Deletion of murine astrocytic vesicular nucleotide transporter increases anxiety and depressive-like behavior and attenuates motivation for reward

Abstract

Astrocytes are multi-functional glial cells in the central nervous system that play critical roles in modulation of metabolism, extracellular ion and neurotransmitter levels, and synaptic plasticity. Astrocyte-derived signaling molecules mediate many of these modulatory functions of astrocytes, including vesicular release of ATP. In the present study, we used a unique genetic mouse model to investigate the functional significance of astrocytic exocytosis of ATP. Using primary cultured astrocytes, we show that loss of vesicular nucleotide transporter (Vnut), a primary transporter responsible for loading cytosolic ATP into the secretory vesicles, dramatically reduces ATP loading into secretory lysosomes and ATP release, without any change in the molecular machinery of exocytosis or total intracellular ATP content. Deletion of astrocytic Vnut in adult mice leads to increased anxiety, depressive-like behaviors, and decreased motivation for reward, especially in females, without significant impact on food intake, systemic glucose metabolism, cognition, or sociability. These behavioral alterations are associated with significant decreases in the basal extracellular dopamine levels in the nucleus accumbens. Likewise, ex vivo brain slices from these mice show a strong trend toward a reduction in evoked dopamine release in the nucleus accumbens. Mechanistically, the reduced dopamine signaling we observed is likely due to an increased expression of monoamine oxidases. Together, these data demonstrate a key modulatory role of astrocytic exocytosis of ATP in anxiety, depressive-like behavior, and motivation for reward, by regulating the mesolimbic dopamine circuitry.

Figure 1.. Vnut deletion in primary astrocytes does not cause major changes in the expression of signature astrocyte marker genes.

(A) Fluorescent images of primary astrocytes expressing mCherry-fused Vnut protein (red) and immunostained with Lamp1 (green). DAPI (blue) labels the nuclei of the astrocytes. Scale bar: 10 μm. (B) Reverse-transcription PCR to examine the expression of the exon 1 of the Vnut mRNA in astrocytes infected with adenovirus encoding GFP or GFP:Cre fusion protein. The expression of Tbp mRNA was used as loading controls. (C) Relative mRNA expression of signature genes for astrocytes in control and VnutKO astrocytes. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05; **, P < 0.01. N = 4. (D) Relative mRNA expression of Lamp1 and Lamp2 in control and VnutKO astrocytes. Data are shown as mean ± SEM. N = 4. (E) Immunoblotting showing the protein expression of Gfap and Lamp1 in control and VnutKO astrocytes. Gapdh is used as loading control. (F) Densitometric analysis of Gfap and Lamp1 in control and VnutKO astrocytes. Data are shown as mean ± SEM. N = 6. (G) Immunostaining of Lamp1 (red) in control and VnutKO astrocytes. DAPI (blue) labels the nuclei of the astrocytes. Scale bar: 10 μm. (H) Mean intensity of Lamp1 immunofluorescent signal of all particles in control and VnutKO astrocytes. Data are shown as mean ± SEM. Two-tailed t-test. **, P < 0.01. N = 5 for Vnut^f/f and 4 for VnutKO coverslips.

Figure 2.. Loss of Vnut impairs ATP loading into secretory lysosomes and ATP release in primary astrocytes.

(A) Representative confocal images showing intracellular localization of ATP analog Mant-ATP (cyan) and Lysotracker Red in control and VnutKO astrocytes. Scale bar: 20 μm. (B) Quantification showing the mean fluorescent intensity of Mant-ATP in Lysotracker Red-labeled vesicles in control and VnutKO astrocytes. Data are shown as mean ± SEM. Two-tailed t-test. ***, P < 0.001. N = 5 coverslips. (C) ATP release from control and VnutKO astrocytes in 1x HBSS for 30 min normalized to total protein content. Data are shown as mean ± SEM. Two-tailed t-test. **, P < 0.01. N = 6. (D) Total intracellular ATP content in control and VnutKO astrocytes normalized to total protein content. Data are shown as mean ± SEM. N = 6. (E) Relative mRNA expression of SNARE proteins enriched in astrocytes in control and VnutKO astrocytes. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05; N = 4. (F) Immunoblotting showing the protein expression of Stx4, Vamp7 and Munc18c in control and VnutKO astrocytes. Gapdh is used as loading control. (G) Densitometric analysis of Stx4, Vamp7 and Munc18c in in control and VnutKO astrocytes. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05; N = 6.

Figure 3.. Loss of Vnut in astrocytes in adult mice shows minor effects on the expression of astrocyte marker genes, as well as genes involved in exocytosis and lysosomes.

