Cytoskeletal microdifferentiation: a mechanism for organizing morphological plasticity in dendrites

Abstract

Experimental evidence suggests that microfilaments and microtubules play contrasting roles in regulating the balance between motility and stability in neuronal structures. Actin-containing microfilaments are associated with structural plasticity, both during development when their dynamic activity drives the exploratory activity of growth cones and after circuit formation when the actin-rich dendritic spines of excitatory synapses retain a capacity for rapid changes in morphology. By contrast, microtubules predominate in axonal and dendritic processes, which appear to be morphologically relatively more stable. To compare the cytoplasmic distributions and dynamics of microfilaments and microtubules we made time-lapse recordings of actin or the microtubule-associated protein 2 tagged with green fluorescent protein in neurons growing in dispersed culture or in tissue slices from transgenic mice. The results complement existing evidence indicating that the high concentrations of actin present in dendritic spines is a specialization for morphological plasticity. By contrast, microtubule-associated protein 2 is limited to the shafts of dendrites where time-lapse recordings show little evidence for dynamic activity. A parallel exists between the partitioning of microfilaments and microtubules in motile and stable domains of growing processes during development and between dendrite shafts and spines at excitatory synapses in established neuronal circuits. These data thus suggest a mechanism, conserved through development and adulthood, in which the differential dynamics of actin and microtubules determine the plasticity of neuronal structures.

Figure 1

Actin and MAP2 differ in both distribution and dynamics in living hippocampal neurons. (A) Distribution of actin and MAP2 in a transfected hippocampal neuron in cell culture for 24 days, simultaneously expressing actin-GFP and MAP2c-YFP. The phase-contrast image (Left) shows the arrangement of the cell body and processes of the transfected cell interspersed with the network of axonal processes of untransfected cells. The original gray-scale images for actin-GFP (Center) and MAP2c-YFP (Right) images were prepared by using appropriate selective filter sets. (Bar = 20 μm.) (B) Comparative distribution of actin and MAP2 in a dendrite segment produced by overlaying pseudocolored images for actin-GFP (green) and MAP2c-YFP (red). The high concentration of actin in dendritic spines (arrowheads) contrasts with the confinement of MAP2 to dendrite shafts. (Bar = 2 μm.) (C and D) Time-dependent changes in the configuration of actin and MAP2 in dendrites. Six frames from a single time-lapse recording for actin-GFP (C) and MAP2-YFP (D) images, recorded alternately 30 s apart, were converted into profile outlines. Each outline was assigned a different color and overlaid onto a single gray-scale image from the same recorded sequence. Variations between the different color outlines indicate regions of morphological change that are evident in the actin images of dendritic spines (C) but are absent from the MAP2 images of the dendrite shaft (D). (Bar = 2 μm.) Refer to supplemental Movie 1 for the original time-lapse sequence.

Figure 2

MAP2 is absent from dendritic spines. (A and B) Like the embryonic low-molecular weight variant MAP2c, high-molecular weight MAP2b is confined to the somatodendritic domain of hippocampal neurons and is absent from the axon (arrow). Shown are both a phase (A) and a fluorescence (B) image of a live neuron transfected with GFP-tagged MAP2b and kept in culture for 14 days. (Bar = 25 μm.) (C and D) This neuron transfected with MAP2b-GFP was maintained in culture for 4 weeks, by which time cells carry many dendritic spines. In the enlarged image (D) of the area outlined in C, the restriction of MAP2b-GFP fluorescence to microtubule bundles in the dendritic shaft is obvious. No fluorescence is detected in spine protuberances from the dendrite. (Bars: C = 15 μm; D = 2 μm.)

Figure 3

Time-lapse recording of MAP2 in hippocampal tissue slices from transgenic mice stably expressing MAP2c-GFP. (A) Confocal GFP fluorescence image taken near the cell body layer of area CA1 in an organotypic slice culture established from an 11-day-old transgenic mouse and maintained in culture for 25 days. Nuclei in cell bodies are marked with *. (Bar = 10 μm.) (B) MAP2 localization in hippocampal neurons is limited to the shafts of dendrites. Single frame taken from a time-lapse recording of MAP2c-GFP fluorescence in the CA1 neuropil of a hippocampal slice maintained in culture for 4 weeks. (Bar = 5 μm.) (C and D) Short-term time-lapse assay for MAP2 dynamics. (C Left) A single confocal gray-scale image of MAP2-GFP fluorescence in area CA1 of a 5-week-old hippocampal slice culture. (C Right) A pseudocolor “difference image” produced by summing gray-scale differences between images taken 30 sec apart over 10 min of time-lapse recording. Compare the overall lack of change in the MAP2 image (dark blue color) during the recording period to the high degree of change (green, yellow, and red) in actin images of similar configuration (Fig. 4A Right). (Bar = 5 μm.) (D) Long-term timelapse assay for MAP2 dynamics. Original gray-scale fluorescence image (Upper) and pseudocolor difference image (Lower) of a dendrite segment followed over a 3-h time period. The dark blue color again indicates an overall lack of change in MAP2 distribution during this longer recording period. (Bar = 5 μm.)

Figure 4

Time-lapse recording of actin dynamics in dendrite spines of hippocampal tissue slices from transgenic mice expressing actin-GFP. (A) (Left) An original fluorescence image in a single frame from a time-lapse recording in which frames were collected 30 sec apart. (Right) Changes in actin distribution over 10 min displayed by difference imaging using a pseudocolor scale (see text for details). Red and yellow patches indicate areas of high motility associated with dendritic spines. (Bar = 10 μm.) (B) Single gray-scale frame (Upper) and pseudocolor difference image at higher magnification. Shape changes are associated with dendritic spines (red and yellow patches) whereas the dendrite shaft shows little dynamic activity (Lower). (Bar = 10 μm.)

Figure 5

Hypothetical scheme for the partitioning of cytoskeletal microdomains between shaft and spine in dendrites. (Left) Part of dendrite in the region of a spine synapse. The axonal component (ax.), with its swollen presynaptic (pre.) bouton containing synaptic vesicles (sv.) is outlined in gray. It forms a synapse at the tip of a dendritic spine head. Inside the spine head the junctional region is marked by the postsynaptic density (psd.), a complex of scaffolding proteins that acts as the platform for assembling functional molecules such as neurotransmitter receptors and ion channels. The cytoskeleton of the dendritic spine is composed of actin filaments (barbed lines) that are inserted into the psd. The cytoskeleton of the underlying dendrite consists predominantly of microtubules (gray rods), which in dendrites are bidirectionally oriented so that some have the plus ends distally and others the minus end distally as indicated. This distribution of cytoskeletal filaments demarcates three cytoplasmic zones, an M zone in the dendrite shaft, where microtubules predominate, an A zone in the dendritic spine, where actin filaments predominate, and a T, or transition, zone. (Right) The expanded diagram shows the relationship of these zones to the delivery of materials to the synaptic domain as suggested by current evidence. Transport vesicles (blue filled circles) carry cargoes of functional molecules, such as NMDA receptors (pale blue symbols), bound for the postsynaptic membrane. These vesicles bear both microtubule-dependent (M, kinesin and dynein) and actin-dependent (A, myosin) motor molecules. Transitory detachment of kinesin and dynein from microtubule tracks provides the opportunity for the myosin motors of transport vesicles to engage with the actin filaments of dendritic spines along which they travel to the synaptic domain. Single chevrons in the vicinity of the postsynaptic membrane represent the presence of labile actin filaments in this zone.