Neurotox Res (2016) 30:122–130 DOI 10.1007/s12640-016-9638-0

ABSTRACT

Abstract from the 2015 Neurotoxicity Society Meeting: Poster Abstracts

Ó Springer Science+Business Media New York 2016

Fragment C Domain of Tetanus Toxin Mitigates Methamphetamine Neurotoxicity in Mice 1,2

1,2

Compensatory Changes to Methamphetamine-Induced Dopaminergic Degeneration

2,3

Liliana Mendieta* , Noelia Granado* , Jose´ Aguilera , Yousef Tizabi4, Rosario Moratalla1,2 1

Instituto Cajal, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Madrid, Spain; 2Centro de Investigacio´n Biome´dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain; 3 Institut de Neurocie`ncies and Departament de Bioquı´mica i de Biologia Molecular, Facultat de Medicina, Universitat Auto`noma de Barcelona (UAB), Cerdanyola del Valle`s, Spain; 4Department of Pharmacology, Howard University College of Medicine, Washington, DC, USA The C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) is a peptide fragment with demonstrated in vitro and in vivo neuroprotective effects against striatal dopaminergic damage induced by MPP+ (1-methyl-4-phenylpyridinium) and 6-hydoxydopamine, suggesting its possible therapeutic potential in Parkinson disease (PD). Methamphetamine (METH), a widely abused psychostimulant, has selective dopaminergic neurotoxicity. This study was undertaken to determine whether Hc-TeTx might also protect against METH-induced neurotoxicity. For this purpose, we treated groups of mice with a toxic regimen of METH (4 mg/kg, 3 consecutive i.p. injections, each 3 h apart), followed by 3 injections of 40 lg/kg of Hc-TeTx into grastrocnemius muscle at 1 h, 24 h, and 48 h post-METH treatment. We found that Hc-TeTx protects against dopaminergic markers loss (TH and DAT) and neurodegeneration (by amino-cupric silver staining) induced by METH in striatum of mice. nNOS, microgliosis and astrocytosis into striatum, were also examined. Furthermore, we found Hc-TeTx prevented the increase of nNOS, but did not affect the inflammatory response by METH. All these improvements could be reflected on motor behavior at one and three days after METH treatment. Regardless of Hc-TeTx did not cause significant protection on number of neurons (TH positive) which decreased by METH in substantia nigra, it causes protective effects in dopaminergic terminals of striatum, which was clearly demonstrated. In summary, we report that Hc-TeTx that reduces the neurotoxic effects induced by METH in striatal terminals and that this reduction is functionally reflected by locomotor improvement. Thus, Hc-TeTx fragment may represent a potent therapeutic drug for METH-abusers.

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Noelia Granado1,2*, Sara Ares-Santos1,2*, Yousef Tizabi3 and Rosario Moratalla1,2,# 1

Instituto Cajal, Consejo Superior de Investigaciones Cientı´ficas, CSIC, Madrid, Spain; 2CIBERNED, ISCIII, Madrid, Spain; 3 Department of Pharmacology, Howard University College of Medicine, Washington, DC, USA *equal contribution Methamphetamine, a psychostimulant drug with high abuse potential, may double the risk of developing Parkinson’s disease (PD). Studies in animals have shown that this drug produces persistent dopaminergic neurotoxicity in the nigrostriatal pathway. Since some compensatory changes to dopaminergic damage have been described after treatment with other dopaminergic neurotoxins like MPTP (1-methyl-4-phenyl1,2,3,6-tetrahydropyridine), 6-OHDA (6-hydoxydopamine), or MDMA (3,4-methylenedioxy-methamphetamine), we were curious to determine whether compensatory responses may also occur following METH treatment. Three established regimens of METH: a single high dose (1 9 30 mg/kg), multiple lower doses (3 9 5 mg/kg) or (3 9 10 mg/kg) of METH with known neurotoxicity were applied. As expected, significant degeneration of striatal dopaminergic fibers was observed a day later in all cases. The damage was highest with the 3 9 10 mg/kg dose followed by 3 9 5 mg/kg, which was followed by 1 9 30 mg/kg. Moreover, regimen-dependent partial recovery was also noticed after 3 days. Interestingly, the recovery was also highest in 3 9 10 mg/kg, followed by 3 9 5 mg/kg, followed by 1 9 30 mg/kg. These partial recoveries were associated with a similar pattern of increase in tyrosine hydroxylase immunoreactivity and some fiber sprouting as evidenced by Gap-43 positive fibers. Additionally, METH treatment resulted in an increase in Iba-1 staining (reflective of microglia activation) after one day that was fully recovered by day 3. However, the increase in GFAP staining (reflective of astroglia activation) that was observed after one day was further increased by day 3. These results confirm that partial striatal recovery occurs following METH treatment and that astro- and microglia may have some role in this compensatory process. Nonetheless, further studies on long-term effects of METH and recovery mechanism(s) are warranted. This work was supported by grants from the Spanish Ministry of Sanidad, Servicios Sociales e Igualdad, PNSD #2012/071, Spanish Ministry of

Neurotox Res (2016) 30:122–130 Economı´a y Competitividad grant # BFU2010-20664, CIBERNED #CB06/05/0055 and Comunidad de Madrid ref S2010/BMD-2336 to RM. NG received a research contract NEUROSTEM-CM S2010/BMD2336; SAS a JAE pre-doctoral fellowship.

Protein Kinase Cd Gene Depletion Inhibits Trimethyltin-Induced Apoptotic Neurodegeneration in the Hippocampus of Mice Thu-Hien Thi Tu1, Yunsung Nam1, Hai-Quyen Tran1, Ji Hoon Jeong2, Eun-Joo Shin1, Hyoung-Chun Kim1,* 1

Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea; 2Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul, 156-756, Republic of Korea We investigated whether protein kinase C (PKC) is involved in trimethyltin (TMT)-induced neurotoxicity. Treatment with TMT resulted in a significant increase of PKCd out of PKC isozymes (i.e., a, bI, bII, d, and 1) in the hippocampus of wild-type (WT) mice. Consistently, treatment with TMT resulted in significant increases in cleaved PKCd expression. Genetic or pharmacological inhibition (PKCd knockout or rottlerin) was less susceptible to TMT-induced seizures than WT mice. TMT treatment increased glutathione oxidation, lipid peroxidation, protein oxidation, and levels of reactive oxygen species. These effects were more pronounced in the WT mice than in PKCd knockout mice. In addition, the ability of TMT to induce nuclear translocation of Nrf2, Nrf2 DNA binding activity, and upregulation of c-glutamyl cysteine ligase was significantly increased in the PKCd knockout mice and rottlerin-treated WT mice. Furthermore, neuronal degeneration (as shown by nuclear chromatin clumping and TUNEL-staining) in WT mice was most pronounced 2 d after TMT. At the same time, TMT-induced inhibition of phosphoinositol 3-kinase (PI3K)/Akt signaling was evident, thereby decreasing phospho-Bad, expression of Bcl-xL and Bcl-2, and the interaction between phospho-Bad and 14-3-3 protein, and increasing Bax expression and caspase-3 cleavage were observed. Rottlerin or PKCd knockout significantly protected these changes in anti- and proapoptotic factors. Importantly, the protective effects (i.e., Nrf-2-dependent glutathione induction and pro-survival phenomenon) of rotterlin were counteracted by the PI3K inhibitor LY294002. Therefore, our results suggest that down-regulation of PKCd and upregulations of Nrf2-dependent glutathione defense mechanism and PI3 K/Akt signaling are critical for attenuating TMT neurotoxicity [TTH Tu, Y Nam, and HQ Tran are involved in BK21 PLUS program, National Research Foundation of Korea. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (#NRF-2013R1A1A2060894 and #NRF2013R1A1A1007378), Republic of Korea]

Genetic Inhibition of p47 Phox Attenuates Methamphetamine-Induced Dopaminergic Neurotoxicity Duy-Khanh Dang1, Yunsung Nam1, The-Vinh Tran1, Choon-Gon Jang2, Eun-Joo Shin1, Hyoung-Chun Kim1,* 1 Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea; 2Department of Pharmacology, School

123 of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea Accumulating evidences suggest that oxidative stress mediates MA-induced dopaminergic neurotoxicity. Since recent reports emphasized emerging role for NADPH oxidases as a source of ROS, we investigated whether involvement of NADPH oxidase in MAinduced proapoptosis (i.e., TUNEL-positive cells), microglial activation, and dopaminergic impairment in the striatum of the mice. As the p47 phox subunit of NADPH oxidase acts as a connector between the components of the membrane and the cytoplasm, we performed intracerebroventricular infusion of p47 phox antisense oligonucleotides (p47 phox ASO) before a single injection of MA (35 mg/kg, i.p). Treatment with MA resulted in a significant increase in TUNEL-positive cells in the striatum of Taconic ICR mice. Application of p47phox ASO significantly attenuated the increase in TUNEL-positive populations induced by MA. Furthermore, p47phox ASO significantly protected reactive microgliosis (as labeled by Iba-1) and reductions in tyrosine hydroxylase-positive immunoreactivity and dopamine level in the striatum of Taconic ICR mice. Consistently, reactive microgliosis and dopaminergic loss induced by MA were less pronounced in p47 phox knockout mice than in wild-type mice. Our results suggest that p47 phox mediates acute dopaminergic neurotoxicity induced by MA [DK Dang, TV Tran, and Y Nam are involved in BK21 PLUS program, National Research Foundation of Korea. This research was supported by a grant (14182MFDS979) from Ministry of Food and Drug Safety in 2014, Republic of Korea].

Protective Effects of Phosphatidylcholine on Oxaliplatin-Induced Neuropathy in Rats Ji Hoon Jeong1, Yoon Hee Chung2, Sung Tae Kim3, Ho Sung Lee1, Yunsung Nam4, Eun-Joo Shin4 and Hyoung-Chun Kim4 1 Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul, 156-756, Republic of Korea; 2Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea; 3Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea; 4Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea

Oxaliplatin is a third-generation platinum-based antineoplastic agent that is widely used in the treatment of colorectal cancer. The present study was designed to investigate the therapeutic potential of phosphatidylcholine (PC) on oxaliplatin-induced peripheral neuropathy. In this study, male Sprague–Dawley rats were randomly divided into three groups: the control group, the oxaliplatin group, and the oxaliplatin + PC group. Here we found that PC showed a protective effect on mechanical and thermal hyperalgesia induced by oxaliplatin in paw pressure and tail-flick tests. Additional experiments were performed to evaluate the effect of PC on the degree of oxidative stress in sciatic nerves by measuring the level of malondialdehyde (MDA), total glutathione (GSH), glutathione peroxidase (GPx) activity, and superoxide dismutase (SOD) activity. The results indicate that PC administration attenuated oxidative stress by increasing antioxidant levels. In the histopathological evaluation, the PC administrated group maintained normal morphologic appearance of sciatic nerves, similar to the control group. In spinal cords, however, no significant difference between the oxaliplatin-alone group and the oxaliplatin + PC group was observed. In immunohistochemical evaluation, PC administration ameliorated oxaliplatin-induced

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124 microglial activation. In conclusion, it is suggested that PC has a therapeutic potential against oxaliplatin-induced peripheral neuropathy due to its antioxidant property and modulation of microglial activities.

Neuropsychotoxic Effect of Paramethoxymethamphetamine (PMMA) in Mice Yunsung Nam1, Eun-Joo Shin1, Duy-Khanh Dang1, Hai-Quyen Tran1, Yong Sup Lee2, Choon-Gon Jang3, Hyoung-Chun Kim1,* 1 Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea; 2Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea; 3Department of Pharmacology, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea

PMMA is para-ring-substituted amphetamine derivatives sold as illegal psychotropic drug of abuse. Although severe intoxication and death of PMMA user have been described worldwide for many years, a very limited information is available up to now. In order to better elucidate neurotoxicity induced by PMMA, we have evaluated whether PMMA affects neurobehavioural and neurochemical parameters in mice. A single injection of PMMA (40 or 80 mg/kg, i.p.) produced a significant hyperthermia, dopaminergic loss, behavioural and memory impairments (i.e., Y-maze test). In addition, multiple doses of PMMA exhibited hyperthermia, serotonergic loss, behavioural and memory impairments (i.e., Y-maze test). Although neuropsychotoxicological profiles mediated by PMMA are less pronounced than those mediated by MA, the neuropsychotoxic potentials were, at least in part, comparable to those mediated by other amphetamine analogs. Therefore, further studies for understanding on the precise pharmacological mechanism are required [Y Nam, DK Dang, and HQ Tran are involved in BK21 PLUS program, National Research Foundation of Korea. This research was supported by a grant (14182MFDS979) from Ministry of Food and Drug Safety in 2014, Republic of Korea].

