysiology to confirm the monosynaptic excitatory connectivity between the PBN and the PVT. Optogenetic or pharmacogenetic activation of the PBN-PVT pathway induced anxiety-like, aversion-like, and fear-like behaviors. Optogenetic inhibition of the PBN-PVT pathway could partially reduce 2-MT-induced aversive behaviors as well as footshock-induced freezing behaviors. The activity of PVT neurons was increased with the presentation of several aversive stimuli and could be further increased by activation of PBN-PVT projections. Consistently, activation of PVTPBN neurons induced anxiety-like behaviors. Taken together, our results reveal the functional role of PBN-PVT projections in modulating negative affective states in mice.

PBN efferents and PBN-PVT monosynaptic excitatory projections

The PBN is a critical hub receiving sensory information from the spinal cord (Todd, 2010). The widespread distribution of PBN efferents contributes to different aspects of behavioral and physiological responses. Previous studies have shown that CGRP-expressing neurons in the PBN project to the CeA and contribute to the affective dimension of pain. In contrast, non-CGRP neurons may transmit sensory pain information (Han et al., 2015). The projections from the PBN to the VMH or PAG are involved in producing escape behaviors to avoid injury, while the projections from the PBN to the BNST or CeA participate in facilitating aversive memory (Chiang et al., 2020). PBN neurons, which receive projections from the spinal cord, form strong functional synaptic connections with ILN neurons but not CeA neurons to process nociceptive signals (Deng et al., 2020). The PVT, which is located in the midline of the brain, is an important area that participates in the processing of affective states (Kirouac, 2015). Although recent research has reported that projecting fibers from the PBN were found in the PVT (Chiang et al., 2020), remarkably little is known about the connectivity information and function of these PBN-PVT projections.

Since we injected the constitutively expressed ChR2 virus into the PBN, some neurons in the LC (which is medial to the PBN) might be infected. LC neurons express type 1 vesicular glutamate transporter (VgluT1) and also project to the PVT (Beas et al., 2018). Although PBN-PVT projections comprise the major portion of the projections, there is still potential contamination from LC-PVT projections. We also observed a small portion of inhibitory connections between the PBN and the PVT by performing slice recording experiments. These results are consistent with a previous study showing that GABAergic neurons in the PBN also send sparse projections to the PVT (Chiang et al., 2020). We speculate that this PBN-PVT GABAergic pathway might play an opposite role in modulating negative affective states. Further in situ hybridization results confirmed that the PBN-PVT neurons were mainly glutamatergic neurons expressing Slc17a6 mRNA. These results suggest that the majority of the PBN-PVT projections appeared to be excitatory. Further optoelectrode experiment results also confirmed that firing rates of some PVT neurons could be directly increased by monosynaptic inputs from PBN.

We also found that the connectivity and density of glutamatergic PBN projection fibers were higher in the middle and pPVT. These results are consistent with various studies supporting the idea that the pPVT is a particularly sensitive region of the PVT to aversive stimuli (Gao et al., 2020; Beas et al., 2018; Barson et al., 2020).

PBN-PVT projections modulate negative affective states

We found that activation of PBN-PVT projections or PBN-PVT neurons induced anxiety-like behaviors and fear-like behaviors in the OFT and EZM. We observed that mice displayed robust running and jumping behaviors mainly in the first minute of optogenetic manipulation, and these phenomena were not observed in the pharmacogenetic experiment. These might have been caused by the instantly increased activity of PBN-PVT projections induced by optogenetic manipulation. Mice might display ‘fight or flight’ during sudden affective state transitions. The pharmacogenetic approach takes several minutes and gradually enhances neural activity, and the resulting affective states change in a relatively mild way. We also observed that anxiety-like behaviors in the OFT still existed several minutes after optogenetic activation of PBN-PVT projections. However, in the RTPA test, aversion appeared when the laser was on and disappeared when the laser was off, indicating that aversion was transient and could not be translated to associative learning. This was further confirmed by the prolonged CPA test and cue-dependent optogenetic conditioning test. These results suggest that activation of PBN-PVT projections induces instant negative affective states but does not drive associative fear memory formation.

The selective optogenetic inhibition of PBN-PVT projections or PBN-PVT neurons could reduce aversion-like and fear-like behaviors. To better examine the behavioral changes, we performed a 10 min test in 2-MT and footshock experiments. We used a relatively long-term protocol in optogenetic inhibition experiments (10 min of constant laser stimulation). Such long-term inhibition protocols have been used in other studies (Zhou et al., 2019; Sun et al., 2020). We also performed the classic fear conditioning test and found that inhibition of PBN-PVT projections did not affect associative fear memory formation or retrieval, suggesting that PBN-PVT projections mainly promote aversion but do not facilitate negative associations.

Our calcium imaging and Fos staining results indicate that PVT neurons are activated after exposure to aversive stimuli, consistent with a previous study (Zhu et al., 2018). The dual Fos staining experiment and optoelectrode experiments confirmed a broad overlap between laser-activated and footshock-activated neurons. The median response latency of laser-activated PVT neurons was 10 ms, indicating that monosynaptic inputs from PBN could directly activate a part of PVT neurons. We also observed that some neurons had relatively long latency (from 20 to 150 ms). That might be caused by cumulative effects of monosynaptic inputs or antidromic activation of PBN and multisynaptic activation of other PVT inputs that are innervated by the PBN. Further analysis showed that activation of PBN-PVT projections enhanced the overall firing rates of PVT neurons in response to footshock. These results suggest that the activation of PBN-PVT projections could enhance neuronal activity in response to aversive stimulation.

Previous studies have reported that activation of the PBN-CeA pathway was sufficient to drive behaviors associated with negative affective states (Bowen et al., 2020; Cai et al., 2018; Han et al., 2015), enable associative learning, and generate aversive memory (Chiang et al., 2020). Distinct from PBN-CeA projections, we found that activation of PBN-PVT projections induced only transient aversion-related behaviors, and inhibition of PBN-PVT projections did not affect fear memory acquisition or retrieval. A study reported that only a few fluoro-gold (FG)/tetramethylrhodamine-dextran (TMR) double-labeled neurons were sparsely distributed in the PBN of mice injected with FG into the PVT and TMR into the CeA (Liang et al., 2016). Our results also showed few collateral projecting fibers in the CeA or VMH from PBN-PVT neurons. These results suggested that the PBN-PVT pathway and the PBN-CeA pathway are two parallel pathways originating from distinct efferent neurons within the PBN that perform distinct functions.

Recently, Chiang et al. found two major efferent pathways from the lateral PBN: one originating from the dorsal division of lateral PBN collateralizes the VMH and PAG and a second arising from the external lateral division of lateral PBN that collateralize to the BNST and CeA. They suggested that activation of the first pathway generates the aversive memory, and activation of the second one drives escape behaviors (Chiang et al., 2020). In our study, the results indicate that the PBN-PVT pathway arises from both dorsal and external lateral divisions of lateral PBN and collateralizes the BNST, LH, PVN, and PAG but not the CeA or VMH. According to the location of originated PBN neurons and collateral projection pattern, we speculated that the PBN-PVT efferent pathway is different from both PBN-VMH/PAG pathway and PBN-BNST/CeA pathway. Since there were broad collateral projection fibers in BNST, PAG, and other brain regions, the possibility of antidromic effects following photoactivation of PBN terminals in PVT should be considered.

The tracing results showed that PVTPBN neurons projected to multiple brain areas, particularly the NAc, BNST, and CeA. The BNST and CeA have been previously implicated in negative affective behaviors (Jennings et al., 2013; Tye et al., 2011). Previous studies have shown that the activation of PVT-CeA projections induced place aversion, and the effect persisted the next day in the absence of photostimulation (Do-Monte et al., 2017). Similarly, long-term depression (LTD)-like stimulation of PVT-CeA projections or inhibition of the same circuit induced a persistent attenuation of fear responses (Chen and Bi, 2019; Do-Monte et al., 2015; Penzo et al., 2015). These results revealed a critical role of PVT-CeA projections in aversive memory formation. In our study, we found that PBN-PVT projections were not crucial for aversive memory formation. The possible reason might be that manipulation of PVT-CeA projections induced direct excitatory inputs to the CeA, and the inputs were strong enough for aversive memory formation. However, activation of PBN-PVT projections might not induce enough excitatory inputs to the CeA via the disynaptic connection.

A previous study also found that the PVT mediates descending pain facilitation underlying persistent pain conditions via the PVT-CeA-PAG circuit (Liang et al., 2020). Different downstream pathways of PVTPBN neurons might have different functions, and deciphering the circuit mechanisms needs further examination.

The potential role of PBN-PVT projections in depression and pain

It is worth noting that although the pharmacogenetic activation of PBN-PVT neurons induced anxiety-like behaviors and fear-like behaviors in hM3Dq group mice, no depression-like symptoms were observed in the TST and FST. On the other hand, chronic pain models, such as the partial sciatic nerve ligation model, spared nerve injury model, and complete Freund’s adjuvant model, generally induce anxiety and depression at least 3–4 weeks after surgery in mice (Dimitrov et al., 2014; Zhou et al., 2019). Our study collected behavioral data 30 min after a single dose of CNO injection. Different behavioral tests were performed at least 3 days apart to eliminate the residual CNO effects. We hypothesized that depression-like behaviors might be observed if we repeatedly activated PBN-PVT projections for weeks. However, whether PBN-PVT projections are involved in depression is still unknown.

A recent study revealed that PBN neurons convey nociceptive information from the spinal cord to the ILN, which is relatively close to the PVT (Deng et al., 2020). In our results, we carefully checked the virus expression and optic fiber locations. We found that pharmacogenetic activation of PBN-PVT neurons did not affect basal nociceptive thresholds or formalin-induced licking behaviors. Moreover, no obvious nociception-related behaviors were found through specific manipulations of the PBN innervated PVT neurons, which suggests that PBN-PVT projections might not be involved in nociceptive information processing.

