Activation of mGluR2/3 underlies the effects of N-acetylcystein on amygdala-associated autism-like phenotypes in a valproate-induced rat model of autism

Yu-Wen Chen, Hui-Ching Lin, Ming-Chong Ng, Ya-Hsin Hsiao, Chao-Chuan Wang, Po-Wu Gean, Po See Chen, Yu-Wen Chen, Hui-Ching Lin, Ming-Chong Ng, Ya-Hsin Hsiao, Chao-Chuan Wang, Po-Wu Gean, Po See Chen

Abstract

Autism-like phenotypes in male valproate (VPA)-exposed offspring have been linked to high glutamatergic neurotransmission in the thalamic-amygdala pathway. Glial cystine/glutamate exchange (system Xc(-)), which exchanges extracellular cystine for intracellular glutamate, plays a significant role in the maintenance of extracellular glutamate. N-acetylcysteine (NAC) is a cystine prodrug that restores extracellular glutamate by stimulating system Xc(-). In this study, we examined the effects of NAC on autism-like phenotypes and neurotransmission in the thalamic-amygdala synapses, as well as the involvement of metabotropic glutamate receptors 2/3 (mGluR2/3). Valproate-treated rats received a single intraperitoneal injection of 500 mg/kg NaVPA on E12.5. On postnatal day 21 (P21), NAC or saline was administered once daily for 10 days. From day 8 to 10, NAC was given 1/2 h prior to behavioral testing. Chronic administration of NAC restored the duration and frequency of social interaction and ameliorated anxiety-like behaviors in VPA-exposed offspring. In amygdala slices, NAC treatment normalized the increased frequency of mEPSCs and decreased the paired pulse facilitation (PPF) induced by VPA exposure. The effects of NAC on social interaction and anxiety-like behavior in the VPA-exposed offspring were blocked after intra-amygdala infusion of mGluR2/3 antagonist LY341495. The expressions of mGluR2/3 protein and mGluR2 mRNA were significantly lower in the VPA-exposed offspring. In contrast, the mGluR3 mRNA level did not differ between the saline- and VPA-exposed offspring. These results provide the first evidence that the disruption of social interaction and enhanced presynaptic excitatory transmission in VPA-exposed offspring could be rescued by NAC, which depends on the activation of mGluR2/3.

Keywords: N-acetylcysteine; amygdala; autistic spectrum disorders; glutamate; valproate.

Figures

Figure 1
Figure 1
The experimental design employed in the present study. On postnatal day 21 (P21), NAC (150 mg/kg, intraperitoneal (i.p.); sigma) or saline was administered once daily for 10 days. From P28 to P30, the social interaction test, elevated plus-maze test, and open field test were conducted each day after NAC treatment. To elucidate whether the effect of NAC on social interaction in VPA-exposed offspring is mediated by mGluR2/3, on P24, the mGluR2/3 antagonist LY341495 (2 μg/0.5 μl/side) was infused into the amygdala 30 min before the administration of NAC once per day for 5 days.
Figure 2
Figure 2
Reversal of the social interaction deficit in VPA-exposed offspring by NAC. Rats of VPA- or saline-expose offspring weaned at postnatal day 21 (P21) were used in this experiment. NAC (150 mg/kg, i.p.) or saline was administered once per day for 10 days (P21 to P30). On P28, the male offspring were tested for social interaction. The duration (A) and the frequency (B) of occurrence of various social behaviors such as sniffing, mounting and grooming partner were measured in the VPA- and saline-exposed offspring for 20 min. Sample sizes (n): Saline/Saline n = 21, Saline/NAC n = 5, VPA/Saline n = 25, VPA/NAC n = 17. ***p < 0.001 vs. VPA/NAC.
Figure 3
Figure 3
Amelioration of anxiety in the VPA-exposed offspring by NAC. On P29 and P30, rats were administered the elevated plus-maze test (EPM) and the open field test (OF). In the EPM test, the time spent in the open arms (A) and the distance traveled (B) were measured for 5 min. ***p < 0.001 vs. VPA/NAC. In the OF test, the time spent in the center (C) and the distance traveled (D) were measured for 15 min. **p < 0.01 vs VPA/NAC.
Figure 4
Figure 4
Effects of NAC on the amplitude and frequency of mEPSCs recorded in the LA of VPA-exposed offspring. (A) Sample traces of mEPSCs taken from slices of Saline/Saline, Saline/NAC, VPA/Saline, and VPA/NAC rats. mEPSCs were recorded in the LA neurons at a holding potential of −70 mV in the presence of TTX (1 μM). Calibration: 30 pA, 100 ms. (B,C) Summary plots of the frequency (B) and amplitude (C) of mEPSCs in the Saline/Saline, Saline/NAC, VPA/Saline, and VPA/NAC rats. **p < 0.01 vs. VPA/saline. ###p < 0.001 vs. Saline/Saline.
Figure 5
Figure 5
Effects of NAC on the PPF of EPSCs at the thalamo-LA synapses of the VPA-exposed offspring. (A) Sample traces of the PPF of EPSCs taken from slices of Saline/Saline, Saline/NAC, VPA/Saline, and VPA/NAC rats. Sample traces were the average of 3–5 successive responses. Calibration; 50 pA, 30 ms. (B) Plot of the PPF in the Saline/Saline, Saline/NAC, VPA/Saline, and VPA/NAC rats. *p < 0.05, **p < 0.01 vs. VPA/saline. ##p < 0.01 vs. Saline/Saline.
Figure 6
Figure 6
NAC-mediated reversal of social interaction deficit in VPA-exposed offspring is blocked by mGluR2/3 antagonist. (A,B) Rats of saline-exposed offspring weaned at postnatal day 21. At postnatal day 24, the rats received an intra-amygdala injection of LY341495 (2 μg/0.5 μl per side) or saline once per day for 5 days. One hour after the last injection of LY341495 or saline, the rats were administered the social interaction test. The duration (A) and frequency (B) of contact between the VPA- and saline-exposed offspring were measured for 20 min. Sample sizes (n): Saline/Saline n = 4, Saline/LY n = 5. (C,D) Rats of VPA-exposed offspring weaned at postnatal day 21 were administered an intraperitoneal injection of NAC (150 mg/kg) once per day for 8 days. At postnatal day 24, the rats received an intra-amygdala injection of LY341495 (2 μg/0.5 μl per side) or saline 30 min before NAC once per day for 5 days. One hour after the last injection of LY341495 or saline, the rats were administered the social interaction test. The duration (A) and frequency (B) of contact between the VPA- and saline-exposed offspring were measured for 20 min. Sample sizes (n): Saline/Saline n = 9, Saline/LY n = 9. ***p < 0.001 vs. NAC/Saline.
Figure 7
Figure 7
NAC-mediated amelioration of anxiety in VPA-exposed offspring is blocked by mGluR2/3 antagonist. (A,B) Rats of saline-exposed offspring weaned at postnatal day 24 received an intra-amygdala injection of LY341495 (2 μg/0.5 μl per side) once per day for 5 days. Twenty-four hours later, the rats were administered the elevated plus-maze test. The time spent in the open arms (A) and the distance traveled (B) were measured for 5 min. (C,D) Rats of VPA-exposed offspring weaned at postnatal day 21 were administered an intraperitoneal injection of NAC (150 mg/kg) once per day for 8 days. At postnatal day 24, the rats received an intra-amygdala injection of LY341495 (2 μg/0.5 μl per side) 30 min before NAC once per day for 4 days. Twenty-four hours after the last injection of NAC and LY341495, the rats were administered the elevated plus-maze test. The time spent in the open arms (C) and the distance traveled (D) were measured for 5 min. *p < 0.05 vs. NAC/Saline.
Figure 8
Figure 8
The expression of the mGluR2/3 receptor is reduced in VPA-exposed offspring. (A,B) Rats of saline- or VPA-exposed offspring weaned at postnatal day 21 were administered NAC (150 mg/kg, i.p.) or saline once per day for 7 days. Twenty-four hours after the last injection of NAC, the amygdala mGluR2/3 (A) and Xc−(B) protein levels were determined by Western blotting analysis. *p < 0.05. (C,D) The mRNA levels of mGluR2 and mGluR3 were determined by RT-PCR analysis.

