HIV-1 Tat and morphine interactions dynamically shift striatal monoamine levels and exploratory behaviors over time

Abstract

Despite the advent of combination anti-retroviral therapy (cART), nearly half of people infected with HIV (PWH) treated with cART still exhibit HIV-associated neurocognitive disorders (HAND). HAND can be worsened by comorbid opioid use disorder (OUD). The basal ganglia are particularly vulnerable to HIV-1 and exhibit higher viral loads and more severe pathology, which can be exacerbated by co-exposure to opioids. Evidence suggests that dopaminergic neurotransmission is disrupted by HIV exposure, however, little is known about whether co-exposure to opioids may alter neurotransmitter levels in the striatum and if this in turn influences behavior. Therefore, we assayed motor, anxiety-like, novelty-seeking, exploratory, and social behaviors, and levels of monoamines and their metabolites following 2 weeks and 2 months of Tat and/or morphine exposure in transgenic mice. Morphine decreased dopamine levels, but significantly elevated norepinephrine, the dopamine metabolites DOPAC and HVA, and the serotonin metabolite 5-HIAA, which typically correlated with increased locomotor behavior. The combination of Tat and morphine altered dopamine, DOPAC, and HVA concentrations differently depending on the neurotransmitter/metabolite and duration of exposure but did not affect the numbers of tyrosine hydroxylase-positive neurons in the mesencephalon. Tat exposure increased the latency to interact with novel conspecifics, but not other novel objects, suggesting the viral protein inhibits exploratory behavior initiation in a context dependent manner. By contrast, and consistent with prior findings that opioid misuse can increase novelty seeking behavior, morphine exposure increased the time spent exploring a novel environment. Finally, Tat and morphine interacted to affect locomotor activity in a time-dependent manner, while grip strength and rotarod performance were unaffected. Together, our results provide novel insight into the unique effects of HIV-1 Tat and morphine on monoamine neurochemistry that may underlie their divergent effects on motor and exploratory behavior.

Graphical Abstract

To examine the relationship between opioid and HIV-1 induced alterations in striatal monoamine concentrations and behavior, morphine was administered to HIV-1 Tat transgenic mice for 2 weeks or 2 months. Morphine and Tat altered levels of dopamine, dopamine metabolites, norepinephrine, and 5-HIAA depending on the duration of exposure and neurotransmitter/metabolite assayed. Co-exposure to Tat and morphine disrupted locomotor activity in a time-dependent manner. Alternatively, Tat alone inhibited social exploratory behavior, whereas morphine alone tended to increase novelty seeking behavior. The data suggest that Tat and morphine effects on monoamine neurochemistry are complex and contribute to motor and exploratory behavioral dysregulation.

Introduction

Although cART has reduced the typical severity of HIV-associated neurocognitive disorders (HAND), milder forms of neurocognitive impairment persist in 60% of people with neurocognitive disorders (Heaton et al., 2011; Lindl et al., 2010; Ozdener, 2005). HAND is characterized by impairments in working memory, processing speed, executive function, learning, and motor skills associated with frontal and subcortical regions which are highly affected by HIV. HAND can also be exacerbated by co-exposure to opioids which has become more prevalent with the recent ubiquitous use and misuse of opioids (Bokhari et al., 2011; Hauser et al., 2012; Hodder et al., 2021). The subcortical region most severely affected by HIV and opioid co-exposure is the dorsal striatum (Fitting et al., 2010a; Fitting et al., 2010b; Hauser et al., 2005). The dorsal striatum is essential for motor behavior and habit in both motor and cognitive realms (Graybiel and Grafton, 2015). Although the ventral striatum is more closely associated with behavioral reinforcement and associated emotional/motivational processes, dopamine receptor-expressing neurons in the dorsal striatum are also implicated in reinforcement learning and decision making (Balleine et al., 2007; Kravitz et al., 2012; Loewke et al., 2021). In addition to direct damage to striatal neurons and glia, there is evidence of HIV-induced disruption to neurochemical systems which modulate neuronal activity in the striatum and guide behavioral outputs (Capó-Vélez et al., 2018; Gaskill et al., 2017; Kumar et al., 2001; Nolan and Gaskill, 2019).

People infected with HIV (PWH) have been found to be extremely sensitive to anti-psychotics which disrupt dopaminergic signaling. While most PWH on cART have low viral loads or are aviremic, over 30% are still exposed to viral proteins such as trans-activator of transcription (Tat) (Henderson et al., 2019; Johnson et al., 2013). Tat per se is directly neurotoxic and can drive inflammation (Dickens et al., 2017; Dong et al., 2019; Johnson et al., 2013; Li et al., 2008; Periyasamy et al., 2019). Several studies have connected HIV-1 Tat or HIV-induced inflammatory mediators to regulatory changes or damage to the dorsal raphe nucleus and the substantia nigra which may disrupt normal monoaminergic signaling in the brain (Heinisch and Kirby, 2009; Zauli et al., 2000). Previous work in HIV-1 Tat transgenic mice found that 3 d of Tat exposure was sufficient to alter dopamine levels; however, dopamine levels normalized by 11 d and this trend continued through d 40 (Kesby et al., 2016a; Kesby et al., 2016b). Like most drugs with abuse liability, opioids acutely enhance dopamine levels. However, opioids can also affect other neurotransmitter systems including serotoninergic, norepinephrinergic, glutamatergic, and GABAergic systems (Covey et al., 2014; Jolas and Aghajanian, 1997; Jolas et al., 2000; Ozdemir et al., 2012; Vander Weele et al., 2014). Despite this evidence, few studies have explored opioid-induced effects on these neurotransmitters in the context of neuroHIV.

To examine the timing and extent to which sustained morphine and HIV-1 Tat-dependent alterations in the levels of monoamines and their metabolites in the striatum correlate with behavioral dysfunction, we co-exposed HIV-1 Tat+ and Tat− (control) transgenic mice to morphine or saline for either 2 weeks or 2 months. Despite an absence of changes in the relative numbers of tyrosine hydroxylase- (TH) immunoreactive neurons in the mesencephalon, our findings revealed prolonged exposure to morphine and Tat caused duration dependent alterations in dopamine metabolites and norepinephrine that tended to correlate with changes in motor, but not other, behaviors.

Materials and methods

Subjects and housing

The HIV-1 tat transgenic (Tat-tg) mouse line was described previously (Bruce-Keller et al., 2008) and were generated in the vivarium at Virginia Commonwealth University. HIV-1 Tat_1–86 is conditionally expressed in a CNS-targeted manner via a glial fibrillary acidic protein-driven Tet-on promoter (activated via consumption of doxycycline (Dox)-containing chow; Dox Diet, 6 g/kg, cat no. 2018, Harlan Laboratories, Indianapolis, IN). Mice (~30 g) were housed 2–5 per cage in a temperature- and humidity-controlled, AAALAC-accredited facility, with ad libitum access to food and water, on a 12:12 light:dark cycle. Mice were 5–8 months old at the completion of these experiments. The use of mice in these studies was approved by The Virginia Commonwealth University Animal Care and Use Committee (protocol AM10175). All experiments were conducted in accordance with the National Institutes of Health (NIH Publication No. 85–23) ethical guidelines.

