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. 2015 Jun;40(7):1569-79.
doi: 10.1038/npp.2015.26. Epub 2015 Feb 9.

Astroglial glutamate transporter deficiency increases synaptic excitability and leads to pathological repetitive behaviors in mice

Tomomi Aida  1 , Junichi Yoshida  1 , Masatoshi Nomura  2 , Asami Tanimura  3 , Yusuke Iino  1 , Miho Soma  1 , Ning Bai  1 , Yukiko Ito  1 , Wanpeng Cui  1 , Hidenori Aizawa  1 , Michiko Yanagisawa  1 , Terumi Nagai  4 , Norio Takata  4 , Kenji F Tanaka  5 , Ryoichi Takayanagi  2 , Masanobu Kano  3 , Magdalena Götz  6 , Hajime Hirase  5 , Kohichi Tanaka  7
Affiliations

Astroglial glutamate transporter deficiency increases synaptic excitability and leads to pathological repetitive behaviors in mice

Tomomi Aida et al. Neuropsychopharmacology. 2015 Jun.

Abstract

An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD), and Tourette's syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLAST(CreERT2/+)/GLT1(flox/flox), iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 has a critical role in controlling the synaptic efficacy at corticostriatal synapses and its dysfunction causes pathological repetitive behaviors.

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Figures

Figure 1
Figure 1
Inducible ablation of GLT1 in adolescent mice. (a) Generation of adolescent-GLASTCreERT2/+/GLT1flox/flox mice (iKO) or control (Ctrl) mice by tamoxifen (Tmx) or oil injections. (b) Western blot analysis of GLT1 of the cerebral cortex, thalamus and striatum from postnatal day (P) 63 mice (n=3). Monomer bands of GLT1, or β-Actin are shown. GLT1 band intensities were normalized with those of β-actin. All data are presented as the mean±SEM. Student's t-tests were used to compare GLT1 protein levels of Ctrl and iKO mice for each brain region. *P<0.05, ***P<0.005. (c) GLT1 immunohistochemistry. Boxed regions were enlarged in the right panels. Scale bar: 1 mm, and 100 μm for inset. (d) Facial lesions in 12- to 16-week-old male iKO mice.
Figure 2
Figure 2
Excessive repetitive behaviors, and normal anxiety levels and social behavior in iKO mice. (a, b) Durations of self-grooming in Ctrl and iKO mice in a novel environment (n=7 for Ctrl and 8 for iKO mice, a) and in the home cage (n=6, b). (c) Hematoxylin-eosin (HE) staining of neck skin from Ctrl and pre-lesion iKO mice. Scale bar: 500 μm. (d) Hot plate test (55 °C, n=7 for Ctrl and 8 for iKO mice). (e) Durations of self-grooming in Ctrl (n=8) and iKO (n=7) mice treated with saline (blue bar) or desloratadine (DL, red bar). (f–k) Behaviors analyses. (f) Number of tic-like movements in a novel environment (n=7 for Ctrl and 8 for iKO mice). (g) Elevated plus maze test (n=22 for Ctrl and 27 for iKO mice). (h) Light-dark box test (n=21 for Ctrl and 29 for iKO mice). (i) Open field test (n=22 for Ctrl and 29 for iKO mice). (j) Reciprocal social interaction test (n=7 for Ctrl and 9 for iKO mice). (j) Three-chamber social interaction test (n=7). All data are presented as the mean±SEM. Statistical significance was calculated by Student's t-test in (a, b, d and j), by Mann–Whitney's u-tests in (f–i), and by two-way repeated measures ANOVA with post hoc t-test in (e) and (k). *P<0.05, **P<0.01, ***P<0.005.
Figure 3
Figure 3
Increased seizure severity in iKO mice. (a) Seizure severity was tested in 8-week-old-Ctrl and iKO mice (n=8 and 12, respectively) by intraperitoneal administration of 30 mg/kg kainic acid. Higher maximum seizure scores represent more severe seizure status; a score of 7 indicates mortality. Statistical significance was calculated by Mann–Whitney's u-test. **P<0.01. (b) EEG recordings from iKO mice (n=4). A representative EEG trace from somatosensory cortex is shown (upper panel). Lines represent periods of grooming bouts. Arrows indicate timing of tic-like movements. EEG traces around grooming onsets are enlarged in lower panels. Lines with gray boxes represent periods of grooming bouts. iKO mice do not show any epileptiform discharge both during resting states and grooming bouts.
Figure 4
Figure 4
Altered synaptic transmission efficacy at corticostriatal synapses in iKO mice. (a) c-fos mapping in the striatum (n=8 for Ctrl and 9 for iKO mice). c-Fos positive cells within each boxed region (red) were counted. Statistical significance was calculated by Student's t-test. Scale bars: 500 μm. (b) Western blot analysis of 16-week-old striatum crude synaptosomal fractions (n=3). Glutamate receptors (NR1, NR2A, NR2B, GluR1, and GluR2), postsynaptic density protein-95 (PSD95), synaptophysin (SYP), or β-actin were shown. Statistical significance was calculated by Student's t-test. (c) Representative traces of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs) from medium spiny neurons (MSNs). Amplitudes were measured at the peaks (leftward arrows for AMPA EPSCs and downward arrows for NMDA EPSCs) (n=8 for Ctrl and 10 for iKO mice). Interpulse intervals: 50 ms. Statistical significance was calculated by Student's t-test and Mann–Whitney's u-tests. (d) (Top) Representative traces of EPSCs from the MSNs during prolonged repetitive stimulation (10 Hz, 100 pulses). Only responses 1–5 (black) and 21–25 (red) are shown. (Bottom) The average normalized response amplitudes of EPSCs (n=5 for Ctrl and 4 for iKO mice). Each point represents the average of 10 consecutive responses. Statistical significance was calculated by two-way ANOVA with Holm-Sidak's post hoc tests. ***P<0.001.
Figure 5
Figure 5
Memantine rescued excessive grooming and tic-like movements in the iKO mice. (a, b) Durations of self-grooming (a) and number of tic-like movements (b) in the Ctrl (n=10) and iKO (n=7) mice after 6-day saline (blue bar) or fluoxetine (SSRI, red bar) treatment. (c–e) Durations of self-grooming (c), number of tic-like movements (d), or traveled distance in open field tests (e) in the Ctrl and iKO mice after 30-min saline (blue bar, n=8) or memantine (red bar, n=9 for Ctrl, and 7 for iKO mice) treatment. Statistical significance was calculated by two-way repeated measure ANOVA with post hoc t-test. *P<0.05, **P<0.01, ***P<0.005.
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