(A) Relative mRNA expression of Vnut in astrocytes FACS sorted from 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05; N = 4. (B) Relative mRNA expression of Vnut in microglia FACS sorted from 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. N = 4. (C) Representative confocal images of 4-month-old female Vnut^GfapKO mouse brain sections stained with Vnut (red) and astrocyte-specific marker S100β (green). Arrows: astrocytes without detectable Vnut expression; Arrowheads: astrocytes with Vnut expression. Scale bar: 20 μm. (D) Relative mRNA expression of astrocytes signature genes in astrocytes FACS sorted from 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05; N = 4 for Vnut^f/f and 5 for Vnut^GfapKO. (E) Relative mRNA expression of SNARE proteins in astrocytes FACS sorted from 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. N = 4 for Vnut^f/f and 5 for Vnut^GfapKO. (F) Relative mRNA expression of Lamp1 and Lamp2 in astrocytes MACS sorted from 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. N = 6. (G) Representative confocal images of nucleus accumbens core of the 4-month-old female Vnut^f/f and Vnut^GfapKO mouse brain stained with Lamp1 (red) and astrocyte-specific marker S100β (green). Scale bar: 10 μm. (H) Quantification showing the percentage of Lamp1⁺ particles in S100β⁺ astrocytes. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05. N = 44 cells from 4 animals.

Figure 4.. Loss of Vnut in astrocytes in adult mice does not affect systemic glucose metabolism and insulin sensitivity.

(A) The body weight of 4-month-old male Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. N = 7 for Vnut^f/f and 8 for Vnut^GfapKO. (B) Serum glucose levels of 4-month-old male Vnut^f/f and Vnut^GfapKO mice after overnight fasting. Data are shown as mean ± SEM. N = 7 for Vnut^f/f and 8 for Vnut^GfapKO. (C) Serum insulin levels of 4-month-old male Vnut^f/f and Vnut^GfapKO mice after overnight fasting. Data are shown as mean ± SEM. N = 7 for Vnut^f/f and 8 for Vnut^GfapKO. (D) Glucose tolerance test of 4-month-old male Vnut^f/f and Vnut^GfapKO mice after overnight fasting. Data are shown as mean ± SEM. N = 7 for Vnut^f/f and 8 for Vnut^GfapKO. (E) Insulin tolerance test of 4-month-old male Vnut^f/f and Vnut^GfapKO mice after 4 h fasting early in the morning. Data are shown as mean ± SEM. N = 6 for Vnut^f/f and 7 for Vnut^GfapKO. (F) The body weight of 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. N = 8 for Vnut^f/f and 7 for Vnut^GfapKO. (G) Serum glucose levels of 4-month-old female Vnut^f/f and Vnut^GfapKO mice after overnight fasting. Data are shown as mean ± SEM. N = 8 for Vnut^f/f and 7 for Vnut^GfapKO. (H) Serum insulin levels of 4-month-old female Vnut^f/f and Vnut^GfapKO mice after overnight fasting. Data are shown as mean ± SEM. N = 8 for Vnut^f/f and 7 for Vnut^GfapKO. (I) Glucose tolerance test of 4-month-old female Vnut^f/f and Vnut^GfapKO mice after overnight fasting. Data are shown as mean ± SEM. N = N = 8 for Vnut^f/f and 7 for Vnut^GfapKO. (J) Insulin tolerance test of 4-month-old female Vnut^f/f and Vnut^GfapKO mice after 4 h fasting early in the morning. Data are shown as mean ± SEM. N = 8 for Vnut^f/f and 7 for Vnut^GfapKO.

Figure 5.. Astrocyte-specific Vnut KO female mice display increased anxiety and depressive-like behavior.

(A) Center zone entries of 4-month-old female Vnut^f/f and Vnut^GfapKO mice during the open field test. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05. N = 12 for Vnut^f/f and 10 for Vnut^GfapKO. (B) Total distance traveled of 4-month-old female Vnut^f/f and Vnut^GfapKO mice during the open field test. Data are shown as mean ± SEM. N = 12 for Vnut^f/f and 10 for Vnut^GfapKO. (C) Percentage of daily consumption of 1% sucrose solution over total liquid consumption in 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05. N = 12 for Vnut^f/f and 10 for Vnut^GfapKO. (D) Time of immobility of 4-month-old female Vnut^f/f and Vnut^GfapKO mice during the forced swimming test. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05. N = 12 for Vnut^f/f and 10 for Vnut^GfapKO. (E) Time of immobility of 4-month-old female Vnut^f/f and Vnut^GfapKO mice during the forced swimming test with pre-treatment of saline or 20 pmol 2-Me-SATP (i.c.v.). Data of individual mouse with or without 2-Me-SATP treatment are plotted and matched. Two-way RM ANOVA followed by Sidak’s multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. N = 10 for Vnut^f/f and 9 for Vnut^GfapKO.

Figure 6.. Astrocytic Vnut in the nucleus accumbens is a critical mediator for depressive-like behavior in female mice in response to inescapable stress.

