Glutamic acid decarboxylase 67 haplodeficiency impairs social behavior in mice Kiran Veer Sandhua, Daniel Langa, Bettina Müllera, Sven Nullmeierb, Yuchio Yanagawac, Herbert Schweglerb,d, Oliver Storka,d,* a
Department of Genetics & Molecular Neurobiology, Institute of Biology, Otto-von-Guericke
University Magdeburg, 39120 Magdeburg, Germany b
Institute of Anatomy, Otto-von-Guericke University Magdeburg, 39120 Magdeburg,
Germany c
Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School
of Medicine and JST, CREST, Maebashi 371-8511, Japan d
Center for Behavioral Brain Sciences, Magdeburg, 39120 Magdeburg, Germany
* Corresponding author at: Department of Genetics and Molecular Neurobiology, Institute of Biology, Otto-vonGuericke-University Magdeburg, Leipziger Str. 44, Hs91, 39120 Magdeburg, Germany Tel ++49-391-6755100 Fax ++49-391-6755101 Email address [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/gbb.12131
This article is protected by copyright. All rights reserved
Abstract Reduced glutamic acid decarboxylase (GAD)67 expression may be causally involved in the development of social withdrawal in neuropsychiatric states such as autism, schizophrenia and bipolar disorder. In this study, we report disturbance of social behavior in male GAD67 haplodeficient mice. GAD67+/- mice, compared to GAD67+/+ littermates, show reduced sociability and decreased intermale aggression, but normal nest building and urine marking behavior, as well as unchanged locomotor activity and anxiety-like behavior. Moreover, the mutants display a reduced sensitivity to both social and non-social odors, indicating a disturbance in the detection and/or processing of socially relevant olfactory stimuli. Indeed, we observed reduced activation of the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, medial and cortical amygdala upon exposure of GAD67+/- mice to social interaction paradigm, as indicated by c-Fos immunohistochemistry. These data suggest a disturbance of stimulus processing in the brain circuitry controlling social behavior in GAD67+/- mice, which may provide a useful model for studying the impact of a reduced GAD67 expression on alterations of social behavior related to neuropsychiatric disorders.
Keywords: GAD67;
GABA;
social
interaction;
aggressive
behavior;
social
odor;
c-Fos
immunohistochemistry; olfactory bulb; amygdala; animal model.
Introduction Glutamate decarboxylase (GAD) is the key enzyme for the synthesis of γ-aminobutyric acid (GABA) and critical for its developmental, homeostatic and activity-dependent regulation (for review, see Obata, 2013). GAD occurs in two major isoforms, GAD65 and GAD67, which are functionally equivalent and co-expressed in the vast majority of GABAergic neurons (Esclapez et al., 1993, 1994). However, they are encoded by different genes and are differentially regulated in their expression, intracellular distribution and activity-dependent
This article is protected by copyright. All rights reserved
regulation (Kanaani et al., 2010). With the generation of mouse mutants for GAD65 and GAD67 it became feasible to discriminate their roles in brain function (Asada et al., 1996, 1997; Tamamaki et al., 2003). Indeed, specific effects of GAD65 and GAD67 mutation have been reported in the development of visual cortex and cerebellum, respectively (e.g., Hensch et al., 1998; Nakayama et al., 2012). While homozygous GAD67-/- mice die early postnatal due to the development of cleft palate (Asada et al., 1997), GAD67+/- mice are viable and show largely normal brain morphology (Tamamaki et al., 2003). These mice carry a knock-in of the green fluorescence protein (GFP), resulting in an allele with non-functional GAD67. They show an overall 40% reduction of GAD67 expression and a 16% reduction of brain GABA levels in the young adult, but normal levels of GAD65 and the vesicular GABA transporter in the brain (Tamamaki et al., 2003; Wang et al., 2009), and allow us to investigate consequences of reduced GAD67-dependent neural functions in vivo. Intriguingly, GAD67+/- mice display an increased vulnerability to maternal and fetal stress (Uchida et al., 2011), which are important factors for various neuropsychiatric disturbances (Markham & Koenig, 2011). Indeed, genetic variation (Addington et al., 2005; Straub et al., 2007) and reduced expression of GAD67 have been associated with schizophrenia, autism and bipolar disorder (Fatemi et al., 2002; Guidotti et al., 2000; Hashimoto et al., 2003; Heckers et al., 2002). Disruption of social behavior