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. Author manuscript; available in PMC: 2026 Aug 23.
Published in final edited form as: Neuropharmacology. 2025 Aug 23;279:110653. doi: 10.1016/j.neuropharm.2025.110653

Hypocretin Signaling in the Central Amygdala Drives Methamphetamine Self-Administration in Male Rats

Tyler A Zarin a, Adam J Brandner b,c, Yanan Zhang d, George F Koob b, Brooke E Schmeichel a,*
PMCID: PMC12447516  NIHMSID: NIHMS2107612  PMID: 40854475

Abstract

Methamphetamine use is growing in the United States and around the world. Hypocretin/orexin (HCRT) is a neuropeptide closely associated with drug-taking behaviors and stress response systems in humans and rodents. HCRT signaling occurs at HCRT-receptor 1 (HCRT-R1) and - receptor 2 (HCRT-R2); the contribution of signaling at each of these receptors to methamphetamine self-administration in rodents is largely unknown. These studies investigate the extent to which pharmacological HCRT-R1 and -R2 antagonists (RTIOX-276 [RTI] and JNJ-10397049 [JNJ], respectively) attenuate methamphetamine intake in adult male Wistar rats allowed either short or long access to intravenous methamphetamine self-administration under fixed and progressive ratio schedules of reinforcement. RTI and JNJ each elicited a dose-dependent attenuation of methamphetamine intake in rats allowed long, but not short, access to methamphetamine. Each antagonist also produced mild sedative-like effects on locomotor activity in drug naïve and short, but not long, access self-administration in rats. To further examine the role of HCRT in methamphetamine long access conditions, we measured HCRT-1 and HCRT-receptor densities in the amygdala, finding increased HCRT-1 and HCRT-R1 in the central amygdala (CeA), suggesting CeA as a target for HCRT activity in methamphetamine addiction-like behavior. Subsequent chemogenetic silencing of HCRT-neuronal projections from the dorsal hypothalamus to the CeA, a stress sensitive brain region implicated in addiction-like behaviors, significantly attenuated methamphetamine self-administration in rats allowed long access. Combined, these results suggest that HCRT signaling in the CeA is necessary for escalated methamphetamine-taking behavior characteristic of methamphetamine addiction.

1. Introduction

Methamphetamine misuse is a rapidly growing health issue in the US, with a reported 2.6 million people having used methamphetamine in 2023 (SAMHSA report 2024). Compulsive use of methamphetamine (or other stimulants such as cocaine or prescription stimulants) by an individual can often lead to the emergence of a stimulant use disorder that is defined by misuse of stimulant drugs or medications despite causing physical, social, or financial harm to the individual (Ingram & Montresor-Lopez 2018; Jones et. al. 2020; Lipari & Park-Lee 2020; Seaman et. al. 2022). It is posited that in the transition to drug addiction, and stimulant use disorders in particular, occurs when the primary motivators for continued drug use move from positive reinforcement (e.g., driven by feelings of euphoria and social disinhibition) to negative reinforcement (e.g., driven by relief of anxiety, anhedonia, and other hyperkatifeia-like symptoms; Daley et. al. 2019; Knopf 2020). The allostatic changes to the reward and stress systems can be modeled through extended access to drug self-administration in rats (Ahmed & Koob 1998; Allain & Samaha 2019; Panlilio & Goldberg 2007; Thomsen & Caine 2005). Under this model, repeated exposures to drug withdrawal and sensitized stress signaling contribute to the negative reinforcement that escalates drug-taking (Koob 2014).

The prevalence of stimulant use disorders in the population is increasing, yet the treatment options remain limited. For stimulants, current interventions primarily involve cognitive behavioral therapies that are associated with high relapse rates, highlighting an urgent need for the development of adjuvant pharmaceutical treatments for stimulant use disorder (Andreassen et. al. 2019; Knopf 2020; Mclellan et. al. 2000). One possible therapeutic target may be found in the hypocretin/orexin (HCRT) neuropeptides, existing in forms HCRT-1 and HCRT-2, acting as ligands for HCRT-receptors 1 and 2 (HCRT-R1 and -R2, respectively; Peyron et. al. 1998; Sakurai et. al. 1998). HCRT signaling from the dorsal hypothalamus, the locus of all HCRT-producing neurons, modulates many aspects of reward seeking behavior (Baimel et. al. 2015; Harris et. al. 2005). HCRT signaling blockade through pharmacological HCRT-R1 antagonism causes significant attenuation of drug-taking behavior selectively in stimulant (i.e., cocaine) extended access rats compared to short access rats, although the effects of HCRT-R2 antagonism on drug-taking behavior has been largely understudied (James et. al. 2018; Schmeichel et. al. 2017; Zhou et al. 2012a, 2012b). However, a recent study determined differential, age-specific effects of HCRT-R1 and -R2 antagonism on methamphetamine-seeking behavior using a short access model of methamphetamine self-administration (Zlebnik et al., 2021). Surprisingly, there are very few studies investigating the role of HCRT-R neurotransmission in methamphetamine self-administration, particularly in the context of methamphetamine addiction-like behavior associated with extended access paradigms. It is notable that higher circulating levels of HCRT have been reported in the plasma of individuals recently abstinent from methamphetamine use (Chen et al., 2016), suggesting persistent activation of the HCRT system during early withdrawal. HCRT modulates motivated drug-taking behaviors through reciprocal projections between the dorsal hypothalamus and the central amygdala (CeA), a stress- and withdrawal-sensitive region that integrates reward signals and undergoes neuroadaptations promoting compulsive drug use and relapse (Fang et al., 2022; Koob 2009; Koob and Volkow, 2010). Our previous research has shown that HCRT-receptor blockade within the stress-sensitive CeA attenuates both escalated cocaine self-administration and stress-induced reinstatement of cocaine seeking in rats given extended access to the drug (Schmeichel et. al. 2017). It remains to be known whether HCRT projections to the CeA contribute to escalated methamphetamine-taking under extended access conditions.

The current studies are designed to examine the effects of HCRT-R-subtype antagonism on extended access methamphetamine self-administration in adult rats. Chemogenetic studies were employed to reveal the HCRT-related neurocircuitry involved in methamphetamine addiction-like drug-taking by investigating HCRT signaling within the CeA. We predicted that HCRT-R1 and - R2 antagonism would attenuate methamphetamine self-administration in extended, but not short, access rats and that chemogenetic inhibition of HCRT signaling to the CeA via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) would be sufficient to attenuate methamphetamine self-administration behavior in long access rats.

2. Material and Methods

2.1. Animals

Adult male Wistar rats (N=70; behavioral pharmacology and immunohistochemistry experiments: Charles River, Raleigh, NC; chemogenetic experiment: Envigo, Raleigh, NC), weighing 225–275 grams at the start of the experiments, were housed in groups of 2 per cage in a temperature controlled (22°C) vivarium on a 12/12-hour reverse light/dark cycle (lights off at 07:00, lights on at 19:00) with ad libitum access to food and water. The animals were allowed at least one week to acclimate to the vivarium before experiments began. All procedures adhered to the National Institute of Health Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of the National Institute of Drug Abuse (behavioral pharmacology studies) and at East Tennessee State University (immunohistochemical and chemogenetic studies).

2.2. Intravenous Catheterization Surgery

Rats were anesthetized with isoflurane (1–3%) and implanted with a silastic catheter (fabricated in lab; 0.51mm i.d. × 0.94mm o.d.; DuPont, Wilmington DE) into the right jugular vein under aseptic conditions. The distal portion of the catheter was routed subcutaneously to the back and passed through the skin of the rat with a metal guide canula (22-gauge; Plastics One Inc., Roanoke, VA) adhered to a piece of subcutaneous mesh. Catheters were flushed daily with 0.2 ml of sterile heparinized (30 USP units/ml) saline and prophylactic cefazolin (20mg/day; Apotex Corp., Weston, Florida). The patency of the catheters in the rats were tested using an ultra-short-acting barbiturate methohexital sodium dissolved in saline (10 mg/ml, 0.2 ml intravenous administration) if catheter failure was suspected during the study. Patency was verified with the loss of muscle tone within 5 seconds of administration.