(A) c-fos immunostaining of the nucleus accumbens of the 4-month-old female Vnut^f/f and Vnut^GfapKO mice at basal condition and 1 h after forced swimming test. Scale bar: 500 μm. (B-C) Quantification of c-fos+ neurons in NAc core (B) and shell (C) regions. Two-way ANOVA followed by Tukey’s multiple comparisons test. **, P < 0.01; ****, P < 0.0001. N = 4 animals for basal groups and 6 animals for FST groups. (D) Schematics of AAV8-hGFAP-Cre:GFP injection into the nucleus accumbens of adult female mice. (E) Center zone entries during the open field test of 4-month-old female Vnut^f/f mice injected with AAV8-hGFAP-Cre:GFP or GFP alone in the NAc. Data are shown as mean ± SEM. N = 9. (F) Total distance traveled during the open field test of 4-month-old female Vnut^f/f mice injected with AAV8-hGFAP-Cre:GFP or GFP alone in the NAc. Data are shown as mean ± SEM. N = 9. (G) Time of immobility during the forced swimming test of 4-month-old female Vnut^f/f mice injected with AAV8-hGFAP-Cre:GFP or GFP alone in the NAc. Data are shown as mean ± SEM. Two-tailed t-test. **, P < 0.01. N = 9. (H) Percentage of daily consumption of 1% sucrose solution over total liquid consumption in 4-month-old female Vnut^f/f mice injected with AAV8-hGFAP-Cre:GFP or GFP alone in the NAc. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05. N = 9.

Figure 7.. The effects of astrocytic Vnut loss in adult female mice on evoked dopamine release in the nucleus accumbens.

(A-C) Total numbers (A), amplitude (B), and T_1/2 (C) of dopamine molecules (DA) released after each electric stimulation in the NAc from 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. Two-tailed t-test. N = 17 for Vnut^f/f and 12 for Vnut^GfapKO. (D) Relative mRNA expression of astrocytes signature genes in the NAc of 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. N = 6. (E) Relative mRNA expression of genes involved in dopamine synthesis, release, signaling and degradation in the NAc of 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05. N = 6. (F) Immunostaining and quantification of tyrosine hydroxylase (Th) in the NAc of 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Scale ar: 100 μm. Data are shown as mean ± SEM. N = 4. (G) Immunoblotting showing the protein expression levels of Gfap, Th, MaoA and MaoB in the NAc tissues of 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Gapdh is used as loading control. (H) Densitometric analysis of Gfap and Th in the NAc of 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. N = 6. (I) Densitometric analysis of MaoA and MaoB in the NAc of 4-month-old female Vnut^f/f and Vnut^GfapKO mice. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05. N = 6.

Figure 8.. Loss of Vnut in Aldh1l1⁺ astrocytes increases depressive-like behavior and reduces motivation for reward in adult female mice.

(A) Center zone entries of 4-month-old female Vnut^f/f and Vnut^Aldh1l1KO mice during the open field test. Data are shown as mean ± SEM. N = 9 for Vnut^f/f and 10 for Vnut^Aldh1l1KO. (B) Total distance traveled of 4-month-old female Vnut^f/f and Vnut^Aldh1l1KO mice during the open field test. Data are shown as mean ± SEM. N = 9 for Vnut^f/f and 10 for Vnut^Aldh1l1KO. (C) Percentage of daily consumption of 1% sucrose solution over total liquid consumption in 4-month-old female Vnut^f/f and Vnut^Aldh1l1KO mice. Data are shown as mean ± SEM. Two-tailed t-test. **, P < 0.01. N = 9 for Vnut^f/f and 8 for Vnut^Aldh1l1KO. (D) Time of immobility of 4-month-old female Vnut^f/f and Vnut^Aldh1l1KO mice during the forced swimming test. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05. N = 9 for Vnut^f/f and 10 for Vnut^Aldh1l1KO. (E) Active nose pokes for the active port of 4-month-old female Vnut^f/f and Vnut^Aldh1l1KO mice during the fixed ratio 1 and 5 training sessions for sucrose pellets. Data are shown as mean ± SEM. N = 9 for Vnut^f/f and 12 for Vnut^Aldh1l1KO. (F) Breakpoints of 4 month-old female Vnut^f/f and Vnut^Aldh1l1KO mice during the progressive ratio schedule for each reinforcement. Data are shown as mean ± SEM. Two-tailed t-test. *, P < 0.05. N = 9 for Vnut^f/f and 12 for Vnut^Aldh1l1KO. (G) Representative Nissl staining showing the position of the implanted guide canula in the NAc for microdialysis. (H) Dopamine concentrations in the dialysates collected from 5-month-old female Vnut^f/f and Vnut^Aldh1l1KO mice. Data are shown as mean ± SEM. N = 9 for Vnut^f/f and 11 for Vnut^Aldh1l1KO.