References

    1. Adolphs R., Tranel D., Damasio H. (2001). Emotion recognition from faces and prosody following temporal lobectomy. Neuropsychology 15, 396–404 10.1037/0894-4105.15.3.396
    1. Aylward E. H., Minshew N. J., Goldstein G., Honeycutt N. A., Augustine A. M., Yates K. O., et al. (1999). MRI volumes of amygdala and hippocampus in non-mentally retarded autistic adolescents and adults. Neurology 53, 2145–2150 10.1212/WNL.53.9.2145
    1. Baharnoori M., Bhardwaj S. K., Srivastava L. K. (2012). Neonatal behavioral changes in rats with gestational exposure to lipopolysaccharide: a prenatal infection model for developmental neuropsychiatric disorders. Schizophr. Bull. 38, 444–456 10.1093/schbul/sbq098
    1. Bailey A., Luthert P., Dean A., Harding B., Janota I., Montgomery M., et al. (1998). A clinicopathological study of autism. Brain 121(pt 5), 889–905 10.1093/brain/121.5.889
    1. Baird G., Simonoff E., Pickles A., Chandler S., Loucas T., Meldrum D., et al. (2006). Prevalence of disorders of the autism spectrum in a population cohort of children in South Thames: the Special Needs and Autism Project (SNAP). Lancet 368, 210–215 10.1016/S0140-6736(06)69041-7
    1. Baker D. A., Xi Z. X., Shen H., Swanson C. J., Kalivas P. W. (2002). The origin and neuronal function of in vivo nonsynaptic glutamate. J. Neurosci. 22, 9134–9141
    1. Baron-Cohen S., Belmonte M. K. (2005). Autism: a window onto the development of the social and the analytic brain. Annu. Rev. Neurosci. 28, 109–126 10.1146/annurev.neuro.27.070203.144137
    1. Baron-Cohen S., Ring H. A., Bullmore E. T., Wheelwright S., Ashwin C., Williams S. C. (2000). The amygdala theory of autism. Neurosci. Biobehav. Rev. 24, 355–364 10.1016/S0149-7634(00)00011-7
    1. Baron-Cohen S., Ring H. A., Wheelwright S., Bullmore E. T., Brammer M. J., Simmons A., et al. (1999). Social intelligence in the normal and autistic brain: an fMRI study. Eur. J. Neurosci. 11, 1891–1898 10.1046/j.1460-9568.1999.00621.x
    1. Bateup H. S., Johnson C. A., Denefrio C. L., Saulnier J. L., Kornacker K., Sabatini B. L. (2013). Excitatory/inhibitory synaptic imbalance leads to hippocampal hyperexcitability in mouse models of tuberous sclerosis. Neuron 78, 510–522 10.1016/j.neuron.2013.03.017
    1. Christensen J., Gronborg T. K., Sorensen M. J., Schendel D., Parner E. T., Pedersen L. H., et al. (2013). Prenatal valproate exposure and risk of autism spectrum disorders and childhood autism. JAMA 309, 1696–1703 10.1001/jama.2013.2270
    1. Dalton K. M., Nacewicz B. M., Johnstone T., Schaefer H. S., Gernsbacher M. A., Goldsmith H. H., et al. (2005). Gaze fixation and the neural circuitry of face processing in autism. Nat. Neurosci. 8, 519–526 10.1038/nn1421
    1. Eichler S. A., Meier J. C. (2008). E-I balance and human diseases - from molecules to networking. Front. Mol. Neurosci. 1:2 10.3389/neuro.02.002.2008
    1. El-Ansary A. K., Ben Bacha A., Kotb M. (2012). Etiology of autistic features: the persisting neurotoxic effects of propionic acid. J. Neuroinflammation 9:74 10.1186/1742-2094-9-74
    1. Farr S. A., Poon H. F., Dogrukol-Ak D., Drake J., Banks W. A., Eyerman E., et al. (2003). The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice. J. Neurochem. 84, 1173–1183 10.1046/j.1471-4159.2003.01580.x
    1. Flagstad P., Mork A., Glenthoj B. Y., Van Beek J., Michael-Titus A. T., Didriksen M. (2004). Disruption of neurogenesis on gestational day 17 in the rat causes behavioral changes relevant to positive and negative schizophrenia symptoms and alters amphetamine-induced dopamine release in nucleus accumbens. Neuropsychopharmacology 29, 2052–2064 10.1038/sj.npp.1300516