is a core feature of these disorders (Abdi & Sharma, 2004; Couture et al., 2006; Pelphrey et al., 2005), but the role of GAD67 in the development of social symptoms remains to be clarified. In healthy subjects, stimulation of GABAergic signaling with benzodiazepines modulates social interaction in a dosage dependent manner (Lane & Gowin, 2009). In mice, disruption of GABAergic interneuron development during the early postnatal period results in social deficits (Levitt, 2005). Moreover, the, GABAA modulator imidazenil has been shown to correct social deficits evoked by hypermethylation of the GAD67 promoter and downregulation of GAD67 expression (Tremolizzo et al., 2005). Further, GABA under various conditions has been associated with aggressive behavior both in humans and rodents (Miczek et al., 2003; Yanowitch & Coccaro, 2011). Therefore, in this study we investigated the potential role of GAD67 in social behavior, using the established model of GAD67+/- mice. In humans, males are particularly susceptible to the development of psychiatric disorders like schizophrenia (Aleman et al; 2003; Castle et al., 1998). As we furthermore tried to minimize potential confounds through hormonal effects on the GAD67 gene, we focused our current analysis on the behavior of male GAD67+/- mice, This article is protected by copyright. All rights reserved
notwithstanding the potential role of GAD67 in female and intergender social behavior. Our data reveal a profound disturbance of social affiliation and aggressive behavior of male haplodeficient GAD67 mutants. This is associated with a reduced sensitivity both for social and non-social odors, and is reflected with reduction of social interaction-induced c-Fos expression in brain circuits controlling social behavior.
Methods Animals GAD67 mutants examined in this study carry insertion of a green fluorescence protein in the GAD67 gene (Tamamaki et al., 2003) and have been widely used as a tool to identify GABAergic neurons and related morphological and physiological functions (e.g., Meis et al., 2008; Mueller et al., 2012). Mutants were backcrossed to C57Bl/6Ola for 12 generations and bred in our animal facility. Fifteen-to-sixteen week old male GAD67+/- mice and their GAD67+/+ male littermates were used in all experiments. Animals were raised and kept in littermate groups of 3-5 under a 12:12 reverse light/dark cycle (lights off at 7:00 with a 30min dawn phase) with food and water ad libitum. Genotypes were determined with allele-specific PCR at the time of weaning as described previously (Meis et al., 2008). Unless stated otherwise animals were experimentally naïve and single caged one week prior to the behavioral test. All behavioral experiments were performed between 10:00 and 16:00, i.e. during the active phase of both genotypes. All procedures were performed under strict observance of German regulations for the use of laboratory animals (animal permission Nr. 42502-2-887 UniMD). Social choice test Sociability was tested in an apparatus consisting of a grey opaque acrylic box (41.5x30.5x61.5cm) that was divided into three interconnected compartments (each 19.8x41.5cm) by two clear acrylic glass partitions with a centrally placed opening in the bottom (6x6cm). Wire mesh tubes (12.5x7.5cm cylinders, closed at the top with a lid) used for controllable exposure to male and female test partners were placed in the center of each outer compartment. Unfamiliar naïve C57BL/6J mice (N=17) of same age were used as stimulus mice (Silverman et al., 2010); they were handled extensively but not habituated to
This article is protected by copyright. All rights reserved
the test apparatus before the experiment. Stimulus mice that did not voluntarily enter the tube or tried to escape were not used for the experiment. During the initial 5min habituation session animals (N=8 male GAD67+/- and N=9 male GAD67+/+ mice) were placed in the center compartment and allowed to explore freely all the three compartments. Then, one interaction partner was randomly placed in either of the tubes and animal behavior was recorded for a period of 10min, using a video camera positioned directly above the apparatus. The number of entries to each compartment and the time spent in each compartment were automatically measured with AnyMaze software (Version 4.50, Stoelting, Wood Dale IL). Additionally, the number of direct contacts and the time of such contact with either of the tubes were scored manually. Preference indices were calculated as: ((tube1-tube2)/(tube1+tube2))*100. After every test, the apparatus was cleaned with 70% alcohol and then thoroughly with water to remove any traces of olfactory stimuli from the previous animal. Another naïve group of animals (N=9 male GAD67+/- and N=8 male GAD67+/+ mice) was exposed simultaneously to one unfamiliar male (N=17) and one unfamiliar female (N=17) (both C57BL/6Ola, 12-13 weeks old), placed in either of the two tubes in the outer chamber. Female stimulus mice were employed to control for the possibility of a selective disturbance in intermale social behavior, and in order to minimize sexually motivated exploration were in diestrus phase as determined by visual inspection of the vagina (Byers et al., 2012). The positioning of stimuli mice was randomized and behavior measurements were performed as described above. Intruder aggression test Resident males (N=8 GAD67+/- and N=10 GAD67+/+ mice, 14-16 weeks old at the time of testing) were housed individually for four weeks prior to the experiment. After 10min of habituation to test conditions, they were confronted with a six-to-eight week old, group housed male C57BL/6 intruder in a single 5min test session. Each intruder was used only in a single experiment to avoid experimentally induced bias; moreover, age and weight difference (20-25g intruder vs. >30g residents) were chosen to ensure that the social status of the intruder in his peer group would not significantly affect the behavior in the test session. Latencies for first frontal contact and anogenital sniffing, mounting and first attack as well as the number of mounts, chases and attack were measured by an experience observer blind to the animal’s genotype (Stork et al., 2000).
This article is protected by copyright. All rights reserved
Home cage activity Home cage activity was measured in single housed animals (N=13 male GAD67+/- and N=10 male GAD67+/+ mice) over a period of 72h. Animals were transferred to a fresh cage and after 24h of habituation, activity was monitored using an infrared detection system in 15s bins (HCA10-V2.1, Coulbourn Instr., Whitehall PA). Activity values were expressed in the 5min periods of activity or inactivity, with a threshold of 3 large movements recorded during each 5min bin. Nest building test Test animals (N=8 male GAD67+/- and N=10 male GAD67+/+ mice) were previously used to assess the home cage activity. 5 days after the test they were transferred to a new individual home cage 1h before the beginning of the dark phase with no bedding material available. After 24h, nestlets (EBECO, Castrop-Rauxel, Germany) of 2.2 g standardized weight was placed in the food hopper, and the weight of the collected nest material was determined. In addition, nest quality was scored qualitatively according to an established scale, rating 1 for primitive flat nest with slight elevated bedding, 2 for a more complex nest with high walls, 3 for nest resembling a cup shape structure and 4 for a hooded nest with high walls around and slightly above resembling a ceiling (Albrecht & Stork, 2012; Deacon, 2006). Urine marking test Animals (N=9 male GAD67+/- and N=9 male GAD67+/+ mice) were single caged one week prior to the test. The test mice were moved into a novel standard mice cage lined with filter paper (32x15.5cm) at the bottom of the cage kept in a separate experiment room. Animals were kept at 5lux dim light in the cage for 60min to leave pheromone marks before the filter papers were collected and sprayed with 0.1% ninhydrin spray (Sigma-Aldrich, St. Louis, MO) under a hood. After 24h of drying the filter papers with urine markings were scanned and quantified with Image-J software. Scoring was done by an experimenter, who was blind with respect to the animals’ genotype (Drickamer, 2001). Open field test Locomotor activity and anxiety related behavior in mice was measured with an open-field test (N=10 male GAD67+/- and N=10 male GAD67+/+ mice). The apparatus comprised of a square arena (50x50x35cm) with a dark floor and black acrylic glass walls. The apparatus was illuminated with red light. Mice were placed in the center (a medial square of 25x25cm) of the apparatus and the distance travelled as well as the number of entries and the time in the This article is protected by copyright. All rights reserved
center and the 12.5cm wide rim were video recorded over 20min. All measurements were automatically measured and evaluated using the AnyMaze software. Light/dark avoidance test The test system comprised of a rectangular chamber with a 19x21cm light compartment (100lux) made of white acrylic glass and equally sized dark compartment (
Department of Genetics & Molecular Neurobiology, Institute of Biology, Otto-von-Guericke
University Magdeburg, 39120 Magdeburg, Germany b
Institute of Anatomy, Otto-von-Guericke University Magdeburg, 39120 Magdeburg,
Germany c
Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School
of Medicine and JST, CREST, Maebashi 371-8511, Japan d
Center for Behavioral Brain Sciences, Magdeburg, 39120 Magdeburg, Germany
* Corresponding author at: Department of Genetics and Molecular Neurobiology, Institute of Biology, Otto-vonGuericke-University Magdeburg, Leipziger Str. 44, Hs91, 39120 Magdeburg, Germany Tel ++49-391-6755100 Fax ++49-391-6755101 Email address [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/gbb.12131
This article is protected by copyright. All rights reserved
Abstract Reduced glutamic acid decarboxylase (GAD)67 expression may be causally involved in the development of social withdrawal in neuropsychiatric states such as autism, schizophrenia and bipolar disorder. In this study, we report disturbance of social behavior in male GAD67 haplodeficient mice. GAD67+/- mice, compared to GAD67+/+ littermates, show reduced sociability and decreased intermale aggression, but normal nest building and urine marking behavior, as well as unchanged locomotor activity and anxiety-like behavior. Moreover, the mutants display a reduced sensitivity to both social and non-social odors, indicating a disturbance in the detection and/or processing of socially relevant olfactory stimuli. Indeed, we observed reduced activation of the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, medial and cortical amygdala upon exposure of GAD67+/- mice to social interaction paradigm, as indicated by c-Fos immunohistochemistry. These data suggest a disturbance of stimulus processing in the brain circuitry controlling social behavior in GAD67+/- mice, which may provide a useful model for studying the impact of a reduced GAD67 expression on alterations of social behavior related to neuropsychiatric disorders.
Keywords: GAD67;
GABA;
social
interaction;
aggressive
behavior;
social
odor;
c-Fos
immunohistochemistry; olfactory bulb; amygdala; animal model.
Introduction Glutamate decarboxylase (GAD) is the key enzyme for the synthesis of γ-aminobutyric acid (GABA) and critical for its developmental, homeostatic and activity-dependent regulation (for review, see Obata, 2013). GAD occurs in two major isoforms, GAD65 and GAD67, which are functionally equivalent and co-expressed in the vast majority of GABAergic neurons (Esclapez et al., 1993, 1994). However, they are encoded by different genes and are differentially regulated in their expression, intracellular distribution and activity-dependent
This article is protected by copyright. All rights reserved
regulation (Kanaani et al., 2010). With the generation of mouse mutants for GAD65 and GAD67 it became feasible to discriminate their roles in brain function (Asada et al., 1996, 1997; Tamamaki et al., 2003). Indeed, specific effects of GAD65 and GAD67 mutation have been reported in the development of visual cortex and cerebellum, respectively (e.g., Hensch et al., 1998; Nakayama et al., 2012). While homozygous GAD67-/- mice die early postnatal due to the development of cleft palate (Asada et al., 1997), GAD67+/- mice are viable and show largely normal brain morphology (Tamamaki et al., 2003). These mice carry a knock-in of the green fluorescence protein (GFP), resulting in an allele with non-functional GAD67. They show an overall 40% reduction of GAD67 expression and a 16% reduction of brain GABA levels in the young adult, but normal levels of GAD65 and the vesicular GABA transporter in the brain (Tamamaki et al., 2003; Wang et al., 2009), and allow us to investigate consequences of reduced GAD67-dependent neural functions in vivo. Intriguingly, GAD67+/- mice display an increased vulnerability to maternal and fetal stress (Uchida et al., 2011), which are important factors for various neuropsychiatric disturbances (Markham & Koenig, 2011). Indeed, genetic variation (Addington et al., 2005; Straub et al., 2007) and reduced expression of GAD67 have been associated with schizophrenia, autism and bipolar disorder (Fatemi et al., 2002; Guidotti et al., 2000; Hashimoto et al., 2003; Heckers et al., 2002). Disruption of social behavior is a core feature of these disorders (Abdi & Sharma, 2004; Couture et al., 2006; Pelphrey et al., 