Sex differences in inflammatory cytokine levels following synthetic cathinone self-administration in rats

Abstract

Synthetic cathinones are used as stimulants of abuse. Many abused drugs, including stimulants, activate nuclear factor-κB (NF-κB) transcription leading to increases in NF-κB-regulated proinflammatory cytokines, and the level of inflammation appears to correlate with length of abuse. The purpose of this study was to measure the profile of IL-1α, IL-1β, IL-6, CCL2 and TNF-α in brain and plasma to examine if drug exposure alters inflammatory markers. Male and female Sprague-Dawley rats were trained to self-administer α-pyrrolidinopentiophenone (α-PVP) (0.1 mg/kg/infusion), 4-methylmethcathinone (4MMC) (0.5 mg/kg/infusion), or saline through autoshaping, and then self-administered for 21 days during 1 hr (short access; ShA) or 6 hr (long access; LgA) sessions. Separate rats were assigned to a naïve control group. Cytokine levels were examined in amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, thalamus, and plasma. Rats acquired synthetic cathinone self-administration, and there were no sex differences in drug intake. Synthetic cathinone self-administration produced sex differences in IL-1α, IL-1β, IL-6, CCL2 and TNF-α levels. There were widespread increases in inflammatory cytokines in the brains of male rats compared to females, particularly for 4MMC, whereas females were more likely to show increased inflammatory cytokines in plasma compared to saline groups than males. Furthermore, these sex differences in cytokine levels were more common after LgA access to synthetic cathinones than ShA. These results suggest that synthetic cathinone use likely produces sex-selective patterns of neuroinflammation during the transition from use to abuse. Consequently, treatment need may differ depending on the progression of synthetic cathinone abuse and based on sex.

1.0. Introduction

Synthetic cathinones are stimulant-like drugs that continue to be widely available throughout the U.S. Identification of new synthetic cathinones (NDEWS, 2019), and identifications of synthetic cathinones in seized drug products (DEA, 2017; Drug Enforcement Administration, 2019; NDEWS, 2018b) is increasing in some U.S. markets. Unintentional ingestion of synthetic cathinones is also rising (Oliver et al., 2019). α-Pyrrolidinopentiophenone (α-PVP) is still used by high school students in the U.S. (Palamar et al., 2019), and continues to be encountered by law enforcement (Drug Enforcement Administration, 2019). Its use has led to multiple medical emergencies (NDEWS, 2015, 2018a).

While sex and gender differences in the pharmacological effects of classic stimulants of abuse such as cocaine and methamphetamine are well established (Lynch et al., 2002), sex and gender differences are less studied, and appear to be less common for effects of synthetic cathinones (Fattore et al., 2020; Lopez-Rodriguez and Viveros, 2019). High school and middle school boys were more likely to use synthetic cathinones than girls (Patrick et al., 2016), whereas 4-methylmethcathinone (4MMC) use patterns were similar for adult men and women (Jones et al., 2016). Women were more likely to quit using synthetic cathinones due to future health consequences than men (Sande, 2016). Animal studies on synthetic cathinones also show inconsistent sex differences. One report demonstrated that α-PVP produced similar locomotor activity in male and female mice (Marusich et al., 2016), while another found female rats showed greater α-PVP-induced locomotor activity than males (Nelson et al., 2019). Self-administration of α-PVP and 4MMC in rats were similar in both sexes (Marusich et al., 2021), while α-PVP showed greater place preference in male rats than females (Nelson et al., 2019). Male rats showed greater α-PVP-induced hyperthermia (Nelson et al., 2019) and greater 3,4-methylenedioxypyrovalerone- (MDPV) induced cardiovascular effects than females (McClenahan et al., 2019). Furthermore, MDPV led to similar place preference in rats of both sexes, whereas females showed less conditioned taste avoidance than males (King et al., 2015). This literature demonstrates that sex differences in effects of synthetic cathinones are not well understood.

Past research on humans established that stimulant use alters peripheral inflammatory cytokine levels in plasma (Araos et al., 2015). Interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) are the major inflammatory cytokines that regulate innate immune system responsivity to insult or injury such as exposure to a drug (Drutskaya et al., 2018). Many abused drugs, including stimulants, activate nuclear factor-κB (NF-κB) transcription (Crews et al., 2011) leading to increases in NF-κB-regulated pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and chemokine (C-C motif) ligand 2 (CCL2) (Ahearn et al., 2021; Clark et al., 2013), and the level of inflammation appears to correlate with length of abuse (Angoa-Perez et al., 2016). TNF-α appears to play a key role in dopaminergic neurotoxicity (Sriram et al., 2006). Furthermore, acute and chronic exposure to stimulants can differentially alter levels of these inflammatory markers in the brain (Harricharan et al., 2017).

The scant information on effects of synthetic cathinones on neuroinflammation shows mixed results. In some studies, repeated experimenter administration of 4MMC had no effect on oxidative stress or microgliosis (Lopez-Arnau et al., 2015; Motbey et al., 2012). In contrast, repeated 4MMC administration increased glial fibrillary acidic protein (GFAP) immunoreactivity in hippocampus (Martinez-Clemente et al., 2014), while repeated 4MMC or MDPV administration had no effect on GFAP in striatum (Anneken et al., 2015; Martinez-Clemente et al., 2014). Antagonism of the chemokine CXCR4 blunted MDPV-induced locomotor activity and place preference, suggesting that CXCR4 plays a pivotal role in the behavioral and rewarding effects of MDPV (Oliver et al., 2018).

While this literature base provides substantial evidence for the role of synthetic cathinones in altering inflammatory levels, these past studies were only conducted in a single sex; all used male rodents except for the study by Annekan and colleagues (2015). The purpose of this study was to measure the profile of inflammatory cytokines in different regions of the brain and plasma to potentially uncover brain region-specific effects due to drug exposure and to examine if there are sex differences in these effects. Rats self-administered synthetic cathinones or saline for different durations of time. Because the development of drug abuse activates nuclear factor-κB (NF-κB) transcription (Crews et al., 2011), NF-κB-regulated proinflammatory cytokines (IL-1α, IL-1β, IL-6, CCL2 and TNF-α) were examined in several brain regions and plasma.

