Mol Neurobiol DOI 10.1007/s12035-014-9024-y
Behavioral Responses in Animal Model of Congenital Muscular Dystrophy 1D Clarissa M. Comim & Aryadnne L. Schactae & Jaime A. Soares & Letícia Ventura & Viviane Freiberger & Francielle Mina & Diogo Dominguini & Mariz Vainzof & João Quevedo
Received: 23 September 2014 / Accepted: 20 November 2014 # Springer Science+Business Media New York 2014
Abstract Congenital muscular dystrophies 1D (CMD1D) present a mutation on the LARGE gene and are characterized by an abnormal glycosylation of α-dystroglycan (α-DG), strongly implicated as having a causative role in the development of central nervous system abnormalities such as cognitive impairment seen in patients. However, in the animal model of CMD1D, the brain involvement remains unclear. Therefore, the objective of this study is to evaluate the cognitive involvement in the Largemyd mice. To this aim, we used adult homozygous, heterozygous, and wild-type mice. The mice underwent six behavioral tasks: habituation to an open field, step-down inhibitory avoidance, continuous multiple trials step-down inhibitory avoidance task, object recognition, elevated plus-maze, and forced swimming test. It was observed that Largemyd individuals presented deficits on the habituation to the open field, step down inhibitory avoidance, continuous multiple-trials step-down inhibitory avoidance, object recognition, and forced swimming. This study shows C. M. Comim (*) : A. L. Schactae : J. A. Soares : L. Ventura : V. Freiberger Laboratory of Experimental Neuropathology, Postgraduate Program in Health Sciences, University of South Santa Catarina, Palhoça, SC, Brazil e-mail: [email protected] F. Mina : D. Dominguini : J. Quevedo Laboratory of Neurosciences, Graduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, Criciúma, SC, Brazil M. Vainzof Human Genome Research Center, Biosciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil J. Quevedo Center for Experimental Models in Psychiatry, Department of Psychiatry and Behavioral Sciences, The University of Texas Medical School at Houston, Houston, TX, USA
the first evidence that abnormal glycosylation of α-DG may be affecting memory storage and restoring process in an animal model of CMD1D. Keywords Congenital muscular dystrophy 1D . Large . Memory and learning . Behavior . Mice
Introduction Congenital muscular dystrophies (CMDs) are a group of clinical and genetically heterogeneous disorders currently untreatable. These diseases are usually severe and often involve other organs, particularly the central nervous system (CNS) [1]. LARGE is one of five genes that encode known or putative glycosylation enzymes and are is found in various forms of muscular dystrophy [2]. Mutations in the human gene (LARGE) have been identified in a patient with congenital muscular dystrophy type 1D (CMD1D). CMD1D is characterized by an abnormal glycosylation of α-dystroglycan (αDG), which is strongly implicated as having a causative role in the development of CNS abnormalities, such as severe mental retardation [3], various degrees of delayed motor and cognitive milestones in patients [4]. α-Dystroglycan appears to play a major role in neuronal migration. Brain magnetic resonance imaging shows extensive white matter abnormalities and subtle structural changes indicative of a neuronal migration defect in patients [3, 5]. In an animal model of CMD1D, Largemyd mice presented abnormal migration of granule cells in the cerebellum and hippocampus and clusters of heterotopic neurons are seen in the cerebellar white matter [6, 7]. Alterations in the dentate gyrus were also observed [8]. Our research group demonstrated that Largemyd mice present oxidative damage and energetic metabolism alterations in low levels of brain-derived neurotrophic factor (BDNF) in brain tissue [9].
Mol Neurobiol
Despite many studies in the area, there are only few that describe the cognitive involvement in CMD1D in both patients and animal models. Thus, the objective of this study is to evaluate the habituation, aversive and object recognition memory, and depression- and anxiety-like behaviors in order to better understand the involvement of the CNS in adult Largemyd mice.