2005), but the role of GAD67 in the development of social symptoms remains to be clarified. In healthy subjects, stimulation of GABAergic signaling with benzodiazepines modulates social interaction in a dosage dependent manner (Lane & Gowin, 2009). In mice, disruption of GABAergic interneuron development during the early postnatal period results in social deficits (Levitt, 2005). Moreover, the, GABAA modulator imidazenil has been shown to correct social deficits evoked by hypermethylation of the GAD67 promoter and downregulation of GAD67 expression (Tremolizzo et al., 2005). Further, GABA under various conditions has been associated with aggressive behavior both in humans and rodents (Miczek et al., 2003; Yanowitch & Coccaro, 2011). Therefore, in this study we investigated the potential role of GAD67 in social behavior, using the established model of GAD67+/- mice. In humans, males are particularly susceptible to the development of psychiatric disorders like schizophrenia (Aleman et al; 2003; Castle et al., 1998). As we furthermore tried to minimize potential confounds through hormonal effects on the GAD67 gene, we focused our current analysis on the behavior of male GAD67+/- mice, This article is protected by copyright. All rights reserved
notwithstanding the potential role of GAD67 in female and intergender social behavior. Our data reveal a profound disturbance of social affiliation and aggressive behavior of male haplodeficient GAD67 mutants. This is associated with a reduced sensitivity both for social and non-social odors, and is reflected with reduction of social interaction-induced c-Fos expression in brain circuits controlling social behavior.
Methods Animals GAD67 mutants examined in this study carry insertion of a green fluorescence protein in the GAD67 gene (Tamamaki et al., 2003) and have been widely used as a tool to identify GABAergic neurons and related morphological and physiological functions (e.g., Meis et al., 2008; Mueller et al., 2012). Mutants were backcrossed to C57Bl/6Ola for 12 generations and bred in our animal facility. Fifteen-to-sixteen week old male GAD67+/- mice and their GAD67+/+ male littermates were used in all experiments. Animals were raised and kept in littermate groups of 3-5 under a 12:12 reverse light/dark cycle (lights off at 7:00 with a 30min dawn phase) with food and water ad libitum. Genotypes were determined with allele-specific PCR at the time of weaning as described previously (Meis et al., 2008). Unless stated otherwise animals were experimentally naïve and single caged one week prior to the behavioral test. All behavioral experiments were performed between 10:00 and 16:00, i.e. during the active phase of both genotypes. All procedures were performed under strict observance of German regulations for the use of laboratory animals (animal permission Nr. 42502-2-887 UniMD). Social choice test Sociability was tested in an apparatus consisting of a grey opaque acrylic box (41.5x30.5x61.5cm) that was divided into three interconnected compartments (each 19.8x41.5cm) by two clear acrylic glass partitions with a centrally placed opening in the bottom (6x6cm). Wire mesh tubes (12.5x7.5cm cylinders, closed at the top with a lid) used for controllable exposure to male and female test partners were placed in the center of each outer compartment. Unfamiliar naïve C57BL/6J mice (N=17) of same age were used as stimulus mice (Silverman et al., 2010); they were handled extensively but not habituated to
This article is protected by copyright. All rights reserved
the test apparatus before the experiment. Stimulus mice that did not voluntarily enter the tube or tried to escape were not used for the experiment. During the initial 5min habituation session animals (N=8 male GAD67+/- and N=9 male GAD67+/+ mice) were placed in the center compartment and allowed to explore freely all the three compartments. Then, one interaction partner was randomly placed in either of the tubes and animal behavior was recorded for a period of 10min, using a video camera positioned directly above the apparatus. The number of entries to each compartment and the time spent in each compartment were automatically measured with AnyMaze software (Version 4.50, Stoelting, Wood Dale IL). Additionally, the number of direct contacts and the time of such contact with either of the tubes were scored manually. Preference indices were calculated as: ((tube1-tube2)/(tube1+tube2))*100. After every test, the apparatus was cleaned with 70% alcohol and then thoroughly