Microdomains in forebrain spines: an ultrastructural perspective

Abstract

Glutamatergic axons in the mammalian forebrain terminate predominantly onto dendritic spines. Long-term changes in the efficacy of these excitatory synapses are tightly coupled to changes in spine morphology. The reorganization of the actin cytoskeleton underlying this spine "morphing" involves numerous proteins that provide the machinery needed for adaptive cytoskeletal remodeling. Here, we review recent literature addressing the chemical architecture of the spine, focusing mainly on actin-binding proteins (ABPs). Accumulating evidence suggests that ABPs are organized into functionally distinct microdomains within the spine cytoplasm. This functional compartmentalization provides a structural basis for regulation of the spinoskeleton, offering a novel window into mechanisms underlying synaptic plasticity.

Figure 1. Appearance of dendritic spines in the electron microscope

A. 3D reconstruction of serial thin sections (from stratum radiatum of rat CA1 shows stubby (s) spines on the same segment of a dendritic shaft as spines with thin (t) and mushroom (m) shapes. Synaptic contacts have been colorized in red. Image is from http://synapses.clm.utexas.edu/anatomy/compare/compare.stm, reprinted with kind permission from J. Spacek. B. Micrograph of a thin (70 nm) section shows a typical mushroom-shaped spine from the rat hippocampus. Note the presynaptic terminal with synaptic vesicles, the synaptic cleft (arrowhead) and the postsynaptic density (PSD). The filamentous material in the spine head (sp) represents the actin cytoskeleton. Scale bars: 200 nm

Figure 2. Appearance of F-actin in spines, demonstrated by high-resolution EM techniques

Left panel shows electron tomography of a thin section from rat neocortex (images adapted with permission from [81]); right panel is from a platinum-shadowed replica of cultured hippocampal neurons ((image adapted with permission from [80]). A shows a virtual slice (~4 nm thick) through a spine (the PSD is at top). A volume containing a long, straight filament (highlighted by the white box) is shown in B₁ (image inverted to highlight features). This filament displays helical periodicity corresponding closely to that predicted for F-actin. B₂ shows a helically-averaged surface representation of the extracted density. B₃ is a low-resolution representation of an atomic model of a canonical actin filament obtained from high-resolution electron microscopy analysis. B₄ shows the fit of this atomic model (blue cartoon representation) into the symmetrized, extracted filament density (chicken wire representation). C is an enlargement of the boxed region in B. Scale bars: 100 nm for A, 10nm for B and C. D illustrates the cytoskeleton of a spine after membranes have been removed. The organization of filaments in the head and neck of a mushroom-shaped spine associated with dendrite (yellow pseudocolor, at bottom of inset) and with axon running along the head (magenta, top of inset) from 14 DIV neurons, treated with Triton X-100 to reveal internal features. Thick fibers in dendritic shaft represent microtubules. Actin filaments (cyan pseudocolor in inset) are the main cytoskeletal elements in the spine head and neck. Inset in D is a shrunk and pseudocolored version of panel D. Scale bar 200 nm.

Figure 3. ABP content of spines, revealed by immunogold

Hippocampal spines (Sp) labeled with immunogold for eight different actin-binding proteins. (Images adapted with permission from [109] (α-actinin), [116] CaMKII, [137] (cofilin), [149] (Arp2/3 complex),[163] (profilin), [157] (cortactin), [174] (drebrin) and [125] (synaptopodin). Shaded area represents the zone of concentration, for each protein. Scale bar: 200 nm.

Figure 4. ABP microdomains in the spinoskeleton

Proteomic studies of forebrain synapses have identified a sizable number of ABPs in the biochemically-defined PSD [110, 182, 183]. Some of these may be contaminants, but several, including α-actinin (blue double ovals) and CaMKII (black hexagons), have been demonstrated with immuno-EM to lie within the morphologically-defined PSD. Imaging studies reveal that actin (small blue circles) is more dynamic in the shell of the spine than in the center, suggesting a functional gradient of activity, from shell to core. Consistent with these data, cofilin (yellow circles) — a protein responsible for depolymerization of filaments — is heavily concentrated in this shell domain. The presence of a distinct “subshell” microdomain within the spinoplasm is suggested by the accumulation of the Arp2/3 complex (green composites), which mediates filament branching. The center (“core”) of the spine contains a relatively stable pool of actin. A heterogeneous pool of ABPs, including cortactin, profilin and drebrin (red circles), concentrate in this core microdomain.