Developmental exposure to non-dioxin-like polychlorinated biphenyls promotes sensory deficits and disrupts dopaminergic and GABAergic signaling in zebrafish
1.Landrigan, P. J. et al. The Lancet Commission on pollution and health. Lancet 391, 462–512 (2018).PubMed
Article
Google Scholar
2.Grandjean, P. & Landrigan, P. J. Neurobehavioural effects of developmental toxicity. Lancet Neurol. 13, 330–338 (2014).CAS
PubMed
PubMed Central
Article
Google Scholar
3.Boix, J., Cauli, O. & Felipo, V. Developmental exposure to polychlorinated biphenyls 52, 138 or 180 affects differentially learning or motor coordination in adult rats mechanisms involved. Neuroscience 167, 994–1003 (2010).CAS
PubMed
Article
Google Scholar
4.Boucher, O., Muckle, G. & Bastien, C. H. Prenatal exposure to polychlorinated biphenyls: a neuropsychologic analysis. Environ. Health Perspect. 117, 7–16 (2009).CAS
PubMed
Article
Google Scholar
5.Ennaceur, S., Gandoura, N. & Driss, M. R. Distribution of polychlorinated biphenyls and organochlorine pesticides in human breast milk from various locations in Tunisia: Levels of contamination, influencing factors, and infant risk assessment. Environ. Res. 108, 86–93 (2008).CAS
PubMed
Article
Google Scholar
6.Lancz, K. et al. Duration of breastfeeding and serum PCB 153 concentrations in children. Environ. Res. 136, 35–39 (2015).CAS
PubMed
Article
Google Scholar
7.Herbstman, J. B. et al. Determinants of prenatal exposure to polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in an urban population. Environ. Health Perspect. 115, 1794–1800 (2007).CAS
PubMed
PubMed Central
Article
Google Scholar
8.Lancz, K. et al. Ratio of cord to maternal serum PCB concentrations in relation to their congener-specific physicochemical properties. Int J. Hyg. Env. Health 218, 91–98 (2015).CAS
Article
Google Scholar
9.Bergonzi, R. et al. Distribution of persistent organochlorine pollutants in maternal and foetal tissues: data from an Italian polluted urban area. Chemosphere 76, 747–754 (2009).CAS
PubMed
Article
Google Scholar
10.Patel, J. F., Hartman, T. J., Sjodin, A., Northstone, K. & Taylor, E. V. Prenatal exposure to polychlorinated biphenyls and fetal growth in British girls. Environ. Int. 116, 116–121 (2018).CAS
PubMed
PubMed Central
Article
Google Scholar
11.Rice, D. & Barone, S. Critical periods of vulnerability for the developing nervous system: Evidence from humans and animal models. Environ. Health Perspect. 108, 511–533 (2000).PubMed
PubMed Central
Google Scholar
12.Trnovec, T. et al. Serum PCB concentrations and cochlear function in 12-year-old children. Environ. Sci. Technol. 44, 2884–2889 (2010).CAS
PubMed
PubMed Central
Article
Google Scholar
13.Jusko, T. A. et al. Prenatal and postnatal serum PCB concentrations and cochlear function in children at 45 months of age. Environ. Health Perspect. 122, 1246–1252 (2014).PubMed
PubMed Central
Article
Google Scholar
14.Ribas-Fitó, N., Sala, M., Kogevinas, M. & Sunyer, J. Polychlorinated biphenyls (PCBs) and neurological development in children: A systematic review. J. Epidemiol. Community Health 55, 537–546 (2001).PubMed
PubMed Central
Article
Google Scholar
15.Walkowiak, J. et al. Environmental exposure to polychlorinated biphenyls and quality of the home environment: effects on psychodevelopment in early childhood. Lancet 358, 1602–1607 (2001).CAS
PubMed
Article
PubMed Central
Google Scholar
