Nociceptive Stimuli Activate the Hypothalamus–Habenula Circuit to Inhibit the Mesolimbic Reward System and Cocaine-Seeking Behaviors

Abstract

Nociceptive signals interact with various regions of the brain, including those involved in physical sensation, reward, cognition, and emotion. Emerging evidence points to a role of nociception in the modulation of the mesolimbic reward system. The mechanism by which nociception affects dopamine (DA) signaling and reward is unclear. The lateral hypothalamus (LH) and the lateral habenula (LHb) receive somatosensory inputs and are structurally connected with the mesolimbic DA system. Here, we show that the LH–LHb pathway is necessary for nociceptive modulation of this system using male Sprague Dawley rats. Our extracellular single-unit recordings and head-mounted microendoscopic calcium imaging revealed that nociceptive stimulation by tail pinch excited LHb and LH neurons, which was inhibited by chemical lesion of the LH. Tail pinch increased activity of GABA neurons in ventral tegmental area, decreased the extracellular DA level in the nucleus accumbens ventrolateral shell in intact rats, and reduced cocaine-increased DA concentration, which was blocked by disruption of the LH. Furthermore, tail pinch attenuated cocaine-induced locomotor activity, 22 and 50 kHz ultrasonic vocalizations, and reinstatement of cocaine-seeking behavior, which was inhibited by chemogenetic silencing of the LH–LHb pathway. Our findings suggest that nociceptive stimulation recruits the LH–LHb pathway to inhibit mesolimbic DA system and drug reinstatement.

SIGNIFICANCE STATEMENT The LHb and the LH have been implicated in processing nociceptive signals and modulating DA release in the mesolimbic DA system. Here, we show that the LH–LHb pathway is critical for nociception-induced modulation of mesolimbic DA release and cocaine reinstatement. Nociceptive stimulation alleviates extracellular DA release in the mesolimbic DA system, cocaine-induced psychomotor activities, and reinstatement of cocaine-seeking behaviors through the LH–LHb pathway. These findings provide novel evidence for sensory modulation of the mesolimbic DA system and drug addiction.

Introduction

Nociceptive stimuli include noxious pressure (e.g., tail pinch), temperature (<10 and >40°C), and chemicals (e.g., acids; Sneddon, 2018). The nociceptive signals are conveyed to the CNS from the periphery via spinal cord circuits and interact with many different brain areas, including those involved in physical sensation, reward, cognition, and emotion (Bushnell et al., 2013). The mesolimbic dopamine (DA) reward system comprises DA neurons in the midbrain ventral tegmental area (VTA) that project to the nucleus accumbens (NAc), the prefrontal cortex, and the amygdala (Settell et al., 2017). This system is critically involved in motivation, reward, and addiction (Settell et al., 2017). Emerging evidence points to a role of nociception in the modulation of the mesolimbic system. Peripheral nerve injury, for instance, reduces morphine-induced conditioned place preference in mice, and this effect is associated with DAergic activity in the NAc and the VTA (Narita et al., 2003). In addition, nociceptive stimuli such as electric foot shocks and chemical injection during the self-administration training period strongly reduce drug-taking behavior of drugs such as fentanyl, cocaine, and methamphetamine in rodents (Pelloux et al., 2007; Wade et al., 2013; Hu et al., 2019). Given the convergence of nociception within the mesolimbic DA system, it is likely that nociception modifies mesolimbic DA transmission that influences drug reinstatement. However, to date, which neural circuit contributes to the delivery of nociceptive information to the mesolimbic DA system has not been fully characterized.

Nociceptive stimuli may require a series of neural circuits to arrive at the mesolimbic DA system (Yam and Loh, 2018). The lateral habenula (LHb), an epithalamic structure, has been reported to be involved in processing information of peripheral sensation and nociceptive events and modulating motivational and cognitive processes (Hu et al., 2020). Once activated, the LHb transmits a glutamatergic projection to the rostromedial tegmental nucleus (RMTg; Baker et al., 2016). The RMTg projects GABAergic inputs to VTA DA neurons, which eventually reduces DA release in the NAc (Baker et al., 2016; Zhao et al., 2020). Studies have addressed the role of the LHb–RMTg–VTA connections in motivated behaviors and drug addiction (Velasquez et al., 2014; Baker et al., 2016; Zhao et al., 2020). The lateral hypothalamus (LH) has also been reported to process nociceptive signals (Dafny et al., 1996; Siemian et al., 2019). The LH and LHb are directly connected with each other by glutamatergic inputs arising from the LH (Poller et al., 2013). Lazaridis et al. (2019) reported that the LH–LHb pathway encodes negative valence showing that optogenetic activation of the LHb-projecting LH glutamate neurons induces mice to switch from reward to nonreward in the probabilistic two-choice switching task. Given these findings, we hypothesized that the LH–LHb pathway conveys nociceptive signals to the mesolimbic DA system via glutamatergic of VTA GABA neurons, which inhibit DA neurons, thereby modulating cocaine-seeking behavior.

To demonstrate this, we performed in vivo extracellular single-unit recordings in the LH, LHb, and VTA and calcium imaging to ascertain the effects of tail pinch on neural activities. In addition, we chemically ablated the LH to examine whether the neural activity of the LHb is influenced by tail pinch in the absence of the LH signals. We next recorded extracellular DA concentration in the NAc ventrolateral shell during application of tail pinch and/or cocaine injection using in vivo fast-scan cyclic voltammetry (FSCV). We investigated effects of tail pinch on behavioral changes induced by a single cocaine injection and cocaine-seeking/taking behaviors in the self-administration paradigm. Then, we investigated the role of the LH–LHb pathway in the effects of tail pinch on cocaine-induced behavioral alterations by chemogenetic inhibition of the LH–LHb pathway.

Materials and Methods

Animals

All experiments were performed with male Sprague Dawley rats weighing 250–320 g (Hyochang). Rats had access ad libitum to food and water and were kept under 12 h light/dark cycle room conditions at a constant temperature (24 ± 1°C) and 50% humidity. All procedures were approved by the Institutional Animal Care and Use Committee at Daegu Haany University (DHU2018-824) and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Chemicals, reagents, and antibodies

Cocaine hydrochloride (Macfarlan Smith), ibotenic acid (5 μg/μl in saline; Sigma-Aldrich), adeno-associated virus (AAV)-hSyn1-GCaMP6s-P2A-nls-dTomato (serotype AAV1, viral titer ≥ 5 × 10¹² vg/ml; Addgene,), pAAV-hSyn-hM4D(Gi)-mCherry (serotype AAV retrograde, viral titer ≥ 7 × 10¹² vg/ml; Addgene), and Clozapine N-oxide (CNO) dihydrochloride (Tocris Bioscience) were used. Anti-c-fos antibody (catalog #ab190289, Abcam), anti-rabbit IgG antibody (Alexa Fluor 488, catalog #A21206, Thermo Fisher Scientific), and Vectashield antifade mounting medium with DAPI (H-1200; Vector Laboratories) were used for the immunohistochemistry.

