Minocycline prevents neuronal hyperexcitability and neuroinflammation in medial prefrontal cortex, as well as memory impairment caused by repeated toluene inhalation in adolescent rats

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Highlights

  • Repeated exposure to 8000 ppm toluene increases neuronal hyperexcitability in mPFC.

  • Toluene activates astrocytes, microglia and NLRP3 inflammasome in mPFC.

  • Toluene also increases mRNA levels of IL-1β and decreases those of TGF-β.

  • Toluene impairs memory in two tests related to PFC function.

  • Minocycline, an antibiotic with anti-inflammatory actions, prevents these effects.

Abstract

Toluene can be intentionally misused by adolescents to experience psychoactive effects. Toluene has a complex mechanism of action and broad behavioral effects, among which memory impairment is reported consistently. We have previously reported that repeated toluene inhalation (8000 ppm) increases layer 5 prelimbic pyramidal cells' excitability in the medial prefrontal cortex (mPFC) of adolescent rats. Toluene also produces reactive oxygen species (ROS), which activate glial cells. Here, we tested the hypothesis that the anti-inflammatory agent minocycline would decrease toluene's effects because it inhibits NF-κB (nuclear factor enhancer of the kappa light chains of activated B cells) and reduces pro-inflammatory cytokine and ROS production. Our results show that minocycline (50 mg/kg, ip, for 10 days) prevents the hyperexcitability of mPFC neurons observed after repeated 8000 ppm toluene exposure (30 min/day, 2×/day for 10 days). Minocycline prevents toluene-induced hyperexcitability by a mechanism that averts the loss of the slow calcium-dependent potassium current, and normalizes mPFC neurons' firing frequency. These effects are accompanied by significant decreased expression of astrocytes and activated microglia in the mPFC, reduced NLRP3 inflammasome activation and mRNA expression levels of the pro-inflammatory cytokine interleukin 1β (IL-1β), as well as increased mRNA expression of the anti-inflammatory cytokine transforming growth factor β (TGF-β). Minocycline also prevents toluene-induced memory impairment in adolescent rats in the passive avoidance task and the temporal order memory test in which the mPFC plays a central role. These results show that neuroinflammation produces several effects of repeated toluene administration at high concentrations, and minocycline can significantly prevent them.

Introduction

Inhalant misuse is the intentional inhalation of high concentrations (several thousand ppm) of volatile compounds to achieve psychoactive effects (Balster et al., 2009; Lubman et al., 2008). This practice is particularly prevalent among adolescents and young adults (Crossin et al., 2019; Hynes-Dowell et al., 2011; Villatoro et al., 2011). Toluene is the most commonly and better characterized misused inhalant in terms of behavioral, molecular and electrophysiological effects (Cruz et al., 2014; Shelton, 2016; Win-Shwe and Fujimaki, 2010; Woodward and Beckley, 2014). Preclinical studies have shown that acute exposure to toluene, at concentrations relevant to solvent misuse, produces a wide range of behavioral responses, most of which are typical of central nervous system depressant drugs (Cruz et al., 2014), including memory impairment (Dick et al., 2014; Huerta-Rivas et al., 2012). As for its mechanism of action, toluene releases dopamine (Beckley and Woodward, 2013), acts as a positive allosteric modulator of GABAA, glycine and 5HT3 receptors (Beckstead et al., 2000; Lopreato et al., 2003), is a non-competitive, highly selective NMDA receptor antagonist (Bale et al., 2005; Cruz et al., 1998), and a sodium (Cruz et al., 2003), calcium (Tillar et al., 2002) and potassium channel blocker (Del Re et al., 2006), among other effects (Beckley and Woodward, 2013; Bowen et al., 2006; Lo et al., 2009).

We have recently shown that repeated exposure to 8000 ppm (30 min, 2×/day, for 10 days), but not to lower concentrations, increases the excitability of medial prefrontal cortex (mPFC) pyramidal neurons (Armenta-Resendiz et al., 2018). Similar findings were reported by Wayman and Woodward after a brief exposure to 10,500 ppm toluene (Wayman and Woodward, 2017). The mPFC is involved in decision-making, working memory, attention, planning, cognitive flexibility, learning and impulse control (Euston et al., 2012). This brain area is particularly vulnerable during adolescence to different insults, including drugs of abuse (Goldstein and Volkow, 2002; Winters and Arria, 2011).

