Figure 1. Two neighboring astrocytes expressing Lck-GFP against a backdrop of GFAP immunohistochemistry (purple). Colocalized signal for GFAP and Lck-GFP is shown in white.
Figure 2. Astrocyte Modeling
Figure 3. Colocalization of Lck-GFP with PSD-95
Figure 4. Astrocyte Embracing a Blood Vessel
Astrocytes are the most abundant cell type in the brain, so named because of their star-like appearance. These beautiful cells play many important roles in the brain, largely through their interactions with other cell types including neurons, blood cells, and oligodendrocytes. For example, astrocytes function critically in axon guidance, synapse formation and refinement, synaptic function and plasticity, neuronal metabolic processes, homeostatic balance of extracellular ions, regulation of blood flow, response to injury or infection. A couple of great reviews for introduction to astrocytes include: Parpura et al (2012) J Neurochem 121(1):4-27 and Khakh & Sofroniew (2015) Nature Neuroscience volume 18, pages 942–952.
In the Reissner lab, we are particularly interested in understanding the roles that astrocytes play in the synaptic and behavioral responses to experience. In particular, we are focused on the structural and physiological effects of cocaine self-administration on astrocytes, and how these effects influence communication between astrocytes and neurons, and accordingly, neuronal function and behavior. To address these effects and the role(s) of astrocytes in the enduring cellular adaptations which may mediate substance use disorders, we utilize behavior, microscopy, molecular biology, and slice electrophysiology.
Measuring Structural Features of Astrocytes
We have developed an approach to assess morphometric features (surface area, volume) as well as synaptic colocalization of astrocyte peripheral processes, following cocaine self-administration and extinction training (Scofield et al 2016). This approach allows for analysis of features of individual astrocytes, including the very fine processes which colocalize and communicate with synapses. Importantly, imaging these fine processes cannot be accomplished with GFAP immunohistochemistry or a soluble astrocyte-expressed GFP, and requires use of the membrane-tagged Lck-GFP expressed under control of the GfaABC1D promoter. Figure 2 illustrates the dramatic differences between GFAP labeling of astrocytes (blue signal) and Lck-GFP labeling of astrocytes (green signal). Using this tool, we found that nucleus accumbens astrocytes are smaller after cocaine self-administration and extinction, and colocalize less with synapses. Figure 3 on the left shows an Lck-GFP labeled astrocyte together with immunohistochemistry for PSD-95. The pixels in white are positive for both the GFP and PSD-95 signals, indicating colocalization and an index of synaptic contact. We are now applying this approach toward studies of astrocytes under a variety of other conditions of cocaine self-administration, development, and experience, and are excited to use this tool to build on our appreciation of astrocyte responses to cocaine. We have shared this reagent and approach with a number of labs with ongoing projects, and we are happy to talk with anyone with interest in this area.
Lck-GFP labeling of astrocytes can also be used to image the physical relationship of astrocytes with blood vessels. Figure 4 shows an Lck-GFP labeled striatal astrocyte together with a DiI-labeled blood vessel. Appreciate how the blood vessel moves through the astrocyte, which is embraced by the astrocyte end feet.
Beyond structural analysis of astrocytes, other projects in the lab are designed to investigate the reciprocal signals sent between astrocytes and neurons within the reward circuitry at different stages in the self-administration model. For example, it is well known that cocaine self-administration leads to reduced glutamate uptake by astrocytes via GLT-1/EAAT2. So, we are very interested in studying the mechanisms by which cocaine impairs GLT-1, and how that relates to the structural changes observed in astrocytes. Relatedly, we are interested in how impaired transmitter uptake, release, and ion flow by astrocytes influences synaptic and behavioral changes associated with cocaine use. This is accomplished using whole cell electrophysiological patch clamp analysis of both neurons and astrocytes.
Developing new tools for monitoring and manipulating astrocytes
All of our available tools and reagents are listed on the Resources page, which we are happy to share. Many of these are from commercial sources, or that we have received from others in the field. We are also eager to develop new tools by which features such as structural properties and calcium dynamics of astrocytes can be effectively measured in rats, and also by which genetic material in astrocytes can be selectively and effectively manipulated. We will update this section as new tools in development become available.
Some of Kate’s favorite astrocyte reviews
M.D. Scofield et al (2018) Exploring the Role of Astroglial Glutamate Release and Association With Synapses in Neuronal Function and Behavior. Biological Psychiatry, S0006-3223(17)32159-5
R.L. Kim, K.L. Healey, M.T. Sepulveda-Orengo, K.J. Reissner. Emerging Evidence for Astroglial Deficits in Neuropsychiatry Disease. Prog Neuropsychopharmacol Biol Psychiatry, S0278-5846(17)30485-2.
M. De Pitta, N. Brunel, A. Volterra (2016) Astrocytes: Orchestrating synaptic plasticity? Neuroscience 323:43-61
B.S. Khakh and MD McCarthy (2015) Astrocyte calcium signaling: from observations to functions and the challenges therein. Cold Spring Harb Perspect Biol. 7(4):a020404
A.Volterra, N Liaudet, I. Savtchouk (2014) Astrocyte Ca²⁺ signalling: an unexpected complexity. Nat Rev Neurosci, 15(5):327-35.