Dynorphinergic lateral hypothalamus to posterior ventral tegmental area pathway matures after adolescence in male rats

1.0: INTRODUCTION

Adolescence is a developmental period marked by increased risk taking, novelty seeking, and reorganization of brain circuits responsible for motivation and reward^1,2. During this transition, new synapses form and unused synapses are pruned, particularly in brain regions important for stress, reward, and executive control^1,3. Long-range projections between these critical regions also form during adolescence^4,5. Adolescent brains are especially attuned to reward and habit formation, rendering them extremely sensitive to drugs of abuse^6,7. Importantly, we and others have repeatedly demonstrated that adolescent nicotine exposure potentiates the rewarding effects of other drugs of abuse later in life^8–11. Nicotine and alcohol are the most commonly used drugs during adolescence¹². Notably, bidirectional associations between nicotine and ethanol have been described across several studies. Nicotine use is often initiated in combination with alcohol and increases its consumption, while alcohol increases nicotine use^13–18. Yet the effects of combined use are not studied as often as either drug alone.

A promising target for both understanding and modulating reward is the ventral tegmental area (VTA)^19–22. The VTA is a critical brain region deeply involved in the neurobiological processes underlying substance use disorders^23–25 along with other regions in the basal ganglia, the hippocampus, and the amygdala. For a comprehensive review of the neurocircuitry of addiction, we refer the reader to an excellent review by George Koob and Nora Volkow²⁶. Evidence shows that VTA pathways are still maturing during adolescence^4,27. These and other late-developing pathways may be important contributors to the unique susceptibility to addiction observed in adolescents, as well as the long-lasting effects of drugs of abuse^28,29. Here, we focus on the dynorphinergic lateral hypothalamus (LH) projection to the posterior VTA (pVTA)^30,31 to identify a potential mechanism for behavioral and functional age differences observed in our previous studies^10,32.

Our previous research indicated that adolescent male rats find intravenous nicotine and alcohol self-administration more reinforcing when combined than either alone, while adult male rats show resilience to this effect¹⁰. Systemic inhibition of kappa opioid receptors (KORs) in adult male rats abolishes their resistance to the reinforcing effects of a low dose of nicotine and ethanol; female rats of either age did not change their preference for nicotine + ethanol with KOR antagonism¹⁰. KORs are G_i/G_o coupled receptors that bind to the endogenous ligand dynorphin and are widespread in the brain, including the LH³². A subsequent study found that a single exposure to a low dose of combined nicotine and ethanol elicits coordinated cFos mRNA expression between LH and the pVTA only in adult male rats²⁷. Thus, we hypothesized that activity in dynorphin neurons projecting from LH to pVTA in adult rats^31,33, protects adults from the reinforcing effects of nicotine + ethanol, and that this projection is either not active or not present in adolescents. We suspect that our previous results showing adolescent-like susceptibility to nicotine + ethanol reward in adults treated with systemic norBNI is due to antagonism of KORs in the pVTA, likely on dopaminergic neurons. In the current study, we test these hypotheses by quantifying LH neurons expressing both prodynorphin and cFos in adolescent and adult male rats following drug exposure and by examining Fluorogold tracing of the LH-pVTA axon tract in drug-naïve adult and adolescent male rats. While dynorphin neurons innervate other brain regions, including the nucleus accumbens and parabrachial nucleus^30,31, our activity results²⁷ and evidence that LH-VTA projections have causal effects on reward behavior led us to focus only on the LH-pVTA dynorphinergic pathway.

While our previous results suggest a primary involvement of the dynorphin system, this is coupled with orexin signaling; many neurons co-release dynorphin and orexin^30,34. Orexin and dynorphin have opposing effects on ³⁵, while KOR agonism increases the threshold for intracranial self-stimulation reward; this effect is rescued by KOR antagonism by norBNI³⁶. Additionally, multiple studies have shown age-dependent changes in orexin expression^37,38, and a recent study showed age-dependent effects of ORX1 and ORX2 antagonism in cue-induced reinstatement³⁹, suggesting that changes in orexin composition with development have behavioral impacts. We therefore examined differences in orexin expression at each timepoint and differences in orexinergic neuronal activity following nicotine + ethanol experience.