2.0. Methods

2.1. Subjects

Adult male and female Sprague-Dawley rats (Envigo, Frederick, MD) (n=72/sex), aged approximately 65–70 days at the start of the experiment, were housed individually in polycarbonate cages with hardwood bedding. Rats were housed in temperature-controlled conditions (20–24°C) with a 12 h standard light-dark cycle (lights on at 0700). Rats had free access to water in the home cage and were lightly food restricted (e.g., 20 g for males and 17 g for females daily). Experiments were approved by the Institutional Animal Care and Use Committee for RTI and complied with the ARRIVE guidelines. All research was conducted as humanely as possible, and followed the principles of laboratory animal care (National Research Council, 2011).

2.2. Drugs

α-Pyrrolidinopentiophenone (α-PVP) and mephedrone (4MMC) were synthesized in house using standard synthetic procedures. They were formulated as recrystalized salt and were > 97% pure. The purity was assessed by several analytical techniques including carbon, hydrogen, nitrogen (CHN) combustion analysis, and proton nuclear magnetic resonance spectroscopy. Compounds were dissolved in saline (Patterson Veterinary Supply, Columbus, OH). Gentamicin and heparin, used for maintaining catheter patency, were purchased from Patterson Veterinary Supply.

2.3. Apparatus

Experimental sessions were conducted in operant chambers for rats (MED Associates, St. Albans, VT) housed inside sound-attenuating chambers (MED Associates). Each chamber contained two retractable levers, with a stimulus light above each lever, and a house light. One lever was designated as the active lever and the other lever was designated as inactive. The side of the chamber associated with the active lever was counterbalanced across subjects. Fans provided ventilation for each chamber and speakers provided white noise. Infusion pumps (Med Associates) were located outside the chamber. Experimental events were arranged and recorded by MED-PC software (Med Associates).

2.4. Surgical Procedures

Rats in most groups were surgically implanted with chronic indwelling jugular catheters under general anesthesia as described previously (Marusich et al., 2021). The external end of the catheter was secured by a quick connect harness. Rats were given a minimum of 7 days to recover from surgery before beginning the experiment. Catheters were flushed daily with saline prior to the session, and with 0.2 mL of a post-flush solution (0.96% gentamicin, 2.88% heparin, 96.2% saline) after the session to maintain patency. All catheters were checked for patency prior to the start of the experiment.

2.5. Drug Self-Administration

Rats were randomly assigned to one of nine experimental groups: α-PVP autoshaping only (AO), 4MMC AO, saline AO, α-PVP short access (ShA; 1 hr sessions), 4MMC ShA, saline ShA, α-PVP long access (LgA; 6 hr sessions), 4MMC LgA, or drug- and experimentally-naïve control (n=8/sex/group). All α-PVP groups had access to 0.1 mg/kg/infusion, and all 4MMC groups had access to 0.5 mg/kg/infusion. Drug doses were chosen based on prior use in studies showing successful acquisition of self-administration in rats using the same schedule of reinforcement (Aarde et al., 2015; Creehan et al., 2015; Marusich et al., 2021; Nguyen et al., 2017; Nguyen et al., 2016; Vandewater et al., 2015), and these doses were at the peak of the dose-effect curves in most past studies (Aarde et al., 2015; Gannon et al., 2017; Nguyen et al., 2016). The naïve groups provided a control for the saline groups, and were not exposed to any experimental conditions, were not implanted with jugular catheters, and were not exposed to any drugs or saline. These rats were food restricted to the same degree as the other rats, weighed daily, and housed in the colony room for 14–15 days to match the duration of time that rats in AO groups were in the colony room. Thus, the naïve groups captured the levels of inflammatory markers in adult Sprague-Dawley rats following brief housing in our colony room. The timeline of experimental events for all groups is shown in Figure 1.

Figure 1.

Timeline of experimental events for all groups.

Self-administration procedures were described in detail previously (Marusich et al., 2021). Briefly, rats in all groups were trained to self-administer through an autoshaping procedure for 7 days (Carroll and Lac, 1993; Marusich et al., 2010). Active lever extension was paired with an infusion based on a random time 60 s schedule during autoshaping sessions. Fifteen seconds after lever extension, or immediately after a lever press, an infusion was delivered. Infusions were followed by a 20-s timeout signaled by illumination of both stimulus lights. Autoshaping sessions delivered 15 infusions over approximately 30 min. Following autoshaping, sessions ceased for rats in all AO groups. ShA and LgA groups continued to self-administer on a fixed ratio 1 schedule of reinforcement (FR1) for an additional 21 days during daily 1 hr or 6 hr sessions, respectively.

2.6. Brain and Plasma Sample Collection

Rats in self-administration groups were euthanized by rapid decapitation approximately 24 hr after their last self-administration session to avoid direct drug effects, and rats in the naïve group were euthanized after 14–15 days in the colony. The hypothalamus was removed from the ventral side of the brain and divided into halves. The brain was then cut down the mid line and each cortical half was opened, and the hippocampus was removed (Spijker, 2011). Next, each half was cut into three coronal slices using midline anatomical markers moving rostral to caudal. The first cut was made at the beginning of the corpus callosum, the second at the fornix, and the third cut at the end of the corpus callosum. PFC was taken from the first section. Striatum was removed from the second slice. Finally, thalamus and amygdala were removed from the third section (Chiu et al., 2007; Honkanen, 1999). Tissue samples were wrapped in aluminum foil, inserted into a cryovial, flash frozen in liquid nitrogen, and stored at −80°C. Whole trunk blood was collected and centrifuged for 10 min at 3,000 rpm. Plasma was extracted and stored at −80°C.

