Introduction

During threatening situations, the inhibition of pain is an essential adaptive response that can be crucial to promote self-preservation^1–4. The inhibition of pain responses to a stimulus that would typically elicit pain is defined as analgesia⁵. In addition, previous studies have described that a fearful event can reduce pain sensitivity, a phenomenon referred to as fear-conditioned analgesia (FCA)⁶. In particular, pharmacological inactivation of the basolateral amygdala abolishes this fear-modulated analgesia and is associated with increased immediate-early gene c-Fos expression in the central amygdala and periaqueductal gray matter (PAG)⁷. The PAG is a midbrain structure that receives many cortical and subcortical inputs from brain structures involved in fear processing^8–11, projecting to the spinal cord primarily through a brainstem relay in the rostral ventromedial medulla (RVM)¹², a key structure involved in nociceptive transmission^13,14. Indeed, the ventrolateral part of the PAG (vlPAG) is crucial for the descending control of pain^13,15 in the dorsal horn of the spinal cord (DH)¹², and several reports indicate that electrical stimulation of the vlPAG selectively inhibits responses to noxious stimuli in a variety of pain test conditions¹². Moreover, analgesia induced upon vlPAG stimulation is opioid-dependent^16–18 and involves direct descending projections to the RVM and the DH^19–22. Furthermore, previous research indicates that activation of the vlPAG induces analgesia through the recruitment of local GABAergic cells²³, which is consistent with our understanding of the role of the GABAergic system in the descending control of pain^24,25. Thus, the vlPAG is ideally positioned to mediate the emotional regulation of pain behavior, yet the underlying neuronal circuits and mechanisms are largely unknown. Here, we investigated the precise neural circuits within the vlPAG that mediate the suppression of pain behavior during threatening situations. We developed a unique behavioral paradigm where a cued-elicited defensive state promotes changes in pain sensitivity. Combining behavioral, anatomical, optogenetic, and electrophysiological approaches, we identified a novel midbrain to brainstem circuit mechanism composed of somatostatin positive (SST⁺) vlPAG cells projecting to the RVM that is critically involved in the suppression of pain behavior during conditioned fear states.

Results

Cued-fear conditioning elicits analgesia

To evaluate the contribution of specific vlPAG cell populations to the fear modulation of pain behavior, we developed a novel cued fear-conditioned analgesia (CFCA) paradigm, consisting of a pain sensitivity assay during the presentation of threat-predicting cues (Figure 1a). In this paradigm, an initially neutral stimulus (the conditioned stimulus, CS⁺) becomes associated with a footshock (the unconditioned stimulus, US), whereas another neutral stimulus serves as a control (CS^-). After conditioning, re-exposure to the CS⁺ elicited freezing behavior, a behavioral readout of associative learned fear²⁶ (Figure 1b and Supplementary Figure 1a). Following the fear retrieval session, male mice were submitted to the pain sensitivity assay. Mice were placed on a Hot-Plate (HP) apparatus in which the floor plate temperature progressively increases while either CS^- or CS⁺ were concomitantly presented. Plate temperature increased, and CSs were continuously played until mice displayed a nociceptive (NC) response reflecting pain behavior (Figure 1c), defined by either jumping or hindpaw licking (Figure 1d). The detection of a NC response terminated the test session (see Methods).

Figure 1.

We observed a significant delay in the NC response performance for CS⁺ compared to CS^-presentations, reflecting that pain-suppression was selectively promoted by the threat-predicting CS (Figure 1e and Supplementary Figure 1b). This increased NC latency during CS⁺ presentation could alternatively be explained by a competition between freezing and pain-related responses. However, freezing probability at the time of the NC response for CS⁺ was remarkably low and comparable to CS^- levels, suggesting that CS⁺ increased NC response latency was not due to competing freezing responses (Figure 1f, see Methods). Instead, these results suggest that fear induced by CS⁺ presentations drives the increase in NC latency. Importantly, such states induced by our CFCA paradigm reflected fear associative processes (Supplementary Figure 2). To demonstrate a causal link between cued-fear and analgesia, mice were submitted to an extinction procedure to devalue the CS⁺-US contingency and abolish the negative emotional state during CS⁺ presentations (Figure 1g). Under these conditions, CS⁺ presentation after extinction had no effects on NC response latency (Figure 1h). Furthermore, the absence of auditory cues, or the presence of auditory cues not associated with a threat, led to NC response latencies comparable to those observed during CS^- presentations (Figure 1i). Finally, previous studies indicate that fear can lead to vasoconstriction, which could result in a redirection of the blood flow to the skeletal musculature, decreasing the temperature in the extremities of the body²⁷. However, monitoring the back and tail temperatures of mice submitted to the HP test did not reveal any differences between the CSs presentations, suggesting that the increased latency was not due to fear-induced vasoconstriction (Supplementary Figure 1c-e). Overall, our results indicate that the presence of a conditioned threat-predicting cue can selectively promote analgesia.