2.3. Methamphetamine self-administration

Methamphetamine self-administration sessions were conducted in standard operant conditioning chambers (Med Associates, St. Albans, VT, USA). The rats were trained with 1 hour of access to two levers: one active lever that delivered methamphetamine in saline solution (0.05 mg/kg/infusion) on a fixed ratio (FR1) schedule of reinforcement where one lever press resulted in one 0.1 ml intravenous delivery and illumination of a cue light for a 20-second time-out period (to prevent potential overdose), and one inactive lever that had no programmed response. Upon stable levels of responding for methamphetamine (≤ 20% variation in the number of reinforcements over three consecutive sessions), rats were split evenly into two groups: short (1 hour/session) and long (6 hours/session) access. In the assigned groups, rats were given respective access to methamphetamine self-administration on an FR1 schedule of reinforcement for 12 sessions to ensure a plateau of escalation in the long access group and conducted 5–7 sessions/week to maintain stable drug intake patterns while avoiding excessive breaks (Recinto et al., 2012; Tunstall et al., 2018; Wee et al., 2007; Whitfield et al., 2015). For the progressive ratio (PR) schedule, each successive drug delivery required greater lever-pressing requirements. The response requirement began at 1 response/delivery and increased according to the following equation: responses/injection = (5 × e(injection number × 0.2))−5 (e.g., ratio schedule: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, etc.) (Richardson & Roberts 1996; Wee et. al. 2007; Whitfield et. al 2015). The PR session ended when the next dose was not achieved within 1 hour, and the maximum session time was 6 hours.

2.4. Experiments

2.4.1. Experiment 1: Behavioral pharmacology of HCRT-R antagonists

Two separate cohorts of rats (n=15 each, one rat in each cohort was dropped due to failed catheter patency) were tested for methamphetamine self-administration under first FR1, and then, PR schedules of reinforcement for one of two HCRT-R-specific antagonists after 12 escalation sessions. Each antagonist was intraperitoneally (i.p.) injected at a volume of 1 mL/kg 30 minutes prior to behavioral testing. For antagonist testing under an FR1 schedule, the rats received all doses of the antagonist (0, 10, and 20 mg/kg) in a within-subject, Latin-Square design. Locomotor activity data (measured by infrared beam crosses in the operant chamber) were collected and analyzed for the first hour of the self-administration session. For antagonist testing under a PR schedule, vehicle and the most effective dose of the HCRT-R antagonist under FR1 was tested as within-subject in a counter-balanced order. A regular FR1 methamphetamine self-administration washout session without antagonist treatment was performed between testing days. A separate third cohort of rats (n=6) was tested for 2 hours with each antagonist (at the most effective dose and vehicle) for locomotor activity in the absence of methamphetamine in a within-subject, counter-balanced order.

2.4.2. Experiment 2: Quantitative HCRT-1, -R1, and -R2 Densities

Two cohorts of rats (n=7 each) were catheterized and allowed either short or long methamphetamine access, under FR1 schedules of reinforcement, as described above. A separate third cohort of rats (n=6) was kept methamphetamine-naïve to serve as control. Following 12 sessions, all rats, including methamphetamine-naïve, were euthanized 24 hours after the last session and transcardially perfused with pre-fixative (heparinized saline) followed by 4% formaldehyde. The brains were collected, post-fixed overnight, and then subjected to a 20–30% sucrose gradient over the following days. Brains were cryosectioned at 40 μm and stored in 0.01M phosphate buffered saline (PBS) containing 0.1% sodium azide at 4°C.

Sections were then processed for HCRT-1 peptide, HCRT-R1, and HCRT-R2 immunolabeling, first rinsed with 0.3% Triton X-100 in 0.01 M PBS, then quenched with 0.75% hydrogen peroxide and blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Sections were then rinsed and incubated for 48 hours at 4°C with rabbit anti-HCRT-1 antibody (1:5000; AB6214, ABCAM, Cambridge, UK; diluted in 0.01M PBS containing 10% Triton X-100), rabbit anti-HCRT-R1 antibody (1:1000; AOR-001, Alomone Labs, Jerusalem, Israel; diluted in 0.01M PBS containing 0.1% Triton X-100), or rabbit anti-HCRT-R2 antibody (1:10000; AOR-002, Alomone Labs, Jerusalem, Israel; diluted in 0.01M PBS with 0.1% Triton X-100). Sections were then rinsed with 0.01 M PBS, incubated with goat anti-rabbit secondary antibody (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 90 minutes. After DAB staining, sections were mounted on microscope slides and cover-slipped with DPX (Sigma-Aldrich, St. Louis, MO).

Immunostained sections were analyzed using an AmScope 40–1000x microscope equipped with an MU2003-BI camera and AmScope software (version 4.11). Staining for HCRT-1, HCRT-R1, and HCRT-R2 in the regions of interest (ROI)s, specifically the full extent of the CeA (including medial, lateral, and capsular subregions) and the basolateral amygdala (BLA) using established landmarks consistent with Paxinos and Watson atlas coordinates, was quantified using brightfield microscopy. Quantification was conducted bilaterally on two coronal sections (approximately at AP level −2.3 to −2.8 mm from Bregma) with optical density measurements calculated using ImageJ/Fiji at 200x magnification. Optical densities for HCRT-1, -R1, and -R2 optical densities were measured within a capture window encompassing the entire ROI. These values were normalized to background levels within the image and averaged across four captured images per ROI (two hemispheres from two coronal sections).

2.4.3. Experiment 3: Gi-DREADD inhibition of HCRT projections to the CeA

2.4.3.1. Gi-DREADD construct and microinjection

The inhibition of HCRT projections to the CeA was conducted using a novel adeno-associated viral vector (AAV) expressing an inhibitory Gi-protein coupled receptor (hM4Di) gene fused with an HA epitope tag under the control of the PLe112 promoter (Addgene plasmid 29004; hypocretin precursor (human)). The plasmid was cloned and DREADD construct (AAV1-Ple112-HA-hM4D(Gi); HCRT-Gi-DREADD) was packaged into an AAV1 serotype (titer of 7.4×1011 GU/mL) by the National Institute on Drug Abuse Genetic Engineering and Viral Vector Core Facility (RRID:SCR_022969).

A separate cohort of rats (n=12) were catheterized and trained to self-administer methamphetamine as described above. Once baseline responding for FR1 methamphetamine self-administration was established, rats were anesthetized with isoflurane (1–3%) and mounted in a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA). The HCRT-Gi-DREADD was injected bilaterally into the field of HCRT neurons in the dorsal hypothalamus (1 μL/hemisphere; flat head, posterior −2.9 mm and mediolateral ±1.2 mm from bregma, and ventral −8.4 mm from dura) using a 30-gauge stainless steel needle and a micro-infusion pump (Kd Scientific Legato 130 pump, Holliston, MA, USA) with a flow rate of 0.33 μL/min over 3 minutes. Needles remained in place for 10 minutes to assure adequate diffusion and prevent backflow along the needle track. Intracranial guide canulae (26 gauge, 11 mm, Protech International Inc., Cornelius, NC) were then implanted bilaterally into the CeA (flat head, posterior −2.3 mm and mediolateral ±4.3 mm from bregma, and ventral −6.8 mm from dura). A head cap was then fashioned from dental acrylic and anchored to the skull via 4 screws to secure canulae position. A 33 gauge stainless-steel stylet with a threaded plastic connector (Protech International Inc.) was inserted into each guide cannula. Rats recovered for 5–7 days before resuming methamphetamine self-administration.

2.4.3.2. HCRT-Gi-DREADD effects on Methamphetamine self-administration

Following recovery, rats were allowed 12 sessions of LgA methamphetamine self-administration (final escalation session methamphetamine intake: mean 108.7 ± 10.2 infusions over 6 hours). One rat overdosed during escalation, another rat lost catheter patency, and three rats had cannulae that were unusable due to chewing. Remaining rats (n=7) were intracranially injected in a counter-balanced order with both clozapine-N-oxide (CNO; MilliporeSigma; Burlington, MA) and vehicle (1% dimethylsulfoxide (DMSO) in artificial cerebral spinal fluid) into the CeA fifteen minutes prior to operant testing under FR1 and PR schedules of reinforcement. A standard FR1 methamphetamine self-administration washout session (without CNO or vehicle injection) was performed between testing days. Injections (0.25 μL of 100 ng CNO or vehicle per hemisphere; Mahler et al., 2014, Patterson et al., 2025, Venniro et al., 2017) were made using a 33 gauge needle with 1 mm projection beyond the guide cannula tip and a micro-infusion pump (Kd Scientific LEGATO 111 pump, Holliston, MA, USA) with a flow rate of 0.25 μL/min over 1 minute.

2.4.3.3. HCRT-Gi-DREADD Immunohistochemistry

At the conclusion of HCRT-Gi-DREADD behavioral testing, approximately 8 weeks after virus injection, rats (n=10) were administered either CNO (3 mg/kg, i.p.) or vehicle (1 ml /kg, i.p.) one hour before euthanasia to assess injection size and functional activity of the HCRT-Gi-DREADD injection in downstream brain regions. Rats were perfused with pre-fixative solution, followed by 4% formaldehyde. Brains were collected, post-fixed overnight, and then subjected to a 20–30% sucrose gradient over the following days. Brains were cryosectioned at 40 μm and stored in 0.01M PBS containing 0.1% sodium azide at 4°C.