2.7. Cytokine Profiling

Secreted protein expression profiling of inflammatory cytokines was performed for plasma and brain tissue. Frozen tissue samples were weighed on dry ice and transferred to tubes with homogenization beads. Lysis Buffer (1X, RayBiotech, Peachtree Corners, GA) was added at a volume of 10 μL buffer/mg of tissue. Samples were homogenized for 2 cycles of 4 m/s for 30 s, with a dwell time of 10 s, and then centrifuged for 10 min at 16,000 rcf at 4°C. Samples were kept on ice to perform BCA protein quantitation (Thermo Fisher Scientific, Waltham, MA) on 2 μL of each sample. Volumes of each brain tissue homogenate were calculated to load 2,000 μg/mL on the protein arrays/sample. The calculated volumes of homogenate were transferred to new tubes and 1X Blocking Buffer (RayBiotech) was added (up to 102 μL), to load 100 μL of each sample/array. Plasma samples were thawed on ice, vortexed, and centrifuged at 16,000 rcf for 5 min to pellet any debris. Plasma samples were diluted 1:1 with 1X blocking buffer prior to array loading.

RayBiotech G2 series rat antibody cytokine slide arrays were used to analyze inflammatory cytokines in plasma and brain tissue (AAR-CYT-G2–8). Arrays were blocked for 60 min on a platform rocker with 1X blocking buffer at room temperature. Blocking buffer was then removed from array wells. Samples were loaded onto arrays and incubated at 4°C overnight on a platform rocker. The following day, slide arrays were washed with wash buffers (RayBiotech) and incubated for 2 hr with secondary biotin-conjugated antibody at room temperature. Slide arrays were washed again, incubated with fluorescent-conjugated, streptavidin antibody in the dark for 2 hr, washed again, and air-dried. Arrays were wrapped in foil and stored at −20°C prior to shipment for scanning (RayBiotech). Arrays were scanned at a wavelength of 532 nm using 2-color fluorescent detection through the GenePix Pro 6.0.1.25 software (Molecular Devices, San Jose, CA).

2.8. Data Analysis

Statistical analyses were conducted using NCSS (Number Cruncher Statistical Systems, Kaysville, UT). For all analyses, α-PVP and 4MMC were analyzed separately. One male rat from each of the α-PVP ShA, 4MMC ShA, and saline ShA groups were dropped from the study due to catheter patency problems or experimenter errors. All data for these rats were excluded from graphs and analyses. Data analyses for self-administration results were described previously (Marusich et al., 2021).

Cytokine signal intensity results for all cytokines were first quantified as relative fluorescence units (RFU), which was calculated by subtracting the signal from the average of the negative controls (background), then normalized to the average of the positive control reactive signals within the slide. Prior to further normalizing data, data from male and female rats within each control condition (e.g. Saline ShA, Saline AO, and Naïve) were analyzed with two-sample t-tests to examine sex differences. All data were then normalized to their respective control groups. To examine drug effects, intensity data for each synthetic cathinone (α-PVP or 4MMC) were converted to percent saline by dividing the drug data by the average of the saline group of the similar duration (AO saline was used for AO drug groups; ShA saline was used for ShA and LgA drug groups) and multiplying by 100 [(drug/saline)*100]. To examine effects of saline self-administration, intensity data for each saline group (ShA or AO) were converted to percent naïve by dividing the saline data by the average of the naïve group and multiplying by 100 [(saline/naïve)*100].

Statistical analyses were then performed on the normalized data. Data from α-PVP and 4MMC groups were analyzed separately. Cytokine data from LgA and ShA groups were analyzed with between-factors condition (LgA drug vs ShA drug vs ShA saline) × sex ANOVAs using percent saline ShA data. Cytokine data from AO groups were analyzed with between-factors condition (AO drug vs AO saline) × sex ANOVAs using percent saline AO data. Cytokine data from saline and naïve groups were analyzed with between-factors condition (ShA saline vs AO saline vs naïve) × sex ANOVAs using percent naïve data. Each anatomical location (e.g. brain region or plasma) and cytokine combination was analyzed separately. All tests were considered significant at p < 0.05 and significant ANOVAs were followed with Tukey’s post hoc tests as appropriate.

3.0. Results

Self-administration data from rats used in the present study were published previously (Marusich et al., 2021), and are briefly summarized here. Rats responded minimally during the autoshaping phase, and thus, most infusions were delivered noncontingently during that phase. In the self-administration phase, rats that self-administered α-PVP and 4MMC in ShA (1 hr) and LgA (6 hr) groups responded more on the active than inactive lever, and saline ShA groups also responded more on the active than inactive lever. Rats in the α-PVP and 4MMC ShA and LgA groups all increased their active responses at the end of the self-administration phase compared to Day 1, indicating escalation of drug intake. There was no main effect of sex or sex × lever interaction on responses for any of the drug self-administration groups (α-PVP ShA, α-PVP LgA, 4MMC ShA, 4MMC LgA) (p > 0.05) (Marusich et al., 2021).

Cytokine RFU signal intensities (mean and SEM) for IL-1α, IL-1β, IL-6, CCL2 and TNF-α in each brain region and in plasma are shown in Supplementary Table S1. There were several differences between drug groups, sex, and durations.

3.1. Effects of ShA and LgA Synthetic Cathinone vs ShA Saline

3.1.1. Effects of α-PVP self-administration.

Cytokine signal intensities for α-PVP ShA and LgA groups expressed as percent Saline ShA values are shown in Figure 2, and results of the corresponding statistical analyses are shown in Table 1. Plasma showed a different pattern of results than the brain for the five cytokines in Figure 2, with increased levels of all cytokines in plasma for female LgA α-PVP groups compared to female ShA saline, female ShA α-PVP, and male LgA α-PVP. In contrast, males were more likely to have higher brain cytokine levels than females. α-PVP had similar effects on IL-1α, IL-6, and CCL2, as shown in Figure 2. LgA exposure to α-PVP increased levels of IL-1α, IL-6, and CCL2 in males relative to ShA Saline in all brain regions except for IL-1α in amygdala, and CCL2 in hippocampus. LgA males also showed higher levels of IL-1α, IL-6, and CCL2 than male ShA α-PVP-exposed rats in all brain regions except for IL-6 in amygdala. In contrast, female LgA groups only showed altered IL-1α, IL-6, and CCL2 levels relative to Saline ShA for IL-1α in hippocampus, IL-6 in striatum, and CCL2 in amygdala. Similarly, female LgA levels only differed from α-PVP ShA for IL-1α in thalamus, IL-6 in striatum, and CCL2 in hypothalamus. α-PVP LgA females showed lower IL-1α, IL-6, and CCL2 levels than LgA males in all locations except plasma.

