Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jan;44(2):344-355.
doi: 10.1038/s41386-018-0151-4. Epub 2018 Jul 13.

Ventral Midbrain Astrocytes Display Unique Physiological Features and Sensitivity to Dopamine D2 Receptor Signaling

Affiliations
Free PMC article

Ventral Midbrain Astrocytes Display Unique Physiological Features and Sensitivity to Dopamine D2 Receptor Signaling

Wendy Xin et al. Neuropsychopharmacology. .
Free PMC article

Abstract

Astrocytes are ubiquitous CNS cells that support tissue homeostasis through ion buffering, neurotransmitter recycling, and regulation of CNS vasculature. Yet, despite the essential functional roles they fill, very little is known about the physiology of astrocytes in the ventral midbrain, a region that houses dopamine-releasing neurons and is critical for reward learning and motivated behaviors. Here, using a combination of whole-transcriptome sequencing, histology, slice electrophysiology, and calcium imaging, we performed the first functional and molecular profiling of ventral midbrain astrocytes and observed numerous differences between these cells and their telencephalic counterparts, both in their gene expression profile and in their physiological properties. Ventral midbrain astrocytes have very low membrane resistance and inward-rectifying potassium channel-mediated current, and are extensively coupled to surrounding oligodendrocytes through gap junctions. They exhibit calcium responses to glutamate but are relatively insensitive to norepinephrine. In addition, their calcium activity can be dynamically modulated by dopamine D2 receptor signaling. Taken together, these data indicate that ventral midbrain astrocytes are physiologically distinct from astrocytes in cortex and hippocampus. This work provides new insights into the extent of functional astrocyte heterogeneity within the adult brain and establishes the foundation for examining the impact of regional astrocyte differences on dopamine neuron function and susceptibility to degeneration.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Morphology of ventral midbrain and telencephalic astrocytes. a Coronal brain section from a P60 Aldh1L1-eGFP mouse immunostained for Aldh1L1 (red). b Images taken from P60 Aldh1L1-eGFP mouse section showing density and tissue coverage by eGFP+ astrocyte processes in the ventral tegmental area (VTA), cortex (CTX), and hippocampus (HPC). c Closeup of individual eGFP+ astrocytes. d Quantification of eGFP+ astrocyte density in P60 animals. One-way ANOVA, F = 0.6611, p = 0.5441, N = 5 animals, 1 section per animal, per region. e Quantification of percent tissue coverage by eGFP+ astrocyte processes in P60 animals (see Methods for details). One-way ANOVA, F = 7.866, p = 0.0025; Bonferroni post hoc tests for CTX vs HPC t = 0.1441, CTX vs VTA t = 3.536, HPC vs VTA t = 3.396 (**p < 0.01). N = 8–9 sections, 4–5 animals per region. f Quantification of eGFP+ astrocyte soma size in P60 animals. One-way ANOVA, F = 19.52, p < 0.0001; Bonferroni post hoc tests for CTX vs HPC t = 1.899, CTX vs VTA t = 4.179, HPC vs VTA t = 6.078 (***p < 0.0001). N = 6–8 sections, 4–6 animals per region. g Example scatter plot of one FACS experiment, with forward scatter values on the x-axis and FITC channel intensity on the y-axis. Circle indicates eGFP+ astrocyte considered in analysis. h Forward scatter values from one FACS experiment in which Aldh1L1-eGFP tissue microdissected from ventral midbrain, CTX, and HPC were dissociated into single-cell suspensions and analyzed by flow cytometry. Graph shows aggregate forward scatter values of eGFP+ astrocytes from the three regions. i Quantification of average forward scatter values (per animal/experiment) of eGFP+ astrocyte from ventral midbrain, CTX, and HPC. One-way ANOVA, F = 31.06, p < 0.0001; Bonferroni post hoc tests for CTX vs HPC t = 0.5599, CTX vs ventral midbrain t = 0.7089, HPC vs ventral midbrain t = 6.529 (***p < 0.001). N = 6 animals/experiments