For pre-proHCRT, HA-tag, MCH, and c-Fos immunolabeling, sections were rinsed with 0.3% Triton X-100 in 0.01 M PBS, then quenched with 0.75% hydrogen peroxide and blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Sections were then rinsed and incubated for 24 hours at room temperature with rabbit anti-rat prepro-HCRT antibody (1:500; AB3096, EMD Millipore, Billerica, MA, USA; diluted in 0.01 M PBS containing 10% Triton X-100 [PBS-TX]), goat anti-HA-tag antibody (1:400; A190–138A, Bethyl, Montgomery, TX, USA; diluted in 0.1 M PBS containing 10% Triton X-100), goat anti-MCH antibody (1:400; sc-293231, Santa Cruz Biotech, Dallas, TX, USA; diluted in PBS-TX), or mouse anti-c-Fos antibody (1:1000; ab208942, ABCAM, Cambridge, UK; diluted in PBS-TX). Tissue was then rinsed with 0.01 M PBS, incubated with fluorescent donkey anti-rabbit secondary antibody (1:200; ab150063, ABCAM, Cambridge, UK), donkey anti-goat secondary antibody (1:200; ab175665, ABCAM, Cambridge, UK), or donkey anti-mouse secondary antibody (1:200; ab150109, ABCAM, Cambridge, UK) for 120 minutes. After staining, sections were mounted on microscope slides and cover-slipped with Vectashield (Vector Laboratories, Newark, CA).

Immunoassayed sections were analyzed using an Olympus BX63 Automated Fluorescence Microscope equipped with two cameras (Olympus U-TV1Xc and DP74) and cellSens Dimension software. HA-tag fluorescence staining was used to visualize injection sites and extent of viral vector transduction; placements of bilateral HCRT-Gi-DREADD injections into the dorsal hypothalamus were confirmed with HA-tag fluorescence and visualization of needle track marks using brightfield microscopy. Quantification of HCRT-immunoreactive (-ir) and MCH-ir cells by manually counting distinct soma bilaterally on two coronal sections (approximate AP level −2.8 mm from Bregma) at 200x magnification from a capture window in the dorsal hypothalamus that encompassed the entire HCRT-neuronal field dorsoventrally and mediolaterally. Counts were totaled across both hemispheres from two coronal sections. Images were layered on top of each other to confirm HCRT cell specificity of HCRT-Gi-DREADD transfection. The tissue was also stained to quantify c-Fos expressing cells to confirm HCRT-Gi-DREADD-induced inhibition of neuronal activity in the CeA. Quantification of c-Fos was conducted bilaterally on two coronal sections (approximately at AP level −2.3 to −2.8 mm from Bregma, using the same anatomical boundaries of CeA established for density measures) at 200x magnification using the ImageJ/Fiji analyze particles function following threshold adjustment to control for background noise. Expression of the HA-tagged HCRT-Gi-DREADD construct was visually confirmed in postmortem tissue in the dorsal hypothalamus and in projection fields within the CeA and BLA using fluorescence microscopy. These observations were used to verify successful targeting, although expression in CeA and BLA was not quantified or included in the final figures.

2.5. Drugs

Methamphetamine HCl was provided by the NIDA Drug Supply program and dissolved in heparinized sterile saline (60 USP units heparin per 1 mL saline). HCRT-R antagonists consisted of the HCRT-R1 antagonist, RTIOX-276 (RTI; 0, 10, 20mg/kg, dissolved in vehicle; RTI International, Research Triangle Park, NC) and the HCRT-R2 antagonist, JNJ-10397049 (JNJ; 0, 10, 20mg/kg, dissolved in vehicle; Cayman Chemical, Ann Arbor, MI). Vehicle for antagonists consisted of 5%v/v Cremaphor, 10%v/v DMSO solution dissolved in sterile water. For Gi-DREADD activation, CNO was dissolved in 1%v/v DMSO in artificial cerebral spinal fluid for intracranial injections and 1%v/v DMSO in sterile saline for systemic injections for immunohistochemical studies.

2.6. Statistical Analyses

All data are expressed as means and standard errors of the mean (+SEM). To directly compare escalation curves and antagonist effect on short access and the first hour of long access methamphetamine infusions and locomotor activity (beam crossovers), means were analyzed using a repeated-measured mixed two-way analysis of variance (ANOVA), with group (short and long-first hour) as the between-subject factor and session (1–12) or antagonist dose (0, 10 and 20 mg/kg) as the within-subjects factor; total long access escalation was analyzed using a one-way ANOVA comparing mean methamphetamine infusions per 1-hour session, as the 6-hour session duration is quantitatively and behaviorally distinct from the 1-hour access condition. Dose response curves for fixed ratio self-administration and locomotor activity means were also analyzed using a repeated-measures one-way ANOVA on each group separately (short access, long access- first hour, long access- 6-hour total). Post hoc comparisons were conducted using Dunnett’s test (comparing escalation to session 1) or Tukey’s Honestly Significant Difference test, as appropriate. Locomotor activity means for methamphetamine-naïve rats were analyzed using a dependent, two-tailed Student’s t test. The mean number of methamphetamine infusions for progressive ratio self-administration were analyzed using a repeated-measures mixed two-way ANOVA with group as a between-subjects factor and antagonist dose as a within-subjects factor. HCRT optical density means were analyzed using a between subjects one-way ANOVA analysis, with post hoc comparisons performed using Tukey’s Honestly Significant Difference test when appropriate. Immunohistochemistry c-Fos counts and HCRT-Gi-DREADD behavioral means were analyzed using an unpaired (c-Fos counts) or paired (HCRT-Gi-DREADD behavior), two-tailed Student’s t test. Analyses were conducted to assess potential order effects in all within-subject comparisons, and no significant order effects were detected. P values less than 0.05 were considered statistically significant for all tests.

3. Results

3.1. Experiment 1: Behavioral Pharmacology of HCRT-R antagonists

3.1.1. Effects of HCRT-R1 antagonism on methamphetamine self-administration

RTIOX-276 (0, 10, and 20 mg/kg) administration was used to assess the effects of HCRT-R1 antagonism on methamphetamine self-administration and locomotor activity (experimental timeline shown in Figure 1A) in short (n= 8) and long (n= 7) access rats, and locomotor activity in methamphetamine-naïve rats (n= 6). In the first hour, long, but not short, access rats showed a significant escalation of methamphetamine taking over 12 sessions (Figure 1B; Group: F(1,13)= 6.58, p< 0.05; Session: F(11,143)= 16.40, p< 0.01; Group × Session: F(11,143)= 6.45, p< 0.01). Long access rats also showed a significant escalation of methamphetamine intake in the full 6-hour session (Figure 1B; long total: F(6,66)= 17.60, p< 0.01).

Figure 1. HCRT-R1 antagonist has dose dependent effects on methamphetamine self-administration and locomotion.

Figure 1.

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A) Experimental timeline. B) Symbols represent mean (±SEM) methamphetamine infusions over 12 sessions for rats given short (open circle; ShA) or long access (first hour, open triangle; LgA (1h); total time, closed triangle; LgA (6h)) self-administration. C) Bars represent mean number (+SEM) of methamphetamine infusions per RTIOX-276 dose (RTI 0, 10, 20 mg/kg) in ShA and LgA rats. D) Bars represent mean number (+SEM) of infrared beam crossovers per RTI dose in ShA, LgA, and methamphetamine-naïve rats. E) Bars represent mean number (+SEM) of methamphetamine infusions (left Y axis) and progressive ratio breakpoint (right Y axis) in ShA and LgA rats following RTI 0 or 20 mg/kg. #p <0.05 and ##p <0.01 vs respective vehicle, using two-way ANOVA. *p <0.05 and **p <0.01 using one-way ANOVA, with ^p <0.05 (post-hoc) vs first session or respective vehicle.

There was a main effect of group and dose of RTIOX-276 on methamphetamine-taking in the first hour (Figure 1C; Group: F(2,19)= 147.1, p< 0.01; Dose: F(2,38)= 15.81, p< 0.01; Group × Dose: F(4,38)= 1.17, p= 0.34). Further dose curve analysis with a repeated-measures one-way ANOVA revealed that treatment with RTIOX-276 antagonist significantly reduced methamphetamine FR1 self-administration in long access rats in both the first hour and over the total 6-hour session (Figure 1C; long first hour: F(2,12)= 7.65, p< 0.05; long total: F(2,12)= 9.02, p< 0.01), but not short access rats (F(2,14)= 1.92, p= 0.18). In long access rats, methamphetamine intake was significantly reduced during both the first hour and during the total 6-hour session at the higher dose (20 mg/kg) compared to vehicle controls, and at the lower dose (10 mg/kg) during the total 6-hour session.