with water to remove any traces of olfactory stimuli from the previous animal. Another naïve group of animals (N=9 male GAD67+/- and N=8 male GAD67+/+ mice) was exposed simultaneously to one unfamiliar male (N=17) and one unfamiliar female (N=17) (both C57BL/6Ola, 12-13 weeks old), placed in either of the two tubes in the outer chamber. Female stimulus mice were employed to control for the possibility of a selective disturbance in intermale social behavior, and in order to minimize sexually motivated exploration were in diestrus phase as determined by visual inspection of the vagina (Byers et al., 2012). The positioning of stimuli mice was randomized and behavior measurements were performed as described above. Intruder aggression test Resident males (N=8 GAD67+/- and N=10 GAD67+/+ mice, 14-16 weeks old at the time of testing) were housed individually for four weeks prior to the experiment. After 10min of habituation to test conditions, they were confronted with a six-to-eight week old, group housed male C57BL/6 intruder in a single 5min test session. Each intruder was used only in a single experiment to avoid experimentally induced bias; moreover, age and weight difference (20-25g intruder vs. >30g residents) were chosen to ensure that the social status of the intruder in his peer group would not significantly affect the behavior in the test session. Latencies for first frontal contact and anogenital sniffing, mounting and first attack as well as the number of mounts, chases and attack were measured by an experience observer blind to the animal’s genotype (Stork et al., 2000).
This article is protected by copyright. All rights reserved
Home cage activity Home cage activity was measured in single housed animals (N=13 male GAD67+/- and N=10 male GAD67+/+ mice) over a period of 72h. Animals were transferred to a fresh cage and after 24h of habituation, activity was monitored using an infrared detection system in 15s bins (HCA10-V2.1, Coulbourn Instr., Whitehall PA). Activity values were expressed in the 5min periods of activity or inactivity, with a threshold of 3 large movements recorded during each 5min bin. Nest building test Test animals (N=8 male GAD67+/- and N=10 male GAD67+/+ mice) were previously used to assess the home cage activity. 5 days after the test they were transferred to a new individual home cage 1h before the beginning of the dark phase with no bedding material available. After 24h, nestlets (EBECO, Castrop-Rauxel, Germany) of 2.2 g standardized weight was placed in the food hopper, and the weight of the collected nest material was determined. In addition, nest quality was scored qualitatively according to an established scale, rating 1 for primitive flat nest with slight elevated bedding, 2 for a more complex nest with high walls, 3 for nest resembling a cup shape structure and 4 for a hooded nest with high walls around and slightly above resembling a ceiling (Albrecht & Stork, 2012; Deacon, 2006). Urine marking test Animals (N=9 male GAD67+/- and N=9 male GAD67+/+ mice) were single caged one week prior to the test. The test mice were moved into a novel standard mice cage lined with filter paper (32x15.5cm) at the bottom of the cage kept in a separate experiment room. Animals were kept at 5lux dim light in the cage for 60min to leave pheromone marks before the filter papers were collected and sprayed with 0.1% ninhydrin spray (Sigma-Aldrich, St. Louis, MO) under a hood. After 24h of drying the filter papers with urine markings were scanned and quantified with Image-J software. Scoring was done by an experimenter, who was blind with respect to the animals’ genotype (Drickamer, 2001). Open field test Locomotor activity and anxiety related behavior in mice was measured with an open-field test (N=10 male GAD67+/- and N=10 male GAD67+/+ mice). The apparatus comprised of a square arena (50x50x35cm) with a dark floor and black acrylic glass walls. The apparatus was illuminated with red light. Mice were placed in the center (a medial square of 25x25cm) of the apparatus and the distance travelled as well as the number of entries and the time in the This article is protected by copyright. All rights reserved
center and the 12.5cm wide rim were video recorded over 20min. All measurements were automatically measured and evaluated using the AnyMaze software. Light/dark avoidance test The test system comprised of a rectangular chamber with a 19x21cm light compartment (100lux) made of white acrylic glass and equally sized dark compartment (
