Research ReportNMDA receptor binding is reduced within mesocorticolimbic regions following chronic inhalation of toluene in adolescent rats
Introduction
Intentional inhalation of volatile organic solvents, such as toluene, is a major health concern worldwide, especially within adolescent populations (Lubman et al., 2006). Abuse during this time is a predictor of substance abuse disorders later in life and has a significant co-morbidity with neuropsychiatric disorders (Wu et al., 2004). Inhalant abuse is also associated with long-term adverse outcomes including behavioural and cognitive dysfunction (Yucel et al., 2008, Dick et al., 2014). Such outcomes may be due to toluene-induced neuroadaptations within discrete neural circuitry following repeated exposure (Beckley et al., 2013a).
Toluene is found in many household products that are commonly abused, such as paints, glues and aerosols. It has high potential for abuse as it can elicit a conditioned place preference (CPP) in rodents (Funada et al., 2002) and is self-administered in mice (Blokhina et al., 2004) and non-human primates (Weiss et al., 1979). Similar to other drugs of abuse, toluene׳s acute hedonic properties are thought to be mediated, at least in part, via regulation of signalling within mesocorticolimbic circuitry (Riegel and French, 1999, Riegel et al., 2007). Toluene׳s effects on behaviour may relate to its ability to regulate both inhibitory and excitatory neurotransmission, and receptor subunit expression, within discrete regions of this circuitry. Notably, exposure to toluene results in consistent regulation of N-methyl-D-aspartate (NMDA) receptors in particular (Cruz et al., 1998, Williams et al., 2005, MacIver, 2009, Beckley and Woodward, 2011).
NMDA receptors are tetrameric complexes consisting of two GluN1 and two GluN2 subunits. The GluN2A and GluN2B subunits predominate, particularly within forebrain structures, and show differential channel kinetics (Watanabe et al., 1993, Monyer et al., 1994, El Gaamouch et al., 2012). Toluene rapidly and reversibly inhibits NMDA receptors in vitro with the GluN1/GluN2B conformation reportedly the most sensitive to toluene-induced inhibition (Cruz et al., 1998). Moreover, toluene exposure in adult rodents can differentially alter GluN2B protein expression in discrete brain regions (Williams et al., 2005) as well as γ-aminobutyric acid type-A (GABAA) receptor subunits. In contrast, neonatal toluene administration (500 mg/kg, i.p., postnatal day [PN] 4–9) in rats results in increased GluN2A expression within the dorsal hippocampus (dHPC) and cerebellum at the onset of adolescence (PN 30), with alterations of GluN2B only observed within the cerebellum (Lee et al., 2005). In a validated rodent model we have previously observed discrete cognitive deficits following chronic intermittent toluene (CIT) exposure during adolescence in rats. These deficits were present within the first week after exposure to toluene with deficits lasting at least 10 weeks suggesting long-term neuroadaptive responses within the mesocorticolimbic circuitry (Dick et al., 2014). Furthermore, changes in cognitive processes parallel with toluene-induced glutamatergic dysfunction mediated by NMDA receptors in our model (Duncan et al., 2014).
Cholinergic signalling is also affected following exposure to toluene with reduced extracellular acetylcholine release observed within the striatum and hippocampus (HPC) in vivo (Stengard, 1994, Honma and Suda, 2004). Neonatal (PN 4–9) and pre/early adolescent (PN 25–30) toluene administration (500 mg/kg, i.p.) in rats also alters the behavioural sensitivity to acute nicotine administration in early adulthood in the absence of alterations neuronal nicotinic acetylcholine (nACh) receptor subunit expression (Chan et al., 2008). Toluene inhibits neuronal nACh receptors in vitro, with the α4β2 subtype being most sensitive (Bale et al., 2002, Bale et al., 2005). Interestingly, α4β2 nACh receptors are inhibited by toluene at similar concentrations as GluN1/GluN2B NMDA receptors emphasising a comparative sensitivity of these two receptor subtypes to toluene-induced regulation in vitro (Cruz et al., 1998, Bale et al., 2002, Bale et al., 2005). Thus, the sensitivity of α4β2 nACh receptors to toluene may implicate a further neurotransmitter system affected via chronic exposure, although this remains to be elucidated especially following adolescent exposure.
Recent evidence suggests that both glutamatergic and cholinergic synapses are labile throughout adolescence, particularly within the medial prefrontal cortex (mPFC) (Counotte et al., 2012, Wang et al., 2013, Flores-Barrera et al., 2014). Thus, these systems may be vulnerable to CIT-induced alteration during this period. Moreover, toluene׳s neurochemical and neuroadaptive properties appear to be concentration-dependent (Gerasimov et al., 2002, Koga et al., 2007, Beckley et al., 2013a) such that high (>5000 ppm) as opposed to moderate (~3000 ppm) concentrations of toluene have differential pharmacological effects. Frequency of exposures must also be considered as initial experimentation with inhalants in humans often progresses from frequent inhalation of moderate concentrations via “sniffing,” to less frequent episodes of extended inhalation of higher vapour concentrations via “huffing” or “bagging” as inhalant misuse escalates (Henretig, 1996, Kurtzman et al., 2001). Together, concentration and frequency of toluene exposure mediate diverse modifications including behavioural outcomes (Beckley et al., 2013a, Batis et al., 2010).
As we have previously observed both cognitive and behavioural deficits following chronic intermittent exposure to toluene during adolescence (Dick et al., 2014), which paralleled with long-term changes in glutamatergic signalling in our model (Duncan et al., 2014), this study examined neurochemical changes within discrete regions of the mesocorticolimbic system 3 days after the last exposure to toluene to investigate potential neurobiological mechanisms subserving these changes. We hypothesised that NMDA receptors would be susceptible to toluene-induced adaptations following adolescent CIT exposure. Based on observations from human settings (Henretig, 1996), we also sought to examine the effects of concentration and/or frequency upon these neurobiological parameters. We hypothesised that models reflecting early (moderate toluene concentration, 3000 ppm, at high frequency, 5 days/week) compared to later (high toluene concentration, 10,000 ppm, less frequently, 3 days/week) stages of inhalant abuse would be less likely to induce neurochemical adaptations. We observed a concentration-dependent decrease in receptor binding specifically to NMDA receptors containing the GluN2B subunit in the caudal anterior cingulate cortex (ACg), dorsal striatum (DS) and nucleus accumbens (NAc); however this was not associated with changes in genes encoding NMDA receptor subunits or GluN2B protein expression. Moreover, no changes to nACh receptor binding, or genes encoding nACh, GABAA or dopamine receptor subunits were evident following the exposure period. Thus, this study suggests that adolescent CIT exposure results in altered NMDA receptor binding within regions of corticostriatal circuitry, which may be mediated via post-translational mechanisms.
Section snippets
Adolescent CIT exposure retards body weight gain throughout the exposure period
Analysis of grouped body weights throughout the exposure period revealed main effects of treatment (F(2, 35)=7.56, p<0.01) and time (F(2, 70)=5227.0, p<0.001) and a treatment x time interaction (F(4, 70)=23.39, p<0.001). Post hoc analysis revealed this effect was due to a significant retardation in weight gain in rats exposed to 10,000 ppm compared to those exposed to air or 3000 ppm, which was evident by the 6th and 12th exposure, respectively (Fig. 1). Body weights of rats exposed to 10,000 ppm
Discussion
The present study investigated the sensitivity of NMDA and nACh receptors within mesocorticolimbic regions following adolescent CIT exposure. In line with previous findings, adolescent CIT exposure (10,000 ppm) attenuated weight gain compared to air-exposed controls (Dick et al., 2014). However, no significant differences were observed between rats exposed to air or CIT at 3000 ppm. Analysis of brains collected 72 h following the exposure period revealed a reduction in [3H]-ifenprodil binding
Animals
Adolescent male Wistar rats (n=41, ~PN 24) were obtained from the Australian Resources Centre (Perth, Australia). Rats were pair housed and maintained on a 12-hour light/dark cycle with food and water ad libitum. Rats were acclimatised for 3 days prior to experimentation. All experiments were performed in accordance with the Prevention of Cruelty to Animals Act, 1986 under the guidelines of the Australian National Health and Medical Research Council Code of Practice for the Care and Use of
Acknowledgments
The authors would like to thank Dr. Despina Ganella for kindly donating the Hprt1, Drd1 and Drd2 primer sets employed in qPCR experiments. This study was supported by The National Health and Medical Research Council of Australia of which AJL is a Principal Research Fellow (1020737), the Australian Research Council (DP 110100379) of which JRD was a Future Fellow during the time of the study (100100235) and the Victorian Government׳s Operational Infrastructure Support Scheme. There are no
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