Methods Animals Adult homozygous (KO-LARGEmyd) (n=60), heterozygous (HT) (n=60) as well as wild-type (WT) littermate mice (n= 60) used in this study were genotyped using PCR tail DNA. We used only male mice weighing 25 and 30 g. They were housed five to a cage with food and water available ad libitum and were maintained on a 12-h light/dark cycle (lights on at 7:00 AM). All experimental procedures were approved by the Animal Care and Experimentation Committee of UNISUL (protocol number 13.005.4.08.IV), Brazil
Behavior Tests The animals (ten animals per group) underwent six behavioral tasks separately: habituation to an open field, step-down inhibitory avoidance, continuous multiple trials step-down inhibitory avoidance task, object recognition, elevated plusmaze, and forced swimming test. All behavioral procedures were conducted between 01:00 and 04:00 PM in a soundisolated room. A single animal performed only one behavior test.
Habituation to an Open Field Task This task evaluates motor performance in the training section and non-associative memory in the retention test session. Habituation to an open field was carried out in a 40×40-cm open field surrounded by 40 cm high walls made of brown plywood with a frontal glass wall. The floor of the open field was divided into 12 equal rectangles by black lines. The animals were gently placed on the left rear quadrant and left to explore the arena for 5 min (training session). Immediately after, the animals were taken back to their home cage and, 24 h later, submitted again to a similar open-field session (test session). Crossing of the black lines and rearings performed in both sessions were counted. The decrease in the number of crossings and rearings between the two sessions were taken as a measure of the retention of habituation [10].
Step-Down Inhibitory Avoidance Task This task evaluates aversive memory. The apparatus and procedures have been described in previous reports. In the training session, animals were placed on the platform, and their latency to step down on the grid with all four paws was measured with an automatic device. Immediately after stepping down on the grid, the animals received a 0.3-mA, 2.0-s foot shock and were returned to their home cage. A retention test session was performed 24 h after training. The retention test session was procedurally identical to training, except that no foot shock was performed. The retention test step-down latency (maximum 180 s) was used as a measure of inhibitory avoidance retention [11]. Continuous Multiple Trials Step-Down Inhibitory Avoidance Task This task evaluates aversive memory in the test section and learning when analyzing the number of training trials required for the acquisition criterion. It was performed in the same stepdown inhibitory avoidance apparatus; however, in the training session, the animal was placed on the platform and immediately after stepping down on the grid, received a 0.3-mA, 2.0-s foot shock. This procedure continued until the rat remained on the platform for 50 s. The animal was then returned to the home cage. The number of training sessions required to reach the acquisition criterion of 50 s on the platform was recorded. The retention test was performed 24 h later [11]. Object Recognition This task evaluates non-aversive, non-spatial memory. The apparatus and procedures for the object recognition task have been described elsewhere. The apparatus used was the same as Habituation to an open field task. All animals were submitted to a habituation session where they were allowed to freely explore the open field for 5 min. No objects were placed in the box during the habituation session. Crossings of the black lines and rearings performed in this session were evaluated as locomotors and exploratory activity, respectively. Different times after habituation, training was conducted by placing individual rats for 5 min in the field, in which two identical objects (objects A1 and A2, both being cubes) were positioned in two adjacent corners, 10 cm from the walls. In a short-term recognition memory test given 1.5 h after training, the rats explored the open field for 5 min in the presence of one familiar (A) and one novel (B, a pyramid with a squareshaped base) object. All objects had similar textures (smooth), colors (blue), and sizes (weight 150–200 g) but distinctive shapes. A recognition index calculated for each animal is reported as the ratio TB/(TA+TB) (TA=time spent exploring the familiar object A; TB=time spent exploring the novel
Mol Neurobiol
object B). In a long-term recognition memory test given 24 h after training, the same rats were allowed to explore the field for 5 min in the presence of the familiar object A and a novel object C (a sphere with a square-shaped base). Recognition memory was evaluated as done for the short-term memory test. Exploration was defined as sniffing (exploring the object 3–5 cm away from it) or touching the object with the nose and/ or forepaws [12]. Elevated Plus-Maze The apparatus used in animal models for anxiety has been described in detail elsewhere. Briefly, the apparatus consisted of two open arms and two closed arms arranged in such a way that the two arms of each type were opposite to each other, with a central platform. The maze’s height was 50 cm, and the tests were conducted under dim red light. Animals were exposed for 5 min to the red light in their own home cages before the testing procedure. Next, they were placed individually on the central platform of the plus-maze facing an open arm. During a 5-min test period, the following measurements were recorded by two observers: the number of entries, the time spent in the open and closed arms, and the total number of arm entries [13]. Forced Swimming Test The test was conducted according to previous reports [14] and was used as a model for depressive behavior. Briefly, the test involves two exposures to a cylindrical water tank in which rats cannot touch the bottom or from which they cannot escape. The tank is made of transparent plexiglass and filled with water (22–23 °C). Water in the tank was changed for each rat. For the first exposure, the rats were placed in the water for 15 min. After 24 h, the rats were placed in the water again for a 5-min session. The periods of immobility were analyzed. The mice were judged to be immobile whenever they stopped swimming and remained floating in the water, with their head just above water level. Statistical Analysis Data for the elevated plus-maze and forced swimming tests are reported as mean±SEM and were analyzed by the Student’s t test. Data from the inhibitory avoidance task, object recognition task, and the number of training trials from continuous multiple trials step-down inhibitory avoidance are reported as median and interquartile ranges, and comparisons among groups were performed using Mann–Whitney U tests. The within-individual groups were analyzed by Wilcoxon’s tests. In all comparisons, p < 0.05 indicated statistical significance.