Figure 2.

Effects of α-PVP on cytokine signal intensity as a function of peripheral or CNS location. Data from ShA and LgA groups are expressed as percent of the Saline (Sal) ShA group of the same sex. Note different y-axis scale for CCL2. Dashed lines represent average Saline ShA values. $ indicates a significant difference from Saline ShA for the same sex, # indicates a significant difference from drug ShA for the same sex, and * indicates a significant difference from male for the same condition (p < 0.05). Plas = plasma, Amyg = amygdala; Hippo = hippocampus; Hypo = hypothalamus; Stri = striatum; Thal = thalamus. n=7–8/group.

Table 1.

F values for significant main effects of condition, sex, and significant drug by sex interactions on cytokine signal intensity within the condition (LgA drug vs ShA drug vs ShA saline) × sex ANOVAs (p < 0.05) for ShA and LgA α-PVP groups. Degrees of freedom for all analyses are 1, 40 for sex, and 2, 40 for condition and the sex × condition interaction.

Location	IL-1α	IL-1β	IL-6	CCL2	TNF-α
Plasma			Sex: 21.57		Sex: 15.10
	Condition: 30.98	Condition: 12.27	Condition: 30.58		Condition: 23.50
	Interaction: 24.69	Interaction: 20.39	Interaction: 49.24	Interaction: 22.61	Interaction: 24.37
Amygdala		Sex: 11.30	Sex: 50.05	Sex: 113.22	Sex: 21.14
	Condition: 6.27	Condition: 8.51	Condition: 3.82	Condition: 45.59	Condition: 5.28
	Interaction: 4.22		Interaction: 13.45	Interaction: 101.77	Interaction: 9.01
Hippocampus	Sex: 33.36	Sex: 7.42	Sex: 78.50
	Condition: 12.62	Condition: 30.12	Condition: 132.88		Condition: 4.76
	Interaction: 64.85	Interaction: 21.83	Interaction: 78.50	Interaction: 10.75
Hypothalamus	Sex: 61.21		Sex: 90.07	Sex: 15.83
	Condition: 190.38		Condition: 35.25	Condition: 20.17
	Interaction: 150.59		Interaction: 42.88	Interaction: 89.46
PFC	Sex: 167.68		Sex: 56.03	Sex: 18.27
	Condition: 196.42		Condition: 93.15	Condition: 53.37	Condition: 67.82
	Interaction: 136.77		Interaction: 70.43	Interaction: 28.42	Interaction: 8.46
Striatum	Sex: 39.02	Sex: 26.25	Sex: 5.41	Sex: 64.39
	Condition: 60.27		Condition: 72.58	Condition: 57.19	Condition: 11.95
	Interaction: 48.67	Interaction: 8.46	Interaction: 25.90	Interaction: 38.48
Thalamus	Sex: 88.42		Sex: 16.87	Sex: 74.66	Sex: 8.91
	Condition: 160.63	Condition: 3.61	Condition: 47.98	Condition: 51.79
	Interaction: 90.46		Interaction: 17.28	Interaction: 28.23

IL-1β and TNF-α showed fewer systematic between-group brain differences in cytokine levels for α-PVP groups than the other cytokines (Figure 2). There were several effects of condition for α-PVP females for IL-1β in brain, with elevated levels of IL-1β observed for LgA α-PVP females compared to Saline ShA or α-PVP ShA females in all locations except hypothalamus and PFC. In contrast, there were fewer effects of condition for IL-1β in brain for males than other cytokines. TNF-α showed a series of small, but significant differences for α-PVP groups in both sexes compared to Saline ShA. Interestingly, both ShA and LgA α-PVP lowered TNF-α levels in PFC relative to Saline ShA for both sexes. Furthermore, females showed greater TNF-α levels than males in thalamus, regardless of condition.

3.1.2. Effects of 4MMC self-administration.

Cytokine signal intensities for 4MMC ShA and LgA groups expressed as percent Saline ShA values are shown in Figure 3, and results of the corresponding statistical analyses are shown in Table 2. 4MMC self-administration primarily altered cytokine levels for males (Figure 3). LgA 4MMC self-administration elevated levels of all five cytokines in plasma for one or both sexes compared to Saline ShA and 4MMC ShA groups. 4MMC had similar effects on IL-1β and IL-6 in the brain with 4MMC LgA males showing large increases in these cytokines in all brain regions compared to male Saline ShA and 4MMC ShA, and LgA females. LgA 4MMC also increased IL-1β and IL-6 in plasma for males compared to male Saline ShA and 4MMC ShA. For females, LgA 4MMC elevated IL-1β compared to 4MMC ShA in hippocampus, whereas ShA 4MMC elevated IL-1β compared to Saline ShA in striatum.

Figure 3.

Effects of 4MMC on cytokine signal intensity as a function of peripheral or CNS location. Data from ShA and LgA groups are expressed as percent of the Saline ShA group of the same sex. Note different y-axis scale for CCL2. Dashed lines represent average Saline ShA values. $ indicates a significant difference from Saline ShA for the same sex, # indicates a significant difference from drug ShA for the same sex, and * indicates a significant difference from male for the same condition (p < 0.05). n=7–8/group. Abbreviations are the same as those in Figure 2.

Table 2.

F values for significant main effects of condition, sex, and significant drug by sex interactions on cytokine signal intensity within the condition (LgA drug vs ShA drug vs ShA saline) × sex ANOVAs (p < 0.05) for ShA and LgA 4MMC groups. Degrees of freedom for all analyses are 1, 40 for sex, and 2, 40 for condition and the sex × condition interaction.

