Abstract
Opioid use disorder constitutes a major health and economic burden, but our limited understanding of the underlying neurobiology impedes better interventions. Alteration in the activity and output of dopamine (DA) neurons in the ventral tegmental area (VTA) contributes to drug effects, but the mechanisms underlying these changes remain relatively unexplored. We used translating ribosome affinity purification (TRAP) and RNA sequencing to identify gene expression changes in mouse VTA DA neurons following chronic morphine exposure. We found that expression of the neuropeptide neuromedin S (NMS) is robustly increased in VTA DA neurons by morphine. Using an NMS-iCre driver line, we confirmed that a subset of VTA neurons express NMS and that chemogenetic modulation of VTA NMS neuron activity altered morphine responses in male and female mice. Specifically, VTA NMS neuronal activation promoted morphine locomotor activity while inhibition reduced morphine locomotor activity and conditioned place preference. Interestingly, these effects appear specific to morphine, as modulation of VTA NMS activity did not affect cocaine behaviors, consistent with our data that cocaine administration does not increase VTA Nms expression. Chemogenetic manipulation of VTA neurons that express glucagon-like peptide, a transcript also robustly increased in VTA DA neurons by morphine, does not alter morphine-elicited behavior, further highlighting the functional relevance of VTA NMS-expressing neurons. Together, our current data suggest that NMS-expressing neurons represent a novel subset of VTA neurons that may be functionally relevant for morphine responses and support the utility of cell-type–specific analyses like TRAP to identify neuronal adaptations underlying substance use disorder.
Significance Statement
The opioid epidemic remains prevalent in the USA, with >70% of overdose deaths caused by opioids. The ventral tegmental area (VTA) is responsible for regulating reward behavior. Although drugs of abuse can alter VTA dopaminergic neuron function, the underlying mechanisms have yet to be fully explored. This is partially due to the cellular heterogeneity of the VTA. Here, we identify a novel subset of VTA neurons that express the neuropeptide neuromedin S (NMS). Nms expression is robustly increased by morphine and alteration of VTA NMS neuronal activity is sufficient to alter morphine-elicited behaviors. Our findings are the first to implicate NMS-expressing neurons in drug behavior and thereby improve our understanding of opioid-induced adaptations in the VTA.
Introduction
The mesocorticolimbic dopamine system is a critical circuit for reward processing. This includes exogenous rewards such as drugs of abuse, which alter the activity and output of dopamine (DA) neurons in the ventral tegmental area (VTA; Nestler, 2005). For example, acute administration of opioids like morphine can increase DA release into the nucleus accumbens (NAc) by disinhibition of VTA DA neurons (Di Chiara and Imperato, 1988; Johnson and North, 1992). Furthermore, chronic opioid exposure changes VTA DA neuron morphology (Sklair-Tavron et al., 1996; Chu et al., 2007; Simmons et al., 2019) which has been linked with opioid-induced changes in neuronal activity and behavior (Russo et al., 2007; Mazei-Robison et al., 2011). However, the molecular mechanisms underlying these changes in VTA DA function remain incompletely understood, in part due to the cellular heterogeneity of the VTA. While we can study mechanisms that impact VTA DA function using a candidate gene approach, an unbiased approach is likely needed to uncover novel mechanisms.
Several studies have profiled gene expression differences in the VTA, including those that arise following chronic morphine administration, but these studies had a variety of limitations. Gene profiling studies that identify changes induced by opioid exposure or withdrawal have utilized whole VTA RNA extraction (McClung et al., 2005; Townsend et al., 2021; Olusakin et al., 2023), potentially obscuring changes that occur in specific VTA cell types, like DA neurons. Single-cell RNA sequencing has identified transcriptionally distinct populations of neurons in the ventral tegmental area of mice (Poulin et al., 2014) and rats (Phillips et al., 2022), including subpopulations of dopaminergic neurons (Poulin et al., 2020). Nonetheless, these studies were conducted in naive mice, so the effects of opioid exposure are unknown. Therefore, there is a need for studies identifying morphine-induced changes in gene expression that occur in specific cell subpopulations within the VTA, such as VTA DA neurons. Our lab took advantage of translating ribosome affinity purification (TRAP; Heiman et al., 2014) to isolate actively translating RNA from VTA DA neurons following chronic morphine administration. As expected, RNA sequencing analysis confirmed morphine-induced gene expression differences in VTA DA neurons. Of particular interest was the neuropeptide neuromedin S (NMS), as its expression in VTA DA neurons was robustly increased in male and female mice following morphine administration.
NMS was first discovered in 2005, when it was isolated from rat brain tissue (Mori et al., 2005). The analysis of the peptide's sequence highlighted its similarity to the structurally related neuropeptide neuromedin U (NMU) placing it in the neuromedin family of peptides (Mori et al., 2008; Gajjar and Patel, 2017). NMS mRNA expression is prominent in the suprachiasmatic nucleus (SCN; Mori et al., 2005) and outside a role for NMS SCN neurons in circadian behavior (Lee et al., 2015; Porcu et al., 2022), little is known about the functional role of NMS in the brain or about its expression outside the SCN. Thus, there is a lack of fundamental information on NMS expression and function in the rest of the brain, making it a highly novel target of study. NMS and NMU are both ligands for the G-protein-coupled NMUR1 and NMUR2 receptors, and NMUR2 is expressed in the NAc (Kasper et al., 2016), one of the prominent projection sites of VTA DA neurons. This, along with reports that NMU alters alcohol (Vallöf et al., 2020) and cocaine responses (Kasper et al., 2016, 2022), suggests that NMS-expressing neurons could be poised to modulate responses to drugs of abuse. Here, we sought to characterize the cells in the VTA that express NMS and their role in morphine behavior. We found that a small subset of VTA DA neurons express NMS and that activation and inhibition of this population are sufficient to promote and inhibit morphine-elicited behavior, respectively.
Materials and Methods
Animals
All experiments were approved by the Institutional Animal Care and Use Committee at Michigan State University. Mice were housed at 22–25°C on a 12:12 h light/dark cycle with ad libitum food and water, and all breeding was completed at MSU. For TRAP studies, heterozygous dopamine transporter (DAT)-IRES-Cre recombinase (Cre) mice (The Jackson Laboratory, 006660) were crossed with homozygous Rosa26-L10a-eGFP reporter mice (The Jackson Laboratory, 024750). For chemogenetic experiments, heterozygous neuromedin S (NMS)-iCre (The Jackson Laboratory, 027205) or glucagon (GCG)-Cre mice (The Jackson Laboratory, 030542) were crossed with wild-type C57BL/6J (The Jackson Laboratory, 000664). DNA was isolated at 3–4 weeks as described previously (Doyle et al., 2020), and genotyping was completed following The Jackson Laboratory protocols (DAT, 23107; NMS, 28624; GCG, 31403).
Drugs
Morphine sulfate was obtained from Sigma-Aldrich (M8777) and Cayman Biochemical (25955), and cocaine hydrochloride was obtained from Sigma-Aldrich (C5776). For all behavioral experiments, morphine and cocaine were dissolved in 0.9% sterile saline and delivered by intraperitoneal injection. For biochemical studies (TRAP and RNA sequencing), subcutaneous morphine (25 mg, 9300-008) and placebo (9300-009) pellets were generously provided by the NIDA Drug Supply Program. Clozapine N-oxide (CNO) was obtained from Thermo Fisher Scientific (NC1044836) and was diluted in DMSO and PBS. Mice received intraperitoneal injections of CNO (0.3 mg/kg) or vehicle (0.05% DMSO); this low dose of CNO does not metabolize to clozapine (Manvich et al., 2018) or exert off-target effects in control mice (Woodworth et al., 2017).
Morphine pelleting surgery
Mice (10–11 weeks) were anesthetized with isoflurane and implanted subcutaneously with either morphine (25 mg) or sham pellets. Pellets were implanted 48 h apart on Days 1 and 3. Tissue was collected on Day 5, as described previously (Heller et al., 2015; Simmons et al., 2019).
Translating ribosome affinity purification (TRAP)
To isolate actively translating mRNA, TRAP was performed according to published protocols (Heiman et al., 2014) with the following minor alterations to isolate RNA from DA VTA neurons. VTA was microdissected from coronal slices of DATL10a-eGFP mice using a 14-gauge blunt needle, and tissue was immediately frozen on dry ice and stored at −80°C until further processing. VTAs from four mice were pooled for each immunoprecipitation (IP) and homogenized in 1 ml of tissue lysis buffer. In addition, 100 µl of this tissue was aliquoted for input control analysis. Affinity matrix beads were incubated with anti-GFP antibodies (Memorial Sloan-Kettering Monoclonal Antibody Facility: Htz-GFP-19F7 and Htz-GFP-19C8, 50 µg/ml each) for 2–4 h at 4°C. Isolation of DA-specific translating mRNA was achieved by incubation of VTA lysate with an anti-GFP affinity matrix overnight at 4°C.
Final RNA purification was performed using the RNeasy Micro Kit (Qiagen, 74004) according to the kit protocol with the following adjustments. Input and pulldown samples were incubated for 10 min at 4°C with 10% β-ME in RLT buffer (Qiagen, RNeasy Micro Kit, 74004) and briefly vortexed. RNA was then precipitated using 70% ethanol and immediately placed in the Qiagen centrifuge isolation tube, and the Qiagen kit protocol was followed for the remaining steps. Final purified RNA was extracted from columns using 12 µl (for qPCR) or 14 µl (for RNAseq) 60°C RNAse-free water.
RNA sequencing
VTA samples were processed from 10–11-week-old female DATL10a-GFP mice (n = 16 sham-treated and 16 morphine-treated mice) identical to the above procedures. As described above, four VTA samples were pooled for each IP, resulting in four replicates for each treatment group (sham input, sham IP, morphine input, morphine IP; note that the final analysis included n = 3 for the morphine input due to loss of one replicate during library processing). RNA quantity and quality were assessed via Illumina Bioanalyzer, and all samples had RIN >8, and IP samples had total RNA yields of at least 9–10 ng total RNA. Final sample processing and RNAseq were performed by the University of Maryland Genomics Core Facility. cDNA libraries were generated using a strand-specific library kit (low input NEB kit with poly(A) enrichment) followed by amplification. Samples were pooled (eight input and eight IP), and RNA was sequenced across two lanes (Illumina HiSeq 4000, 75 bp paired-end read). RNAseq alignment statistics confirmed an average of 47 million total reads per sample with a high proportion (90%) of unique mapped reads per sample. Differential gene expression was assessed by Q.H. (Heller Lab, University of Pennsylvania) using edgeR/DEseq program software (Bioconductor 3.7). RNA sequencing data are available in Extended Data Figure 1-1.
Real-time polymerase chain reaction (RT-PCR)
RNA quality was immediately assessed using nanodrop, and 8–10 ng of RNA was reversed transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). Final cDNA samples were diluted with DNAse/RNAse-free water to ∼140 ng/µl and stored at −20°C until further processing. RT-PCR analysis was performed using SYBR-green (CFX Connect, Bio-Rad). Samples were run in triplicate and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the ΔΔCt method (Tsankova et al., 2006). Four microliters of cDNA were used for most genes, except for low-abundance genes (Gcg) where cDNA volume was increased to 6 µl. Primers were designed using Primer-BLAST (Ye et al., 2012) and validated via RT-PCR, and primer specificity and product size were confirmed by DNA gel electrophoresis (primer sequences, all listed 5′-3′: Nms-F, CCA ACC TAA GGA AAA CCA GGA TGT A; Nms-R, CCC CAG GCT GGT AGT AGG AT; Gcg-F, GAT GAG ATG AAT GAA GAC AAA CG; Gcg-R, AAC TCA CAT CAC TAA AGG; Sgk1-F, CGT CAA AGC CGA GGC TGC TCG AAG C; Sgk1-R, GGT TTG GCG TGA GGG TTG GAG GAC; Gapdh-F, AGG TCG GTG TGA ACG GAT TTG; Gapdh-R, TGT AGA CCA TGT AGT TGA GGT CA).
Viral vectors
The following AAV viral vectors were obtained from Addgene: AAV2-hSyn-DIO-hM3Dq-mCherry (Gq: excitatory DREADD, 44361-AAV2), AAV2-hSyn-DIO-hM4Di-mCherry (Gi: inhibitory DREADD, 44362-AAV2), and AAV2-hSyn-DIO-mCherry (control, 50459-AAV2).
Stereotaxic surgery
Mice for behavioral studies underwent stereotaxic surgeries following established procedures (Doyle et al., 2021). Briefly, adult male and female mice (8–10 weeks old) were anesthetized using 90 mg/kg ketamine and 10 mg/kg xylazine. All mice received bilateral intra-VTA infusions (0.4 µl) of the corresponding virus at established coordinates (−3.2 A/P, +1.0 M/L, −4.6 D/V, 7° angle). Mice were allowed to recover for 2 weeks following surgery to allow for peak transgene expression. Controls consisted of both wild-type mice that received the Cre-dependent DREADD virus and Cre+ mice that received a non-DREADD virus (AAV2-hSyn-DIO-mCherry).
Viral targeting and immunohistochemistry (IHC) to verify viral expression
After experimental assays were completed, mice were deeply anesthetized via chloral hydrate (4 g/kg). Mice were transcardially perfused with ice-cold phosphate-buffered saline (PBS) followed by 10% formalin–PBS. Brains were collected and postfixed for 24 h in formalin–PBS and then cryopreserved in 30% sucrose–PBS. Brains were sectioned at 30 µm using a freezing microtome, and slices were stored in 0.01% sodium azide–PBS until use.
Immunohistochemistry was completed following published procedures (Simmons et al., 2019). Briefly, sections were washed using PBS and blocked using 3% normal donkey serum (Jackson ImmunoResearch, 017-000-121) with 0.3% Triton X-100. Next, slices were incubated in primary antibody in 0.3% Tween 20–PBS overnight. The primary antibodies used were mouse anti-tyrosine hydroxylase (TH, Sigma-Aldrich, MAB318, 1:2,000), rat anti-mCherry (Invitrogen, M11217, 1:5,000), goat anti-GFP (Abcam, A11122, 1:4,000), and rabbit anti-c-Fos (Cell Signaling Technology, 2250S, 1:1,000). Following PBS washes, the sections were incubated for 2 h with a secondary antibody (1:500 in PBS). All secondary antibodies were obtained from Jackson ImmunoResearch. The secondary antibodies used were anti-rat-cy3 (712-165-153), anti-rat-594 (712-585-153), anti-mouse-488 (715-545-150), anti-mouse-cy3 (715-165-150), anti-rabbit-cy2 (711-225-152), anti-rabbit-cy3 (711-165-152), and anti-goat-488 (705-545-147). Sections were mounted on Superfrost microscope slides (Fisherbrand, 12-550-15), dehydrated with 70–100% ethanol, coverslipped with DPX mounting media (VWR, 100503-834), and stored protected from light.
Images were acquired via fluorescent microscopy (Nikon 600HL Eclipse Ni-U upright microscope with a Lumencor Sola light engine and Photometrics CoolSNAP DYNO camera, 10×/0.3 Plan Fluor DIC LN1 objective) using the NIS-Elements software (version 4.6) and analyzed using standard protocols (Simmons et al., 2019; Bali et al., 2021). FITC, Texas Red, and Cy5 filters were used to detect c-Fos, mCherry, and TH expression, and within each experiment, identical microscope settings were used to acquire images. All slides were coded, and images were scored via blinded investigators. Bilateral targeting of VTA was confirmed using mCherry expression in 4× images. Animals with unilateral VTA expression were excluded, except for mice used in the NMS-Gi morphine conditioned place preference (CPP) experiment, since there was no difference in the reduction of CPP between unilaterally and bilaterally targeted mice. Colocalization was assessed in 10× images to validate DREADD-mediated cellular activation (mCherry- and c-fos-positive) and the percentage of NMS-expressing cells (mCherry-positive) that were dopaminergic (TH-positive).
Behavioral testing
Behavioral tests took place during the light phase
Open field
The open-field (OF) task was performed as previously described (Doyle et al., 2020; Eagle et al., 2020). Briefly, mice were placed in the center of an open-field box (38 cm × 38 cm) and allowed to explore for 10 min under red light. Sessions were video recorded, and time spent in the center of the box (50% size of the whole box, centered) and total distance traveled were assessed using CleverSys TopScan video-tracking software.
Elevated plus maze
Elevated plus maze (EPM) testing was performed following established procedures (Doyle et al., 2020; Eagle et al., 2020). Mice were placed in the center of a plus maze (5 cm × 35 cm) with two open arms and two closed arms and allowed to explore for 5 min under red light. Sessions were video recorded, and time spent in different zones (open arms, closed arms, and center) and total distance traveled were assessed using CleverSys TopScan video-tracking software.
Conditioned place preference (CPP)
Morphine CPP was performed following established procedures with slight modifications (Koo et al., 2012) to allow for chemogenetic manipulation during pairing. Briefly, mice were placed in the center chamber of a three-chamber CPP box with distinct walls, floors, and lighting conditions (San Diego Instruments). On the first day, mice underwent a 20 min pretest where they could freely explore all three chambers. Mice next underwent four pairing days with 45 min morning and afternoon sessions. During the morning session, mice received intraperitoneal injections of vehicle 20 min before receiving a saline injection and being placed in one chamber. In the afternoon, mice received CNO (0.3 mg/kg) 20 min before receiving morphine (15 mg/kg) and being placed in the opposite chamber. A 20 min posttest was performed 2 d after the last pairing, and the CPP score was calculated. The CPP score was defined as the time spent in the morphine plus CNO-paired chamber minus the time spent in the vehicle plus saline-paired chamber during the pre- or posttest. For cocaine CPP, mice underwent 2 pairing days with 30 min morning and afternoon sessions. As in morphine experiments, mice received vehicle or CNO (0.3 mg/kg) 20 min before saline or cocaine (12.5 mg/kg, i.p.) injections in the morning and afternoon, respectively. A 20 min posttest was performed the following day.
CNO CPP was performed with slight modifications to the above procedure. Mice underwent 3 pairing days with 30 min morning and afternoon sessions. Mice received vehicle and CNO (0.3 mg/kg) 20 min before the morning and afternoon sessions, respectively. The posttest was conducted the day after the last pairing, and the CPP score was calculated as the time spent in the CNO-paired chamber minus the time spent in the vehicle-paired chamber during the pre- and posttests.
Locomotor activity
Morphine (15 mg/kg) or cocaine (10 mg/kg) locomotor activity was assessed following established procedures with slight modifications (Doyle et al., 2021). Locomotor activity was recorded using a three-chambered CPP box (San Diego Instruments) and calculated as total beam breaks [4 × 16 (x–y) photobeam array). During daily testing sessions (8 d), mice received intraperitoneal injections of saline (Day 1) for habituation, CNO plus saline (Days 2–3), and CNO plus drug (Days 4–8); the control group received CNO plus saline (no morphine) on Days 4–8. CNO (or vehicle) was administered 20 min before the start of the assay, and then mice were given morphine (or saline) and placed in the boxes; locomotor activity was immediately recorded for 60 min. Mice underwent forced abstinence for 5 d before receiving a challenge injection of CNO plus morphine (Day 14) to assess morphine locomotor sensitization.
Statistics
GraphPad Prism was used for all statistical analyses, and all values are represented as mean ± SEM. For experiments with two experimental groups (OF, EPM, and RT-PCR), unpaired t tests were performed. For drug locomotor and CPP experiments, two-way repeated-measures ANOVAs were performed, followed by Šidák's post hoc test to compare differences. Significance was defined as p < 0.05. Male and female mice were used in all experiments. No sex differences were observed, so combined data are presented with sex indicated by symbol (males, black circle; females, white square). For RNA sequencing data, differential gene expression was assessed using the edgeR/DEseq program, and padj < 0.05 was considered significant. All genes identified were included in the volcano plot analysis. Venn diagram analysis was completed using https://bioinformatics.psb.ugent.be/webtools/Venn/. Schematics and diagrams were generated using BioRender.
Results
Ventral tegmental area dopamine neurons have altered gene expression following chronic morphine
To identify morphine-induced changes in VTA DA gene expression, we utilized translating ribosome affinity purification (TRAP). DAT-L10-eGFP mice were generated by crossing DAT-Cre and Rosa26-L10-eGFP mice, and VTA was microdissected from female mice following morphine or sham pelleting (Fig. 1A), an administration paradigm that maintains a steady level of morphine for days mimicking human use and produces morphine dependence (Fischer et al., 2008), decreased VTA DA soma size (Mazei-Robison et al., 2011; Simmons et al., 2019), and increased VTA DA activity (Mazei-Robison et al., 2011). mRNA was isolated from DAT-expressing neurons as well as whole VTA following published TRAP protocols (Heiman et al., 2014). Briefly, actively translating mRNA from DA neurons was isolated from VTA homogenates using affinity purification. RNA sequencing was performed on both the input (whole VTA) and the pulldown (VTA DA IP). Data from sham-treated animals were used to validate the DAergic profile. Specifically, markers of DAergic neurons (i.e., Th and Dat) were enriched in the pulldown (VTA DA IP) whereas markers of GABAergic neurons (i.e., Gad1 and vGAT), glutamatergic neurons (i.e., vGlut2), and glia (i.e., Gfap and Glast1) were depleted, compared with input (whole VTA) as expected (all padj < 0.0001, Fig. 1B). Next, we identified differential gene expression caused by morphine treatment in whole VTA samples (Fig. 1C). In total, 23,062 genes were identified, of which 2,103 genes were significantly regulated by chronic morphine (Extended Data Fig. 1-1, padj < 0.05). Specifically, there were 948 significantly upregulated and 1,155 significantly downregulated genes. Similarly, we identified gene expression changes induced by morphine specifically in VTA DA neurons by comparing sham and morphine IP samples (Fig. 1E). Of the 21,754 total genes identified, 1,792 genes were significantly regulated by chronic morphine (Extended Data Fig. 1-1, padj < 0.05). Specifically, 998 were significantly upregulated, and 794 were significantly downregulated. Interestingly, when comparing the morphine-induced changes in gene expression, there was little overlap between those identified in whole VTA and those from DA-specific analyses (Fig. 1D). Only 370 of the significantly morphine-regulated genes were identified in both input and IP analyses, while 3,525 of the chronic morphine-regulated genes were unique to the input and DA-specific IP fractions. Of the 370 common genes, the neuropeptide neuromedin S (Nms) was of particular interest as it was one of the most highly and consistently induced genes in the DA-specific fraction (Extended Data Fig. 1-1, VTA DA sham vs VTA DA morphine, padj = 1.75 × 10−6) and was enriched in the VTA DA fraction (Extended Data Fig. 1-1, sham VTA input vs sham VTA DA pulldown, padj = 0.0046).
Neuromedin S (Nms) gene expression is increased in ventral tegmental area (VTA) dopamine (DA) neurons following morphine administration. A, VTA were pooled from four female DAT-L10-eGFP mice that received sham or morphine (25 mg) pellets for immunoprecipitation (IP) and RNA sequencing. B, The VTA DA IP fraction was validated by enrichment of DA neuron transcripts (DAergic) and depletion of glutamatergic, GABAergic, and non-neuronal transcripts (other) compared with whole VTA input. C, E, The volcano plots illustrate morphine-induced gene expression changes in whole VTA (C) and VTA DA neurons (E). D, Morphine-induced gene expression changes are relatively distinct between whole VTA and VTA DA neurons. F, RT-PCR confirms that morphine increases Nms expression in whole VTA and VTA DA neurons, *p < 0.05, ***p < 0.0001, n = 7 mice/group. Square, female; circle, male. See Extended data Figure 1-1 for more details.
Figure 1-1
RNA sequencing data. VTA were pooled from four female DATL10-EGFP mice that received sham or morphine (25 mg) pellets for immunoprecipitation (IP) and RNA sequencing. RNA sequencing was completed on both input and IP from sham- and morphine-treated mice. Differential gene expression results for three analyses are entered in separate tabs, sorted by padj values. Tab A, sham IP vs. morphine IP results, Tab B, sham input vs. morphine input results, Tab c, sham input vs. sham IP results. Download Figure 1-1, XLSX file.
Ventral tegmental area neuromedin S expression is induced by repeated morphine, but not cocaine administration
To validate the RNA sequencing results, we performed RT-PCR on mRNA from an independent VTA DA TRAP pulldown. Using both male and female mice, we saw a significant increase in Nms expression in both whole VTA samples (Fig. 1F; unpaired t test, t(12) = 2.49, p = 0.03) and VTA DA samples (Fig. 1F; unpaired t test, t(12) = 8.79, p < 0.0001) in morphine-pelleted mice. Combined data are shown (male, black circle; female, white square), as there were no sex differences observed. The morphine-induced increase in NMS expression was not observed in similar TRAP pulldowns from VTA GABA neurons (vGAT-L10-eGFP: data not shown) supporting cell-type–specific regulation. To determine whether Nms expression is also increased by repeated morphine injections, male and female mice were given daily morphine injections (15 mg/kg) for 1 week. Similar to morphine pellet results, we found that Nms expression is increased in whole VTA samples following repeated morphine injections (Fig. 2A; unpaired t test, t(27) = 2.27, p = 0.03). We next assessed whether Nms expression is induced by cocaine exposure, as drugs of abuse can induce both similar and unique gene expression changes in the VTA (Heller et al., 2015). In contrast to our morphine results, we found that male and female mice treated with daily cocaine injections (20 mg/kg) had no change in Nms expression (Fig. 2B; unpaired t test, t(18) = 0.28, p = 0.79). Given that opioids are often administered chronically, we examined whether increased VTA Nms expression was maintained over an extended exposure. Similar to our 7 d results, we found that 4 weeks of daily morphine treatment (15 mg/kg) significantly increased VTA Nms expression (Fig. 2C; unpaired t test, t(26) = 2.47, p = 0.021). Finally, we determined whether acute morphine exposure was sufficient to induce VTA Nms expression. In contrast to repeated treatment, a single morphine injection did not increase VTA Nms expression (Fig. 2D; unpaired t test, t(17) = 0.15, p = 0.88). We found that VTA Sgk1 expression was increased in all drug treatment conditions as expected (7 d morphine, t(30) = 7.86, p < 0.0001; 7 d cocaine, t(18) = 2.60, p = 0.02; 1 d morphine, t(17) = 3.57, p = 0.002), as it was previously shown to increase with both acute and chronic drug treatment (Heller et al., 2015). Together, these data indicate that repeated morphine exposure increases VTA Nms expression in DA neurons and that this regulation may be opioid-specific.