In summary, we identified the functional role of PBN-PVT projections in modulating negative affective states. Our study paves the way for further deciphering the distinct roles of the PBN neural circuit in affective behaviors.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Ai9)Jackson LaboratoryStock# 007909;RRID:MGI:3813511Dr. Hua-Tai Xu (Institutes of Neuroscience, Chinese Academic of Sciences)
Genetic reagent (M. musculus)STOCK Slc17a6tm2(cre)Lowl/J (Vglut2-ires-Cre)Jackson LaboratoryStock# 016963;RRID:MGI:5300532Dr. Yan-Gang Sun (Institutes of Neuroscience, Chinese Academic of Sciences)
Genetic reagent (Dependoparvovirus)AAV2/8-hSyn-ChR2-mCherryObio TechnologyCat# AG269764 × 1012 v.g./mL
Genetic reagent (Dependoparvovirus)AAV2/8-EF1α-DIO-EGFPTaitool BioscienceCat# S02704 × 1012 v.g./mL
Genetic reagent (Dependoparvovirus)retroAAV2/2-hSyn-CreTaitool BioscienceCat# S0278-2RP-H204 × 1012 v.g./mL
Genetic reagent (Dependoparvovirus)AAV2/9-EF1α-DIO-ChR2-mCherryTaitool BioscienceCat# S0170-9-L204 × 1012 v.g./mL
Genetic reagent (Dependoparvovirus)AAV2/9-EF1α-DIO-mCherryObio TechnologyCat# AG202994 × 1012 v.g./mL
Genetic reagent (Dependoparvovirus)AAV2/9-hSyn-DIO-hM3Dq-mCherryBrainVTACat# PT-00194 × 1012 v.g./mL
Genetic reagent (Dependoparvovirus)AAV2/9-EF1α-DIO-NpHR3.0-EYFPObio TechnologyCat# AG269664 × 1012 v.g./mL
Genetic reagent (Dependoparvovirus)AAV2/8-hSyn-GCaMP6sTaitool BioscienceCat# S0225-84 × 1012 v.g./mL
Genetic reagent (Dependoparvovirus)AAV2/1-hSyn-CreTaitool BioscienceCat# S0278-1-H501.5 × 1013 v.g./mL
Sequence-based reagentRNAscope Probe-fos-C2Advanced Cell DiagnosticsCat# 316921-C2
Sequence-based reagentRNAscope Probe-Tac1-C2Advanced Cell DiagnosticsCat# 410351-C2
Sequence-based reagentRNAscope Probe-Tacr1-C2Advanced Cell DiagnosticsCat# 428781-C2
Sequence-based reagentRNAscope Probe-PdynAdvanced Cell DiagnosticsCat# 318771
Sequence-based reagentRNAscope Probe- Slc17a6-C2Advanced Cell DiagnosticsCat# 319171-C2
AntibodyAnti-Fos (rabbit polyclonal)AbcamCat# ab190289; RRID:AB_2737414IF (1:4000)
AntibodyAnti-CGRP (goat polyclonal)AbcamCat# ab36001; RRID:AB_725807IF (1:1000)
AntibodyAnti-DsRed (goat polyclonal)Takara BioCat# 632496; RRID:AB_10013483IF (1:500)
AntibodyAlexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L)Jackson ImmunoResearch LabsCat# 711-545-152; RRID:AB_2313584IF (1:400)
AntibodyCy3 AffiniPure Donkey Anti-Rabbit IgG (H+L)Jackson ImmunoResearch LabsCat# 711-165-152; RRID:AB_2307443IF (1:400)
AntibodyAlexa Fluor 488 AffiniPure F(ab’)2 Fragment Donkey Anti-Goat IgG (H+L)Jackson ImmunoResearch LabsCat# 705-546-147; RRID:AB_2340430IF (1:400)
Commercial assay or kitRNAscope Multiplex Fluorescent Reagent Kit v2Advanced Cell DiagnosticsCat# 320293
Software, algorithmImageJNIH
Software, algorithmLabStateAniLab

Animals

Male C57Bl/6J wild-type mice, Rosa26-tdTomato mice (Jax Stock# 007909, gifted from Dr. Hua-Tai Xu, Institutes of Neuroscience, Chinese Academic of Sciences), and Vglut2-ires-Cre mice (Jax Stock# 016963, gifted from Dr. Yan-Gang Sun, Institutes of Neuroscience, Chinese Academic of Sciences) were used. Animals were housed in standard laboratory cages in a temperature (23–25°C)-controlled vivarium with a 12:12 light/dark cycle, free to food and water. For tracing and behavioral experiments, the mice were injected with the virus at 7–8 weeks old and performed the behavioral tests at 11–12 weeks old. For the electrophysiological experiments, the mice were injected with the virus at 4–6 weeks old to accomplish the electrophysiological experiments at 7–9 weeks old. For in vivo fiber photometry and optoelectrode experiments, the mice were injected with the virus at 7–8 weeks old to accomplish the experiments at 10–11 weeks old. All animal experiment procedures were approved by the Animal Care and Use Committee of Shanghai General Hospital (2019AW008).

Stereotaxic surgery

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Mice were anesthetized by vaporized sevoflurane (induction, 3%; maintenance, 1.5%) and head-fixed in a mouse stereotaxic apparatus (RWD Life Science Co.).

For electrophysiological experiments, the AAV2/8-hSyn-ChR2-mCherry virus (300 nL, 4 × 1012 v.g./mL, AG26976, Obio Technology) was injected into the PBN nucleus of WT mice in the stereotaxic coordinate: anteroposterior (AP) −5.2 mm, mediolateral (ML) +1.3 mm, and dorsoventral (DV) −3.4 mm.

For tracing studies, the AAV2/8-EF1α-DIO-EGFP virus (300 nL, S0270, Taitool Bioscience) was injected into the PBN (mentioned above) of Vglut2-ires-Cre mice.

For the retrovirus injection surgery, the retrograde transport Cre recombinase retroAAV2/2-hSyn-Cre virus (150 nL, 4 × 1012 v.g./mL, S0278-2RP-H20, Taitool Bioscience) was injected in the Rosa26-tdTomato mice at two locations of PVT, respectively: (1) AP −1.22 mm, ML 0 mm, DV −2.9 mm; (2) AP −1.46 mm, ML 0 mm, DV −2.9 mm.

For optogenetic activation of PVT-projecting PBN fibers, the AAV2/9-EF1α-DIO-ChR2-mCherry virus (300 nL, 4 × 1012 v.g./mL, S0170-9-L20, Taitool Bioscience) or the AAV2/9-EF1α-DIO-mCherry virus (300 nL, 4 × 1012 v.g./mL, AG20299, Obio Technology) was bilaterally injected into the PBN (mentioned above) of Vglut2-ires-Cre mice, and a 200 μm diameter optic fiber was implanted over the PVT (AP −1.46 mm, ML 0 mm, DV −2.9 mm) with a 20° angle towards the midline.

For the pharmacogenetic activation of PBN-PVT neurons, the retroAAV2/2-hSyn-Cre virus (150 nL, 4 × 1012 v.g./mL, S0278-2RP-H20, Taitool Bioscience) was injected into the PVT (AP −1.46 mm, ML 0 mm, DV −2.9 mm), and the AAV2/9-hSyn-DIO-hM3Dq-mCherry virus (300 nL, 4 × 1012 v.g./mL, PT-0019, BrainVTA) or the control AAV2/9-EF1α-DIO-mCherry virus was bilaterally injected into the PBN (mentioned above) of the WT mice.

For optogenetic inhibition of PVT-projecting PBN fibers, AAV2/9-EF1α-DIO-NpHR3.0-EYFP virus (300 nL, 4 × 1012 v.g./mL, AG26966, Obio Technology) or the AAV2/8-EF1α-DIO-EGFP virus was bilaterally injected into the PBN (mentioned above) of Vglut2-ires-Cre mice, and a 200 μm diameter optic fiber was implanted over the PVT (AP −1.46 mm, ML 0 mm, DV −2.9 mm) with a 20° angle towards the midline.

For optogenetic inhibition of PBN-PVT neurons, retroAAV2/2-hSyn-Cre was injected into the PVT (AP −1.46 mm, ML 0 mm, DV −2.9 mm), and AAV2/9-EF1α-DIO-NpHR3.0-EYFP virus (300 nL, 4 × 1012 v.g./mL, AG26966, Obio Technology) or the AAV2/8-EF1α-DIO-EGFP virus was bilaterally injected into the PBN of WT mice, the left optic fiber was implanted over the PBN vertically, and the right one was placed over the PBN with a 20° angle towards the midline.

For in vivo fiber photometry experiments, the AAV2/8-hSyn-GCaMP6s virus (200 nL, 4 × 1012 v.g./mL, S0225-8, Taitool Bioscience) was injected into the PVT nucleus (AP −1.46 mm, ML 0 mm, DV −2.90 mm) of the WT mice, and the optic fiber was implanted above the PVT with a 20° angle towards the midline.

For optoelectrode experiments, the AAV2/9-EF1α-DIO-ChR2-mCherry virus (300 nL, 4 × 1012 v.g./mL, S0170-9-L20, Taitool Bioscience) was bilaterally injected into the PBN (mentioned above) of Vglut2-ires-Cre mice. Three weeks later, the homemade optoelectrode was implanted into the PVT nucleus (AP −1.46 mm, ML 0 mm, DV −2.90 mm).

For pharmacogenetic activation of PVTPBN neurons, the AAV2/1-hSyn-Cre virus (300 nL, 1.5 × 1013 v.g./mL, S0278-1-H50, Taitool Bioscience) was bilaterally injected into the PBN nucleus, and the AAV2/9-hSyn-DIO-hM3Dq-mCherry virus or the control AAV2/9-EF1α-DIO-mCherry virus was injected into the PVT (AP −1.46 mm, ML 0 mm, DV −2.9 mm) of the WT mice.

The virus was infused through a glass pipette (10–20 μm in diameter at the tip) at the rate of 50–100 nL/min. The injection pipette was left in place for additional 8 min. After the surgeries, the skin was closed by the sutures, and the optic fiber was secured through the dental acrylic. Generally, tracing, electrophysiological, or behavioral experiments were performed at least 3 weeks later. After experiments, histological analysis was used to verify the location of viral transduction and the optic fiber. The mice without correct transduction of virus or correct site of optic fiber were excluded for analysis.