Tail-pinch stimulation

Tail-pinch stimulation was conducted as described previously with some modifications (Goebel-Stengel et al., 2014). Binder clips (19 mm; WHASHIN) were used for pinching the tails with a pressing force of 1.0 ∼ 1.2 kg. Before experiments, the pressing force of the binder clips was further ensured by using a force sensor (SW-02, CAS). The stimulation was applied ∼10–20 mm apart to the tips of the tails.

Chemical or electrolytic lesion of the LH

As performed in our previous study (Chang et al., 2017), ibotenic acid (5 μg/μl) was injected 7 d before experiments for in vivo extracellular single-unit recordings, calcium imaging, and FSCV. In brief, rats were anesthetized by intraperitoneal injection (i.p.) with pentobarbital sodium (50 mg/kg) and placed in a stereotaxic frame, and two holes were drilled in the skull for access to the LH (anterior, −2.6 mm; lateral, ±1.7 mm; depth, −8.3 mm). Ibotenic acid or saline was injected into the LH at a rate of 0.25 μl/min for 2 min using a 26 gauge Hamilton syringe and a microinjection pump (Pump 22, Harvard Apparatus). The syringe was left in place for at least 5 min to prevent reflux after injection. For electrolytic lesion of LH, insulated tungsten electrodes, except at 0.5 mm tip, were inserted in the LH, and lesions were made by passing ±0.35 mA of DC current for 8 s, as performed in our laboratory (Chang et al., 2017).

In vivo extracellular single-unit recording

Rats were anesthetized by urethane (1.5 g/kg, i.p.), and a carbon-filament glass microelectrode (0.4–1.2 MΩ; Carbostar-1, Kation Scientific) was stereotaxically inserted into the LH (anterior, −2.6 mm; lateral, ±1.7 mm; depth, −8.3 mm) or the LHb (anterior, −3.5 mm; lateral, ±0.7 mm; depth, −4.9 mm) or VTA (anterior −6.6 to about −7.2 mm; lateral, ±0.8 to about ±1.0 mm; depth, −6.5 to about −7.5 mm). GABA neurons in the tail of VTA, called rostromedial tegmental nucleus (RMTg), were identified by discharge activity characteristics including relatively fast firing rate, phasic nonbursting activity, and negative spike with duration (<200 ms; Steffensen et al., 1998). Spontaneous discharges were amplified and filtered at 0.1–10 kHz (ISO-80; World Precision Instruments). Single-unit activity was discriminated from noise, binned at 1 s intervals, and analyzed via a CED 1401 Micro3 device and Spike2 software (Cambridge Electronic Design). After recording a stable baseline, if the single-unit discharges rates did not show drifts or fluctuations for at least 10 min (<10% variation in baseline), the rats received a brush, light pressure, or tail pinch for each 10 s and recorded for a further 10 min. After recording a stable baseline of at least 10 min, unilateral electrolytic lesion of LH was made and then the responses of VTA GABA neurons following tail pinch were compared before and after electrolytic lesion of LH.

In vivo microendoscopic calcium imaging

Calcium sensor GCaMP6s (AAV-hSyn1-GCaMP6s-P2A-nls-dTomato) was injected at a rate of 0.25 μl/min for 2 min (0.5 μl per side) into the left or the right side of the LH (anterior, −2.6 mm; lateral, ±1.7 mm; depth, −8.3 mm) or the LHb (anterior, −3.5 mm; lateral, ±0.7 mm; depth, −4.9 mm) in the rats anesthetized with pentobarbital sodium. An imaging cannula with a 500 μm diameter gradient index (GRIN) lens (Doric Lenses) was then placed into the LH or the LHb and anchored to the skull using dental cement and stainless steel screws. Four to 6 weeks after the surgery, the rats were connected to the microscope body via the imaging cannulas implanted on the heads, and then calcium imaging process was conducted. The fluorescent calcium transients were recorded and processed via Doric Neuroscience Studio software (version 5.2.2.3; 10 frames per second and 20 ± 5% of illumination power, Doric Lenses). After recording basal calcium activity for 10 s, the rats were given a tail pinch for 10 s, and recordings were continued for a further 10 s. The GCaMP-based calcium imaging was analyzed using Doric Neuroscience Studio software. Time series movies were motion corrected by aligning each image with a single reference image. The background fluorescence was eliminated from the motion-corrected images. Regions of interest (ROIs) were determined using an automated cell-finding algorithm of principal component analysis (PCA)/independent component analysis (ICA), in which PCA was used as the input of ICA to extract the distinctive cellular signals from imaging datasets. Finally, the relative fluorescence change of each ROI was analyzed by an algorithm calculating ΔF(F–F₀)/F₀, with F₀ corresponding to the temporal average of fluorescence intensity. Mean ΔF/F₀ during a 10 s tail pinch was compared with that during 10 s before the stimulation.

In vivo FSCV for monitoring DA release

Electrically evoked DA release in the NAc ventrolateral shell was measured by in vivo FSCV as performed in our previous study (Jang et al., 2015). A custom-made carbon fiber electrode (CFE; 7 μm diameter, 200 μm length of exposed tip) was used. The electrode potential was scanned with a triangular waveform from –0.4 to +1.3 V and back to –0.4 V versus Ag/AgCl at a scan rate of 400 V/s. Cyclic voltammograms were recorded every 100 ms by ChemClamp voltage-clamp amplifier (Dagan). Recording and analyzing were performed using LabVIEW-based (National Instruments) customized Demon voltammetry software. Under urethane anesthesia (1.5 g/kg, i.p.), bipolar stainless steel electrode and CFE were stereotaxically placed into the medial forebrain bundle (MFB; anterior, −2.5 mm; lateral, ±1.9 mm; depth, −8.0 to about −8.5 mm) and the NAc ventrolateral shell (anterior, +1.6 mm; lateral, ±1.9 mm; depth, −8.0 to about −8.5 mm), respectively. The MFB was stimulated with 60 monophasic pulses at 60 Hz (4 ms pulse width) every 2 min. After a stable baseline was established (<10% variability in peak heights of five consecutive collections), the changes of DA release in the NAc ventrolateral shell following tail pinch and/or cocaine injection (15 mg/kg in saline, i.p.) were monitored for a further 30 min. To identify the recording sites, the rats were killed at the end of the experiments and perfused with 4% formaldehyde. The brains were extracted, stored in 30% sucrose solution, and cryosectioned coronally at a thickness of 30 μm. The slices were stained with toluidine blue and observed under a light microscope (Microscopesmall).

Chemogenetics and cannula implantation

Under pentobarbital anesthesia (50 mg/kg, i.p.), a retrograde viral vector encoding an inhibitory designer receptors exclusively activated by designer drugs (DREADD), hM4Di, was bilaterally injected at a rate of 0.25 μl/min for 2 min (0.5 μl per side) into the LHb by using a 26 gauge Hamilton syringe mounted on a microinjection pump. Four weeks after the viral injection, guide cannulas (26 gauge; Plastics One) were implanted bilaterally into the LH (anterior, −2.6 mm; lateral, ±1.7 mm; depth, −7.3 mm) to locally infuse the DREADD agonist, CNO, or artificial CSF (aCSF). A week after the surgery, experiments for locomotor activity and 50 kHz ultrasonic vocalizations (USVs) were conducted. Internal cannulas (33 gauge; Plastics One) which protruded beyond the length of guide cannulas by 1 mm were inserted into the guide cannulas implanted on the heads. CNO was dissolved in aCSF to 1 mm, as previously described (Mahler et al., 2014). CNO or vehicle (VEH; aCSF) was intracranially infused at a rate of 0.15 μl/min for 2 min through the internal cannulas connected to a 26 gauge Hamilton syringe and a microinjection pump by polyethylene tubing, and an additional 1 min was allowed for further diffusion.

