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. 2006 Aug 30;26(35):8881-91.
doi: 10.1523/JNEUROSCI.1302-06.2006.

Cooperative astrocyte and dendritic spine dynamics at hippocampal excitatory synapses

Michael Haber  1 , Lei Zhou, Keith K Murai
Affiliations

Cooperative astrocyte and dendritic spine dynamics at hippocampal excitatory synapses

Michael Haber et al. J Neurosci. .

Abstract

Accumulating evidence is redefining the importance of neuron-glial interactions at synapses in the CNS. Astrocytes form "tripartite" complexes with presynaptic and postsynaptic structures and regulate synaptic transmission and plasticity. Despite our understanding of the importance of neuron-glial relationships in physiological contexts, little is known about the structural interplay between astrocytes and synapses. In the past, this has been difficult to explore because studies have been hampered by the lack of a system that preserves complex neuron-glial relationships observed in the brain. Here we present a system that can be used to characterize the intricate relationship between astrocytic processes and synaptic structures in situ using organotypic hippocampal slices, a preparation that retains the three-dimensional architecture of astrocyte-synapse interactions. Using time-lapse confocal imaging, we demonstrate that astrocytes can rapidly extend and retract fine processes to engage and disengage from motile postsynaptic dendritic spines. Surprisingly, astrocytic motility is, on average, higher than its dendritic spine counterparts and likely relies on actin-based cytoskeletal reorganization. Changes in astrocytic processes are typically coordinated with changes in spines, and astrocyte-spine interactions are stabilized at larger spines. Our results suggest that dynamic structural changes in astrocytes help control the degree of neuron-glial communication at hippocampal synapses.