Astaxanthin Provides a Neuroprotection Against Methamphetamine-Induced Neurotoxicity Yunsung Nam, Duy-Khanh Dang, Huynh-Nhu Mai, Eun-Joo Shin, Hyoung-Chun Kim* Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea It has been recognized that Astaxanthin (ASX), a natural carotenoid antioxidant, exerts various pharmacological activities. Since recent study reported that Nrf2-mediated antioxidant effects play a role in pharmacological action of ASX, we examined the effect of ASX on the methamphetamine (MA)-induced dopaminergic toxicity. In order to enhance bioavailability of ASX, it was applied as a form of liposome-encapsulated ASX (L-ASX). Treatment with L-ASX resulted in

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Neurotox Res (2016) 30:122–130 a significant protection against MA-induced dopaminergic neurotoxicity. Especially, Nrf2-dependent system was positively modulated by L-ASX, leading to recover glutathione (GSH) synthesis and glutathione peroxidase-1 (GPx-1) expression. To confirm L-ASX on GPx-1 induction, GPx-1 overexpression transgenic (GPx1-Tg) mice were applied. GPx1-Tg mice showed equipotent protections as compared to L-ASX-treated nontrangenic (Non-Tg) mice. However, L-ASX did not alter neuroprotection as shown in GPx1-Tg mice against MA insult, suggesting that GPx-1 gene is essential for protective mechanism mediated by ASX. These results suggest that GPx1 is important for producing neuroprotective effects of L-ASX, and that L-ASX can be a potential candidate for pharmacological intervention in response to MA-induced dopaminergic neurotoxicity via modulations in GPx-1 gene and Nrf2-dependent GSH-synthesis [Y Nam, DK Dang, and HN Mai are involved in BK21 PLUS program, National Research Foundation of Korea].

Role of Glutathione Peroxidase in Cocaine-Induced Neurotoxicity in Mice Yoon Hee Chung1, Thuy-Ty Lan Nguyen2, Huynh-Nhu Mai2, Yunsung Nam2, Dae-Joong Kim3, Choon-Gon Jang4, Eun-Joo Shin2, Hyoung-Chun Kim2,* 1

Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea; 2 Neuropsychopharmacology and Toxicology Program, College of Pharmacy, and 3Department of Anatomy and Cell Biology, Medical School, Kangwon National University, Chunchon 200-701, Republic of Korea; 4Department of Pharmacology, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea Many evidence indicated that oxidative stress contributes to central and peripheral toxicities induced by cocaine. To better elucidate underlying mechanism on neurotoxicity induced by cocaine, the present study was performed. As the glutathione peroxidase is a major one out of enzymatic antioxidants, and glutathione peroxidase1 (GPx1) is a main one out of GPx subtypes, it was employed both GPx1 knockout mice (GPx1 KO) and GPx1 overexpressing transgenic mice (GPx1 TG) in this study. A single high dose or multiple high doses of cocaine showed early increase in oxidative damages (i.e., reactive oxygen species, lipid peroxidation and protein carbonyl) in the brain of wild-type mice (WT) or non-TG mice (non-TG). This phenomenon was more pronounced in GPx1 KO than WT and was less pronounced in GPx1 TG than non-TG. Cocaine-induced decreased in astrogliosis as labeled by GFAP immunoreactivity was noted in GPx1 KO, while increase in reactive microgliosis as labeled by Iba-1 immunoreactivity was observed in GPx1 KO. However, other species did not show any specific changes in these immunoreactivities of GFAP and IBa-1. In addition, these cocaine-induced neuroexcitotoxicities were inhibited by AG490, but not by pyrrolidone dithiocarbamate (PDTC), suggesting that JAK2/STAT3 signaling, but not NFkB signaling, is involved in cocaine-induced neuroexcitotoxicity. Taken together, GPx1 gene is a potential protectant against neurotoxicity induced by cocaine [HN Mai and Y Nam are involved in BK21 PLUS program, National Research Foundation of Korea. This research was supported by a grant (14182MFDS979) from Ministry of Food and Drug Safety in 2014, Republic of Korea].

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Glutathione Peroxidase1 Plays a Protective Role on Cocaine-Induced Convulsion And Kindling in Mice Thuy-Ty Lan Nguyen1, Huynh-Nhu Mai1, Yunsung Nam1, Choon-Gon Jang2, Eun-Joo Shin1, Hyoung-Chun Kim1,* 1

Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea; 2Department of Pharmacology, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea Oxidative stress and impaired antioxidant system are strongly implicated in a number of neurological conditions. Many studies have demonstrated that oxidative stress might be involved in the pathophysiology of neurodegenerative disease including convulsive disorders. Although glutathione peroxidase (GPx) is the most important hydrogen peroxide scavenger enzyme in brain, the etiologic role of GPx in convulsive conditions still remain to be established. In the previous report, cocaine-treated mice showed significant increases of oxidative stress markers (reactive oxygen species, lipid peroxidation, and protein oxidation). The present study was designed to investigate the potential influence of GPx1 in response to cocaineinduced convulsion and kindling in mice. A single dose of cocaine (60 mg/kg, i.p.) revealed more severe convulsion in the GPx1 gene knockout mice (as shown by increases in convulsive ratio and convulsive score, while decrease in recovery ratio) than wild-type mice. On the other hand, we observed that GPx1 overexpressing transgenic (GPx1-Tg) mice induced less severe convulsion compared to genetically GPx1-non-Tg mice. Consistently, this finding from cocaineinduced convulsion paralleled that from cocaine-induced kindling in our experimental condition. Our results suggest that GPx1 activation is, in part, a defensive factor against convulsive behaviors induced by cocaine [HN Mai and Y Nam are involved in BK21 PLUS program, National Research Foundation of Korea. This research was supported by a grant (14182MFDS979) from Ministry of Food and Drug Safety in 2014, Republic of Korea].

Ceruloplasmin Plays a Protective Role in Kainate-Induced Excitotoxicity via Maintaining Iron Homeostasis and Antioxidant Defense System The-Vinh Tran1, Eun-Joo Shin1, Ji Hoon Jeong2, Wei-Yi Ong3, Dae-Joong Kim4, Myung-Bok Wie5, Eon Sub Park6, Yoon Hee Chung7, Yunsung Nam1, Hwa-Jung Kim8, Chun Kee Chung9, Hyoung-Chun Kim1,* 1

Neuropsychopharmacology and Toxicology Program, College of Pharmacy, 4Department of Anatomy and Cell Biology, Medical School, and 5Department of Veterinary Medicine, Kangwon National University, Chunchon 200-701, Republic of Korea; 2Department of Pharmacology, 6Department of Pathology, and 7Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea. 3Department of Anatomy, National University of Singapore, Singapore 119260, Singapore; 8College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea; 9Department of Neurosurgery, Seoul National University College of Medicine, Seoul National University Hospital, Seoul 110-799, Republic of Korea To determine the role of ceruloplasmin (Cp) in epileptic seizures, we used a kainate (KA) seizure animal model and examined hippocampal