Results Habituation to an Open-Field Task In the open-field task, there were no differences in the number of crossings and rearings between groups in the habituation to the open-field training session (p>0.05), demonstrating no difference in motor and exploratory activity between groups. In the test session, there was a significant reduction in both crossings and rearings in the test when compared with training in the WT and HT groups. In the KO group, there was no reduction in the number of crossings and rearings in the test when compared with training, suggesting memory impairment (p0.05) (Fig. 2), suggesting impaired aversive memory. Continuous Multiple Trials Step-Down Inhibitory Avoidance Task In the continuous multiple trials step-down inhibitory avoidance, it was demonstrated a significant increase in the number of training trials required to reach the acquisition criterion (50 s on the platform) in the KO group as compared with the WT and HT groups (p0.05) (Fig. 3b). Object Recognition The KO mice presented impairment of novel object recognition memory, i.e., they did not spend a significantly higher percentage of time exploring the novel object (p>0.05). The
Fig. 1 Habituation on the open field. Number of crossings and rearings are presented as mean±SEM, *p
Behavioral Responses in Animal Model of Congenital Muscular Dystrophy 1D Clarissa M. Comim & Aryadnne L. Schactae & Jaime A. Soares & Letícia Ventura & Viviane Freiberger & Francielle Mina & Diogo Dominguini & Mariz Vainzof & João Quevedo
Received: 23 September 2014 / Accepted: 20 November 2014 # Springer Science+Business Media New York 2014
Abstract Congenital muscular dystrophies 1D (CMD1D) present a mutation on the LARGE gene and are characterized by an abnormal glycosylation of α-dystroglycan (α-DG), strongly implicated as having a causative role in the development of central nervous system abnormalities such as cognitive impairment seen in patients. However, in the animal model of CMD1D, the brain involvement remains unclear. Therefore, the objective of this study is to evaluate the cognitive involvement in the Largemyd mice. To this aim, we used adult homozygous, heterozygous, and wild-type mice. The mice underwent six behavioral tasks: habituation to an open field, step-down inhibitory avoidance, continuous multiple trials step-down inhibitory avoidance task, object recognition, elevated plus-maze, and forced swimming test. It was observed that Largemyd individuals presented deficits on the habituation to the open field, step down inhibitory avoidance, continuous multiple-trials step-down inhibitory avoidance, object recognition, and forced swimming. This study shows C. M. Comim (*) : A. L. Schactae : J. A. Soares : L. Ventura : V. Freiberger Laboratory of Experimental Neuropathology, Postgraduate Program in Health Sciences, University of South Santa Catarina, Palhoça, SC, Brazil e-mail: [email protected] F. Mina : D. Dominguini : J. Quevedo Laboratory of Neurosciences, Graduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, Criciúma, SC, Brazil M. Vainzof Human Genome Research Center, Biosciences Institute, University of São Paulo, São Paulo, São Paulo, Brazil J. Quevedo Center for Experimental Models in Psychiatry, Department of Psychiatry and Behavioral Sciences, The University of Texas Medical School at Houston, Houston, TX, USA
the first evidence that abnormal glycosylation of α-DG may be affecting memory storage and restoring process in an animal model of CMD1D. Keywords Congenital muscular dystrophy 1D . Large . Memory and learning . Behavior . Mice
