Brain Ultrastructure: Putting the Pieces Together

Abstract

Unraveling the fine structure of the brain is important to provide a better understanding of its normal and abnormal functioning. Application of high-resolution electron microscopic techniques gives us an unprecedented opportunity to discern details of the brain parenchyma at nanoscale resolution, although identifying different cell types and their unique features in two-dimensional, or three-dimensional images, remains a challenge even to experts in the field. This article provides insights into how to identify the different cell types in the central nervous system, based on nuclear and cytoplasmic features, amongst other unique characteristics. From the basic distinction between neurons and their supporting cells, the glia, to differences in their subcellular compartments, organelles and their interactions, ultrastructural analyses can provide unique insights into the changes in brain function during aging and disease conditions, such as stroke, neurodegeneration, infection and trauma. Brain parenchyma is composed of a dense mixture of neuronal and glial cell bodies, together with their intertwined processes. Intracellular components that vary between cells, and can become altered with aging or disease, relate to the cytoplasmic and nucleoplasmic density, nuclear heterochromatin pattern, mitochondria, endoplasmic reticulum and Golgi complex, lysosomes, neurosecretory vesicles, and cytoskeletal elements (actin, intermediate filaments, and microtubules). Applying immunolabeling techniques to visualize membrane-bound or intracellular proteins in neurons and glial cells gives an even better appreciation of the subtle differences unique to these cells across contexts of health and disease. Together, our observations reveal how simple ultrastructural features can be used to identify specific changes in cell types, their health status, and functional relationships in the brain.

Keywords: aging; brain; disease; electron microscopy; glial cells; health; neurons; organelles.

FIGURE 1

Light micrographs of a toluidine blue-stained plastic section from the adult mouse somatosensory brain region seen at low (A) and high (B) magnification. The cortex is arranged into layers that contain different types of neurons, with the largest neurons (pyramidal) in layers 4–6 which contain large euchromatic nuclei with nucleoli. Pyramidal neurons have prominent dendrites that project toward the outer cortex. At the surface of the cortex is a thin pia mater layer with underlying blood vessels (BV) that are present throughout the cortex. Deep to the cortex are myelinated axons of the white matter (WM). (B) At high magnification, capillaries (Caps) are evenly distributed in the brain parenchyma amongst the neurons, along with glial cells, which are less obvious, and contain smaller nuclei. Dispersed myelinated axons stain deeper blue and appear as wormlike structures in the parenchyma.

FIGURE 2

Low (A) and high (B) magnification EM images of a pyramidal neuron in layer 4/5 of the somatosensory cortex. The neuron contains a large euchromatic nucleus with two nucleoli (*) and a large primary dendrite emanates from the cell body. Surrounding the neuronal cell body is the neuropil consisting of a mixture of glial and neuronal processes, including synapses. The nuclear envelope contains numerous nuclear pores (NPs) and the perinuclear cytoplasm has a rich collection of organelles including mitochondria (Mito), RER, and Golgi complexes (GC) cisternae, vesicles, multivesicular bodies (MVBs), and lysosomes (Ly). Free ribosomes and neurofilaments (NFs) are dispersed in between the organelles.

FIGURE 3

A pyramidal neuron in layer 5 with a satellite cell that displays ultrastructural features of an oligodendrocyte precursor cell closely abutting its cell body. The pyramidal neuron contains a large euchromatic nucleus with a centrally located nucleolus (*). Nuclear envelope invaginations (arrow) are occasionally seen in highly active neurons (Wittmann et al., 2009). Some neurons have intimately associated satellite cells (microglia and oligodendrocyte precursor cells) with a smaller ovoid nucleus. The surrounding neuropil contains synapses, axons (A) and glial cell processes.

FIGURE 4

Protoplasmic astrocyte in the mouse cerebral cortex (A) and high magnification view of a fibrous astrocyte in the CA1 region of a rat hippocampus (B). (A) In the cortex, astrocytes are branched and contain a relatively clear cytoplasm with a variety of organelles including mitochondria, RER, lysosomes, perinuclear Golgi complexes. In rodent astrocytes, mitochondria (Mi) stain relatively lighter after postfixation with reduced osmium. De, dendrite; Sp, dendritic spine; *, presynaptic terminal.

FIGURE 5

Components of the blood-brain barrier (BBB) and neurovascular unit. (A) Transverse section through a mouse cortical capillary showing the highly attenuated endothelium with small branches of pericytes sitting on their abluminal surface. (B) The BBB consists of the capillary endothelium with their tight junctions, underlying pericytes, both surrounded by a prominent amorphous basement membrane (BM) and astrocytic end-feet. End-feet connect with neighboring end-feet of other astrocytes via gap junctions. Ast, astrocyte; De, dendrite.