2.0: METHODS

Subjects

Male Sprague-Dawley rats were purchased from Charles River and arrived at postnatal day (P)17 or P75. Although shipping during adolescence is a known stressor that can disrupt drug effects^40,41, the ages during shipment are outside of this sensitive period, and this shipping timeline has been used successfully across several studies^8–11. Juveniles were housed with a dam until weaning at P21. Since age-specific nicotine + ethanol reinforcement effects were not observed in females in our previous study¹⁰, females were not included. Weaned juveniles and adults were group-housed in an AALAC-accredited vivarium on a 12-h light-dark cycle with food and water available ad libitum. Animals were weighed daily to ensure maintenance of normal growth. Starting at P21 or P79, all animals were handled for two minutes daily prior to surgery and throughout experimentation. All handling occurred during the light cycle. All procedures followed NIH guidelines and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.

Surgical Procedures

Intravenous catheterization.

Adolescent and adult male rats were surgically prepared before drug treatment. Adolescent and adult rats underwent surgery at P28 and P86, respectively. Animals were anesthetized with 0.035 mg/kg Equithesin injected intraperitoneally and were surgically implanted with a catheter into their right jugular vein⁴². Rats were given 4 days to recover before drug exposure. Cannulae were flushed daily with sterile heparinized saline to maintain catheter patency. Propofol (5mg/kg, i.v.) was injected the day before nicotine + ethanol administration. Animals that did not display rapid (5–10s) anesthesia were excluded from analysis.

Intracranial injections.

Adolescent and adult male rats underwent intracranial injections of the retrograde neuronal tracer Fluoro-Gold⁴³ at P28 and P79, respectively. Fluoro-Gold was dissolved in sterile saline to a 3% weight/volume concentration. Pressure injections of 0.25μl were targeted to the left hemisphere posterior VTA (Adults: AP −6.4, ML +0.4, DV −8.1 from brain surface; Adolescents: AP −6.4+0.005(300-weight(g)), ML +0.4, DV −8.1) in equithesin-anesthetized animals. Coordinates were visually confirmed using cresyl violet injection before proceeding with Fluoro-Gold injection. Fluoro-Gold treated animals were returned to the home cage to recover. Animals were sacrificed 10 days later, the recommended incubation period for the tracer, and tissue was prepared for immunohistochemistry. Posterior VTA coordinates in Fluoro-Gold treated animals were confirmed in 3 pilot injections; spread in these injections was minimal, not extending to any other brain region. We did not find any reports of differences in neuronal uptake or transport of Fluorogold between adolescent and adult rats.

Drugs

Nicotine tartrate was purchased from Glentham Life Sciences (London, UK). The appropriate mass was calculated as a base and converted to the molecular weight of the salt, dissolved in sterile saline, and adjusted to pH 7.2–7.4. Ethanol was purchased from Sigma (St. Louis, MO) and prepared at concentrations no greater than 20% (v/v). All drugs were filtered through sterile filters. Propofol was purchased from Zoetis Animal Health (Parsippany-Troy Hills, New Jersey). Adolescent and adult rats were given two injections, spaced 1 minute apart, of either saline (1 ml/kg, i.v.) or combined nicotine and ethanol (2 × 15 μg/kg nicotine plus 2 × 2 mg/kg ethanol, i.v.). This procedure and dose mirrors was used in the previous brain mapping analysis²⁷ that identified the differences between adult and adolescent LH-pVTA connectivity in male rats. This dose also reflects the average responses on the reinforced lever during the first minute of self-administration of nicotine + ethanol in our previous study¹⁰ that demonstrated combined nicotine + ethanol is more reinforcing in adolescent than adult male rats. Given the behavioral and connectivity differences has only been observed in male rats with this dose thus far, we only included this dose and used male rats in the current study. All animals were allowed to habituate to the experimental room for 30 minutes in their home cages before drug exposure. One hour after drug injection, animals were sacrificed and tissue was prepared for immunohistochemistry. The time chosen for tissue collection (1 hour after drug injection) corresponds to the peak of stimulus-induced cFos protein expression⁴⁴.