125 samples from epileptic patients. Treatment with KA resulted in a time-dependent decrease in Cp protein expression in the hippocampus of rats. Cp-positive cells were co-localized with neurons or reactive astrocytes in KA-treated rats and epileptic patient samples. KA-induced seizures, initial oxidative stress (i.e., hydroxyl radical formation, lipid peroxidation, protein oxidation, and synaptosomal reactive oxygen species), altered iron status (increasing Fe2+ accumulation and L-ferritin-positive reactive microglial cells, while decreasing H-ferritin-positive neurons), impaired glutathione homeostasis and neurodegeneration (i.e., Fluoro-Nissl and Fluoro-jade B staining analyses) were more pronounced in Cp antisense oligonucleotide (ASO) than Cp sense oligonucleotide-treated rats. Consistently, Cp ASO facilitated KA-induced LDH release, Fe2+ accumulation, and GSH loss in neuron-rich and mixed cultures; however, Cp ASO did not alter KA-induced LDH release, Fe2+ accumulation in the astroglial culture, but did facilitate impairments in glutathione homeostasis in the same culture. Importantly, treatment with human Cp resulted in a significant attenuation against these neurotoxicities induced by Cp ASO. Our results suggest that Cpmediated neuroprotection occurs via the inhibition of seizure-associated oxidative damage (including impairments in glutathione homeostasis), Fe2+ accumulation, and alterations in ferritin immunoreactivity, and that interactive modulation between neurons and glia is important for Cp up-regulation in attenuation of epileptic damage in both animals and humans [TV Tran and Y Nam are involved in BK21 PLUS program, National Research Foundation of Korea. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (#NRF2013R1A1A2060894 and #NRF-2013R1A1A1007378), Republic of Korea]

Behavioral Changes and Dopaminergic Neurodegeneration Following Metabolic Impairment in Rats Justine Renaud1, Carole Lavoie2, Karine Dufresne1, Giulia Costa3, Nicola Simola3, Micaela Morelli3, Maria-Grazia Martinoli1 Cellular Neurobiology and 2Metabolism and Diabetes Laboratories, Dept. of Medical Biology, U. Que´bec, Trois-Rivie`res, Canada; 3Dept. Biomedical Sciences, U. Cagliari, Cagliari, Italy

1

Hyperglycemia is recognized as a cause of oxidative stress also reported to be harmful for the nervous system. Accumulating epidemiological evidence displays a relationship between diabetes and neurodegenerative disorders, including Alzheimer’s disease and Parkinson’s disease. We have recently shown that dopaminergic neurons in culture are vulnerable to high—although physiologically sustainable—levels of glucose by exhibiting increased cell death, expression of pro-apoptotic markers, and production of reactive oxygen/nitrogen species. In this context, this study aimed to characterize dopaminergic neurodegeneration in a streptozotocinnicotinamide rat model of chronic hyperglycemia and metabolic impairment. Male Sprague–Dawley rats were injected with nicotinamide followed by streptozotocin, and metabolic parameters were monitored for five months. During the last three weeks, ultrasonic vocalizations and social behavior were recorded in pairs of unacquainted rats in a novel environment. At 5 months, rats were sacrificed, and brain and intestinal tissues were harvested for immunoblotting or immunohistochemical analyses. Metabolic measurements show that streptozotocin-nicotinamide-injected rats were hyperglycemic and metabolically impaired, as demonstrated by

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126 modulation of weight, glucose tolerance, plasma insulin levels, polyuria, polydipsia, and polyphagia, as compared with control rats. In addition, ultrasonic vocalizations, which are at least partly regulated by dopaminergic pathways, were disrupted in pairs of hyperglycemic rats compared to pairs of control rats. Indeed, the mean number of 22 kHz vocalizations, associated with aversive stimulation, was significantly enhanced while that of 50 kHz vocalizations, related with appetitive situations, was reduced in hyperglycemic rats. Freezing and aggressive incidents were also notably increased in these rats compared to control animals. Further experiments were performed to detect dopaminergic neurodegeneration in hyperglycemic rat midbrains and mesenteric plexus, where up to 50 % of total dopamine in the body resides. Preliminary results show a definite modulation of dopaminergic function and neurodegeneration in hyperglycemic rats. Altogether, our data evoke a correlation between hyperglycemia and dopaminergic neurodegeneration, which provides new insight on the higher occurrence of PD in diabetic patients.

Role of Mutations N370S and L444P of the GBA Gene in Autophagy and Its Involvement in Parkinson’s Disease P. Garcı´a-Sanz1,2, I. Espadas1,2, L. Orgaz1,2, G. Bueno1,2, E. Rodrı´guez-Traver1,2, J. Kulisevsky3,2, R.A. Gonza´lez-Polo4,2, J.M. Fuentes4,2, A. Gutierrez2,5, C. Vicario1,2 and R. Moratalla1,2 1 Instituto Cajal, CSIC, Madrid; 2CIBERNED, Madrid; 3Servicio de Neurologı´a, Hospital de la Santa Creu i Sant Pau, Universitat Auto´noma de Barcelona, Institut d’Investigacions Biosanita´ries Sant Pau, Barcelona; 4Departamento de Bioquı´mica y Biologı´a Molecular y Gene´tica, F. Enfermerı´a y T.O., Universidad de Extremadura, Ca´ceres; 5Departamento de Biologı´a Celular, Gene´tica y Fisiologı´a, Facultad de Ciencias, Universidad de Ma´laga, Ma´laga

There is now a well-established clinical association between mutations in the glucocerebrosidase gene (GBA) that encodes the lysosomal hydrolase glucocerebrosidase (GCase) and the development of Parkinson’s disease (PD) in the general population. The mechanisms by which GBA mutations predispose to neurodegeneration remain unclear. To study the role of heterozygous GBA mutations in the pathology of PD, we generated fibroblast lines from skin biopsies of four PD patients with heterozygous GBA mutation carriers (N370S and L444P) and three controls. Both GBA mutations demonstrated a significantly reduced level of GCase protein and enzyme activity and retention of GCase isoforms within the endoplasmic reticulum (ER). We have observed that this ER retention overload activates ER stress pathway leading to the activation of signaling events known as the unfolded protein response (UPR), resulting in increased autopaghy demand. Our results also showed that this was associated with enhanced expression of the lysosomal markers Lamp1 and Lysotracker and a significant accumulation of lamellar bodies (as pathology maker for lysosomal storage disorder) detected by electron microscopy. In addition, using flow cytometry, we demonstrated that GBA mutations not only were associated with evidence of ER but also oxidative stress. Related to this, these mutations showed significant decline in mitochondria membrane potential and Golgi apparatus (GA) fragmentation. To sum up, our results demonstrated that N370S and L444P induce

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Neurotox Res (2016) 30:122–130 autophagic flux and carry out impairment in lysosomal function due to an accumulation of misfolded proteins in ER.