    1. Foley K. A., Ossenkopp K. P., Kavaliers M., Macfabe D. F. (2014). Pre- and neonatal exposure to lipopolysaccharide or the enteric metabolite, propionic acid, alters development and behavior in adolescent rats in a sexually dimorphic manner. PLoS ONE 9:e87072 10.1371/journal.pone.0087072
    1. Fu A. L., Dong Z. H., Sun M. J. (2006). Protective effect of N-acetyl-L-cysteine on amyloid beta-peptide-induced learning and memory deficits in mice. Brain Res. 1109, 201–206 10.1016/j.brainres.2006.06.042
    1. Gkogkas C. G., Khoutorsky A., Ran I., Rampakakis E., Nevarko T., Weatherill D. B., et al. (2013). Autism-related deficits via dysregulated eIF4E-dependent translational control. Nature 493, 371–377 10.1038/nature11628
    1. Hadjikhani N., Joseph R. M., Snyder J., Tager-Flusberg H. (2007). Abnormal activation of the social brain during face perception in autism. Hum. Brain Mapp. 28, 441–449 10.1002/hbm.20283
    1. Hardan A. Y., Fung L. K., Libove R. A., Obukhanych T. V., Nair S., Herzenberg L. A., et al. (2012). A randomized controlled pilot trial of oral N-acetylcysteine in children with autism. Biol. Psychiatry 71, 956–961 10.1016/j.biopsych.2012.01.014
    1. Hascup E. R., Hascup K. N., Pomerleau F., Huettl P., Hajos-Korcsok E., Kehr J., et al. (2012). An allosteric modulator of metabotropic glutamate receptors (mGluR(2)), (+)-TFMPIP, inhibits restraint stress-induced phasic glutamate release in rat prefrontal cortex. J. Neurochem. 122, 619–627 10.1111/j.1471-4159.2012.07784.x
    1. Hsia A. Y., Malenka R. C., Nicoll R. A. (1998). Development of excitatory circuitry in the hippocampus. J. Neurophysiol. 79, 2013–2024
    1. Kalivas P. W. (2009). The glutamate homeostasis hypothesis of addiction. Nat. Rev. Neurosci. 10, 561–572 10.1038/nrn2515
    1. Khan M., Sekhon B., Jatana M., Giri S., Gilg A. G., Sekhon C., et al. (2004). Administration of N-acetylcysteine after focal cerebral ischemia protects brain and reduces inflammation in a rat model of experimental stroke. J. Neurosci. Res. 76, 519–527 10.1002/jnr.20087
    1. Kupchik Y. M., Moussawi K., Tang X. C., Wang X., Kalivas B. C., Kolokithas R., et al. (2012). The effect of N-acetylcysteine in the nucleus accumbens on neurotransmission and relapse to cocaine. Biol. Psychiatry 71, 978–986 10.1016/j.biopsych.2011.10.024
    1. Levitt P., Campbell D. B. (2009). The genetic and neurobiologic compass points toward common signaling dysfunctions in autism spectrum disorders. J. Clin. Invest. 119, 747–754 10.1172/JCI37934
    1. Lin H. C., Gean P. W., Wang C. C., Chan Y. H., Chen P. S. (2013). The amygdala excitatory/inhibitory balance in a valproate-induced rat autism model. PLoS ONE 8:e55248 10.1371/journal.pone.0055248
    1. Macfabe D. F., Cain D. P., Rodriguez-Capote K., Franklin A. E., Hoffman J. E., Boon F., et al. (2007). Neurobiological effects of intraventricular propionic acid in rats: possible role of short chain fatty acids on the pathogenesis and characteristics of autism spectrum disorders. Behav. Brain Res. 176, 149–169 10.1016/j.bbr.2006.07.025
    1. Markram K., Rinaldi T., La Mendola D., Sandi C., Markram H. (2008). Abnormal fear conditioning and amygdala processing in an animal model of autism. Neuropsychopharmacology 33, 901–912 10.1038/sj.npp.1301453
    1. Nacewicz B. M., Dalton K. M., Johnstone T., Long M. T., McAuliff E. M., Oakes T. R., et al. (2006). Amygdala volume and nonverbal social impairment in adolescent and adult males with autism. Arch. Gen. Psychiatry 63, 1417–1428 10.1001/archpsyc.63.12.1417
    1. Ornstein P. L., Bleisch T. J., Arnold M. B., Kennedy J. H., Wright R. A., Johnson B. G., et al. (1998). 2-substituted (2SR)-2-amino-2-((1SR,2SR)-2-carboxycycloprop-1-yl)glycines as potent and selective antagonists of group II metabotropic glutamate receptors. 2. Effects of aromatic substitution, pharmacological characterization, and bioavailability. J. Med. Chem. 41, 358–378 10.1021/jm970498o