Location	IL-1α	IL-1β	IL-6	CCL2	TNF-α
Plasma		Sex: 40.83			Sex: 7.90
	Condition: 4.72	Condition: 70.31	Condition: 4.90	Condition: 26.37	Condition: 14.81
	Interaction: 6.73	Interaction: 39.96
Amygdala		Sex: 70.74	Sex: 125.09	Sex: 33.63	Sex: 20.12
	Condition: 11.98	Condition: 53.68	Condition: 44.39	Condition: 60.84
		Interaction: 52.30	Interaction: 72.56	Interaction: 11.95	Interaction: 8.13
Hippocampus	Sex: 7.58	Sex: 93.21	Sex: 302.91
	Condition: 5.50	Condition: 140.80	Condition: 250.59
	Interaction: 8.25	Interaction: 72.58	Interaction: 245.45	Interaction: 22.98
Hypothalamus		Sex: 38.88	Sex: 372.60	Sex: 30.70	Sex: 8.45
		Condition: 48.62	Condition: 315.81	Condition: 16.73	Condition: 6.92
		Interaction: 54.83	Interaction: 319.65	Interaction: 68.27	Interaction: 19.03
PFC	Sex: 4.18	Sex: 31.10	Sex: 35.70
		Condition: 46.08	Condition: 37.55
	Interaction: 8.54	Interaction: 67.92	Interaction: 45.75	Interaction: 9.18	Interaction: 7.93
Striatum	Sex: 13.82	Sex: 9.19	Sex: 134.13	Sex: 67.83	Sex: 14.24
	Condition: 31.66	Condition: 32.01	Condition: 255.00	Condition: 70.13	Condition: 24.37
	Interaction: 19.04	Interaction: 48.23	Interaction: 185.43	Interaction: 56.47	Interaction: 26.32
Thalamus			Sex: 23.24	Sex: 26.92	Sex: 33.09
	Condition: 18.01	Condition: 9.38	Condition: 37.06	Condition: 17.33	Condition: 11.80
	Interaction: 5.02	Interaction: 9.68	Interaction: 25.79	Interaction: 7.75	Interaction: 8.31

4MMC LgA self-administration also increased IL-1α, CCL2, and TNF-α levels in males in PFC and striatum compared to LgA females and male Saline ShA and/or male 4MMC ShA (Figure 3). 4MMC LgA also elevated male CCL2 in hypothalamus compared to LgA females, Saline ShA males, and 4MMC ShA males. Effects of 4MMC were also confined to LgA males for IL-1α in thalamus, and TNF-α in hypothalamus. Other than these instances, IL-1α, CCL2, and TNF-α showed fewer male-specific alterations than IL-1β and IL-6. 4MMC self-administration decreased IL-1α in amygdala and hippocampus for some groups, and deceased CCL2 in amygdala for some groups compared to Saline or 4MMC ShA groups. Thalamus showed a particularly interesting profile of effects with 4MMC males showing increased CCL2 and 4MMC females showing increased TNF-α, both of which were evident when compared to Saline ShA and the opposite sex 4MMC groups.

3.1.3. Sex differences in Saline ShA groups.

Figure 4 shows cytokine signal intensities for Saline ShA groups, and results of the corresponding statistical analyses are shown in Table 3. There were sex differences in the absence of drug in levels of IL-1α and CCL2 in all locations that were measured, whereas sex differences occurred in most, but not all, locations for IL-1β, IL-6, and TNF-α. Females showed higher levels of cytokines in brain than males in most instances, while males showed higher cytokine levels in plasma than females. This may account for some proportion of the sex differences observed in Figures 1 and 2 because female synthetic cathinone data were normalized to higher brain levels of cytokines than male synthetic cathinone data, and vice versa for plasma. Although male Saline ShA groups had higher levels of IL-1β in striatum than females, male LgA 4MMC IL-1β levels were still higher than female LgA 4MMC levels, indicating that the sex difference for IL-1β levels in striatum for 4MMC LgA groups was substantial.

Figure 4.

Mean cytokine signal intensity as a function of peripheral or CNS location for Saline ShA groups. Note different y-axis scales in each panel. * indicates a significant difference from male (p < 0.05). n=7–8/group. Abbreviations are the same as those in Figure 2.

Table 3.

T values for significant effects of sex within the two-sample t-test for Saline ShA groups (p < 0.05). Degrees of freedom for all analyses are 13.

Location	IL-1α	IL-1β	IL-6	CCL2	TNF-α
Plasma	−4.0564	−2.6744	−3.8533	−3.0102	−3.3146
Amygdala	15.2223	3.2613	12.1400	20.5579	8.7259
Hippocampus	7.7250		9.0253	5.6770	3.1731
Hypothalamus	14.9516		25.6249	15.3287	3.9994
PFC	9.1434		5.9434	5.7561	2.5871
Striatum	3.7688	−4.0117		3.3474
Thalamus	2.3668			3.3965	−10.5575

3.2. Effects of AO Synthetic Cathinone vs AO Saline

3.2.1. Effects of synthetic cathinones.

Cytokine signal intensities for α-PVP and 4MMC AO groups expressed as percent Saline AO values are shown in Figure 5, and results of the corresponding statistical analyses are shown in Table 4. Despite the low drug quantity which was delivered largely noncontingently during autoshaping for α-PVP and 4MMC AO groups, there were still several changes in cytokine levels compared to Saline AO, although the magnitude of these drug effects was smaller than the effects of LgA and ShA drug. Of the five cytokines, IL-6, CCL2, and TNF-α showed the most widespread effects of synthetic cathinone exposure versus saline. Both α-PVP and 4MMC altered levels of all three of these cytokines in hippocampus and altered levels of 2–3 of these cytokines in hypothalamus and amygdala. Across all cytokines, drug effects occurred fairly equally for both sexes for α-PVP groups, whereas 4MMC’s drug effects were observed for both sexes or only for males, except for TNF-α in plasma. Interestingly, synthetic cathinones consistently lowered cytokine levels in amygdala when drug effects were present, and there were no drug effects on cytokine levels in thalamus. Regarding sex differences, female α-PVP levels were higher than those of males in hippocampus for all cytokines. Other sex differences varied by cytokine and brain region, and sex differences for AO groups (Figure 5) were less common than in ShA and LgA groups (Figures 1 and 2).