Histology

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Animals were deeply anesthetized with vaporized sevoflurane and transcardially perfused with 20 mL saline, followed by 20 mL paraformaldehyde (PFA, 4% in PBS). Brains were extracted and soaked in 4% PFA at 4°C for a minimum of 4 hr and subsequently cryoprotected by transferring to a 30% sucrose solution (4°C, dissolved in PBS) until brains were saturated (for 36–48 hr). Coronal brain sections (40 μm) were cut using a freezing microtome (CM1950, Leica). The slices were collected and stored in PBS at 4°C until immunohistochemical processing. Nuclei were stained with DAPI (Beyotime, 1:10000) and washed three times with PBS.

The brain sections undergoing immunohistochemical staining were washed in PBS three times (10 min each time) and incubated in a blocking solution containing 0.3% TritonX-100 and 5% normal donkey serum (Jackson ImmunoResearch, USA) in PBS for 1 hr at 37°C. Sections were then incubated (4°C, 24 hr) with primary antibodies dissolved in 1% normal donkey serum solution. Afterward, sections were washed in PBS four times (15 min each time), then incubated with secondary antibodies for 2 hr at room temperature. After DAPI staining and washing with PBS, sections were mounted on glass microscope slides, dried, and covered with 50% glycerin (Thermo Fisher). The images were taken with the Leica Dmi8 microscope and by the Leica SP8 confocal microscopy. The images were further processed using Fiji and Photoshop.

RNAscope in situ hybridization

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Mice were anesthetized with isoflurane and rapidly decapitated. Brains were roughly dissected from perfused mice and post-fixed in 4% PFA at 4°C overnight, dehydrated in 30% sucrose 1× PBS at 4°C for 2 days. Mouse brains were embedded, cryosectioned in 15 µm coronal slices, and mounted on SuperFrost Plus Gold slides (Fisher Scientific). In situ hybridization was performed according to the protocol of the RNAscope Multiplex Fluorescent Reagent Kit v2 (Cat# 320293). Probes were purchased from Advanced Cell Diagnostics: fos (Cat# 316921-C2), Tac1 (Cat# 410351-C2), Tacr1 (Cat# 428781-C2), Pdyn (Cat# 318771), and Slc17a6 (Cat# 319171-C2). Primary antibodies include rabbit anti-Fos (Abcam, Cat# ab190289, 1:4000), goat anti-CGRP (Abcam, Cat# ab36001, 1:1000), and rabbit anti-DsRed (Takara Bio, Cat# 632496, 1:500). All secondary antibodies were purchased from Jackson ImmunoResearch and used at 1:400 dilution. Secondary antibodies include Alexa 488 donkey anti-rabbit (Cat# 711-545-152), Cy3 donkey anti-rabbit (Cat# 711-165-152), and Alexa 488 donkey anti-goat (Cat# 705-546-147). Images were collected on a Leica fluorescence microscope and Leica LAS Software.

Fos induction

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The mice were habituated for 3 days and performed gentle grabbing and holding for 1 min, five times every day, to minimize background Fos expression.

To study the effect of pharmacogenetic manipulations on PBN-PVT neurons, we intraperitoneally injected 0.5 mg/kg CNO (Sigma). 90 min later, the brain tissues were processed.

To assess 2-MT-evoked Fos expression in the PVT, the mice were kept in a chamber with a floor covered with cotton containing 100 mL 1:1000 diluted 2-MT for 90 min. Then the mice were perfused.

To assess footshock-induced Fos expression in the PVT, we placed the mice into the chamber and delivered 30 times inevitable footshock (0.5 mA, 1 s) with a variable interval (averaging 60 s). After stimulation, animals were kept in the same apparatus for another 60 min, and brain tissues were then processed.

For the dual Fos experiments, we first delivered 20 min 473 nm laser pulses (20 Hz, 5 mW, 5 ms) and left the mice to rest in the homecage for 60 min. Then we delivered the 20 min footshock stimulus (0.5 mA, 1 s, 30 times, variable interval) and perfused the mice.

Electrophysiology

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The electrophysiological experiment was performed as previously described (Mu et al., 2017). Mice were anesthetized with sevoflurane and perfused by the ice-cold solution containing (in mM) 213 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, 11 glucose (300–305 mOsm). Brains were quickly dissected, and the coronal slices (250 μm) containing the PBN or PVT were chilled in ice-cold dissection buffer using a vibratome (V1200S, Leica) at a speed of 0.12 mm/s. The coronal sections were subsequently transferred to a chamber and incubated in the artificial cerebrospinal fluid (ACSF, 34°C) containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 26 NaHCO3, 10 glucose (300–305 mOsm) to recover for at least 40 min, then kept at room temperature before recording. All solutions were continuously bubbled with 95% O2/5% CO2.

All experiments were performed at near-physiological temperatures (30–32°C) using an in-line heater (Warner Instruments) while perfusing the recording chamber with ACSF at 3 mL/min using a pump (HL-1, Shanghai Huxi). Whole-cell patch-clamp recordings were made from the target neurons under IR-DIC visualization and a CCD camera (Retiga ELECTRO, QIMAGING) using a fluorescent Olympus BX51WI microscope. Recording pipettes (2–5 MΩ; Borosilicate Glass BF 150-86-10; Sutter Instrument) were prepared with a micropipette puller (P97; Sutter Instrument) and backfilled with potassium-based internal solution containing (in mM) 130 K-gluconate, 1 MgCl2, 1 CaCl2, 1 KCl, 10 HEPES, 11 EGTA, 2 Mg-ATP, 0.3 Na-GTP (pH 7.3, 290 mOsm), or cesium-based internal solution containing (in mM) 130 CsMeSO3, 1 MgCl2, 1 CaCl2, 10 HEPES, 2 QX-314, 11 EGTA, 2 Mg-ATP, 0.3 Na-GTP (pH 7.3, 295 mOsm). Biocytin (0.2%) was included in the internal solution.

In PBN-PVT ChR2 experiments, whole-cell recordings of PBN neurons with current-clamp (I = 0 pA) were obtained with pipettes filled with the potassium-based internal solution. The 473 nm laser (5 Hz, 10 Hz, 20 Hz pulses, 0.5 ms duration, 2 mW/mm2) was used to activate PBN ChR2-positive neurons. Light-evoked EPSCs and IPSCs of PVT neurons recorded with voltage-clamp (holding voltage of –70 mV or 0 mV) were obtained with pipettes filled with the cesium-based internal solution. The 473 nm laser (20 Hz paired pulses, 1 ms duration, 4 mW/mm2) was used to activate ChR2-positive fibers. The light-evoked EPSCs were completely blocked by 1 μM TTX, rescued by 100 μM 4-AP, and blocked by 10 μM (6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX)). NBQX and TTX were purchased from Tocris Bioscience. All other chemicals were obtained from Sigma.

Voltage-clamp and current-clamp recordings were carried out using a computer-controlled amplifier (MultiClamp 700B; Molecular Devices, USA). During recordings, traces were low-pass filtered at 4 kHz and digitized at 10 kHz (DigiData 1550B1; Molecular Devices). Data were acquired by Clampex 10.6 and filtered using a low-pass Gaussian algorithm (–3 dB cutoff frequency = 1000 Hz) in Clampfit 10.6 (Molecular Devices).

Optogenetic manipulation

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For activating the PBN-PVT projection, a 473 nm laser (20 Hz, 5 mW, 5 ms pulse duration) was delivered. For inhibition of the PBN-PVT projection and the PBN-PVT neurons, a constant laser (589 nm, 10 mW) was delivered.

Pharmacogenetic manipulation

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All behavioral tests were performed 30 min after i.p. injection of 0.5 mg/kg CNO in pharmacogenetic manipulation. Different behavior tests were performed at least 3 days apart.

Open field test

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The OFT was used to assess locomotor activity and anxiety-related behavior in an open field arena (40 × 40 × 60 cm) with opaque plexiglass walls. The mouse was placed in the center of the box and recorded with a camera attached to a computer. The movement was automatically tracked and analyzed using AniLab software (Ningbo AnLai, China). The total distance traveled, the total velocity, the total immobility time (the mice were considered to be immobile if immobility time lasts more than 1 s), and time spent in the center area (20 × 20 cm) were measured. The box was cleaned with 70% ethanol after each trial.

To assess the effect of optogenetic activation of the PBN-PVT projection, 15 min sessions consisting of 5 min pre-test (laser OFF), 5 min laser on test (laser on), and 5 min post-test (laser off) periods. Laser (473 nm, 20 Hz, 5 mW, 5 ms) was delivered during the laser on phase.

To assess the effect of pharmacogenetic manipulations of PBN-PVT neurons on locomotor activity and affective behaviors, we recorded the movement 30 min after i.p. injection with CNO.

To assess the effect of inhibition of the PBN-PVT projection on the aversive behaviors induced by 2-MT, one cotton ball containing 5 mL 2-MT (1:1000) solution was placed on the center of the upper-left quadrant to disseminate fear-odor, then a constant laser (589 nm, 10 mW) was delivered during the 10 min test. The time spent in the 2-MT paired quadrant was calculated.

Elevated zero maze

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The EZM was an opaque plastic circle (60 cm diameter), which consisted of four sections with two opened and two closed quadrants. Each quadrant had a path width of 6 cm. The maze was elevated 50 cm above the floor. The animals were placed into an open section facing a closed quadrant and freely explored the maze for 5 min.

Real-time place aversion test

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Mice were habituated to a custom-made 20 × 30 × 40 cm two-chamber apparatus (distinct wall colors and stripe patterns) before the test. Each mouse was placed in the center and allowed to explore both chambers without laser stimulation for 10 min on day 1. The movement was recorded for 10 min as a baseline. The mice performed a slight preference for the black chamber according to the fact that the mice have innate aversion to brightly illuminated areas. On day 2, 473 nm laser stimulation (20 Hz, 5 mW, 5 ms) was automatically delivered when the mouse entered or stayed in the black chamber and turned off when the mouse exited the black chamber for 10 min. Finally, the mouse was allowed to freely explore both chambers without laser stimulation for another 10 min. The RTPA location plots and total time on the stimulated side were recorded and counted with the AniLab software.