Photostimulation of SST⁺ vlPAG cells alters pain and fear behavior

To identify the cell types in the vlPAG mediating the analgesic effect in the CFCA task, we focused on GABAergic interneurons, whose activity is suggested to be inhibited during analgesia through µ-opioid receptor-dependent mechanisms^12,25. Using single-molecule fluorescent in situ hybridization (smFISH, see Methods), we confirmed that the majority of vlPAG SST mRNAs were expressed in inhibitory neurons (Supplementary Figure 3). As such, since somatostatin-expressing interneurons (SST) are the most abundant interneuron class in the vlPAG²⁸, we focused on this cell population. Thus, to evaluate the contribution of SST⁺ vlPAG cells in CFCA, SST-Cre mice were injected in the vlPAG with a Cre-dependent AAV expressing the Channelrhodopsin (ChR2), Archaerhodopsin (ArchT), or GFP while optic fibers were bilaterally implanted targeting this area (Figure 2a, Supplementary Figure 11). Next, mice were submitted to the CFCA task while photostimulating SST⁺ cells in the vlPAG (Figure 2b; see Methods). After fear retrieval (Figure 2c, e; Supplementary Figure 4a-d), the optogenetic activation of SST⁺ vlPAG cells during the HP test had no effect on CS^- presentation, whereas the same manipulation performed during CS⁺ blocked the increase in NC response latency compared to GFP controls (Figure 2d and Supplementary Figure 5a). In contrast, optogenetic inhibition of SST⁺ vlPAG cells increased NC response latency for both CS^- and CS⁺ conditions compared to GFP controls (Figure 2f and Supplementary Figure 5b). Importantly, these changes in NC response latencies were not due to motor or aversive effects of the optogenetic manipulations, since photoactivation of SST⁺ vlPAG cells did not induce changes in distance traveled in an open field nor place aversion in a real-time place preference test (Supplementary Figure 5c-f). In addition, we did not observe changes in fear learning or expression when the footshock was replaced or paired with photoactivation of the SST⁺ vlPAG cells during the fear conditioning procedure (Supplementary Figure 6).

Figure 2.

Altogether, these experiments confirmed that SST⁺ vlPAG activation did not promote pain responses and instead suggested that the SST⁺ vlPAG cells are key elements of an endogenous pain-suppression circuit. Additionally, the optogenetic effect observed during CFCA was not due to alteration of somatostatin levels in our homozygous SST-Cre mice²⁹, as we observed the same effect in heterozygous SST-Cre mice (Supplementary Figure 7a-d). Finally, in contrast with STT⁺ vlPAG cells, the optogenetic inhibition of another important class of vlPAG inhibitory cells expressing the vasoactive intestinal peptide (VIP⁺) did not change the NC response latencies during CS^- or CS⁺ presentations (Supplementary Figure 7e-h, Supplementary Figure 10).

Because our CFCA paradigm is dependent on fear associative processes (Figure 1 and Supplementary Figure 1-2), it is possible that the optogenetic manipulation of SST⁺ vlPAG cells may have altered the expression of fear behavior. To control for this possibility, we optogenetically activated or inhibited SST⁺ vlPAG cells during a fear retrieval session following auditory fear conditioning (Figure 3a, b). Our data indicate that whereas optogenetic inhibition of SST⁺ vlPAG cells had no effect on freezing behavior relative to GFP controls, their optogenetic activation reduced freezing levels (Figure 3c, d). Altogether, our data show that the local SST⁺ vlPAG network can modulate both fear expression and the analgesia promoted during defensive states.

Figure 3.

Photostimulation of SST⁺ vlPAG cells regulates spinal cord nociceptive transmission

Consistent with our observations, previous studies found that the vlPAG is involved in fear-conditioned analgesia⁷. It is conceivable that the SST⁺ vlPAG cells are indeed part of a circuit involved in the fear regulation of endogenous pain-suppression. To evaluate the effect of the manipulation of SST⁺ vlPAG cells on analgesia independently of the expression of fear behavior, we evaluated the impact of SST⁺ vlPAG cells manipulation on the control of nociceptive transmission directly at the level of the spinal cord DH. Therefore, we performed extracellular local field potential and single-unit recordings in the spinal cord DH during optogenetic stimulation of SST⁺ vlPAG cells in anesthetized mice (Figure 4a). Nociceptive stimulation induces early and late neuronal responses at the level of the spinal cord DH³⁰ (A- comprising non-nociceptive Aβ and nociceptive A8 and C- fiber mediated, respectively; Figure 4a). While optogenetic activation of SST⁺ vlPAG cells during suprathreshold electrical stimulation of the paw increased the magnitude of late nociceptive field potentials, the optogenetic inhibition reduced it (Figure 4b, c). To determine whether this effect was specific to the nociceptive network, we recorded wide dynamic range (WDR) cells in the DH, which receive both tactile (Aβ) and nociceptive (Aδ and C) information³⁰. We observed that photoactivation of SST⁺ vlPAG cells increases both fast nociceptive (A8-fiber) and slow nociceptive (C-fiber) responses without affecting non-nociceptive (Aβ-fiber) responses (Figure 4d). Moreover, optogenetic inhibition of SST⁺ vlPAG cells specifically inhibited nociceptive responses, whereas non-nociceptive responses (Aβ-fiber) remained unaffected (Figure 4e). In addition, we performed subliminal electrical stimulations that did not elicit nociceptive neuronal responses. In this condition, we observed that removing analgesia by activating SST⁺ vlPAG cells elicited WDR responses at latencies corresponding to nociceptive A8- and C-fibers (Figure 4f). Finally, the optogenetic activation of SST⁺ vlPAG cells had no effect on purely tactile fast latency spinal neurons (Supplementary Figure 8). Consistent with our optogenetic manipulations during the CFCA task (Figure 2), these data suggest that, under analgesia promoted by isoflurane, activation and inhibition of SST⁺ vlPAG cells increased and decreased nociceptive transmission, respectively, by specifically promoting pronociceptive and antinociceptive responses in the spinal cord DH.