There was a no main effect of group or dose of RTIOX-276 on locomotor activity in the first hour (Figure 1D; Group: F(1,11)= 0.97, p= 0.36; Dose: F(2,22)= 1.52, p= 0.24; Group × Dose: F(2,22)= 1.07, p= 0.36). However, further dose curve analysis with a repeated-measures one-way ANOVA revealed that there was a significant reduction in locomotor activity at both RTIOX-276 doses in short access rats (Figure 1D; F(2,12)= 8.43, p< 0.01), but not in long access rats (Figure 1C; F(2,10)= 0.68, p= 0.53) or in methamphetamine-naïve rats (Figure 1D; t(5)= 0.58, p= 0.59). This suggests that in short access rats, HCRT-R1 antagonism may have some effect on locomotor activity, likely due to a moderate, though statistically non-significant, reduction in methamphetamine intake, as RTIOX-276 had no effect on locomotor activity in methamphetamine-naïve rats.

There was a main effect of group but not dose of RTIOX-276 on PR infusions (Figure 1E; Group: F(1,12)= 5.19, p< 0.05; Dose: F(1,12)= 0.48, p= 0.50; Group × Dose: F(1,12)= 0.30, p= 0.59).

3.1.2. Effects of HCRT-R2 antagonism on Methamphetamine self-administration

JNJ-10397049 (0, 10, and 20 mg/kg) was used to assess the effects of HCRT-R2 antagonism on methamphetamine self-administration and locomotor activity (experimental timeline shown in Figure 2A) in short (n= 7) and long (n= 8) access rats, and locomotor activity in methamphetamine-naïve rats (n= 6). In the first hour, long, but not short, access rats showed a significant escalation of methamphetamine taking over 12 sessions (Figure 2B; Group: F(1,13)= 1.77, p= 0.21; Session: F(11,143)= 9.24, p< 0.01; Group × Session: F(11,143)= 2.27, p< 0.05). Long access rats also showed a significant escalation of methamphetamine intake in the full 6-hour session (Figure 2B; long total: F(7,77)= 18.82, p< 0.01).

Figure 2. HCRT-R2 antagonist has dose dependent effects on methamphetamine self-administration and locomotion.

Figure 2.

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A) Experimental timeline. B) Symbols represent mean (±SEM) methamphetamine infusions over 12 sessions for rats given short (open circle; ShA) or long access (first hour, open triangle; LgA (1h); total time, closed triangle; LgA (6h)) self-administration. C) Bars represent mean number (+SEM) of methamphetamine infusions per JNJ-10397049 dose (JNJ 0, 10, 20 mg/kg) in ShA and LgA rats. D) Bars represent mean number (+SEM) of infrared beam crossovers per JNJ dose in ShA, LgA, and methamphetamine-naïve rats. E) Bars represent mean number (+SEM) of methamphetamine infusions (left Y axis) and progressive ratio breakpoint (right Y axis) in ShA and LgA rats following JNJ 0 or 20 mg/kg. #p <0.05 and ##p <0.01 vs respective vehicle, using two-way ANOVA. *p <0.05 and **p <0.01 using one-way ANOVA, with ^p <0.05 (post-hoc) vs first session or respective vehicle.

There was a main effect of group and dose of JNJ-10397049 on methamphetamine-taking in the first hour (Figure 2C; Group: F(2,20)= 143.30, p< 0.01; Dose: F(2,40)= 4.50, p< 0.05; Group × Dose: F(4,40)= 1.74, p= 0.16). Further dose curve analysis with a repeated-measures one-way ANOVA revealed that treatment with JNJ-10397049 antagonist significantly reduced methamphetamine FR1 self-administration in long access rats (Figure 2C; F(2,14)= 5.25, p< 0.05) at both doses (10 and 20mg/kg) compared to vehicle during the 6-hour period. There was no significant difference at either dose during the first hour in long or short access rats (Figure 2C; long first hour: F(2,14)= 1.21, p= 0.33; short: F(2,12)= 4.0, p= 0.68). These results indicate that HCRT-R2 antagonism attenuates methamphetamine-seeking behavior in long access animals over a longer time frame.

There was a no main effect of group or dose of JNJ-10397049 on locomotor activity in the first hour (Figure 2D; Group: F(1,13)= 1.09, p= 0.31; Dose: F(2,26)= 0.05, p= 0.95; Group × Dose: F(2,26)= 1.21, p= 0.31). However, further dose curve analysis with a repeated-measures one-way ANOVA revealed that there was a significant reduction in locomotor activity at the high dose (20mg/kg) in methamphetamine-naïve rats (Figure 2C; t(6)= 3.02, p< 0.05), but not in long or short access rats (Figure 2C; long: F(2,14)= 0.60, p= 0.56; short: F(2,12)= 0.87, p= 0.44). This suggests that HCRT-R2 antagonism may affect locomotor activity but is likely counteracted by the powerful stimulant effects of methamphetamine.

There was a main effect of dose but not group of JNJ-10397049 on PR infusions (Figure 2E; Group: F(1,12)= 2.98, p= 0.11; Dose: F(1,12)= 16.07, p< 0.01; Group × Dose: F(1,12)= 1.33, p= 0.27).

3.2. Experiment 2: Quantitative HCRT-1, -R1, and -R2 densities.

Methamphetamine-naïve (n=6), short access (n=7), and long access rats (n=7) were used to assess the effects of methamphetamine on HCRT-1 peptide, HCRT-R1, and HCRT-R2 densities in two amygdalar regions: the CeA and BLA (microphotographs seen in Figure 3A–I). HCRT-1 peptide optical density was significantly increased in the CeA in both long and short access rats compared methamphetamine-naïve (Figure 3J; F(2,17)= 14.66, p< 0.01). HCRT-1 peptide optical density was significantly increased in the BLA in both long and short access rats (Figure 3M; F(2,17)= 12.47, p< 0.01) compared to methamphetamine-naïve. HCRT-R1 optical density was significantly increased in the CeA in long, but not in short, access rats (Figure 3K; F(2,17)= 3.47, p< 0.05) compared to methamphetamine-naïve but had no significant change in the BLA (Figure 3N; F(2,17)= 2.23, p= 0.14). HCRT-R2 optical density was not significantly different in the CeA in either long or short access rats (Figure 3L; F(2,17)= 1.32, p= 0.29) compared methamphetamine-naïve, nor in the BLA (Figure 3O; F(2,17)= 1.67, p= 0.22). These results verify that HCRT-1 signaling was increased in both brain regions in methamphetamine-exposed rats, but that HCRT-R1 upregulation in the CeA was specific to long access rats. This indicates potential long-term homeostatic alterations in the CeA of long access rats, further supporting the importance of HCRT signaling in that region during methamphetamine dependence.

Figure 3. ShA and LgA methamphetamine self-administration has differential effects on HCRT-1, -R1, and -R2 density in the amygdala.

Figure 3.

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A-I) Representative microphotographs (2x) of amygdala regions (dashed outlines) with staining for HCRT-1, -R1, and -R2 immunoreactivity in naïve (A/D/G), short (ShA; B/E/H) and long (LgA; C/F/I) access rats. J-O) Bars represent mean (+SEM) optical density (left axis) and percentage of methamphetamine-naïve control (right Y axis) for HCRT-1 peptide (J/M), HCRT-R1 (K/N), and HCRT-R2 (L/O) in the CeA (J-L, purple) or BLA (M-O, green) of methamphetamine-naïve, ShA, and LgA rats. ^p<0.05 and ^^p<0.01 vs naïve.

3.3. Experiment 3: Gi-DREADD inhibition of HCRT projections to the CeA

To test the sufficiency of HCRT signaling in the CeA, we examined the effects of HCRT-signaling blockade in the dorsal hypothalamus-CeA pathway in long access methamphetamine rats (n= 7; experimental timeline shown in Figure 4A). Analyses of the HCRT-Gi-DREADD infected tissue revealed an 87% infection rate of HCRT-positive neurons (Figures 4B,F) and a 0% infection rate of control MCH-positive neurons (Figure 4C). There was a significant reduction in c-Fos expression in the CeA following systemic CNO injection, compared to vehicle (Figures 4D,E,G; t(5)= 3.28, p< 0.01). These analyses indicate adequate HCRT-neuron specificity of the HCRT-Gi-DREADD and functional downstream inhibition of general cell activity via intra-CeA CNO injection. Long access rats showed a significant escalation of methamphetamine intake in the full 6-hour session (Figure 4H; long first hour: F(11,66)= 1.82, p= 0.07; long total: F(11,66)= 10.27, p< 0.01).

Figure 4. CNO activated HCRT-Gi-DREADD reduces local neuronal activity and methamphetamine drug-taking behavior in LgA rats.

Figure 4.

