The Journal of Neuroscience, June 15, 2011 • 31(24):8905–8919 • 8905 Development/Plasticity/Repair Astrocytes Display Complex and Localized Calcium Responses to Single-Neuron Stimulation in the Hippocampus Yann Bernardinelli, Chris Salmon, Emma V. Jones, W. Todd Farmer, David Stellwagen, and Keith K. Murai Centre for Research in Neuroscience, Department of Neurology and Neurosurgery, The Research Institute of the McGill University Health Centre, Montreal General Hospital, Montreal, Quebec, Canada, H3G 1A4 Astrocytes show a complex structural and physiological interplay with neurons and respond to neuronal activation in vitro and in vivo with intracellular calcium elevations. These calcium changes enable astrocytes to modulate synaptic transmission and plasticity through various mechanisms. However, the response pattern of astrocytes to single neuronal depolarization events still remains unresolved. This information is critical for fully understanding the coordinated network of neuron–glial signaling in the brain. To address this, we developed a system to map astrocyte calcium responses along apical dendrites of CA1 pyramidal neurons in hippocampal slices using single-neuron stimulation with channelrhodopsin-2. This technique allowed selective neuronal depolarization without invasive manip- ulationsknowntoaltercalciumlevelsinastrocytes.Light-evokedneuronaldepolarizationwaselicitedandcalciumeventsinsurrounding astrocytes were monitored using the calcium-sensitive dye Calcium Orange. Stimulation of single neurons caused calcium responses in populations of astrocytes along the apical axis of CA1 cell dendrites. Calcium responses included single events that were synchronized with neuronal stimulation and poststimulus changes in calcium event frequency, both of which were modulated by glutamatergic and purinergic signaling. Individual astrocytes near CA1 cells showed low ability to respond to repeated neuronal depolarization events. However, the response of the surrounding astrocyte population was remarkably accurate. Interestingly, the reliability of responses was graded with respect to astrocyte location along the CA1 cell dendrite, with astrocytes residing in the primary dendrite subregion being most responsive. This study provides a new perspective on the dynamic response property of astrocyte ensembles to neuronal activity. Introduction vivo (Fellin et al., 2009). However, a few studies have questioned Astrocytes are important for information processing in the brain the importance of astrocyte calcium signaling in synaptic trans- (Volterra and Meldolesi, 2005). This concept gained support mission and plasticity (Fiacco et al., 2007; Petravicz et al., 2008; when astrocytes were shown to respond to glutamate by elevating Agulhon et al., 2010). Thus, additional studies addressing the 2ϩ complex nature of neuron–glial interactions and the properties intracellular calcium ([Ca ]i) (Cornell-Bell et al., 1990). Astro- cyte calcium signaling triggers gliotransmitter release that can of neuron-evoked astrocyte calcium events are needed. regulate NMDA receptors (Parri et al., 2001), AMPA receptors Astrocytes show intricate morphological interactions with (Fiacco and McCarthy, 2004), and inhibitory synapses (Kang et neurons and other astrocytes. A single astrocyte associates with 5 al., 1998) and modulates heterosynaptic depression (Serrano et hundreds of dendrites (Halassa et al., 2007) and up to 10 syn- al., 2006), presynaptic glutamate release (Jourdain et al., 2007), apses (Bushong et al., 2002) and is coupled with other astrocytes and long-term potentiation (Henneberger et al., 2010). Neuron– through gap junctions (Giaume and Venance, 1998). At the same glial signaling also scales synaptic weight in the hypothalamus time, a single neuron passes through the territory of many astro- 2 (Gordon et al., 2009) and modulates cortical network activity in cytes (ϳ5 astrocytes/8000 ␮m ) in the hippocampus (Nixdorf- Bergweiler et al., 1994). Thus, a population of astrocytes is likely responsive to the activity of an individual neuron. Indeed, Received Dec. 6, 2010; revised April 4, 2011; accepted April 26, 2011. 