Impaired Associative and Spatial Learning Related with Changes in Hippocampal CA3-CA1 Synaptic Plasticity in Drd22/2 Mice Isabel Espadas1,2, Oskar Ortiz1,2, *,Patricia Garcı´a-Sanz1,2, Jose´ Marı´a Delgado-Garcı´a3, Agne`s Gruart3 and Rosario Moratalla1,2*,# 1

Instituto Cajal, CSIC, Madrid, 28002 and 2CIBERNED, ISCIII, Madrid-28002, Spain, 3Divisio´n de Neurociencias, Univ. Pablo de Olavide, Sevilla-41013, Spain Recent studies demonstrate that dopamine plays an important role in learning and memory. Moreover, integration of glutamate and dopamine-mediated signals at the cellular level is required for long-term potentiation (LTP), learning, and memory process. We have previously shown that dopamine D1 receptor is required for associative learning and the CA3-CA1 synaptic plasticity; however, the role of dopamine D2 receptor (D2R) is less clear. In this study, we used two models of D2R loss, D2R knock-out mice (Drd2-/-) and mice with intrahippocampal injections of Drd2-siRNA (small interfering RNA), to study the role of D2R in different models of learning and hippocampal long-term potentiation (LTP). We found that inactivation of D2Rs impairs spatial memory, associative learning, and classical conditioning of eyelid response, as well as the associated activitydependent synaptic plasticity in the hippocampal CA1-CA3 synapse. These results provide the first experimental demonstration that D2R is required for trace eyeblink conditioning and associated changes in synaptic strength in hippocampus of behaving mice. Drd2-siRNA mice show similar results than Drd2-/- mice in all experiments. Our data reveal a functional relationship between acquisition of associative learning, increase in synaptic strength at the CA3-CA1 synapse in hippocampus by demonstrating that all three are dramatically impaired when D2R is eliminated or reduced.

The ‘‘UCM1212’’ A New Allosteric Positive Dopamine Modulator Garcia-Montes J.R.1, Escobar-Verduga J.H.1 Lope´z-Rodriguez M.2, Behamu´ B.2, Moratalla-Villalba R.1 1

Departamento de Neurobiologı´a funcional y de sistemas, Instituto Cajal. Madrid, Espan˜a; 2Departamento de Quı´mica Orga´nica I, Universidad Complutense de Madrid. Madrid, Espan˜a The discovery of new allosteric modulators is allowing the possibility to avoid side effects of drugs; this is due to the regulation of the receptor activity by coupling to a site distinct to the orthosteric agonist site. Currently dopaminergic allosteric modulators have been proposed as a pharmacological strategy to control the side effects of levodopa. The aim of this study is to test in vivo the effects of UCM1212, a positive allosteric dopaminergic modulator on dyskinesia. To address this issue, we measured the motor behavior and striatal FosB an immuno-histochemical marker of Dyskinesia in the Aphakia mouse model of ‘‘Parkinson´s disease.’’

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Different Neurotoxic Effects Induced by Combined Administration of MDMA Plus Caffeine, in Adult and Adolescent Mice Pier Francesca Porceddu1, Lucia Frau1, Camilla Madeddu1, Maria Grazia Ennas1, M. Paola Castelli1 and Micaela Morelli1,2 1

Department of Biomedical Sciences, University of Cagliari, Italy; CNR, Institute of Neuroscience, Cagliari, Italy

2

The use of amphetamine-related drugs and their association with caffeine-containing beverage is very common among adolescents and young adults. On this base, we studied the neurotoxic proprieties of MDMA and caffeine association in adolescent (28 days) or adult (3 months) mice. Previous results showed that acute repeated MDMA treatment induced a decrease in dopaminergic neurons in substantia nigra pars compacta (SNc) of adult and adolescent mice, whereas THpositive fibers in striatum were decreased in adult mice only. However, MDMA were associated with caffeine, a more pronounced degeneration in adolescent compared with adult mice was observed. To better clarify the molecular mechanism at the base of the different neurotoxic effects of this drug association at different ages, we evaluated the nNOS expression, which plays a critical role in the integration of dopaminergic and glutamatergic transmissions, in the striatum of adolescent or adult mice treated with MDMA (4 9 20 mg/kg), alone or in combination with caffeine (2 9 10 mg/ kg). To confirm the neurodegeneration induced by MDMA + caffeine, we evaluated the dopamine transporter (DAT) expression in the striatum of adolescent and adult mice. nNOS immunohistochemistry revealed that MDMA induced an increase in striatal nNOS-positive neurons only in adult mice. Notably, MDMA + caffeine association induced nNOS expression in adolescents. The DAT immunohistochemical analysis showed a decrease of DAT-positive fibers in both adult and adolescent mice treated with MDMA alone or MDMA + caffeine. These data confirm the previous results concerning neurodegeneration and suggest that the use of caffeine in association with MDMA during adolescence may worsen the toxicity elicited by MDMA.

Effect of Some Psychoactive Drugs Used as ‘Legal Highs’ on Brain Neurotransmitters Krystyna Gołembiowska, Katarzyna Kamin´ska, Karolina Noworyta-Sokołowska, Alexandra Jurczak, Anna Go´rska Institute of Pharmacology, Polish Academy of Science, 31-343 Krako´w, 12 Sme˛tna, Poland The term ‘legal highs’ describes a growing range of synthetic substances that have become popular as recreational drugs of abuse. Recently, popular ‘legal highs’ such as cathinones and amphetamines have been placed under legal control in Europe and North America. There is little or no data on pharmacology and neurotoxic impact of ‘legal highs’. Even the long-term effects of the best known amphetamine derivatives—methamphetamine and 3,4-methylenedioxymetham phetamine (MDMA‘ecstasy’)—are as yet not fully explained. The aim of present study was to assess the acute effects of widely advertised on a number of websites amphetamine derivatives paramethoxyamphetamine (PMA) and para-methoxymethamphetamine (PMMA) and synthetic cathinone mephedrone on release of dopamine (DA) and serotonin (5-HT) in the striatum of freely moving rats using microdialysis. PMA and PMMA are widespread among young people and consumed without any safety testing. PMA is often sold as

127 ‘ecstasy’ and has been linked to an apparent higher incidence of hyperthermia and toxicity than seen for MDMA. Acute effects of PMA and PMMA are thought to be mediated by their interaction with serotonin transporter SERT, while mephedrone produces elevation in extracellular DA concentration and in high doses evokes hyperthermia and motor stimulation with a profile comparable to MDMA. In our study, PMA and PMMA at doses of 5 and 10 mg/kg, respectively, increased release of DA and 5-HT with a potency similar to MDMA. Mephedrone at doses of 10 and 20 mg/kg enhanced release of 5-HT stronger than that of DA. These data indicate potential for abuse and addiction of the mephedrone and both amphetamines studied.