HIV-1 Tat induction and morphine administration

All studies were conducted on male mice 5–8 months of age (n = 120 total; 5–9 per group). Values from previous studies using HIV-1 Tat mice to examine behavioral (Nass et al., 2020; Nass et al., 2023; Nass et al., 2021; Nass et al., 2022) and mass spectrometry analyses (Hermes et al., 2020a; Kesby et al., 2017) were used to determine the sample sizes needed to sufficiently power our analyses. A posteriori power calculations were conducted using G*Power 3.1.9.7 (Faul et al., 2007) and suggest that the study was sufficiently powered to discern a medium effect size (d = 0.50) for 3-way ANOVA neurochemistry and behavioral assays and a large effect size (d = 0.80) for 2-way ANOVA two-week monoamine metabolite data and dopamine cell data. For 3-way ANOVAs with 3 (genotype × drug × time/exposure duration) independent variables with 2 levels each, results showed that a total sample of 34 subjects with 8 equal-sized groups of n = 4 was required to achieve a power of 0.80 with a medium effect size (d = 0.50) and an alpha of 0.05. For 2-way ANOVAs with 2 (genotype x drug) independent variables with 2 levels each, results showed that a total sample of 15 subjects with 4 equal sized groups of n = 4 was required to achieve a power of 0.80 with a large effect size (d = 0.80) and an alpha of 0.05. Standard mouse chow (5.8% fat, cat no. 7012, Envigo Teklad, Indianapolis, IN) was replaced with Dox Diet (6 g/kg, Harlan Laboratories) at the start of the 2-week or 2-month treatment period to induce Tat expression. Mice within each genotype (Tat– or Tat+) were randomly assigned using a stratified randomization design within either Tat– or Tat+ strata into 4 treatment groups for each level of the (saline or morphine) treatment and (2-week or 2-month) treatment duration variables (Fig. 1). Specifically, all the male Tat-tg mice born within a 3-month interval were randomly assigned an ordinal number within the colony when they were weaned and genotyped. Mice were then divided into 2 strata based on genotype and assigned to a treatment group, in cardinal order (starting with the saline/2-week exposure duration) until each mouse was assigned to a treatment group. The cardinal order of birth was used to ensure Tat− and Tat+ mice from the same litter and similar age were distributed equally across the 4 treatment groups for each genotype. Morphine solution was prepared in sterile saline and all solutions were administered at room temperature at a volume of 10 μL/g body weight. Tat induction and morphine administration were initiated concurrently. Morphine or saline injections were administered s.c., b.i.d. at 10–12-hour intervals for 2-weeks or 2-months (Nass et al., 2021; Ohene-Nyako et al., 2021). During the 2-week treatment protocol, the morphine dosage was increased by 10 mg/kg, b.i.d., at 2-day intervals until reaching the final dose of 40 mg/kg which was administered for 8 days. During the 2-month treatment protocol, morphine was initially administered at 10 mg/kg, b.i.d. (~ 1 week), and then increased to 20 mg/kg, b.i.d. (~ 1 week), 30 mg/kg, b.i.d. (~ 1 week), and 40 mg/kg, b.i.d., thereafter (32 days). Four cohorts of mice were used (Figure 1). Three cohorts of mice were treated with morphine for 2-weeks: (1) n = 36 mice (9 per group) used for neurochemical analysis were run through the full course of behavior (described below), (2) n = 21 mice (5–6 per group) used for monoaminergic analysis were run through the abbreviated course of behavior (described below), and (3) n = 27 mice (6–7 per group) was used for immunohistochemical analysis. The fourth cohort of n = 36 mice (9 per group) were treated with morphine (b.i.d.) for 2-months and analyzed behaviorally and neurochemically. The morphine doses were escalated to allow mice to develop tolerance and thereby avoid the weight loss, respiratory depression, and other consequences of immediately starting mice on a high, 40 mg/kg, b.i.d., dose of morphine (Nass et al., 2023; Ohene-Nyako et al., 2021; Ohene-Nyako et al., 2023). All mice completed the study experiments. Sixteen mice were eliminated from the novelty-induced hypophagia analyses, and one mouse was eliminated from the reciprocal social interaction analyses due to meeting exclusion criteria for those paradigms during test, as described further in methods sections. No mice died prior to the completion of the study.

Figure 1. In vivo experimental timeline.

Tat+ and Tat– (control) mice were divided into 4 cohorts and administered doxycycline (DOX)-containing chow to induce Tat expression for the entire experimental length, either 2 weeks or 2-months. Cohort 1 was administered a ramping dose of morphine (10 – 40 mg/kg, increasing by 10 mg/kg/2 day, s.c., b.i.d.) or saline for 2 weeks, humanely euthanized, and tissues were assessed histologically. Cohort 2 mice were administered 2 weeks of ramping morphine or saline and assessed in an abbreviated course of behavior consisting of Day 1: open field, Day 2: novelty-induced hypophagia and exploratory hole-board, and Day 3: adhesive removal task, before tissues were assessed for monoamine oxidase (MAO)-A and MAO-B expression. Mice in cohort 3 were assessed in a full course of behavior consisting of Day 1: open field and light dark box, Day 2: novelty-induced hypophagia and exploratory hole-board, and Day 3: reciprocal social interaction, grip strength, and rotor rod after receiving 2 weeks of ramping morphine or saline. Tissues were then assessed neurochemically. Cohort 4 mice were administered 2 months of ramping morphine (10 – 40 mg/kg, increasing by 10 mg/kg/1 week, s.c., b.i.d.), assessed in the full behavior course, and tissues were assessed neurochemically.

Neurochemical analyses

The day after behavioral testing, mice were humanely euthanized via rapid cervical dislocation, and striatal tissue was dissected from whole brains, snap-frozen in liquid nitrogen, and stored at −80 °C. The coded samples were sent on dry ice to researchers who were unaware of group assignments for analysis of neurotransmitters (Vanderbilt University Neurochemistry Core).

Tissue Extraction.

Tissues were kept frozen at −80 °C and were held on dry ice prior to the addition of homogenization buffer to prevent degradation of biogenic amines. Tissues were homogenized using a handheld sonic tissue dismembrator in 100–750 μl of 0.1 M trichloroacetic acid (TCA) containing 0.01 M sodium acetate, 0.1 mM EDTA, and 10.5% methanol (pH 3.8). The homogenization buffer contained isoproterenol (0.100 ng/μL), which was used as the internal standard during analysis by ultra performance liquid chromatography-electrochemical detection (UPLC-ECD). Ten microliters of homogenate were used for the protein assay. The samples were then spun in a microcentrifuge at 10,000 × g for 20 min prior to analysis.

Protein assay.

The protein concentration in the cell pellets is determined by the BCA Protein Assay Kit (cat. no. 23227, Pierce Biotechnology, Rockford, IL). A ten-microliter volume of tissue homogenate was distributed into each well of a 96-well plate and 200 μl of mixed BCA reagent (25 ml of Protein Reagent A was mixed with 500 μL of Protein Reagent B). The plate was incubated at room temperature for 2 hours for color development. A BSA standard curve is conducted at the same time. Absorbance is measured using a plate reader (POLARstar Omega), purchased from BMG LABTECH Company.

UPLC-ECD.

Biogenic amines were quantified in tissue supernatant using a Vanquish UPLC (Thermo Scientific) fitted with a Kinetix 2.6 μm C18 column (3.0 × 50 mm, Phenomenex, Torrance, CA) and connected to a Dionex Ultimate 3000 electrochemical detector (ECD). The UPLC mobile phase was 0.1 M trichloroacetic acid (TCA) containing 0.01 M sodium acetate, 0.1 mM EDTA, and 10.5% methanol (pH 3.8) and operated at 0.700 ml/min.

LC-MS.

Amino acid neurotransmitters were quantified using liquid chromatography/mass spectrometry (LC/MS) methodology following derivatization with benzoyl chloride (BZC). 20 μL of either cell extract or media is added to a 1.5 mL microcentrifuge tube containing 60 μL acetonitrile:water (80:20) and vortexed. To 5 μL of that solution is added 10 μL each of 500 mM NaCO3 (aq) and 2% BZC in acetonitrile. After two minutes, the reaction is stopped by the addition of 10 μL ¹³C₆-BZC internal standard solution.

Stock solutions of amino acids (5 ng/μL each) were made in DI water and stored at −80 °C. To prepare internal standards, stock solutions were derivatized in a similar manner to samples using isotopically labeled benzoyl chloride. 50 μL of the amino acid stock solution was diluted with 200 μL acetonitrile. 100 μL each of 500 mM NaCO₃ (aq) and 2% ¹³C₆-BZC in acetonitrile was added to the solution. After two minutes, the reaction was stopped by the addition of 200 μL 20% acetonitrile in water containing 3% sulfuric acid and 400 μL water. This solution was stored in 10 μL aliquot at −80 ° C. One aliquot was diluted 100× with 20% acetonitrile in water containing 3% sulfuric acid to make the working internal standard solution used in the sample analysis.

LC was performed on a Waters Acquity UPLC using an Acquity BEH C18 column (2.0 × 50 mm, 1.7 μm particle, Waters Corporation, Milford, MA). Mobile phase A was 15% aqueous formic acid and mobile phase B was acetonitrile. Samples were separated by a gradient of 98–5% of mobile phase A over 22 min at a flow rate of 600 μL/min prior to delivery to a Waters Xevo TQ-XS triple quadrupole mass spectrometer.

Monoamine oxidase activity assay

Briefly, harvested tissue was homogenized using in Pierce^™ IP Lysis buffer (cat. no. 87787, Thermo Fischer Scientific), containing Halt phosphatase inhibitor cocktail (cat. no. 78444, Thermo Fischer Scientific). Protein concentrations were then quantified for each sample using BCA assay (cat. no. 23227, Pierce Biotechnology). MAO-A and MAO-B enzyme activity in each sample was assayed using the Sigma-Aldrich monoamine oxidase assay kit (cat. no. MAK136, St. Louis, MO) according to manufacturer’s instructions. MAO enzyme activity was calculated as directed by manufacturer’s instructions: MAO Activity (units/L) = Sample Fluorescence –(Sample + MAO A/B inhibitor) Fluorescence/slope (of 4 standards) × incubation time. Samples were run in duplicate (on two plates due to the number of samples) reported as μg protein/mL normalized to BCA protein standards.

