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, 34 (49), 16273-85

Glial Dysfunction in the Mouse Habenula Causes Depressive-Like Behaviors and Sleep Disturbance

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Glial Dysfunction in the Mouse Habenula Causes Depressive-Like Behaviors and Sleep Disturbance

Wanpeng Cui et al. J Neurosci.

Abstract

The lateral habenula (LHb) regulates the activity of monoaminergic neurons in the brainstem. This area has recently attracted a surge of interest in psychiatry because studies have reported the pathological activation of the habenula in patients with major depression and in animal models. The LHb plays a significant role in the pathophysiology of depression; however, how habenular neurons are activated to cause various depression symptoms, such as reduced motivation and sleep disturbance, remain unclear. We hypothesized that dysfunctional astrocytes may cause LHb hyperactivity due to the defective uptake activity of extracellular glutamate, which induces depressive-like behaviors. We examined the activity of neurons in habenular pathways and performed behavioral and sleep analyses in mice with pharmacological and genetic inhibition of the activity of the glial glutamate transporter GLT-1 in the LHb. The habenula-specific inhibition of GLT-1 increased the neuronal firing rate and the level of c-Fos expression in the LHb. Mice with reduced GLT-1 activity in the habenula exhibited a depressive-like phenotype in the tail suspension and novelty-suppressed feeding tests. These animals also displayed increased susceptibility to chronic stress, displaying more frequent avoidant behavior without affecting locomotor activity in the open-field test. Intriguingly, the mice showed disinhibition of rapid eye movement sleep, which is a characteristic sleep pattern in patients with depression. These results provide evidence that disrupting glutamate clearance in habenular astrocytes increases neuronal excitability and depressive-like phenotypes in behaviors and sleep.

Keywords: astrocytes; depression; glutamate transporters; habenula; monoamines; rapid eye movement sleep.