16.Chen, Y.-C. J., Guo, Y.-L., Hsu, C.-C. & Rogan, W. J. Cognitive development of Yu-Cheng (‘Oil Disease’) children prenatally exposed to heat-degraded PCBs. JAMA 268, 3213 (1992).CAS
PubMed
Article
Google Scholar
17.Berger, D. F. et al. Hyperactivity and impulsiveness in rats fed diets supplemented with either Aroclor 1248 or PCB-contaminated St. Lawrence river fish. Behav. Brain Res. 126, 1–11 (2001).CAS
PubMed
Article
Google Scholar
18.Johansen, E. B. et al. Behavioral changes following PCB 153 exposure in the spontaneously hypertensive rat—an animal model of attention-deficit/hyperactivity disorder. Behav. Brain Funct. 10, 1–19 (2014).PubMed
PubMed Central
Article
CAS
Google Scholar
19.Crofton, K. M., Ding, D.-L., Padich, R., Taylor, M. & Henderson, D. Hearing loss following exposure during development to polychlorinated biphenyls: a cochlear site of action. Hear. Res. 144, 196–204 (2000).CAS
PubMed
Article
Google Scholar
20.Goldey, E. S., Kehn, L. S., Lau, C., Rehnberg, G. L. & Crofton, K. M. Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Toxicol. Appl. Pharmacol. 135, 77–88 (1995).CAS
PubMed
Article
PubMed Central
Google Scholar
21.Lilienthal, H., Korkalainen, M., Andersson, P. L. & Viluksela, M. Developmental exposure to purity-controlled polychlorinated biphenyl congeners (PCB74 and PCB95) in rats: Effects on brainstem auditory evoked potentials and catalepsy. Toxicology 327, 22–31 (2015).CAS
PubMed
Article
PubMed Central
Google Scholar
22.Lilienthal, H., Heikkinen, P., Andersson, P. L., van der Ven, L. T. M. & Viluksela, M. Auditory effects of developmental exposure to purity-controlled polychlorinated biphenyls (PCB52 and PCB180) in rats. Toxicol. Sci. 122, 100–111 (2011).CAS
PubMed
Article
PubMed Central
Google Scholar
23.Poon, E., Bandara, S. B., Allen, J. B., Sadowski, R. N. & Schantz, S. L. Developmental PCB exposure increases susceptibility to audiogenic seizures in adulthood. Neurotoxicology 46, 117–124 (2015).CAS
PubMed
Article
PubMed Central
Google Scholar
24.Stewart, A. M., Braubach, O., Spitsbergen, J., Gerlai, R. & Kalueff, A. V. Zebrafish models for translational neuroscience research: From tank to bedside. Trends Neurosci. 37, 264–278 (2014).CAS
PubMed
PubMed Central
Article
Google Scholar
25.Fonnum, F. & Mariussen, E. Mechanisms involved in the neurotoxic effects of environmental toxicants such as polychlorinated biphenyls and brominated flame retardants. J. Neurochem. 111, 1327–1347 (2009).CAS
PubMed
Article
PubMed Central
Google Scholar
26.Dervola, K. S. N., Johansen, E. B., Walaas, S. I. & Fonnum, F. Gender-dependent and genotype-sensitive monoaminergic changes induced by polychlorinated biphenyl 153 in the rat brain. Neurotoxicology 50, 38–45 (2015).CAS
PubMed
Article
PubMed Central
Google Scholar
27.Campagna, R. et al. Cerebellum Proteomics addressing the cognitive deficit of rats perinatally exposed to the food-relevant polychlorinated biphenyl 138. Toxicol. Sci. 123, 170–179 (2011).CAS
PubMed
Article
PubMed Central
Google Scholar
28.Enayah, S. H., Vanle, B. C., Fuortes, L. J., Doorn, J. A. & Ludewig, G. PCB95 and PCB153 change dopamine levels and turn-over in PC12 cells. Toxicology 394, 93–101 (2018).CAS
PubMed
Article
PubMed Central
Google Scholar
29.Langeveld, W. T., Meijer, M. & Westerink, R. H. S. Differential effects of 20 non-dioxin-like PCBs on basal and depolarization-evoked intracellular calcium levels in PC12 cells. Toxicol. Sci. 126, 487–496 (2012).CAS
PubMed
Article
PubMed Central