Conditioned place aversion

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After habituation, mice were placed in the center of the two-chamber apparatus and allowed to explore either chamber for 15 min on day 1. On day 2, mice were restricted to one chamber (laser-paired chamber) with photostimulation (473 nm, 20 Hz, 5 mW, 5 ms) for 30 min in the morning and restricted to the other chamber (unpaired chamber) without photostimulation in the afternoon. On day 3, mice were restricted to the unpaired chamber without photostimulation in the morning and restricted to the laser-paired chamber with photostimulation in the afternoon. On day 4, mice were allowed to explore both chambers without laser stimulation for another 15 min. The time in the laser-paired chamber was calculated on days 1 and 4.

2-MT-induced aversion

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To assess the effect of optogenetic inhibition of the PBN-PVT projection or PBN-PVT neurons on the aversive state, three cotton balls containing 15 mL 2-MT (1:1000) solution were placed in the black chamber. A constant laser (589 nm, 10 mW) was delivered during the 10 min test.

Cue-dependent optogenetic conditioning test

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Video Freeze fear conditioning system with optogenetic equipment (MED Associates, MED-VFC-OPTO-USB-M) and Video Freeze software were used.

On day 1, mice were habituated to the fear conditioning chambers and allowed to explore for 2 min freely, then three tones (75 dB, 4 kHz, 30 s duration) separated by a variable interval with a range of 60–120 s and the average of 90 s were delivered.

On day 2, mice were trained with the sound cue (75 dB, 4 kHz, 30 s) paired with a simultaneous 30 s laser pulse train (20 Hz, 5 ms, 5 mW) for six times separated by a variable interval (averaging 90 s). The mice were kept in the conditioning chamber for another 60 s before returning to the home cages.

On day 3, mice were placed back into the original training chamber for 3 min to perform the contextual test. After 2–3 hr, the conditioning chamber was modified by changing its metal floor and sidewalls. Mice were placed in the altered chamber for 3 min to measure the freezing level in the altered context. A tone (75 dB, 4 kHz) was delivered for 30 s to perform the cue test.

The behavior of the mice was recorded and analyzed with the Video Freeze software. Freezing was defined as the complete absence of movement for at least 0.5 s. On the conditioning day, the freezing percentages were calculated for 30 s during each tone/laser stimulus. For the contextual test, the freezing percentages were calculated for 3 min. For the cue test, the freezing percentages were calculated for 30 s during tone.

Auditory fear conditioning test

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On day 1, mice were habituated to the fear conditioning chambers. On day 2, mice were conditioned by seven trials of sound tone (75 dB, 4 kHz, 30 s) co-terminated with footshock (0.6 mA, 2 s) averagely separated by 90 s. Laser (589 nm, 10 mW) was delivered 1 s before the footshock and lasted for 4 s at each trial. On day 3, mice were placed back into the original training chamber for 3 min to perform the contextual test, and the laser was delivered during the second minute. After 2–3 hr, the mice were placed into a modified chamber to perform the cue test. Three tones were given averagely separated by 90 s. The laser was delivered during the second tone.

The behavior of the mice was recorded and analyzed with the Video Freeze software. The freezing percentages of the 27 s tone before laser (to avoid the influence of laser) for each trial were summarized to indicate fear memory acquisition in the conditioning test. For the contextual test, the freezing percentages were calculated for every minute. For the cue test, the freezing percentages were calculated for 30 s during tone.

Freezing behavior

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For analyses of freezing behavior induced by pharmacogenetic activation of PBN-PVT neurons, we injected CNO and recorded the mouse behavior using the Video Freeze fear conditioning system 30 min later.

The Video Freeze fear conditioning system (MED Associates, MED-VFC-OPTO-USB-M) was also used to assess the effect of optogenetic inhibition of PBN-PVT projection and the PBN-PVT neurons on the fear-like behavior induced by footshock. After free exploration of the chamber for 2 min, 15 times footshocks (0.6 mA, 1 s) were delivered within 10 min with a constant 589 nm laser (10 mW). The freezing percentages during 10 min were analyzed.

The Video Freeze fear conditioning system was also used to assess the effect of optogenetic inhibition of the PBN-PVT neurons on the fear-like behavior induced by 2-MT. 10 mL 2-MT (1:1000) dissolved in the ddH2O was soaked into the cotton ball on the bottom of the training box. A constant laser (589 nm, 10 mW) was delivered during the tests.

Tail suspension test

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Mice were individually suspended by an adhesive tape placed roughly 2 cm from the tip of the tail and videotaped for 6 min. Mice were considered immobile without initiated movements, and the immobility time was scored in the last 3 min by an observer unknown of the treatments.

Forced swimming test

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Mice were individually placed for 6 min in clear cylinders (45 cm height, 20 cm internal diameter) containing freshwater (25°C, 15 cm depth). The swimming activity was videotaped, and immobility time in the last 3 min was counted manually by an investigator unaware of animal grouping. The mice were considered immobile when they stopped swimming/struggling or only slightly moved to keep the nose above the surface.

Von Frey test

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The von Frey test was used to assess the mechanical sensitivity (Mu et al., 2017). The mice were acclimated to the observation chambers for 2 days (2 hr for each day) before the test. A series of von Frey hairs with logarithmically incrementing stiffness (0.16–2.0 g) were used to stimulate the mouse hind paw perpendicularly. The 50% paw withdrawal threshold was determined using the up-down method.

Hargreaves test

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Hargreaves tests were performed as described previously (Mu et al., 2017). Mice were placed in an individual plexiglass box with a glass floor. A radiant heat beam was exposed directly to the hind paw until the paw was withdrawn. The trials were repeated three times with an interval of at least 15 min. To avoid potential damage, the test was executed with a 20 s cutoff time.

Formalin test

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In the formalin test, the mice received an intraplantar injection of formalin (5%, 20 μL/mouse) and were placed into a plexiglass box (width: 10 cm; length: 10 cm; height: 15 cm) individually to record the pain-related licking behaviors for 1 hr. All videos were analyzed by trained investigators blinded to the experimental treatment of the animals.

Rotarod test

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Mice were trained twice on a rotarod apparatus (MED Associates) with a rod accelerated 5–20 revolutions per minute (rpm) for 5 min before the experimental day. On the second day, each mouse underwent three trials with a rod programmed to accelerate from 0 to 40 rpm over 300 s, then the average rpm at the point of falling was recorded.

Fiber photometry

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In vivo fiber photometry experiments were performed as previously described (Zhu et al., 2020). After 2 weeks of virus expression, the mice were gently handled to be familiar with the calcium signal recording experiments (Thinker-Biotech). A signal (for synchronization) was manually tagged with the shock and air puff to evaluate the activity of PVT neurons. The calcium transient was recorded at 50 Hz. The fluorescence values change (ΔF/F) was calculated from the formula of (F−F0)/F0, where the F0 represents the median of the fluorescence values in the baseline period (−1 to −0.5 s relative to the stimulation onset). To precisely quantify the change of the fluorescence values across the shock or air puff stimulation, we defined 0.5–1.0 s after the onset as the post-stimulus period.

Optoelectrode recording and analysis

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The homemade optoelectrode consisted of an optic fiber (200 mm in diameter) glued to 16 individually insulated nichrome wires (35 μm internal diameter, 300–900 Kohm impedance, Stablohm 675, California Fine Wire). The 16 microwire arrays were arranged in a 4 + 4 + 4 + 4 pattern and soldered to an 18-pin connector (Mil-Max). Three weeks after virus injection, the optoelectrode was implanted to the PVT nucleus (AP −1.46 mm, ML 0 mm, DV −2.90 mm). After 1 week of recovery, two trials were performed continuously. Trial 1 contained 10 sweeps of 2 s laser pulse trains (473 nm, 5 ms, 20 Hz, 8 mW). The interval of sweeps was 60 s. Trial 2 contained 20 sweeps of 2 s footshock (0.5 mA). The interval of sweeps was 60 s. In the even time sweeps (2, 4, 6, 8,10, 12, 14, 16, 18, 20), 2 s laser pulse trains were delivered spontaneously with the 2 s footshock. Neuronal signals were recorded using a Zeus system (Zeus, Bio-Signal Technologies, McKinney, TX), and spike signals were filtered online at 300 Hz. At the end of the experiment, all animals were perfused to confirm the optical fiber sites. Only the data of animals with correct optical fiber sites and virus expression regions were analyzed.

The spikes were sorted by the valley-seeking method with Offline Sorter software (Plexon, USA) and analyzed with NeuroExplorer (Nex Technologies, Boston, MA). Firing rates of the neurons and timestamps were exported for further analysis using customized scripts in MATLAB. The Kolmogorov–Smirnov (K-S) test was used to compare the spike firing rate of PVT during 2 s baseline (before stimulus) and 2 s after each stimulus. p<0.001 indicates statistical significance. The code generated during this study is available on github (Xiang, 2021a, copy archived at swh:1:rev:ce467c67c1c21f424c92a6e189c7cd96ea938e89; Xiang, 2021b). Z-score normalization maps were constructed from normalized firing rates.

To calculate the latency of response of PVT neurons to laser and shock activation, we extracted the data in small bins of 2 ms. The onset of the response was calculated as the first of at least five consecutive bins higher than 20% above the baseline.

Quantification of the fiber intensity

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For quantification of fluorescence of PVTPBN efferents, the downstream targets of PVTPBN neurons were imaged using the identical character, and the mean fluorescence value in each ROI (400 × 400 pixels) of each brain region was analyzed using Fiji. The fiber intensity was calculated as the fluorescence value of each brain region divided by that of the NAc. All data came from at least three different mice and are presented as mean ± SEM.

Analysis

Statistical detection methods include unpaired Student’s t-test, paired Student’s t-test, one-way ANOVA with Bonferroni’s correction for multiple comparisons, two-way ANOVA with Bonferroni’s correction for multiple comparisons. A value of p<0.05 is considered statistically significant. All data are represented as mean ± SEM.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file. The behavioral data and imaging analysis results have been made available on Dryad Digital Repository. All MATLAB code has been deposited on Github (copy archived at swh:1:rev:ce467c67c1c21f424c92a6e189c7cd96ea938e89) and is publicly available.