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Figures

Figure 1.
Figure 1.
Targeting complex neuron–glial interactions in the hippocampus using organotypic hippocampal slices and viral gene delivery. a, SFV constructs used in the study. EGFPf or RFPf were cloned after the genes encoding viral nonstructural proteins and a subgenomic promoter (arrow) on SFV plasmids. b, c, Confocal images showing examples of apical dendrites with prominent spines on CA1 pyramidal neurons infected with SFV PD EGFPf (b, green) or SFV PD RFPf (c, red). d–f, Low-magnification epifluorescence images showing the selectivity of SFV A7 for protoplasmic astrocytes in the stratum radiatum of area CA1. Infected astrocytes expressing EGFPf (d, green; arrowheads) are labeled for GFAP (e, red). g–i, Single-plane confocal images of an SFV A7-infected astrocyte showing EGFPf expression (g, green) and GFAP immunoreactivity (h, red). Note how the GFAP immunolabeling only labels the main processes of the infected protoplasmic astrocyte, whereas EGFPf reveals the fine processes (i). Scale bars: b, c, 5 μm; d–i, 10 μm.
Figure 2.
Figure 2.
Astrocytic elaboration and domain spacing in area CA1 of the adult mouse hippocampus and in organotypic slice culture. a–c, Diolistic labeling of protoplasmic astrocytes in fixed hippocampal tissue. Maximum projections of confocal images of neighboring astrocytes labeled with DiI (a, red) and DiO (b, green) reveals their distinct domain spacing. d–f, Astrocytes in organotypic hippocampal slice culture also occupy distinct territories. Maximum projections of confocal images of adjacent astrocytes expressing RFPf (d, red) and EGFPf (e, green) after 1 week in vitro. The dashed lines in c and f indicate the boundary between neighboring astrocytes. Scale bars, 10 μm.
Figure 3.
Figure 3.
Complex neuron–glial interactions in the hippocampal CA1 neuropil. a–c, Confocal maximum projections of a DiI-labeled apical dendrite from a CA1 pyramidal cell (a, red) extending through a DiO-labeled astrocyte domain (b, green) in a fixed slice of the adult hippocampus. Areas of overlap show the complexity of neuron–glial interactions. d–f, Confocal maximum projections of a dendrite from an SFV PD RFPf-infected CA1 pyramidal neuron (d, red) projecting through an SFV A7 EGFPf-infected astrocyte domain (e, green) after 1 week in vitro. g, h, Three-dimensional reconstructions of the boxed areas in f, demonstrating the complex interplay between astrocytic processes and dendritic spines. Inset in h shows the neuron–glial association rotated 90°. Arrowheads show examples of astrocytic processes extending toward spines. Scale bars, 5 μm.
Figure 4.
Figure 4.
Dendritic spines are innervated in hippocampal slice culture. a, b, SFV PD RFPf-infected slices at 1 and 3 weeks in vitro and immunolabeled for synaptophysin (white) to label presynaptic terminals. At both time points, the majority of CA1 pyramidal cell dendritic spine heads (red) are innervated by presynaptic terminals (arrowheads), whereas only a small proportion are not (arrows). Spines that show association of synaptophysin punctae on the neck are not labeled. Shown are images of the synaptophysin labeling before and after performing a three-dimensional “masking” procedure to preserve only the synaptophysin punctae that are associated with the dendrite (see Materials and Methods). Scale bars, 1 μm.
Figure 5.
Figure 5.
Astrocytes undergo rapid structural modifications. a, An example showing astrocytic process retraction and extension over a 10 min imaging period (confocal maximum projections of the first 4 time points are shown). b, Three-dimensional reconstructions of the area outlined in a over the 10 min period. c, Volume measurements of retracting (red circles) and extending (blue squares) processes plotted over time. Scale bars: a, 10 μm; b, 4 μm.
Figure 6.
Figure 6.
Astrocyte–spine interactions show dynamic structural interplay. a, Example of an astrocytic process (green) extending toward a dendritic spine (red). b, Example of an interaction that is lost between a dendritic spine (red) and a retracting astrocytic process (green). Insets show 90° (a) or top-down (b) views of the astrocyte–spine interactions. Scale bars, 1 μm.
Figure 7.
Figure 7.
Properties of astrocyte–spine interactions. a, Astrocytic processes have significantly higher motility indices (MI) than spines (n = 71 spine–astrocyte pairs from 7 experiments; p < 0.007, paired Student's t test; error bars represent SEM). b, Plot of astrocyte versus spine motility reveals three populations of interactions: astrocytes with similar (51%, astrocyte MI/spine MI is >0.5 but <1.5; circles, line represents best fit), greater (32%, astrocyte MI/spine MI is >1.5; squares), or less (17%, astrocyte MI/spine MI is <0.5; triangles) motility than dendritic spines. c, Approximately 50% of astrocytic process–spine pairs show coordinated changes in size over time, 27% are not coordinated, and 23% show opposite changes (10% cutoff for increases or decreases in size; see Materials and Methods; error bars represent SEM). d, Plot of spine motility index versus average spine area. Spines with greater area show less motility. Curve represents a power trend. e, Cytochalasin-D treatment (5 μm) reduces both spine and astrocyte motility. Motility indexes calculated before (circles) and after (squares) the start of cytochalasin-D (Cyto-D) treatment for individual spines and astrocytic processes (spines, p < 0.003, paired Student's t test, n = 20 spines; astrocytic processes, p < 0.006, paired Student's t test, n = 25 astrocytic processes). f, Perfusion of bicuculline (20 μm) significantly reduces the motility of dendritic spines but not astrocytic processes. Motility indexes calculated before (circles) and after (squares) the start of bicuculline application for spines and astrocytic process (spines, p = 0.002, paired Student's t test, n = 31 spines; astrocytic processes, p > 0.05, paired Student's t test, n = 49 astrocytic processes).
Figure 8.
Figure 8.
Stability of astrocyte–spine interactions. a, Example of an interaction between a stable spine and its associated astrocytic process during a 15 min imaging period. b, Plot of percentage spine volume change versus average spine volume showing that larger spines have smaller changes in volume over time and more stable contacts with astrocytic processes (n = 32 pairs from 3 experiments). Curves represent power trends. Scale bar, 0.5 μm.
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