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A) Experimental Timeline. B and C) Representative microphotographs of coronal sections showing B) HCRT-positive (green) and HA-positive (red; HCRT-Gi-DREADD exhibiting) neurons with overlap (golden) and C) MCH-positive (blue) and HA-positive (green) neurons in the hypothalamus. D and E) Representative coronal sections showing c-Fos-positive nuclei (green) in the CeA in vehicle (VEH) or CNO treated rat. F) Bars represent the mean number (+SEM) of HCRT-positive neurons (white bar) and dual HCRT- and HA-positive (orange bar) neurons. G) Bars represent the total number (+SEM) of c-Fos particles in the CeA of methamphetamine LgA rats that were treated with either systemic VEH (white bar) or CNO (orange bar). H) Symbols represent mean (±SEM) methamphetamine infusions over 12 sessions for rats given long access (first hour, open triangle; LgA (1h); total time, closed triangle; LgA (6h)) self-administration. I) Representative drawings showing needle-tip placements (matching symbols indicate bilateral injection placement for each rat) of CNO (0 and 100ng) injection sites within the CeA. J and K) Bars represent the mean number (+SEM) of self-administered methamphetamine rewards for the first hour (J) and full 6-hour session (K) in LgA rats that received intra-CeA microinjection of either VEH (white bar) or CNO (orange bar). *p < 0.05 and **p < 0.01 versus first session; ^p < 0.05 using Student’s t test. 3V, third ventricle; BLA, basolateral amygdala; CeC, central amygdaloid nucleus, central; CeL, central amygdaloid nucleus, lateral; CeM, central amygdaloid nucleus, medial; fx, fornix; ic, internal capsule; LH, lateral hypothalamus; mfb, medial forebrain bundle; opt, optic tract.

HCRT-signaling blockade in the CeA (Figure 4I) significantly attenuated methamphetamine self-administration (FR1) in long access rats versus vehicle treated controls during the first hour (Figure 4J,K; long access first hour: t(6)= 2.79, p< 0.05; long access total: t(6)= 2.96, p< 0.05). These data suggest that HCRT-signaling in the CeA significantly contributes to methamphetamine addiction-like behavior in rats, and that CeA specific HCRT-signaling blockade is sufficient for attenuating methamphetamine self-administration.

4. Discussion

The effects of HCRT-R antagonism on methamphetamine addiction are not yet fully understood, and the specific neural circuits underlying these effects remains to be identified. We investigated the role of HCRT-receptor subtypes and HCRT signaling in the CeA in methamphetamine self-administration. Our results showed HCRT-R1 antagonism attenuated FR methamphetamine-taking during the first hour in long access male rats, and under a PR schedule of reinforcement. Both HCRT-R1 and -R2 antagonism attenuated methamphetamine-taking behavior throughout the full 6-hour session in long access rats, independent of any locomotor suppression. Furthermore, both HCRT-1 peptide and HCRT-R optical densities were increased in the CeA of long access rats, and chemogenetically inactivating HCRT signaling in the CeA significantly attenuated methamphetamine-taking behavior. Altogether, these data provide novel insights into the role of HCRT in methamphetamine addiction, suggesting functional role for HCRT-R neurotransmission in methamphetamine-taking and the CeA as a site of HCRT action contributing to methamphetamine self-administration behavior in male rats.

4.1. Pharmacological Blockade of HCRT Signaling

With the rise of dual HCRT-R antagonists (e.g., suvorexant, daridorexant, and lemborexant) gaining FDA approval for the treatment of insomnia, very few preclinical studies have directly compared the effects of subtype-specific HCRT-R antagonists on drug-taking and seeking behavior, despite indications that HCRT-R-specific treatments could be tailored to individual or drug class characteristics. However, there have been inconsistent reports on HCRT-R2 efficacy in stimulant (e.g., cocaine) drug administration models despite a reported upregulation of HCRT-R2 levels in the nucleus accumbens following cocaine exposure (Gentile et. al. 2017; Smith et al., 2009; Zhang et al., 2007). The specific contributions of HCRT-R subtypes to methamphetamine drug-taking and seeking behaviors are still unknown. In the current study, experiment 1 compared the effects of two HCRT-R subtype-specific antagonists, RTIOX-276 and JNJ-10397049, on drug-taking and locomotor activity in rats allowed short or long access to methamphetamine self-administration. In short access rats, neither antagonist significantly affected methamphetamine self-administration on either FR or PR schedules of reinforcement, suggesting that HCRT-R signaling plays a limited role in the immediate, positive reinforcement associated with acute drug reward. The absence of an effect in short access rats is consistent with previous HCRT-R antagonism research in stimulants which also found minimal impact of HCRT-R antagonism on acute reward response under low FR schedules of reinforcement (España et. al. 2010; Hutcheson et. al. 2011 Lawrence et. al. 2006; Schmeichel et. al., 2017; Smith et. al. 2009; Zhou et. al., 2012). In contrast, for long access rats, HCRT-R1 antagonism attenuated methamphetamine-taking behavior at the 20 mg/kg dose during the initial hour, and both HCRT-R1 and -R2 antagonist at the 10 and 20 mg/kg doses attenuated methamphetamine-taking during the full 6-hour session under the low FR1 schedule of reinforcement. These results show that HCRT-R1 antagonism had a faster onset of effect than HCRT-R2 antagonism, but both had a significant effect on methamphetamine self-administration indicating a role for HCRT signaling at both HCRT-R-subtypes in the maintenance of escalated methamphetamine-taking behavior. Only the HCRT-R1 antagonist reduced effortful drug-taking on the PR schedule of reinforcement. Overall, the findings demonstrate a differential effect of HCRT-R1 and -R2 antagonism on methamphetamine self-administration, depending on access condition; HCRT-R1 antagonism showed selective efficacy of attenuating methamphetamine self-administration in both low effort FR1 and more effortful PR schedules of reinforcement in escalated methamphetamine-taking rats.

It is well known that HCRT-R antagonism can induce somnolence in humans and animals. Thus, in experiment 1, we additionally measured locomotor activity to determine whether the effect of HCRT-R antagonism on drug-taking could be attributed to an antagonist-specific disruption of general behavioral performance. While locomotion was not affected in methamphetamine-naïve rats by the HCRT-R1 antagonist, there was a significant reduction in locomotor activity in short access rats at both doses of the HCRT-R1 antagonist, and in methamphetamine-naïve rats at the 20 mg/kg dose of the HCRT-R2 antagonist. These results suggest a mild sedative-like effect caused by HCRT-R antagonism, which has also been observed in prior research (Smith et. al. 2009). In contrast, in long access rats, there was no effect of either HCRT-R antagonist locomotor behavior, suggesting the sedative-like effect shown in short access and methamphetamine-naïve rats was likely counteracted by the robust stimulant locomotor-enhancing effects of methamphetamine exposure in long access rats.

Altogether, these results from the behavioral pharmacology studies in experiment 1 show that signaling at both HCRT-R1 and -R2 plays a significant role in sustaining methamphetamine-taking behavior in long access rats but has no effect on methamphetamine-taking in short access rats. This finding enhances our understanding of how subtype-specific HCRT-R antagonism could be targeted to mitigate drug-taking behaviors in stimulant addiction. Due to the faster onset effects from HCRT-R1 antagonism, one might hypothesize that HCRT-R1 antagonism is a more effective pharmacotherapy target for stimulant use disorders in comparison to HCRT-R2 antagonism, although HCRT-R2 antagonism did show mild effectiveness. Additional preclinical studies comparing the effectiveness of dual-HCRT-R antagonism versus single-HCRT-R antagonism on methamphetamine and other drug-seeking behavior could further enhance our understanding of HCRT signaling on addiction-like behavior across various drug classes. It is also important to note that these findings are limited to male rats, and future studies should include both sexes, particularly given evidence that sex differences influence methamphetamine self-administration (Reichel et al., 2012).