Histological analysis of dopamine neurons at 2 weeks

Tissue harvesting.

A third, cohort of mice expressing (Tat+), or lacking the tat transgene (Tat−), were placed on a 6 g/kg doxycycline diet and received ramping, b.i.d., injections of saline or morphine for 2-weeks. Mice were anesthetized via 4% isoflurane inhalation for >3 min and maintained on isoflurane anesthesia until they were transcardially perfused with 4% paraformaldehyde (cat. no. P6148, Sigma-Aldrich). Fixed brains were dissected and placed in 4% PFA in PBS overnight, then washed 3× 2 h with PBS the next day. Brains were then transferred to 10% sucrose solution overnight, incubated in 20% sucrose solution for 24 h and embedded in Tissue-Tek O.C.T. compound (cat. no. 4583, Sakura Finetek, Torrance, CA). Embedded brains were sectioned in the coronal plane at a thickness of 20 μm for analysis of MSN populations. A Leica CM1850 cryostat was used to cut the sections (Leica Biosystems, Buffalo Grove, IL). Sections were mounted on slides (Fisherbrand^™ Tissue Path Superfrost^™ Plus Gold, Thermo Fisher) and stored at −80 °C until use.

Immunohistochemistry.

Neuronal Counts:

Slides stored at −80 °C were briefly warmed to room temperature for ~5 min prior to rinsing in PBS (3 × 10 min unless noted otherwise). Tissue sections were outlined with a PAP pen (Thermo-Fisher) and gently rehydrated with 1× PBS, then permeabilized for 30 min in 0.1% Triton X-100 in PBS, blocked in 2% donkey serum in 1× PBS (2 h), and incubated with primary mouse anti-NeuN (cat no. MAB 377, Millipore Sigma, Burlington, MA; 1:200), goat anti-Iba-1 (cat no. AB5076, Abcam, Boston, MA;1:500), and rabbit anti-tyrosine hydroxylase (TH) (cat no. 213 102, Synaptic Systems, Gӧttingen, Germany; 1:1000) antibodies in blocking solution overnight at 4 °C. Sections were rinsed and incubated in fluorescently labeled, secondary antibodies including donkey anti-mouse Alexa Fluor^® 594 (cat no. A21203, Invitrogen, Waltham, NA; 1:1000), donkey anti-goat Alexa Fluor^® 405 (cat no. ab 175664, Abcam; 1:200), and donkey anti-rabbit-Alexa Fluor^® 488 (cat no. 711–546-152, Jackson ImmunoResearch, West Grove, PA; 1:500) conjugated antibodies for 1 h. Lastly, sections were counterstained with Hoechst 33342 dye (#H3570, Invitrogen, 1:20,000) for 10 min to identify cell nuclei followed by 3 × 10 min rinses in 1× PBS to remove excess dye. Slides were mounted in ProLong Gold Antifade reagent (cat no. P36930, Invitrogen). Single plane images (40×) were taken at 6 separate locations across the VTA (2) and substantia nigra (4) using the Keyence BZ-X800 (Keyence, Itasca, IL). The identity of dopamine neurons was confirmed by colocalizing TH and NeuN immunoreactivity. The TH-positive neurons are reported as a percentage of total NeuN immunoreactive cells. The samples were coded, and a separate experimenter unaware of the experimental conditions performed the ‘blind’ cell counts.

Behavioral assays

To explore the behavioral consequences of short-term and long-term Tat and/or morphine exposure, mice underwent a battery of behavioral assays during the 3 days prior to humane euthanasia and tissue collection for neurochemical or MAO enzyme activity analysis. Over the course of three days, 2-week and 2-month groups were assessed for changes in motor, exploratory, novelty-seeking, anxiety-like, and social behaviors. Behavioral tasks were executed during light period and tests were ordered such that the presumed least intrusive tests preceded increasingly intrusive tests. Full course Day 1: open field and light dark box, Day 2: novelty-induced hypophagia and exploratory hole-board, and Day 3: reciprocal social interaction, grip strength, and rotarod. An abbreviated course was run with a smaller cohort used for monoamine consisting of Day 1: open field, Day 2: novelty-induced hypophagia and exploratory hole-board, and Day 3: adhesive removal task. Mice were habituated to the testing room for at least 1 h prior to behavioral testing and mice were tested at a minimum of 3 h following morphine/saline injections to reduce confounds due to morphine-induced hyperactivity (Babbini and Davis, 1972; Hecht and Schiørring, 1979). Experimenters were blinded to treatment conditions throughout the studies. All assays except the adhesive removal, grip strength, and rotarod tasks were monitored and analyzed using a digital camera and ANY-maze software (Stoelting Co.).

Open field test:

The open field test is used to assess several qualities including locomotor activity levels, anxiety-like, and exploratory behavior (Gould et al., 2009). Motor behavior was assessed by measuring the speed, distance travelled, and time spent active. Anxiety-like behavior was assessed using thigmotaxis—the tendency of mice to stay by walls (Simon et al., 1994), by measuring the latency to enter the center of the chamber, the number of center entries, and time spent in the center. Exploratory behavior was assessed through rearing, center entries, and distance travelled. As previously described (Barbour et al., 2021; Paris et al., 2016) mice were placed in the corner of the open field chamber (40 × 40 × 35 cm; Stoelting Co., Wood Dale, IL, USA) and allowed to explore for 20 min. Activity (speed and distance traveled) and location within the chamber were recorded and analyzed.

Light:Dark test:

The light:dark chamber primarily assesses anxiety-like behavior by creating a dark, closed space where mice can retreat to avoid the brightly lit area and presumably feel safer from predation. Anxious mice tend to avoid exposure by spending more time in the dark (Bourin and Hascoët, 2003). The light:dark chamber was partitioned with a divider into two equal compartments with an opening in the middle: a black plastic roof covers the dark portion of the arena. As previously described (Hahn et al., 2015), mice were placed in the corner of the light side of the light:dark chamber (40 × 40 × 35 cm; Stoelting Co.) and allowed to freely move between the light and dark areas for 10 min. Transitions, latency to enter the dark and light portions of the chamber were recorded and analyzed.

Exploratory hole-board:

This test measures the innate tendency of mice to explore a novel environment (Kliethermes and Crabbe, 2006; Pogorelov et al., 2005; Takeda et al., 1998). Engaging in more frequent nose-pokes into the novel holes is interpreted as an increase in exploratory behavior, whereas fewer nose-pokes is assessed as increased anxiety-like behavior. A hole-board insert (Stoelting Co.) with 16 equidistant holes was placed into an open field chamber (40 × 40 × 35 cm). As previously described, mice were placed in the center of the board and allowed to freely explore for 10 min. The initiation, number of nose-pokes into holes, and time exploring holes (time spent nose-poking) were recorded and analyzed.

Novelty-Induced hypophagia:

The novelty-induced hypophagia test was also used to assess novelty exploration by assessing the competing motivation to explore a novel, colorful, and sweet food source placed in the center of the open field versus avoidance of open spaces (Dulawa and Hen, 2005). In mice, reduced time with food is interpreted as increased anxiety-like behavior; whereas increased time spent with food in a novel environment suggests increased drive for novelty exploration (Kliethermes and Crabbe, 2006; Loos et al., 2009). In this test, a 60 mm-diameter Petri dish was filled with pieces of Fruit Rings cereal (Kroger, Cincinnati, OH) divided into 5 mm x 5 mm pieces and placed in the center of an open field chamber (40 × 40 × 35 cm). As previously described (Nass et al., 2020; Nass et al., 2023) mice were placed in the corner of the chamber and allowed to freely explore for 10 min. Both movements and time spent with the cereal were recorded and analyzed. If mice moved the dish substantially from the center of the testing chamber or scattered food about the arena, the results were discarded from the analysis.

Social interaction:

Assessments of social interaction with a novel, same-sex, conspecific as a final measure of novelty exploration have been previously described (Jamain et al., 2008; Morris et al., 2016). As previously described (Nass et al., 2020; Nass et al., 2021), mice were placed in the corner of the open field chamber (40 × 40 × 35 cm) and allowed to explore and interact with a novel, same sex conspecific for 10 min. The time spent interacting (i.e., direct physical contact, sniffing, following) initiated by the test mouse was video-recorded using ANY-maze software (Stoelting Co.) and coded by an experimenter unaware of group assignment. Measures such as the latency to interact, the total number of interactions, and the time spent interacting were assessed to identify changes in social behavior in mice exposed to Tat and or morphine. Exclusion criteria included excessively aggressive behavior from either novel conspecific or study subject including biting with aggressive chasing for < 1 min.

