Visualizing the brain's astrocytes

Abstract

Astrocytes are the most abundant cell type in the brain and are a crucial part of solving its mysteries. Originally assumed to be passive supporting cells, astrocyte's functions are now recognized to include active modulation and information processing at the neural synapse. The full extent of the astrocyte contribution to neural processing remains unknown. This is, in part, due to the lack of methods available for astrocyte identification and analysis. Existing strategies employ genetic tools like the astrocyte specific promoters glial fibrillary acidic protein (GFAP) or Aldh1L1 to create transgenic animals with fluorescently labeled astrocytes. Recently, small molecule targeting moieties have enabled the delivery of bright fluorescent dyes to astrocytes. Here, we review methods for targeting astrocytes, with a focus on a recently developed methylpyridinium targeting moiety's development, chemical synthesis, and elaboration to provide new features like light-based spatiotemporal control of cell labeling.

Keywords: Astrocytes; Brain imaging; Cationic fluorophore; Euroimaging; Glia imaging; Photoactivatable; Small molecules.

Figure 1.. Role of astrocytes in the brain.

(A) Depiction of the tripartite synapse, which is magnified for B–D. (B) Release of the neurotransmitter glutamate from a presynaptic neuron can bind to the astrocytic glutamate receptor, GLT1, and cause Ca²⁺ excitability, increasing intracellular [Ca²⁺]. (C) Ca²⁺ excitability also causes the release of gliotransmitter glutamate, which activates post-synaptic NMDA receptors, regulating synaptic signaling. (D) Ca²⁺ excitability releases the gliotransmitter ATP, where it is converted to adenosine in the synapse and binds the presynaptic A1 receptor to induce synaptic suppression.

Figure 2.. Differences in expression by astrocyte-specific promoters and proteins.

(A) GFP expression driven by a truncated 1740-bp GFAP promoter (green) with immunostaining against GFAP (red) showing incomplete overlap between promoter-driven expression and protein labeling in mouse cortex. (B) Inset of (A) showing GFP⁺ astrocytes in hippocampal stratum. Arrowheads indicate GFP-expressing astrocytes. (C, D) GFP expression driven by the astrocyte-specific Aldh1L1 promoter (green), with S100β (C) or GFAP (D) co-immunostaining (red) in mouse hippocampal astrocytes. Aldh1L1 reveals the very fine astrocytic processes that S100β or GFAP cannot. Panels (A, B): Reprinted with permission from Meng, X., Yang, F., Ouyang, T., Liu, B., & Jiang, W. (2015). Specific gene expression in mouse cortical astrocytes is mediated by a 1740bp-GFAP promoter-driven combined adeno-associated virus. Neuroscience Letters, 593, 45–50. Panels (C, D): Srinivasan, R., Lu, T.-Y., Chai, H., Xu, J., Huang, B. S., Golshani, P., … Khakh, B. S. (2016). New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo. Neuron, 92(6), 1181–1195. https://doi.org/10.1016/J.NEURON.2016.11.030.

Figure 3.. Fluorescent small molecules to label astrocytes.

(A) Structure of the small molecule SR101. (B) SR101 can label oligodendrocytes in the mouse hippocampus seen through co-localization between SR101 and a marker for oligodendrocytes, proteolipid-protein (PLP-GFP mice). Colocalization denoted with arrows. (C) Structure of the small molecule β-Ala-Lys-N_ε-AMCA. (D) β-Ala-Lys-N_ε-AMCA is capable of labeling astrocytes in the supraoptic nucleus of the rat hypothalamus as seen by colocalization with GFAP immunolabeling. Panel (B): Reprinted with permission from Hagos, L., & Hülsmann, S. (2016). Unspecific labelling of oligodendrocytes by sulforhodamine 101 depends on astrocytic uptake via the thyroid hormone transporter OATP1C1 (SLCO1C1). Neuroscience Letters, 631, 13–18. https://doi.org/10.1016/j.neulet.2016.08.010. Panel (D): Reprinted with permission from Espallergues, J., Solovieva, O., Técher, V., Bauer, K., Alonso, G., Vincent, A., & Hussy, N. (2007). Synergistic activation of astrocytes by ATP and norepinephrine in the rat supraoptic nucleus. Neuroscience, 148(3), 712–723. http://doi.org/10.1016/J.NEUROSCIENCE.2007.03.043.

Figure 4.. Cationic methylpyridinium markers label primary rat hippocampal astrocytes and astroglia in the larval zebrafish.

(A) Structure of Rhodamine B Methylpyridinium marker. (B) Structure of NVOC₂-Q-Rhodamine Methylpyridinium photolabile marker. (C–D) Primary rat hippocampal astrocytes are robust labeled by Rhodamine B Methylpyridinium (C) while after illumination with 365 nm light, NVOC₂-Q-Rhodamine Methylpyridinium shows moderate, punctate labeling (D). (E–F) Rhodamine B Methylpyridinium labels astroglia throughout entire larval zebrafish brain (E) while after illumination with 365 nm light, NVOC₂-Q-Rhodamine Methylpyridinium shows labeling in a small subset of the zebrafish midbrain (F). Scale bar 50 μm.