    1. Palmer R. F., Blanchard S., Wood R. (2009). Proximity to point sources of environmental mercury release as a predictor of autism prevalence. Health Place 15, 18–24 10.1016/j.healthplace.2008.02.001
    1. Parachikova A., Green K. N., Hendrix C., Laferla F. M. (2010). Formulation of a medical food cocktail for Alzheimer's disease: beneficial effects on cognition and neuropathology in a mouse model of the disease. PLoS ONE 5:e14015 10.1371/journal.pone.0014015
    1. Reissner K. J., Kalivas P. W. (2010). Using glutamate homeostasis as a target for treating addictive disorders. Behav. Pharmacol. 21, 514–522 10.1097/FBP.0b013e32833d41b2
    1. Roberts E. M., English P. B., Grether J. K., Windham G. C., Somberg L., Wolff C. (2007). Maternal residence near agricultural pesticide applications and autism spectrum disorders among children in the California Central Valley. Environ. Health Perspect. 115, 1482–1489 10.1289/ehp.10168
    1. Rojas D. C., Smith J. A., Benkers T. L., Camou S. L., Reite M. L., Rogers S. J. (2004). Hippocampus and amygdala volumes in parents of children with autistic disorder. Am. J. Psychiatry 161, 2038–2044 10.1176/appi.ajp.161.11.2038
    1. Santini E., Huynh T. N., Macaskill A. F., Carter A. G., Pierre P., Ruggero D., et al. (2013). Exaggerated translation causes synaptic and behavioural aberrations associated with autism. Nature 493, 411–415 10.1038/nature11782
    1. Schumann C. M., Amaral D. G. (2006). Stereological analysis of amygdala neuron number in autism. J. Neurosci. 26, 7674–7679 10.1523/JNEUROSCI.1285-06.2006
    1. Shultz S. R., Macfabe D. F., Martin S., Jackson J., Taylor R., Boon F., et al. (2009). Intracerebroventricular injections of the enteric bacterial metabolic product propionic acid impair cognition and sensorimotor ability in the Long-Evans rat: further development of a rodent model of autism. Behav. Brain Res. 200, 33–41 10.1016/j.bbr.2008.12.023
    1. Todd R. M., Anderson A. K. (2009). Six degrees of separation: the amygdala regulates social behavior and perception. Nat. Neurosci. 12, 1217–1218 10.1038/nn1009-1217
    1. Tyzio R., Nardou R., Ferrari D. C., Tsintsadze T., Shahrokhi A., Eftekhari S., et al. (2014). Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring. Science 343, 675–679 10.1126/science.1247190
    1. Wang A. T., Dapretto M., Hariri A. R., Sigman M., Bookheimer S. Y. (2004). Neural correlates of facial affect processing in children and adolescents with autism spectrum disorder. J. Am. Acad. Child Adolesc. Psychiatry 43, 481–490 10.1097/00004583-200404000-00015
    1. Wang C. C., Lin H. C., Chan Y. H., Gean P. W., Yang Y. K., Chen P. S. (2013). 5-HT1A-receptor agonist modified amygdala activity and amygdala-associated social behavior in a valproate-induced rat autism model. Int. J. Neuropsychopharmacol. 16, 2027–2039 10.1017/S1461145713000473
    1. Washburn M. S., Moises H. C. (1992). Electrophysiological and morphological properties of rat basolateral amygdaloid neurons in vitro. J. Neurosci. 12, 4066–4079
    1. Windham G. C., Zhang L., Gunier R., Croen L. A., Grether J. K. (2006). Autism spectrum disorders in relation to distribution of hazardous air pollutants in the san francisco bay area. Environ. Health Perspect. 114, 1438–1444 10.1289/ehp.9120
    1. Wood A. (in press). Prenatal exposure to sodium valproate is associated with increased risk of childhood autism and autistic spectrum disorder. Evid. Based Nurs. 10.1136/eb-2013-101422
    1. Zucker R. S., Regehr W. G. (2002). Short-term synaptic plasticity. Annu. Rev. Physiol. 64, 355–405 10.1146/annurev.physiol.64.092501.114547

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