Immunohistochemistry

All animals were transcardially perfused with ice-cold phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. Brains were extracted and post-fixed overnight at 4°C, then transferred to PBS. Tissue was cryoprotected in 30% sucrose and rapidly frozen in −30 °C 2-methylbutane. Frozen tissue was trimmed to remove the cerebellum, olfactory bulb, and rostral portion of the brain (~bregma +1.5 mm), then cut on a cryostat (Leica Biosystems, Wetzlar, Germany) into 40μm sagittal sections. LH sections were collected from 2mm lateral of bregma to 0.5 mm lateral of bregma on each hemisphere, then either stored in PBS overnight or immediately rinsed and blocked in 3% normal donkey serum in PBS with 0.2% Triton X-100. Sections spanning the whole LH from each hemisphere were selected for staining, prioritizing the highest tissue quality. Tissue was incubated in primary antibody overnight (guinea pig anti-prodynorphin, Thermo Fisher PA1–27974, 1:500; rabbit anti-cFos, Synaptic Systems #226 008, 1:1000) with 1% normal donkey serum in PBS at 4 °C, then rinsed in PBS and incubated in secondary antibody (Thermo Fisher A10040, A21448, Jackson Labs 706–545-148, 1:500) for two hours at room temperature. Sections 1.4mm, 1mm, and 1.8mm lateral of bregma from each hemisphere were mounted onto slides and coverslipped with Fluoromount-G with DAPI (Invitrogen), then imaged on an epifluorescence microscope at 20x (Keyence, Itasca Illinois). No differences between hemispheres were observed; cell counts are pooled between hemispheres.

Fluorogold-traced tissue, from the injected left hemisphere, was prepared in the same way. Tissue was incubated in primary antibody overnight (guinea pig anti-prodynorphin, Thermo Fisher PA1–27974, 1:500; anti-Fluorogold, Fluorochrome, Denver Colorado, 1:500; anti-orexin-A, Bio-Rad, California, 1:500) with 1% normal donkey serum in PBS. Following secondary antibody incubation, sections were mounted onto slices and coverslipped with Fluoromount-G without DAPI. DAPI was omitted due to shared excitation channel with Fluoro-Gold to allow direct detection if necessary. Orexin staining was used for regional and cell identification within the LH.

Imaging

Images used for quantification were collected on a Keyence BZ-X810 epifluorescence microscope at 20X magnification, using the same illumination settings for all images. Representative images were taken using a Zeiss LSM 900 confocal microscope at 20X magnification.

Images were manually counted in ImageJ (NIH). Six 20x fields of view (725 × 546 μm) in the LH were analyzed in each brain, counting all cells with PDYN or cFos staining per field of view. Total PDYN+, total cFos+, and total PDYN+/cFos+ neurons were summed for each brain. The same approach was used for Fluorogold-traced tissue. Cells were identified manually by inspection. Experimenters were blind to experimental groups. Individual channels were adjusted to minimize background without fully saturating any pixel. Cells were identified by DAPI signal and morphology in the dynorphin study or by morphology alone and orexin signal in the Fluoro-Gold tracing study. Co-labeled neurons were identified by the presence of signal above background in both PDYN and FG or cFos channels and cellular morphology.

Statistical Analysis

All statistical analysis was performed using GraphPad Prism 9 (GraphPad, Boston Massachusetts). Animals which did not display immediate propofol-induced anesthesia after drug treatment were excluded from analysis. Outliers of box and whisker plot separated by all groups were excluded from analysis.

For drug treated animals, cell counts were collected as described above in Imaging for each treatment group separately and then analyzed by two-way ANOVA to identify effects of age and drug treatment. Bonferroni corrected post hoc analysis was applied for significant main or interactive effects with one or two tailed t-tests, as appropriate. For Fluoro-Gold tracing data, cells were counted manually for single and double staining of prodynorphin and fluorogold. Orexin was included in analysis since it is an abundant cell type within the LH. Cell types were calculated as a percentage of all labeled cells and compared between adolescents and adults. Multiple t-tests were performed to investigate age differences for each cell type and T-test results were adjusted for multiple comparisons by the Bonferonni-Dunn method.

3.0: RESULTS

3.1: LH – pVTA dynorphinergic projection develops after adolescence

LH dynorphin neuron activity is significantly higher in nicotine + ethanol treated adult rats than in nicotine + ethanol treated adolescent rats.

