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Abnormal Accumulation Of Manganese In The Brain Impairs Motor Behavior In Mice

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Title:
Abnormal Accumulation Of Manganese In The Brain Impairs Motor Behavior In Mice
Series Title:
19th Annual Undergraduate Research Symposium
Creator:
Garcia, Armin
Schrier, Jessica
Language:
English
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Undetermined

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Center for Undergraduate Research
Center for Undergraduate Research
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Conference papers and proceedings
Poster

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Abstract:
Manganese (Mn) is a cofactor in many enzymes, making it an essential nutrient required for normal growth and physiological processes. This trace element is available in most plant-derived foods, whereas animal-based foods are nearly free of Mn. While dietary deficiency has not been reported, exposure to higher levels of Mn and its abnormal accumulation in the brain leads to a parkinsonian-like disorder known as manganism. We recently published a characterization of a mouse model of manganism, which shows compromised Mn homeostasis due to a loss of function mutation in the Slc39a14 gene (Jenkitkasemwong 
et al. (2018) PNAS). The knockout Slc39a14-/- mice show dysfunction in Mn homeostasis with consequent aggregation of Mn in the brain and impairment in motor behavior. In the present study, we extended the characterization of this model focusing on anxiety and cognitive behavioral domains. We confirmed that the Slc39a14-/- mice showed compromised motor performance compared to their wild type controls at older ages than previously published. Additionally, we report that the mice manifested decreased anxiety-like behaviors and faster acquisition of Pavlovian associative learning as evaluated in fear conditioning tests. ( en )
General Note:
Research authors: Armin Garcia, Jessica Schrier, Genesys Giraldo, Mitchell Knutson, Christopher Janus - University of Florida
General Note:
Faculty Mentor: Manganese (Mn) is a cofactor in many enzymes, making it an essential nutrient required for normal growth and physiological processes. This trace element is available in most plant-derived foods, whereas animal-based foods are nearly free of Mn. While dietary deficiency has not been reported, exposure to higher levels of Mn and its abnormal accumulation in the brain leads to a parkinsonian-like disorder known as manganism. We recently published a characterization of a mouse model of manganism, which shows compromised Mn homeostasis due to a loss of function mutation in the Slc39a14 gene (Jenkitkasemwong 
et al. (2018) PNAS). The knockout Slc39a14-/- mice show dysfunction in Mn homeostasis with consequent aggregation of Mn in the brain and impairment in motor behavior. In the present study, we extended the characterization of this model focusing on anxiety and cognitive behavioral domains. We confirmed that the Slc39a14-/- mice showed compromised motor performance compared to their wild type controls at older ages than previously published. Additionally, we report that the mice manifested decreased anxiety-like behaviors and faster acquisition of Pavlovian associative learning as evaluated in fear conditioning tests. - Center for Undergraduate Research,

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Copyright Armin Garcia. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Introduction Manganese (Mn) is a naturally occurring element and an essential nutrient for humans and animals alike Mn plays an important role in energy metabolism, cellular protection from damaging free radical species, and bone mineralization, however, an accumulation of excess Mn in the body and brain can lead to serious adverse health effects and is neurotoxic 1 Toxic overexposure to manganese, known as manganism, is most commonly seen across industrial workers, albeit genetic loss of function mutations also result in neurotoxicity by affecting the homeostasis of manganese 2 3 One of the genes regulating the homeostasis and excretion of Mn is Slc 39 a 14 which encodes transmembrane metal transporter Slc 39 a 14 protein 3 We recently published a characterization of a mouse Slc 39 a 14 knockout (Slc 39 a 14 / ) model 4 The Slc 39 a 14 / mice showed dysfunction in Mn homeostasis with consequent aggregation of Mn in the brain and impairment in motor behavior In this project, we focused on characterizing the behavioral consequences of elevated levels of manganese in the brains of the Slc 39 a 14 / mice a ) at older ages, and b ) extending the phenotyping characterization to anxiety and cognitive behavioral domains We hypothesized that Slc 39 a 14 / mice would be differentiated from their control, wild type counterparts in anxiety responses and cognitive domain, additionally to already reported motor impairments To this end, the mice were put through a battery of behavioral tests