Toluene is metabolized to hippuric acid mainly via cytochrome P450 2E1 (CYP2E1) and produces reactive oxygen species (ROS) through various mechanisms (Kodavanti et al., 2015; Tamie and Rui-Sheng, 1994). ROS can be damaging to protein structure and DNA material. Moreover, oxidative stress leads to a reactive state of microglia and astrocytes, as well as the release of pro-inflammatory mediators (Qin and Crews, 2012), including interleukin-1β (IL-1β) (Gustafson-Vickers et al., 2008; He et al., 2019). IL-1β synthesis depends on the activation of transcription factor NF-κB (nuclear factor enhancer of the kappa light chains of activated B cells). ROS overproduction modulates NF-κB, while ROS inhibition prevents inflammasome expression (Abais et al., 2015; Juliana et al., 2012; Morgan and Liu, 2011). Inflammasomes are cytosolic multiprotein complexes with a sensor protein of the NOD-like receptor (NLR) family, needed for inflammatory cytokines' activation and caspase-1 processing (Guo et al., 2015). In particular, activation of NLRP3 (NLR containing pyrin domain 3) is needed for maturation of pro-IL-1β into IL-1β (Cullen et al., 2015; Martin, 2016). In experiments simulating toluene as an occupational hazard, long-exposure periods to 1500 ppm for 4 h per day for 7 days (Gotohda et al., 2002) or 0–500 ppm toluene for 6 weeks (Win-Shwe et al., 2011) produce an increase in pro-inflammatory cytokines and NF-κB mRNA in rodent hippocampus and cerebellum. In addition, Kanter and colleagues found evidence of dilated cisternae of the endoplasmic reticulum and swelling of intracellular mitochondria (which may signify neuroinflammation) in rats exposed to 3000 ppm toluene, 8 h per day, 6 days per week for 12 weeks (Kanter, 2008). To our knowledge, there are no studies addressing the possibility that binge patterns of repeated toluene exposure, mimicking solvent misuse, can also produce neuroinflammatory effects.

Minocycline (MN) is a potent anti-inflammatory agent (Blum et al., 2004; Choi et al., 2005; Dean et al., 2012; Ray et al., 2014) that inhibits the NF-κB pathway and confers neuroprotection by reducing cytokine and ROS production (Blum et al., 2004; Henry et al., 2008; Tikka and Koistinaho, 2001). By utilizing this pharmacological agent, we can indirectly assess oxidative stress produced by repeated toluene exposure. Based on these findings, the aim of this work was two-fold: i) to evaluate if neuroinflammation occurs in the mPFC of adolescent rats repeatedly exposed to a high toluene concentration; and ii) to test the hypothesis that MN would prevent the effects of repeated toluene exposure on neuronal hyperexcitability, neuroinflammation and memory impairment.

Section snippets

Animals

We used a total of 124 adolescent male Wistar rats (25 days of age at the beginning of the experiment) from our own vivarium. Animals were socially housed, had ad libitum access to food and water, and were kept under a 12:12 h light/dark cycle in a climate-controlled room at 22 ± 2 °C. Experimental procedures were carried out in strict accordance with the Mexican Official Norm for utilization and care of laboratory animals “NOM-062-ZOO-1999” and the Institutional Ethics Committee (Protocols

Effect of minocycline on the intrinsic excitability and firing frequency of mPFC layer V prelimbic neurons

Using whole cell recordings, we first aimed to establish whether the daily administration of MN modified the neurophysiological properties of prelimbic mPFC neurons and found that there were no significant differences between the resting membrane potential (RMP) of control and MN-treated neurons (RMP in control = −72.5 ± 1.3 mV; MN group = −70.8 ± 1.9 mV; P = .49, Student's t-test). Then, we constructed a series of I-V relationships. The trajectory and rectification properties of the I-V plots