Measurement of USVs and locomotor activity

USVs and locomotor activity were recorded simultaneously in customized sound-attenuating chambers. The chamber consisted of two boxes to minimize exterior noise (inside box, 60 × 42 × 42 cm; outside box, 68 × 50 × 51 cm). A condenser ultrasonic microphone (Ultramic 250K, Dodotronic) and a digital camera were positioned at the center of the ceiling of the chamber. As performed in our laboratory (Kim et al., 2021), 22 and 50 kHz USVs were recorded using the ultrasonic microphone with an UltraSoundGate 416H data acquisition device (Avisoft Bioacoustics). Ultrasonic vocal signals were band filtered between 30 and 80 kHz for the 50 kHz USVs and between 20 and 30 kHz for the 22 kHz USVs, respectively. The total number of emitted 22 and 50 kHz USVs was automatically counted by using pulse train detection analysis from Avisoft-SASLab Pro (version 4.2, Avisoft Bioacoustics), hold time of 10 ms and minimum duration of 4 ms. Locomotor activity was measured with a video-tracking system (Ethovision XT, Noldus Information Technology). Rats were habituated for 30 min in the chambers. After recording basal USVs and basal activity for 30 min, the rats received cocaine injection, tail pinch, and/or either CNO (1 mm) or aCSF through the implanted guide cannulas. The recordings were continued for another 60 min.

Cocaine self-administration, extinction, and reinstatement procedures

Food training and cocaine self-administration were performed in operant chambers equipped with active and inactive levers (Med Associates), as described previously with slight modifications (Chang et al., 2019). Initially, rats were food restricted with 16 g of lab chow per day and trained to press the active lever to gain 45 mg food pellets (Bio-Serve). Rats that achieved the criterion for food responding (100 food pellets for 3 consecutive days) were chosen for the cocaine self-administration procedure and were surgically implanted with chronically indwelling intravenous catheters under pentobarbital anesthesia. After a recovery period of at least 7 d, the rats were trained to self-administer cocaine intravenously by pressing the active lever under a fixed-ratio schedule in a daily 2 h session. Once the active lever was pressed, 0.5 mg/kg/0.1 ml cocaine was infused over 5 s. When the intravenous cocaine infusion was initiated, the house light was extinguished for 20 s, and a cue light located above the active lever was illuminated for 5 s, concomitant with a 15 s time-out period. During the time-out period, the active lever responses were recorded in an automated counting program (Schedule Manager, Med Associates), but no cocaine infusion was made. Pressing the inactive lever produced no scheduled responses, but signals were recorded in the program (Schedule Manager, Med Associates). After 10 sessions of the cocaine self-administration training, the rats underwent 7 sessions of the extinction task during which saline was delivered to the rats without an illumination of the cue light when they had pressed the active lever. After the extinction task, the rats received a single priming intravenous injection of 0.5 mg/kg cocaine, and the experiment was performed with the same experimental conditions as the extinction task.

Immunohistochemistry

The immunohistochemistry for c-fos was conducted as described previously (Jin et al., 2018). As it is known that the c-fos mRNA accumulation reaches a maximum between 30 and 45 min after the onset of stimulation (Müller et al., 1984), and the c-fos protein synthesis is detected 20 ∼ 90 min after stimulation, the rats were killed 40 min after cocaine injection and perfused with 4% formaldehyde. The brains were taken out, postfixed, cryoprotected and cryosectioned into 30-μm-thick slices. The brain slices were incubated with anti-c-fos rabbit polyclonal antibodies (1:500), followed by donkey anti-rabbit IgG antibodies (1:500; Alexa Fluor 488). The slices were then mounted on gelatin-coated slides, photographed, and examined under a confocal laser scanning microscope (LSM700, Carl Zeiss). The number of c-fos-positive cells in the LHb or the LH was blindly counted, and five to seven slices per animal were analyzed.

Statistical analysis

Data were presented as the mean ± SEM and analyzed by one-way or two-way ANOVA, one-way or two-way repeated measures ANOVA, followed by post hoc testing using Tukey's method, unpaired t test, or paired t test, where appropriate. Values of p < 0.05 were regarded as statistically significant.

Results

Excitation of the LHb and the LH by tail pinch

To examine whether nociceptive stimulation excites the LH–LHb pathway, neural activities of the LHb and the LH following tail pinch were investigated using in vivo extracellular single-unit recording or in vivo microendoscopic calcium imaging. Three different stimuli of brush, light pressure, and tail pinch were sequentially given to the tails for each 10 s (Fig. 1A–C). Although firing rates of the LHb neurons were not changed by brush and light pressure (8.5 g von Frey filament), application of tail pinch evoked firing rates of the LHb neurons up to 182.25 ± 7.22% compared with the basal activity for 10 s before tail pinch (Fig. 1B,C; n = 16 cells, one-way repeated measures ANOVA, F_(2,18) = 79.491).

Figure 1.

Effects of tail pinch on the neural activities of the LHb and the LH. A, Schematic diagram of in vivo extracellular single-unit recording in the LHb. B, C, Neural activities of the LHb in response to brush, light pressure, and tail pinch for each 10 s; n = 16 cells; ***p < 0.001, Pre versus Tail-pinch. D–G, In vivo calcium imaging in the LHb following tail pinch. Timeline of in vivo microendoscopic calcium imaging for neural activities of the LHb following tail pinch in the rats (D, top) GCaMP6s expression in the LHb (D, bottom-left) and representative images of the GRIN lens track in the LHb (D, bottom-middle). Scale bar, 400 μm. Illustration of a rat with a fluorescence microscope (D, bottom-right). Representative field of view in the LHb with ROIs (E, top; n = 27 cells). Scale bar, 40 μm. ΔF/F₀ traces from the ROIs in the LHb (E, bottom). Normalized calcium transients of the ROIs in the LHb neurons (F). Bar graph (G) of the averages of ΔF/F₀ before and during tail pinch for each 10 s in the LHb (n = 6 rats; **p < 0.005, Pre vs Tail-pinch). H–K, In vivo calcium imaging in the LH following tail pinch. GCaMP6s expression in the LH. Scale bar, 400 μm (H). Representative field of view in the LH with ROIs (I, top; n = 32 cells). Scale bar, 40 μm. ΔF/F₀ traces from the ROIs in the LH (I, bottom). Normalized calcium transients of the ROIs in the LH neurons (J). Bar graph (K) of the averages of ΔF/F₀ before and during tail pinch for each 10 s in the LH (n = 5 rats, **p < 0.009, Pre vs Tail-pinch). LH, lateral hypothalamus; LHb, lateral habenula; VTA, ventral tegmental area; NAc, nucleus accumbens; Pre, pre-stimulus; GRIN lens, gradient index lens; MHb, medial habenula; EP, entopeduncular nucleus; opt, optic tract.