2ϩ Author contributions: Y.B. and K.K.M. designed research; Y.B., C.S., E.V.J., and W.T.F. performed research; D.S. contrib- Schaeffer collateral stimulation causes [Ca ]i changes in 15% uted unpublished reagents/analytic tools; Y.B., C.S., and K.K.M. analyzed data; Y.B. and K.K.M. wrote the paper. (Pasti et al., 1997) to 62% (Porter and McCarthy, 1996) of astro- This work was supported by the Canadian Institutes of Health Research, Canada Research Chairs Program, Cana- cytes within a microscope field of view. Groups of cortical astro- dian Foundation for Innovation, and the EJLB Foundation (K.K.M.). Y.B. was supported by Fellowships PBLAB- cytes also respond to visual stimuli (Schummers et al., 2008) and 118155 and PA00A3-121419 from the Swiss National Science Foundation. C.S. was supported by a fellowship through the Canadian Institutes of Health Research, and E.V.J. and W.T.F. were partially supported through post- show activation patterns coordinated with neuronal subpopula- doctoral fellowships through the Research Institute of the McGill University Health Centre. We thank Dr. Karel tions (Schipke et al., 2008), suggesting that astrocytes form func- SvobodaforthepCAGGS–ChR2–Venusconstruct,Dr.RogerTsienfortheoriginalmCherryvector,andthelaboratory tional domains near neurons (Fellin, 2009). However, the spatial of Dr. Charles Bourque for their technical assistance with acute slice electrophysiology. relationship between a single neuron and a responding astrocyte CorrespondenceshouldbeaddressedtoDr.KeithK.Murai,CentreforResearchinNeuroscience,MontrealGeneral Hospital, 1650 Cedar Avenue, L7-212, Montreal, QC, Canada, H3G 1A4. E-mail: [email protected]. population has not been reported. DOI:10.1523/JNEUROSCI.6341-10.2011 To understand how neuron–astrocyte domains are organized, Copyright © 2011 the authors 0270-6474/11/318905-15$15.00/0 a method for single-neuron activation needs to be used. Depolar- 8906 • J. Neurosci., June 15, 2011 • 31(24):8905–8919 Bernardinelli et al. • Astrocyte Responses to Single-Neuron Stimulation ization of interneurons (Kang et al., 1998) or CA1 cells (Na- open chamber (Warner Instruments) on the stage of an upright Leica varrete and Araque, 2008) using patch-clamp techniques can DMS microscope connected to an Ultraview confocal spinning disk sys- trigger astrocyte calcium responses. However, direct mechanical tem (PerkinElmer Life and Analytical Sciences) and perfused with 34°C and electrical stimulation of astrocytes can cause glial calcium ACSF. The ACSF solution contained the following (in mM): 130 NaCl, 3 KCl, 26 NaHCO , 2 CaCl , 1.48 MgCl , 1.23 NaH PO , and 10 glucose events (Hassinger et al., 1996; Angulo et al., 2004; Bernardinelli et 3 2 2 2 4 ϩ (bubbled with 5% CO /95% oxygen). CaO was excited by a 568 nm al., 2004) and complicate [Ca 2 ] measurements. Recent ad- 2 i Kr/Ar laser band, and emission was collected through a rhodamine emis- vances in optogenetics have provided powerful approaches to sion filter (Chroma Technology) with a 40ϫ Achroplan, 0.8 NA water- non-invasively map neuronal circuits (Wang et al., 2009). Here immersion objective (Leica). Single-plane images were acquired at 0.5 Hz we applied this technology using channelrhodopsin-2 (ChR2) using Metafluor software (Molecular Devices). CaO fluorescence inten- (Nagel et al., 2003) to control single-neuron activation in situ and sities were monitored offline in every astrocyte soma in the field of view probe neuron–glial interactions without acute physical pertur- using Metafluor, and calcium events were analyzed in Clampfit (Molec- bation of cells. Stimulation of a single hippocampal CA1 cell ular Devices). All calcium increases Ͼ2 SDs were collected. Events oc- 2ϩ curring 0–15 s after neuronal stimulation were considered as induced [Ca ]i changes in astrocytes with a reliability that was related to astrocyte position and dependent on glutamatergic and synchronized calcium events. This time window corresponds to the time needed for an intercellular calcium wave to propagate over a distance of purinergic pathways. Furthermore, astrocyte ensembles accu- ␮ rately responded to neuronal stimulation, suggesting that astro- 275 m [mean distance of propagation of calcium waves (Kang and Othmer, 2009) with a speed of 21 ␮m/s (mean calcium wave speed) cytes organize into functional domains and respond as a network (Kang and Othmer, 2009)] plusa2slatency (Dani et al., 1992). The of cells. This type of coordination is likely essential for modulat- calculation made here corresponds to the time window previously used ing neuronal activity within hippocampal microcircuits. by others (Navarrete and Araque, 2008). For uncoupling experiments, the time window was reduced to 5 s. This is the time needed for an Materials and Methods intracellular calcium wave to cross a single astrocyte having a 50 ␮m Animals. For all experiments, both male and female mice (C57BL/6J) diameter and witha2slatency. Astrocytes diameter was calculated by were used. Animal procedures were performed in accordance with the approximating their shape as a sphere and having a volume of 65,900 guidelines of the Canadian Council for Animal Care and the Montreal ␮m 3 (Bushong et al., 2002). General Hospital Facility Animal Care Committee. Immunofluorescence labeling. For immunofluorescence after astrocyte Organotypic hippocampal slice preparation. Organotypic hippocampal loading