Adhesive removal task:

The adhesive removal task is used to assess speed and agility of motor/grooming response to a sensory stimulus. It has been used in mouse models of Parkinson’s Disease and was originally adapted from rat models (Fleming et al., 2004). Briefly, the mouse was gently restrained while a small adhesive sticker (Avery adhesive-backed labels, one-quarter inch round) was placed on the snout of the mouse. The mouse was then placed in its home cage. An observer unaware of group assignment used a stopwatch to measure the length of time it takes the mouse to remove the sticker. There was a cut-off time of 60 s in which the experiment was stopped if a mouse failed to remove the sticker (Fleming et al., 2013).

Statistical analyses

All statistical analyses were performed using GraphPad Prism software version 9.1 (La Jolla, CA, USA). The neurochemical and behavioral data were assessed via three-way ANOVA followed by planned comparisons using Bonferroni’s post hoc test with each P value adjusted to account for multiple comparisons. The two-week monoamine metabolite data and dopamine cell data were assessed via two-way ANOVA followed by planned comparisons using Bonferroni’s post hoc test with each P value adjusted to account for multiple comparisons. The relationship between neurochemicals and behavioral outcomes significantly altered by morphine or Tat were assessed using the Pearson’s correlation with false discovery rate (FDR) corrections to adjust for multiple analyses. Data were not assessed for normality or outliers and all data points were included in the analyses except 16 in the novelty-induced hypophagia measures and 1 in the reciprocal social interaction measures due to the mice engaging in exclusionary behavior (discussed above) for each respective assay. All data are presented as the mean ± the S.E.M. Differences were considered statistically significant if p < 0.05.

Results

Neurochemistry

Glutamate and GABA levels were not significantly altered by Tat or morphine.

A three-way ANOVA was performed to analyze the effects of Tat and morphine on neurotransmitter levels within the striatum after 2 weeks and 2 months of exposure. Tat and/or morphine did not affect GABA or glutamate levels at either 2 weeks or 2 months. There were, however, significant time-dependent reductions in glutamate (main effect; F_{(1, 64)} = 322.3, p < 0.0001) and GABA (main effect; F_{(1, 64)} = 235.1, p < 0.0001) concentrations in all mice at 2 months irrespective of genotype or opioid exposure (Table 1).

Table 1.

Effects of Tat and morphine on glutamate and GABA levels in the striatum following 2 weeks and 2 months of exposure

		2-weeks				2-months
		Tat−		Tat+		Tat−		Tat+
		Saline	Morphine	Saline	Morphine	Saline	Morphine	Saline	Morphine
Glutamate (ng/mg protein)	Mean	2289.53	2155.18	2212.43	2186.78	958.00*	1026.23*	1181.07*	1043.99*
	SEM	169.70	64.15	89.32	95.81	55.34	50.68	81.36	65.81
GABA (ng/mg protein)	Mean	930.23	866.51	842.86	1037.22	317.58*	348.84*	364.84*	398.36*
	SEM	86.13	56.47	73.44	60.13	6.79	19.05	27.09	26.77

Glutamate and GABA levels, which significantly declined in all animals after 2 months, were unaffected by Tat and/or morphine exposure. Results from n = 72 mice (9 per group). Post hoc analyses revealed significant differences in the levels of glutamate and GABA between 2-week and 2-month samples (p < 0.0001) irrespective of treatment.

^*p < 0.05 vs. levels at 2 weeks.

Effects of Tat and morphine on the levels of catecholamines and their metabolites.

Tat did not affect striatal dopamine, norepinephrine, 3-methoxytyramine (3-MT), or homovanillic acid (HVA) levels (Fig. 2), but markedly decreased striatal 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations at 2 weeks and 2 months (main effect; F_{(1, 64)} =5.402, p = 0.0233: Fig. 2E). In contrast, morphine significantly reduced dopamine levels (main effect; F_{(1, 64)} = 5.276, p = 0.0249; Fig. 2A); whereas, morphine elevated DOPAC (main effect; F_{(1, 64)} = 21.90, p < 0.0001; Fig. 2E) and HVA (main effect; F_{(1, 64)} = 24.03, p < 0.0001; Fig. 2G) levels primarily in Tat− groups, and norepinephrine (main effect; F_{(1, 64)} = 4.153, p = 0.0457; Fig. 2B) levels across all groups, but did not alter 3-MT levels (p = 0.0828; Fig. 2C). A three-way ANOVA revealed statistically significant interactions between the effects of Tat and morphine on levels of dopamine (F_{(1, 64)} = 7.112, p = 0.0097; Fig. 2A) and its metabolites 3-MT (F_{(1, 64)} = 5.182, p = 0.0262; Fig. 2C), DOPAC (F_{(1, 64)} = 16.92, p =0.0001; Fig. 2E), and HVA (F_{(1, 64)} = 7.454, p = 0.0082: Fig. 2G), but not norepinephrine (p = 0.1262; Fig. 2B). In Tat− (but not Tat+) mice, there were significant morphine-dependent increases in DOPAC (p < 0.0001, p = 0.0141; Fig 2E) and HVA (p < 0.0004, p = 0.0263; Fig 2G) levels. In mice exposed to morphine for two-weeks, co-exposure to Tat resulted in a significant decline in the levels of DOPAC (p = 0.0197; Fig 2E). However, post hoc analyses revealed no significant differences in 3-MT levels between groups.

Figure 2. Morphine has widespread effects on catecholamines and interacts with Tat to alter levels of dopamine and its metabolites in the striatum.

(A-H). Morphine overall decreases dopamine (A) and increases norepinephrine (B) concentrations.3-MT concentrations were largely unaffected by morphine and Tat (C), although Tat caused an overall increase in the ratio of 3-MT/dopamine concentrations at 2 weeks and 2 months (D). Morphine significantly increased DOPAC (E) and HVA (G) levels in Tat– mice at 2 weeks and 2 months, while Tat interacted with morphine to negate the morphine-dependent increases in DOPAC and HVA seen in control (Tat−) mice, particularly at 2 weeks. Morphine significantly increased the ratio of both DOPAC/dopamine (F) and HVA/dopamine (H) concentrations, while planned comparisons revealed that morphine co-exposure at 2 weeks, resulted in significantly higher DOPAC/dopamine and HVA/dopamine ratios in Tat+ mice –mice (H); whereas at 2 months morphine co-exposure increased DOPAC/dopamine ratios in Tat– mice. Results from n = 72 mice (9 per group) are presented as the mean ± the SEM; ^#p < 0.05, main effect of morphine; *p < 0.05, main effect of Tat; ^Ɛp < 0.05, morphine vs vehicle with respective Tat exposure and duration; ^ψp < 0.05 significant difference of Tat+ vs Tat– in respective drug treatment and duration.

When the ratio of metabolites to their precursors was examined to identify shifts in metabolism, Tat exposure by itself significantly enhanced the ratio of 3-MT/dopamine (main effect; F_{(1, 64)} = 6.706, p = 0.0119; Fig. 2D). Morphine on the other hand significantly enhanced the ratios of DOPAC/dopamine (main effect; F_{(1, 64)} = 38.87, p < 0.0001; Fig. 2F) and HVA/dopamine (main effect; F_{(1, 64)} = 21.39, p < 0.0001; Fig. 2H). Planned comparisons revealed that morphine significantly increased the ratio of DOPAC/dopamine in Tat+ mice after 2-weeks (p = 0.0002; Fig. 2F) and in Tat− mice after 2-months exposure (p = 0.0271; Fig. 2F) and HVA/dopamine in Tat+ after 2 weeks of exposure (p = 0.0006, Fig. 2H), but failed to do so after 2 months of exposure. Finally, in mice exposed to morphine for two weeks, Tat induction significantly increased the ratio of HVA/dopamine (p = 0.0352, Fig. 2H). Because morphine markedly affected dopamine metabolism in Tat+ mice, we assayed the drug’s effects on monoamine oxidase A (MAO A) and MAO B enzyme activity. Neither Tat nor morphine affected MAO A (Fig. 3A) or MAO B activity (Fig. 3B).

Figure 3. Monoamine oxidase A (MAO-A) and MAO-B enzyme activities were unaffected by Tat or morphine in the striatum.

MAO-A (A) or MAO-B (B) activity was assessed following 2 weeks of Tat induction and/or morphine exposure. Results from n = 21 mice (5–6 per group). Data are presented as the mean ± the SEM.