Our previous studies suggested that the age-dependent difference in susceptibility to reinforcement evoked by low doses of nicotine + ethanol is mediated by kappa opioid receptor function/dynorphinergic signaling¹⁰. Additional investigation using the same model of drug administration identified age differences in behavioral responses to a single low dose of nicotine + ethanol with a distinct brain activity network between LH to VTA present in adult and not adolescent male rats²⁷. These behavioral findings suggest that KOR inhibition in adult male rats can induce adolescent-like drug responses, potentially driven by blockade of dynorphinergic signaling. To test our hypothesis that LH dynorphin neurons respond to nicotine + ethanol in an age-dependent manner, we quantified both LH dynorphin⁺ and dynorphin⁺/cFos⁺ double-positive neurons one hour after nicotine + ethanol administration in adult and adolescent rats (Figure 1) using the same drug administration protocol as in previous studies. The number of dynorphin⁺ neurons did not differ between saline or nicotine + ethanol treatment groups; however, we identified a trend toward adults having more dynorphin⁺ neurons per sample than adolescents. The number of dynorphin⁺ neurons did not vary with saline or nicotine-ethanol treatment. Most notably, the portion of dynorphin⁺ neurons which were also cFos⁺ nearly doubled in the adult rats that received nicotine + ethanol. A two-way ANOVA was performed to analyze the effect of drug and age on neuronal activation. The main effect of age was the most influential variable, with the interaction between age and drug strongly trending. Main effect of age F(1,15) = 4.66, p = 0.0473; main effect of drug F(1,15) = 3.77, p = 0.709, interaction F(1,15) = 4.46, p = 0.0517. While no difference in cFos activity is observed in adolescent LH dynorphin⁺ neurons following drug administration, significantly more adult LH dynorphin⁺ neurons express cFos after drug experience (p = 0.0248, Bonferroni-corrected one-tailed t-test).

Figure 1: Activity in LH PDYN+ cells increases after nicotine + ethanol exposure in adult rats.

A: Schematic of paradigm with saline or nicotine + ethanol exposure and tissue processing. B: Representative image of cFOS and PDYN staining in the saline treated adolescent LH. C: Representative image of cFOS and PDYN staining in the nicotine + ethanol treated adolescent LH. Arrows show cFOS⁺-PDYN⁺ colocalization. D: Representative image of cFOS and PDYN staining in the saline treated adult LH. E: Representative image of cFOS and PDYN staining in the nicotine+ ethanol treated adult LH. Arrows show cFOS⁺-PDYN⁺ colocalization. F: Total number of PDYN⁺ cells in LH of all adolescent and adult brains imaged. G: Percentage of PDYN⁺ cells which are double positive for PDYN⁺/cFOS⁺ cells in LH of saline and nicotine + ethanol treated adolescent and adult rats. (*p < 0.05). Scale bar is 50 μm. Data represent mean + SEM. n = 3–6/group

Retrograde tracers reveal a late-developing dynorphinergic LH-pVTA connection

We next assessed whether developmental differences in axonal projections between LH and pVTA could be identified between adolescent and adult drug-naïve male rats using retrograde tracing. We performed intracranial injections of Fluoro-Gold into pVTA and quantified Fluoro-Gold⁺ neurons in the LH. We calculated Fluoro-Gold⁺, prodynorphin⁺, and orexin⁺ cell counts as a percent of total cells and compared between adolescents and adults with t-tests and appropriate Bonferroni corrections. Double staining for prodynorphin⁺ / Fluoro-Gold⁺ was also compared to confirm dynorphinergic axonal projections. Adults had a significantly greater number of cells labeled with Fluoro-Gold (t(15)=3.56, p=0.01), with prodynorphin (t(15) = 2.60, p = 0.04), and double positive for prodynorphin and Fluoro-Gold (t(15) = 3.34, p = 0.01). Adolescents had significantly greater number of cells labeled with orexin (t(15) = −2.82, p = 0.05) (Figure 2).

Figure 2: Fluoro-Gold traced dynorphinergic projections from the LH to the VTA develop after adolescence in rats.

A: Schematic of paradigm. Below left image depicting Fluoro-Gold injection location in posterior VTA and right image depicting staining and imaging in the LH. B: Representative image of PDYN and Fluoro-Gold staining in adolescent LH. Orange arrows indicate double positive cells for prodynorphin and Fluoro-Gold. C: Representative image of PDYN and Fluoro-Gold staining in adult LH. Orange arrows indicate double positive cells for prodynorphin and Fluoro-Gold. D: Adults had significantly greater number of cells labeled with Fluoro-Gold (t(15) = 3.56, p = 0.012), with prodynorphin (t(15) = 2.60, p = 0.04), and double positive for prodynorphin and Fluoro-Gold (t(15) = 3.34, p = 0.01). Adolescents had significantly greater number of cells labeled with orexin (t(15) = −2.82, p = 0.05).* p < 0.05; ** p < 0.01. Scale bar is 150 μm. Data represent mean + SEM. n = 8/group.

