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.
Highlights
The ventral midbrain, comprised of the ventral tegmental area (VTA) and the substantia nigra pars compacta, houses the majority of dopamine-releasing neurons in the brain
We used the Aldh1L1-enhanced green fluorescent protein (eGFP) line of transgenic mice, which labels all astrocytes as well as their fine processes [24, 25], and imaged astrocytes in the VTA—one of the two dopaminergic nuclei in the ventral midbrain—as well as cortex and hippocampus of 2-month-old mice. eGFP expression is confined to cells that express endogenous Aldh1L1 (Fig. 1a) and does not overlap with cell-type-specific markers for oligodendrocytes, NG2 cells, or neurons (Figure S1a)
The density of astrocytes was Ventral midbrain astrocytes are molecularly distinct from telencephalic astrocytes To investigate whether differences in astrocyte morphology are accompanied by differences in other aspects of cell phenotype, we isolated eGFP+ astrocytes from the ventral midbrain, cortex, and hippocampus of adult Aldh1L1-eGFP mice via flow cytometry and performed RNA sequencing to examine the astrocyte transcriptome in each of these three regions (Figure S2a, b)
Summary
The ventral midbrain, comprised of the ventral tegmental area (VTA) and the substantia nigra pars compacta, houses the majority of dopamine-releasing neurons in the brain. Astrocytes are ubiquitous throughout the central nervous system (CNS) These abundant cells perform numerous critical tissue functions, including buffering extracellular potassium, supporting glutamate homeostasis, regulating synaptic connectivity and plasticity, providing metabolic support to neurons, and participating in injury responses following pathological insults [7, 9,10,11,12,13,14]. These aspects of astrocyte biology have been extensively studied in hippocampus and cortex and are widely assumed to be equivalent in astrocytes throughout the CNS. Using a combination of histology, RNA sequencing, electrophysiology, and calcium
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