Histological analysis of dopamine neurons at 2 weeks

To assess whether there was a loss of TH immunoreactive, dopaminergic neurons which may have contributed to the altered dopamine and metabolite levels in the striatum of Tat and morphine exposed mice, we performed immunohistochemical analysis of TH-positive neurons in the ventral tegmental area (VTA) and substantia nigra (Fig. 4A). We found no significant differences in the percentage of dopaminergic neurons among mice exposed to Tat and/or morphine (Figs. 4B-E) in the VTA (p = 0.3542; p = 0.1554; Fig. 4F) or substantia nigra (p = 0.9650; p = 0.7498; Fig. 4G).

Figure 4. The percentage of tyrosine hydroxylase immunopositive (TH+), dopaminergic neurons is unaffected by 2 weeks of Tat and/or morphine exposure.

TH (green fluorescence) antigenicity was co-localized with neuronal nuclear marker (NeuN) (red fluorescence) immunoreactive neurons and cell nuclei were visualized by counterstaining with Hoechst 33342 dye (blue fluorescence). (A) Appearance of the ventral tegmental area and substantia nigra in the coronal plane of saline-treated, Tat− mice. (B-E) higher magnification images of TH-immunoreactive, dopaminergic neurons in Tat− and saline (B), Tat− and morphine (C), Tat+ and saline (D), and Tat+ and morphine treatment groups. (E) The percent of TH+ neurons in the VTA (F) and substantia nigra (G) were not significantly affected by exposure to Tat or morphine. Results from n = 27 mice (6–7 per group). Data are presented as the mean ± the SEM.

Effects of Tat and morphine on levels of serotonin and its 5-HIAA metabolite.

A three-way ANOVA revealed no effect of morphine (p = 0.4409) and/or Tat (p = 0.9165) on striatal serotonin levels at 2 weeks or 2 months, although serotonin levels in all groups were increased at 2 months compared to 2 weeks (main effect; F_{(1, 64)} = 5.659; p = 0.0204; Fig. 5A). Unlike its effect on serotonin, morphine significantly elevated striatal 5-HIAA levels (main effect; F_{(1, 63)} = 30.45, p < 0.0001; Fig. 5B) and the increases in 5-HIAA levels were especially evident in Tat+ morphine-treated mice following 2 weeks of exposure (p = 0.0029). 5-HIAA levels were unaffected by Tat alone (p = 0.7423; Fig. 5B).

Figure 5. Morphine increases the serotonin metabolite 5-HIAA and the ratio of 5-HIAA to serotonin (5-HT), while Tat negated the effects of morphine on the HIAA to 5-HT ratio.

(A-C). The duration of exposure increased levels of serotonin across all treatment groups (A). Morphine caused overall increases in 5-HIAA levels (B) in Tat− and Tat+ mice. In Tat+ mice, morphine increased 5-HIAA levels following 2 weeks (B), whereas in Tat– mice the ratio of 5-HIAA/5-HT increased following 2 months of exposure (C). Tat expression tended to counter the increases in the 5-HIAA/5-HT ratio caused by morphine after 2 months of exposure (C). Results from n = 72 mice (9 per group) are presented as the mean ± the SEM, ^% p < 0.05 main effect of time, ^Ɛp < 0.05 significant difference with morphine treatment at respective Tat exposure and duration, ^πp < 0.05 vs 2 weeks Tat+ and morphine treated samples.

We then explored the ratio of 5-HIAA to its serotonin precursor to identify shifts in metabolism. Analyses of main effects revealed that morphine significantly altered the 5-HIAA/serotonin ratio (F_{(1, 64)} = 10.34, p = 0.0020; Fig. 5C), while the ratio showed no effect of Tat-exposure alone. The three-way ANOVA revealed an interactive effect (F_{(1, 64)} = 8.516, p = 0.0049; Fig. 5C). Specifically, morphine significantly increased the ratio of 5-HIAA/serotonin in Tat− mice at 2 months (p = 0.0224) but not after 2 weeks of exposure (p = 0.0634). The 5-HIAA/serotonin ratio also declined in Tat+ mice following 2 months of morphine co-exposure compared to Tat+ mice co-exposed for 2 weeks (p = 0.0204). Interestingly we also observed a main effect of time enhancing the ratios of 5-HIAA/serotonin in mice at 2 weeks compared to 2 months across all groups (F_{(1, 64)} = 22.53, p < 0.0001; Fig. 5C).

Rotarod, grip strength and adhesive removal performance were unaffected by Tat and/or morphine exposure.

Motor function was initially assessed using rotarod and grip strength (Fig. 6). Both the time spent on the rotarod (main effect; F_{(1, 64)} = 9.684, p = 0.0028; Fig. 6A) and grip strength (main effect; F_{(1, 64)} = 38.12, p < 0.0001; Fig. 6B) were lower in the 2-month groups. Morphine (main effect; p = 0.0550; Fig. 6B) tended, albeit not significantly to decrease grip strength. An additional adhesive removal assay was completed in a smaller 2-week cohort to assess more targeted motor functioning but performance on this assay was not significantly altered by Tat (p = 0.4847) or morphine (p = 0.0711) exposure (Fig. 6C).

Figure 6. Motor coordination measures are not altered by Tat or morphine.

The time on the rotarod and grip strength decreased in 2-month samples (A,B). The latency of mice to remove an adhesive sticker was unaffected by Tat but tended to be increased by morphine (p = 0.071) (C), which also strongly trended toward reduce grip strength (p = 0.055) (B). Results from rotarod and grip strength have n = 72 mice (9 per group) and adhesive removal n = 21 mice (5 to 6) per group are presented as the mean ± the SEM, ^%p < 0.05 main effect of time.

Open field behavior was altered by Tat and morphine depending on the duration of exposure.

Motor function was further assessed by examining behavior in the open field test. Main effects analysis showed that morphine significantly decreased travel speed (F_{(1, 85)} = 4.402, p = 0.0389; Fig. 7A) and the total distance traveled (F_{(1, 85)} = 4.520, p = 0.0364; Fig. 7C). Overall, Tat exposure increased travel speed (F_{(1, 85)} = 15.09, p = 0.0002; Fig. 7A), rearing events (F_{(1, 85)} = 12.23, p = 0.0007; Fig. 7B), distance travelled (F_{(1, 85)} = 15.62, p = 0.0002; Fig. 7C), and the time active (F_{(1, 85)} = 19.65, p < 0.0001; Fig. 7D). A three-way ANOVA revealed significant interactions between the duration of exposure, Tat, and morphine in the open field parameters including travel speed (F_{(1, 85)} = 9.424, p = 0.0029; Fig. 7A), rearing events (F_{(1, 85)} = 9.470, p =0.0028; Fig. 7B), distance traveled (F_{(1, 85)} = 9.79, p = 0.0024; Fig. 6C), and the time actively moving (F_{(1, 85)} = 9.478, p = 0.0028; Fig. 7D). After 2 weeks of Tat exposure, morphine-naïve mice showed significant increases in the distance traveled and rearing events (p = 0.0412 and p = 0.0164). By contrast, 2-months of Tat exposure in morphine-naïve mice significantly decreased the distance traveled, travel speed, and amount of time active (p = 0.0132, p = 0.0204, p = 0.0322; respectively; Fig. 7) compared to the 2-week exposure time. After 2 months of Tat and morphine co-exposure, mice exhibited an increase in travel speed, rearing events, distance travelled, and time active (p = 0.0020, p = 0.0178, p = 0.0017, p < 0.0001; respectively; Fig. 7).

Figure 7. Morphine and Tat altered locomotor activity in a time dependent manner.

Tat appeared to enhance activity measures while morphine subtly decreased them (A-D). Tat+ saline-treated mice at 2-weeks traveled a further distance and exhibited more rearing events than Tat− saline-treated mice (C, D). Two months of Tat exposure significantly decreased the speed, distance traveled, and time spent active compared to 2 weeks in saline-treated mice (A,C,D). Two months of morphine exposure also significantly reduced the speed and distance traveled in Tat− vs. Tat+ mice (A,C). By contrast, morphine significantly increased all measures of open field activity in Tat+ vs. Tat– mice (A-D). Results from n = 93 mice (9–15 per group). Data are presented as the mean ± the SEM; ^Ɛp < 0.05 saline vs morphine treatment with respective Tat exposure and duration, ^Ψp < 0.05 Tat– vs Tat+ in respective treatment and duration, ^θp < 0.05, 2 weeks vs 2-months of Tat exposure in saline treated mice.