Introduction Congenital muscular dystrophies (CMDs) are a group of clinical and genetically heterogeneous disorders currently untreatable. These diseases are usually severe and often involve other organs, particularly the central nervous system (CNS) [1]. LARGE is one of five genes that encode known or putative glycosylation enzymes and are is found in various forms of muscular dystrophy [2]. Mutations in the human gene (LARGE) have been identified in a patient with congenital muscular dystrophy type 1D (CMD1D). CMD1D is characterized by an abnormal glycosylation of α-dystroglycan (αDG), which is strongly implicated as having a causative role in the development of CNS abnormalities, such as severe mental retardation [3], various degrees of delayed motor and cognitive milestones in patients [4]. α-Dystroglycan appears to play a major role in neuronal migration. Brain magnetic resonance imaging shows extensive white matter abnormalities and subtle structural changes indicative of a neuronal migration defect in patients [3, 5]. In an animal model of CMD1D, Largemyd mice presented abnormal migration of granule cells in the cerebellum and hippocampus and clusters of heterotopic neurons are seen in the cerebellar white matter [6, 7]. Alterations in the dentate gyrus were also observed [8]. Our research group demonstrated that Largemyd mice present oxidative damage and energetic metabolism alterations in low levels of brain-derived neurotrophic factor (BDNF) in brain tissue [9].
Mol Neurobiol
Despite many studies in the area, there are only few that describe the cognitive involvement in CMD1D in both patients and animal models. Thus, the objective of this study is to evaluate the habituation, aversive and object recognition memory, and depression- and anxiety-like behaviors in order to better understand the involvement of the CNS in adult Largemyd mice.
Methods Animals Adult homozygous (KO-LARGEmyd) (n=60), heterozygous (HT) (n=60) as well as wild-type (WT) littermate mice (n= 60) used in this study were genotyped using PCR tail DNA. We used only male mice weighing 25 and 30 g. They were housed five to a cage with food and water available ad libitum and were maintained on a 12-h light/dark cycle (lights on at 7:00 AM). All experimental procedures were approved by the Animal Care and Experimentation Committee of UNISUL (protocol number 13.005.4.08.IV), Brazil
Behavior Tests The animals (ten animals per group) underwent six behavioral tasks separately: habituation to an open field, step-down inhibitory avoidance, continuous multiple trials step-down inhibitory avoidance task, object recognition, elevated plusmaze, and forced swimming test. All behavioral procedures were conducted between 01:00 and 04:00 PM in a soundisolated room. A single animal performed only one behavior test.
Habituation to an Open Field Task This task evaluates motor performance in the training section and non-associative memory in the retention test session. Habituation to an open field was carried out in a 40×40-cm open field surrounded by 40 cm high walls made of brown plywood with a frontal glass wall. The floor of the open field was divided into 12 equal rectangles by black lines. The animals were gently placed on the left rear quadrant and left to explore the arena for 5 min (training session). Immediately after, the animals were taken back to their home cage and, 24 h later, submitted again to a similar open-field session (test session). Crossing of the black lines and rearings performed in both sessions were counted. The decrease in the number of crossings and rearings between the two sessions were taken as a measure of the retention of habituation [10].