Figure 4.

SST⁺ vlPAG-RVM-spinal cord pathway activation abolishes analgesia induced during defensive states

We next evaluated whether SST⁺ vlPAG cells mediate their analgesia modulation by contacting WDR cells directly in the spinal cord or through the rostro ventromedial medulla (RVM), a structure known to receive direct projections from vlPAG and mediate pain modulation^19,20 (Figure 5a). Anatomical tracing revealed large labeling of SST⁺ fibers in RVM (Figure 5b) and only sparse labeling within the spinal cord (SC; data not shown). By combining fluorogold retrolabeling from the SC and GFP anterograde labeling from SST⁺ vlPAG cells (Figure 5c), we observed RVM cells projecting to the DH with close apposition of SST⁺ putative boutons (Figure 5d), suggesting that SST⁺ vlPAG cells project to the RVM and contact DH-projecting RVM cells. Optogenetic activation of SST descending fibers above the SC has no effect on WDR neural activity upon paw electrical stimulation, suggesting that SST⁺ vlPAG long-range projections to SC did not play a major role in endogenous pain-suppression (Figure 5e). In contrast, photoactivation of SST⁺ vlPAG inputs to the RVM increased both nociceptive (A8 and C-fiber) responses (Figure 5f, Supplementary Figure 9). Altogether, these data revealed that the SST⁺ vlPAG-RVM-DH pathway mediates endogenous pain-suppression.

Figure 5.

We next addressed whether the SST⁺ vlPAG-RVM-DH pathway was specific to mediating endogenous pain-suppression induced by defensive states. Thus, SST-Cre mice were injected in the vlPAG with a Cre-dependent AAV expressing the ChR2 or GFP, and a single optic fiber was implanted above the RVM (Figure 6a). While photoactivation of the SST⁺ vlPAG-RVM pathway promoted nociception (Figure 5f), it did not impact fear expression as freezing levels remained unchanged (Figure 6b). Consistently, activation of the SST⁺ vlPAG-RVM pathway during CFCA had no impact on CS^- presentation, whereas the same manipulation performed during CS⁺ blocked the increase in NC response latency compared to GFP controls (Figure 6c, d). Overall, these data suggest the existence of two populations of SST⁺ vlPAG cells mediating fear expression and analgesia induced by defensive states. (Figure 6e). Altogether, our results indicate that the SST⁺ vlPAG-RVM-DH pathway is critically involved in the analgesia induced during defensive states.

Figure 6.

Discussion

In this study, we established that SST⁺ vlPAG cells control analgesia during defensive states in male mice. Endogenous pain-suppression was mediated by long-range SST⁺ vlPAG cells projecting to the RVM, which, in turn, transmitted the pain signal to the DH of the spinal cord (Figures 2 and 5). Finally, our data indicated that SST⁺ vlPAG cells can regulate both fear expression and the analgesia promoted during defensive states, and that the expression of these two behaviors can be segregated by precisely targeting the SST⁺ vlPAG-RVM pathway (Figure 6). Together, these results identify a novel midbrain to brainstem circuit mechanism composed of long-range SST⁺ vlPAG cells projecting to the RVM that regulate analgesia elicited during defensive states. Pain mechanisms and their modulation are known to exhibit sex-dependent differences³¹, and thus we cannot exclude the possibility that the circuits described here may differ in females. Nevertheless, recent studies of RVM-spinal cord descending control suggest broadly similar organization and function across sexes^32,33. Direct experimental investigation in female animals will be required to determine the extent to which the present findings generalize across sexes.

Our observations of the involvement of vlPAG in our cued-based fear conditioned analgesia paradigm are consistent with previous studies of contextual-based FCA involving this region⁷. In such FCA studies⁶, animals undergo contextual fear conditioning followed by pain behavior testing in the same conditioning context, thereby limiting the temporally precise elicitation of aversive states. Instead, our behavioral paradigm goes beyond these limitations by promoting defensive states through a cued conditioned stimulus, advancing toward temporally specific manipulations such as optogenetics, and allowing us to study the phenomena with a within-subject experimental design through subsequent CS^+/- presentations. Nevertheless, since the circuits involved in contextual and cued fear may differ^30,34, this might also imply different circuits involved in the emotional modulation of pain-suppression behavior. To our knowledge, this is the first demonstration of a cued fear-conditioned analgesia model in rodents. Addressing potential differences in the circuitry involved in contextual and cued fear-induced analgesia will require further investigation.

