CHAPTER 6 Neurons and Neuroglia
Neurons
Webster’s dictionary defines a neuron as “the structural and functional unit of the nervous system, consisting of the nerve cell body and all its processes, as the dendrites and axon.”1 One may quibble with the attribution of structural properties to the neuron, but otherwise, this is a relatively concise and accurate definition. The problem of defining the neuron in any more detail is curiously difficult. This difficulty arises because more than any other cell type in the body, there is an enormous diversity of structural and functional characteristics possessed by the various cells that go by this name. Consider as three examples the Purkinje cell, the retinal photoreceptor cell, and the dorsal root ganglion neuron (Fig. 6-1). All three are considered neurons, yet the commonalities among them are difficult to pin down. The Purkinje cell is the most straightforward of the three (Fig. 6-1A). There is a clear cellular domain that is the neuronal dendrite, a prominent cell body, and an obvious single axon. The parts of the retinal photoreceptor (Fig. 6-1B) are less easily categorized. The apical portion serves as a receptor for light, yet it is hardly a normal dendrite. The cell body is apparent, but the axon is unconventional in its thickness and appearance. The dorsal root ganglion neuron (Fig. 6-1C) also diverges from the classic neuronal form. A cell body is clear, but the process that emanates from it bifurcates; one branch connects to a peripheral target, and the other branch connects to a target within the central nervous system (CNS). Functionally, the two branches are distinct, but morphologically, they are nearly identical; under most definitions, however, neither would qualify as a dendrite.
Neuronal Function
The emphasis in the previous section was on the diversity of nerve cell types. Discussion of the functioning of the adult neuron, however, requires a focus on commonalities. The goal of this section is not to describe the biochemical and biophysical mechanisms that the cell uses to create, store, and transmit electrical signals; this information is provided in Chapter 49. Rather, this section follows a packet of information as it moves through a typical nerve cell in the brain, with the goal of introducing the biology of the cell. Figure 6-2A is a diagram of one typical neuron. Synaptic input occurs on all synaptic spines, as shown in the enlarged spines at the far left. Synaptic spines can receive input from a single (Fig. 6-2B) or multiple (Fig. 6-2C) axons.
The information arrives at the nerve cell at a highly specialized structure known as a synapse. At this site, a process from the previous neuron in the circuit approaches to within a few hundred angstroms of the next cell but does not touch it. The gap between the cells remains continuous with the extracellular space. The presence of the gap requires a specialized mechanism for transferring an electrical signal from the presynaptic to the postsynaptic cell. This transfer is achieved by way of the presynaptic cell secreting a chemical transmitter substance into the gap. The transmitter is usually a small molecule such as acetylcholine or noradrenaline, but it can also be a peptide such as substance P or vasointestinal peptide. Diffusion carries this pulse of chemicals across the gap to the membrane of the postsynaptic cell, which is covered with receptor proteins. These receptors recognize the secreted chemicals and transform the information from the chemical pulse into an electrical event that can be propagated down the neuron. This mechanism is discussed in more detail later. A synapse can occur virtually anywhere on the postsynaptic cell, but the most common location is on the neuronal dendrite. The dendrite is usually the first part of the neuron to initiate an electrical response to a signal from the presynaptic neuron. On many neurons, the dendrites have specialized adornments known as spines, and it is with these structures that the presynaptic neuron forms a synapse. Spines can vary in size and shape, but they are generally no more than a few micrometers in length, with a bulb-like shape at the end of a tapered shaft (see Fig. 6-2). An electrical signal that initiates in a spine travels to the dendritic branch on which it occurs and moves down the dendrite toward the cell body. Although many neuritic processes look similar (especially in culture), a dendrite can usually be distinguished from an axon because it is tapered—decreasing in diameter as distance from the cell body increases. A nerve cell can have a single dendritic shaft emanating from its cell body (as in the Purkinje cell in Fig. 6-1A), or it can have several. A typical pyramidal cell in the cerebral cortex, for example, has a single apical dendrite but also several basal dendrites; a cerebellar granule cell has four to six short dendrites in a star-shaped configuration around the cell body.
The nerve cell body is the most prominent feature of the neuron in most basophilic stain preparations (e.g., hematoxylin-eosin). If one considers size alone, however, the cell soma is usually considerably smaller than the dendrite in both surface area and volume.2 This relatively small soma-to-process volume ratio represents a logistic problem for the cell body: the genetic machinery is located exclusively in the nucleus, yet the products of this machinery must be transported to sites on the cell that are up to a meter away.
The electrical signal reaching the cell body from the dendrite travels to the point on the cell where the axon emerges. The axon of a neuron is the structure that captures the summed electrical information in the dendrites and cell body and routes it to the next neuron (or target organ) in the circuit. Morphologically, the axon can be distinguished from the dendrite by the fact that it has a typically constant caliber over its entire length. Soon after the axon leaves the cell body, a specialized region known as the initial segment (or axon hillock) can be identified. This part of the cell is the biochemical boundary of the axon and the point of initiation of the action potential. