Social deficits during protracted morphine withdrawal

To model the sociability deficits that are frequently observed in humans during protracted opioid withdrawal (Strang et al., 2020;Volkow and Blanco, 2021), wild-type mice were administered escalating doses of morphine (MOR) (Zhu et al., 2016) and tested for prosocial behavior three weeks after cessation of MOR (Figure 1A). Consistent with previous reports (Bravo et al., 2020;Goeldner et al., 2011;Lalanne et al., 2017;Lutz et al., 2014), during this protracted opioid withdrawal, MOR-treated mice exhibited robust decreases in two well-established assays of prosocial behavior: the juvenile interaction test and the 3-chamber social preference test (Figures 1B–1E). To measure the acute responses to MOR, these same mice were placed into one side of a conditioned place preference (CPP) chamber following each MOR injection and tested for place preference 24 hr and 3 weeks later (Figure 1A). Consistent with the reinforcing nature of the MOR experience, a large preference for the MOR-paired side was observed at both time points (Figures 1F,S1A-S1C). Furthermore, MOR elicited a robust locomotor sensitization (Figures S1D andS1E), a behavioral adaptation that is associated with key features of the addicted state (Robinson and Berridge, 2003). To examine the specificity of the sociability deficits, we performed several additional behavioral assays: time spent interacting with a novel inanimate object, preference for an appetitive, palatable food pellet in the 3-chamber assay, and time spent in the center of an open field. Protracted MOR withdrawal produced no changes in any of these assays (Figures S1F-S1I).

The magnitude of the sociability deficits during protracted opioid withdrawal was highly correlated with the magnitude of the memory for the MOR experience. Specifically, the higher the MOR context preference, measured 3 weeks after cessation of MOR administration, the less time spent interacting with a juvenile (Figures 1G and1H) and the lower the preference for the social chamber in the 3-chamber social preference assay (Figures 1I and1J). Even numbers of female and male subjects were included in each group and sub-analyses revealed no sex differences (Figures S1J-S1Q). Correlations were also preserved when analyzed for females and males separately (Figures S1R-S1U). These results suggest that social avoidance behavior during protracted opioid withdrawal strongly correlates with the “memory” of the drug experience and therefore may contribute to an individual’s propensity to relapse.

NAc medial shell KORs are necessary for sociability deficits during withdrawal

KOR activation contributes to stress-induced cocaine seeking and appears to be necessary for some of the social deficits during protracted opioid withdrawal (Lalanne et al., 2017;Land et al., 2008;Land et al., 2009;Lutz et al., 2014;Redila and Chavkin, 2008;Schindler et al., 2012). However, it is unknown which specific brain regions express the KORs that are required for the social behavior changes caused by chronic opioid administration. Given that the NAc contains dynorphin-expressing neurons and receives inputs from DA and 5-HT neurons that play a role in prosocial behavior (Dai et al., 2022;Gunaydin et al., 2014;Heifets et al., 2019;Walsh et al., 2018), we tested if KORs specifically in the NAc medial shell are necessary for social deficits during protracted opioid withdrawal. Guide cannula were implanted bilaterally in the NAc medial shell and following baseline sociability testing, mice were subjected to the chronic MOR administration regimen (Figure 1K). Three weeks after cessation of MOR administration, sociability assays were performed and then mice were randomly split into groups receiving either a microinjection of saline or the long-lasting KOR antagonist norbinaltorphimine (norBNI; 2.5 μg/hemisphere). Sociability testing 24 hr later revealed that the saline injected subjects still exhibited robust deficits in both social assays while these deficits were reversed by the norBNI treatment (Figures 1L–1O).

We next addressed whether a KOR antagonist that is being evaluated in humans, Aticaprant (JNJ-67953964, previously CERC-501 and LY2456302;Krystal et al., 2020), was also effective in ameliorating social deficits during protracted withdrawal. We systemically administered Aticaprant (3 mg/kg, ip) or vehicle in a counterbalanced crossover design before both social assays that were performed 48 hours apart during withdrawal as well as before a CPP posttest (Figure S2A). Consistent with the effects of infusing norBNI into the NAc, Aticaprant reversed sociability deficits during withdrawal and also reduced MOR chamber preference (Figures S2B-S2F).

A potential source for the dynorphin that activates KORs in the NAc during opioid withdrawal are D1 receptor-expressing medium spiny neurons (MSNs), a major proportion of which express dynorphin (NAc^Pdyn neurons) (Al-Hasani et al., 2015). To test if dynorphin release from NAc^Pdyn neurons is necessary for the sociability deficits during protracted opioid withdrawal, we injected adeno-associated virus (AAV) carrying a Cre-dependent tetanus toxin (AAV-DIO-TeTx-p2A-eGFP) or eGFP alone as a control (AAV-DIO-eGFP) into the NAc medial shell ofPdyn-Cre mice (Figures 2A and2B). Tetanus toxin (TeTx) “silences” neurons by preventing SNARE-dependent release of transmitters due to cleavage of the critical SNARE protein VAMP/synaptobrevin (Kim et al., 2009). Surprisingly, mice expressing TeTx in NAc^Pdyn neurons showed normal baseline social behaviors (Figures S2H andS2I) and still exhibited robust sociability deficits during protracted withdrawal, which were similar to those in mice expressing eGFP in NAc^Pdyn neurons (Figures 2C–2F). The TeTx animals also exhibited normal CPP both 24 hr and 3 weeks after MOR administration (Figures 2G,S2J andS2K). However, MOR-induced locomotor sensitization was significantly reduced (Figures S2L andS2M), indicating that the TeTx manipulation was effective in these same animals. Consistent with the results from the initial cohort of animals, there was an inverse correlation between the magnitude of the sociability deficits and the magnitude of the preference for the chamber in which MOR was experienced (Figures S2N andS2O). To further examine if TeTx expression adequately reduced transmitter release from NAc MSNs we recorded stimulation evoked and spontaneous inhibitory postsynaptic currents (IPSCs) from uninfected and infected NAc MSNs in slices prepared from animals that had received unilateral injections of AAV-TeTx-eGFP in the NAc medial shell (Figure S2P). Consistent with the established effects of TeTx, evoked and spontaneous IPSCs were almost entirely absent in NAc MSNs expressing TeTx and recorded in the NAc medial shell compared to recordings in uninfected NAc MSNs from the opposite hemisphere (Figures S2P-S2U).

DR^Pdyn neurons regulate sociability through KOR activation in NAc

Where might the dynorphin that is presumably released in the NAc during protracted opioid withdrawal derive? Examination of the Allen Mouse Brain Atlas revealed thatPdyn is expressed in many brain regions that send projections to the NAc including the DR (Lein et al., 2007). Because 5-HT release in the NAc from DR neurons is critical for prosocial behavior (Heifets et al., 2019;Walsh et al., 2018), we were interested in the possibility that there may be a population of NAc-projecting dynorphin neurons in the DR (DR^Pdyn neurons). Consistent with this prediction, injection of AAV expressing Cre-dependent eYFP into the DR ofPdyn-Cre mice (Figure 2H) revealed a robust projection to the NAc medial shell, particularly in the dorsomedial region and more diffusely in the ventromedial shell (Figure 2I). Injection of AAV expressing Cre-dependent mGFP-p2A-synaptophysin-mRuby into the DR confirmed the presence of many putative release sites in DR^Pdyn axons in the NAc medial shell (Figures S3A andS3B).

To test whether transmitter release from DR^Pdyn neurons is necessary for the sociability deficits during protracted opioid withdrawal,Pdyn-Cre mice were injected with AAV-DIO-TeTx-p2A-eGFP into the DR and run through the same MOR administration and withdrawal procedures (Figure 2J). In marked contrast to expression of TeTx in NAc^Pdyn neurons, expressing TeTx in DR^Pdyn neurons prevented the decrease in prosocial behaviors that routinely occurs during protracted withdrawal (Figures 2K–2N). These same TeTx mice still exhibited a strong initial preference for the MOR-paired chamber and locomotor sensitization (Figures S3D-S3G). However, they showed a reduced preference for the MOR-paired context during the withdrawal CPP test (Figure 2O). Correlation analyses revealed a significant relationship between MOR CPP and sociability deficits in eGFP mice but this correlation was disrupted in TeTx mice (Figures S3H andS3I). Together these results suggest that DR^Pdyn neurons are a critical source for the dynorphin in the NAc that is required for the sociability deficits during protracted opioid withdrawal and also contribute to the prolonged memory of the MOR experience.

In addition to preventing sociability deficits during protracted opioid withdrawal, TeTx expression in DR^Pdyn increased baseline sociability in the juvenile intruder assay with a trend toward an increase in the 3-chamber task (Figures S3J andS3K), suggesting that dynorphin release from this cell population may normally reduce or limit prosocial behavior. To test this prediction, we injected AAV expressing Cre-dependent ChR2 (AAV-DIO-ChR2) or eYFP (AAV-DIO-eYFP) as a control into the DR ofPdyn-Cre mice and implanted an optical fiber in the same location (Figure 3A). Optogenetic activation of DR^Pdyn neurons robustly reduced social interaction and social preference (Figures 3B–3E), but produced no reinforcement or aversion on its own in a real time place preference test (Figure 3F). Importantly, the same stimulation in the same mice did not affect investigation of a novel object, preference for a chamber containing a high-fat food pellet, or time spent in the center and locomotor activity in the open field (Figures S4A-S4E). We next evaluated the effect of stimulating DR^Pdyn inputs specifically in the NAc medial shell by placing fibers above this NAc subregion (Figure 3G). This manipulation mimicked the effects of DR^Pdyn neuron cell body stimulation in that it again reduced prosocial behavior in both assays (Figures 3H–3K) and also did not produce a reinforcement or aversion in a real time place preference assay (Figure S4G). Furthermore, stimulation of this pathway did not affect center time or locomotor activity in the open field (Figures S4H andS4I).

To examine if dynorphin release in NAc medial shell from DR^Pdyn neurons and the consequent activation of NAc KORs are required for the behavioral effects of activating this cell population, we combined optogenetic activation of DR^Pdyn cell bodies with microinfusion of norBNI or saline into the NAc medial shell (Figure 3L). Since norBNI can inhibit KOR signalingin vivo for several weeks (Bruchas et al., 2007), we implemented a procedure that enabled within-subject comparisons (Figure 3L). All mice initially received NAc medial shell infusions with saline, which 24 hours later did not influence the reductions in prosocial behaviors elicited by cell body optogenetic activation of DR^Pdyn neurons (Figures 3M–3Q). A week later, half the mice again received saline infusions while the other half received norBNI, a manipulation which blocked the sociability deficits generated by optogenetic activation of DR^Pdyn neurons (Figures 3M–3Q). Importantly, in the saline treated mice, this same manipulation again caused robust sociability deficits (Figures 3M–3Q). These results demonstrate that DR^Pdyn cell stimulation reduces prosocial behaviors due to KOR signaling in the NAc medial shell.

DR^5-HT and DR^Pdyn neurons send parallel projections to NAc medial shell

The DR contains multiple different cell types with a majority of these being 5-HTergic as evidenced by their expression of tryptophan hydroxylase 2 (TPH2) (Zhang et al., 2004). The DR also has a small population of DA neurons, which have been implicated in social behavior, arousal, pain, and reward memory (Cho et al., 2017;Lin et al., 2020;Matthews et al., 2016;Yu et al., 2021). Given the importance of 5-HT and DA in social behaviors, an important question is whether DR^Pdyn neurons are capable of co-releasing 5-HT or DA. To address this issue, we crossedPdyn-Cre mice with Ai14 reporter mice so thatPdyn cells expressed tdTomato (tdTom) and then stained DR tissue from these mice for TPH2 and tyrosine hydroxylase (TH) (Figure 4A). While <1% of DR^Pdyn neurons co-localized with TH (data not shown), consistent with a previous report (Lemos et al., 2012) ~42% of DR^Pdyn neurons were 5-HTergic as defined by their co-expression of TPH2 (Figure 4B). The complimentary analysis revealed that ~30% of 5-HTergic neurons (i.e. TPH2+ neurons) werePdyn+ (i.e. tdTomato+) (Figure 4C). Sub-analyses revealed no sex differences in these expression profiles (Figures S5A-S5D). The distribution of DR^Pdyn neurons was similar across the AP axis of the DR, whilePdyn+/5-HT+ cells were clustered medially in the mid and caudal aspects of the DR (Figures 4D and4E).Pdyn neurons not expressing TPH2 were clustered laterally in the anterior DR, with a gradient of cells spreading into the neighboring vlPAG. The caudal DR, on the other hand, contained a large cluster of intermingled 5-HT+ and 5-HT-Pdyn neurons. Overall, thisPdyn expression pattern in 5-HT neurons is consistent with single-cell RNA sequencing studies reporting this gene in several major 5-HT cell clusters (Huang et al., 2019;Okaty et al., 2020;Ren et al., 2019).

To map the projection fields of these DR populations, we crossedPdyn-Cre mice toSert-Flp mice, which express Flp in 5-HT neurons (Ren et al., 2019). Injection of a cocktail of AAV-DIO-mCherry and AAV-fDIO-eYFP into the DR ofPdyn-Cre:Sert-Flp mice (Figure 4F) revealedSert+ andPdyn+ projections to the ventral tegmental area (VTA), basolateral amygdala (BLA), bed nucleus of the stria terminalis (BNST), NAc, and anterior prelimbic cortex (Figures S5E-S5J), consistent with previous work (Cardozo Pinto et al., 2019). Axonal projection patterns of the two major cell-types were generally similar withPdyn axons more medially biased and restricted. In the NAc,Sert+ inputs were diffuse across the medial shell, lateral shell, and core whilePdyn inputs were heavily concentrated in the medial shell (Figure 4G). To further examine this anatomical specificity,Pdyn-Cre:Sert-Flp mice were injected in the DR with either AAV-CreON-FlpOFF-eYFP to labelPdyn only cells, AAV-CreOFF-FlpON-eYFP to labelSert only cells, or AAV-CreON-FlpON-eYFP to label neurons that are bothPdyn+ andSert+ (Figures 4H–4P). Inputs fromPdyn only DR neurons in the NAc were found innervating the medial border of the medial shell from the dorsal cone to the ventral region (Figure 4I), whereas inputs fromSert only DR neurons encompassed the entire NAc shell and encroached into the core (Figure 4L).Pdyn+/Sert+ DR inputs were found throughout the medial shell, with the densest axon field in the dorsal region (Figure 4O). Viral infection of cell bodies in the DR matched the predicted distribution across the AP axis from our co-localization data withPdyn-Cre:Ai14 mice (Figures 4J,4M, and4P). These tracing data demonstrate three parallel projections to the NAc emanating from 3 subtypes of DR neurons withPdyn inputs preferentially innervating the dorsomedial shell, compared to broader 5-HT inputs innervating the entire shell and core.

KORs in 5-HT neurons are necessary for sociability deficits during opioid withdrawal

The finding that norBNI infusion into the NAc medial shell reverses the sociability deficits during protracted opioid withdrawal indicates that cellular elements within the NAc must express the requisite KORs but does not distinguish between KORs on local NAc neurons/glia versus KORs on presynaptic inputs. To examine whether KORs on intrinsic NAc neurons are necessary for the sociability deficits, we selectively ablated local NAc KORs by injecting AAV-Cre-eGFP into the NAc of floxed KOR (Oprk1^fl/fl) mice and compared their behaviors during protracted opioid withdrawal withOprk1^fl/fl mice that were injected with AAV-ΔCre-eGFP (Figure 5A). Mice expressing the active Cre and therefore lacking KORs in the NAc medial shell exhibited sociability deficits that were essentially identical to those exhibited byOprk1^fl/fl mice injected with the control virus (Figures 5B–5E). These same animals also exhibited normal locomotor sensitization and MOR reward learning in the CPP procedure (Figures S6A-S6E), which persisted 3 weeks into withdrawal (Figure 5F), as well as an inverse correlation between MOR CPP and sociability (Figures S6F andS6G).

5-HT and DA inputs to the NAc play key roles in social behaviors (Gunaydin et al., 2014;Walsh et al., 2018) and activation of KORs in the NAc reduces extracellular 5-HT (Tao and Auerbach, 2002) and DA (Spanagel et al., 1992). Moreover, KOR activation suppresses 5-HT release from synaptosome preparations extracted from the NAc (Schindler et al., 2012). We therefore hypothesized that KORs on 5-HT or DA inputs to the NAc are critical for the sociability deficits during protracted opioid withdrawal. While KOR expression in DA neurons is well established (Tejeda and Bonci, 2019), KOR expression in 5-HT neurons is still unclear. To determine if NAc-projecting DR 5-HT neurons express KORs, we injected fluorescent cholera toxin B (CTB-647) into the NAc medial shell of wild-type mice and harvested tissue for fluorescencein situ hybridization (FISH) (Figure 5G). CTB-647 preferentially labeled a population of neurons that localized to the dorsomedial aspect of the caudal DR (cDR;Figures 5H–5J). Few retro-labeled cells were found in the anterior or central DR (Figures 5I). FISH revealed that >90% of these NAc-projecting neurons in the cDR expressedSert (Slc6a4), identifying them as 5-HTergic (Figure 5J).Oprk1, the mRNA encoding KORs, was detected in cells throughout the DR but many of these wereSert- and not labelled by CTB-647 (Figures 5I and5J). The vast majority of the NAc-projectingSert+ neurons that were alsoOprk1+ were in the cDR (Figures 5H and5J). Reanalysis of a recent single-cell RNAseq dataset from DR cells (Huang et al., 2019) revealed a similar result in thatOprk1 was expressed in all cell-types in the DR with the highestOprk1 expression in 5-HT neurons found in the 5-HT neuron cluster localized to the caudal DR (data not shown). Consistent with an independent DR single-cell RNAseq paper (Ren et al., 2019),Oprk1 expression was minimal in all other 5-HT neuron subtypes, which project to other brain areas. These anatomical analyses identify a special NAc-projecting subpopulation of 5-HT neurons that express KORs and therefore may be influenced by dynorphin.

To directly test whether KORs expressed specifically in 5-HT neurons are necessary for the sociability deficits during protracted opioid withdrawal, we crossedOprk1^fl/fl mice withSert-Cre mice (Figure 5K). Homozygous 5-HT KOR knockout mice generated by this cross exhibited basal levels of social interaction and social preference that were similar to wild-type littermates (Figures 5L and5N). However, the decreases in these measures that normally occur during protracted opioid withdrawal were absent (Figures 5L–5O). These same KOR knockout mice also exhibited a blunted preference for the MOR-paired chamber during withdrawal (Figure 5P) although locomotor sensitization and the initial MOR CPP were normal (Figures S6H-S6K). Furthermore, while the wild-type control mice exhibited the expected inverse correlation between the magnitude of MOR CPP and sociability, the KOR knockout mice did not (Figures S6L andS6M). FISH confirmed genetic deletion ofOprk1 from 5-HT neurons (Figures S6N-S6P).

To test whether KORs in DA neurons (Figures 5Q and5R) are also required for the withdrawal sociability deficits, we crossedOprk1^fl/fl mice withDat-Cre mice (Figure 5S). These homozygous DA KOR knockout mice did not differ from littermate controls in that they exhibited comparable sociability deficits during withdrawal (Figures 5T–5W), normal locomotor sensitization (Figures S6Q andS6R) as well as normal place preference initially (Figures S6S andS6T) and during withdrawal (Figure 5X). Furthermore, there was an inverse correlation between the magnitude of MOR CPP and sociability during withdrawal (Figures S6U andS6V).Oprk1 knockout was verified with FISH (Figures S6W-S6Y). Together, these results identify a requisite, selective role for KORs in 5-HT neurons in the social avoidance phenotype associated with protracted opioid withdrawal.

KORs suppress 5-HT release in the NAc during protracted opioid withdrawal

Given the importance of 5-HT release in the NAc for prosocial behavior (Heifets et al., 2019;Walsh et al., 2018), all of the results presented thus far are consistent with the hypothesis that prolonged opioid withdrawal leads to increased dynorphin release in the NAc, which in turn reduces 5-HT release during social interactions due to activation of KORs on DR^5-HT inputs. To further examine this hypothesis, we directly measured 5-HT release dynamics by performing fiber photometry in wild-type mice expressing the genetically encoded fluorescent 5-HT sensor GRAB_5-HT (Wan et al., 2021) in the NAc medial shell. To test and presumably confirm the utility of this approach, we initially performed pharmacological manipulations (Figure 6A). Administration of MDMA (3,4-Methylenedioxymeth-amphetamine, 7.5 mg/kg, ip), which is known to cause large increase in 5-HT levels (Green et al., 2003), generated a robust increase in NAc medial shell GRAB_5-HT fluorescence (Figures S7A-S7C). In contrast, the KOR agonist U-50488 (5 mg/kg, ip) decreased GRAB_5-HT fluorescence (Figures 6B and6C), concomitant with a reduction in transient event frequency and amplitude, all of which were blocked by pretreatment with norBNI (10 mg/kg, ip) (Figures 6B–6E).

We next recorded NAc medial shell GRAB_5-HT fluorescence during social interactions using the juvenile intruder assay (Figure 6F). During baseline measurements, transient increases in GRAB_5-HT fluorescence occurred during initial social contact in two independent groups of mice (Figure 6G), a result consistent with the increases in DR^5-HT neuron activity that occur during similar social interactions (Walsh et al., 2018). These same mice then received our standard saline or escalating MOR treatments and three weeks into protracted withdrawal were imaged again during an identical juvenile interaction test. MOR treated mice exhibited a decrease in GRAB_5-HT fluorescence during their social interactions while saline treated mice exhibited increases that were similar to those observed during baseline measurements (Figures 6H–6J). Subsequent administration of norBNI in the MOR treated mice resulted in recovery of GRAB_5-HT fluorescence to normal levels during social contact (Figures 6K–6O) and, as expected, an increase in the total duration of the social interactions (Figure S7D). Because DA levels in the NAc also increase during social interactions (Dai et al., 2022;Wang et al., 2021), we repeated these same experiments in mice in which GRAB_DA (Sun et al., 2020) was expressed in NAc medial shell (Figure S7E). Similar to GRAB_5-HT fluorescence, NAc medial shell GRAB_DA fluorescence increased during baseline social interactions (Figure S7F) but this increase was not significantly reduced during social interactions three weeks into protracted opioid withdrawal (Figures S7G-S7I).

To further test if KOR signaling specifically in 5-HT neurons mediates the reduction in 5-HT release that occurs during social interactions in mice experiencing protracted withdrawal, we expressed GRAB_5-HT in the NAc medial shell of homozygous 5-HT KOR knockout mice (Figure 7A). Knockout mice and controls displayed the expected increase in NAc GRAB_5-HT fluorescence during baseline social contact (Figure 7B). However, during protracted opioid withdrawal, the knockout mice continued to exhibit a clear increase in GRAB_5-HT fluorescence during social contact while, consistent with our previous results, the control mice did not (Figures 7C-7E). Furthermore, as expected, the sociability deficits during withdrawal were present in the control mice but absent in the knockout mice (Figure S7J).

Because the KOR-mediated reduction of 5-HT release during social interactions is happening within the NAc, changes in the physiology of DR^5-HT neurons themselves during withdrawal are not required to account for any of our results, but nevertheless could importantly contribute (Walsh et al., 2018), especially given that KOR activation can suppress DR^5-HT neuron excitability (Lemos et al., 2012). To address this topic, we injected AAV-DIO-GCaMP6m into the DR ofSert-Cre mice and recorded DR^5-HT neuron activity during baseline social interactions and when the mice were experiencing protracted opioid withdrawal (Figure 7F). During prolonged opioid withdrawal, in contrast to the dramatic decreases in NAc GRAB_5-HT fluorescence during social interactions, there was no detectable change in the increase in DR^5-HT neuron GCaMP6m fluorescence (Figures 7G-7J) even though there was the expected decrease in social interaction times (Figure S7K). Thus, sociability deficits during protracted opioid withdrawal and in an autism model (Walsh et al., 2018) both appear due to a decrease in NAc 5-HT release during prosocial interactions but these decreases are mediated via different mechanisms.

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by thelead contact, Dr. Robert C. Malenka (malenka@stanford.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The datasets generated and/or analyzed in the current study are available from thelead contact upon reasonable request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from thelead contact upon request.

Subjects

Female and male C57BL/6J (Jackson Laboratory; stock #00664), heterozygousPdyn^{tm1.1(cre)Mjkr}/LowlJ (Pdyn-IRES-Cre, Jackson Laboratory; stock #027958), Tg(Slc6a4-Cre)ET33Gsat (Sert-Cre, Jackson Laboratory; MGI: 3836639),Slc6a3^{tm1.1(cre)bkmn}/J (Dat-IRES-Cre, Jackson Laboratory; stock #006660),Slc6a4^{tm1.1(flop)Luo}/J (Sert-IRES-Flp, Jackson Laboratory; stock #034050;Ren et al., 2019), homozygousOprk1^tm2.1Kff/J (Oprk1^fl/fl, Jackson Laboratory; stock #030076), and Gt(ROSA)^{26Sortm14(CAG-tdTomato)Hze}/J (Ai14, Jackson Laboratory; stock #007914) mice were used. All mice (8–18 weeks old) were kept on a C57BL/6J background and group housed on a 12-hr light/dark cycle with food and waterad libitum. All procedures complied with animal care standards set forth by the National Institute of Health and were approved by the Stanford University’s Administrative Panel on Laboratory Animal Care and Administrative Panel of Biosafety.

Viral vectors and stereotaxic surgery

AAVs purchased from Stanford Neuroscience Gene Vector and Virus Core: AAVdj-hSyn-Cre-eGFP, AAVdj-hSyn-ΔCre-eGFP, AAVdj-CMV-DIO-eGFP-2A-TeTx, AAVdj-CMV-eGFP-2A-TeTx, AAVdj-CMV-DIO-eGFP, AAVdj-EF1a-DIO-hChR2(H134R)-eYFP, AAVdj-EF1a-DIO-eYFP, AAVdj-EF1a-DIO-mCherry, AAVdj-EF1a-fDIO-eYFP, AAVdj-hSyn-CreON-FlpOFF-eYFP-WPRE, AAVdj-hSyn-CreOFF-FlpON-eYFP-WPRE, AAVdj-hSyn-CreON-FlpON-eYFP-WPRE, AAVdj-hSyn-FLEX-mGFP-2A-Synaptophysin-mRuby, and AAVdj-EF1a-DIO-GCaMP6m. AAV9-hSyn-GRAB_5HT 2h (5-HT 3.5) and AAV9-hSyn-GRAB_DA 2m (DA 4.4) were purchased from WZ Biosciences (Columbia, MD). All viruses were injected 4–6 · 10¹² infectious units per mL.

Mice of at least 7 weeks of age were anesthetized with isoflurane (1–2% v/v) and secured in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Viruses were injected into the DR (AP −4.6; ML ±0; DV −3.1 from skull) or bilaterally into the NAc medial shell (AP +1.1; ML ±0.7; DV −3.6 from dura) at a rate of 150 nL min⁻¹ (500 nL total volume) with a borosilicate pipette coupled to a pump-mounted 5 μL Hamilton syringe. Injector pipettes were slowly retracted after a 5 min diffusion period. For optogenetic behavioral experiments, mice were also implanted with optical fibers above the DR (AP −4.6; ML ±0; DV −3.0 from skull) or bilateral NAc (AP +1.1; ML ±1.3; DV −3.1 from dura, 10°). Optical fibers were constructed in-house using 1.25 mm diameter multimode ceramic ferrules (ThorLabs), 200 μm core fiber optic cable with 0.39 numerical aperture (NA) (ThorLabs), and blue dye epoxy (Fiber Instrument Sales). For photometry recordings, optical fibers (Doric Lenses) with 400 μm core and 0.66 NA were unilaterally implanted over the NAc (AP +1.1; ML +0.7; DV −3.6 from dura). Optical fibers were secured to the skull with stainless steel screws (thread size 00–90 × 1/16, Antrin Miniature Specialties), C&B Metabond, and light-cured dental adhesive cement (Geristore A&B paste, DenMat).

For drug microinfusions, a 26-gauge bilateral cannula (P1 Technologies), 3.5 mm in length from the cannula base, was implanted over the NAc (AP +1.1; ML ±0.75; DV −3.1 from dura). Mice that received drug infusions with optogenetic stimulation were implanted with an optical fiber targeting the DR and a guide cannula targeting the NAc in the same surgery. Mice were group housed to recover for 3 weeks before experiments began.

Drug administration

For MOR withdrawal experiments, mice were made MOR dependent with 6 injections of 10–50 mg/kg MOR (Sigma Aldrich, M8777, ip) over a period of 6 days: i.e. 10 mg/kg on day 1, 20 mg/kg on day 2, etc. Mice received a second injection of 50 mg/kg MOR on day 6 (Zhu et al., 2016). Mice were then left undisturbed in homecages for three weeks before behavioral testing or recordings.

For systemic administration of the KOR antagonist Aticaprant, wild-type mice were subjected to protracted withdrawal procedures and then tested in a counterbalanced crossover design. Thirty min before behavioral testing, mice were injected with Aticaprant (3 mg/kg, ip, Med Chem Express) or vehicle (10% ethanol in corn oil, 5 mL/kg) and then administered the opposite solution before an identical test 48 hours later. This protocol was repeated for the juvenile intruder test, the 3-chamber social preference test, and the CPP posttest. Within-subject comparisons of social behavior and MOR chamber preference after vehicle and Aticaprant injections were made for each subject. Mice that failed to show deficits in at least one social assay during withdrawal were excluded from the study.

For local NAc inhibition of KORs, mice were administered an intra-NAc infusion of norBNI (2.5 μg/hemisphere, Tocris Bioscience) 24 hr prior to behavioral testing. Drugs were infused through an injector cannula coupled to a 5 μL Hamilton syringe using a microinfusion pump (Harvard Apparatus) at a continuous rate of 100 nL min⁻¹ to a total volume of 0.3 μL per hemisphere. Injector cannulas were removed 2 min after infusions were complete. Mice were left undisturbed in homecages before behavioral testing. Mice in the experiment displayed inFigures 1L–1O were initially tested for social behavior while in MOR withdrawal. One week later, mice were administered a microinjection of norBNI or saline and then tested in a second round of social assays 24 hr later. Mice in the experiment displayed inFigures 3N–3Q were microinjected with saline 24 hr prior to the first round of sociability testing with optogenetic stimulation. One week later, mice were split into norBNI or saline groups and received a second microinjection of the respective drug into the NAc. 24 hr later all mice were tested in a second round of social assays with optogenetic stimulation in an identical manner to round one.

During fiber photometry experiments, mice were injected with saline or norBNI (10 mg/kg, ip) 24 hr before the final recording. For pharmacology recordings, mice were injected with U-50488 (5 mg/kg, ip, Tocris Bioscience) or MDMA (7.5 mg/kg, NIDA) 5 min into the recording. Some mice administered U-50488 were pretreated with norBNI (10 mg/kg, ip) 24 hr prior to the recording.

Protracted withdrawal behavior

All mice in withdrawal experiments were subjected to a standardized behavioral and drug administration procedure. Mice were initially tested for baseline social behaviors in the juvenile intruder and 3-chamber preference assays. Mice were then administered 6 ascending doses of MOR while confined in a conditioned place preference (CPP) chamber. 24 hr after the last dose of MOR, mice were tested for MOR CPP. Mice were then left undisturbed in homecages for three weeks. After the incubation period, mice were then tested again for social behavior in the same assays. Finally, mice were placed back into the CPP chamber and tested for long-term MOR preference. As much as possible, all behavioral assays were performed blind, without knowledge of the treatment history of the subject.

Optogenetic stimulation

For optogenetic experiments, optical fibers were connected to a 473 nm laser diode (OEM Laser Systems) through a FC/PC adapter connected to a fiber optic rotary joint commutator (Doric Lenses). BIOBSERVE was programmed to control laser output using a Master-8 pulse stimulator (A.M.P.I.), which delivered 5 ms light pulses at 20 Hz (Walsh et al., 2018). Light output through the fibers was adjusted to ~5 mW for somatic stimulation and ~15 mW for terminal stimulation using a digital power meter console (ThorLabs).

Fiber photometry

Fiber photometry was performed as previously described (Heifets et al., 2019;Walsh et al., 2018;Wu et al., 2021). AAVdj-EF1a-DIO-GCaMP6m was injected into the DR with a fiber directed above the DR. AAV9-hSyn-GRAB_5HT or AAV9-hSyn-GRAB_DA were injected into the NAc medial shell with a fiber directed above. After at least 3 weeks, mice were habituated to the photometry and behavioral setups before any recordings took place. On the test day, mice were connected to patch cables and allowed to habituate alone in the homecage for 2 min. A novel, sex-matched juvenile conspecific was then introduced into the cage for free interaction for 3 min. After the baseline interaction test, all mice were subjected to the MOR administration regimen in their homecages and left undisturbed. Three weeks into protracted withdrawal, the fiber photometry and social test procedure was performed again, identically to the baseline test. One week later, mice treated with MOR were randomly split into saline or norBNI groups and injected ip with the respective compound. 24 hr later, mice were tested in the same fiber photometry and social test procedure. A cohort of homozygous 5-HT KOR knockout mice were prepared and tested in an identical procedure but received no drug injections after MOR administration. Mice used for pharmacology recordings were prepared with GRAB_5HT and allowed to recover for at least 3 weeks. 5-HT release was recorded in the homecage during a 5 min baseline. Then the respective drug was injected ip and recordings continued for 40 min.

Data were acquired using Synapse software controlling an RZ5P lock-in amplifier (Tucker-Davis Technologies). GCaMP6m and GRAB sensors were excited by frequency-modulated 465 and 405 nm LEDs (Doric Lenses). Optical signals were band-pass-filtered with a fluorescence mini cube (Doric Lenses) and signals were digitized at 6 kHz. Signal processing was performed with custom scripts in MATLAB (MathWorks). Briefly, signals were de-bleached by fitting with a mono-exponential or bi-exponential decay function and the resulting fluorescence traces were z-scored. Videos were manually analyzed for the first social contact. The first social contact was used in all recordings because of the predicted decrease in sociability during opioid withdrawal. Peristimulus time histograms were constructed by taking the average of 15 sec epochs of fluorescence consisting of 5 sec before and 10 sec after social contact, which was defined as time = 0. Before averaging, each epoch was offset such that the z-score averaged from −5 to −1 sec equaled 0. Peak z-scored fluorescence was determined for each peristimulus time histogram as the maximal z-score value between 0 and +10 sec. Area under the curve was defined as the integral between 0 and +10 sec. Peak z-score and area under the curve were calculated with Prism 9.

Slice electrophysiology

Wild-type mice were unilaterally injected with AAV-CMV-eGFP-p2A-TeTx into the NAc medial shell. Three weeks later, mice were decapitated following deep isoflurane anesthesia. The forebrain was quickly removed and placed in ice cold cutting solution (bubbled with 95%O₂/5%CO₂) consisting (in mM): 228 sucrose, 2.5 KCl, 1.0 NaH₂PO₄, 8 MgSO₄, 26 NaHCO₃, 20 glucose, and 0.5 CaCl₂. The forebrain was then mounted on a cutting platform and hemisected prior to slicing. Coronal slices (200 μm) containing the NAc were cut with a vibratome (Leica VT1200s) and transferred to a holding chamber containing warmed (30°C, bubbled with 95%O₂/5%CO₂) ASCF consisting of (in mM): 119 NaCl, 2.5 KCl, 1 NaH₂PO₄, 1.3 MgSO₄, 26 NaHCO₃, 10 glucose, and 2.5 CaCl₂. Slices were incubated for 45 min and then kept at room temperature for 1–1.5 hr before being transferred to a fixed recording chamber of an upright microscope (BX51WI, Olympus). Slices were perfused with ACSF (bubbled with 95%O₂/5%CO₂) and maintained at 29–30°C. TeTx infection in the NAc medial shell was identified by eGFP epifluorescence. Whole-cell recordings from neurons in the medial shell were obtained with patch electrodes containing (in mM): 135 CsMeSO₄, 10 HEPES, 5 Na-phosphocreatine, 8 NaCl, 0.25 EGTA, 2 MgCl₂, 4 Mg-ATP, 0.3 GTP, and 1 spermine (300 mOsM, pH 7.35). Electrode resistances ranged from 2.5–3 MΩ. Series resistance was continually monitored and experiments were discarded if it changed by >20%. Whole-cell voltage clamp recordings were made with a MultiClamp 700B (Molecular Devices). Signals were filtered at 2 KHz and digitized at 10 KHz (National Instruments BNC-209 or Digidata 1320A, Axon Instruments).