The following data sets were generated
    1. Mu D
    (2022) Dryad Digital Repository
    PBN-PVT projections modulate negative affective states in mice.
    https://doi.org/10.5061/dryad.1rn8pk0w4

References

Decision letter

  1. Mario Penzo
    Reviewing Editor; National Institute of Mental Health, United States
  2. Andrew J King
    Senior Editor; University of Oxford, United Kingdom
  3. Mario Penzo
    Reviewer; National Institute of Mental Health, United States
  4. Fabricio H Do Monte
    Reviewer; The University of Texas Health Science Center at Houston, United States

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "PBN-PVT projection modulates negative emotions in mice" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Mario Penzo as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Andrew King as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Fabricio do Monte (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

While individual assessments and recommendations from each of the reviewer is included below, here we provide you with a brief list of items that we collectively consider to be essential revisions that must be addressed in order for the manuscript to be considered further for publication at eLife.

1) Revise the writing and overall framing of the study. The authors should avoid anthropomorphizing rodents by using terms typically associated with human's subjective experience of emotions like sadness and misery.

2) The authors should provide a more detailed discussion and present the temporal profiles of the behaviors elicited by photo stimulating PBN-PVT projections (see comments from Reviewer # 2).

3) While the authors showed that VGLUT2 expressing neurons of the PBN project to the PVT, we ask that the authors perform a more complete characterization of the molecular identity of PVT-projecting PBN neurons. The authors are not expected to identify new markers, but rather use known PBN markers as reference. The objective should be to determine what fraction of PBN projections to the PVT arise from VGLUT2+ neurons vs other PBN markers.

4) The authors should demonstrate that PBN input shapes PVT neuronal responses to aversive stimuli by expanding on their findings presented in Figure 5.

Reviewer #1 (Recommendations for the authors):

1) I recommend that the authors reconsider their overall framing of the term emotion and its evaluation in animal models. While in general the writing of the manuscript could be improved, I found the use of subjective terms of emotion like sadness and misery particularly problematic. Framing of the study within the constraints of aversion may be more appropriate here.

2) In Figure 1 it appears that the density of PBN projections is higher in the middle and posterior PVT (pPVT). The authors should better highlight this observation in the text, especially considering that the notion of the pPVT being a particularly aversive region of the PVT is now supported by various studies. The overall framing of the PBN-PVT pathway as involved in aversion fits well with the anatomical distribution of these projections.

3) Justification for the use of different genetic strategies to target the PBN-PVT pathway is required. When using the constitutively expensed ChR2 in the PBN to demonstrate glutamatergic projections to the PVT, please show whether there is contamination from adjacent brain stem structures like the LC and DRN, which project to the PVT and are known to contain glutamatergic neurons (vglut1 and vglut3, respectively). In figure 4 why were Vglut2-cre mice not used and PBN terminals in the PVT inhibited as in Figure 2?

4) In the retrograde labeling experiment (Figure 1) please show what fraction of retrogradely label cells are VGLUT2+. You should also ensure that a similar population of PBN-PVT neurons has been assessed throughout the study.

5) Please reassess the statistical tests used, as outlined in the Public Review section.

6) Please consider whether associative memory (e.g. fear memory retrieval) is impacted by manipulations of the PBN-PVT pathway.

7) Please provide better evidence for whether increased activity of PVT neurons is mediated by input from the PBN.

Reviewer #2 (Recommendations for the authors):

1. This manuscript has numerous grammatical issues and typos. It would benefit enormously if the text is carefully proofread. It also misuses terminologies and concepts in several cases, including the use of the word "affection" presumably to refer to "affective states", or the use of the word "rescue" to describe the reduction of "fear-like behaviors". These issues should be corrected.

2. The authors frequently refer to Chiang et al. 2020 (PMID: 32289251) paper to support their observation that PBN-PVT projections are exclusively glutamatergic. However, the same paper showed that the PBN also sends a smaller number of GABAergic fibers to PVT. While the authors showed only EPSCs in PVT neurons when optogenetically activating PBN inputs, it is not clear whether the recordings were also performed after clamping the voltage at 0mV to allow the detection of possible IPSCs after stimulation of PBN fibers. Alternatively, the authors could use immunohistochemistry to identify the cell-type specificity of PVT-projecting neurons in PBN and reconcile their findings with the current literature.

3. Similarly, Chiang et al. 2020 described the role of PBN-CeA/BNST projections in regulating aversion. Although the authors showed that PVT-projecting neurons in PBN do not project to CeA, it would be very informative for the field if the authors could also provide the data for other brain regions to which PVT-projecting neurons also send collateral, especially those regions that are important for anxiety- and fear- like behaviors. This would help the authors to exclude (or include) the possible contribution of collateral projections to the reported behaviors.

4. For the PBN-PVT photoactivation experiments in Figure 2, a deeper discussion about the lack of effects on aversive memory formation (RTPA and fear conditioning) is missing. The authors should compare their results with prior studies in the literature. For example, activation of PVT-CeA projections induces place aversion and the effect persists in the next day in the absence of illumination (Do Monte et al. 2017, PMID: 28426970). Similarly, LTD-like stimulation of PVT-CeA projections or optogenetic/chemogenetic inhibition of the same circuit induces a persistent attenuation of fear responses (Chen and Bi, 2019, PMID: 30406427; Do Monte et al. 2015, PMID: 25600268; Penzo et al. 2015, PMID: 25600269)

5. In Figure 4, the authors should justify why they have inhibited the soma of PVT-projecting PBN neurons instead of axonal terminals in the PVT. They should also make it very clear in their schematic drawing by adding an optical fiber on the top of the PVT, similar to what they have done in other figures.

6. During optogenetic manipulations, the authors used a very long period of laser illumination (5 uninterrupted minutes of pulses for photoactivation and 10 minutes of constant laser for photoinhibition). These laser durations are much longer than the traditional intervals used for optogenetic manipulations in rodents. An explanation of the rationale for using such long intervals is needed. Also, regarding the photoactivation experiment (illustrated in Supplementary Video 1), it is very clear that the animals exhibited a wide range of different behaviors along the 5-min period of stimulation. These behaviors varied from no effect to increased speed of locomotion, to jumping episodes, to freezing responses by the end of the stimulation, thereby making it hard to define the correct function of this pathway in behavioral regulation. A breakdown of the behaviors by time and a deeper discussion of the different responses would probably help the readers to better understand the results.

7. The authors should provide a Cre-negative control for Figure 5G-N, given that tdTomato Ai14 mice can have baseline levels of tdTomato expression even in the absence of Cre. This is highly important because the transneuronal transduction of AAV1-Cre is generally limited and has never been tested in PVT.

Reviewer #3 (Recommendations for the authors):

1. It has been shown that PBN-VMH/PAG drive escape behaviors, whereas PBN-BNST/CEA generate aversive memory. The authors need to examine the relationship between PVT-projection and VMH/BNST-projections.

2. The authors need to examine the cellular identity of PVT-projecting PBN neurons, whether it originate from known PBN neuronal subtypes such as CGRP, Tac1, Pdyn, Nts et al.

3. As authors mentioned in discussion part, a recent study revealed that the PBN neurons project to ILN, which is relatively closed to the PVT. The authors need to carefully examine their manipulation experiments especially optogenetic stimulation. They need to make sure that the effect they observed was not due to the activation of PBN-ILN pathway. Since activation of PBN-ILN pathway induced pain-related behavior which could be very similar to negative emotions.

4. For the cue-dependent optogenetics conditioning test: activation of the PBN-PVT projection induces instant aversion and freezing but not drives associative fear learning. Could it be simply because the conditioning (6 times 30 seconds light stimulation) was too weak? The authors should try a prolong conditioning protocol that resemble drug CPP such as 30 min light stimulation at paired side.

5. Optogenetics and Pharmacogenetics are nouns while optogenetic and pharmacogenetic are adjectives. There are many places in the manuscript where the noun was misused.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "PBN-PVT projections modulate negative affective states in mice" for further consideration by eLife. Your revised article has been evaluated by Andrew King (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The reviewers collectively agree that, in the present revision, the authors have addressed the most relevant criticism raised following the initial submission. As such, we consider that the manuscript is a strong candidate for publication in eLife. However, before reaching a final decision on the manuscript, we recommend that the authors address the remaining recommendations, particularly from Reviewer #2. No additional experiments are requested at this time. Revisions are strictly for clarity purposes.

Reviewer #1 (Recommendations for the authors):

In their revised manuscript, the authors have adequately addressed the previous concerns and recommendations raised by this reviewer. Importantly, doing so has improved the overall quality of the manuscript.

The manuscript could benefit from additional copy editing to avoid statements such as "PVT neurons innervated by PBN innervation" (Line 39).

Reviewer #2 (Recommendations for the authors):

In this revised version of the manuscript, the authors did a good job fully addressing all the concerns raised during the first submission. They have now re-framed the manuscript with careful interpretation and discussion of the results. They also carried out new experiments to address some important questions that were unclear in the first version of the manuscript. As a result, the clarity and quality of the manuscript has significantly improved compared to the previous version.

Reviewer #3 (Recommendations for the authors):

The authors have carefully addressed all my questions and I appreciate their efforts in revising manuscript. I have no further comments.

https://doi.org/10.7554/eLife.68372.sa1

Author response

Essential revisions:

While individual assessments and recommendations from each of the reviewer is included below, here we provide you with a brief list of items that we collectively consider to be essential revisions that must be addressed in order for the manuscript to be considered further for publication at eLife.

1) Revise the writing and overall framing of the study. The authors should avoid anthropomorphizing rodents by using terms typically associated with human's subjective experience of emotions like sadness and misery.

We have revised the text to avoid using subjective terms of emotions. We used terms including anxiety-like, depression-like, fear-like behaviors, freezing, aversion-like behaviors. We replaced the terms “emotions” “affections” with “affective states”. We also replaced the terms “rescue” “relieve” with “reduce”.

