Mol. Cells 2015; 38(6): 540-547
Published online May 22, 2015
https://doi.org/10.14348/molcells.2015.0041
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: wonmah@knu.ac.kr
Attention deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders, affecting approximately 5% of children. However, the neural mechanisms underlying its development and treatment are yet to be elucidated. In this study, we report that an ADHD mouse model, which harbors a deletion in the
Keywords ADHD, astrocytosis, basal ganglia,
Attention-deficit/hyperactivity disorder (ADHD) is a prevalent psychiatric disorder that affects approximately 5% of children worldwide. It is characterized by inattention, hyperactivity, and impulsivity. Psychostimulants, such as amphetamine and methylphenidate, are frequently used to treat individuals with ADHD. These medications increase the levels of monoamine neurotransmitters at synapses, suggesting that a deficit in monoamines may contribute to ADHD (Biederman, 2005; Swanson et al., 1998; 2007).
In addition, both clinical and genetic studies support the involvement of monoamines in the etiology of ADHD. Several clinical studies have reported dopamine depletion and decreased activity of monoamine-related brain circuits in ADHD (Shaywitz et al., 1977; Volkow et al., 2007). Genome-wide association studies have revealed several chromosomal loci containing dopamine and norepinephrine-related genes that are associated with ADHD (Ogdie et al., 2004). In line with these findings, several ADHD animal models, such as the Spontaneously Hypertensive Rat (SHR), Coloboma mouse with a
The dopaminergic signaling pathway incorporates interconnected brain regions that form the basal ganglia (cortico-striato-pallido-thalamic) circuit (DeLong and Wichmann, 2007). Dopaminergic neurons in the striatum can regulate thalamic nuclei
In addition to the dopamine deficit hypothesis, there is evidence to suggest a role for astrocytes in ADHD pathophysiology. Astrocytes are important for modulation of synaptic transmission, GABA-mediated tonic inhibition, glutamate metabolism, and supply of nutrients to neurons (Araque et al., 1998; Lee et al., 2010; Sonnewald et al., 1997). Their impairment is related to various neurological disorders (De Keyser et al., 2008). Impaired astrocytic modulation of the neuronal energy metabolism has been postulated as a candidate for the etiology of ADHD (Todd and Botteron, 2001). In addition, a recent study has demonstrated that an astrocyte-specific perturbation of SynCAM induces ADHD-like behavioral symptoms in mice (Sandau et al., 2012). This implies that impaired communication between astrocytes and neurons may cause ADHD-related symptoms.
Astrocytosis refers to an abnormal increase in the number of reactive astrocytes resulting from the death of nearby neurons. Reactive astrocytes are distinguished by their swollen cell body and altered expression of various proteins, including glial fibrillary acidic protein (GFAP), vimentin, and glutamine synthetase (Eid et al., 2004; Halassa and Haydon, 2010). The expression of glutamine synthetase, a key enzyme that mediates the conversion of glutamate into glutamine, is decreased in patients with epileptic seizures (Eid et al., 2004), which indicates that impaired astrocytic function may cause abnormal neuronal activity.
G protein-coupled receptor kinase-interacting protein-1 (
In the present study, we performed immunohistochemical analysis in the brains of
Antibodies against GluR2 (1:1000; #1195) were previously generated in our laboratory (Kim et al., 2009). The following antibodies were purchased: GFAP (1:1000), GABA (1:1000; Abcam); Iba1 (0.5 μg·ml?1; Wako); vGlut1 (2 μg·ml?1), vGAT (1:500; Synaptic Systems); NeuN (2.5 μg·ml?1), GAD67 (2 μg·ml?1), glutamine synthetase (2 μg·ml?1), parvalbumin (1:1000; Millipore); Bassoon (1:1000; Stressgen). The optimal concentration of antibodies was determined according to the manufacturer’s recommended protocols.
Brains were isolated from adult mice (4 weeks or 2?3 months old) after cardiac perfusion (4% paraformaldehyde). Following post-fixation for 12 h, 50-μm sections were obtained using a vibratome (Leica). Sections were washed 3 times with PBS for 10 min, permeabilized with 0.5% Triton X-100 for 30 min, blocked with 5% bovine serum albumin (BSA) for 1 h, stained with primary antibodies at 4°C for 12 h, stained with secondary antibodies for 1 h, and mounted with VECTASHIELD. For quantitative analysis, images were captured with a confocal microscope (63× and 20× objectives; Leica Microsystems) and analyzed using Metamorph (Molecular Devices). We measured the strength of immunoreactivity from both the cell soma and processes.