Google Scholar
30.Keil, K. P., Sethi, S. & Lein, P. J. Sex-dependent effects of 2,2′,3,5′,6-pentachlorobiphenyl on dendritic arborization of primary mouse neurons. Toxicol. Sci. 168, 95–109 (2019).CAS
PubMed
Article
Google Scholar
31.Pruitt, D. L., Meserve, L. A. & Bingman, V. P. Reduced growth of intra- and infra-pyramidal mossy fibers is produced by continuous exposure to polychlorinated biphenyl. Toxicology 138, 11–17 (1999).CAS
PubMed
Article
Google Scholar
32.Yang, D. et al. Developmental exposure to polychlorinated biphenyls interferes with experience-dependent dendritic plasticity and ryanodine receptor expression in weanling rats. Environ. Health Perspect. 117, 426–435 (2009).CAS
PubMed
Article
Google Scholar
33.Lein, P. J. et al. Ontogenetic alterations in molecular and structural correlates of dendritic growth after developmental exposure to polychlorinated biphenyls. Environ. Health Perspect. 115, 556–563 (2007).CAS
PubMed
PubMed Central
Article
Google Scholar
34.Tropepe, V. & Sive, H. L. Can zebrafish be used as a model to study the neurodevelopmental causes of autism? Genes. Brain Behav. 2, 268–281 (2003).CAS
Google Scholar
35.Maximino, C. & Herculano, A. M. A review of monoaminergic neuropsychopharmacology in zebrafish. Zebrafish 7, 359–378 (2010).CAS
PubMed
Article
Google Scholar
36.Howe, K. et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 496, 498–503 (2013).CAS
PubMed
PubMed Central
Article
Google Scholar
37.Wolman, M. A. A. et al. A genome-wide screen identifies PAPP-AA-mediated IGFR signaling as a novel regulator of habituation learning. Neuron 85, 1200–1211 (2015).CAS
PubMed
PubMed Central
Article
Google Scholar
38.Meserve Id, J. H. et al. A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1. PLoS Genet. 17, e1008943 (2021).CAS
PubMed
PubMed Central
Article
Google Scholar
39.Voesenek, C. J., Muijres, F. T. & Van Leeuwen, J. L. Biomechanics of swimming in developing larval fish. J. Exp. Biol. 221, jeb149583 (2018).40.Roberts, A. Early functional organization of spinal neurons in developing lower vertebrates. Brain Res. Bull. 53, 585–593 (2000).CAS
PubMed
Article
Google Scholar
41.Liu, Y. C. & Hale, M. E. Local spinal cord circuits and bilateral mauthner cell activity function together to drive alternative startle behaviors. Curr. Biol. 27, 697–704 (2017).CAS
PubMed
Article
Google Scholar
42.Burgess, H. A. & Granato, M. Sensorimotor gating in larval zebrafish. J. Neurosci. 27, 4984–4994 (2007).CAS
PubMed
PubMed Central
Article
Google Scholar
43.Troconis, E. L. et al. Intensity-dependent timing and precision of startle response latency in larval zebrafish. J. Physiol. 595, 265–282 (2017).CAS
PubMed
Article
Google Scholar
44.Smith, N. L. & Kimelman, D. Establishing the body plan: The first 24 h of zebrafish development. The Zebrafish in Biomedical Research: Biology, Husbandry, Diseases, and Research Applications, https://doi.org/10.1016/B978-0-12-812431-4.00007-5 (Elsevier, 2019).45.Meyers, J. R. et al. Lighting up the senses: FM1-43 loading of sensory cells through nonselective ion channels. J. Neurosci. 23, 4054–4065 (2003).CAS
PubMed
PubMed Central
Article
Google Scholar
46.Tabor, K. M. et al. Direct activation of the Mauthner cell by electric field pulses drives ultrarapid escape responses. J. Neurophysiol. 112, 834–844 (2014).PubMed
PubMed Central
Article
Google Scholar