Figure 5.

Effects of synthetic cathinones on cytokine signal intensity as a function of peripheral or CNS location. Data from AO groups are expressed as percent of the Saline AO group of the same sex. Dashed lines represent average Saline AO values. $ indicates a significant difference from Saline AO for the same sex, and * indicates a significant difference from male for the same condition (p < 0.05). n=8/group. Abbreviations are the same as those in Figure 2.

Table 4.

F values for significant main effects of condition, sex, and significant drug by sex interactions on cytokine signal intensity within the condition (AO drug vs AO saline) × sex ANOVAs (p < 0.05) for AO synthetic cathinone groups. Degrees of freedom for all analyses are 1, 28 for sex, condition, and the sex × condition interaction.

Location	IL-1α	IL-1β	IL-6	CCL2	TNF-α
α-PVP
Plasma					Sex: 7.27
	Condition: 6.09
					Interaction: 7.27
Amygdala		Condition: 16.14	Condition: 18.44	Condition: 18.30	Condition: 4.76
Hippocampus	Sex: 11.68	Sex: 20.83	Sex: 154.35	Sex: 25.15	Sex: 32.60
	Condition: 96.71	Condition: 8.42	Condition: 23.74	Condition: 71.30	Condition: 14.11
	Interaction: 11.68	Interaction: 20.83	Interaction: 154.35	Interaction: 25.15	Interaction: 32.60
Hypothalamus			Sex: 9.83	Sex: 22.82
			Condition: 14.08	Condition: 37.80
			Interaction: 9.83	Interaction: 22.82
PFC					Sex: 9.44
				Condition: 4.29	Condition: 24.63
					Interaction: 9.44
Striatum		Condition: 16.59	Condition: 10.05		Condition: 23.59
Thalamus	Sex: 5.71
	Interaction: 5.71
Location	IL-1α	IL-1β	IL-6	CCL2	TNF-α
4MMC
Plasma					Sex: 9.63
			Condition: 5.47		Condition: 7.17
					Interaction: 9.63
Amygdala	Condition: 5.66		Condition: 26.36	Condition: 33.11
Hippocampus			Condition: 9.40	Condition: 4.63	Condition: 12.20
				Interaction: 4.21
Hypothalamus			Sex: 4.48	Sex: 49.25	Sex: 12.82
	Condition: 4.32			Condition: 20.31
			Interaction: 4.48	Interaction: 49.25	Interaction: 12.82
PFC				Condition: 4.80
Striatum	Sex: 5.96	Sex: 8.73	Sex: 13.73
	Condition: 49.55			Condition: 12.56	Condition: 11.20
	Interaction: 5.96	Interaction: 8.73	Interaction: 13.73

3.2.2. Sex differences in Saline AO groups.

Figure 6 shows cytokine signal intensities for Saline AO groups, and results of the corresponding statistical analyses are shown in Table 5. There were significant sex differences in levels of IL-6 in all measured locations, and for IL-1α, CCL2, and TNF-α levels in 6 of 7 locations. Similar to the ShA Saline groups (Figure 4), females in the AO saline group showed higher levels of cytokines in brain than males in most instances, while males in the AO saline group showed higher cytokine levels in plasma than females (Figure 6). Interestingly, IL-1β levels in brain showed more within-group variability than the other cytokines, regardless of sex or brain region. This may explain some of the variability seen for IL-1β following AO synthetic cathinone exposure (Figure 5) because the synthetic cathinone data were normalized to Saline AO data.

Figure 6.

Mean cytokine signal intensity as a function of peripheral or CNS location for Saline AO groups. Note different y-axis scales in each panel. * indicates a significant difference from male (p < 0.05). n=8/group. Abbreviations are the same as those in Figure 2.

Table 5.

T values for significant effects of sex within the two-sample t-test for Saline AO groups (p < 0.05). Degrees of freedom for all analyses are 14.

Location	IL-1α	IL-1β	IL-6	CCL2	TNF-α
Plasma	−4.6632	−5.0961	−15.0816	−3.6673	−4.9627
Amygdala	15.3655	3.0478	11.3491	7.6024	4.8552
Hippocampus	13.8021	−4.3310	4.5582	18.8067
Hypothalamus	15.6009	−3.4608	11.4862	25.6655	5.4754
PFC	8.9084		6.0133	4.0185	−2.2146
Striatum	15.5742		2.6954	14.2765	12.0205
Thalamus			3.2718		−5.1327

3.3. Effects of Saline AO and Saline ShA vs Naive

3.3.1. Effects of Saline AO and Saline ShA.

Cytokine signal intensities for Saline AO and ShA groups expressed as percent Naïve values are shown in Figure 7; results of the corresponding statistical analyses are shown in Table 6. Although exposure to saline and/or jugular implantation surgery produced several alterations in levels of cytokines, the magnitude of these effects were notably smaller than those produced by ShA and LgA synthetic cathinone self-administration (compare y-axis scales for Figures 1 and 2 to Figure 7). Exposure to an operant contingency for saline and/or jugular catheter surgery in saline groups lowered levels of all cytokines in plasma for both durations of saline exposure. IL-1α levels were higher for female Saline AO and ShA groups than males in all measured locations. IL-6 levels were higher for female Saline AO and/or ShA groups than males in brain, but lower for female Saline AO and ShA than males in plasma. Furthermore, effects of an operant contingency for saline and/or jugular catheter surgery on IL-6 levels were more common for males than females. Like IL-6, CCL2 levels in brain were higher for female than male saline groups in most instances, and the operant contingency for saline and/or jugular catheter surgery was more likely to alter CCL2 levels for males than females. For TNF-α levels, several sex differences and effects of the saline contingency and/or jugular catheter surgery occurred, but the direction of these effects varied by location. TNF-α also displayed the largest impact of the saline contingency and jugular catheter surgery of the cytokines.

Figure 7.