To confirm the excitatory effect of tail pinch on the LHb neurons, in vivo calcium imaging was performed in the LHb (Fig. 1D–G). The rats with calcium sensor GCaMP6s in the LHb were head mounted with fluorescence microscope, and then calcium transients following tail pinch were recorded (Fig. 1D). When tail pinch was applied for 10 s, the calcium indicator GCaMP6s showed an initial rise followed by a sustained decrease in response to tail pinch in the LHb neurons (Fig. 1E,F). The average of ΔF/F₀ was 6.28 ± 1.43% during tail pinch, whereas the value before tail pinch was 0.76 ± 0.09% (Fig. 1G; n = 175 cells from 6 rats, one-way repeated measures ANOVA, F_(1,5) = 23.769). We repeated this experiment in the LH neurons by using in vivo calcium imaging (Fig. 1H–K). The rats were given application of tail pinch for 10 s. Immediately after tail pinch, fluorescence intensity of the calcium indicator GCaMP6s in the LH neurons markedly increased and declined steadily (Fig. 1I,J). The average of ΔF/F₀ increased to 4.97 ± 0.80% during tail pinch from 1.61 ± 0.21% (Fig. 1K; n = 153 cells from 5 rats, one-way repeated measures ANOVA, F_(1,4) = 22.198). These data indicate that nociceptive stimulation excites both LHb and the LH neurons. In the comparison of mean latencies of the LH and the LHb in response to tail pinch, LH neurons (n = 153) revealed a significantly more rapid response than LHb neurons (n = 175; 130.20 ± 22.63 ms vs 312.21 ± 89.25 ms; data not shown), suggesting that nociceptive signals were delivered to LHb after reaching LH.

The LH mediation in activation of the LHb by tail pinch

To identify the mediation of the LH in the transduction of nociceptive signals of tail pinch to the LHb neurons, we ablated the LH by intracranial injection of neurotoxic ibotenic acid and measured a neural activity of the LHb following tail pinch using in vivo extracellular single-unit recording or in vivo calcium imaging. Ibotenic acid was injected into the LH 7 d before the examinations (Fig. 2A,D). In in vivo extracellular single-unit recording, tail pinch failed to induce excitation of the LHb neurons in the LH-lesioned rats (Fig. 2B,C; n = 52 cells, one-way repeated measures ANOVA, F_(2,18) = 1.697). In addition, there was no significant difference between the baseline activities of the LHb in intact versus LH-lesioned rats (3.45 ± 0.14 impulse/s versus 4.97 ± 0.09 imp/s, respectively), indicating that LH lesion did not affect the baseline activity of LHb neurons (data not shown). To perform in vivo calcium imaging, the LH-lesioned rats were injected with the calcium indicator GCaMP6s in the LHb, and calcium-induced fluorescence changes following tail pinch were monitored in the LHb (Fig. 2D). Although tail pinch induced a consistent increase in the fluorescence in intact rats (Fig. 1D), such an increase was not observed in the LH-lesioned rats (Fig. 2E,F). In addition, the averages of ΔF/F₀ before (Pre) and during tail pinch (Tail-pinch) were 1.86 ± 0.46% and 2.37 ± 0.24%, respectively, but there was no significant difference between the two groups (Fig. 2G; n = 121 cells from four rats, one-way repeated measures ANOVA, F_(1,3) = 1.353). We found that most of the LH neurons were activated by tail pinch (Fig. 1H–K), and ablation of the LH blocked tail-pinch-induced neural activity of the LHb. These data suggest that the LH neurons that activate the LHb neurons would convey nociceptive signals to the LHb. In addition, the neural activity of GABA neurons in the tail of VTA (called RMTg) was strongly evoked by tail pinch, which was blocked by electrolytic lesions of the LH (Fig. 2H–J; n = 12 cells, one-way repeated measures ANOVA, F_(2,18) = 623.111), suggesting that the LH–LHb–RMTg connections play a significant role in the transit of nociceptive signals.

Figure 2.

Effect of the LH lesion on the neuronal activity of the LHb following tail pinch. A, Timeline and diagrams of chemical lesion of the LH and in vivo extracellular single-unit recording in the LHb. B, C, Neural activities of the LHb following tail pinch in the rats with a chemical lesion of the LH; n = 52 cells, p = 0.211. D, Top, Timeline of in vivo calcium imaging for neural activities of the LHb following tail pinch in the rats with a chemical lesion of the LH. Bottom, Diagram and a representative image of the chemical lesion of the LH. Scale bar, 400 μm. E–G, Representative field of view in the LHb with ROIs (E, top; n = 29 cells).” Scale bar, 40 μm. ΔF/F₀ traces from the ROIs in the LHb (E, bottom). Normalized calcium transients of the ROIs in the LHb neurons (F). Bar graph (G) of the averages of ΔF/F₀ before and during tail pinch for each 10 s in the LHb (n = 4 rats; p = 0.329). H–J, Neural activities of the RMTg following tail pinch before (H) and after electrolytic lesion of the LH in rats (I); n = 12 cells, ***p < 0.001, Pre versus Tail-pinch and Tail-pinch versus Tail-pinch (LH lesion). n.s., not significant; GABA, Gamma-aminobutyric acid.

A reduction of DA release in the NAc shell by tail pinch and its reversal by the LH lesion

To determine whether nociceptive stimuli have an influence on mesolimbic DA release, and whether it was mediated via the LH, effects of tail pinch on evoked DA release in the NAc ventrolateral shell were measured using in vivo FSCV in intact or LH-lesioned rats. Ibotenic acid was injected into the LH a week before the experiments, and then DA efflux was evoked by stimulating the MFB (Fig. 3A,H). Recordings were conducted in the ventrolateral part of the NAc shell (Fig. 3B). Dopamine levels gradually decreased to 78.5 ± 4.85% 10 min after application of tail pinch and slowly recovered to the level of baseline when tail pinch was removed (Fig. 3C; n = 6 rats, one-way repeated measures ANOVA, F_(10,50) = 4.817). When tail pinch was applied continuously for 30 min, DA levels steadily decreased to 63.9 ± 4.74% at the end of the stimulation compared with the values before tail pinch (Fig. 3D,E; Tail-pinch group, n = 7 rats) or Control (normal rats, n = 6 rats). The sustained decrease of DA release by tail pinch was not observed in the rats with a chemical lesion of the LH (LH lesion+Tail-pinch group, n = 8 rats; two-way repeated measures ANOVA, F_(28,140) = 3.943). These data suggest the mediation of the LH in nociceptive modulation of the mesolimbic DA system. Additionally, we explored whether nociceptive stimulation can inhibit cocaine-induced NAc DA release. Acute injection of cocaine evoked DA release in the NAc ventrolateral shell (Fig. 3F,G; n = 6 rats), which was prevented by application of tail pinch (n = 5 rats, two-way repeated measures ANOVA, F_(14,56) = 14.060).

Figure 3.

