Research ArticleDISEASES AND DISORDERS

Microglia morphology and proinflammatory signaling in the nucleus accumbens during nicotine withdrawal

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Science Advances  09 Oct 2019:
Vol. 5, no. 10, eaax7031
DOI: 10.1126/sciadv.aax7031
  • Fig. 1 Nicotine withdrawal alters microglial morphology and induces reactive oxygen species in the nucleus accumbens.

    (A) Experimental design. (B) Representative images of IBA1-positive microglia. (i) White dotted traces delineate the caudate putamen (dorsal) and nucleus accumbens (ventral); black dotted traces indicate the area where (ii) to (vii) were taken. Scale bar, 1 mm. Caudate putamen of (ii) saline (Sal) mice, (iii) nicotine (Nic) mice, and (iv) nicotine withdrawal (WD) mice. Nucleus accumbens of (v) Sal mice, (vi) Nic mice, and (vii) WD mice. Scale bar, 100 μm. (C) Quantitation of (B, ii to vii). (i) Cell area (soma size), (ii) cell perimeter, and (iii) process length. Bar charts showing expression of (D) TNFα mRNA and (E) IL-1β mRNA in the nucleus accumbens and caudate putamen. (F) Schematics showing mechanism of reactive oxygen species detection by protein carbonylation assay (protein and arrow icons were downloaded from Reactome (31). (G) Representative immunoblot showing carbonylated protein levels: (i) nucleus accumbens and (ii) caudate putamen. (H) Quantitation of (G). [Compared to Sal—*P < 0.05, **P < 0.01, ****P < 0.0001; compared to Nic—#P < 0.05, ##P < 0.01; (C) n = minimum of 116 cells were quantified from four to eight animals per treatment group; (D and E) n = 12 to 24 per treatment group; (H) n = 6 to 19 per treatment group.]

  • Fig. 2 Nicotine withdrawal induces anxiety-like phenotype in mice.

    (A) MB test: Bar graph showing the mean value of marbles buried by Sal, Nic, and WD mice. (B) OF test: Bar chart showing the time spent in the center of the OF arena by Sal, Nic, and WD mice. (C) Locomotor activity: Bar chart showing the average distance moved in the OF arena by Sal, Nic, and WD mice. (D) Representative OF traces of Sal, Nic, and WD mice. [Compared to Sal—**q < 0.01; compared to Nic—##q < 0.01; (A to C) n = 9 to 23 per treatment group.]

  • Fig. 3 Nicotine withdrawal increases Nox2 expression in the nucleus accumbens.

    (A) Bar graph showing qPCR analysis of Nox isoforms (primarily expressed in the brain) in the nucleus accumbens of Sal, Nic, and WD mice. (i) Nox1, (ii) Nox2, and (iii) Nox4. (B) Bar graph showing qPCR analysis of Nox2 mRNA expression in the caudate putamen of Sal, Nic, and WD mice. [Compared to Sal—**P < 0.01; compared to Nic—###P < 0.001; (A and B) n = 12 to 24 per treatment group.]

  • Fig. 4 Nox2 is expressed in microglia.

    (A) Bar graph showing qPCR analysis of microglia markers in the liver, brain cell types, and tissue (total homogenate). (i) CD11b, (ii) Tmem119, and (iii) P2ry12. (B) Bar graph showing qPCR analysis of Nox isoforms in the liver, brain cell types, and tissue. (i) Nox1, (ii) Nox4, and (iii) Nox2. [Compared to total homogenate—*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; (A and B) n = 2 to 9 per treatment group.]

  • Fig. 5 Inhibition of CSF1R depletes microglia in the nucleus accumbens.

    (A) Experimental design. (B) (i) Representative images of IBA1-positive microglia in the nucleus accumbens of control chow and PLX5622 chow Sal, as well as their WD equivalents. (ii) Quantitation of (B, i). (C) Bar graph showing qPCR analysis of microglia markers in the nucleus accumbens of control chow and PLX5622 chow Sal, as well as their WD equivalents. (i) Tmem119 and (ii) P2ry12. Heatmap showing normalized expression profile of Tmem119, P2ry12, and Ccr2 in the nucleus accumbens of control chow and PLX5622 chow Sal, as well as their WD equivalents. [Compared to Sal control—***P < 0.001, ****P < 0.0001; compared to Sal PLX5622—###P < 0.001, ####P < 0.0001; compared to WD control—$P < 0.05, $$$$P < 0.0001; (B, ii) n = 5 per treatment group; (C and D) n = 10 per treatment group.]

  • Fig. 6 Microglia depletion attenuates nicotine withdrawal–related anxiety.

    (A) Bar graph showing qPCR analysis of Nox2 in the nucleus accumbens of control chow and PLX5622 chow Sal, as well as their WD equivalents. (B) (i) Representative immunoblot showing the expression of carbonylated proteins in the nucleus accumbens of control chow and PLX5622 chow Sal, as well as their WD equivalents. (ii) Quantitation of (B, i). (C) MB test: Bar graph showing the mean amount of marbles buried by control chow and PLX5622 chow Sal, as well as their WD equivalents. (D) OF test: Bar chart showing the time spent in the center of the OF arena by control chow and PLX5622 chow Sal, as well as their WD equivalents. (E) Locomotor activity: Bar chart showing the average distance moved in the OF arena by control chow and PLX5622 chow Sal, as well as their WD equivalents. (F) Representative OF traces of control chow and PLX5622 chow Sal, as well as their WD equivalents. [Compared to Sal control—*P < 0.05, **P < 0.01; compared to Sal PLX5622—##P < 0.01, ###P < 0.001; compared to WD control—$P < 0.05, $$$P < 0.001; (A, Bii, C, D, and E) n = 9 to 10 per treatment group.]

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