Effects of exposure to experimental procedures on cytokine signal intensity as a function of peripheral or CNS location. Data from saline exposure groups are expressed as percent of the Naïve group of the same sex. Note different y-axis scale for CCL2 and IL-6. Dashed lines represent average Naïve values. $ indicates a significant difference from Naïve for the same sex, # indicates a significant difference from Saline AO for the same sex, and * indicates a significant difference from male for the same condition (p < 0.05). n=7–8/group. Abbreviations are the same as those in Figure 2.

Table 6.

F values for significant main effects of condition, sex, and significant drug by sex interactions on cytokine signal intensity within the condition (ShA Saline vs AO Saline vs Naïve) × sex ANOVAs (p < 0.05) for saline groups. Degrees of freedom for all analyses are 1, 41 for sex, and 2, 41 for condition and for the sex × condition interaction.

Location	IL-1α	IL-1β	IL-6	CCL2	TNF-α
Plasma			Sex: 27.50		Sex: 10.50
	Condition: 27.80	Condition: 6.84	Condition: 98.80	Condition: 13.65	Condition: 9.32
			Interaction: 8.27
Amygdala	Sex: 276.66	Sex: 41.61	Sex: 165.60	Sex: 77.37	Sex: 15.01
			Condition: 21.77
	Interaction: 69.73	Interaction: 11.16	Interaction: 41.49	Interaction: 24.97	Interaction: 5.27
Hippocampus	Sex: 11.41		Sex: 30.66		Sex: 21.54
	Condition: 6.12		Condition: 7.60	Condition: 3.29	Condition: 9.58
		Interaction: 9.40	Interaction: 16.88		Interaction: 12.01
Hypothalamus	Sex: 297.59		Sex: 241.63	Sex: 225.03	Sex: 43.52
	Condition: 46.06		Condition: 58.89	Condition: 50.24
	Interaction: 78.00		Interaction: 61.52	Interaction: 65.78	Interaction: 12.03
PFC	Sex: 124.94	Sex: 4.79	Sex: 50.74	Sex: 29.85
	Condition: 130.32	Condition: 7.67	Condition: 76.16	Condition: 21.13	Condition: 62.28
	Interaction: 31.32		Interaction: 17.46	Interaction: 8.92	Interaction: 6.04
Striatum	Sex: 39.46		Sex: 14.36	Sex: 51.73	Sex: 37.60
	Condition: 78.06	Condition: 6.34	Condition: 14.00	Condition: 16.93	Condition: 38.37
	Interaction: 10.30	Interaction: 5.70	Interaction: 3.70	Interaction: 14.22	Interaction: 33.74
Thalamus	Sex: 5.52	Sex: 12.70	Sex: 14.47	Sex: 5.70	Sex: 59.99
	Condition: 27.66		Condition: 15.54	Condition: 24.70	Condition: 4.05
			Interaction: 3.64		Interaction: 16.04

3.3.2. Sex differences in Naïve groups.

Figure 8 shows cytokine signal intensities for Naïve groups, and results of the corresponding statistical analyses are shown in Table 7. Sex differences were less common in Naïve groups (Figure 8) compared to saline groups (Figures 3 and 5), but sex differences still occurred in three variable measured locations for each cytokine, except for the PFC. Sex differences were most common in amygdala and hippocampus, but the direction of these sex differences varied by cytokine.

Figure 8.

Mean cytokine signal intensity as a function of peripheral or CNS location for Naïve groups. Note different y-axis scales in each panel. * indicates a significant difference from male (p < 0.05). n=8/group. Abbreviations are the same as those in Figure 2.

Table 7.

T values for significant effects of sex within the two-sample t-test for Naïve groups (p < 0.05). Degrees of freedom for all analyses are 14.

Location	IL-1α	IL-1β	IL-6	CCL2	TNF-α
Plasma			−4.1707	−2.9672	−2.4658
Amygdala		−2.9039	2.5602	3.1526	5.0993
Hippocampus	3.8572	−5.1858		4.2198	−2.5322
Hypothalamus	−2.1924	−6.2351
Striatum	3.1156
Thalamus			−4.0310

4.0. Discussion

Synthetic cathinone exposure altered protein levels of all cytokines measured, and these changes, when they occurred, were primarily characterized by elevations in cytokine levels compared to the respective saline control groups; however, some decreases in cytokine levels were also observed. Relative changes in protein levels occurred at some point during the study in every measured brain region and in plasma. Most of the changes in cytokine levels for synthetic cathinone groups were observed in LgA (6 hr) but not ShA (1 hr) groups, although there were some exceptions. LgA self-administration was particularly impactful for 4MMC, for which drug effects occurred almost exclusively in LgA groups but not ShA groups. Furthermore, ShA and LgA exposure to synthetic cathinones produced more robust changes in cytokine levels than AO exposure when data were compared to the respective saline groups. These data combined suggest that greater synthetic cathinone exposure leads to higher levels of neuroinflammatory marker perturbations.

Many sex differences in cytokine levels were observed throughout this study. The previously published self-administration data from the rats in this study show that there were no sex differences in drug intake for any groups (Marusich et al., 2021). In contrast, α-PVP and 4MMC self-administration led to profound sex differences in IL-1α, IL-1β, IL-6, CCL2 and TNF-α in the same rats, which cannot be explained by sex differences in amount of drug exposure. There were widespread drug-induced increases in inflammatory cytokines in the brains of male rats compared to females when data were normalized to control groups, whereas female rats were more likely to show increased inflammatory cytokines in plasma than males (Figures 1 and 2). Furthermore, these sex differences in cytokine levels were more common after LgA than ShA synthetic cathinone self-administration, indicating that sex differences in neuroinflammatory markers may only emerge after dysregulated drug intake has occurred. Sex differences in cytokine levels were also noted in some instances for the naïve groups (Figure 8) but were more common for saline groups (Figures 3 and 5). This suggests that exposure to experimental procedures (e.g. jugular catheter surgery and contingent exposure to saline) may cause sex differences in levels of inflammatory markers even in the absence of drug exposure.