IPSCs were evoked with a bipolar electrode fabricated from platinum-iridium wire (A-M systems). To increase the likelihood of stimulating TeTx-infected axons, the stimulating electrode was placed in areas of high eGFP epifluorescence. Whole-cell recordings were made from neurons in the infected area in close proximity (<100 μm) to the stimulating electrode. This arrangement was also applied to recordings from uninfected slices. Evoked and spontaneous IPSCs were recorded at a holding potential of 5 mV in the presence of 10 μM NBQX, 50 μM D-AP5, and 5 μMCGP55845 (all from Tocris Bioscience). IPSCs were evoked at 0.2 Hz with stimulation intensities ranging from 10–50 μA. To generate input-output graphs each point on the graphs represents the average amplitude of ten consecutive IPSCs recorded at each stimulation intensity. sIPSCs were recorded “gap-free” for 5 min and data were analyzed with MiniAnalysis software (Synaptosoft). Recordings performed in the TeTx-infected hemisphere were compared to those in the uninfected hemisphere.

Histology

Mice were anesthetized with isoflurane and perfused transcardially with 1X PBS followed by 4% paraformaldehyde in PBS, pH 7.4. Brains were extracted and post-fixed overnight in the same fixative. Brains were sectioned at 40 μm on a vibratome and collected in PBS. Free-floating sections were washed three times in PBS for 10 min at room temperature and then incubated in PBS with 0.5% Triton X-100, 10% normal goat serum, and 0.2% bovine serum albumin (BSA) for 1 hr. After a 5 min wash in PBS, sections were next incubated in primary antibodies rabbit anti-TPH2 (1:1000, Novus Biologicals, NB100–74555), mouse anti-TH (1:1000, Millipore, MAB318), rat anti-mCherry (1:1000, Millipore, MAB131873), or chicken anti-GFP (1:1000, Aves Labs, GFP-1010) in carrier solution containing 0.5% Triton X-100, 1% normal goat serum, and 0.2% BSA in PBS shaking at room temperature for 24 hr. After four 10 min washes in PBS, sections were incubated in species-specific secondary antibodies Alexa Fluor 488, 594, or 647 (1:750, Invitrogen, Carlsbad, CA, A-11039, A-11058, and A-31573) in carrier solution for 2 hr at room temperature. Finally, sections were washed four times for 10 min in PBS, mounted onto SuperFrost Plus glass slides, and coverslipped with Fluoromount-G with DAPI (Southern Biotech, Birmingham, AL, 0100–20). Fluorescent images were collected on a Nikon A1 confocal microscope or a Keyence BZ-X800 fluorescence microscope.

Fluorescencein situ hybridization

For examination of gene expression in the DR and VTA, wild-type brains were flash frozen in isopentane on dry ice, sectioned on a cryostat at 16 μm, and processed for fluorescencein situ hybridization (FISH) with the RNAscope Multiplex Fluorescent v2 assay according to the manufacturer’s guidelines (Advanced Cell Diagnostics). Transcripts examined wereSlc6a4 (Sert) (ACDBio cat# 315851),Slc6a3 (Dat) (ACDBio cat# 315441), andOprk1 (ACDBio cat# 316111). Slides were coverslipped with Fluoromount-G with DAPI (Southern Biotech, 0100–20) and stored at 4°C in the dark before imaging. For verification ofOprk1 deletion, midbrain sections fromSert-Cre andDat-Cre mice crossed toOprk1^fl/fl mice were processed as described above. Cre- mice from the same litters served as wild-type controls. For analysis of NAc-projecting DR neurons, wild-type mice were bilaterally injected with 200 nL of CTB-647 (4 μg/μL) into the NAc and sacrificed 14 days later. To preserve CTB fluorescence, the only modification to the RNAscope protocol was the application of protease III for 10 min, as opposed to protease IV for 30 min. Fluorescent images were collected on a Nikon A1 confocal microscope. Each image was acquired using identical pinhole, gain, offset, and laser settings (1024 × 1024 pixels). Cells were counted and co-localization was measured using custom macros in Fiji (Schindelin et al., 2012). For measuringOprk1 deletion, analysis was restricted to regions of interest (ROIs) specifically within theSert orDat cell populations. The mean pixel intensity ofOprk1 within these ROIs was calculated. The number ofSert orDat neurons expressingOprk1 was also calculated and normalized to the total number ofSert orDat neurons for each section and averaged for each subject.

Table 1.

INCLUSION AND DIVERSITY.

We worked to ensure sex balance in the selection of non-human subjects. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper self-identifies as a member of the LGBTQIA+ community. One or more of the authors of this paper received support from a program designed to increase minority representation in their field of research. While citing references scientifically relevant for this work, we also actively worked to promote gender balance in our reference list.

Highlights.

• Protracted opioid withdrawal leads to robust social interaction deficits in mice.

• Kappa opioid receptor activation in the NAc is necessary for withdrawal social deficits

• Dynorphin-producing neurons in the DR promote withdrawal social deficits

• KORs reduce 5-HT release in the NAc during withdrawal to mediate social deficits

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Supplementary Materials

Data Availability Statement

The datasets generated and/or analyzed in the current study are available from thelead contact upon reasonable request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from thelead contact upon request.