Neuromedin S (Nms) gene expression is increased in the ventral tegmental area (VTA) following repeated morphine injections. A–D, VTA Nms expression is increased following repeated morphine injections (7 d or 4 weeks, 15 mg/kg, i.p.), but not following repeated cocaine (7 d, 20 mg/kg, i.p.) or acute morphine injection (1 d, 15 mg/kg, i.p.), *p < 0.05, n = 9–17 mice/group. Square, female; circle, male.
Neuromedin S-expressing neurons are a subset of ventral tegmental area neurons that are largely dopaminergic
To identify the population of neurons that express Nms in the VTA, we stereotaxically injected a Cre-dependent viral vector (AAV2-DIO-hM3Dq-mCherry) into the VTA of NMS-iCre and wild-type mice. We observed a small proportion of mCherry-positive cells (NMS-expressing) distributed throughout the VTA of NMS-iCre mice and no mCherry-positive cells in wild-type mice (NMS-iCre, 21.4 ± 1.6 cells/hemisphere, n = 4 mice; wild-type, 0 cells, n = 3 mice). In sections costained with tyrosine hydroxylase (TH) to label dopamine cells, we found that NMS-expressing cells (mCherry-positive) were also positive for TH, consistent with NMS expression in dopaminergic cells (Fig. 3A). However, NMS-expressing cells represented a small proportion of dopaminergic neurons (<5% TH-positive cells). We validated the specificity of NMS labeling by crossing NMS-iCre mice with Rosa26-L10-eGFP reporter mice to label all NMS-expressing neurons. As expected, robust eGFP expression was noted in the SCN consistent with published data of NMS-expressing neurons in this region (Fig. 3B). We also observed eGFP-positive cells in the VTA and found similar numbers of NMS-expressing cells, including those colabeled with TH, as determined using viral reporters (Fig. 3C). Notably, both labeling methods also identified NMS-expressing cells that were nondopaminergic, the majority of which were in midline structures (CLi, IF). While this population may be of interest, we were particularly interested in the VTA DA neurons coexpressing NMS given the increase in Nms expression in this population following morphine exposure (Fig. 1F). To determine whether we could virally target and activate VTA NMS neurons, we utilized chemogenetics and bilaterally injected the excitatory DREADD (AAV2-DIO-hM3Dq-mCherry) into the VTA of NMS-iCre mice. Following CNO administration (0.3 mg/kg), we confirmed NMS neuron activation via increased c-Fos labeling of mCherry-labeled VTA NMS cells compared with vehicle-treated mice (Fig. 3D,E; t(6) = 12.83, p < 0.0001). In a separate cohort of mice, we repeatedly activated VTA NMS neurons in the presence or absence of morphine (0.3 mg/kg CNO in the absence or presence of 15 mg/kg morphine, daily i.p., 7 d) and isolated RNA 1 h following the last injection (Fig. 3F,G). As expected, we found that morphine increased Nms and Sgk1 expression in the VTA. In contrast, repeated CNO treatment robustly increased VTA Nms expression, without affecting Sgk1 (one-way ANOVA and Dunnett's multiple-comparisons test, comparison to Con-CNO: Nms, F(3,31) = 10.83, p < 0.0001; Sgk1, F(3,31) = 11.61, p < 0.0001). Together, these data support the ability to label and activate a novel NMS-expressing cell population in the VTA.
Neuromedin S (NMS) neurons are a small subset of dopamine (DA) VTA neurons. A, A small population of NMS-expressing neurons (purple) was identified in the VTA using a conditional viral vector [tyrosine hydroxylase (TH), green; mCherry, purple; 10× representative image]. B, NMS neurons were genetically labeled with eGFP and were abundant in the suprachiasmatic nucleus (SCN). C, NMS neurons in the VTA (eGFP-positive) are both dopaminergic (TH-positive) and nondopaminergic. The majority of NMS-expressing cells in the lateral VTA [parabrachical (PBP) and paranigral (PN)] are dopaminergic, while the NMS-expressing cells in the midline VTA subregions [caudal linear nucleus (CLi) and interfascicular (IF)] are largely nondopaminergic, n = 6 mice. D, Chemogenetic activation [clozapine N-oxide (CNO)] increases c-fos expression (green) in VTA NMS neurons (purple), consistent with increased neuronal activity (20× representative image). Representative c-Fos-positive and c-Fos-negative VTA NMS neurons are highlighted by closed and open arrows, respectively. E, c-Fos colocalization was significantly increased following NMS CNO (0.3 mg/kg) compared with vehicle administration. n = 3 and 5 mice/group. ***p < 0.001. F, Repeated CNO administration (7 d) increased VTA Nms expression in NMS-Gq mice compared with controls, n = 8–10 mice/group, **p < 0.01. G, Repeated CNO administration (7 d) did not alter VTA Sgk1 expression in NMS-Gq mice, but morphine increased Sgk1 expression in both control and NMS-Gq mice, as expected, n = 8–10 mice/group, *p < 0.05, **p < 0.01. Square, female; circle, male.