Anxiety-related behaviors: Open field and light-dark box

Anxiety-like behavior was assessed in the open field and light-dark box assays. The three-way ANOVA revealed interactions between the duration of Tat and morphine exposure on open field measures including the latency to enter the brightly lit center (F_{(1, 845)} = 7.077, p = 0.0093; Fig. 8A) and time spent in the center (F_{(1, 85)} = 5.947, p = 0.0168; Fig. 8B). Tat alone did not have an overall main effect on anxiety-like behavior in the open field, but 2 months of Tat exposure in morphine-naive mice significantly increased the latency to enter the center of the open field compared to 2-weeks of Tat exposure (p = 0.0032; Fig. 8A). Changes in anxiety-like behaviors in the light:dark box were less robust. The three-way ANOVA revealed some tendency for Tat to interact with duration of exposure in the latency for mice to enter the dark portion of the chamber (p = 0.0718; Fig. 8C). Morphine exposure tended to interact with duration of exposure in the time spent in the light portion of the chamber (p = 0.0567; Fig. 8D). However, planned comparisons revealed no significant differences between treatment groups.

Figure 8. Tat and morphine had modest, time-dependent effects on anxiety-related behaviors.

Open field measures related to thigmotaxis, and light dark box anxiety-like levels. Open field measures center latency (A) center time (B) revealed a significant interaction between morphine, Tat, and time. Saline-treated Tat+ mice exhibited an increased latency to enter the dark chamber at 2 months vs 2 weeks of exposure (A), whereas there were no significant differences in the time spent in the center of the open field (B). There were no significant differences in the latency to enter the dark chamber (C) or time spent in the light chamber (D) in the light:dark assay. Results in open field from n = 93 mice (9–15 per group) results in light dark box n = 72 mice (9 per group). Data are presented as the mean ± the SEM; ^%p < 0.05, main effect of time; ^θp < 0.05 vs 2 weeks Tat and saline exposure.

Exploratory-related behaviors: Novelty-induced hypophagia and exploratory hole-board

Overall, 2-months compared to 2-weeks duration decreased the number of entries into the area or zone where food is given (main effect; F_{(1, 69)} = 1.822, p = 0.0388; Fig. 9B). There was a significant interaction between Tat exposure, morphine exposure, and duration of exposure in the latency to seek sugar cereal food (main effect; F_{(1, 69)} = 4.483, p = 0.0378; Fig. 9A), but post hoc analyses did not reveal any significant comparisons. Morphine tended to increase the time spent with food (main effect; F_{(1, 69)} = 2.802, p = 0.0987; Fig. 9C); whereas 2-months duration overall tended to decrease food time (main effect; F_{(1, 69)} = 3.56, p = 0.0634; Fig. 9C). Analyses of exploratory hole-board behaviors showed that morphine significantly increased the total number of nose-pokes (main effect; F_{(1, 85)} = 3.988, p = 0.0490; Fig. 9E) and the overall time spent nose-poking (main effect; F_{(1, 85)} = 8.671, p = 0.0042; Fig. 9F), the opioid 8F), but did not affect the latency to nose-poke (p = 0.1407; Fig. 9D).

Figure 9. Morphine increased the amount of time mice explored a hole-board.

Changes to exploratory behavior were measured using novelty-induced hypophagia and exploratory hole-board assays. Morphine and Tat did not significantly affect the latency of mice to approach novel sugar food (A), the number of times mice approached the novel food (zone entries; B), or duration of time spent with the novel food (C). However, the number of food approaches was decreased in the 2-month samples (B). Exploratory hole-board measures showed modest effects of morphine (D,E,F). There was no change in the latency to engage in exploratory nose-pokes (D), although morphine increased the number of nose-pokes (E) and the duration of time spent engaging in exploratory nose-poking (F). Results from n = 77 mice (5–13 per group; 93 mice initially; 16 mice that moved the dish out of the center zone defined in ANY-maze were removed from novelty-induced hypophagia analyses) in the novelty-induced hypophagia test and n = 93 mice (9–15 per group) in the exploratory hole-board test. Data are presented as the mean ± the SEM; ^#p < 0.05 main effect of morphine, ^%p < 0.05 main effect of time.

Mice exposed to Tat exhibit enhanced latency to approach a novel, same sex conspecific

Tat exposure significantly delayed the onset of interactions with a novel mouse (main effect; F_{(1, 63)} = 4.104, p = 0.0470; Fig. 10A), while morphine did not affect the social behaviors we assayed. Irrespective of the duration of exposure, Tat and morphine did not interact to affect social behavior among same-sex novel mice, including the latency to interact (Fig. 10A), the total time interacting (Fig. 10B), the average interaction length (Fig. 10C), and the number of interactions (Fig. 10D) as revealed by three-way ANOVA.

Figure 10. Tat increased the latency to interact with a novel mouse but did not affect the duration or number of social interactions.

Reciprocal social interactions were assessed between mice exposed to Tat and/or morphine for 2 weeks or 2 months. Tat expression delayed the onset of interactions with a novel mouse (A); however, neither morphine nor Tat affected the total amount of time in which mice interacted socially (B), the number of interactions (C), or average duration of the interactions (D). Results from n = 71 mice (8–9 per group; except one mouse from the group treated with Tat+ saline for 2-weeks that was removed from analyses due to excessive aggression) are presented as the mean ± the SEM, *p < 0.05, main effect of Tat.

Neurotransmitters and their metabolites correlate with locomotor activity in morphine-treated mice

The relationship between neurochemicals and behavioral outcomes that were altered in Tat or morphine exposed mice were assessed via correlation coefficient z-tests with all hypothesized correlations equal to 0. Statistical parameters for correlations with significant outcomes are presented in Table 2 (statistical parameters for correlations of all tested outcomes are presented in Supplementary Table 1) including correlation coefficients, z-values, and p-values (uncorrected and FDR corrected). Increased levels of norepinephrine, the HVA/dopamine, and the HIAA/serotonin ratio positively correlated with increased locomotor behaviors in the open field test (Table 2). In contrast, decreased levels of dopamine and DOPAC negatively correlated with increased open field test locomotor activity (Table 2).

Table 2.

Significant Tat and/or morphine-dependent correlations between monoamine concentrations and alterations in behavior ^a

Morphine-treated mice b
Variables	Correlation	df	z-value	p-value	FDR p-value
DA, Distance	−0.347	34	−2.082	0.0374*	0.0941
DA, Speed	−0.343	34	−2.053	0.0400*	0.0941
DA, Time Active	−0.333	34	−1.990	0.0466*	0.0941
DA, Rearing Events	−0.344	34	−2.060	0.0394*	0.0941
NE, Distance	0.369	34	2.226	0.0260*	0.0556
NE, Speed	0.366	34	2.204	0.0275*	0.0556
NE, Time Active	0.379	34	2.293	0.0219*	0.0556
NE, Rearing Events	0.470	34	2.931	0.0034*	0.0275*
DOPAC, Distance	−0.387	34	−2.348	0.0189*	0.0574
DOPAC, Speed	−0.381	34	−2.303	0.0213*	0.0574
DOPAC, Time Active	−0.406	34	−2.473	0.0134*	0.0574
HVA/DA, Distance	0.343	34	2.051	0.0403*	0.1681
HVA/DA, Speed	0.341	34	2.038	0.0416*	0.1681
5-HIAA/5-HT, Time Active	−0.341	34	−2.041	0.0413*	0.1669
5-HIAA/5-HT, Rearing Events	−0.350	34	−2.099	0.0358*	0.1669

^aOnly significant correlations are presented in Table 2 for brevity. All tested correlations (significant and non-significant) are presented in Supplementary Table 1.

^bThe correlation coefficients, degrees of freedom (df), z-values, and uncorrected and false discover rate (FDR) corrected p-values from the z-test correlation results of dopamine (DA), norepinephrine (NE), DOPAC, 5-HIAA, HVA, DOPAC/DA, HVA/DA, 5-HIAA/5-HT (serotonin), and 3-MT/DA with 12 behavioral outcomes. Hypothesized correlation coefficient = 0

^*p < 0.05.

Distance, speed, time active, rearing events, and center latency were measured in the open field assay. Nose-pokes and time nose-poking were assessed in the exploratory hole board test. Latency to interact was assessed by reciprocal social interaction. Results from n = 72 mice (9 per group).

Discussion

Although morphine and Tat by themselves affected some key aspects of striatal neurochemistry and behaviors associated with striatal function, the most pronounced changes were evident with when mice were exposed to morphine and Tat in combination. Tat and morphine interactions resulted in widespread alterations in levels of catecholamines, indoleamines and/or their metabolites. The effects of morphine and Tat tended to be selective for monoamines since morphine nor Tat alone or in combination had nominal effects on glutamate and GABA levels (Table 1).