2) The authors should provide a more detailed discussion and present the temporal profiles of the behaviors elicited by photo stimulating PBN-PVT projections (see comments from Reviewer # 2).

We have now provided a detailed analysis (one-minute time window) of optogenetic activation of PBN-PVT projections in OFT experiments. We further divided the laser ON period (5−10 minutes) into five one-minute periods and analyzed the velocity, non-moving time, center time, moving distance, and jumping behaviors. We found that the velocity and non-moving time were increased, and the center time was decreased in the ChR2 mice during most periods. Furthermore, we observed that the distance and jumping behaviors were increased mainly in the first one-minute period in ChR2 mice. This detailed analysis indicated that optogenetic activation induced brief and robust running, jumping behaviors, and persistent anxiety-like behaviors, such as less time spent in the center. These new results have been included in Figure 2−figure supplement 2 and were described in the text. Please see Page 7 Line 179 to Line 189. We also discussed this on Page 14 Line 396 to Line 403.

3) While the authors showed that VGLUT2 expressing neurons of the PBN project to the PVT, we ask that the authors perform a more complete characterization of the molecular identity of PVT-projecting PBN neurons. The authors are not expected to identify new markers, but rather use known PBN markers as reference. The objective should be to determine what fraction of PBN projections to the PVT arise from VGLUT2+ neurons vs other PBN markers.

To examine the molecular identity of PVT-projecting PBN neurons, we have now performed the RNAscope experiments detecting VgluT2, Tac1, Tacr1, Pdyn mRNA, and fluorescent immunostaining detecting CGRP protein in the PBN. We injected the retroAAV-Cre virus into the PVT nucleus on Rosa26-tdTomato mice and found that tdTomato+ neurons were located in the PBN. We found that about 94.4% of tdTomato+ neurons express VgluT2 mRNA. These results indicate that the majority of PVT-projecting PBN neurons are glutamatergic. These new results have been included in Figure 1R−U and were described in the text. Please see Page 5 Line 129 to Line 132.

We further determined the identity of PVT-projecting PBN neurons as suggested by Reviewer #3. We found that tdTomato+ neurons were only partially co-labeled with Tacr1, Tac1, or Pdyn mRNA, but not with CGRP. These new results have been included in Figure 1−figure supplement 1 and were described in the text. Please see Page 5 Line 132 to Line 140.

4) The authors should demonstrate that PBN input shapes PVT neuronal responses to aversive stimuli by expanding on their findings presented in Figure 5.

We have now performed the optoeletrode experiments to examine the effects of activation of PBN-PVT projections on PVT neuronal responses to footshock.

We injected AAV-DIO-ChR2 virus into the PBN and implanted the optoelectrode into the PVT of VgluT2-ires-Cre mice. We first recorded the spiking signals in response to 10 sweeps of 2-second laser pulse trains. Then we recorded the spiking signals in response to 20 sweeps of 2-second footshock without laser in the odd number sweeps or with laser in the even number sweeps. We found that there was also a broad overlap between laser-activated and footshock-activated neurons. Next, we analyzed the firing rates of PVT neurons during footshock with laser sweeps and footshock without laser sweeps. We found that the footshock stimulus with laser activated 30 of 40 neurons and increased the overall firing rates of 40 neurons compared with the footshock without laser result. These results indicated that activation of PBN-PVT projections could enhance PVT neuronal responses to aversive stimulation. These new results have been included in Figure 6 and were described in the text. Please see Page 11 Line 302 to Line 319.

Reviewer #1 (Recommendations for the authors):

1) I recommend that the authors reconsider their overall framing of the term emotion and its evaluation in animal models. While in general the writing of the manuscript could be improved, I found the use of subjective terms of emotion like sadness and misery particularly problematic. Framing of the study within the constraints of aversion may be more appropriate here.

We agree with the reviewer. Now we have reframed this manuscript. We have revised the text to avoid using subjective terms of emotions. We used terms including anxiety-like, depression-like, fear-like behaviors, freezing, aversion-like behaviors. We replaced the terms “emotions” “affections” with “affective states”. We also replaced the terms “rescue” “relieve” with “reduce”.

2) In Figure 1 it appears that the density of PBN projections is higher in the middle and posterior PVT (pPVT). The authors should better highlight this observation in the text, especially considering that the notion of the pPVT being a particularly aversive region of the PVT is now supported by various studies. The overall framing of the PBN-PVT pathway as involved in aversion fits well with the anatomical distribution of these projections.

We injected AAV2/8-EF1α-DIO-EGFP into PBN of VgluT2-ires-Cre mice. It is worth noting that the density of EGFP+ fibers was higher in the middle and posterior PVT (Figure 1−figure supplement 2E−H), considering the notion that the posterior PVT is particularly sensitive to aversion (Gao et al., 2020). Please see Page 5 Line 145 to Page 6 Line 148. We also discussed this on Page 14 Line 387 to Line 391.

We also checked the recording sites of the PVT neurons in the PBN-PVT connectivity experiment. Some neurons should be marked as posterior PVT neurons rather than medial PVT, and we have revised the result. Please see Figure 1D−F, Page 4 Line 108 to Line 112.

3) Justification for the use of different genetic strategies to target the PBN-PVT pathway is required. When using the constitutively expensed ChR2 in the PBN to demonstrate glutamatergic projections to the PVT, please show whether there is contamination from adjacent brain stem structures like the LC and DRN, which project to the PVT and are known to contain glutamatergic neurons (vglut1 and vglut3, respectively). In figure 4 why were Vglut2-cre mice not used and PBN terminals in the PVT inhibited as in Figure 2?

We agree with the reviewer. Now we have reframed this manuscript. We first presented the slice recording results from wild-type mice (Figure 1). We found that light-induced EPSCs in 34 of 52 neurons and light-induced IPSCs in 4 of 52 neurons. Please see Page 5 Line 119 to Line 121.

We carefully examined the ChR2 virus infection area. The raphe nucleus was not infected. Some neurons in the LC were infected with the AAV-hSyn-ChR2 virus. We agreed with the reviewer that there could be potential contamination from the LC, which releases dopamine and norepinephrine to the PVT by LC-PVT projection. We have discussed this on Page 13 Line 375 to Line 380.

We performed tdTomato staining with VgluT2 mRNA in situ hybridization and found that about 94.4% of tdTomato+ neurons express VgluT2 mRNA. These new results indicate that the majority of PVT-projecting PBN neurons are glutamatergic. Please see Figure 1R−U and Page 5 Line 129 to Line 132.

Then we used VgluT2-ires-Cre mice to perform tracing (Figure1−figure supplement 2) and behavioral tests (optogenetic activation in Figure 2, optogenetic inhibition in Figure 4). We also performed the pharmacogenetic activation of PVT-projecting PBN neurons on wild-type mice (Figure 3). We observed that pharmacogenetic activation of the PVT-projecting PBN neurons reduced the center duration in the OFT, similar to the optogenetic activation OFT result. We also observed that pharmacogenetic activation of the PVT-projecting PBN neurons induced freezing behaviors. Our pharmacogenetic activation experiment supported the hypothesis that PBN-PVT projections modulate negative affective states.

Now we have performed the optogenetic inhibition of the PBN-PVT projections using VgluT2-ires-Cre mice. We found that inhibition of PBN-PVT projections reduces 2-MT-induced aversion-like behaviors and footshock-induced freezing behaviors. These new results have been included in Figure 4, Figure 4−figure supplement 1 and 2, and were described in the text. Please see Page 9 Line 254 to Page 10 Line 274.

4) In the retrograde labeling experiment (Figure 1) please show what fraction of retrogradely label cells are VGLUT2+. You should also ensure that a similar population of PBN-PVT neurons has been assessed throughout the study.

We performed tdTomato staining with VgluT2 mRNA in situ hybridization and found that about 94.4% of tdTomato+ neurons express VgluT2 mRNA. These results indicate that the majority of PVT-projecting PBN neurons are glutamatergic. These new results have been included in Figure 1R−U and described in the text. Please see Page 5 Line 129 to Line 132.

5) Please reassess the statistical tests used, as outlined in the Public Review section.

We have revised the statistics (Unpaired student’s t-test) and calculated the percentage of freezing behaviors in two groups during 10-minute experiment, which matched the constant optogenetic inhibition. Please see Figure 4K and related figure legend. Similar changes have been made in the Figure 4−figure supplement 3K.

6) Please consider whether associative memory (e.g. fear memory retrieval) is impacted by manipulations of the PBN-PVT pathway.

We have now performed several experiments to examine the effects of the PBN-PVT projections on aversion formation and memory retrieval.

We first performed a prolonged conditioned place aversion that mimics drug-induced place aversion. And we found that optogenetic activation of PBN-PVT projections did not induce aversion in the postconditioning test on Day 4. These new results have been included in Figure 2−figure supplement 2H−I and described in the text. Please see Page 7 Line 196 to Line 199.

Then, we performed the classical auditory fear conditioning test and found that optogenetic inhibition of PBN-PVT projections during footshock in the conditioning period did not affect freezing levels in contextual test or cue test (Laser OFF trials). And inhibition of the PBN-PVT projection during contextual test or cue test (Laser On trials) did not affect freezing levels either. These data suggest that PBN-PVT projections are not crucial for associative fear memory formation or retrieval. These new results have been included in Figure 4−figure supplement 2 and described in the text. Please see Page 10 Line 268 to Line 274. We also discussed this on Page 15 Line 430 to Page 16 Line 473.

7) Please provide better evidence for whether increased activity of PVT neurons is mediated by input from the PBN.

We have now performed the dual Fos staining experiment and the optoeletrode experiment.

In the dual Fos staining experiment, we found that there was a broad overlap between optogenetic stimulation-activated neurons (expressing the Fos protein) and footshock-activated neurons (expressing the fos mRNA) (Figure 6−figure supplement 1B−E).