evaluating their anxiety levels, motor coordination, and Pavlovian associative learning We report that Slc 39 a 14 / mice showed compromised motor behavior in comparison to their wild type counterparts In the anxiety assessments, the Slc 39 a 14 / mice exemplified decreased anxiety like behaviors Additionally, they manifested faster acquisition of associative learning and conditioned memories as seen in fear conditioning tests Methods The study began with 7 wild type (WT) mice and 6 Slc 39 a 14 / mice Due to illness, 2 Slc 39 a 14 / mice died throughout testing Mice were housed in same sex groups of 2 4 under standard laboratory conditions ( 12 h light/dark cycle, lights on at 0600 hours) with room temperature of 22 ¡ C, and water and food available ad libitum All tests were performed during the light phase between 09 : 00 and 14 : 00 hours in accordance with AAALAC and institutional guidelines IACUC UF approved the husbandry conditions and all experimental protocols used in the study Experimental Design The mice were handled prior to the first behavioral test Testing began at 19 weeks (nearly 5 months) of mice age and continued through 23 weeks The tests were run in the following order : Open Field, Light/Dark, Pole, Beam, Rota Rod, and Fear Conditioning Open Field Test (see pic 2 ) The Open Field test is used to evaluate general locomotor activity levels in rodents as well as their anxiety and emotionality Mice were placed in the center of the open field to begin and their movement and time spent in each quadrant of the field was recorded for 5 minutes This test is run one time in one single session prior to all other motor and anxiety testing Light/Dark Test (see pic 3 ) The Light/Dark test is used to measure anxiety like behaviors in rodents Rodents are placed in the light compartment of the apparatus and are free to move around This test was run twice, once after the open field test prior to any motor tests and again after all motor tests had been administered Time spent in both light and dark sides of the box was recorded The time index was found by dividing the recorded time spent in the light side by the total test time Vertical Pole Descending Test (see pic 4 ) The pole test evaluates spontaneous motor behavior and coordination of mice descending a vertical pole Mice were placed head up near the top of a vertical wooden pole The pole was 50 cm in height, 1 cm in diameter, and wrapped with wire mesh to provide grip and prevent sliding The time taken for mice to reorient themselves downward and time taken to descend the pole were recorded The test was terminated at 120 seconds Elevated Beam Traversing Test (see pic 5 ) The beam traversing test is used to assess motor balance and coordination in mice while they cross an elevated beam 120 cm in length with small obstacles measuring 6 x 6 mm every 10 cm After the habituation period, the mice were tested two trials per day over two consecutive days on wide ( 2 5 cm) and narrow ( 1 7 cm) beams, respectively The time to reach the goal cage, and the number of slips, falls, and pauses, were recorded Rotarod Locomotor Test (see pic 6 ) The Rotarod test examines the motor coordination and balance of rodents The mice were placed on a horizontal rod that rotates along its long axis, forcing the mice to continue moving forward to prevent from falling The rod gradually increases speed from 4 to 40 rpm in intervals of 4 9 seconds After two initial days of exposure to the apparatus, the mice were tested for three consecutive days in five minute trials The time taken for each mouse to fall from the rotating rod was recorded Fear Conditioning Test (see pics 7 a, 7 b, 7 c) The Fear Conditioning test evaluates Pavlovian associative learning and memory Mice were trained to associate the context of the training cage, and a tone with a following mild foot shock over a five day training period (Days 1 5 ) A context test was administered on Day 7 and a tone test on Day 8 Learning was assessed by "freezing" behavior, which was manifested by complete cessation of all body movements, except respiration Conclusions The Slc 39 a 14 / mice showed compromised motor behavior, encompassing slower times in descending a vertical pole, slower times in crossing an elevated beam with obstacles along the path, and inability to remain as long on a rotating rod as compared to their control wild type littermates Additionally, they manifested decreased anxiety like behaviors, expressed as spending more time in bright, open areas as seen in light dark box and open field tests respectively, and faster acquisition of associative learning and conditioned memories measured as increased freezing when exposed to conditioned stimuli that was paired with a mild foot shock in fear conditioning tests 0 50 100 150 200 day 1 day 2 day 3 Latency to fall (s) WT Slc39a14 / Figure 6 Slc 39 a 14 / mice on average had a lower latency to fall meaning they were unable to remain on the rotating rod for as long as the WT mice The mice were tested in the rotarod test