We hypothesized that the ADHD-like symptoms in
This feature could represent reactive astrocytosis, which is often caused by neuronal damage or loss and can lead to neuroinflammation. However, the number of neurons and microglia were unchanged in the affected regions, including the GP and TRN (Figs. 1C and 4). In addition, glutamine synthetase, which is typically downregulated during reactive astrocytosis (Eid et al., 2004; Ortinski et al., 2010), was unchanged or increased in these regions (Fig. 5). This excluded the possibility of neuroinflammation and reactive astrocytosis.
Astrocytes play an active role in neuronal transmission in the tripartite synapse model (Volterra and Meldolesi, 2005). There was no sign of neuroinflammation in the regions showing severe astrocytosis of
Notably, there was a significant increase in expression of the inhibitory neurotransmitter, GABA, in the GP, but not the TRN (Figs. 6A?6D). In addition, GABA was colocalized with GFAP-positive astrocytes but not Iba1-positive microglia in both 2?3-month-old and 4-week-old
In addition, two thalamic regions (ventrobasal and ventrolateral) showed reduced signals of parvalbumin (Figs. 8C?8F); however, no changes were observed in the striatum, including the caudate nucleus and putamen (Figs. 8A and 8B). Taken together, the astrocytosis and immunohistochemical data suggested that the GP, and related brain regions involved in the basal ganglia pathway, may have functional defects in
We have reported severe astrocytosis in specific brain regions of
The basal ganglia comprise a collection of subcortical nuclei that are associated with modulation of motor activities, and their dysfunction is linked to motor defects in both Parkinson’s and Huntington’s disease (DeLong and Wichmann, 2007; Graybiel, 2000; Kravitz et al., 2010). Due to the severe astrocytosis and increased GABA levels in the GP of
Astrocytes are a well-known source of glutamine for neurons, which regulates their excitability
Increased GABA in the GP may arise
Although the source of the increased GABA is unclear, it is likely to significantly inhibit GP GABAergic neurons in
Our finding of increased GABA in the GP is in contrast to a previous study that shows decreased inhibition in the hippocampus of
It is unclear why severe astrocytosis was observed in specific brain regions and how this affected parvalbumin-positive GABAergic interneuron functioning. The astrocytosis observed differed from conventional astrocytosis, which is associated with neuronal loss and neuroinflammation, suggesting that it occurred in a cell-autonomous manner.
It is important to address how astrocytosis might affect behavioral manifestations in
In conclusion, we have observed severe astrocytosis and an altered expression of GABA and parvalbumin in the brain regions related to basal ganglia circuitry in
Mol. Cells 2015; 38(6): 540-547
Published online June 30, 2015 https://doi.org/10.14348/molcells.2015.0041
Copyright © The Korean Society for Molecular and Cellular Biology.
Soo-Yeon Lim1, and Won Mah1,2,*
1Department of Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu 700-412, Korea, 2Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea
Correspondence to:*Correspondence: wonmah@knu.ac.kr
Attention deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders, affecting approximately 5% of children. However, the neural mechanisms underlying its development and treatment are yet to be elucidated. In this study, we report that an ADHD mouse model, which harbors a deletion in the
Keywords: ADHD, astrocytosis, basal ganglia,
Attention-deficit/hyperactivity disorder (ADHD) is a prevalent psychiatric disorder that affects approximately 5% of children worldwide. It is characterized by inattention, hyperactivity, and impulsivity. Psychostimulants, such as amphetamine and methylphenidate, are frequently used to treat individuals with ADHD. These medications increase the levels of monoamine neurotransmitters at synapses, suggesting that a deficit in monoamines may contribute to ADHD (Biederman, 2005; Swanson et al., 1998; 2007).
In addition, both clinical and genetic studies support the involvement of monoamines in the etiology of ADHD. Several clinical studies have reported dopamine depletion and decreased activity of monoamine-related brain circuits in ADHD (Shaywitz et al., 1977; Volkow et al., 2007). Genome-wide association studies have revealed several chromosomal loci containing dopamine and norepinephrine-related genes that are associated with ADHD (Ogdie et al., 2004). In line with these findings, several ADHD animal models, such as the Spontaneously Hypertensive Rat (SHR), Coloboma mouse with a
The dopaminergic signaling pathway incorporates interconnected brain regions that form the basal ganglia (cortico-striato-pallido-thalamic) circuit (DeLong and Wichmann, 2007). Dopaminergic neurons in the striatum can regulate thalamic nuclei
In addition to the dopamine deficit hypothesis, there is evidence to suggest a role for astrocytes in ADHD pathophysiology. Astrocytes are important for modulation of synaptic transmission, GABA-mediated tonic inhibition, glutamate metabolism, and supply of nutrients to neurons (Araque et al., 1998; Lee et al., 2010; Sonnewald et al., 1997). Their impairment is related to various neurological disorders (De Keyser et al., 2008). Impaired astrocytic modulation of the neuronal energy metabolism has been postulated as a candidate for the etiology of ADHD (Todd and Botteron, 2001). In addition, a recent study has demonstrated that an astrocyte-specific perturbation of SynCAM induces ADHD-like behavioral symptoms in mice (Sandau et al., 2012). This implies that impaired communication between astrocytes and neurons may cause ADHD-related symptoms.