Step-Down Inhibitory Avoidance Task This task evaluates aversive memory. The apparatus and procedures have been described in previous reports. In the training session, animals were placed on the platform, and their latency to step down on the grid with all four paws was measured with an automatic device. Immediately after stepping down on the grid, the animals received a 0.3-mA, 2.0-s foot shock and were returned to their home cage. A retention test session was performed 24 h after training. The retention test session was procedurally identical to training, except that no foot shock was performed. The retention test step-down latency (maximum 180 s) was used as a measure of inhibitory avoidance retention [11]. Continuous Multiple Trials Step-Down Inhibitory Avoidance Task This task evaluates aversive memory in the test section and learning when analyzing the number of training trials required for the acquisition criterion. It was performed in the same stepdown inhibitory avoidance apparatus; however, in the training session, the animal was placed on the platform and immediately after stepping down on the grid, received a 0.3-mA, 2.0-s foot shock. This procedure continued until the rat remained on the platform for 50 s. The animal was then returned to the home cage. The number of training sessions required to reach the acquisition criterion of 50 s on the platform was recorded. The retention test was performed 24 h later [11]. Object Recognition This task evaluates non-aversive, non-spatial memory. The apparatus and procedures for the object recognition task have been described elsewhere. The apparatus used was the same as Habituation to an open field task. All animals were submitted to a habituation session where they were allowed to freely explore the open field for 5 min. No objects were placed in the box during the habituation session. Crossings of the black lines and rearings performed in this session were evaluated as locomotors and exploratory activity, respectively. Different times after habituation, training was conducted by placing individual rats for 5 min in the field, in which two identical objects (objects A1 and A2, both being cubes) were positioned in two adjacent corners, 10 cm from the walls. In a short-term recognition memory test given 1.5 h after training, the rats explored the open field for 5 min in the presence of one familiar (A) and one novel (B, a pyramid with a squareshaped base) object. All objects had similar textures (smooth), colors (blue), and sizes (weight 150–200 g) but distinctive shapes. A recognition index calculated for each animal is reported as the ratio TB/(TA+TB) (TA=time spent exploring the familiar object A; TB=time spent exploring the novel
Mol Neurobiol
object B). In a long-term recognition memory test given 24 h after training, the same rats were allowed to explore the field for 5 min in the presence of the familiar object A and a novel object C (a sphere with a square-shaped base). Recognition memory was evaluated as done for the short-term memory test. Exploration was defined as sniffing (exploring the object 3–5 cm away from it) or touching the object with the nose and/ or forepaws [12]. Elevated Plus-Maze The apparatus used in animal models for anxiety has been described in detail elsewhere. Briefly, the apparatus consisted of two open arms and two closed arms arranged in such a way that the two arms of each type were opposite to each other, with a central platform. The maze’s height was 50 cm, and the tests were conducted under dim red light. Animals were exposed for 5 min to the red light in their own home cages before the testing procedure. Next, they were placed individually on the central platform of the plus-maze facing an open arm. During a 5-min test period, the following measurements were recorded by two observers: the number of entries, the time spent in the open and closed arms, and the total number of arm entries [13]. Forced Swimming Test The test was conducted according to previous reports [14] and was used as a model for depressive behavior. Briefly, the test involves two exposures to a cylindrical water tank in which rats cannot touch the bottom or from which they cannot escape. The tank is made of transparent plexiglass and filled with water (22–23 °C). Water in the tank was changed for each rat. For the first exposure, the rats were placed in the water for 15 min. After 24 h, the rats were placed in the water again for a 5-min session. The periods of immobility were analyzed. The mice were judged to be immobile whenever they stopped swimming and remained floating in the water, with their head just above water level. Statistical Analysis Data for the elevated plus-maze and forced swimming tests are reported as mean±SEM and were analyzed by the Student’s t test. Data from the inhibitory avoidance task, object recognition task, and the number of training trials from continuous multiple trials step-down inhibitory avoidance are reported as median and interquartile ranges, and comparisons among groups were performed using Mann–Whitney U tests. The within-individual groups were analyzed by Wilcoxon’s tests. In all comparisons, p < 0.05 indicated statistical significance.
Results Habituation to an Open-Field Task In the open-field task, there were no differences in the number of crossings and rearings between groups in the habituation to the open-field training session (p>0.05), demonstrating no difference in motor and exploratory activity between groups. In the test session, there was a significant reduction in both crossings and rearings in the test when compared with training in the WT and HT groups. In the KO group, there was no reduction in the number of crossings and rearings in the test when compared with training, suggesting memory impairment (p0.05) (Fig. 2), suggesting impaired aversive memory. Continuous Multiple Trials Step-Down Inhibitory Avoidance Task In the continuous multiple trials step-down inhibitory avoidance, it was demonstrated a significant increase in the number of training trials required to reach the acquisition criterion (50 s on the platform) in the KO group as compared with the WT and HT groups (p0.05) (Fig. 3b). Object Recognition The KO mice presented impairment of novel object recognition memory, i.e., they did not spend a significantly higher percentage of time exploring the novel object (p>0.05). The
Fig. 1 Habituation on the open field. Number of crossings and rearings are presented as mean±SEM, *p