47.Jain, R. A. et al. A forward genetic screen in zebrafish identifies the G-protein-coupled receptor CaSR as a modulator of sensorimotor decision making. Curr. Biol. 28, 1357–1369.e5 (2018).CAS
PubMed
PubMed Central
Article
Google Scholar
48.Mariussen, E. & Fonnum, F. The effect of polychlorinated biphenyls on the high affinity uptake of the neurotransmitters, dopamine, serotonin, glutamate and GABA, into rat brain synaptosomes. Toxicology 159, 11–21 (2001).CAS
PubMed
Article
Google Scholar
49.Wigestrand, M. B., Stenberg, M., Walaas, S. I., Fonnum, F. & Andersson, P. L. Non-dioxin-like PCBs inhibit [3H]WIN-35,428 binding to the dopamine transporter: a structure–activity relationship study. Neurotoxicology 39, 18–24 (2013).CAS
PubMed
Article
Google Scholar
50.Oikonomou, G. et al. The serotonergic raphe promote sleep in zebrafish and mice. Neuron 103, 686–701.e8 (2019).CAS
PubMed
PubMed Central
Article
Google Scholar
51.Fosque, B. F. et al. Labeling of active neural circuits in vivo with designed calcium integrators. Science 347, 755–760 (2015).CAS
PubMed
Article
PubMed Central
Google Scholar
52.Klocke, C. & Lein, P. J. Evidence implicating non-dioxin-like congeners as the key mediators of polychlorinated biphenyl (PCB) developmental neurotoxicity. Int. J. Mol. Sci. 21, 1013 (2020).53.Puel JL, Pujol R, Ladrech S, Eybalin M. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid electrophysiological and neurotoxic effects in the guinea-pig cochlea. Neuroscience. Nat. Rev. Neurosci. 45, 63–72. https://doi.org/10.1016/0306-4522(91)90103-u (1991).CAS
Article
Google Scholar
54.Sebe JY, et al. Ca2+-Permeable AMPARs Mediate Glutamatergic Transmission and Excitotoxic Damage at the Hair Cell Ribbon Synapse. J Neurosci. 37, 6162–6175. https://doi.org/10.1523/JNEUROSCI.3644-16.2017 (2017).CAS
Article
PubMed
PubMed Central
Google Scholar
55.Coleman, M. Axon degeneration mechanisms: commonality amid diversity. Nat. Rev. Neurosci. 6, 889–898 (2005).CAS
PubMed
Article
PubMed Central
Google Scholar
56.Lacoste, A. M. B. et al. A convergent and essential interneuron pathway for mauthner-cell-mediated escapes. Curr. Biol. 25, 1526–1534 (2015).CAS
PubMed
PubMed Central
Article
Google Scholar
57.Kang, K. S., Wilson, M. R., Hayashi, T., Chang, C. C. & Trosko, J. E. Inhibition of gap junctional intercellular communication in normal human breast epithelial cells after treatment with pesticides, PCBs, and PBBs, alone or in mixtures. Environ. Health Perspect. 104, 192–200 (1996).CAS
PubMed
PubMed Central
Google Scholar
58.Bager, Y., Lindebro, M. C., Martel, P., Chaumontet, C. & Wärngård, L. Altered function, localization and phosphorylation of gap junctions in rat liver epithelial, IAR 20, cells after treatment with PCBs or TCDD. Environ. Toxicol. Pharmacol. 3, 257–266 (1997).CAS
PubMed
Article
PubMed Central
Google Scholar
59.Machala, M. et al. Inhibition of gap junctional intercellular communication by noncoplanar polychlorinated biphenyls: Inhibitory potencies and screening for potential mode(s) of action. Toxicol. Sci. 76, 187–195 (2003).Article
CAS
Google Scholar
60.Nyffeler, J. et al. A structure–activity relationship linking non-planar PCBs to functional deficits of neural crest cells: new roles for connexins. Arch. Toxicol. 92, 1225–1247 (2018).CAS
PubMed
Article
PubMed Central
Google Scholar
61.Kang, K.-S., Park, J.-E., Ryu, D.-Y. & Lee, Y.-S. Effects and neuro-toxic mechanisms of 2,2’,4,4’,5,5’-hexachlorobiphenyl and endosulfan in neuronal stem cells. J. Vet. Med. Sci. 63, 1183–1190 (2001).CAS