4.2. HCRT signaling in the central amygdala and the effects of site-specific blockade

Systemic pharmaceutical antagonism of HCRT-Rs in experiment 1 determined the effects of HCRT-R-specific blockade on drug-taking behavior in methamphetamine exposed rats. The results support our hypothesis that signaling at HCRT-Rs is necessary for methamphetamine-taking behavior in rats with extended access to methamphetamine self-administration, a model for compulsive-like responding. We further hypothesized that highly motivated methamphetamine drug-taking behavior is modulated, in part, by recruitment of HCRT neuropeptide to the CeA, a stress-sensitive brain region closely associated with drug withdrawal (Boutrel et. al. 2005; Koob and Volkow 2010; Koob et. al. 2014; Schmeichel et. al. 2017; Volkow et. al. 2019). HCRT plays a key role in the homeostatic regulation of arousal and stress responses, with signaling amplified throughout the brain during periods of anxiety or duress (Baldo et. al. 2003; James et. al. 2018; Johnson et. al. 2009; Koob 2013; Suzuki et. al. 2005). We hypothesize that the transition from moderate, controlled methamphetamine use to compulsive methamphetamine use associated with stimulant use disorders is driven by negative reinforcement, mediated in part by stress-related HCRT activity. Prior research demonstrates that stress peptides, including HCRT, dynorphin, and corticotropin releasing factor, in the CeA contribute to compulsive drug-taking in rats (Koob et. al. 2014; Matzeu & Martin-Fardon 2018; Schmeichel et. al. 2017; Zorilla et. al. 2001). In experiment 2, we observed a significant increase in the optical density of both HCRT-1 peptide and HCRT-R1 in the CeA methamphetamine-exposed rats, suggesting neuroplasticity of long-term HCRT signaling in the CeA. In the BLA, we detected increased HCRT-1 peptide without changes in receptor expression, suggesting that although HCRT inputs to the BLA may be modulated by methamphetamine exposure, the absence of receptor upregulation limits downstream signaling. In contrast, the CeA exhibits both ligand and receptor adaptations in long access rats, highlighting its unique role in mediating stress-related aspects of drug dependence. Future studies should investigate whether HCRT peptide changes contribute to BLA-modulated cue-based behaviors (Keefer et al., 2021; Koob 2021; Koob and Volkow, 2009; Li et al., 2023) or require concurrent receptor adaptations to influence drug seeking. To examine the specific role of HCRT signaling in the CeA in methamphetamine self-administration, we targeted the dorsal hypothalamus-CeA HCRT circuit using localized chemogenetic methods.

In experiment 3, we utilized a novel inhibitory HCRT-Gi-DREADD system to target the HCRT neuronal field in the dorsal hypothalamus. The Gi-DREADD was activated via CNO, a selective exogenous DREADD ligand that lacks endogenous binding sites and therefore interacts exclusively with the DREADD receptor (Cope et. al. 2019; Smith et. al. 2016). First, to verify that the HCRT-Gi-DREADD construct specifically transfected HCRT neurons, we performed fluorescence immunohistochemical analysis of dorsal hypothalamus brain tissue, staining for the viral marker (HA-tag), prepro-HCRT and melanin-concentrating hormone (MCH). We observed an 87% overlap between virus-tagged and prepro-HCRT-tagged neurons, with no overlap between virus-tagged and MCH-tagged neurons also in the dorsal hypothalamus, confirming precise targeting of HCRT neurons. We also verified the functionality of the HCRT-Gi-DREADD system using c-fos, a marker for neuronal activity, in the CeA. Systemic CNO administration significantly reduced CeA neuronal activity compared to vehicle-treated controls, demonstrating that CeA activation depends on excitatory input from HCRT neurons.

Next, to test the causal role of CeA HCRT neuronal activity in methamphetamine-taking behavior, we administered bilateral CNO microinjections into the CeA of long access rats prior to the self-administration session. Short access rats were excluded from this experiment, as HCRT-R blockade had no effect on methamphetamine-taking behavior in Experiment 1, suggesting limited involvement of this system under non-escalated conditions. However, future studies directly comparing the effects of chemogenetic inhibition in both short and long access animals will be important to more definitively determine whether CeA-directed HCRT projections are uniquely engaged in escalated, dependence-like methamphetamine use. In experiment 3, CeA-specific inhibition of HCRT neurons via Gi-HCRT-DREADD activation significantly decreased methamphetamine-taking behavior during both the first hour and full six hours session compared to vehicle. These findings suggest that CeA HCRT signaling plays a role in the elevated drug-seeking behavior associated with methamphetamine dependence in the long-access model. However, we acknowledge that the absence of a control viral group lacking inhibitory DREADD expression represents a limitation in our study. Future studies should include a non-DREADD viral control to confirm that observed behavioral effects are solely due to HCRT neuron inhibition. Furthermore, while minor CNO diffusion into adjacent regions cannot be entirely ruled out, the small injection volume and post-mortem verification of cannula placement make this unlikely. We also acknowledge that we did not include a separate CNO-only control group in this study. Although CNO was administered locally in a small volume to limit non-specific effects, we cannot fully exclude the possibility of off-target actions. Future studies using appropriate CNO-only control groups or alternative DREADD ligands (e.g., compound 21) will help further isolate DREADD-specific effects.

Although the Gi-HCRT-DREADD was designed to target HCRT-producing neurons specifically, CNO-mediated inhibition of HCRT release may also trigger local neuropeptide adaptations affecting drug-taking behavior. In the dorsal hypothalamus, HCRT and dynorphin are nearly completely co-localized (Chou et. al. 2001; Muschamp et. al. 2014; Schmeichel et. al. 2018), so reducing HCRT release likely diminished dynorphin co-release as well. Given that dynorphin signaling blockade decreases drug-taking in long access models (Schlosburg et. al. 2013; Whitfield et. al. 2015), our observed attenuation of methamphetamine self-administration in long access rats may reflect combined reductions in HCRT and dynorphin signaling. Intra-CeA HCRT-R antagonist microinjections while controlling dynorphin activity will be essential to establish HCRT receptor-level specificity. Nonetheless, our findings extend a growing literature implicating the CeA as a critical brain region recruited during not only methamphetamine addiction-like behavior, but addiction-like behavior across multiple drug classes (Baldo et. al. 2003; Gilpin et. al. 2011; Roberto et. al. 2010; Wrase et. al. 2008). Future studies with larger sample sizes and independent cohorts will be important to replicate and further validate these findings, strengthening their translational relevance.

4.3. Summary

In conclusion, HCRT-R1 or -R2 antagonism had no effect on methamphetamine-taking in short access rats, but significantly reduced intake in long access rats. Additionally, HCRT-Gi-DREADD-mediated inhibition of HCRT projections to the CeA significantly attenuated methamphetamine-taking behavior in long access rats, highlighting the critical role of CeA HCRT signaling in modulating dependent-like methamphetamine use. Combined, these findings support HCRT-R antagonism as a promising therapeutic strategy for the treatment of stimulant use disorders and underscore the HCRT-CeA circuit as a key component of addiction-like behavior.

Highlights.

  • HCRT-R1 and -R2 blockade reduces methamphetamine intake in long access rats

  • Long access to methamphetamine increases HCRT-1 and -R1 in the central amygdala

  • Silencing HCRT pathways to the central amygdala lowers methamphetamine intake

  • HCRT signaling in the central amygdala promotes escalated methamphetamine taking

Acknowledgements

AAV viral vectors were produced by the National Institute on Drug Abuse Genetic Engineering and Viral Vector Core Facility (RRID:SCR_022969).

Funding Sources

This work was supported by the National Institutes of Health (DA042110) and the National Institute of Drug Abuse Intramural Research Program.

Footnotes

Declaration of Competing Interests

The authors have nothing to declare.

