Bulk Loading of Calcium Indicator Dyes to Study Astrocyte Physiology: Key Limitations and Improvements Using Morphological Maps

Calcium signaling has been studied in astrocyte cell bodies using bulk loading of calcium indicator dyes, and astrocytes are known to display intracellular calcium transients. An assumption in recent data on the neuronal impact of somatic astrocyte calcium transients has been that bulk loading reflects signaling in relevant astrocyte compartments such as processes. We assessed bulk loading using Sholl analysis (Sholl, 1953) of astrocytes loaded with common calcium indicator dyes and compared these data with Sholl analysis of astrocyte morphology. In the CA1 region of the hippocampus from rats, we found that bulk loading of calcium indicator dyes only reports on calcium signals within the soma and in the most proximal processes, leaving ∼90% of the area of an astrocyte and its extensive processes unsampled. By using morphological reconstructions as “maps” after the imaging session, we present simple procedures that remedy these shortfalls and permit reliable detection of calcium transients in distal astrocyte processes. The data thus reveal limitations in the interpretation of astrocyte calcium imaging data gathered with bulk loading and provide refinements to minimize these shortcomings.

[1]  Michael M. Halassa,et al.  Synaptic Islands Defined by the Territory of a Single Astrocyte , 2007, The Journal of Neuroscience.

[2]  A. Kocharyan,et al.  Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo , 2009, Journal of Neuroscience Methods.

[3]  A. Araque,et al.  Endocannabinoids Mediate Neuron-Astrocyte Communication , 2008, Neuron.

[4]  Baljit S Khakh,et al.  A genetically targeted optical sensor to monitor calcium signals in astrocyte processes , 2010, Nature Neuroscience.

[5]  P. Haydon,et al.  Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Neuronal Synchrony Mediated by Astrocytic Glutamate through Activation of Extrasynaptic NMDA Receptors , 2005, Neuron.

[7]  K. Harris,et al.  Three-Dimensional Relationships between Hippocampal Synapses and Astrocytes , 1999, The Journal of Neuroscience.

[8]  M. C. Angulo,et al.  Glutamate Released from Glial Cells Synchronizes Neuronal Activity in the Hippocampus , 2004, The Journal of Neuroscience.

[9]  G. Perea,et al.  Properties of Synaptically Evoked Astrocyte Calcium Signal Reveal Synaptic Information Processing by Astrocytes , 2005, The Journal of Neuroscience.

[10]  D. Attwell,et al.  Glial and neuronal control of brain blood flow , 2022 .

[11]  J. Schoenen.  The dendritic organization of the human spinal cord: The dorsal horn , 1982, Neuroscience.

[12]  H. Parri,et al.  Spontaneous astrocytic Ca2+ oscillations in situ drive NMDAR-mediated neuronal excitation , 2001, Nature Neuroscience.

[13]  M. Zonta,et al.  Cytosolic Calcium Oscillations in Astrocytes May Regulate Exocytotic Release of Glutamate , 2001, The Journal of Neuroscience.

[14]  J. Lacaille,et al.  GABAergic Network Activation of Glial Cells Underlies Hippocampal Heterosynaptic Depression , 2006, The Journal of Neuroscience.

[15]  Giorgio Carmignoto,et al.  Glutamate‐mediated astrocyte‐to‐neuron signalling in the rat dorsal horn , 2010, The Journal of physiology.

[16]  T. Pozzan,et al.  Intracellular Calcium Oscillations in Astrocytes: A Highly Plastic, Bidirectional Form of Communication between Neurons and Astrocytes In Situ , 1997, The Journal of Neuroscience.

[17]  G. Carmignoto,et al.  Enhanced Astrocytic Ca 2 + Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus , 2010 .

[18]  Sholl Da.  Dendritic organization in the neurons of the visual and motor cortices of the cat. , 1953 .

[19]  C. Rose,et al.  Developmental profile and properties of sulforhodamine 101—Labeled glial cells in acute brain slices of rat hippocampus , 2008, Journal of Neuroscience Methods.

[20]  Todd A Fiacco,et al.  Selective Stimulation of Astrocyte Calcium In Situ Does Not Affect Neuronal Excitatory Synaptic Activity , 2007, Neuron.

[21]  Todd A Fiacco,et al.  Supporting Online Material Materials and Methods Som Text Figs. S1 to S4 Hippocampal Short-and Long-term Plasticity Are Not Modulated by Astrocyte Ca 2+ Signaling , 2022 .

[22]  K. McCarthy,et al.  GFAP‐positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca2+]i , 1995, Glia.

[23]  Michael M. Halassa,et al.  Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior. , 2010, Annual review of physiology.

[24]  G. Carmignoto,et al.  Enhanced Astrocytic Ca2+ Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus , 2007, The Journal of Neuroscience.

[25]  Todd A Fiacco,et al.  Loss of IP3 Receptor-Dependent Ca2+ Increases in Hippocampal Astrocytes Does Not Affect Baseline CA1 Pyramidal Neuron Synaptic Activity , 2008, The Journal of Neuroscience.

[26]  S. Oloff,et al.  Hippocampal astrocytes in situ exhibit calcium oscillations that occur independent of neuronal activity. , 2002, Journal of neurophysiology.

[27]  A. Araque,et al.  Calcium Elevation in Astrocytes Causes an NMDA Receptor-Dependent Increase in the Frequency of Miniature Synaptic Currents in Cultured Hippocampal Neurons , 1998, The Journal of Neuroscience.

[28]  G. Perea,et al.  Astrocytes Potentiate Transmitter Release at Single Hippocampal Synapses , 2007, Science.

[29]  Grant R. Gordon,et al.  Astrocyte-Mediated Distributed Plasticity at Hypothalamic Glutamate Synapses , 2009, Neuron.

[30]  C. J. Lee,et al.  Astrocytic control of synaptic NMDA receptors , 2007, The Journal of physiology.

[31]  E. Shigetomi,et al.  Two Forms of Astrocyte Calcium Excitability Have Distinct Effects on NMDA Receptor-Mediated Slow Inward Currents in Pyramidal Neurons , 2008, The Journal of Neuroscience.

[32]  K. McCarthy,et al.  Sorting out astrocyte physiology from pharmacology. , 2009, Annual review of pharmacology and toxicology.