In addition to sex, mechanism of drug action may be a pivotal factor in stimulant-induced neuroinflammation. Self-administration of α-PVP and 4MMC, a dopamine uptake inhibitor and a dopamine releaser, respectively, led to divergent inflammatory profiles (Figures 1 and 2). 4MMC produced larger magnitude elevations in cytokine levels than α-PVP, and sex differences in cytokine levels were more widespread across brain regions for 4MMC than α-PVP. Furthermore, IL-1β showed elevations for the female α-PVP LgA group in plasma, hippocampus, and striatum, while the male 4MMC LgA group had increased IL-1β levels in these locations compared to their respective Saline ShA. This suggests that mechanism of action may play a role in the development of sex-specific neuroinflammation.

Another potentially noteworthy finding of the present study is that synthetic cathinone exposure decreased proinflammatory cytokine levels in some instances. These were most notable for TNF-α levels in PFC for α-PVP (Figure 2), CCL2 levels in amygdala for 4MMC (Figure 3), and for all cytokine levels in amygdala for rats that were in α-PVP or 4MMC AO groups (Figure 5). Downregulated proinflammatory markers have also been found in abstinent cocaine users, and are thought to be a result of compensatory processes that were invoked after cocaine use ceased (Araos et al., 2015; Irwin et al., 2007). Interestingly, decreases in cytokines in the present study were more common during initial drug exposure (AO groups; Figure 5) than following self-administration (ShA and LgA groups; Figures 1 and 2). For example, IL-1β in amygdala and IL-6 in hypothalamus were downregulated in the α-PVP AO group, but significantly upregulated in the α-PVP LgA group when data were normalized to saline. Thus, duration of drug exposure may impact the processes involved in downregulation of inflammatory cytokines.

Previous studies have also demonstrated changes in protein levels of inflammatory cytokines following cocaine or methamphetamine self-administration in rodents, and some of the results are consistent with the present study. Effects of LgA methamphetamine self-administration on IL-1β levels in male rat striatum were dependent on length of drug abstinence, with increases in IL-1β at 1 day of abstinence, the same timepoint and same result as the present study, but decreases at 7 days of abstinence (Gonçalves et al., 2017). Cocaine self-administration also increased IL-1β levels in mouse striatum (Burkovetskaya et al., 2020). Greater methamphetamine seeking was associated with greater CCL2 levels in rat frontal cortex (Loftis et al., 2020), while cocaine self-administration in mice increased levels of CCL2 in striatum but decreased CCL2 in medial PFC (Burkovetskaya et al., 2020). The results from the current study are somewhat similar in that CCL2 levels are increased in male LgA groups for both synthetic cathinones in the PFC and striatum. LgA methamphetamine self-administration increased TNF-α levels in rat hippocampus and striatum (Gonçalves et al., 2017), which was also observed here with the α-PVP LgA groups vs. ShA groups. Self-administration also altered peripheral cytokines in plasma, with cocaine self-administration decreasing IL-1α levels (Calipari et al., 2018), and increasing TNF-α levels (Kubera et al., 2008). In contrast, methamphetamine self-administration decreased TNF-α levels in plasma (Mata et al., 2015). Although some of these specific effects were not found in the present study, they provide further support for the theory that exposure to stimulants of abuse influences drug-induced changes in peripheral and neurochemical inflammatory markers. Importantly, all these previous studies only used male rodents, therefore, potential sex differences in effects of cocaine and methamphetamine self-administration on protein levels of inflammatory cytokines remain unexplored.

Comparison of the present results with past cocaine and methamphetamine self-administration studies should be interpreted with caution because the timepoint at which brain and blood samples were collected varied across studies. Some past studies examined inflammatory markers in samples collected within 2 hrs of the last drug administration, and therefore, while rodents were under the direct effects of stimulants (Brown et al., 2018; Kubera et al., 2008). The present study collected biological samples approximately 24 hrs after the last drug administration in accordance with some previous studies (Burkovetskaya et al., 2020; Calipari et al., 2018; Gonçalves et al., 2017; Mata et al., 2015). Due to the lack of information on whether synthetic cathinone self-administration produces withdrawal, and the latency to development of withdrawal, it is unclear if rats in the present study were experiencing withdrawal when brain tissue was collected. Yet other studies analyzed levels of inflammatory markers two or more days after the last drug exposure, when drug withdrawal was more likely to be occurring (Gonçalves et al., 2017; Loftis et al., 2020).

In the present study, saline groups also showed changes in cytokine levels compared to the naïve group. Interestingly, saline groups were more likely to show downregulation in cytokine levels compared to the naïve groups, unlike the effect in drug groups relative to saline groups. One limitation of the present study is that it did not isolate a specific cause of the changes in cytokine levels in saline groups vs naïve group. Future studies are needed to determine whether the changes observed for saline groups relative to naïve groups were attributable to learning, since even saline rats learned that pressing the active lever turned on the stimulus lights, or if they were caused by jugular catheter implantation or chemicals used to maintain catheter patency.

5.0. Conclusion

In summary, this study shows that self-administration of synthetic cathinones produced profound sex differences in proinflammatory cytokines that are linked to stimulant use disorders (IL-1α, IL-1β, IL-6, CCL2 and TNF-α). There were widespread increases in measured cytokine levels in the brains of male rats compared to females, particularly for 4MMC, whereas females were more likely to show increased inflammatory cytokines in plasma compared to saline groups than males. Furthermore, these sex differences in cytokine levels were more common after LgA access to synthetic cathinones than ShA, indicating that sex differences in neuroinflammation may only emerge after dysregulated drug intake has occurred. The changes in proinflammatory cytokine levels observed in the present study suggest that synthetic cathinone use likely produces sex-selective patterns of neuroinflammation during the transition from use (ShA) to abuse (LgA). Consequently, treatment need may differ depending on the progression of synthetic cathinone abuse and based on sex.

Highlights.

• Stimulants of abuse increase NF-κB-regulated pro-inflammatory cytokines.

• Rats self-administered synthetic cathinones or saline.

• IL-1α, IL-1β, IL-6, CCL2 and TNF-α levels were examined in brain and plasma.

• There were no sex differences in drug intake.

• Synthetic cathinones produced sex differences in cytokine levels.