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Reference List

  1. Ahmed SH, & Koob GF (1998). Transition from moderate to excessive drug intake: change in hedonic set point. Science, 282, 298–300. https://www.science.org/doi/10.1126/science.282.5387.298 [DOI] [PubMed] [Google Scholar]
  2. Allain F, & Samaha AN (2019). Revisiting long-access versus short-access cocaine self-administration in rats: intermittent intake promotes addiction symptoms independent of session length. Addiction Biology, 24(4), 641–651. 10.1111/adb.12629 [DOI] [PubMed] [Google Scholar]
  3. Andreassen Ø, Brønnick K, Njå A-L, Furulund E, & Nesvåg S (2019). The Effect of High-Intensity Interval/Circuit Training on Cognitive Functioning and Quality of Life During Recovery From Substance Abuse Disorder. A Study Protocol. Frontiers in Psychology, 0, 2564. 10.3389/FPSYG.2019.02564 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baimel C, Bartlett SE, Chiou LC, Lawrence AJ, Muschamp JW, Patkar O, Tung LW, & Borgland SL (2015). Orexin/hypocretin role in reward: implications for opioid and other addictions. British Journal of Pharmacology, 172(2), 334–348. 10.1111/BPH.12639 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baldo BA, Daniel RA, Berridge CW, & Kelley AE (2003). Overlapping distributions of orexin/hypocretin- and dopamine-β-hydroxylase immunoreactive fibers in rat brain regions mediating arousal, motivation, and stress. Journal of Comparative Neurology, 464(2), 220–237. 10.1002/CNE.10783 [DOI] [PubMed] [Google Scholar]
  6. Boutrel B, Kenny PJ, Specio SE, Martin-Fardon R, Markou A, Koob GF, & De Lecea L (2005). Role for hypocretin in mediating stress-induced reinstatement of cocaine-seeking behavior. Proceedings of the National Academy of Sciences of the United States of America, 102(52), 19168–19173. 10.1073/PNAS.0507480102 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen WY, Kao CF, Chen PY, Lin SK, Huang MC Orexin-A level elevation in recently abstinent male methamphetamine abusers. Psychiatry Res. 2016. May 30;239:9–11. 10.1016/j.psychres.2016.02.059 [DOI] [PubMed] [Google Scholar]
  8. Chou TC, Lee CE, Lu J, Elmquist JK, Hara J, Willie JT, Beuckmann CT, Chemelli RM, Sakurai T, Yanagisawa M, Saper CB, & Scammell TE (2001). Orexin (Hypocretin) Neurons Contain Dynorphin. The Journal of Neuroscience, 21(19), RC168. 10.1523/JNEUROSCI.21-19-J0003.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cope ZA, Vazey EM, Floresco SB, & Aston Jones GS (2019). DREADD-mediated modulation of locus coeruleus inputs to mPFC improves strategy set-shifting. 10.1016/j.nlm.2019.02.009 [DOI] [PubMed] [Google Scholar]
  10. Daley J, Elliott FJ, Gibbs TM, & Kitteringham G (2019). Substance abuse. In The Professional Protection Officer: Practical Security Strategies and Emerging Trends (pp. 233–252). Butterworths. 10.1016/B978-0-12-817748-8.00022-5 [DOI] [Google Scholar]
  11. España RA, Oleson EB, Locke JL, Brookshire BR, Roberts DCS, & Jones SR (2010). The hypocretin–orexin system regulates cocaine self-administration via actions on the mesolimbic dopamine system. European Journal of Neuroscience, 31(2), 336–348. 10.1111/J.1460-9568.2009.07065.X [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fang Yehong, Sun Yunkai, Liu Yi, Liu Tieqiao, Hao Wei, Liao Yanhui, Neurobiological mechanisms and related clinical treatment of addiction: a review, Psychoradiology, Volume 2, Issue 4, December 2022, Pages 180–189, 10.1093/psyrad/kkac021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gentile TA, Simmons SJ, Barker DJ, Shaw JK, España RA, & Muschamp JW (2018). Suvorexant, an orexin/hypocretin receptor antagonist, attenuates motivational and hedonic properties of cocaine. Addiction Biology, 23(1), 247. 10.1111/ADB.12507 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gentile TA, Simmons SJ, Watson MN, Connelly KL, Brailoiu E, Zhang Y, & Muschamp JW (2017). Effects of Suvorexant, a Dual Orexin/Hypocretin Receptor Antagonist, on Impulsive Behavior Associated with Cocaine. Neuropsychopharmacology 2018 43:5, 43(5), 1001–1009. 10.1038/npp.2017.158 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gilpin NW, Misra K, Herman MA, Cruz MT, Koob GF, & Roberto M (2011). Neuropeptide y opposes alcohol effects on gamma-aminobutyric acid release in amygdala and blocks the transition to alcohol dependence. Biological Psychiatry, 69(11), 1091–1099. 10.1016/j.biopsych.2011.02.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harris GC, Wimmer M, & Aston-Jones G (2005). A role for lateral hypothalamic orexin neurons in reward seeking. 10.1038/nature04071 [DOI] [PubMed] [Google Scholar]
  17. Hutcheson DM, Quarta D, Halbout B, Valerio E, & Heidbreder C (2011). Orexin-1 receptor antagonist SB-334867 reduces the acquisition and expression of cocaine-conditioned reinforcement and the expression of amphetamine-conditioned reward. Behavioural Pharmacology, 22, 173–181. 10.1097/FBP.0b013e328343d761 [DOI] [PubMed] [Google Scholar]
  18. Ingram DD, & Montresor-Lopez JA (2018). Drug Overdose Deaths in the United States, 1999–2017. U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Centers, 329, 1–8. https://www.cdc.gov/nchs/data/databriefs/db329_tables-508.pdf#3. [Google Scholar]
  19. James MH, Stopper CM, Zimmer BA, Koll NE, Bowrey HE, & Aston-Jones G (2018). Increased number and activity of a lateral subpopulation of hypothalamic orexin/hypocretin neurons underlies the expression of an addicted state in rats. In bioRxiv. 10.1101/356220 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Johnson PL, Truitt W, Fitz SD, Minick PE, Dietrich A, Sanghani S, Träskman-Bendz L, Goddard AW, Brundin L, & Shekhar A (2009). A key role for orexin in panic anxiety. Nature Medicine, 16(1), 111–115. 10.1038/NM.2075 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones CM, Compton WM, & Mustaquim D (2020). Patterns and Characteristics of Methamphetamine Use Among Adults — United States, 2015–2018. MMWR. Morbidity and Mortality Weekly Report, 69(12), 317–323. 10.15585/MMWR.MM6912A1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Keefer SE, Gyawali U, Calu DJ. Choose your path: Divergent basolateral amygdala efferents differentially mediate incentive motivation, flexibility and decision-making. Behav Brain Res. 2021. Jul 9;409:113306. 10.1016/j.bbr.2021.113306 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Knopf A (2020). NIDA: Plethora of treatments for SUDs, regardless of substance. Alcoholism & Drug Abuse Weekly, 32(24), 3–4. 10.1002/ADAW.32751 [DOI] [Google Scholar]
  24. Koob GF (2009). Brain stress systems in the amygdala and addiction. Brain Research, 2009 Mar 28;1293:61–75. 10.1016/j.brainres.2009.03.038 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koob GF (2013). Negative reinforcement in drug addiction: The darkness within. Current Opinion in Neurobiology, 23(4), 559–563. 10.1016/j.conb.2013.03.011 [DOI] [PubMed] [Google Scholar]
  26. Koob GF. A Basolateral Amygdala Microcircuit for Drug Craving: Is There a Craving Engram? Biol Psychiatry. 2021. Feb 15;89(4):323–325. 10.1016/j.biopsych.2020.11.008 [DOI] [PubMed] [Google Scholar]
  27. Koob GF, Buck CL, Cohen A, Edwards S, Park PE, Schlosburg JE, Schmeichel B, Vendruscolo LF, Wade CL, Whitfield TW, & George O (2014). Addiction as a stress surfeit disorder. Neuropharmacology, 76(PART B), 370–382. 10.1016/J.NEUROPHARM.2013.05.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Koob GF, & Volkow ND (2010). Neurocircuitry of Addiction. Neuropsychopharmacology, 35(1), 217. 10.1038/NPP.2009.110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lawrence AJ, Cowen MS, Yang HJ, Chen F, & Oldfield B (2006). The orexin system regulates alcohol-seeking in rats. British Journal of Pharmacology, 148(6), 752. 10.1038/SJ.BJP.0706789 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li JY, Yu YJ, Su CL et al. Modulation of methamphetamine memory reconsolidation by neural projection from basolateral amygdala to nucleus accumbens. Neuropsychopharmacol. 48, 478–488 (2023). 10.1038/s41386-022-01417-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lipari RN, & Park-Lee E (2020). Key Substance Use and Mental Health Indicators in the United States: Results from the 2019 National Survey on Drug Use and Health.
  32. Mahler S, Vazey E, Beckley J et al. Designer receptors show role for ventral pallidum input to ventral tegmental area in cocaine seeking. Nat Neurosci 17, 577–585 (2014). 10.1038/nn.3664 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matzeu A, & Martin-Fardon R (2018). Drug Seeking and Relapse: New Evidence of a Role for Orexin and Dynorphin Co-transmission in the Paraventricular Nucleus of the Thalamus. Frontiers in Neurology, 9(AUG). 10.3389/FNEUR.2018.00720 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Matzeu A, & Martin-Fardon R (2020). Blockade of Orexin Receptors in the Posterior Paraventricular Nucleus of the Thalamus Prevents Stress-Induced Reinstatement of Reward-Seeking Behavior in Rats With a History of Ethanol Dependence. Frontiers in Integrative Neuroscience, 14, 599710. 10.3389/FNINT.2020.599710/BIBTEX [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mclellan AT, Lewis DC, O’brien CP, & Kleber HD (2000). Drug Dependence, a Chronic Medical Illness Implications for Treatment, Insurance, and Outcomes Evaluation. JAMA, 284(13), 1689. www.jama.com [DOI] [PubMed] [Google Scholar]