Our observation that photoactivation of SST⁺ vlPAG cells not only abolished analgesia during defensive states but also decreased freezing expression has far-reaching functional consequences. Two independent previous studies found that long-range inhibitory inputs from the central medial amygdala contact inhibitory cells within the vlPAG, implicated in different roles: the modulation of fear behavior²³ and nociceptive transmission³⁵. Notably, both studies describe the involvement of vlPAG inhibitory cells in a disinhibitory circuit mechanism of long-range projecting excitatory cells as the underlying neural substrates responsible for such modulations^23,35. We observed that SST⁺ cells are the most abundant inhibitory cell type in the vlPAG (Supplementary Figure 3). Thus, it is conceivable that the SST⁺ vlPAG cells are indeed part of a circuit involved in the emotional regulation of endogenous pain-suppression. Notably, global manipulation of SST⁺ vlPAG cells cannot disentangle their role in analgesia and fear behavior at the local level (Figures 2 and 3). Because the studies mentioned above exclusively addressed the contribution of vlPAG circuits to either pain³⁵ or fear²³ behavior, these studies may have been oblivious to alternative behavioral readout interpretations. Here, we demonstrated that SST⁺ vlPAG cells projecting to the RVM modulate analgesia induced during defensive states without impacting the behavioral expression of fear (Figure 6). Thus, our results suggest that fear and analgesia signals rely on overlapping neuronal circuits and that the discrimination between these two states could be mediated within the vlPAG network. Altogether, our data are consistent with the existence of different populations of SST⁺ cells within the vlPAG that mediate fear behavior and analgesic information, defined by their output connectivity.

The lateral inhibition model of analgesia within the PAG relies on the activation of PAG excitatory cells projecting to the RVM. More precisely, in this model, analgesia is thought to occur through an opioid-dependent inhibition of GABAergic cells, ultimately disinhibiting excitatory cells projecting to the RVM^12,20,21. Our data indicate that, in addition to this disinhibitory mechanism, analgesia is also mediated by the inhibition of a direct long-range SST⁺ neurons projection onto RVM cells projecting to the DH (Figure 6). In line with this, inhibition of vlPAG SST cells induced analgesia in our experiments, consistent with previous findings obtained using chemogenetic approaches³⁶. However, important differences emerge when comparing our results with Zhang et al.³⁴. In that study, activation of vlPAG SST cells produced hyperalgesia, whereas in our study, activation of SST⁺ vlPAG cells did not induce hyperalgesia but instead suppressed fear-conditioned analgesia. One key distinction between the two studies is the experimental context: Zhang et al. examined this circuit in a neuropathic pain model, whereas our work focuses on acute nociceptive processing and fear-dependent modulation of pain. Thus, the functional contribution of SST⁺ vlPAG cells may depend on the behavioral and pathological state, with potentially distinct roles in chronic versus acute pain conditions³⁵. In addition, methodological differences, such as our nociceptive test that uses a slow temperature ramp, may impose a floor effect that limits the detection of thermal hyperalgesia. Our results are therefore consistent with the parallel inhibition-excitation model, in which inhibitory and excitatory cells form two distinct, parallel descending pathways for pain modulation²⁴. Indeed, previous studies have identified inhibitory projections within the PAG–RVM-spinal cord dorsal horn neuraxis³⁷, and our results suggest that SST⁺ vlPAG cells contribute to analgesia within this broader framework. At the same time, our data indicate that vlPAG SST⁺ cells are not exclusively inhibitory, as approximately one third co-localize with excitatory markers, and recent work has shown that excitatory SST⁺ vlPAG cells project to the RVM³⁶. Together, these observations raise the possibility that distinct SST⁺ subpopulations contribute differently to descending pain control, with long-range vlPAG SST⁺ cells participating in the excitatory pathway projecting throughout the PAG–RVM-spinal cord dorsal horn neuraxis, and local GABAergic vlPAG SST⁺ cells contributing to local circuit dynamics related to fear response, such as freezing²³. We identified a novel midbrain to brainstem circuit mechanism composed of SST⁺ vlPAG cells that project to the RVM, thereby regulating analgesia during defensive states. Substantial progress has been made in outlining the midbrain circuitry of the endogenous pain-suppression pathway^12,24,26. However, only more recently have high cortical areas been shown to be involved in analgesia^7,38. Previous reports describe connections between CeL and vlPAG inhibitory neurons in the modulation of pain behavior^35,39. Future work is required to elucidate whether the exact circuit that regulates analgesia is mediated by different negative emotional states, such as fear, depression, or stress.

Recent human reports reveal strong comorbidity between mood and anxiety disorders with and pain-related disorders⁴⁰, suggesting that the deregulation of the circuits mediating the interplay between emotional and pain behavior could be at the core of such psychiatric conditions. Comprehensive knowledge about the circuits mediating the emotional regulation of pain behavior is urgently needed. We identified an SST⁺ cell type-and pathway-specific involvement in the mechanisms that mediate the emotion-dependent regulation of pain behavior. Thus, these findings may empower the development of more effective analgesic therapies by the improvement of further refined pharmacological targets.