By itself, Tat exposure did not alter dopamine, serotonin, or norepinephrine levels in the striatum in the present study, which is similar to previous findings in Tat transgenic mice (Kesby et al., 2016a; Kesby et al., 2016b; Kesby et al., 2017). Intrastriatal injections of Tat alone tend to result in slight, non-significant, reductions in striatal dopamine concentrations in rats after 1 week exposure, although in combination with methamphetamine, Tat can synergistically reduce striatal dopamine levels and evoke its overflow in ex vivo slices (Cass et al., 2003; Maragos et al., 2002). By contrast, Tat selectively decreases tyrosine hydroxylase (TH) expression and neuronal firing rates without increasing neuronal death in the substantia nigra pars compacta (SNpc), but not in the VTA, suggesting SNpc neurons are functionally vulnerable and a likely target of Tat-dependent alterations in dopamine release or metabolism (Miller et al., 2018).

In the prefrontal cortex (PFC), acute (7 day) Tat induction inhibits both the dopamine and norepinephrine transporters, increasing the concentrations of both catecholamines (Strauss et al., 2020). Fast-scan cyclic voltammetry reveals reduced dopamine reuptake in the nucleus accumbens of male and female HIV-1 transgenic rats that chronically express multiple HIV proteins including Tat (Denton et al., 2019). In the cerebrospinal fluid of simian immunodeficiency virus (SIV)-infected rhesus macaques, concentrations of dopamine metabolites fluctuate with disease progression (Koutsilieri et al., 1997). Moreover, the inhibitory effects of Tat on dopamine and norepinephrine transporters and reuptake are well established (Davis et al., 2023; Kesby et al., 2017; Strauss et al., 2020; Strauss et al., 2022). Collectively, these findings suggest that catecholamine levels can change dynamically depending on the brain region involved, the onset and duration of HIV infection, and with co-exposure to opioids.

Unlike Tat, which affects dopamine transporter expression and function (Gaskill et al., 2017; Midde et al., 2012; Sun et al., 2019) and perhaps dopamine receptors (Gaskill et al., 2017), morphine alone typically increased the levels of dopamine and serotonin metabolites following 2 weeks of exposure. Our findings of increased dopamine metabolites agree with findings that acute morphine exposure can elevate levels of dopamine and its metabolites in the striatum (Moleman et al., 1984), while chronic morphine exposure causes modest reductions in dopamine, similar to our findings (Fadda et al., 2005; Kish et al., 2001; Pozzi et al., 1995; Tjon et al., 1994). Collectively, this and other evidence suggest that morphine and Tat by themselves act largely independently to effect dopamine function and metabolism.

In combination, morphine and Tat interacted to markedly alter the concentrations of most of the neurotransmitters or their metabolites. Interestingly, at 2 weeks, combined Tat and morphine exposure tended to lower levels of striatal dopamine and its metabolites compared to morphine-treated Tat− mice. Moreover, decreases in dopamine levels inversely correlated with increases in locomotor activity in morphine-treated mice. Given this trend in Tat+ mice exposed to short-term morphine, we speculate that morphine and Tat co-exposure may accelerate the decline in dopamine levels typically seen after chronic morphine exposure. While the underlying mechanisms are unclear, Tat exposure can decrease morphine’s potency and efficacy, in part, by increasing inflammation and the activation of specific chemokine receptors such as CCR5, which can heterologously cross-desensitize μ-opioid receptors (MOR) (Gonek et al., 2018). Conversely, morphine can exacerbate the neuroinflammatory and pathophysiologic effects of Tat in astro- and microglia—dysregulating morphine’s normal effect on neuronal function(Fitting et al., 2020; Hauser et al., 2007; Hauser et al., 2023). Moreover, with sustained exposure to morphine or HIV/Tat, less-well understood maladaptive processes become operative making it even more challenging to elucidate possible underlying mechanisms. For example, following 1–2 weeks of exposure to opioids, e.g., the expression of dopamine D2 receptors increases, and they become supersensitized—markedly affecting striatal function (Strickland et al., 2022). With sustained Tat exposure and inflammation (1–10 months), key innate immune mechanisms appear to become tolerant, and there is an allostatic shift in a variety of metabolic and physiologic processes (Dickens et al., 2017; Hermes et al., 2020b; Nass et al., 2023).

Analysis of early postmortem changes from male PWH revealed that while HIV alone lowers numbers of substantia nigra neurons assessed stereologically, comorbid intravenous drug use can cause a significant additional loss of nigral neurons (Reyes et al., 1991). Although previous studies in another HIV-1 Tat transgenic model show decreased TH immunoreactivity following 7 days of Tat induction in SNpc neurons suggesting Tat directly inhibits the rate limiting enzyme for dopamine synthesis (Miller et al., 2018), decreases in the number (or immunofluorescent intensity) of TH neurons in SNpc were not evident following 2 weeks of Tat induction in the present study. The reasons for the discrepancy are uncertain but may relate to gene dosing. The transgenic mouse line used by Miller et al. (Miller et al., 2018) expresses 3–7 copies of the tat gene (see Kim et al., 2003), whereas the mice used the current study only express a single copy of the tat transgene and typically display a slower onset, less severe pathology (Dickens et al., 2017; Nass et al., 2020). Alternatively, we speculate that certain aspects of innate immune responsiveness become tolerant to Tat exposure in a time-dependent manner (Hermes et al., 2020b; Nass et al., 2023), though the reasons why specific innate immune responses become tolerant are not well understood (see Divangahi et al., 2021). Since there are deficits in dopamine in our model, but no decreases in TH-positive neurons, we speculate that reductions in dopamine biosynthesis likely precede frank losses of TH neurons. This would occur when neurons are exposed to lower levels of Tat over a more prolonged exposure period, whether in an experimental model or in PWH who are virally suppressed. It is also important to note that neither levels of dopamine nor its metabolites were assessed in the VTA or outside the striatum where their metabolism is likely to be differentially affected by Tat. Prior studies indicate there are regional differences in Tat expression within the CNS (Duncan et al., 2008; Fitting et al., 2012) that may cause unique regional differences in the metabolism of dopamine and other catecholamines. In Parkinson’s disease, patients may lose up to 30–50% of their nigral neurons prior to developing motor signs (Cheng et al., 2010). Since catecholamine and indoleamine metabolism mainly occurs within presynapses and synaptic clefts in the striatum (Meiser et al., 2013) and we see greater pathology in the striatum than the mesencephalon, we anticipated that levels of metabolites would be more greatly perturbed than their parent compounds, which was confirmed by our findings. Lastly, given the elevated synaptodendritic injury seen with opioid co-exposure (Fitting et al., 2020; Fitting et al., 2010a), we speculated that opioid co-exposure would further disrupt Tat-dependent alterations in levels of catecholamines, serotonin, and their metabolites, which was frequently observed. Importantly, our results suggest even relatively brief opioid exposure might further deplete dopamine in the substantial subpopulations of PWH who express the HIV protein Tat.

Interestingly, like dopamine, significant Tat and morphine interactions were also found in the dopamine metabolites DOPAC AND HVA. In agreement with prior work, morphine elevated DOPAC and HVA levels in Tat− mice. Importantly, because there were nominal changes in DOPAC and HVA concentrations in Tat+ mice co-administered morphine, any reductions in dopamine levels caused by Tat are unlikely to be attributable to enhanced dopamine metabolism by catechol-O-methyltransferase (COMT) or MAO. In fact, we found that MAO-A and B activities were unaffected by Tat, which suggests that any reductions in dopamine and/or its metabolites likely result from Tat-induced inhibition of dopamine synthesis and/or release. Analysis of the ratio of metabolites to their precursors revealed that morphine greatly increased the conversion of dopamine to DOPAC and HVA in all mice: however, the increases were particularly dramatic following shorter-term (2-week) morphine co-exposure with Tat. Lastly, because we observed elevations in DOPAC and 5-HIAA metabolites after morphine exposure, we tested whether monoamine oxidases A and B might be altered by morphine. Similar to Tat, there were no effects of morphine on either enzyme following two weeks of exposure.

There is substantial evidence that norepinephrine and serotonin contribute to multiple aspects of drug addiction (Müller and Homberg, 2015; Sofuoglu and Sewell, 2009; Weinshenker and Schroeder, 2007). Norepinephrine levels and release from the locus coeruleus are increased with addiction and thought to contribute to some of the negative aspects of drug withdrawal including heightened anxiety (nervousness and trembling; “the jitters”) (Kosten and George, 2002) and can elevate motor activity and other behaviors (Geyer et al., 1972). β-adrenergic receptor antagonists can attenuate anxiety and stress induced by withdrawal and can restrict cocaine self-administration in male and female rats (Beldjoud et al., 2023). Interestingly, we found morphine increased norepinephrine levels, but only in the presence of Tat, which may contribute to differences in naloxone-precipitated morphine withdrawal between Tat− and Tat+ mice (Fitting et al., 2016). Indeed, increased norepinephrine concentrations directly correlated with heightened locomotor behavior in morphine and Tat co-exposed mice. Finally, morphine-dependent elevation in 5-HIAA concentrations paralleled the increases seen with the dopamine metabolites DOPAC and HVA. Changes in the 5-HIAA/5-HT ratio suggest that Tat can attenuate morphine’s effects on serotonin metabolism, and this is more pronounced following 2-months.