Effect of the LH lesion on the mesolimbic DA release following tail pinch. A, Schematics for in vivo FSCV in the rats with ibotenic acid lesion of the LH. Diagrams of ibotenic acid lesion of the LH and electrically evoked DA release in the NAc by stimulating the MFB. B, Representative images of the recording site. Black dashed line indicates the track of a CFE. Scale bar, 400 μm. C, Effect of tail pinch for 10 min on the NAc DA release in naive rats (n = 6 rats, ***p < 0.001, **p = 0.002, 0.002, and 0.005, *p = 0.03 before tail pinch versus after tail pinch). D, Comparison of the NAc DA release between Control group (n = 6 rats), Tail-pinch group (n = 7 rats), and LH lesion plus Tail-pinch group (n = 8 rats; *p = 0.012, ***p < 0.001, Tail-pinch vs Control; ^#p = 0.016, ^###p < 0.001, Tail-pinch vs LH lesion+Tail-pinch). E, Representative pseudo-color plots with color bars indicating the current (top). Time series plots indicate the current versus time traces for DA release in each group (bottom-right). Each cyclic voltammogram corresponds to the pseudo-color plots (bottom-left). F, Comparison of the NAc DA release between Coc. group (n = 6) and Coc.+Tail-pinch group (n = 5; ***p < 0.001, Coc. vs Coc.+Tail pinch). G, Representative pseudo-color plots with color bars indicating the current (top). Time series plots indicate the current versus time traces for DA release in each group (bottom-right). Each cyclic voltammogram corresponds to the pseudo-color plots (bottom-left). H, Schematic illustration (top) and a representative image (bottom) showing the locations of the MFB (stimulating site) and the LH lesion. Scale bar, 500 μm. MFB, medial forebrain bundle; CFE, carbon fiber electrode;DA, dopamine; Coc., cocaine.

Suppression of cocaine-induced psychomotor activities by tail pinch and mediation of the LH–LHb pathway

On the basis of our electrophysiological and microendoscopic findings that tail pinch activated the LH–LHb pathway and thus suppressed extracellular DA release in the NAc ventrolateral shell, we further explored the effects of tail pinch on acute cocaine-enhanced locomotor activity and the emission of 22 and 50 kHz USVs, known to be associated with mesolimbic DA levels (Sanchez et al., 2022). Locomotor activity and USVs were recorded in custom-built chambers simultaneously (Fig. 4A). Cocaine administration rapidly increased locomotion with a peak at 10 min, followed by a steady decrease over 60 min [Fig. 4B; Cocaine (Coc.) group, n = 6 rats]. Tail pinch significantly inhibited the cocaine-enhanced locomotion (Coc.+Tail-pinch group, n = 6 rats), compared with the Coc. group, whereas tail pinch itself did not affect locomotor activity in normal rats (Tail-pinch group, n = 6 rats; two-way repeated measures ANOVA, F_(10,50) = 31.814). Furthermore, we analyzed the number of 22 and 50 kHz USVs, detected during the locomotor activity (Fig. 4C). Cocaine administration evoked a large number of 50 kHz USVs, compared with the basal value before cocaine injection, which was strongly reduced by application of tail pinch (two-way ANOVA, F_(2,24) = 64.629). On the other hand, application of tail pinch produced robust emissions of 22 kHz USVs, indicating the generation of nociceptive pain by tail pinch (Fig. 4C). These data suggest a reversal of the cocaine-induced psychomotor activities by nociception.

Figure 4.

Effect of chemogenetic silencing of the LH–LHb pathway on tail-pinch inhibition of cocaine-induced locomotion and emission of 50 kHz USVs. A, Illustration of a freely moving rat in a customized USV chamber (top) and a representative spectrogram of 50 and 22 kHz USVs (bottom). B, C, Effect of tail pinch on cocaine-induced locomotion in rats (B, left; ***p < 0.001, Coc. vs Tail-pinch; ^##p = 0.005, ^###p < 0.001, Coc. vs Coc.+Tail-pinch; n = 6/group). Representative locomotion tracks for 30 min after tail pinch and/or cocaine injection (B, right). Average of 22 and 50 kHz USVs for 30 min before and after tail pinch and/or cocaine injection (C); ***p < 0.001 (50 kHz), Coc. versus Pre, Tail-pinch, and Coc.+Tail-pinch; ^###p < 0.001 (22 kHz), Coc.+Tail-pinch versus Pre and Coc.; p = 0.335 (22 kHz), Coc.+Tail-pinch versus Tail-pinch; Tail-pinch, n = 5; Coc., n = 5; Coc.+Tail-pinch, n = 5. Tail-pinch, Tail pinch in naive rats; Coc., cocaine injection only; Coc.+Tail-pinch, tail pinch in cocaine-treated rats. D, Timeline and diagrams of hM4Di injection in the LHb and guide cannula implantation in the LH (top). Representative images of hM4Di expression in the LHb and the LH (bottom). Scale bar, 800 μm. E, F, Effect of chemogenetic silencing of the LH–LHb pathway on tail-pinch inhibition of cocaine-induced locomotion and emission of USVs. Effect of chemogenetic silencing of the LH–LHb pathway on tail-pinch inhibition of cocaine-induced locomotion (E, left; ***p < 0.001, Coc.+Tail-pinch+CNO vs Coc., Coc.+CNO, and Coc.+Tail-pinch+hM4Di/CNO; ^###p < 0.001, Coc.+Tail-pinch+hM4Di/VEH vs Coc., Coc.+CNO, and Coc.+Tail-pinch+hM4Di/CNO; Coc., n = 7; Coc.+Tail-pinch+hM4Di/VEH, n = 8; Coc.+Tail-pinch+hM4Di/CNO, n = 6) and representative locomotion tracks for 30 min after tail pinch, cocaine injection, and/or either CNO or aCSF infusion (E, right). A retrograde viral vector encoding an inhibitory DREADD (hM4Di) was injected into the LHb, and the CNO was intracranially infused into the LH. Average of 22 and 50 kHz USVs for 30 min before and after tail pinch, cocaine injection, and/or either CNO or aCSF infusion (F); ***p < 0.001 (50 kHz), Coc. versus Coc.+Tail-pinch+CNO, Coc.+Tail-pinch+hM4Di/VEH, and Coc.+Tail-pinch+hM4Di/CNO; ^###p < 0.001 (22 kHz), Coc.+Tail-pinch+CNO versus Coc., Coc.+CNO, and Coc.+Tail-pinch+hM4Di/CNO; Coc., n = 5; Coc.+Tail-pinch+hM4Di/VEH, n = 6; Coc.+Tail-pinch+hM4Di/CNO, n = 5. Coc., Cocaine injection only in hM4Di-expressed rats; Coc.+CNO, cocaine injection, and CNO infusion into the LH; Coc.+Tail-pinch+CNO, cocaine injection, tail pinch, and CNO infusion into the LH; Coc.+Tail-pinch+hM4Di/VEH, cocaine injection, tail pinch, and aCSF infusion into the LH in hM4Di-expressed rats; Coc.+Tail-pinch+hM4Di/CNO, cocaine injection, tail pinch, and CNO infusion into the LH in hM4Di-expressed rats. CNO, clozapine-N-oxide; VEH, vehicle.