Activation or inhibition of VTA NMS-expressing neurons does not alter baseline behavior
Given that activation or inhibition of the whole population of VTA DA neurons can alter locomotion and conditioned place preference (Tsai et al., 2009; Boekhoudt et al., 2016), and because the NMS subpopulation of VTA neurons has not been previously described, we first sought to determine whether alteration of their activity is inherently rewarding or changes baseline behaviors. To do this, we injected Cre-dependent excitatory (AAV2-hSyn-DIO-hM3Dq) or inhibitory (AAV2-hSyn-DIO-hM4Di) DREADDs into the VTA of male and female NMS-iCre mice. To determine whether VTA NMS neuron manipulation alters general locomotor activity, we measured the distance traveled in open-field testing following CNO (0.3 mg/kg) administration. Neither acute VTA NMS neuron activation (NMS-Gq) nor inhibition (NMS-Gi) altered locomotor activity (Fig. 4A,E) compared with controls (NMS-Gq, t(30) = 1.31, p = 0.20; NMS-Gi, t(19) = 1.22, p = 0.24). Additionally, we saw no differences in time spent in the center of the chamber between NMS-activated (Fig. 4B) or inhibited (Fig. 4F) mice (NMS-Gq, t(30) = 0.31, p = 0.76; NMS-Gi, t(19) = 0.38, p = 0.71). To confirm a lack of effect of VTA NMS neurons on anxiety-like behaviors, the mice also underwent elevated plus maze (EPM) testing. Activation or inhibition of VTA NMS neurons didn't alter open arm duration (Fig. 4C,G; NMS-Gq, t(30) = 0.87, p = 0.39; NMS-Gi, t(20) = 0.48, p = 0.63). Finally, we sought to determine whether activation or inhibition of VTA NMS neurons was inherently rewarding or aversive. To do this, we conducted a CNO conditioned place preference (CPP) assay, where chambers were paired with either vehicle (morning) or CNO (0.3 mg/kg, afternoon). Neither NMS VTA activation (Fig. 4D) nor NMS VTA inhibition (Fig. 4H) induced preference for either chamber in the posttest (NMS-Gq, t(30) = 1.57, p = 0.13; NMS-Gi, t(25) = 0.50, p = 0.62), suggesting that alteration of VTA NMS neuronal activity alone is not sufficient to promote rewarding or aversive conditioning. As with our molecular datasets, we did not observe differences between males and females, so data from all chemogenetic experiments are combined (female, white square; male, black circle).
Chemogenetic activation and inhibition of neuromedin S (NMS) ventral tegmental area (VTA) neurons do not alter baseline behavior. A, B, Chemogenetic activation of VTA NMS neurons does not alter total locomotion (A) or center time (B) in open-field testing, n = 15, 17 mice/group. C, Chemogenetic activation of VTA NMS neurons does not alter open arm time in elevated plus maze testing, n = 15, 17 mice/group. D, Chemogenetic activation of VTA NMS neurons does not induce a conditioned place preference (CPP) response, n = 15, 17 mice/group. E, F, Chemogenetic inhibition of VTA NMS neurons does not alter total locomotion (E) or center time (F) in open-field testing, n = 10, 11 mice/group. G, Chemogenetic inhibition of VTA NMS neurons does not alter open arm time in elevated plus maze testing, n = 12, 10 mice/group. D, Chemogenetic inhibition of VTA NMS neurons does not induce a CPP response, n = 16, 11 mice/group. Square, female; circle, male.
Activation and inhibition of VTA NMS-expressing neurons alters morphine-induced locomotor activity
Given that morphine increases locomotor activity in mice (Murphy et al., 2001; Han et al., 2010; Masukawa et al., 2020), we sought to determine whether alteration of VTA NMS neuronal activity affected morphine-induced locomotor activity and sensitization. To identify the role of VTA NMS neurons in morphine behaviors, we used male and female NMS-iCre mice and wild-type littermates bilaterally injected with excitatory or inhibitory DREADDs to allow for selective activation or inhibition of NMS neurons and assessed locomotor activity. Animals received intraperitoneal injections of saline (Day 1), saline and CNO (Days 2–3, 0.3 mg/kg), and CNO and morphine (Days 4–8, 0.3 and 15 mg/kg, respectively). Following 5 d of abstinence, control and NMS-Gq mice were given a challenge injection of CNO and morphine (Day 14, 0.3 and 15 mg/kg, respectively). While morphine increased locomotor activity in control animals as expected, mice with concomitant VTA NMS neuron activation had a significantly greater increase in morphine-induced locomotion (Fig. 5A; mixed-effects model: day, F(2.885,163.4) = 31.39, p < 0.0001; virus, F(1,61) = 10.12, p = 0.0023; day–virus interaction, F(8,453) = 4.991, p < 0.0001; Šidák's multiple-comparisons post hoc test Con vs NMS-Gq: Day 7 p = 0.005, Day 8 = 0.011). Repeated VTA NMS activation in the absence of morphine did not affect locomotor activity, as repeated CNO did not increase locomotor activity compared with controls (Fig. 5B; two-way repeated-measures ANOVA: day, F(2.489,27.38) = 11.02, p = 0.0001; virus, F(1,11) = 0.60, p = 0.45; day–virus interaction, F(7,77) = 1.45, p = 0.20). In inhibitory experiments, there were significant main effects of day and virus, representing overall decreased locomotor activity in NMS-Gi mice; however, there were no significant differences between control and NMS-Gi on a single day (Fig. 5C; two-way repeated-measures ANOVA: day, F(2.438,126.8) = 35.30, p < 0.0001; virus, F(1,52) = 4.16, p = 0.046; day–virus interaction, F(8,416) = 1.89, p = 0.06). To determine whether locomotor effects were generalizable to other drugs of abuse, we also assessed the ability of VTA NMS neuronal activity to affect cocaine locomotor activity in a separate cohort of mice. In contrast to morphine, cocaine responses in mice with VTA NMS neuronal activation or inhibition did not differ from controls (Fig. 5D; two-way repeated-measures ANOVA: day, F(3.072,58.36) = 62.93, p < 0.0001; virus, F(2,19) = 0.42, p = 0.66; day–virus interaction, F(12,114) = 1.60, p = 0.10). Overall, these data support the hypothesis that VTA NMS neuronal activity is sufficient to increase morphine-induced locomotor activity.
Chemogenetic modulation of ventral tegmental area (VTA) neuromedin S (NMS) neuronal activity alters morphine-induced locomotor activity. A, Chemogenetic activation of VTA NMS neurons significantly increases morphine-induced locomotor activity, n = 31 and 32 mice/group, n = 14 mice/group for Day 14, **p < 0.01, *p < 0.05. B, Repeated activation of VTA NMS neurons in the absence of morphine does not alter locomotor activity, n = 6 and 7 mice/group. C, Chemogenetic inhibition of VTA NMS neurons decreases locomotor activity, n = 29 and 25 mice/group. D, Chemogenetic modulation of VTA NMS neuronal activity does not alter cocaine-induced locomotor activity, n = 5–10 mice/group.