PubMed
Article
PubMed Central
Google Scholar
62.Aluru, N., Krick, K. S., Mcdonald, A. M. & Karchner, S. I. Developmental exposure to PCB153 (2,2′,4,4′,5,5′-hexachlorobiphenyl) alters circadian rhythms and the expression of clock and metabolic genes. Toxicol. Sci. 173, 41–52 (2020).PubMed
Article
PubMed Central
Google Scholar
63.GEO Accession viewer. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM2450663 (2017).64.Serrano-Velez, J. L. et al. Abundance of gap junctions at glutamatergic mixed synapses in adult Mosquitofish spinal cord neurons. Front. Neural Circuits 8, 66 (2014).PubMed
PubMed Central
Article
Google Scholar
65.Tanaka, Y. et al. Aroclor 1254 and BDE-47 inhibit dopaminergic function manifesting as changes in locomotion behaviors in zebrafish embryos. Chemosphere 193, 1207–1215 (2018).CAS
PubMed
Article
PubMed Central
Google Scholar
66.Rungta, R. L. et al. The cellular mechanisms of neuronal swelling underlying cytotoxic edema. Cell 161, 610–621 (2015).CAS
PubMed
Article
PubMed Central
Google Scholar
67.Cesetti, T., Ciccolini, F. & Li, Y. GABA not only a neurotransmitter: osmotic regulation by GABA AR signaling. Front Cell Neurosci. 6, 3 (2012).68.Fernandes, E. C. A. et al. Potentiation of the human GABAA receptor as a novel mode of action of lower-chlorinated non-dioxin-like PCBs. Environ. Sci. Technol. 44, 2864–2869 (2010).CAS
PubMed
Article
PubMed Central
Google Scholar
69.Magnusson, O., Mohringe, B., Thorell, G. & Lake-Bakaar, D. M. Effects of the dopamine D2 selective receptor antagonist remoxipride on dopamine turnover in the rat brain after acute and repeated administration. Pharmacol. Toxicol. 60, 368–373 (1987).CAS
PubMed
Article
PubMed Central
Google Scholar
70.Seegal, R. F., Bush, B. & Shain, W. Lightly chlorinated ortho-substituted PCB congeners decrease dopamine in nonhuman primate brain and in tissue culture. Toxicol. Appl. Pharmacol. 106, 136–144 (1990).CAS
PubMed
Article
Google Scholar
71.Toro, C. et al. Dopamine modulates the activity of sensory hair cells. J. Neurosci. 35, 16494–16503 (2015).CAS
PubMed
PubMed Central
Article
Google Scholar
72.Gafni, J., Wong, P. W. & Pessah, I. N. Non-coplanar 2,2’,3,5’,6-pentachlorobiphenyl (PCB 95) amplifies ionotropic glutamate receptor signaling in embryonic cerebellar granule neurons by a mechanism involving ryanodine receptors. Toxicol. Sci. 77, 72–82 (2003).PubMed
Article
CAS
Google Scholar
73.Ta, T. A., Feng, W., Molinski, T. F. & Pessah, I. N. Hydroxylated xestospongins block inositol-1,4,5-trisphosphate-induced Ca2+ release and sensitize Ca2+-induced Ca2+ release mediated by ryanodine receptors. Mol. Pharmacol. 69, 532–538 (2006).CAS
PubMed
Article
Google Scholar
74.Brunelli, L. et al. Insight into the neuroproteomics effects of the food-contaminant non-dioxin like polychlorinated biphenyls. J. Proteomics. 75, 2417–2430 (2012).CAS
PubMed
Article
Google Scholar
75.McCormick, M. I., Fakan, E. & Allan, B. J. M. Behavioural measures determine survivorship within the hierarchy of whole-organism phenotypic traits. Funct. Ecol. 32, 958–969 (2018).Article
Google Scholar
76.Lai, Z. et al. Residual distribution and risk assessment of polychlorinated biphenyls in surface sediments of the Pearl River Delta, South China. Bull. Environ. Contam. Toxicol. 95, 37–44 (2015).CAS
PubMed
PubMed Central
Article
Google Scholar
77.Gräns, J. et al. Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor. Aquat. Toxicol. 159, 198–207 (2015).PubMed