at 22 weeks of age The mice were trained in the rotarod test for 2 days and then tested for 3 days ( Slc 39 a 14 / n= 4 ; WT n= 7 ) Data represent mean S D *p < 0 05 0 10 20 30 40 genotype Freezing (%) WT Slc39a14 / 0 5 10 15 20 wide (2.5cm) narrow (1.7cm) Time to Cross(s) WT Slc39a14 / Figure 5 Slc 39 a 14 / mice showed longer times than WT mice to cross both wide and narrow beams The mice were trained in crossing the beams over 2 days and then tested first on a wider 2 5 cm beam and the following day on a narrower 1 7 cm beam at 21 weeks of age ( Slc 39 a 14 / n= 4 ; WT n= 7 ) Data represent mean S D *p < 0 05 0 5 10 15 20 day 1 day 2 day 3 Time to Descent (s) WT Slc39a14 / Figure 4 Slc 39 a 14 / mice showed longer times than WT mice to climb down the pole in the pole test The mice were trained to descend the pole over 2 sessions and then tested in the task across 3 consecutive days at 20 weeks of age ( Slc 39 a 14 / n= 4 ; WT n= 7 ) Data represent mean S D *p < 0 05 0 25 50 75 100 125 day 1 day 2 day 3 day 4 day 5 Freezing (%) WT Slc39a14 / Figure 7 a Slc 39 a 14 / mice acquired a freezing response to the fear conditioning training more quickly than did WT mice The mice were trained over 5 days in the chamber using the same environmental context with an 80 dB tone immediately preceding a mild foot shock Data represent mean S D *p < 0 05 Figure 7 b Slc 39 a 14 / mice showed greater freezing than WT mice in context test At the end of training, the mice were exposed to just the original context without receiving any shock The amount of freezing was recorded as a measure of how well the mice remembered and learned to associate the original training environment with the mild foot shock ( Slc 39 a 14 / n= 4 ; WT n= 7 ) Data represent mean S D *p < 0 05 4 5 6 0 10 20 30 40 50 pre tone after tone % Freezing WT Slc39a14 / 7b 7c 15 20 25 30 35 40 19 20 21 22 23 Body weight (g) Age (weeks) WT Slc39a14 / Rotarod Locomotor Test Elevated Beam Traversing Test Vertical Pole Descending Test Context Test Tone Test Fear Condition Training Figure 2 Slc 39 a 14 / mice showed less anxiety like behaviors than the WT mice in the open field test Mice were tested at 20 weeks of age for anxiety like behaviors by measuring the amount of time spent in the center of the open field test When placed in a new environment, mice have a tendency to seek the edges of the field for safety therefore spending time in center is correlated with decreased anxiety ( Slc 39 a 14 / n= 5 ; WT n= 7 ) Data represent mean S D *p < 0 05 Figure 1 Body weight of Slc 39 a 14 / and wild type (WT) mice shown between 19 and 23 weeks of age ( Slc 39 a 14 / n= 4 6 ( 2 mice died during the study) ; WT n= 7 ) Data represents mean S D *p < 0 05 0 10 20 30 40 50 60 70 week 20 week 23 % Time Spent in Light Side WT Slc39a14 / Figure 3 Slc 39 a 14 / mice showed less anxiety like behaviors than the WT mice in the light/dark test The test was administered before at the onset (week 20 ) and at the end (week 23 ) of the study In the first test the Slc 39 a 14 / mice spent significantly greater time in the light side of the box, thus showing lower anxiety than the WT mice In the second test the levels of both groups were comparable ( Slc 39 a 14 / n= 4 ; WT n= 7 ) Data represent mean S D *p < 0 05 0 10 20 30 genotype Center/Total Time Ratio (%) WT Slc39a14 / 1 2 3 Light/Dark Test Open Field Test Assessment of Physical Condition Picture 1 Picture 2 Picture 3 Picture 4 Picture 5 Picture 6 Picture 7c Picture 7a Picture 7b Figure 7 c Slc 39 a 14 / mice showed greater freezing than WT mice in tone test At the end of training, the mice were exposed to just the original tone in a new context The amount of freezing was recorded as a measure of how well the mice remembered and learned to associate the tone with the mild foot shock ( Slc 39 a 14 / n= 4 ; WT n= 7 ) Data represent mean S D *p < 0 05 7a Abnormal accumulation of manganese in the brain impairs motor behavior in mice Armin Garcia Jessica Schrier Genesys Giraldo, Mitchell Knutson, Christopher Janus Center for Translational Research in Neurodegenerative Diseases, Department of Neuroscience, University of Florida, Gainesvil le, FL References 1 Chen, Pan et al ( 2015 ), Toxicology Research 4 19 2 B Bowman, G F Kwakye E Herrero Hernandez and M Aschner ( 2015 ), Journal of Trace Elements in Medicine and Biology, 25 191 203 3 Hojyo S, Fukada T, Shimoda S, Ohashi W, Bin B H, et al ( 2011 ), PLoS ONE 6 3 4 Supak Jenkitkasemwong, Adenike Akinyode, Elizabeth Paulus, Ralf Weiskirchen, Shintaro Hojyo, Toshiyuki Fukada, Genesys Giraldo, Jessica Schrier, Armin Garcia, Christopher Janus, Benoit Giasson, Mitchell D Knutson ( 2018 ), Proceedings of the National Academy of Sciences doi : 10 1073 /pnas 1720739115 Acknowledgments We thank Denisse Cancel, Shaina Wallach, Jeffry Berrier and Kristi Douglas (UF ACS) for excellent husbandry care of the mice We also thank Genesys Giraldo for lending a helping hand during testing Motor Coordination Tests Associative Learning Tests Anxiety Assessments