Fig. 2
Fig. 2
Transcriptomic analysis of ventral midbrain and telencephalic astrocytes. a RPKM values of cell-type-specific genes obtained from RNA sequencing of Aldh1L1-eGFP+ astrocyte transcripts (see Methods for details). b Degree of overlap among the top 1500 or top 500 expressed genes in each region, using mean RPKM. c Number of significantly up- or down-regulated genes in each region (EDGE test p < 0.05, mean RPKM >2 and norm. SEM < 0.5 in the more highly expressed region). d Degree to which genes in various functional families are conserved or differentially expressed (see Methods for details). e Heat maps comparing RPKM fold change in VM vs. HPC or CTX astrocytes for genes related to calcium dynamics (top) or electrical membrane properties (bottom). Asterisks above genes indicate EDGE test p values (*p < 0.05, **p < 0.01)
Fig. 3
Fig. 3
Electrophysiological properties of ventral midbrain and telencephalic astrocytes. a Trace from one astrocyte recording in voltage clamp, in which a brief negative voltage step was delivered. b Quantification of astrocyte resting potential. One-way ANOVA, F = 1.91, p = 0.152. N = 19 CTX cells (12 animals, 14 slices), 50 HPC cells (20 animals, 22 slices), and 70 VTA cells (28 animals, 32 slices). c Quantification of astrocyte membrane resistance. One-way ANOVA, F = 49.3, p < 0.0001; Bonferroni post hoc tests for CTX vs HPC t = 1.764, CTX vs VTA t = 5.366, HPC vs VTA t = 5.362 (***p < 0.001). N = 17 CTX cells (12 animals, 14 slices), 50 HPC cells (20 animals, 22 slices), and 46 VTA cells (22 animals, 23 slices). d Example traces of one VTA astrocyte and one HPC astrocyte in voltage clamp, stepped from −130 mV to + 30 mV holding potential in 10 mV increments (voltage step command depicted in inset, top right). Left, current measurements in ACSF; middle, current measurements in 100 µM barium; right, digital subtraction of current measured in barium from current measured in ACSF. e Quantification of IV relationship of all cells in ACSF. One-way ANOVA, F = 0.09728, p = 0.983. N = 8 CTX (8 animals, 8 slices), 22 HPC (13 animals, 14 slices), and 37 VTA (16 animals, 19 slices). f Quantification of IV relationship of barium-sensitive current for all cells. One-way ANOVA, F = 0.1091, p = 0.0035; Bonferroni post hoc tests for CTX vs HPC t = 0.7489, CTX vs VTA t = 3.397, HPC vs VTA t = 2.648. N = 7 CTX (5 animals, 7 slices), 13 HPC (10 animals, 13 slices), and 8 VTA (7 animals, 8 slices). g Quantification of barium-sensitive current at +30 mV holding potential. One-way ANOVA, F = 14.23, p < 0.0001; Bonferroni post hoc tests for CTX vs HPC t = 0.05326, CTX vs VTA t = 4.343, HPC vs VTA t = 4.947 (***p < 0.001). N = 7 CTX (5 animals, 7 slices), 13 HPC (10 animals, 13 slices), and 8 VTA (7 animals, 8 slices)
Fig. 4
Fig. 4
Extensive coupling between astrocytes and oligodendrocytes in the ventral midbrain. a Confocal image of a horizontal midbrain section in which one astrocyte was patched and filled with biocytin. Biocytin was visualized by post hoc staining with streptavidin conjugated to Alexa 594. b Confocal image of a horizontal midbrain section in which one astrocyte was patched and filled with biocytin, then stained for aspartoacylase (ASPA), a mature oligodendrocyte marker. White arrowheads indicate biocytin+, ASPA+, eGFP− oligodendrocytes. c Quantification of the percentage of astrocytes (left) or oligodendrocytes (right) within the biocytin diffusion radius that are biocytin+. N = 8 CTX, 5 HPC, and 7 VTA sections. d Percentage of network occupied by astrocytes or oligodendrocytes in each region. N = 8 CTX, 5 HPC, and 7 VTA sections