FIGURE 6

A fortuitous section of a mouse cortical capillary showing an endothelial cell and a pericyte at the level of their nuclei. Notice how the crescent-shaped heterochromatic nuclei take on the shape of the capillary. Tight junctions (TJs) link neighboring endothelial cells. The pericyte embraces the endothelium and resides within the same basement membrane. The position and branching nature of its processes are strategically situated to change the capillary lumen diameter. The relatively clear astrocyte end-feet (Ast) occupy the surrounding region around the capillary in the lower part of the image. Some electron dense glycogen granules are visible within the astrocyte.

FIGURE 7

Low (A) and medium (B) magnification images of a typical oligodendrocyte (Oligo) sectioned at the level of its small heterochromatic nucleus in layer 5 of the mouse cortex. Several neurons are seen in the surrounding region for comparison of nuclear size and chromatin density. A neighboring capillary (Cap) and numerous dendrites (De) are seen in the surrounding neuropil. (B) A collection of myelinated axons (A) juxtapose and appear partially embedded within the relatively electron-dense cytoplasm of the oligodendrocyte. The dense cytoplasm contains a prominent perinuclear Golgi complex (GC), scattered mitochondria and small segments of rough endoplasmic reticulum. A spine head (*) synapses on a presynaptic terminal in the lower right of the image.

FIGURE 8

Low (A) and high (B) magnification views of a perivascular microglial cell in the cerebral cortex of an aged mouse (18 mo old). The nuclei of microglia are small and pleomorphic, and contain relatively more heterochromatin than neurons. Large, tertiary lysosomes with undigestible debris occupy the cytoplasm. Long stretches of rough endoplasmic reticulum (RER) characterize microglia that are active, in terms of producing inflammatory cytokines and other mediators. A dendritic spine (Sp) forms a synapse with a presynaptic terminal near the microglial cell, and an astrocytic branch (Ast) is in close proximity. Microglia are strategically situated between neurons and capillaries (Cap), and function as the resident immune cell and phagocyte required for maintaining brain health throughout life.

FIGURE 9

Low (A) and high (B) magnification views of a perineuronal microglial cell closely abutting a pyramidal neuron in the aged mouse cortex. Microglia are now believed to play an integral part in maintaining the synapse (aka quadpartite synapse, encircled) which includes the presynaptic terminal (*), dendritic spine (Sp), astrocyte process (Ast), and microglia. A, axon; De, dendrite; Ly, lysosome.

FIGURE 10

Examples of synapses in the mouse substantia nigra (A) and dentate gyrus (B). (A) Three types of presynaptic terminals are evident in the substantia nigra: Cholinergic (Ach) with large (50–70 nm) vesicles, glutamatergic (Glut) with medium sized (35–50 nm) vesicles, and GABAergic (inhibitory) with small ovoid (20–35 nm) vesicles. (B) High magnification view of dendritic spines (Sp) emanating from dendrites (De) and synapsing with glutamatergic pre-synaptic terminals (Pre). post-synaptic densities (PSD) characterize glutamatergic synapses. Astrocytic processes (Ast) occupy the intervening spaces between these structures. Microtubules (MT) are seen running along the length of the dendrite. A, axon.

FIGURE 11

Dystrophic neurites closely surrounded by glial cells in the hippocampal CA1 region of a 20 mo old APP-PS1 mouse, a model of Alzheimer disease pathology (A). Magnified regions are shown in panels (B) and (C). An abnormally large astrocyte (Ast) contains distinctive intermediate filaments, while a dark microglia (DM) is recognized by the condensation state of its cytoplasm and nucleoplasm, in addition to its microglial features (e.g., long stretches of endoplasmic reticulum). (C) The dark microglia contains lipidic inclusions and extends a process (arrowheads) that contacts a synapse and encircles a dystrophic neurite (*). Cap, capillary.

FIGURE 12

Normal (A) and 3 day post-stroke capillaries (B) in the peri-infarct zone of the mouse cortex. Astrocytes (shaded red) are pseudocolored to illustrate the drastic increase in volume after ischemia. The endothelium (E) and pericytes (P) are also enlarged after stroke. Note the accumulation of glycogen granules in the perivascular end-feet of the astrocytes. De, dendrite; Micro, microglial cell.

FIGURE 13

Low (A) and high (B) magnification views of changes to neurons and dendritic branches following ischemic stroke (3 days post-stroke) deep in the peri-infarct zone of the mouse cortex. A portion of a degenerating neuron (Degen neuron) is seen in the upper left and contains swollen mitochondria. Dendrites in the peri-infart zone swell and appear to absorb the spinous processes with evidence of the synapses apparent at the edges of the dendrite; note the post-synaptic densities (PSD) at the presynaptic terminal (Pre) contacts in (A). Mitochondria in the swollen dendrite undergo dysplastic changes and exhibit dilated, loosely arranged cristae. Note the increase in size of the pericyte surrounding the capillary and the swollen astrocyte (Ast). Compare to a normal capillary in Figure 6.