In optoelectrode experiment, there was also a broad overlap between laser-activated and footshock-activated neurons. This result was consistent with the dual Fos staining result, suggesting that PVTPBN neurons were activated by aversive stimulation. Next, we analyzed the firing rates of PVT neurons during footshock with laser sweeps and footshock without laser sweeps. We found that the footshock stimulus with laser activated 30 of 40 neurons and increased the overall firing rates of 40 neurons compared with the footshock without laser result (Figure 6I). These results indicated that activation of PBN-PVT projections could enhance PVT neuronal responses to aversive stimulation.

These new results have been included in Figure 6, Figure 6−figure supplement 1, and described in the text. Please see Page 10 Line 295 to Page 11 Line 319. We also discussed these results on Page 15 Line 422 to Line 429.

Reviewer #2 (Recommendations for the authors):

1. This manuscript has numerous grammatical issues and typos. It would benefit enormously if the text is carefully proofread. It also misuses terminologies and concepts in several cases, including the use of the word “affection” presumably to refer to “affective states”, or the use of the word “rescue” to describe the reduction of “fear-like behaviors”. These issues should be corrected.

We have revised the text to avoid using subjective terms of emotions. We used terms including anxiety-like, depression-like, fear-like behaviors, freezing, aversion-like behaviors. We replaced the terms “emotions” “affections” with “affective states”. We also replaced the terms “rescue” “relieve” with “reduce”.

2. The authors frequently refer to Chiang et al. 2020 (PMID: 32289251) paper to support their observation that PBN-PVT projections are exclusively glutamatergic. However, the same paper showed that the PBN also sends a smaller number of GABAergic fibers to PVT. While the authors showed only EPSCs in PVT neurons when optogenetically activating PBN inputs, it is not clear whether the recordings were also performed after clamping the voltage at 0mV to allow the detection of possible IPSCs after stimulation of PBN fibers. Alternatively, the authors could use immunohistochemistry to identify the cell-type specificity of PVT-projecting neurons in PBN and reconcile their findings with the current literature.

Now we have revised the overall framing of the study. We first presented the slice recording results from wild-type mice (Figure 1). We found that light-induced EPSCs in 34 of 52 neurons and light-induced IPSCs in 4 of 52 neurons. Please see Page 5 Line 119 to Line 121 in the result section. The small portion of inhibitory connections is consistent with the previous anatomical result. We also discussed this on Page 13 Line 380 to Line 383.

We also detected several molecular markers (Vglut2, Tac1, Tacr1, Pdyn, CGRP) in the PVT-projecting PBN neurons on Ai9 mice and found that about 94% of tdTomato+ neurons expressed VgluT2 mRNA. These new results have been included in Figure 1 and Figure 1−figure supplement 1. Please see Page 5 Line 129 to Line 140.

3. Similarly, Chiang et al. 2020 described the role of PBN-CeA/BNST projections in regulating aversion. Although the authors showed that PVT-projecting neurons in PBN do not project to CeA, it would be very informative for the field if the authors could also provide the data for other brain regions to which PVT-projecting neurons also send collateral, especially those regions that are important for anxiety- and fear- like behaviors. This would help the authors to exclude (or include) the possible contribution of collateral projections to the reported behaviors.

We have now provided the distribution pattern of collateral projections from PVT-projecting PBN neurons. The collateral projections were found in BNST, LH, PVN, PAG but not CeA or VMH. These new results have been included in Figure 1−figure supplement 3 and described in the text. Please see Page 6 Line 148 to Line 151.

Recently, Chiang et al. found two major efferent pathways from the lateral PBN: one originating from the dorsal division of lateral PBN collateralizes the VMH and PAG and a second arising from the external lateral division of lateral PBN that collateralize to the BNST and CeA. They suggested that activation of the first pathway generates the aversive memory and activation of the second one drives escape behaviors (Chiang et al., 2020).

In our study, the results indicate that the PBN-PVT pathway arises from both dorsal and external lateral divisions of lateral PBN and collateralizes the BNST, LH, PVN, and PAG but not the CeA or VMH. According to the location of originated PBN neurons and collateral projection pattern, we speculated that the PBN-PVT efferent pathway is different from both PBN-VMH/PAG pathway and PBN-BNST/CeA pathway. We discussed this on Page 15 Line 444 to Page16 Line 457.

4. For the PBN-PVT photoactivation experiments in Figure 2, a deeper discussion about the lack of effects on aversive memory formation (RTPA and fear conditioning) is missing. The authors should compare their results with prior studies in the literature. For example, activation of PVT-CeA projections induces place aversion and the effect persists in the next day in the absence of illumination (Do Monte et al. 2017, PMID: 28426970). Similarly, LTD-like stimulation of PVT-CeA projections or optogenetic/chemogenetic inhibition of the same circuit induces a persistent attenuation of fear responses (Chen and Bi, 2019, PMID: 30406427; Do Monte et al. 2015, PMID: 25600268; Penzo et al. 2015, PMID: 25600269)

We have now performed two new experiments: the prolonged conditioned place aversion test and the classical auditory fear conditioning test. The results suggested that PBN-PVT projections are not crucial for associative aversive memory formation. We also discussed this on Page 15 Line 430 to Page 16 Line 473.

We first performed a prolonged conditioned place aversion that mimics drug-induced place aversion. And we found that optogenetic activation of PBN-PVT projections did not induce aversion in the postconditioning test on Day 4. These new results have been included in Figure 2−figure supplement 2 and described in the text. Please see Page 7 Line 196 to Line 199.

Then, we performed the classical auditory fear conditioning test and found that optogenetic inhibition of PBN-PVT projections during footshock in the conditioning period did not affect freezing levels in contextual test or cue test (Laser OFF trials). And inhibition of PBN-PVT projections during contextual test or cue test (Laser On trials) did not affect freezing levels either. These data suggest that PBN-PVT projections are not crucial for associative fear memory formation or retrieval. These new results have been included in Figure 4−figure supplement 2 and described in the text. Please see Page 10 Line 268 to Page Line 274.

5. In Figure 4, the authors should justify why they have inhibited the soma of PVT-projecting PBN neurons instead of axonal terminals in the PVT. They should also make it very clear in their schematic drawing by adding an optical fiber on the top of the PVT, similar to what they have done in other figures.

we have now performed the optogenetic inhibition of the PBN-PVT projection using VgluT2-ires-Cre mice. These new results have been included in Figure 4 and described in the text. Please see Page 9 Line 254 to Page 10 Line 274.

6. During optogenetic manipulations, the authors used a very long period of laser illumination (5 uninterrupted minutes of pulses for photoactivation and 10 minutes of constant laser for photoinhibition). These laser durations are much longer than the traditional intervals used for optogenetic manipulations in rodents. An explanation of the rationale for using such long intervals is needed. Also, regarding the photoactivation experiment (illustrated in Supplementary Video 1), it is very clear that the animals exhibited a wide range of different behaviors along the 5-min period of stimulation. These behaviors varied from no effect to increased speed of locomotion, to jumping episodes, to freezing responses by the end of the stimulation, thereby making it hard to define the correct function of this pathway in behavioral regulation. A breakdown of the behaviors by time and a deeper discussion of the different responses would probably help the readers to better understand the results.

There are several studies using relatively short-term optogenetic activation protocols in fear conditioning experiments (Do-Monte et al., 2015; Gao et al., 2020). And there are also studies using relatively long-term optogenetic manipulation protocols in OFT, TST, sucrose preference, and pain-related experiments. For example, Zhou et al. used 30 minutes constant yellow laser (594 nm, 5-8 mW) and 5-minute pulse of blue light (473 nm, 1-3 mW, 15 ms pulses, 20 Hz) (Zhou et al., 2019). Another group used 5 min OFF/5 min ON/5 min OFF protocol to manipulate PBN neurons (Sun et al., 2020). They used 473 nm (20 Hz, 5 ms pulse width, 5 mW) for activating ChR2 and used 589 nm (1 Hz, 999-ms pulse width, 10 mW) for activating NpHR.

Compared with these long-term optogenetic studies, our optogenetic protocol for ChR2 (5 minutes) is acceptable, but the protocol for NpHR (10 minutes constant inhibition) is relatively long. The main reason was the effect of inhibiting the PBN-PVT projection was not as significant as the effect of optogenetic activation. We needed to perform relatively long-term behavior tests (10 minutes in the 2-MT CPA aversion test and 10 minutes in the footshock test) to analyze the results. We discussed this on Page 14 Line 413 to Page 15 Line 418.

We have provided a detailed analysis (one-minute time window) of optogenetic activation of PBN-PVT projections in OFT experiments. We further divided the laser ON period (5−10 minutes) into five one-minute periods and analyzed the velocity, non-moving time, center time, travel distance, and jumping behaviors (Figure 2−figure supplement 2C−G). We found that the velocity and non-moving time were increased, and the center time was decreased in the ChR2 mice during most periods (Figure 2−figure supplement 2C−E). Furthermore, we observed that the distance and jumping behaviors were increased mainly in the first one-minute period in ChR2 mice (Figure 2−figure supplement 2F−G). This detailed analysis indicated that optogenetic activation induced brief and robust running, jumping behaviors, and persistent anxiety-like behaviors, such as less time spent in the center. These new results have been included in Figure 2−figure supplement 2 and described in the text. Please see Page 7 Line 179 to Line 189. We also discussed this on Page 14 Line 396 to Line 403.

7. The authors should provide a Cre-negative control for Figure 5G-N, given that tdTomato Ai14 mice can have baseline levels of tdTomato expression even in the absence of Cre. This is highly important because the transneuronal transduction of AAV1-Cre is generally limited and has never been tested in PVT.

We agree with the reviewer. We used the Ai9 allele (Stock No. 007909). “Both Ai14 and Ai9 may exhibit low levels of tdTomato expression prior to exposure to Cre recombinase – but the tdTomato expression levels after Cre recombination are greater than those baseline levels. As such, it is recommended that researchers include Cre-negative controls to establish the baseline tdTomato levels in their experiments” (from Jax website).

We perfused one Ai9 mouse and carefully checked the baseline tdTomato expression. We found that there were no obvious tdTomato+ neurons in the PVT. Please see Figure 1B. We speculated that the baseline level of tdTomato expression was relatively poor, and we could not detect the tdTomato fluorescence signal in the absence of Cre.