The Effect of Prefrontal Cortex Infusion of Allopregnanolone on BDNF mRNA Expression in the Prefrontal Cortex in Rats Felipe Borges Almeida1; Agnes G.2; Gomez R.3; Nin, M.S.1,4; Barros, H.M.T.1 1

Laborato´rio de Neuropsicofarmacologia, UFCSPA, RS; 2Laborato´rio de Biologia Molecular, UFCSPA, RS; 3Departamento de Farmacologia, UFRGS, RS; 4Disciplina de Bioestatı´stica, Centro Universita´rio Metodista IPA, RS, Brasil BDNF is a neurotrophic protein that has been associated with behavioral effects of allopregnanolone (ALLO) in rats. The present study aimed to verify the effect of bilateral intra-prefrontal cortex (PFC) administration of ALLO in the BDNF mRNA expression in both hemispheres of the same region. Wistar male rats were bilaterally treated with ALLO 1.25, ALLO 2.5 or ALLO 5 g/rat, 24, 5, and 1 h before behavioral tests. At the end of the experiment, the brains were removed and frozen for histological analysis and real-time quantitative PCR protocol. Two-Way ANOVA followed by Tukey test was used when P \ 0.05. The treatment with ALLO did not change the BDNF mRNA expression when analyzing both hemispheres combined (P = 0.065). However, the hemispherical analysis within treatment groups revealed a higher expression of BDNF mRNA in the right hemisphere of controls (P \ 0.001), which was not changed by the low dose of ALLO (P \ 0.001) but was extinguished on groups ALLO 2.5 (P = 0.946) and 5 (P = 0.888). Analyzing the effect of treatments in each hemisphere, no change was observed in the right hemisphere, but the BDNF mRNA expression in the left hemisphere was increased on groups ALLO 2.5 (P = 0.030; P = 0.003) and 5 (P = 0.006; P \ 0.001) when compared with controls and group ALLO 1.25. This study points to an asymmetric role of ALLO on the regulation of BDNF mRNA expression in the PFC, apparently equalizing an asymmetry between the hemispheres. Financial support: CAPES; CNPq.

The Influence of 1METIQ on Disturbed Dopamine Release Induced by 1BNTIQ: In Vivo Microdialysis Study Agnieszka Wa˛sik, Irena Roman´ska, Lucyna Antkiewicz-Michaluk Department of Neurochemistry, Institute of Pharmacology, Polish Academy of Sciences, Sme˛tna 12, 31-343 Krako´w, Poland 1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) is an endogenous neurotoxin which is present in mammalian brain. 1BnTIQ strongly

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affected dopamine metabolism and produced the activation of the oxidative MAO-dependent catabolic pathway [Antkiewicz-Michaluk et al. 2001]. Chronic 1BnTIQ administration induced symptoms of Parkinson‘s disease (PD) in both rodents and monkeys [Kotake et al. 1996]. So, 1BnTIQ may be a good animal model for Parkinson’s disease. In contrast to this, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) exhibits the neuroprotective and MAO-inhibiting properties. The aim of the present study was to investigate the impact of 1MeTIQ on disturbance release of dopamine induced by 1BnTIQ administration using in vivo microdialysis methodology. 1BnTIQ was given multiple during 14 days in a dose 25 mg/kg i.p. Then, 1MeTIQ was administered acute or chronic (during 14 days) in a dose 50 mg/ kg i.p. 20 minutes before 1BnTIQ injections. The present study showed that chronic administration of 1BnTIQ caused pathological increase of the release of dopamine into the extracellular space of about 300 % in the basal samples, continuing until the end of experiment. This effect of 1BnTIQ was completely blocked by both acute and chronic treatments with 1MeTIQ. In summary, chronic 1BnTIQ administration disturbed the function of dopaminergic system in the brain. Simultaneous administration of 1MeTIQ completely antagonized 1BnTIQ-produced not physiological dopamine transmission. We suggest that proper physiological activity of the dopamine neurons may be dependent from balance between the concentration of these two endogenous compounds in the brain.

(from 35 % to 60 %; P \ 0.01) the immobility time od rats and antagonized locomotor depression produced by the model drugs. Depressive-like action of reserpine (0.2 mg/kg i.p.), clonidine (0.1 mg/kg i.p.), and 1BnTIQ (25 mg/kg i.p.) was observed in neurochemical study by significant decline of dopamine, noradrenaline, and serotonin concentrations in the brain structures. 1MeTIQ completely antagonized the monoamines depression in the brain produced by these drugs. Conclusion: The obtained data indicate that 1MeTIQ beyond neuroprotective effect may also offer a clear antidepressantlike activity in animal models of depression.

Antidepressant-Like Effect of 1MeTIQ, an Endogenous Neuroprotective Amine in Different Animal Models of Depression

Background: The ankyrin repeat and kinase domain (ANKK1) TaqIA polymorphism has been extensively studied as a marker of the gene for dopamine receptor 2 (DRD2) in addictions and other dopamineassociated traits. In vitro mRNA and protein studies have shown a potential connection between ANKK1 and the dopaminergic system functioning; however, it remains unknown the molecular mechanisms by which ANKK1 could be associated with the dopaminergic system. Here we investigated whether Ankk1 expression is linked to dopaminergic functioning. Methods: We used quantitative RT-PCR of total brain and Western blots of specific brain areas to study Ankk1 in murine brain after dopaminergic treatments. Results: The D2R-like agonist quinelorane has no effect upon Ankk1 mRNA (P = 0.9885). We found that Ankk1 was upregulated after activation of D1R-like dopamine receptors with SKF38393 (P = 0.002). In contrast, mice treatment with the D2R-like agonist 7-OH-DPAT caused an Ankk1 mRNA downregulation (P = 0.02). Regarding Ankk1 proteins profile, no effects were found after SKF38393 (P = 0.643) and Quinelorane (P = 0.782) treatments. In contrast, the D2R-like agonist 7-OH-DPAT (±) caused a significant increment of Ankk1 protein in the striatum (P = 0.03). Conclusions: These results show that dopaminergic modulation has an impact upon Ankk1 expression in the mice brain.