Methods

Subject details

We used either male C57BL6/J mice (Janvier), heterozygous or homozygous Sst-IRES-Cre mice (Jackson laboratory), or heterozygous VIP-IRES-Cre mice (Jackson Laboratory) age 8-14 weeks, that were individually housed under a 12 h light-dark cycle and provided with food and water ad libitum. All procedures were performed in accordance with standard ethical guidelines (European Communities Directive 86/60-EEC) and were approved by the committee on Animal Health and Care of Institut National de la Santé et de la Recherche Médicale and the French Ministry of Agriculture and Forestry (agreement #A3312001).

Behavioral apparatus

Cued fear-conditioned analgesia task was performed in three different contexts (Figure 1a). Context A was used for Habituation and Retrieval and consisted of a plexiglass cylinder (25 x 24 cm diameter) with a grey, smooth plastic floor and house lights (16 lux). Context B was used for Fear conditioning and consisted of a square plexiglass (25 x 40 cm) with a grid floor connected to a shocker (Coulbourn Instruments) and brighter house lights (40 lux). A total of 5 scrambled footshocks of 1 s duration and intensity of 0.8 mA were delivered via the grid floor and served as the unconditioned stimulus (US). Contexts A and B were cleaned, respectively, with 70% ethanol or 1 % acetic acid between different mice. Both contexts contained infrared beam detection that automatically scored freezing periods, determined by no change in infrared beam crossing for at least 2 s.

Context C was used for the FC Hot Plate (HP) test. A steady increase in temperature was controlled by the Incremental Hot/Cold Plate Analgesia Meter (IITC) device. To restrain the mice’s movement, the HP apparatus had a testing surface (plate) enclosed in a square plexiglass surface (20.3 x 10 x 20.5 cm) and house lights (100 lux). The mice’s temperature and surroundings were recorded using an infrared digital thermographic camera (Testo 885) placed ∼ 50 cm above the testing surface. The thermal camera had a spatial resolution of 320 × 240 pixels, a sampling rate of 25 Hz, and thermal sensitivity of 0.03 °C at 30 °C. The testing surface of context C was cleaned with water between different mice. All three contexts were enclosed in an acoustic foam isolated box with speakers mounted on the top of each compartment. The auditory conditioned stimulus (CS) consists of either 7.5 kHz or white-noise 50 ms pips at 1 Hz, repeated 27 times, with 2 ms rise and fall, and a sound pressure level of 80 dB.

Open field task was performed in a square plexiglass arena (36 x 36 x 25cm). A LED mounted on the top-right side of the arena signaled the start and end of different epochs for offline analyses. A video camera recorded from above the arena at 30 fps for offline video-tracking purposes.

Real-Time Place Preference (RTPP) task was performed in a shuttlebox consisting of a plexiglass box (40 x 10 x 30 cm) with a floor grid, where a small plastic hurdle (1 cm height) divided the arena into two equal compartments, while infrared beam detection automatically monitored the mice shuttling between compartments (Imetronic). A video camera recorded from above the arena at 30 fps for offline video-tracking purposes.

For both the open field and RTPP tasks, a free user video-tracking software (idTracker: Tracking individuals in a group by automatic identification of unmarked animals) together with in-house codes in Matlab (The MathWorks, Inc., Natick, MA, USA) was used to analyze each condition.

Behavioral paradigm

Cued fear-conditioned analgesia (CFCA). C57BL6/J mice (n = 12) were habituated to the context and tones (day 1). Four white-noise (CS^-) and four 7.5 kHz (CS⁺) tones were presented sequentially and without US reinforcement. In Fear conditioning (day 2), five CS^- and five CS⁺ were presented in an intermingled fashion. The CS⁺ presentations were paired with a mild footshock (US) at tone offset, whereas the CS^- was never reinforced. The Fear retrieval session (Day 3) was done 24h after conditioning and in the same context as the habituation session. As in habituation, four CS^- and four CS⁺ were presented sequentially and without US reinforcement (Figure 1a).

Since the focus of this study was the emotional modulation of pain sensitivity, it was compulsory to evaluate the associative fear levels before measuring their impact on pain sensitivity. Two indices were computed: the discrimination index (DI), to assess the discrimination between CS^-and CS⁺, and the conditioning index (CI), which indicated the level of freezing to the tone predicting the US. These indexes were calculated as follows: and CI = DI × (Freezing to CS⁺). Based on preliminary data, mice were only submitted to the FC HP test if DI ≥ 0.4 & CI ≥ 0.3. Mice that did not fit the criteria were conditioned a second time. There was a minimum interval of 2 h between the retrieval and the FC HP test.