Astrocytosis refers to an abnormal increase in the number of reactive astrocytes resulting from the death of nearby neurons. Reactive astrocytes are distinguished by their swollen cell body and altered expression of various proteins, including glial fibrillary acidic protein (GFAP), vimentin, and glutamine synthetase (Eid et al., 2004; Halassa and Haydon, 2010). The expression of glutamine synthetase, a key enzyme that mediates the conversion of glutamate into glutamine, is decreased in patients with epileptic seizures (Eid et al., 2004), which indicates that impaired astrocytic function may cause abnormal neuronal activity.
G protein-coupled receptor kinase-interacting protein-1 (
In the present study, we performed immunohistochemical analysis in the brains of
Antibodies against GluR2 (1:1000; #1195) were previously generated in our laboratory (Kim et al., 2009). The following antibodies were purchased: GFAP (1:1000), GABA (1:1000; Abcam); Iba1 (0.5 μg·ml?1; Wako); vGlut1 (2 μg·ml?1), vGAT (1:500; Synaptic Systems); NeuN (2.5 μg·ml?1), GAD67 (2 μg·ml?1), glutamine synthetase (2 μg·ml?1), parvalbumin (1:1000; Millipore); Bassoon (1:1000; Stressgen). The optimal concentration of antibodies was determined according to the manufacturer’s recommended protocols.
Brains were isolated from adult mice (4 weeks or 2?3 months old) after cardiac perfusion (4% paraformaldehyde). Following post-fixation for 12 h, 50-μm sections were obtained using a vibratome (Leica). Sections were washed 3 times with PBS for 10 min, permeabilized with 0.5% Triton X-100 for 30 min, blocked with 5% bovine serum albumin (BSA) for 1 h, stained with primary antibodies at 4°C for 12 h, stained with secondary antibodies for 1 h, and mounted with VECTASHIELD. For quantitative analysis, images were captured with a confocal microscope (63× and 20× objectives; Leica Microsystems) and analyzed using Metamorph (Molecular Devices). We measured the strength of immunoreactivity from both the cell soma and processes.
We hypothesized that the ADHD-like symptoms in
This feature could represent reactive astrocytosis, which is often caused by neuronal damage or loss and can lead to neuroinflammation. However, the number of neurons and microglia were unchanged in the affected regions, including the GP and TRN (Figs. 1C and 4). In addition, glutamine synthetase, which is typically downregulated during reactive astrocytosis (Eid et al., 2004; Ortinski et al., 2010), was unchanged or increased in these regions (Fig. 5). This excluded the possibility of neuroinflammation and reactive astrocytosis.
Astrocytes play an active role in neuronal transmission in the tripartite synapse model (Volterra and Meldolesi, 2005). There was no sign of neuroinflammation in the regions showing severe astrocytosis of
Notably, there was a significant increase in expression of the inhibitory neurotransmitter, GABA, in the GP, but not the TRN (Figs. 6A?6D). In addition, GABA was colocalized with GFAP-positive astrocytes but not Iba1-positive microglia in both 2?3-month-old and 4-week-old
In addition, two thalamic regions (ventrobasal and ventrolateral) showed reduced signals of parvalbumin (Figs. 8C?8F); however, no changes were observed in the striatum, including the caudate nucleus and putamen (Figs. 8A and 8B). Taken together, the astrocytosis and immunohistochemical data suggested that the GP, and related brain regions involved in the basal ganglia pathway, may have functional defects in
We have reported severe astrocytosis in specific brain regions of
The basal ganglia comprise a collection of subcortical nuclei that are associated with modulation of motor activities, and their dysfunction is linked to motor defects in both Parkinson’s and Huntington’s disease (DeLong and Wichmann, 2007; Graybiel, 2000; Kravitz et al., 2010). Due to the severe astrocytosis and increased GABA levels in the GP of
Astrocytes are a well-known source of glutamine for neurons, which regulates their excitability
Increased GABA in the GP may arise
Although the source of the increased GABA is unclear, it is likely to significantly inhibit GP GABAergic neurons in
Our finding of increased GABA in the GP is in contrast to a previous study that shows decreased inhibition in the hippocampus of
It is unclear why severe astrocytosis was observed in specific brain regions and how this affected parvalbumin-positive GABAergic interneuron functioning. The astrocytosis observed differed from conventional astrocytosis, which is associated with neuronal loss and neuroinflammation, suggesting that it occurred in a cell-autonomous manner.
It is important to address how astrocytosis might affect behavioral manifestations in
In conclusion, we have observed severe astrocytosis and an altered expression of GABA and parvalbumin in the brain regions related to basal ganglia circuitry in
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