  36. Muschamp JW, Hollander JA, Thompson JL, Voren G, Hassinger LC, Onvani S, Kamenecka TM, Borgland SL, Kenny PJ, & Carlezon WA (2014). Hypocretin (orexin) facilitates reward by attenuating the antireward effects of its cotransmitter dynorphin in ventral tegmental area. Proceedings of the National Academy of Sciences of the United States of America, 111(16), E1648. 10.1073/PNAS.1315542111/-/DCSUPPLEMENTAL [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. National Center for Health Statistics. (2019). United States Health. https://www.cdc.gov/nchs/hus/index.htm. [Google Scholar]
  38. Panlilio LV, & Goldberg SR (2007). Self-administration of drugs in animals and humans as a model and an investigative tool. Addiction, 102(12), 1863–1870. 10.1111/j.1360-0443.2007.02011.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Patterson D, Khan N, Collins EA, Stewart NR, Sassaninejad K, Yeates D, et al. (2025) Ventral hippocampus to nucleus accumbens shell circuit regulates approach decisions during motivational conflict. PLoS Biol 23(1): e3002722. 10.1371/journal.pbio.3002722 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, & Kilduff TS (1998). Neurons containing hypocretin (orexin) project to multiple neuronal systems. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 18(23), 9996–10015. 10.1523/JNEUROSCI.18-23-09996.1998 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Recinto P, Samant A, Chavez G et al. Levels of Neural Progenitors in the Hippocampus Predict Memory Impairment and Relapse to Drug Seeking as a Function of Excessive Methamphetamine Self-Administration. Neuropsychopharmacology 37, 1275–1287 (2012). 10.1038/npp.2011.315 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Reichel CM, Chan CH, Ghee SM et al. Sex differences in escalation of methamphetamine self-administration: cognitive and motivational consequences in rats. Psychopharmacology 223, 371–380 (2012). 10.1007/s00213-012-2727-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Richardson NR, & Roberts DCS (1996). Progressive ratio schedules in drug self-administration studies in rats: a method to evaluate reinforcing efficacy. 66, 1–11. 10.1016/0165-0270(95)00153-0 [DOI] [PubMed] [Google Scholar]
  44. Roberto M, Cruz MT, Gilpin NW, Sabino V, Schweitzer P, Bajo M, Cottone P, Madamba SG, Stouffer DG, Zorrilla EP, Koob GF, Siggins GR, & Parsons LH (2010). Corticotropin Releasing Factor-Induced Amygdala Gamma-Aminobutyric Acid Release Plays a Key Role in Alcohol Dependence. Biological Psychiatry, 67(9), 831–839. 10.1016/j.biopsych.2009.11.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JRS, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, … Yanagisawa M (1998). Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell, 92(4), 573–585. 10.1016/S0092-8674(00)80949-6 [DOI] [PubMed] [Google Scholar]
  46. SAMHSA Report; Center for Behavioral Health Statistics. (2024). Key Substance Use and Mental Health Indicators in the United States: Results from the 2023 National Survey on Drug Use and Health. https://www.samhsa.gov/data/report/2023-nsduh-annual-national-report
  47. Schlosburg JE, Whitfield TW, Park PE, Crawford EF, George O, Vendruscolo LF, & Koob GF (2013). Long-Term Antagonism of κ Opioid Receptors Prevents Escalation of and Increased Motivation for Heroin Intake. The Journal of Neuroscience, 33(49), 19384. 10.1523/JNEUROSCI.1979-13.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schmeichel BE, Herman MA, Roberto M, & Koob GF (2017). Hypocretin Neurotransmission Within the Central Amygdala Mediates Escalated Cocaine Self-administration and Stress-Induced Reinstatement in Rats. Biological Psychiatry, 81(7), 606–615. 10.1016/J.BIOPSYCH.2016.06.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schmeichel BE, Matzeu A, Koebel P, Vendruscolo LF, Sidhu H, Shahryari R, Kieffer BL, Koob GF, Martin-Fardon R, & Contet C (2018). Knockdown of hypocretin attenuates extended access of cocaine self-administration in rats. Neuropsychopharmacology, 43(12), 2373. 10.1038/S41386-018-0054-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Seaman RW, Lordson C, & Collins GT (2022). Modeling Stimulant and Opioid Co-use in Rats Provided Concurrent Access to Methamphetamine and Fentanyl. Frontiers in Psychiatry, 13, 814574. 10.3389/FPSYT.2022.814574 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Smith KS, Bucci DJ, Luikart BW, & Mahler SV (2016). DREADDs: Use and application in behavioral neuroscience. In Behavioral Neuroscience (Vol. 130, Issue 2, pp. 137–155). 10.1037/bne0000135 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Smith RJ, See RE, & Aston-Jones G (2009). Orexin/hypocretin signaling at the orexin 1 receptor regulates cue-elicited cocaine-seeking. European Journal of Neuroscience, 30(3), 493–503. 10.1111/j.1460-9568.2009.06844.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Suzuki M, Beuckmann CT, Shikata K, Ogura H, & Sawai T (2005). Orexin-A (hypocretin-1) is possibly involved in generation of anxiety-like behavior. Brain Research, 1044(1), 116–121. 10.1016/J.BRAINRES.2005.03.002 [DOI] [PubMed] [Google Scholar]
  54. Thomsen M, & Caine SB (2005). Chronic intravenous drug self-administration in rats and mice. Current Protocols in Neuroscience / Editorial Board, Jacqueline N. Crawley ... (et Al.), Chapter 9, 1–40. 10.1002/0471142301.ns0920s32 [DOI] [PubMed] [Google Scholar]
  55. Tunstall BJ, Ho CP, Cao J, Vendruscolo JCM, Schmeichel BE, Slack RD, Tanda G, Gadiano AJ, Rais R, Slusher BS, Koob GF, Newman AH, & Vendruscolo LF (2018). Atypical dopamine transporter inhibitors attenuate compulsive-like methamphetamine self-administration in rats. Neuropharmacology, 131, 96–103. 10.1016/j.neuropharm.2017.12.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Venniro M, Caprioli D, Zhang M, Whitaker LR, Zhang S, Warren BL, Cifani C, Marchant NJ, Yizhar O, Bossert JM, Chiamulera C, Morales M, Shaham Y. The Anterior Insular Cortex→Central Amygdala Glutamatergic Pathway Is Critical to Relapse after Contingency Management. Neuron. 2017. Oct 11;96(2):414–427.e8. 10.1016/j.neuron.2017.09.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Volkow ND, Michaelides M, & Baler R (2019). The Neuroscience of Drug Reward and Addiction. Physiological Reviews, 99(4), 2115. 10.1152/PHYSREV.00014.2018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wee S, Specio SE, & Koob GF (2007). Effects of dose and session duration on cocaine self-administration in rats. Journal of Pharmacology and Experimental Therapeutics, 320(3), 1134–1143. 10.1124/jpet.106.113340 [DOI] [PubMed] [Google Scholar]
  59. Wee S, Wang Z, Woolverton WL, Pulvirenti L, & Koob GF (2007). Effect of Aripiprazole, a Partial Dopamine D2 Receptor Agonist, on Increased Rate of Methamphetamine Self-Administration in Rats with Prolonged Session Duration. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology, 32(10), 2238. 10.1038/SJ.NPP.1301353 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Whitfield TW, Schlosburg JE, Wee S, Gould A, George O, Grant Y, Zamora-Martinez ER, Edwards S, Crawford E, Vendruscolo LF, & Koob GF (2015). κ Opioid Receptors in the Nucleus Accumbens Shell Mediate Escalation of Methamphetamine Intake. The Journal of Neuroscience, 35(10), 4296. 10.1523/JNEUROSCI.1978-13.2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wrase J, Makris N, Braus DF, Mann K, Smolka MN, Kennedy DN, Caviness VS, Hodge SM, Tang L, Albaugh M, Ziegler DA, Davis OC, Kissling C, Schumann G, Breiter HC, & Heinz A (2008). Amygdala volume associated with alcohol abuse relapse and craving. American Journal of Psychiatry, 165(9), 1179–1184. 10.1176/APPI.AJP.2008.07121877/ASSET/IMAGES/LARGE/T519F1.JPEG [DOI] [PubMed] [Google Scholar]
  62. Zhang GC, Mao LM, Liu XY, & Wang JQ (2007). Long-lasting up-regulation of orexin receptor type 2 protein levels in the rat nucleus accumbens after chronic cocaine administration. Journal of Neurochemistry, 103(1), 400–407. 10.1111/J.1471-4159.2007.04748.X [DOI] [PubMed] [Google Scholar]
  63. Zhou L, Ghee SM, Chan C, Lin L, Cameron MD, Kenny PJ, See RE (2012a) Orexin-1 receptor mediation of cocaine seeking in male and female rats. Journal of Pharmacology and Experimental Therapeutics, Mar; 340(3), 801–9. 10.1124/jpet.111.187567 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhou L, Smith RJ, Do PH, Aston-Jones G, & See RE (2012b). Repeated orexin 1 receptor antagonism effects on cocaine seeking in rats. Neuropharmacology, 63(7), 1201–1207. 10.1016/J.NEUROPHARM.2012.07.044 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zlebnik NE, Holtz NA, Lepak VC, Saykao AT, Zhang Y, & Carroll ME (2021). Age-specific treatment effects of orexin/hypocretin-receptor antagonism on methamphetamine-seeking behavior. Drug and Alcohol Dependence, 224, 108719. 10.1016/J.DRUGALCDEP.2021.108719 [DOI] [PMC free article] [PubMed] [Google Scholar]

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