The FC HP test consisted of two trials, one in which the CS⁺ was presented and another in which the CS^- was presented. The two trials were counterbalanced within the group. The testing surface was set at 30 °C. After a 60 s acclimatization period, its temperature gradually increased at 6 °C per minute (HP start). Tone presentation (CS⁺ or CS^-) started 130 s after the HP start. Temperature increase and tone presentation terminated concomitantly with the display of a nociception response. Valid nociception readout responses included jumping or licking the hindpaw. The effect of the negative emotional modulation on pain sensitivity was assessed by comparing the latency (or temperature) of the NC response between the two HP trials. There was a minimum of 30 min interval between the two trials for each mouse, during which mice returned to their home cage.

Freezing during the HP test was assessed by automatically analyzing thermographic camera videos using custom scripts (available at: https://github.com/djercog/TestoFreez). Briefly, individual video frames were binarized to define a mask for the mouse within each frame. Consecutive binarized frames were then subtracted, and the number of non-zero pixels in the resultant image determined if the mouse was considered immobile when below a certain threshold (defined to approximate manual freezing estimation on random videos). Immobilization episodes of at least 0.5 s were considered as freezing periods. In contrast, non-immobilization episodes shorter than 0.1 s were merged together with the neighbouring detected freezing episodes.

Extinction training. On days 3 and 4, mice (n =10) were submitted to an extinction training protocol established by Courtin et al. 2014⁴¹. Briefly, there were 4 CS^- and 12 CS⁺, presented in a non-reinforced manner (Figure 1g). Mice were considered to have successfully extinguished the fear expression if the level of freezing in the last 4 CS⁺ was not statistically different from the CS^-. After fear extinction, mice were submitted to the FC HP test, using the same parameters as in the CFCA paradigm.

Stability training. The first three days consisted of the classical CFCA paradigm. On day 4, mice (n = 10) repeated the protocol applied on day 3 (Supplementary Figure 2e-i). All mice that met the fear conditioning criteria (DI ≥ 0.4 & CI ≥ 0.3) on day 3 were kept for the following day, independent of their level of Conditioning on day 4.

Vasoconstriction assay. To determine whether vasoconstriction was a confounding factor in the FC HP test outcomes, we used a modified version of the CFCA paradigm (Figure 1f). During the HP test (n = 13), the temperature was not gradually increased but maintained at 30 °C while CSs were presented. The tones (CS⁺ or CS^-) were presented for 120 s, and mice were kept for an additional 50 s before the HP trial terminated. Offline, with the infrared videos, mice’s back and tail were measured at 30 s intervals. For each measurement point, the temperatures of three spots on the back and tail were averaged to obtain the body temperature for each body part at that time point and mouse.

Basal nociception assay (No tone). Naïve mice (n = 10) were submitted to the HP test (Figure 1i and Supplementary Figure 2a-d). Each mouse underwent two identical trials. The HP test was similar to the one described in the CFCA paradigm, except no tones were presented. There was a minimum of 30 min interval between the two trials, during which mice returned to a restful state in their home cage. Since the two trials were not significantly different, for simplicity, in panel i of Figure 1, the two trials were pooled together.

Tone-specific nociception assay. Naïve mice (n = 12) were submitted to the HP test without auditory fear conditioning (Figure 1i and Supplementary Figure 2a-d). Each animal underwent two trials, one trial with a 7.5 kHz tone presentation and another with a WN tone presentation. The tone presentation was counterbalanced. The HP test parameters were similar to those described for the CFCA, except that both tones were unconditioned. Since the two trials were not significantly different, for simplicity, in panel i of Figure 1, the two trials were pooled together.

Open Field assay. This test was used to determine the effect of optogenetic stimulation on locomotion. Therefore, only mice from optogenetic experiments were submitted to this assay. Mice could freely move during the entire test. The locomotion assay had a total duration of 9 min and was divided into 3 min epochs. The first and third epochs were OFF periods in which no optical stimulation occurred. During the second epoch, mice received photostimulation (ON period). The optogenetic stimulation effect was analyzed by comparing the overall distance traveled between the OFF and ON epochs (Supplementary Figure 5c, d). Mice injected with GFP were used to test the effect of heat and light on the stimulation itself. Mouse tracking data were extracted from video recordings using idTracker⁴² and imported into Matlab for further analysis with custom scripts to calculate the total distance traveled during stimulation.

Real-time Place Preference assay. Only mice from optogenetic experiments were submitted to this assay. During the entire duration of the assay, mice could freely shuttle between the two compartments. Under closed-loop stimulation, mice received photo-stimulation upon entry into one of the two compartments. The stimulated compartment was counterbalanced within the group. The optical stimulation effect was assessed by comparing the time spent in the stimulated compartment between the ChR2 and GFP groups (Supplementary Figure 5e, f). Permanence time in the stimulated compartment was automatically assessed by the apparatus software (Imetronic).