Inhibition of VTA NMS neuronal activity reduces morphine-conditioned place preference (CPP)
Given that alteration of VTA NMS neuronal activity was sufficient to alter morphine locomotor activity and sensitization, we next sought to determine whether it could also impact morphine CPP. Male and female NMS-iCre mice and wild-type littermates were bilaterally injected with excitatory or inhibitory DREADDs to allow for selective activation or inhibition of NMS neurons. Mice received intraperitoneal injections of vehicle and saline in one chamber in the morning and CNO plus morphine (0.3 and 15 mg/kg, respectively) in the opposite chamber in the afternoon (Fig. 6A). As expected, mice showed a preference for the morphine-paired chamber during the posttest; however, VTA NMS neuron activation did not affect CPP compared with controls (Fig. 6B; two-way repeated-measures ANOVA: test date, F(1,23) = 5.63, p = 0.026; virus, F(1,23) = 0.80, p = 0.38; test date–virus interaction, F(1,23) = 0.002, p = 0.96). In contrast, inhibition of VTA NMS neurons significantly blunted morphine CPP (Fig. 6C; two-way repeated-measures ANOVA: test date, F(1,23) = 5.64, p = 0.026; virus, F(1,23) = 4.55, p = 0.044; test–virus interaction, F(1,23) = 4.53, p = 0.044; followed by Fisher's LSD post hoc test: Con pretest vs posttest, p = 0.0035; Con posttest vs NMS-Gi posttest, p = 0.0045) without affecting cocaine CPP (Fig. 6D; test date, F(1,6) = 16.24, p = 0.007; virus, F(1,6) = 1.92, p = 0.12; test date–virus interaction, F(1,6) = 1.42, p = 0.28). Together, these results support a critical role in the activity of VTA NMS neurons in morphine CPP.
Chemogenetic inhibition of ventral tegmental area (VTA) neuromedin S (NMS) neuronal activity decreases morphine-conditioned place preference (CPP). A, Mice received VTA infusions of control, excitatory (NMS-Gq), or inhibitory (NMS-Gi) viruses and underwent morphine CPP testing where mice received vehicle and saline in one chamber in the morning and CNO (0.3 mg/kg) and drug (morphine, 15 mg/kg; cocaine, 12.5 mg/kg) in the opposite chamber in the afternoon. B, Chemogenetic activation of VTA NMS neurons does not alter morphine CPP, n = 12 and 13 mice/group. C, Chemogenetic inhibition of VTA NMS neurons significantly decreases morphine CPP, n = 13 and 12 mice/group, **p < 0.01. D, Chemogenetic inhibition of VTA NMS neurons does not alter cocaine CPP, n = 2 and 6 mice/group. Square, female; circle, male.
Morphine increases gene expression of the neuropeptide glucagon (Gcg) in VTA DA neurons, but modulation of VTA GCG neuron activity does not alter morphine-elicited behavior
NMS was not the only neuropeptide whose expression was significantly increased in VTA DA neurons following chronic morphine exposure. We also found that glucagon (Gcg) expression was increased in VTA DA neurons (Fig. 1E) which was validated by RT-PCR of mRNA from an independent VTA DA TRAP pulldown (Fig. 7A; unpaired t test, t(6) = 12.87, p < 0.001). Utilizing a similar strategy to that described for NMS, we performed chemogenetic studies in GCG-Cre mice to determine whether modulation of VTA GCG neuronal activity was sufficient to alter morphine-elicited behavior (Fig. 7D). In contrast to VTA NMS neuronal modulation, we found that chemogenetic excitation of VTA GCG neurons modestly decreased locomotor activity without affecting morphine CPP (Fig. 7B,C) and inhibition did not significantly alter either behavior (Fig. 7E,F; all two-way repeated-measures ANOVAs, Fig. 7B: day, F(7,350) = 23.26, p < 0.0001; virus, F(1,50) = 1.94, p = 0.17; day–virus interaction, F(7,350) = 2.19, p = 0.035; Fig. 7C: test day, F(1,12) = 20.00, p = 0.0008; virus, F(1,12) = 0.81, p = 0.38; test day–virus interaction, F(1,12) = 3.19, p = 0.10; Fig. 7E: day, F(7,259) =16.37, p < 0.0001; virus, F(1,37) = 3.01, p = 0.09; day–virus interaction, F(7,259) = 0.56, p = 0.78; Fig. 7F: test day, F(1,21) = 19.83, p = 0.0002; virus, F(1,21) = 2.76, p = 0.11; test day–virus interaction, F(1,21) = 0.003, p = 0.95). These data suggest that while there may be multiple subsets of VTA neuropeptide-expressing neurons recruited during morphine exposure, NMS-expressing cells may play a unique role in morphine-elicited behavior.
Glucagon (Gcg) gene expression is increased in the ventral tegmental area (VTA) following repeated morphine injections but modulation of VTA GCG neuronal activity does not alter morphine-elicited behavior. A, RT-PCR confirms that morphine increases Gcg expression in VTA DA neurons, n = 4 samples/group, ***p < 0.0001. B, Chemogenetic activation of VTA GCG neurons modestly decreases morphine-induced locomotor activity, n = 26 mice/group. C, Chemogenetic activation of VTA GCG neurons does not alter morphine CPP, n = 7 mice/group. D, Mice received VTA infusions of control, excitatory (GCG-Gq), or inhibitory (GCG-Gi) viruses and underwent morphine locomotor or CPP testing; representative image shows mCherry expression (purple) in both dopaminergic (green, TH) and nondopaminergic cells of the VTA (20×). E, Chemogenetic inhibition of VTA GCG neurons does not alter morphine-induced locomotor activity, n = 29 and 10 mice/group. F, Chemogenetic inhibition of VTA GCG neurons does not alter morphine CPP, n = 16 and 7 mice/group. Square, female; circle, male.
Discussion
The VTA is a heterogeneous brain region whose function in drug behaviors has been widely studied. While VTA DA neuron activity and DA release are altered by chronic opioid exposure (Sklair-Tavron et al., 1996; Simmons et al., 2019), there is a lack of studies identifying the molecular mechanisms underlying these changes. Here, we sought to use an unbiased approach to identify gene expression changes that occur in VTA DA neurons following chronic morphine. We found that morphine induces an increase in the expression of Nms in both whole VTA and VTA DA neurons specifically. Furthermore, we found that activation of NMS-expressing neurons in the VTA increases morphine-induced locomotor activity, whereas inhibition of these neurons blunts morphine CPP and morphine locomotor activity. Together, these data support VTA NMS-expressing neurons as a novel opioid-responsive subpopulation.