Opioids inhibit opioid receptor-expressing GABAergic interneurons in the VTA, which disinhibit dopaminergic neuronal afferents into the striatum—thereby increasing dopamine levels (Johnson and North, 1992; Klitenick et al., 1992). By contrast, infusing morphine directly into the striatum activates opioid receptors on the presynaptic terminals of the dopaminergic projection neurons from the VTA, which inhibits dopamine release locally within the striatum (Piepponen et al., 1999). Despite the local feedback inhibition of dopamine release by opioids in the striatum, the overriding effect of systemic morphine results in net increases in striatal dopamine. Furthermore, repeated exposure prevents morphine inhibition of striatal dopamine release indicating a loss of normal feedback inhibition with opioid tolerance (Piepponen et al., 1999). Besides altering the expression and function of dopamine-transporters, Tat reduces KCC2 levels and GABA_A receptor-mediated hyperpolarization and inhibition (Barbour et al., 2020), which may alter effects of opioids on GABA-dependent dopamine neurotransmission in region specific manner. Although we do not observe alterations in overall/net levels of GABA in Tat- and/or morphine-exposed mice, more dynamic fluctuations in dopamine release may occur at specific synapses. The simultaneous decline in dopamine with increases in norepinephrine in opioid addiction is thought to be extremely unpleasant (Kosten and George, 2002). We speculate that the concurrent decreases in dopamine metabolites and norepinephrine concentrations, respectively, seen at 2 weeks and 2 months with combined morphine and Tat are similarly distressing and may accelerate the risk of opioid misuse to counteract the negative physiological and resultant behavioral consequences of withdrawal. Furthermore, to the extent that findings in mice can be generalized to humans, our results suggest that similar neurochemical imbalances are likely to contribute to the increased risk of opioid misuse in PWH.

One of the clinical features of HAND is motor impairment, which has been identified as an important predictor of cognitive decline (Arendt et al., 1994). Previous studies find divergent results in mice with regards to motor impairment assessed using the rotarod, open field, and grip strength assays following 2 weeks or 3-months of Tat exposure (Barbour et al., 2020; Hahn et al., 2015). Here, we found minimal evidence of motor impairment on the rotarod or in grip strength, while there were significant interactions between morphine and Tat and the duration of exposure on activity in the open field, indicating distinct differences in morphine and Tat effects at 2 weeks and 2-months. Broadly, Tat enhanced the speed of travel and other open field activities similar to previous observations (Barbour et al., 2020), while morphine after 3 h exposure typically decreased these behaviors. However, at 2-months following morphine co-exposure, Tat+ mice show a robust enhancement of all activities in the open field assay (distance, time active, speed, and rearing) compared to Tat− mice, while the same activities did not differ among saline-treated Tat− and Tat+ mice. This may indicate a Tat-dependent progressive decline in overall activity over 3-months resulting from a hypokinetic state (Hahn et al., 2015) that appears to be reversed by concurrent morphine administration. Regarding morphine, it is important to note that it was administered at least 3 hours prior to behavioral testing to avoid the confounding effects of a morphine-induced hyperkinetic state (Babbini and Davis, 1972; Hecht and Schiørring, 1979). Given this paradigm, it is also possible that mice may be more lethargic after a period of enhanced locomotor activity induced by morphine.

The measures associated with anxiety-like behavior, such as aversion to open brightly lit areas and thigmotaxis, did not appear to be strongly or universally influenced by Tat or morphine exposure in the current study. This was somewhat unexpected since twice daily injections of escalating morphine doses for 2 weeks as in the present study, but not saline, increase corticosteroid levels in Tat transgenic mice and presumably an adaptation to additional stress (Paris et al., 2020), which may ultimately affect the latency to enter the center of the open field. Alternatively, the same study found that exposure to Tat, but not morphine, increases glucocorticoid resistance in splenocytes indicating sustained Tat exposure decreases the response glucocorticoids and alters the response to stress (Paris et al., 2020). In other studies, chronic Tat induction in saline treated mice increased the latency to enter the brightly lit center of the open field chamber. Similarly, in the absence of repeated twice daily injections, 1 month of Tat exposure induces anxiety-like behavior across a variety of behavioral paradigms (Hahn et al., 2016; Paris et al., 2016). Overall, we found modest anxiety-like effects, which make it challenging to fully disentangle the effects of morphine and/or Tat from other inherent stressors in the present experiment.

Tat did not affect the length of time mice spent interacting during reciprocal social interaction in a novel environment and there was no effect of morphine on social behavior. In prior studies, however, Tat and morphine reduced reciprocal social interactions when mice were in their home-cage, but not in a novel environment (Nass et al., 2020; Nass et al., 2021), suggesting that environment influences Tat and morphine’s effects on social interactions. We did find an increase in latency to interact across all Tat+ groups suggesting Tat induction delays the initiation of reciprocal social behaviors in a novel environment. Whether the delay in behavioral onset relates to procrastination, perseveration, or other behavioral impairments is uncertain. However, this pattern of delayed behavioral onset in Tat+ mice is similar to the increased latencies associated with heightened anxiety-like behavior in more traditional assays (e.g., open field and elevated plus maze) and hesitancy to enter the center of the open field following prolonged Tat and saline exposure in the current study. Moreover, we previously found that Tat exposure increased the latency to approach sucrose solution, but not Dox chow, in the center of a novel environment (Nass et al., 2023), suggesting the delayed behavior might be stimulus (e.g., environment, flavor or food) or motivation (e.g., novelty-seeking, hunger, anxiety) specific (Nass et al., 2020). Recent work suggests a more sophisticated dissociation of the underlying components of decision-making behaviors such as exploratory drive, fear, and anxiety involve three systems. The first relates to responding to potential reward—referred to as the behavioral activating system (BAS), the second relates to responding to potential danger—referred to as the flight or fight system (FFS), and a third involves responding to conflicting reward and danger signals—referred to as the behavioral inhibition system (BIS) (Heinz et al., 2021; Loewke et al., 2021). The BIS system is theorized to delay action permitting the collection or integration of more information regarding potential risk/reward (Gray and McNaughton, 2000; Smillie et al., 2006). Further studies examining circuits mediating this process suggest that while the ventromedial PFC-amygdalar circuit may mediate fear/anxiety behaviors (associated with the FFS), the dorsomedial PFC-dorsal-medial striatal pathway may mediate risk/reward assessment during anxiety-like behavior (associated with BIS) (Loewke et al., 2021). While the striatum is well established as a mediator of risk/reward decision making (Balleine et al., 2007), this conceptualization may help explain how Tat, which preferentially disrupts the striatum, may also drive delayed action in the initiation of exploratory behaviors involving social partners, nose-pokes, and sugar food in a novel environment, without similar effects on the latency in simpler, anxiety-based behaviors that may be mediated by ventromedial PFC-amygdalar circuits.

Abbreviations

3-MT

3-methoxytyramine

5-HIAA

5-hydroxyindoleacetic acid

5-HT

5-hydroxytryptamine or serotonin

BAS

behavioral activating system

BIS

behavioral inhibition system

cART

combination anti-retroviral therapy

COMT

catechol-O-methyltransferase

DA

dopamine

DOPAC

dihydroxyphenylacetic acid

Dox

doxycycline

FDS

false discovery rate

FFS

flight or fight system

HAND

HIV-associated neurocognitive disorder(s)

HIV

human immunodeficiency virus

HVA

homovanillic acid

LC

liquid chromatography

MAO

monoamine oxidase

MOR

μ opioid receptor

MS

mass spectrometry3-MT- 3-methoxytyramine

neuroHIV

neuro-acquired human immunodeficiency virus

OUD

opioid use disorder

PFC

prefrontal cortex

PWH

people infected with HIV

SIV

simian immunodeficiency virus

SNpc

substantia nigra pars compacta

Tat

trans-activator of transcription

TH

tyrosine hydroxylase

UPLC-ECD

ultra performance liquid chromatography-electrochemical detection

VTA

ventral tegmental area

Availability of Data.

The data that support the findings of this study are available from the corresponding author Kurt Hauser upon reasonable request.