Virus injections and optogenetics

For optogenetic manipulation of SST⁺ cells in the vlPAG, 0.15-0.2 μL of either ChR2 (AAV5-EF1a-DIO-hChR2(H134R)-EYFP, titer: 3.2×10¹² - Vector Core, University of North Carolina), ArchT (AAV9-CAG-FLEX-ArchT-GFP, titer: 4.7×10¹² - Vector Core, University of North Carolina) or GFP (AAV5-FLEX-GFP, titer: 4.5×10¹² - Vector Core, University of North Carolina) were bilaterally injected into the vlPAG of 8/9 weeks old Sst-IRES-Cre mice from glass pipettes (tip diameter 20-30 μm) at the following coordinates relative to bregma: - 4.4 mm AP; ± 1.5 mm ML;-2.45 mm DV from dura, with a 20 degrees angle. Injection coordinates for manipulating VIP⁺ vlPAG cells were as follows: - 4.4 mm AP; ± 1.35 mm ML;-2.5 mm DV from dura, with a 20 degrees angle.

At two weeks after the injections, mice were bilaterally implanted with custom-built optic fibers (diameter: 200 µm; numerical aperture: 0.39; Thorlabs) above the vlPAG at the following coordinates relative to bregma: i) Sst-IRES-Cre mice: - 4.4 mm AP; ± 1.0 mm ML;-1.8 mm DV from dura, with a 10 degrees angle; ii) VIP-IRES-Cre mice: - 4.4 mm AP; ± 0.8 mm ML;-2.0 mm DV from dura, with a 10 degrees angle. For RVM manipulations, mice were implanted at the following coordinates relative to bregma:-5.8 mm AP; 0.0 mm ML;-5.2 DV from the dura. All implants were secured using three stainless steel screws and Super-Bond cement (Sun Medical). During surgery, long-and short-lasting analgesic agents were injected (Metacam, Boehringer; Lurocaïne, Vetoquinol). After surgery, mice were allowed to recover for at least five days. Afterward, mice were handled daily to familiarize themselves with being restrained for the optic fibers connection. Behavioral experiments were performed at least four weeks after viral injections. Only mice with correct placement of optic fibers and virus expression were included in the analyses.

For optogenetic excitation, light stimulation consisted of blue light (473 nm, ∼8-10 mW at fiber tip) delivered with 2 Hz frequency and 5 ms pulse duration. In contrast, light stimulation consisted of green light (532 nm, ∼8-10 mW at fiber tip) delivered continuously for optogenetic inhibition. For optogenetic manipulations during the CFCA paradigm of either SST⁺ or VIP⁺ vlPAG cells, the light was delivered during the FC HP test and paired with the tone presentation.

Two sets of experiments were performed sequentially for stimulation during the fear conditioning. First, the footshock US was replaced by optical stimulation. Then, the US became the optical stimulation combined with the footshock (Supplementary Figure 6). For both conditions, optogenetic stimulation began 5 s before and lasted until 5 s after CS⁺ offset. The same mice were used for both experiments. For the manipulations during the fear retrieval, there were 12 CS⁺ presentations divided into blocks of 4 CS⁺. The optogenetic stimulation was paired with the second block of CS⁺.

Single-molecule in situ hybridization

Expression of Sst, Slc31a1, Slc17a6, and Cre transcripts was detected in C57/BL6J mice using single-molecule fluorescent in situ hybridization (smFISH) as previously described (Castell et al., 2024 PMID: 37858736). Briefly, coronal PAG sections (16 µm) were collected directly onto Superfrost Plus slides. Probes for Sst (ACDBio; Cat#404631), Slc32a1 (ACDBio; Cat#319191-C2), Slc17a6 (ACDBio; Cat#319171-C2), and Cre (ACDBio; Cat#312281-C2) were used with the RNAscope Fluorescent Multiplex Kit (ACDBio; Cat# 323110). Fluorescence was captured using sequential laser scanning confocal microscopy (Leica SP8), and quantification was performed in ImageJ.

Anatomical tracing

Fluorogold injection. An incision between one to two cm was made slightly caudal to the peak of the dorsal hump to expose the lumbar spinal region. The vertebra of interest was identified. Then, a small incision was made between the tendons and the vertebral column on either side. The vertebra was then secured using spinal adaptor clamps, and all tissue was removed from the surface of the bone. Pulled borosilicate glass capillaries (Ringcaps, disposable capillary pipettes with ring mark, DURAN, Hirschmann Laborgeräte, Germany) were inserted between 2 vertebrae and allowed to microinject 0.5 μL of fluorogold 2% in the dorsal horn of the spinal cord on both sides.

In vivo electrophysiology

Mice were anesthetized with isoflurane 4% for induction, then 1.5% maintenance. The experiment was started as soon as there was no longer any reflex. The colorectal temperature was kept at 37 °C with a heating blanket. Two metal clamps were used to stabilize the animal’s spine in a stereotactic frame (M2E, France) during electrophysiological recordings. Then, a laminectomy was performed at T13-L1 to expose the lumbar part of the spinal cord. The dura mater was carefully removed. Custom-made optical fibers were placed 1mm above the dorsal spinal cord for optogenetic manipulations. C-fiber-evoked field potentials were recorded in the deep lamina of the DH (at a depth range of 250 and 500 µm) with borosilicate glass capillaries (2 MΩ, filled with NaCl 684 mM; Harvard Apparatus, Cambridge, MA, USA). Field potentials were recorded with an ISODAM-amplifier (low filter: 0.1Hz to high filter: 0.1 kHz; World Precision Instruments, USA) in response to electrical stimulation of the ipsilateral paw. Single unit recordings of WDR DH cells were made with the same borosilicate glass capillaries mentioned above and placed in the dorsal part of the spinal cord. The criterion for selecting a cell was the presence of an A-fiber-evoked response (0-80 ms) followed by a C-fiber-evoked response (80-150 ms) to electrical stimulation of the ipsilateral sciatic nerve.