Lucyna Antkiewicz-Michaluk, Agnieszka Wa˛sik, Irena Roman´ska and Jerzy Michaluk Department of Neurochemistry, Institute of Pharmacology Polish Academy of Sciences, 31-343 Krakow, Poland 1,2,3,4-Tetrahydroisoquinoline derivatives (TIQs) are endogenous amines present in human and mammalian brains and may play important physiological role in the brain as the regulators of the activity of dopamine system. It was found that the action of various members of this group ranges from neurotoxicity to neuroprotection. Among them, 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) has been regarded as neurotoxic substance, because of its reserpine-like mechanism of action (as an inhibitor of VMAT2) in the brain. On the other hand, especially, interesting compound from this group is 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), an endogenous neuroprotective amine with a reversible inhibitory activity on MAO and a clear effect on the dopamine system as the partial agonist of dopamine receptors in the central nervous system. The main goal of our study was to determine the potential of antidepressant-like properties in the forced swimming test (FST) and locomotor activity test of an endogenous neuroprotective compound, 1MeTIQ in the animal models of depression (reserpine, clonidine, 1BnTIQ). FST is widely used as an animal test to assess the antidepressant action. Locomotor activity was measured in actometer (Opto-Varimex activity monitors) linked on-line to a compatible IBM-PC. The biochemical ex vivo study was carried out to determine the monoamines and their metabolites levels by HPLC with ED in the brain structures. All experiments were performed on male Wistar Han rats weighed 220 – 240 g. Results: The data have shown depressive-like effect of reserpine, clonidine and 1BnTIQ in behavioral and neurochemical studies. 1MeTIQ (25 and 50 mg/kg i.p.) produced antidepressant-like effect in FST and in dose-dependent manner significantly decreased

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The Addiction-Related Gene ANKK1 is Oppositely Regulated by D1R- and D2R-like Dopamine Receptors Guillermo Ponce 1,2, Adolfo Quin˜ones-Lombran˜a 1,3, Noelia ´ ngel Guerra Martı´n-Palanco1,3, Estrella Rubio-Solsona4, Miguel A Jime´nez-Arriero1,3, Toma´s Palomo1,3 and Janet Hoenicka1,3,4 * Instituto de Investigacio´n Sanitaria del Hospital Universitario 12 de Octubre, Laboratory of Neurosciences, Psychiatry Department, Avda. Andalucı´a s/n, 28041 Madrid1, Red de Trastornos Adictivos (RTA)2, Centro de Investigacio´n Biome´dica en Red de Salud Mental (CIBERSAM)3, Program of Rare and Genetic Diseases, Centro de Investigacio´n Prı´ncipe Felipe, C/Eduardo Primo Yu´fera 3, Valencia 46012, Spain4

The Effects of Environmental Enrichment and Music in MSG Toxicity Rat Model Gabor Horvath, Gyongyver Vadasz, Peter Kiss, Andrea Tamas, Dora Reglodi Department of Anatomy, MTA-PTE ‘‘Lendulet’’ PACAP Research Team, University of Pecs, Pecs, Hungary Background: Environmental enrichment is a popular strategy to enhance motor and cognitive performance and to counteract the effects of various harmful stimuli. We have shown, for the first time, that environmental enrichment has a protective effect in neonatal

Neurotox Res (2016) 30:122–130 lesion of the retina. The effects of musical enrichment are much less known and there are no data about the effects of musical enrichment on early neurobehavioral development. Aims: The aim of our study was to investigate whether environmental enrichment alters neurobehavioral development and whether it provides protection against the neurobehavioral consequences of neonatal monosodium-glutamate (MSG) toxicity. Our other aim was to investigate whether musical enriched environment has any effects on the neurobehavioral development of newborn rats. Methods: For environmental enrichment, we placed newborn Wistar rats in larger cages, supplemented with different toys that were altered daily. For the musical enrichment, we used two types of music: classic and heavy-metal from 6PM till 6AM daily. For both models, physical parameters such as weight, day of eye opening, incisor eruption, and ear unfolding were recorded. Animals were observed for appearance of reflexes such as negative geotaxis, righting reflexes, fore- and hindlimb grasp, fore- and hindlimb placing, sensory reflexes, and gait. In cases of negative geotaxis, surface righting, and gait, the time to perform the reflex was also recorded daily. For examining motor coordination, we performed grid walking, footfault, rope suspension, rota-rod, inclined board, and walk initiation tests. Results: We found that enriched environment alone did not lead to marked alterations in the course of development. On the other hand, MSG treatment caused a slight delay in reflex development and a pronounced delay in weight gain and motor coordination maturation. This delay in most signs and tests could be reversed by enriched environment: MSG-treated pups kept under enriched conditions showed no weight retardation, no reflex delay in some signs, and performed better in most coordination tests. In the musical model, we found slight differences between the two music groups: the heavy-metal group showed appearance of some reflexes earlier than the classic one. This finding could show that metal music could be a stress factor, accelerating somatic and reflex development. Conclusions: Our results show that while raising pups in enriched environment does not considerably alter neurobehavioral development, it can decrease the delaying effects of the excitotoxic MSG treatment. Musical environmental factors only slightly influence the early somatic and reflex development. This study was supported by OTKA K104984, Arimura Foundation, MTA-PTE ‘‘Lendulet’’ Program, NIH/NIGMS 2 SO6 GM08016-39, Hungarian Brain Research Program - Grant No. KTIA_13_NAP-A-III/5.

PACAP Protects Human Retinal Pigment Epithelial Cells Against Oxidative Stress Fabian E.*1, Kovacs K.3, Horvath G.1, Szereday L.2, Tamas A.1, Reglodi D.1 Departments of 1Anatomy, MTA-PTE ‘‘Lendulet’’ PACAP Research Team, 2Medical Microbiology and Immunology, 3Biochemistry and Medical Chemistry, Medical School, University of Pecs, Hungary Angiogenesis plays a critical role in many retinal diseases, such as diabetic retinopathy and macular degeneration. Under these conditions, the integrity of the pigment epithelial cells is disrupted; thus, photoreceptor survival and normal vision is impossible. The retinal pigment epithelial cells are very important elements of the bloodretina barrier, and they are known to express different angiogenic factors, such as VEGF (vascular endothelial growth factor); so these cells are most likely key factors in the process of neovascularisation. PACAP is known to exert retinoprotective effects, against several types of retinal injuries in vivo, including optic nerve transection, retinal ischemia, excitotoxic injuries, UV-A-induced lesion, and diabetic retinopathy. We have shown that PACAP activates

129 antiapoptotic pathways and inhibits proapoptotic signaling in retinal lesions in vivo. Recently, we proved that PACAP is also protective in oxidative stress-induced injury in human pigment epithelial cells (ARPE), but not in retinoblastoma cell line (Y79). According to these findings, this could be a cell-specific protection of the PACAP. In this study, we also examined the possible antiangiogenic effect of PACAP on ARPE cells exposed to oxidative stress. Cells were treated with H2O2 and the expression of angiogenic markers was investigated by specific arrays and flow cytometry. Our results showed that H2O2 administration increased different proangiogenic factors like VEGF, angiogenin, endothelin, and TIMP-1 while PACAP treatment could decrease most of them. This study was supported by OTKA K104984, Arimura Foundation, MTA-PTE ‘‘Lendulet’’ Program, NIH/NIGMS 2 SO6 GM08016-39, Hungarian Brain Research Program - Grant No. KTIA_13_NAP-A-III/5.