Minocycline does not significantly alter basal properties of mPFC neurons

Consistent with previous reports (Armenta-Resendiz et al., 2018; Cruz et al., 2019), we showed that repeated exposure to toluene, at 8000 ppm, a concentration relevant to solvent intoxication, increases the firing output of mPFC neurons in the adolescent rat brain. This effect was prevented when animals exposed to toluene were consecutively treated with MN. This drug is an antibiotic and a very effective anti-inflammatory agent (Garrido-Mesa et al., 2013; Möller et al., 2016). Because of its

Conclusion

A binge pattern of toluene exposure produces neuroinflammation and hyperexcitability of layer 5 neurons of prelimbic mPFC, as well as memory impairment in two assays related to PFC function more generally. The finding that the anti-inflammatory antibiotic MN can prevent these effects suggests that neuroinflammation causes several chronic toluene effects most probably via ROS generation and NF-κB pathway activation. These results highlight the risks of repeated exposure to high toluene

Disclosure statement

Silvia L. Cruz reports grant 239192 from Conacyt (National Council of Science and Technology), Mexico. Monserrat Armenta-Reséndiz and César J. Carranza-Aguilar report scholarships from Conacyt, Mexico. Emilio J. Galván has nothing to disclose.

Declaration of Competing Interest

Funding was provided by grant 239192 (SLC); CB 2016-281617 (EJG) and scholarships 261795 (MAR) and 338376 (CJCA) from Conacyt (National Council of Science and Technology) Mexico.

Acknowledgements

This paper includes data from MAR's Doctoral Dissertation. We thank Araceli Hernández-Mendoza for technical support and Eduardo Violante for some artwork design.

References (100)

  • S.P. Cullen et al.

    Diverse activators of the NLRP3 Inflammasome promote IL-1β secretion by triggering necrosis

    Cell Rep.

    (2015)
  • A.M. Del Re et al.

    Effects of the abused inhalant toluene on ethanol-sensitive potassium channels expressed in oocytes

    Brain Res.

    (2006)
  • O. Devinsky et al.

    Glia and epilepsy: excitability and inflammation

    Trends Neurosci.

    (2013)
  • D.R. Euston et al.

    The role of medial prefrontal cortex in memory and decision making

    Neuron

    (2012)
  • T. Gotohda et al.

    Toluene inhalation induces glial cell line-derived neurotrophic factor, transforming growth factor and tumor necrosis factor in rat cerebellum

    Legal Med.

    (2002)
  • A. Huerta-Rivas et al.

    Toluene impairs learning and memory, has antinociceptive effects, and modifies histone acetylation in the dentate gyrus of adolescent and adult rats

    Pharmacol. Biochem. Behav.

    (2012)
  • M. Imbesi et al.

    Minocycline increases phosphorylation and membrane insertion of neuronal GluR1 receptors

    Neurosci. Lett.

    (2008)
  • I. Izquierdo et al.

    Separate mechanisms for short- and long-term memory

    Behav. Brain Res.

    (1999)
  • C. Juliana et al.

    Non-transcriptional priming and deubiquitination regulate NLRP3 inflammasome activation

    J. Biol. Chem.

    (2012)
  • P.R. Kodavanti et al.

    Toluene effects on oxidative stress in brain regions of young-adult, middle-age, and senescent Brown Norway rats

    Toxicol. Appl. Pharmacol.

    (2011)
  • P.R.S. Kodavanti et al.

    Acute and subchronic toxicity of inhaled toluene in male Long–Evans rats: oxidative stress markers in brain

    Neurotoxicology

    (2015)
  • H.-K. Lee et al.

    Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory

    Cell

    (2003)
  • N. Liu et al.

    Minocycline inhibits hyperpolarization-activated currents in rat substantia gelatinosa neurons

    Neuropharmacology

    (2015)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and

    Methods

    (2001)
  • P.S. Lo et al.

    Acute neurobehavioral effects of toluene: involvement of dopamine and NMDA receptors

    Toxicology

    (2009)
  • G.F. Lopreato et al.

    Inhaled drugs of abuse enhance serotonin-3 receptor function

    Drug Alcohol Depend.