The role of NMS within the reward circuitry has yet to be explored. However, a role for NMS in circadian behavior (Mori et al., 2005) and ingestive behaviors (Ida et al., 2005) has been demonstrated through NMS intracerebroventricular infusion, where NMS-induced phase shifts in locomotor activity and decreased food intake, respectively. NMS action is mediated by binding to NMUR1 and NMUR2, GPCRs expressed in the periphery and brain, respectively. Intriguingly, NMUR2 is expressed in output regions of the VTA, most notably the NAc (Kasper et al., 2016), suggesting a potential VTA NMS circuit contributing to morphine CPP and sensitization effects. While a role for NMUR2 in centrally mediated NMS effects is supported by the ability of NMUR2 knock-out to reverse the anorexigenic effects of intracerebroventricular NMS (Peier et al., 2009), most of what is known about NMUR2 function in the NAc is in the context of NMU. NMU is a structurally similar peptide to NMS that has been implicated in motivated behaviors in rodents, including food reward, alcohol (Vallöf et al., 2020), and cocaine (Kasper et al., 2016, 2022) responses. Specifically, NMU acts through the NAc NMUR2 receptors (predominately localized to presynaptic GABAergic terminals from the dorsal raphe nucleus) to decrease cocaine sensitization (Kasper et al., 2016). This NMU response appears opposite to our results, as we find that repeated activation of VTA NMS neurons increases morphine locomotor activity and sensitization. The differences between our observed responses in drug-induced locomotor activity might be due to multiple factors. We know that opioids and psychostimulants can have differing and sometimes opposite effects (Badiani et al., 2011), including changes in NAc MSN spine density and dendrite branching. Consistent with this idea, while we observe that alteration of VTA NMS activity is sufficient to alter morphine behavior, we do not observe similar effects on cocaine behavior. Notably, NMU hasn't been investigated in opioid responses, so it is possible that NMU effects are also specific to drug type. Secondly, in Kasper et al.'s experimental design, they administered NMU directly into the NAc. While there is a solid rationale to support VTA NMS neurons are exerting their actions via NMUR2 signaling in the NAc, it is possible that VTA NMS neurons project to additional brain regions and behavioral effects could be dependent on output to sites outside the NAc. Finally, because we are using a chemogenetic approach, it is possible that our effects are due to increased DA release from VTA NMS neurons and that this is driving our observed morphine responses. However, we think this last explanation is less likely given that VTA NMS neuronal activation was not sufficient to alter cocaine-elicited locomotor activity, which would be expected of a dopaminergic response, and that activation of a separate small population of VTA DA cells (those that express GCG) was also insufficient. This, in combination with our finding that repeated cocaine treatment does not increase NMS expression in the VTA, suggests that VTA NMS neurons may be more selectively recruited by opioid exposure. However, a clear limitation of our current study is that while our chemogenetic experiments support a role for the activity of VTA NMS-expressing neurons in morphine-elicited behavior, they do not implicate a functional role for the NMS peptide itself. Thus, future studies should address whether expression of NMS in VTA NMS neurons is critical for mediating morphine responses. Given the paucity of literature examining NMS, these experiments will require development of genetic and/or pharmacological tools to specifically alter NMS expression and signaling, respectively.
Here, we show that VTA NMS-expressing neurons play an important role in morphine behavior, given that inhibition of this population decreases morphine CPP. A limitation of this experiment, however, is that we do not know whether inhibition of VTA NMS neuronal activity completely abrogates morphine CPP or just reduces sensitivity, since a single morphine dose (15 mg/kg) was used. While this is a fairly high morphine dose that we have previously used to produce a robust CPP response (Koo et al., 2012), higher doses of morphine during conditioning have been reported to produce greater locomotor responses, although this does not necessarily translate to increased morphine CPP (Hámor et al., 2023). To address this caveat, as well as to allow more a thorough understanding of potential differences between chemogenetic activation and inhibition effects on morphine-elicited behavior, a large dose–response study is necessary. For example, conducting comprehensive morphine dose–response curves for morphine CPP and morphine locomotor activity may help to resolve why we observe that morphine CPP is robustly regulated by chemogenetic inhibition of VTA NMS neurons, but morphine locomotor activity appears more strongly regulated via chemogenetic activation.
As stated above, it is possible that our chemogenetic effects on morphine-elicited behavior are not dependent on increased Nms expression, but instead that NMS is a marker for a functionally relevant subset of VTA neurons for opioid response. This was the case in the SCN where Nms expression was first identified, as researchers found that SCN NMS neurons were required for circadian rhythmicity at a behavioral and molecular level, but the deletion of NMS wasn't sufficient to disrupt circadian rhythms (Lee et al., 2015). Because we know that the VTA contains combinatory neurons (Morales and Margolis, 2017), it will be important to identify the entire signaling complement of VTA NMS neurons. Given the small population and low basal expression of NMS, this subpopulation has not been defined in single-cell datasets. Nevertheless, the identification of NMS as a marker for a functionally relevant neuronal population for opioid response may be significant in and of itself. For example, similar identification of genetically distinct VTA subpopulations, those that express Crhr1 or Cck, has been used to interrogate the role of different VTA-NAc subcircuits in reinforcement behavior (Heymann et al., 2020). Specifically, these populations have distinct projections within the NAc (core vs shell), and while optogenetic activation of Crhr1 neurons was sufficient to drive the acquisition of an operant response, activation of Cck neurons was not. This highlights the relevance of studying genetically isolated subpopulations of DA neurons in the VTA using Cre-driver mouse lines, as we have done with the NMS-iCre mouse model. While a role for VTA NMS-expressing neurons in morphine-elicited locomotor activity and morphine CPP is established here, it is unclear whether these neurons play a role in morphine seeking or withdrawal. Studies examining VTA NMS neuronal activity, and VTA Nms expression, in self-administration and relapse models with morphine and additional commonly used and abused opioid drugs such as oxycodone, heroin, and fentanyl, would determine whether this neural population also contributes to those aspects of drug behavior.
This is the first study to identify the expression of Nms in the VTA and its robust induction in DA neurons following morphine exposure. We also identify a role for NMS-expressing neurons in morphine locomotor activity and place preference. Future studies would benefit from VTA DA neuron-specific Nms deletion to assess the role of peptide release itself in morphine behaviors. Furthermore, identifying the projection sites of VTA NMS neurons and interfering with target signaling via receptor disruption could provide further details about these neurons' mechanism(s) of action. Taken together, our work is the first to implicate NMS in drug behaviors and to identify this peptide as a marker of a functionally relevant neuronal subpopulation in the VTA.
Footnotes
This work was supported by NIDA (R21 DA057418, M.S.M.-R.) and the Peter F. McManus Charitable Trust (M.S.M-R.), and trainees were funded by fellowships and training grants (National Science Foundation Graduate Research Fellowship and HHMI Gilliam Fellowship, C.R.Q.; F31 DA042502, S.C.S.; R25 NS090989, M.A. and N.C.F.). We thank Ken Moon for his assistance with mouse breeding and genotyping, Gizem Kurt and Gina Leinninger for their guidance on viral labeling studies, AJ Robison for his advice and editing of the manuscript, and Chiho Sugimoto and Katie McGrath for their general support of surgeries, behavior, and immunohistochemistry.
The authors declare no competing financial interests.
- Correspondence should be addressed to Michelle S. Mazei-Robison at mazeirob{at}msu.edu.