To evaluate mediation of the LH–LHb pathway in the inhibitory effects of tail pinch on the cocaine-induced psychomotor activities, a retrograde viral vector encoding an inhibitory DREADD (hM4Di) was injected into the LHb, CNO was intracranially infused into the LH (LH-LHb:hM4Di/CNO) to silence the LH–LHb pathway, and then effects of tail pinch on cocaine-induced behaviors were explored (Fig. 4D). As shown in Figure 4E, cocaine administration enhanced locomotor activity (Coc. group, n = 7 rats; Coc.+CNO, n = 8), which was suppressed by tail pinch in the rats with either intracranial CNO infusion in the LH (Coc.+Tail-pinch+CNO group, n = 8) or LH-LHb:hM4Di/VEH (Coc.+Tail-pinch+hM4Di/VEH group, n = 8 rats). The inhibitory effects of tail pinch on the cocaine-induced locomotion were almost completely blocked by intracranial CNO infusion in the rats with LH-LHb:hM4Di (Coc.+Tail-pinch+hM4Di/CNO group, n = 6 rats; two-way repeated measures ANOVA, F_(20,100) = 11.098). These effects were further confirmed by measuring the number of 50 kHz USVs (Fig. 4F). The increased emission of 50 kHz USVs by cocaine injection was suppressed by tail-pinch stimulation in the rats with either intracranial CNO infusion (Coc.+Tail-pinch+CNO group) or LH-LHb:hM4Di/VEH (Coc.+Tail-pinch+hM4Di/VEH group). In contrast, in the rats with LH-LHb:hM4Di, intracranial CNO infusion significantly alleviated the inhibitory effects of tail pinch on cocaine-induced emission of 50 kHz USVs, compared with aCSF infusion (two-way ANOVA, F_(4,54) = 75.864). CNO itself did not disturb the effects of cocaine and/or tail pinch on psychomotor responses. Meanwhile, the number of emitted 22 kHz USVs was significantly higher in the groups given application of tail pinch (Fig. 4F). These results indicate that the effects of tail pinch on the cocaine-induced hyperlocomotion and positive affective states are mediated via the LH–LHb pathway.

Attenuation of cocaine-taking/seeking behaviors by tail pinch and mediation of the LH–LHb pathway

To further examine whether tail pinch could suppress cocaine-taking behavior and reinstatement of cocaine-seeking behavior, we used the cocaine self-administration paradigm (Fig. 5A,E). Mediation of the LH–LHb pathway was investigated by using chemogenetic inhibition (LH-LHb:hM4Di/CNO; Fig. 5B–D). Initially, effects of tail pinch on a natural reward were investigated using food training (Fig. 5F; one-way ANOVA, F_(3,32) = 1.000). Application of tail pinch throughout the test session (3 h; tail-pinch session) did not affect consumption of food pellets. During the cocaine self-administration training (Fig. 5G), rats were trained to administer intravenous cocaine (0.5 mg/kg/infusion; Coc. group, n = 6 rats). Application of tail pinch during the cocaine self-administration training inhibited the establishment of cocaine self-administration (Coc.+Tail-pinch group, n = 5 rats; two-way repeated measures ANOVA, F_(9,36) = 1.355). As shown in Figure 5H, after rats were trained for establishing the cocaine self-administration (Coc. group, n = 9 rats; Coc.+Tail-pinch group, n = 9 rats; Coc.+Tail-pinch+CNO group, n = 7; Coc.+Tail-pinch+hM4Di/VEH group, n = 9 rats; Coc.+Tail-pinch+hM4Di/CNO group, n = 10 rats), we tested the effects of tail pinch on cocaine intakes (Fig. 5H–J, test 1; I, one-way ANOVA, F_(4,39) = 0.0149; J, one-way ANOVA, F_(4,39) = 1.344). Next, the rats were trained for extinguishing cocaine self-administration, and we investigated the effects of tail pinch on reinstatement of cocaine-seeking behavior (Fig. 5K–N, test 2). In test 1, although tail pinch tended to reduce the number of cocaine infusions, there were no statistically significant differences between the groups in the number of cocaine infusions as well as active/inactive lever responses (Fig. 5H–J). The rats were then subjected to extinction sessions in which the cocaine solution was replaced with saline, and test 2 was conducted (Fig. 5K–N). Although a single priming injection of cocaine (0.5 mg/kg/ml) produced robust active lever responses that indicate reinstatement of cocaine-seeking behavior (Coc. group; Fig. 5L), tail pinch suppressed the cocaine-primed active lever responses (Coc.+Tail-pinch group, Coc+Tail-pinch+CNO group, and Coc.+Tail-pinch+hM4Di/VEH group; one-way ANOVA, F_(4,38) = 8.832). The inhibitory effect of tail pinch on the reinstatement of cocaine-seeking behavior was suppressed by pretreatment of intracranial CNO infusion in the rats expressing hM4Di in the LH–LHb pathway. However, tail pinch did not affect the number of cocaine intakes and inactive lever responses (Fig. 5K,N; N, one-way ANOVA, F_(4,39) = 2.376). CNO itself had no effect on cocaine-taking and operant lever-pressing behaviors (Fig. 5H–N). These data indicate that nociceptive stimulation attenuated the cocaine-taking/seeking behaviors via the LH–LHb pathway.

Figure 5.

Effects of tail pinch on cocaine-taking/seeking behaviors and its reversal by chemogenetic silencing of the LH–LHb pathway. A, Experimental procedures for cocaine self-administration. B–D, Viral expression and location of the guide cannula. Example image showing injection of retrograde AAV into the LH using double guide cannula (B). Representative images of hM4Di expression in the LHb (C) and the LH (D). Scale bar, 800 μm. E, Illustration of self-administration operant chamber. F, Effect of tail pinch on the consumption of food pellets during the food training session. The total number of food pellets was limited to 100 in each experiment (n = 9 rats, p = 1.000). G, Effect of tail pinch on cocaine intakes during cocaine self-administration training. Coc., Cocaine self-administration training (n = 6); Coc.+Tail-pinch, tail pinch during cocaine self-administration training (n = 5); *p = 0.034, 0.024, and 0.012, **p = 0.008, 0.005, 0.007, 0.006, 0.004, and 0.006, Coc. versus Coc.+Tail-pinch. H–J, Effect of tail pinch on cocaine intakes after acquisition of cocaine self-administration over 10 sessions. Time courses of cocaine infusions (H). The numbers of active lever responding following tail pinch (I); p = 1.000. The number of inactive lever responding following tail pinch (J); p = 0.271. K–M, Inhibition by tail pinch of cocaine-primed reinstatement of cocaine-seeking behavior and its reversal by chemogenetic silencing of the LH–LHb pathway. Time courses of cocaine infusions (K). The numbers of active lever responding following tail pinch (L); **p = 0.002 and ***p < 0.001, Coc. versus Coc.+Tail-pinch, Coc.+Tail-pinch+CNO, and Coc.+Tail-pinch+hM4Di/VEH; p = 0.452, Coc. versus Coc.+Tail-pinch+hM4Di/CNO). The number of inactive lever responding (M); p = 0.069. Coc., cocaine priming injection only (n = 5); Coc.+Tail-pinch, cocaine priming injection+Tail-pinch; Coc.+Tail-pinch+hM4Di/VEH, cocaine priming injection, tail pinch, and aCSF infusion into the LH in hM4Di-expressed rats (n = 5); Coc.+Tail-pinch+hM4Di/CNO, cocaine priming injection, tail pinch, and CNO infusion into the LH in hM4Di-expressed rats (n = 6). Cocaine SA, cocaine self-administration; LPO, lateral preoptic area.

Elevation of c-fos expression by tail pinch and its reversal by chemogenetic silencing of the LH–LHb pathway

Finally, neuronal activities of the LH and the LHb following cocaine, tail pinch, and/or either CNO or aCSF were evaluated by immunohistochemistry for c-fos (Fig. 6A,E). Tail pinch increased c-fos expression in the LH and the LHb in cocaine naive rats (Tail-pinch group) and the cocaine-injected rats (Coc.+Tail-pinch group), compared with the Control group and the Coc. group (Fig. 6B,C,F,G). In the rats with LH-LHb:hM4Di, the increased c-fos expression by tail pinch was significantly inhibited by CNO administration (Coc.+Tail-pinch+hM4Di/CNO group) but not by aCSF administration (Coc.+Tail-pinch+hM4Di/VEH group; one-way ANOVA for LHb or LH group, F_(5,25) = 19.763 and F_(5,31) = 77.686, respectively). Furthermore, although the ratios of the c-fos-expressing hM4Di-infected neurons to all the hM4Di-infected neurons were 59.44 ± 1.33% and 65.04 ± 2.10% in the LHb and the LH in the Coc.+Tail-pinch+hM4Di/VEH group, the ratios were 14.16 ± 1.25% and 10.66 ± 0.97% in the LHb and the LH in the Coc.+Tail-pinch+hM4Di/CNO group (Fig. 6D,H), indicating that compared with aCSF, CNO infusion significantly decreases c-fos expression induced by tail pinch in the hM4Di-infected LH (two-tailed unpaired t test, t₍₈₎ = 26.521, p < 0.001) and LHb neurons (two-tailed unpaired t test, t₍₈₎ = 27.653, p < 0.001).

Figure 6.

Effects of tail pinch and chemogenetic silencer on c-fos expression in LH and LHb neurons. A, Representative image of c-fos expression in the LHb of rats. Scale bar, 400 μm. B, Representative images of c-fos expression in the LHb in Control (n = 6), Coc. (n = 6), Tail-pinch (n = 5), Coc.+Tail-pinch (n = 5), Coc.+Tail-pinch+hM4Di/VEH (n = 7), and Coc.+Tail-pinch+hM4Di/CNO (n = 6) groups. C, The number of the c-fos-positive neurons in the LHb in each group; ***p < 0.001. D, Representative images of c-fos immunoreactivity, hM4Di viral expression, and c-fos expression in hM4Di-infected neurons (indicated by arrowheads) in the LHb of Coc.+Tail-pinch+hM4Di/VEH and Coc.+Tail-pinch+hM4Di/CNO groups (left) and ratios of c-fos-positive hM4Di-infected neurons to hM4Di-infected neurons in the LHb (right, ***p < 0.001, Coc.+Tail-pinch+hM4Di/VEH vs Coc.+Tail-pinch+hM4Di/CNO). E, Representative image of c-fos expression in the LH of rats. Scale bar, 200 μm. F, Representative images of c-fos expression in the LH in Control (n = 6), Coc. (n = 6), Tail-pinch (n = 5), Coc.+Tail-pinch (n = 5), Coc.+Tail-pinch+hM4Di/VEH (n = 7), and Coc.+Tail-pinch+hM4Di/CNO (n = 6) groups. Scale bar, 40 μm. G, The number of c-fos-positive neurons in the LH in each group; ***p < 0.001. H, Representative images of c-fos immunoreactivity, hM4Di viral expression, and c-fos expression in the hM4Di-infected neurons (indicated by arrowheads) in the LH of the Coc.+Tail-pinch+hM4Di/VEH and Coc.+Tail-pinch+hM4Di/CNO groups (left) and ratios of the c-fos-positive hM4Di-infected neurons to all the hM4Di-infected neurons in the LH (right, ***p < 0.001, Coc.+Tail-pinch+hM4Di/VEH vs Coc.+Tail-pinch+hM4Di/CNO). Scale bar, 40 μm.

Discussion

In the present study, tail pinch excited LH and LHb neurons, which were blocked by a chemical lesion of the LH. Tail pinch decreased DA levels in the NAc ventrolateral shell, which was disrupted by a chemical lesion of the LH. In addition, tail pinch attenuated cocaine-increased extracellular DA concentration, cocaine-induced locomotor activity, emission of 22 and 50 kHz USVs, development of cocaine-taking behavior, and reinstatement of cocaine-seeking behavior, which was reversed by chemogenetic silencing of the LH–LHb pathway. Tail pinch increased c-fos expression of the LH and the LHb neurons, which was inhibited by chemogenetic silencing of the LH–LHb pathway with CNO. These results suggest that nociceptive stimulation activates VTA GABA neurons and thereby suppresses the mesolimbic DA system and cocaine-reinforcing effects through the LH–LHb pathway.

The LH projects glutamatergic inputs to the LHb area, which afferents from the LH directly connect to the VTA-projecting LHb neurons (LHb→VTA neurons; Poller et al., 2013). Previous reports have revealed that an anterograde tracer injected into the LH was expressed in the LHb and strongly overlaid with immunocytochemical localization of vesicular-glutamate transporter 2 in the LHb (Poller et al., 2013). The anterogradely traced LH axons overlapped with the LHb neurons, which were retrogradely traced by the VTA (Poller et al., 2013). Furthermore, whole-brain mapping of neurons projecting to the LHb revealed that the LH is the most prominent input region to the LHb (Lazaridis et al., 2019). In the present study, excitation of the LHb by tail pinch was blocked by the LH lesion, and a retrograde viral vector encoding hM4Di that was injected into the LHb was found in the LH area. It suggests that the LHb is directly innervated by the LH, and the LH–LHb circuit conveys nociception.