Article
CAS
Google Scholar
78.Hudspeth, A. The cellular basis of hearing: the biophysics of hair cells. Science 230, 745–752 (1985).CAS
PubMed
Article
Google Scholar
79.Nakayama, H. Common sensory inputs and differential excitability of segmentally homologous reticulospinal neurons in the hindbrain. J. Neurosci. 24, 3199–3209 (2004).CAS
PubMed
PubMed Central
Article
Google Scholar
80.Pujol-Martí, J. & López-Schier, H. Developmental and architectural principles of the lateral-line neural map. Front. Neural Circuits 7, 47 (2013).PubMed
PubMed Central
Article
Google Scholar
81.Panlilio, J. M., Jones, I. T., Salanga, M. C., Aluru, N. & Hahn, M. E. Developmental exposure to domoic acid disrupts startle response behavior and circuitry in zebrafish. Toxicol. Sci. 182, 310–326 (2021).PubMed
Article
Google Scholar
82.Park, H.-C., Shin, J., Roberts, R. K. & Appel, B. An olig2 reporter gene marks oligodendrocyte precursors in the postembryonic spinal cord of zebrafish. Dev. Dyn. 236, 3402–3407 (2007).CAS
PubMed
Article
Google Scholar
83.Shin, J., Park, H.-C., Topczewska, J. M., Mawdsley, D. J. & Appel, B. Neural cell fate analysis in zebrafish using olig2 BAC transgenics. Methods Cell Sci. 25, 7–14 (2003).CAS
PubMed
Article
Google Scholar
84.Takada, N., Kucenas, S. & Appel, B. Sox10 is necessary for oligodendrocyte survival following axon wrapping. Glia 58, 996–1006 (2010).PubMed
PubMed Central
Google Scholar
85.Almeida, R. G., Czopka, T., Ffrench-Constant, C. & Lyons, D. A. Individual axons regulate the myelinating potential of single oligodendrocytes in vivo. Development 138, 4443–4450 (2011).CAS
PubMed
PubMed Central
Article
Google Scholar
86.Kimmel, C. B. Patterning the brain of the zebrafish embryo. Annu. Rev. Neurosci. 16, 707–732 (1993).CAS
PubMed
Article
Google Scholar
87.Ranasinghe, P. et al. Embryonic exposure to 2,2′,3,5′,6-pentachlorobiphenyl (PCB-95) causes developmental malformations in zebrafish. Environ. Toxicol. Chem. 39, 162–170 (2019).Article
CAS
Google Scholar
88.Jönsson, M. E., Kubota, A., Timme-Laragy, A. R., Woodin, B. & Stegeman, J. J. Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish. Toxicol. Appl. Pharmacol. 265, 166–174 (2012).PubMed
PubMed Central
Article
CAS
Google Scholar
89.Panlilio, J. M., Aluru, N. & Hahn, M. E. Developmental neurotoxicity of the harmful algal bloom toxin domoic acid: Cellular and molecular mechanisms underlying altered behavior in the zebrafish model. Environ. Health Perspect. 128, 117002 (2020).CAS
PubMed
PubMed Central
Article
Google Scholar
90.Inoue, D. & Wittbrodt, J. One for all-a highly efficient and versatile method for fluorescent immunostaining in fish embryos. PLoS One 6, 1–7 (2011).Article
CAS
Google Scholar
91.Schindelin, J. et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).CAS
Article
Google Scholar
92.Edwards KA, Hoppa MB, Bosco G. The Photoconvertible Fluorescent Probe, CaMPARI, Labels Active Neurons in Freely-Moving Intact Adult Fruit Flies. Front Neural Circuits. 14, 22 https://doi.org/10.3389/fncir.2020.00022. (2020).CAS
Article
PubMed
PubMed Central
Google Scholar
93.Zhao, Y. et al. Rare earth elements lanthanum and praseodymium adversely affect neural and cardiovascular development in zebrafish (Danio rerio). Environ. Sci. Technol. https://doi.org/10.1021/acs.est.0c06632 (2020).94.Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67, 1–48 (2015).Article
Google Scholar More