Fig. 5
Fig. 5
Ventral midbrain and hippocampal astrocyte calcium responses to norepinephrine and D2 receptor modulation. a Average (top trace, SEM in gray) and individual (bottom, color raster plot) HPC astrocyte calcium responses to bath application of 40 μM norepinephrine. Raw intensity values were normalized to baseline period as Z-scores. b Average Z-scores of HPC astrocytes 1 min prior to NE (‘ACSF’) and 1 min after onset of NE (‘NE’). Paired t-test, t = 5.301, p < 0.001. N = 5 slices, 2 animals. c Average and individual ventral midbrain astrocyte calcium responses to bath application of 40 μM norepinephrine. d Average Z-scores of ventral midbrain astrocytes 1 min prior to NE (‘ACSF’) and 1 min after onset of NE (‘NE’). Paired t-test, t = 0.866, p = 0.3936. N = 9 slices, 3 animals. e Average and individual hippocampal astrocyte calcium responses to 10 μM quinpirole and 300 nM sulpiride. f Average Z-scores of HPC astrocytes during 5 min of baseline (‘ACSF’), 5 min of quinpirole (‘Q’), and 5 min of sulpiride (‘S’). One-way ANOVA, F = 1.167, p = 0.3187. N = 6 slices, 2 animals. g Average (top) and individual (bottom) ventral midbrain astrocyte calcium responses to 10 μM quinpirole and 300 nM sulpiride. h Average Z-scores of ventral midbrain astrocytes during 5 min of baseline (‘ACSF’), 5 min of quinpirole (‘Q’), and 5 min of sulpiride (‘S’). One-way ANOVA, F = 14.07, p < 0.0001. Bonferroni post hoc tests for ACSF vs Q t = 4.858, p < 0.001; ACSF vs S t = 0.5823, p > 0.05; Q vs S t = 4.276, p < 0.001. N = 7 slices, 2 animals. i Calcium response of an individual ventral midbrain astrocyte (inset) and average response from all imaged ventral midbrain astrocytes to 1 mM glutamate (duration of application indicated by red bar), in the presence of 500 μM lidocaine, 100 μM kyneurinic acid, 100 μM picrotoxin, and 1 μM CGP35348. j Average Z-scores of ventral midbrain astrocytes 2 min prior to glutamate (‘ACSF’) and 2 min after onset of glutamate (‘Glu’). Paired t-test, t = 1.001, p = 0.3228. N = 13 slices, 4 animals. k Calcium responses of an individual ventral midbrain astrocyte (inset) and average response from all imaged to 1 mM glutamate (duration of application indicated by red bar), in the presence of 300 nM sulpiride, 500 μM lidocaine, 100 μM kyneurinic acid, 100 μM picrotoxin, and 1 μM CGP35348. l Average Z-scores of ventral midbrain astrocytes 2 min prior to glutamate (‘ACSF’) and 2 min after onset of glutamate (‘Glu’). Paired t-test, t = 3.145, p = 0.0056. N = 6 slices, 2 animals

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

References

    1. Wise RA. Dopamine, learning and motivation. Nat Rev Neurosci. 2004;5:483–94. doi: 10.1038/nrn1406. - DOI - PubMed
    1. Chong TT-J, Husain M. Chapter 17 - The role of dopamine in the pathophysiology and treatment of apathy. In: Studer B, Knecht S, editors. Progress in brain research, vol. 229. Amsterdam: Elsevier; 2016. p. 389–426. - PubMed
    1. Grace AA. Dysregulation of the dopamine system in the pathophysiology of schizophrenia and depression. Nat Rev Neurosci. 2016;17:524–32. doi: 10.1038/nrn.2016.57. - DOI - PMC - PubMed
    1. Mackin LA. Understanding Parkinson’s disease: detection and early disease management. Lippincotts Prim Care Pract. 2000;4:595–607. - PubMed
    1. Zhou QY, Palmiter RD. Dopamine-deficient mice are severely hypoactive, adipsic, and aphagic. Cell. 1995;83:1197–209. doi: 10.1016/0092-8674(95)90145-0. - DOI - PubMed

Publication types

Feedback