Figures

Figure 1.
Figure 1.
Inhibition of the glutamate transporter GLT-1 activates lateral habenular neurons. A–C, Coronal sections of the mouse habenula showing the expression of the glial markers S100β (red in A–C), GFAP (green in A), GLT-1 (green in B), and GLAST (green in C) in the medial (white asterisks) and LHb (white brackets). D, Gene expression analysis of the glial glutamate transporters in the habenula using the quantitative RT-PCR. Bar graphs represent mean values for the absolute amount of Slc1a2 (synonym for GLT-1, blue) and Slc1a3 (synonym for GLAST, red) expressed in the habenula (N = 4). E–H, High magnification of coronal sections of the mouse LHb showing the localization of GLT-1 (green in E–H), NeuN (red in E), neurofilament (red in F), GLAST (red in G), and S100β (red in H). E, H, Asterisks indicate the neuronal and astrocytic cell bodies, respectively. I, K, Representative activities of the LHb neurons in wild-type animals, which were recorded with electrodes filled with PBS (I) or with 10 mm DHK (K). J, L, Average spike shape of the neurons shown in I (blue solid line in J) and K (red solid line in L). Shaded areas represent SEM. M, N, Bar graphs showing the mean firing rate (M) and mean spike width (N) of the spikes recorded with PBS (blue, N = 14) and with DHK (red, N = 10). *Significant difference (p < 0.05, two-tailed Mann–Whitney U test). Error bars indicate SEM. NS, Not significant. Scale bars: A (applies to B, C), 200 μm; E (applies to F–H), 10 μm.
Figure 2.
Figure 2.
Effect of the activated LHb on c-Fos expression in the brainstem nuclei. A–J, Coronal sections of the LHb (A, F), the RMTg (B, G), the MR (C, H), the DR (D, I), and the VTA/SNc (E, J) of the animals that received injections of PBS (A–E) or DHK (F–J) to the LHb showing the expression of c-Fos (A–J, black), serotonin (5-HT, C, D, H, I, brown), and tyrosine hydroxylase (TH, E, J, brown). C–E, H–J, Insets, Representative cells labeled by 5-HT (C, D, H, I) or by TH (E, J). A, F, Asterisks indicate the medial habenula. G, H, Brackets indicate the RMTg and the MR, respectively. A, B, F, G, Sections were counterstained with neutral red. Aq, Cerebral aqueduct; IPN, interpeduncular nucleus. K–R, Bar graphs represent the mean number of c-Fos(+) cells in the LHb (K) and in the RMTg (L); c-Fos(+)/5-HT(+) cells (M) and c-Fos(+)/5-HT(−) (N) in the MR; c-Fos(+)/5-HT(+) cells (O) and c-Fos(+)/5-HT(−) (P) in the DR; and c-Fos(+)/TH(+) cells (Q) and c-Fos(+)/TH(−) cells (R) in the VTA/SNc of the animals that received injections of PBS (blue, N = 6) or DHK (red, N = 7) to the LHb. *p < 0.05 (two-tailed Mann–Whitney U test). **p < 0.01 (two-tailed Mann–Whitney U test). Error bars indicate SEM. Scale bars: A (applies to B–J), 200 μm; C, inset (applies to D–E, H–J), 10 μm.
Figure 3.
Figure 3.
Acute inhibition of GLT-1 activity in habenular astrocytes exacerbates despair behavior in the tail suspension test. A, A schematic diagram showing the experimental design. After implantation of the receptacle for head restraint and recovery, DHK was bilaterally injected into the LHb via stereotaxic injection under awake conditions 30 min before the tail suspension test (TST). B, C, Coronal sections showing c-Fos expression (black) in the LHb after injections of PBS (B) or DHK (C). Sections were counterstained with neutral red. D, E, Bar graphs showing the mean immobility duration (D) and the mean latency to the first immobility episode (E) of the animals injected with PBS (blue, N = 9) or with DHK (red, N = 5) in the tail suspension test. *p < 0.05 (two-tailed Mann–Whitney U test). Error bars indicate SEM. Scale bars: B (applies to C), 400 μm.
Figure 4.
Figure 4.
Virus-mediated deletion of the GLT-1 gene in habenular astrocytes elicited depressive-like behaviors in the tail suspension test, with increased neuronal excitability. A, A schematic diagram showing the strategy for GLT-1 gene deletion in cells transduced with an AAV vector expressing an EGFP-Cre fusion protein under the control of a GFAP promoter (Pgfap). B–G, Coronal sections of the LHb of GLT-1flox/flox mice with the injection of an EGFP-expressing vector (Hb-control in B–D) or EGFP-Cre-expressing vector (Hb-GLT-1 cKO in E–G) showing the expression of GLT-1 (C, D, F, G, red) and EGFP (B, D, E, G, green). Sections were counterstained with DAPI (D, G, blue). C, F, White brackets indicate the LHb. H, J, Representative activities of the LHb neurons in the Hb-control (H) and Hb-GLT-1 cKO mice (J). I, K, Average spike shape of the neurons shown in H (blue solid line in I) and J (red solid line in K). Shaded areas represent SEM. L, M, Bar graphs represent the mean firing rate (L) and mean spike width (M) of the spikes recorded from the Hb-control (blue, N = 51) and Hb-GLT-1 cKO mice (red, N = 46). N, O, Bar graphs showing the mean immobility duration (N) and latency to the first immobility episode (O) of the Hb-control mice (blue, N = 12), Hb-GLT-1 cKO mice without treatment (red, N = 16), and Hb-GLT-1 cKO mice with vehicle (pink, N = 10) or fluoxetine (purple, N = 11) treatment in the tail suspension test. P, Q, Coronal sections of the LHb of Hb-control (P) and Hb-GLT-1 cKO (Q) mice showing c-Fos expression (black). Sections were counterstained with neutral red. R, Bar graph represents the mean number of c-Fos-expressing cells in the LHb of Hb-control (blue, N = 8) and Hb-GLT-1 cKO (red, N = 6) mice. *p < 0.05 (Tukey's post hoc test for multiple comparisons following one-way ANOVA for four group data and two-tailed Mann–Whitney U test for two group data). **p < 0.01 (Tukey's post hoc test for multiple comparisons following one-way ANOVA). ***p < 0.0001 (two-tailed Mann-Whitney U test). NS, Not significant. Error bars indicate SEM. Scale bars: B (applies to C–G), 200 μm; and P (applies to Q), 200 μm.
Figure 5.
Figure 5.
Habenula-specific GLT-1 deletion leads to depressive-like behaviors under stress without affecting locomotion. A, B, Bar graphs represent the mean latency to feeding in novelty-suppressed feeding of Hb-control (blue, N = 15) and Hb-GLT-1 cKO mice (red, N = 15) (A) and the total distance traveled during the open-field test of the Hb-control (blue, N = 9) and Hb-GLT-1 cKO mice (red, N = 11) (B). C–F, Representative traces of movement during the social avoidance test in the presence of aggressors (white asterisks) of the Hb-control (C, D, blue lines) and Hb-GLT-1 cKO mice (E, F, red lines) after nondefeated (C, E) and defeated (D, F) treatments. Dark and light gray areas represent the mesh cage and interaction zone, respectively. G, H, Bar graphs represent the mean interaction ratio during the social avoidance test after nondefeated (G) and defeated (H) treatments of the Hb-control (blue, N = 9 and 11 for nondefeated and defeated groups, respectively) and Hb-GLT-1 cKO mice (red, N = 10 for nondefeated and defeated groups). *p < 0.05 (two-tailed Mann–Whitney U test). **p < 0.01 (two-tailed Mann–Whitney U test). Error bars indicate SEM. NS, not significant.
Figure 6.
Figure 6.
Disinhibition of REM sleep by GLT-1 deletion in the habenular astrocytes. A, B, Representative sleep pattern in Hb-control (A) and Hb-GLT-1 cKO (B) mice, which is classified into the awake (A), non-REM (NREM), and REM sleep periods according to the spectrogram of EEG (top) and EMG power (bottom). C–F, Bar graphs represent the mean duration spent awake (C), in NREM sleep (D), and in REM sleep (E) and the mean latency to the onset of REM sleep (F) in Hb-control (blue, N = 7) or Hb-GLT-1 cKO (red, N = 6) mice. G–I, Line plots represent the mean probability for being awake (G), in NREM sleep (H), and in REM sleep (I) along Zeitgeber time in Hb-control (blue, N = 7) and Hb-GLT-1 cKO (red, N = 6) mice. The probability for each stage category was calculated for each 2 h bin. *p < 0.05 (two-tailed Mann–Whitney U test). **p < 0.01 (two-tailed Mann–Whitney U test). C–F, Error bars indicate SEM. G–I, Shaded areas represent SEM. NS, not significant.

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