Reviewer #3 (Recommendations for the authors):

1. It has been shown that PBN-VMH/PAG drive escape behaviors, whereas PBN-BNST/CEA generate aversive memory. The authors need to examine the relationship between PVT-projection and VMH/BNST-projections.

We have provided the distribution pattern of collateral projections from PVT-projecting PBN neurons. The collateral projections were found in BNST, LH, PVN, PAG but not CeA or VMH. These new results have been included in Figure 1−figure supplement 3 and described in the text. Please see Page 6 Line 148 to Line 151.

Recently, Chiang et al. found two major efferent pathways from the lateral PBN: one originating from the dorsal division of lateral PBN collateralizes the VMH and PAG and a second arising from the external lateral division of lateral PBN that collateralize to the BNST and CeA. They suggested that activation of the first pathway generates the aversive memory, and activation of the second one drives escape behaviors (Chiang et al., 2020).

In our study, the results indicate that the PBN-PVT pathway arises from both dorsal and external lateral divisions of lateral PBN and collateralizes the BNST, LH, PVN, and PAG but not the CeA or VMH. According to the location of originated PBN neurons and collateral projection pattern, we speculated that the PBN-PVT efferent pathway is different from both PBN-VMH/PAG pathway and PBN-BNST/CeA pathway. We discussed this on Page 15 Line 444 to Page 16 Line 457.

2. The authors need to examine the cellular identity of PVT-projecting PBN neurons, whether it originates from known PBN neuronal subtypes such as CGRP, Tac1, Pdyn, Nts et al.

To examine the molecular identity of PVT-projecting PBN neurons, we have now performed the RNAscope experiments detecting VgluT2, Tac1, Tacr1, Pdyn mRNA, and fluorescent immunostaining detecting CGRP protein in the PBN. We found that about 94.4% of tdTomato+ neurons expressed VgluT2 mRNA. We also found that tdTomato+ neurons were only partially co-labeled with Tacr1 mRNA (38.0%), Tac1 mRNA (6.4%), or Pdyn mRNA (23.0%), but not with CGRP.

These results indicate that the majority of PVT-projecting PBN neurons are glutamatergic neurons and these neurons do not specifically originate from subpopulations labeled by Tacr1, Tac1, Pdyn, or CGRP. These new results have been included in Figure 1, Figure 1−figure supplement 1, and were described in the text. Please see Page 5 Line 129 to Line 140.

3. As authors mentioned in discussion part, a recent study revealed that the PBN neurons project to ILN, which is relatively closed to the PVT. The authors need to carefully examine their manipulation experiments especially optogenetic stimulation. They need to make sure that the effect they observed was not due to the activation of PBN-ILN pathway. Since activation of PBN-ILN pathway induced pain-related behavior which could be very similar to negative emotions.

We have now provided the virus expression and optic fiber tips locations. Please see Figure 2−figure supplement 1 (ChR2 experiments) and Figure 4−figure supplement 1 (NpHR3.0 experiments). The virus was expressed in the PBN, and the optic fibers were above the PVT. These results confirmed that we manipulated the PBN-PVT pathway without affecting the PBN-ILN pathway.

We also performed pharmacogenetic activation of PVT-projecting PBN neurons and found that basal nociceptive thresholds or the formalin-induced pain behaviors were not affected (Figure 3−figure supplement 2). We speculated that PBN-PVT pathway might not be involved in nociceptive information processing. We also discussed this on Page 17 Line 494 to Line 502.

4. For the cue-dependent optogenetics conditioning test: activation of the PBN-PVT projection induces instant aversion and freezing but not drives associative fear learning. -- Could it be simply because the conditioning (6 times 30 seconds light stimulation) was too weak? The authors should try a prolong conditioning protocol that resemble drug CPP such as 30 min light stimulation at paired side.

We agree with the reviewer. We have now performed a prolonged conditioned place aversion that mimics drug-induced condition place aversion (Figure 2−figure supplement 2H−I), and we found that optogenetic activation of the PBN-PVT projection did not induce aversion in the postconditioning test on Day 4.

These new results have been included in Figure 2−figure supplement 2H−I, and were described in the text. Please see Page 7 Line 196 to Line 199. We speculated that PBN-PVT projections might not be crucial for associative fear learning. We also discussed this on Page 15 Line 430 to Page 16 Line 473.

5. Optogenetics and Pharmacogenetics are nouns while optogenetic and pharmacogenetic are adjectives. There are many places in the manuscript where the noun was misused.

We have revised the text.

References

Do-Monte FH, Quinones-Laracuente K, Quirk GJ. (2015). A temporal shift in the circuits mediating retrieval of fear memory. Nature, 519:460-463. DOI: https://doi.org/10.1038/nature14030, PMID:25600268

Gao C, Leng Y, Ma J, Rooke V, Rodriguez-Gonzalez S, Ramakrishnan C, Deisseroth K, Penzo MA. (2020). Two genetically, anatomically and functionally distinct cell types segregate across anteroposterior axis of paraventricular thalamus. Nat Neurosci, 23:217-228. DOI: 10.1038/s41593-019-0572-3. PMID: 31932767

Sun L, Liu R, Guo F, Wen MQ, Ma XL, Li KY, Sun H, Xu CL, Li YY, Wu MY, Zhu ZG, Li XJ, Yu YQ, Chen Z, Li XY, Duan SM. (2020). Parabrachial nucleus circuit governs neuropathic pain-like behavior. Nature Communications, 11. DOI: https://doi.org/10.1038/s41467-020-19767-w, PMID:33239627

Zhou WJ, Jin Y, Meng Q, Zhu X, Bai TJ, Tian YH, Mao Y, Wang LK, Xie W, Zhong N, Luo M, H., Tao WJ, Wang HT, Li J, Qiu BS, Zhou JN, Li XY, Xu H, Wang K, Zhang XC, et al. (2019). A neural circuit for comorbid depressive symptoms in chronic pain. Nature Neuroscience, 22:1649-1658. DOI: https://doi.org/10.1038/s41593-019-0468-2, PMID:31451801

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #1 (Recommendations for the authors):

In their revised manuscript, the authors have adequately addressed the previous concerns and recommendations raised by this reviewer. Importantly, doing so has improved the overall quality of the manuscript.

The manuscript could benefit from additional copy editing to avoid statements such as "PVT neurons innervated by PBN innervation" (Line 39).

We have revised the text. Please see Page 2 Line 40 to 41.

https://doi.org/10.7554/eLife.68372.sa2

Article and author information

Author details

  1. Ya-Bing Zhu

    Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Software, Writing - original draft
    Contributed equally with
    Yan Wang and Xiao-Xiao Hua
    Competing interests
    No competing interests declared
  2. Yan Wang

    Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    Contribution
    Formal analysis, Investigation, Validation
    Contributed equally with
    Ya-Bing Zhu and Xiao-Xiao Hua
    Competing interests
    No competing interests declared
  3. Xiao-Xiao Hua

    The First Rehabilitation Hospital of Shanghai, Tongji University School of Medicine, Shanghai, China
    Contribution
    Investigation
    Contributed equally with
    Ya-Bing Zhu and Yan Wang
    Competing interests
    No competing interests declared
  4. Ling Xu

    Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Ming-Zhe Liu

    Department of Respiratory, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  6. Rui Zhang

    Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    Contribution
    Formal analysis, Investigation, Validation
    Competing interests
    No competing interests declared
  7. Peng-Fei Liu

    Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    Contribution
    Investigation, Validation
    Competing interests
    No competing interests declared
  8. Jin-Bao Li

    Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  9. Ling Zhang

    The First Rehabilitation Hospital of Shanghai, Tongji University School of Medicine, Shanghai, China
    Contribution
    Funding acquisition, Project administration
    For correspondence
    lzhang0808@tongji.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8308-6553
  10. Di Mu

    1. SUSTech Center for Pain Medicine, School of Medicine, Southern University of Science and Technology, Shenzhen, China
    2. Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    Contribution
    Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - original draft, Writing - review and editing
    For correspondence
    damonmu@163.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1209-9311

Funding

National Natural Science Foundation of China (31900717)

  • Di Mu

China Association for Science and Technology (2019QNRC001)

  • Di Mu

Shanghai Association for Science and Technology (19YF1438700)

  • Di Mu

National Natural Science Foundation of China (31571086)

  • Ling Zhang

Shanghai Natural Science Foundation (21ZR1468600)

  • Ling Zhang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr. Yan-Gang Sun for comments on the manuscript and for providing Vglut2-ires-Cre mice. We thank Dr. Hua-Tai Xu for providing Rosa26-tdTomato mice. We thank all the lab members of DM for their helpful discussion. This work was supported by the National Natural Science Foundation of China (No. 31900717, 31571086), the Shanghai Sailing Program (19YF1438700 to DM), and the Young Elite Scientists Sponsorship Program of China Association for Science and Technology (2019QNRC001 to DM), the Shanghai Natural Science Foundation (21ZR1468600 to LZ).

Ethics

All animal experiment procedures were approved by the Animal Care and Use Committee of Shanghai General Hospital (2019AW008).

Senior Editor

  1. Andrew J King, University of Oxford, United Kingdom

Reviewing Editor

  1. Mario Penzo, National Institute of Mental Health, United States

Reviewers

  1. Mario Penzo, National Institute of Mental Health, United States
  2. Fabricio H Do Monte, The University of Texas Health Science Center at Houston, United States

Publication history

  1. Preprint posted: March 12, 2021 (view preprint)
  2. Received: March 13, 2021
  3. Accepted: January 30, 2022
  4. Accepted Manuscript published: February 15, 2022 (version 1)
  5. Version of Record published: March 17, 2022 (version 2)

Copyright

© 2022, Zhu et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Ya-Bing Zhu
  2. Yan Wang
  3. Xiao-Xiao Hua
  4. Ling Xu
  5. Ming-Zhe Liu
  6. Rui Zhang
  7. Peng-Fei Liu
  8. Jin-Bao Li
  9. Ling Zhang
  10. Di Mu
(2022)
PBN-PVT projections modulate negative affective states in mice
eLife 11:e68372.
https://doi.org/10.7554/eLife.68372

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