The paw of the mice was stimulated by trains (every 30 s) of electrical stimulation at two times the threshold for C-fibers, which were performed before, during, and after optogenetic stimulations with an optic fiber placed above the recording site. Subthreshold stimulations were performed below the threshold for C-fiber and A-fiber, respectively.

Histology analyses

Mice were administered a lethal dose of Exagon and underwent transcardial perfusions via the left ventricle with 4% w/v paraformaldehyde (PFA) in 0.1 M PB. Following dissection, brains were post-fixed for 24 h at 4°C in 4% PFA. Brain sections of 80 μm-thick were cut on a vibratome, mounted on gelatin-coated microscope slides, and dried. To verify correct viral injections and optic fiber location, serial 80 μm-thick slices containing the regions of interest were mounted in VectaShield (Vector Laboratories) and imaged using an epifluorescence system (Leica DM 5000) fitted with a 10-x dry objective. The location and the extent of the injections/infections were visually controlled. Only infections targeting the vlPAG and optic fibers terminating, depending on the experiment, above the vlPAG or RVM were included in the analyses.

Statistics

All statistical details are contained in Supplementary Table 1. Box-whisker plots indicate median, interquartile range, and 5th - 95th percentiles of the distribution. All statistics are indicated where used. Statistical analyses were performed with Prism software (GraphPad). We tested the normality of all data with the Kolmogorov–Smirnov normality test in Prism software (GraphPad), and used nonparametric statistical tests for nonnormally distributed datasets, as indicated where used. No statistical methods were used to predetermine sample sizes, but sample sizes were based on previous studies in our laboratory. Blinding for the opsin was done for optogenetics experiments. Animals used for the CFCA paradigm that did not satisfy the DI and CI criteria after two conditioning sessions were discarded from the study. Mice with an incorrect injection of the opsins or misplaced location of the optic fibers were discarded. For in vivo electrophysiology, field potentials were measured as the area above the curve in the C-fiber range (80-300 ms). Absolute values were used to compare values in each condition (OFF vs. ON optogenetic manipulation). In single-unit recordings, the number of A-and C-fiber induced spikes in WDR cells was measured after each electrical stimulation, during, and after optogenetic manipulation. An average of the four stimuli for each WDR recorded was used for statistical analysis. Outliers were removed using the Tukey method.

No other mice were excluded. Significance levels are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001.

Supplementary figures

Supplementary Figure 1.

Supplementary Figure 2.

Supplementary Figure 3.

Supplementary Figure 4.

Supplementary Figure 5.

Supplementary Figure 6.

Supplementary Figure 7.

Supplementary Figure 8.

Supplementary Figure 9.

Supplementary Figure 10.

Supplementary Figure 11.

Supplementary table

Supplementary Table 1

Data availability

Data supporting the findings of this study, including analysis codes, are available from the corresponding author upon request.

Acknowledgements

We thank Joshua Johansen, David Finn, and Nadine Gogolla for critical and stimulating discussions; K. Deisseroth and E. Boyden for generously sharing material, S. Laumond, J. Tessaire, and the technical staff of the housing and experimental animal facility of the Neurocentre Magendie. We thank Claire Francioni for her technical support. Microscopy was performed in the Bordeaux Imaging Center of the CNRS-INSERM and Bordeaux University, member of France BioImaging. This work was supported by grants from the French National Research Agency (ANR-FEARLESSPAIN, ANR-10-EQPX-08 OPTOPATH), the Fondation pour la Recherche Médicale (FRM-PhD grant to NW 2020-2021) and the French Society for the Study and Treatment of Pain (SFETD – grant to NW 2022).

Additional information

Author Contributions

N.W performed behavioral experiments and optogenetic experiments on freely moving animals.

F.A, J.V, performed electrophysiological and optogenetic experiments on anesthetized animals. E.V, L.C, N.W, F.A, Z.G, C.R, R.B, and D.G performed histology. N.W, M.L, S.V. P.F, and C.H. designed the experiments. N.W, F.A, D.J, and E.V analyzed the data, N.W, D.J. and C.H wrote the paper.

Funding

Agence Nationale de la Recherche (ANR) (ANR-19-CE16-0016)

• Cyril Herry

• Fossat Pascal

Agence Nationale de la Recherche (ANR) (ANR-23-CE37-0015)

• Cyril Herry

• Fossat Pascal

Agence Nationale de la Recherche (ANR) (ANR-10-EQPX-08-optopath)

• Cyril Herry

Fondation pour la Recherche Médicale (FRM) (FRM-PhD)

• Nanci Winke