4.0: DISCUSSION AND CONCLUSIONS

Our experiments confirm our hypothesis that the dynorphin projection from LH to pVTA is more developed in adult male rats than early adolescent rats at the ages tested. This projection has been described in adult rats(31,43), but its developmental timeline has not been studied. Our tracing data demonstrate a dynorphin pathway from LH to pVTA in adult rats which is not present in adolescents. Furthermore, adult rats have more dynorphin⁺ neurons than adolescent rats, suggesting that new neurons may migrate to the LH to form the LH-pVTA pathway, or that existing neurons in the LH begin expressing dynorphin and extend axons to the pVTA. Repeated tracing experiments at intermediate timepoints between the adolescent and adult timepoints that we tested would identify the precise developmental schedule of this pathway. Retrograde tracing from the nucleus accumbens and basolateral amygdala would also help to understand the dynorphinergic architecture of younger animals; the amygdala is another important nucleus of dynorphin cell bodies⁴⁵.

We demonstrate that nicotine + ethanol activates LH dynorphin⁺ neurons in adult male rats, but not adolescent male rats at the ages included in our studies. The role of dynorphin and KORs in the consumption of these drugs in adults is well-documented. Dynorphin activity is highly aversive⁴⁶ and increases reward threshold⁴⁷. Both nicotine and ethanol increase dynorphin signaling in the brain, particularly in areas associated with reward^48–52. Multiple studies have demonstrated that the aversive effects of dynorphin drive increased consumption of addictive drugs, or increase the dosage needed to establish reinforcement^{50,51,53–55}. Our previous results suggest that KOR activity is key to the reward threshold of combined nicotine and ethanol¹⁰, with adolescents, who we now show do not exhibit LH dynorphin activation during nicotine + ethanol experience, finding nicotine and ethanol to be reinforcing whereas adults do not. Our results suggest that in adult male rats, the rewarding effects of nicotine + ethanol are tempered by dynorphin signaling in the pVTA; therefore, adults do not find low doses of nicotine + ethanol reinforcing. In adolescent males, the tempering effect of dynorphin is absent and drug administration is reinforced. This is consistent with an earlier study in which adolescents were found to be less sensitive to KOR-agonism induced aversion⁵⁶. Interestingly, our 2017 results showed that female nicotine + ethanol reward is insensitive to KOR antagonism, suggesting that this pathway may be less active or overridden by other activating pathways in females. A follow-up study to investigate the effect of posterior VTA-specific KOR antagonism on intravenous self-administration of low-dose nicotine + ethanol in adult male rats would clarify if the developmental difference in low-dose nicotine + ethanol reinforcement is mediated by dynorphin in this pathway, and if this pathway solely drives the response. Collecting brains of animals in this experiment and quantifying cFos in both LH dynorphin neurons and pVTA neurons would enable verification that norBNI antagonism of pVTA KORs prevents inhibition of pVTA by LH activity in adult rats. Additionally, other brain regions such as amygdala and nucleus accumbens, which may also be involved^57,58, could be evaluated by this step⁵⁹.

We show an age-related decline in orexin neurons, which are known to contribute to reward, motivation, and addiction^60–65. Other investigators have reported a decline in orexin populations between childhood and adulthood (3 weeks and 34 weeks in rats)^37,38. A potential shift of the reward/aversion balance toward reward in adolescents would explain the increased reinforcement observed at low doses of nicotine + ethanol¹⁰. Heightened orexinergic signaling and reduced KOR activation in adolescents compared to adults could be a factor influencing adolescent susceptibility to addiction-like behavior. Together with our identification of the LH-pVTA dynorphinergic pathway, the age difference in orexin positivity in LH indicates that adolescence is marked by even more profound brain changes as long-range projections develop.

Our study does have limitations and avenues for further investigations. Only males were included in the current study because previous work demonstrated that combined nicotine + ethanol was more reinforcing in adolescent male rats when compared to adult male rats and this effect was not observed in female rats¹⁰. Furthermore, our chosen dosing paradigm and the specific LH-pVTA targeted in this study was informed by functional network data established in male rats²⁶. It is possible that multiple developmental timepoints and different dose/drug combinations would provide a detailed understanding of the maturation of this pathway in both sexes. Establishing a causal relationship between this pathway and the reinforcing effects of combined nicotine and ethanol will require future studies using approaches such as self-administration or two-bottle choice paradigm.

The development of the dynorphin pathway and its impact on behavior underscore adolescence as a crucial period for the maturation of neural circuits governing reward and executive control. Multiple long-range pathways connecting important reward, stress, and executive control centers, represent crucial neurobiological events underlying behavioral changes between adolescence and adulthood. These developing pathways also suggest mechanisms of adolescent susceptibility and opportunities for therapeutic or preventive intervention during adolescence.