The Neuroprotective Effect of PACAP in Models of Parkinson’s Disease is Evolutionarily Conserved: Insight from Invertebrate and Vertebrate Models Ga´bor Maa´sz1, Zita Zrı´nyi1, Do´ra Regl} odi2, Do´ra Petrovics3, ´ da´m Rivnya´k2, La´szlo´ Ma´rk3, Tibor Kiss1, Zsolt Pirger1, A Andrea Tama´s2 ¨ K BLI, Adaptive Neuroethology, MTA-CER National Brain MTA O Project, Tihany, Hungary; 2Department of Anatomy, MTA-PTE, Lendulet PACAP Team, University of Pe´cs, Hungary; 3Department of Biochemistry and Medical Chemistry, University of Pe´cs, Hungary 1

PACAP is a widespread neuropeptide with diverse physiological functions. The structure of PACAP is relatively well conserved during phylogeny, reflecting a basic biological function. One important function of PACAP is its neuroprotective and neurotrophic effect. This has been shown both in numerous in vitro studies and in various in vivo models of brain pathologies. Among others, PACAP has strong protective effects against dopaminergic neuronal loss in vitro and in vivo. Some basic neuronal mechanisms can be very well mimicked in invertebrates. Considering the invertebrate nervous system, we have shown earlier that PACAP and its receptors are not only present in molluscs but some vertebrate functions can also be mimicked: we have described that PACAP exerts antiapoptotic effects in snails. Therefore, the primary aim of the present study was to investigate whether PACAP protects dopaminergic functions in both vertebrates and invertebrates, in models of dopaminergic neuronal degeneration. We used rotenone (commercially organic pesticide)-treated snails and 6-hydroxy-dopamine (6-OHDA)-treated rats, as models of Parkinson’s disease with or without PACAP treatments. We measured dopamine and serotonin levels in the brain. A further aim was to map the proteomic profile in the substantia nigra of control, 6-OHDA- and 6-OHDA ? PACAP-injected rats. Finally, our goal was to investigate changes in dopamine metabolic enzymes (S-COMT, MB-COMT and MAO-B) after treatments in both the invertebrate and the vertebrate models. The induced dopamine decreasing effect of rotenone and 6-OHDA was attenuated by *50 % in the PACAP-injected invertebrate and vertebrate samples. Proteomic mapping showed a significant difference in the expression of the DJ-1 (PARK7). The decreased level of the protective DJ-1 protein induced by the 6-OHDA injection was counteracted by PACAP treatment. Changes in the metabolic enzymes were also observed. Highest intensity of MB-COMT was in the toxin-treated groups. MAO-B decreased in 6-OHDA-treated samples, while in the PACAP-injected group it had an intensity similar to that of untreated

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130 controls. MAO-B was not present in the snail central nervous system. Based on our results, we conclude that PACAP is a neuroprotective agent in dopamine-based neurodegenerative disorders, and that the rotenone-model is also suitable for developing and testing some aspects and mechanisms of neurodegenerative disorders. This work was supported by PD-109099 OTKA, Hungarian Brain Research Program KTIA_NAP_13-2-2014-0006 and KTIA_13_NAP-A-III/5; Arimura Foundation and PTE-MTA Lendu¨let Program.

L-DOPA Treatment Selectively Restores Spine Density in D2R-Expressing Projection Neurons in Dyskinetic Mice Samuel Alberquilla1, Luz M Sua´rez1,2, Oscar Solı´s1,2, and Rosario Moratalla1,2 1

Instituto Cajal, Consejo Superior de Investigaciones Cientı´ficas, CSIC, 28002, Madrid; 2CIBERNED, Instituto de Salud Carlos III, Madrid, Spain Background: L-DOPA-induced dyskinesia is an incapacitating complication of L-DOPA therapy which affects most patients with Parkinson’s disease. Previous work indicating that molecular sensitization to D1 dopamine receptor (D1R) stimulation is involved in dyskinesias prompted us to perform electrophysiological recordings of striatal projection ‘‘medium spiny neurons’’ (MSN). Moreover, because enhanced D1R signaling in drug abuse induces changes in

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Neurotox Res (2016) 30:122–130 spine density in striatum, we investigated whether the dyskinesia is related to morphological changes in MSNs. Methods: Wild-type and BAC transgenic mice (D1R-tomato and D2R-GFP) were lesioned with 6-hydroxydopamine and subsequently treated with L-DOPA to induce dyskinesia. Functional, molecular, and structural changes were assessed in corticostriatal slices. Individual MSNs injected with Lucifer-Yellow were used for DAB-derived 3-D reconstructions with Neurolucida software. Intracellular current-clamp recordings with high-resistance micropipettes were used to characterize electrophysiological parameters. Results: Both D1R-MSNs and D2R-MSNs showed diminished spine density in totally denervated striatal regions in parkinsonian mice. Chronic L-DOPA treatment, which induced dyskinesia and aberrant FosB expression, restored spine density in D2R-MSNs but not in D1R-MSNs. In basal conditions, MSN are more excitable in parkinsonian than in sham mice, and excitability decreases towards normal values following L-DOPA treatment. Despite this normalization of basal excitability, in dyskinetic mice, the selective D1R agonist SKF38393 increased the number of evoked action potentials in MSNs, compared to sham animals. Conclusions: Chronic L-DOPA induces abnormal spine re-growth exclusively in D2R-MSNs and robust supersensitization to D1R-activated excitability in denervated striatal MSNs. These changes might constitute the anatomical and electrophysiological substrates of dyskinesia. This work was supported by grants from the Spanish MINECO, ISCIII (BFU2010-20664; SAF2013-48532-R), PNSD, CIBERNED (CB06/05/0055) and CAM (S2011/BMD-2336), RedRTA (RD06/0001/1011).