Previous studies revealed that the LH is involved in drug-taking behaviors, reinstatement of drug-seeking behavior, and drug-induced synaptic plasticity (Aston-Jones et al., 2010; Blacktop and Sorg, 2019). Blacktop and Sorg (2019) reported that degradation of LH structures inhibits cocaine cue-induced reinstatement of drug-seeking behavior in rats (Blacktop and Sorg, 2019). The LHb also plays a critical role in drug-induced craving and aversion (Velasquez et al., 2014). The LH–LHb pathway encodes negative valence and controls motivational behaviors (Lecca et al., 2017; Lazaridis et al., 2019). For example, Lecca et al. (2017) reported that chemogenetic silencing of the LH–LHb pathway disrupts escape behaviors against a compartment paired with electric foot shocks and against the abrupt presentation of shadows mimicking an attack of predators. In our findings, both LH and LHb neurons were excited by tail pinch, and chemogenetic silencing of the LH–LHb pathway alleviated inhibitory effects of tail pinch on cocaine-enhanced locomotor activity and reinstatement of cocaine-seeking behavior, suggesting that nociceptive stimulation inhibits cocaine addictive behaviors through activation of the LH–LHb pathway. In the present study, although tail pinch induced a significant decrease in cocaine-induced psychomotor activities and relapse of cocaine-seeking behavior, food consumption behaviors were not affected by tail pinch. It was reported that modulation of the LH or the LH–LHb pathway does not affect normal feeding behaviors, and inhibition of the LH–LHb preferentially increases the consumption of the high-fat food or palatable liquid associated with reward (Stamatakis et al., 2016). The brain circuits of drugs and natural rewards are partially separated, and drugs and natural rewards differ in capacity for leading neuroadaptation in the NAc core (Nall et al., 2021). The brain circuits comprising the LH, the VTA, and the NAc core are critical for the reward effect of drugs, whereas the brain regions shared between drugs and natural rewards include the basolateral amygdala and the hippocampus (Nall et al., 2021). Furthermore, the dorsal region of the LH controls the expression of cocaine-seeking behavior but not food self-administration (Blacktop and Sorg, 2019). Solecki et al. (2020) revealed that optogenetic inhibition of VTA DA neurons reduced cue-induced reinstatement of cocaine without affecting food seeking (Solecki et al., 2020). Based on these observations, drug-specific actions of mesolimbic DA systems comprising the LH, the LHb, and the VTA may account for nociceptive modulation of cocaine-motivated behaviors but not food consumption behaviors.

The LH has been reported to be critically involved in nociceptive processing (Dafny et al., 1996; Siemian et al., 2019). In our study, in vivo electrophysiological and calcium imaging data supported that the LH neurons are activated by nociception. Previous studies have suggested that the LHb is involved in nociceptive processing (Gao et al., 1996; Hu et al., 2020). It was reported that the LHb responds to noxious but not to non-noxious stimuli (Benabid and Jeaugey, 1989). Consistent with previous studies, our in vivo electrophysiological data showed that firing rates of the LHb neurons were evoked by noxious tail pinch but not by non-noxious stimuli such as brush and light pressure. We further confirmed excitation of the LHb neurons in response to tail pinch by using in vivo calcium imaging. Furthermore, the tail-pinch-induced activation of the LHb neurons was completely blocked by a chemical lesion of the LH. It suggests that nociceptive signals of tail pinch are conveyed to the LHb via the LH. On the other hand, as shown in the raw Ca²⁺ traces of Figure 1I, most LHB neurons were excited during tail pinch, but a few LHb neurons were depressed slightly during tail pinch (Fig. 1I). LH contains at least four types of neurons—glutamatergic, GABAergic, orexinergic, and melanin-concentrating hormone-expressing neurons (Fakhoury et al., 2020). It is noted that although LH neurons projecting to LHb are predominantly glutamatergic, sparce GABAergic projections to the LHb also exist (Stamatakis et al., 2016), which may explain slight depressions of a few LHb neurons during tail pinch.

Cumulative evidence has suggested that the reward system links to the external somatosensory system (Brischoux et al., 2009; Bills et al., 2020). Somatosensory stimuli such as noxious stimulation influence the activity of DAergic neurons in the reward system (Brischoux et al., 2009). Activation of non-nociceptive cervical spine mechanoreceptors with a vibrational stimulus for 1–2 min inhibits the firing of VTA GABA neurons for min and DA release for hours (Bills et al., 2020). Furthermore, we and others have shown that somatosensory stimuli reduce drug-induced craving behaviors through modulating the mesolimbic DA systems in rats (Yang et al., 2010; Chang et al., 2019). Application of acupuncture, widely accepted as a form of peripheral sensory stimulation (Andersson and Lundeberg, 1995), decreases DA release in the NAc by activating GABA interneurons in the VTA and thus suppresses addiction-related behaviors caused by drugs such as cocaine, morphine, and ethanol (Yang et al., 2010; Chang et al., 2019). How the somatosensory stimulation affects mesolimbic DA systems is largely unknown. In the present study, tail pinch reduced NAc DA release, which was ablated by the LH lesion. Tail pinch also attenuated cocaine-enhanced locomotor activity and 50 kHz USVs, known to be associated with an increase of the NAc DA level (Kim et al., 2021), which were inhibited by chemogenetic silencing of the LH–LHb pathway. These findings suggest that somatosensory stimulation such as tail pinch suppresses the mesolimbic DA system via the LH–LHb pathway and activation of VTA GABA neurons. On the other hand, previous tracing studies demonstrated the ipsilateral predominance of the LH–LHb pathways (Sheth et al., 2017; Zahm and Root, 2017) Our results showed that the LH lesion completely blocked LHb neuronal response and NAc DA release following tail pinch. Activity of RMTg GABA neurons, a downstream target of LHb (Hong et al., 2011), by tail pinch was completely blocked by unilateral electrolytic lesion of LH. The present findings may suggest that the LH predominates ipsilaterally over the LHb–RMTg innervating mesolimbic DA system. As it was reported that the LH has sparse connectivity to contralateral LHb (Sheth et al., 2017), bilateral LH lesions in our behavioral experiments might prevent the relay of other information through the LH, which is a limitation of our present study.

Nociceptive stimulation affects the mesolimbic DA system, although there is controversy on whether the mesolimbic DA release is reduced or increased by nociceptive stimuli. A large body of studies has reported that nociception decreases mesolimbic DA release (Di Chiara et al., 1999; Ungless et al., 2004), whereas other studies have shown opposite results, reporting an increase of the mesolimbic DA release by nociceptive stimulation (Abercrombie et al., 1989; Budygin et al., 2012). As mentioned above, we have reported that non-nociceptive sensory stimuli inhibit VTA GABA neurons, excite DA neurons, and enhance DA release in the NAc core, which is opposite to what we demonstrate here with nociceptive stimuli (Bills et al., 2020). Interestingly, new insights and attempts have emerged for subtyping the NAc shell according to neuroanatomical or functional features, reporting that the subtypes of the NAc might explain the opposing functions of nociceptive stimuli on the mesolimbic DA release (de Jong et al., 2019; Yuan et al., 2019). According to a previous study, tail pinch decreased DA release and the activity of DAergic fibers in the ventrolateral part of the NAc shell (Yuan et al., 2019). On the contrary, tail pinch increases DA release and the activity of DAergic fibers in the ventromedial part of the NAc shell (Yuan et al., 2019). de Jong et al. (2019) reported that an electric foot shock suppresses the activity of DAergic fibers in the lateral part of the NAc shell, whereas it enhances the activity in the ventromedial part of the NAc shell. The present study recorded DA efflux in the ventrolateral part of the NAc shell using FSCV and found that tail pinch significantly decreases the firing rate of VTA GABA neurons and decreases mesolimbic DA release. Thus, we assumed that discrepancy in the NAc DA release by non-nociceptive and nociceptive stimulation in previous studies might be because of different DA recording sites in the NAc.

In conclusion, our findings suggest that the LH–LHb pathway plays an important role in transmitting nociceptive inputs to the mesolimbic DA system and thus inhibits cocaine-taking/seeking behaviors.