    (2003)
  • S. Marty et al.

    Neurotrophins and activity-dependent plasticity of cortical interneurons

    Trends Neurosci.

    (1997)
  • N. Tamie et al.

    Induction of cytochrome P450 by toluene

    Int. J. BioChemiPhysics

    (1994)
  • R. Tillar et al.

    Toluene inhibits voltage-sensitive calcium channels expressed in pheochromocytoma cells

    Neurochem. Int.

    (2002)
  • A. Vezzani et al.

    Neuromodulatory properties of inflammatory cytokines and their impact on neuronal excitability

    Neuropharmacology

    (2015)
  • C. Villanueva-Castillo et al.

    Aging-related impairments of hippocampal mossy fibers synapses on CA3 pyramidal cells

    Neurobiol. Aging

    (2017)
  • N. Wang et al.

    Minocycline inhibits brain inflammation and attenuates spontaneous recurrent seizures following pilocarpine-induced status epilepticus

    Neuroscience

    (2015)
  • E.C. Warburton et al.

    Neural circuitry for rat recognition memory

    Behav. Brain Res.

    (2015)
  • J.M. Williams et al.

    Effects of repeated inhalation of toluene on ionotropic GABA a and glutamate receptor subunit levels in rat brain

    Neurochem. Int.

    (2005)
  • T.T. Win-Shwe et al.

    Neurotoxicity of toluene

    Toxicol. Lett.

    (2010)
  • T.-T. Win-Shwe et al.

    Role of hippocampal TLR4 in neurotoxicity in mice following toluene exposure

    Neurotoxicol. Teratol.

    (2011)
  • V.W. Yong et al.

    The promise of minocycline in neurology

    Lancet Neurol.

    (2004)
  • F. Zhu et al.

    Minocycline alleviates behavioral deficits and inhibits microglial activation induced by intrahippocampal administration of granulocyte-macrophage Colony-stimulating factor in adult rats

    Neuroscience

    (2014)
  • J.M. Abais et al.

    Redox regulation of NLRP3 Inflammasomes: ROS as trigger or effector?

    Antioxid. Redox Signal.

    (2015)
  • R. Arezoomandan et al.

    Minocycline increases firing rates of accumbal neurons and modifies the effects of morphine on neuronal activity

    Addict. Biol.

    (2018)
  • R.L. Balster et al.

    Classification of abused inhalants

    Addiction

    (2009)
  • G.R.I. Barker et al.

    Recognition memory for objects, place, and temporal order: a disconnection analysis of the role of the medial prefrontal cortex and Perirhinal cortex

    J. Neurosci.

    (2007)
  • J.T. Beckley et al.

    Volatile solvents as drugs of abuse: focus on the cortico-mesolimbic circuitry

    Neuropsychopharmacology

    (2013)
  • M.J. Beckstead et al.

    Glycine and γ-aminobutyric acid(a) receptor function is enhanced by inhaled drugs of abuse

    Mol. Pharmacol.

    (2000)
  • N.G. Carlson et al.

    Inflammatory cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha impart neuroprotection to an excitotoxin through distinct pathways

    J. Immunol.

    (1999)
  • Y.C. Chang et al.

    Chronic NMDA administration increases neuroinflammatory markers in rat frontal cortex: cross-talk between excitotoxicity and neuroinflammation

    Neurochem. Res.

    (2008)
  • S.H. Choi et al.

    Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo

    J. Neurochem.

    (2005)
  • R. Crossin et al.

    The effect of adolescent inhalant abuse on energy balance and growth

    Pharmacol. Res. Perspect.

    (2019)
  • S.L. Cruz et al.

    Effects of the abused solvent toluene on recombinant N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors expressed in Xenopus oocytes

    J. Pharmacol. Exp. Ther.

    (1998)
  • S.L. Cruz et al.

    Inhibition of cardiac sodium currents by toluene exposure

    Br. J. Pharmacol.

    (2003)
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    1

    SLC and EJG contributed equally to this work.

    2

    Present address: Department of Neuroscience, Medical University of South Carolina, SC, USA.

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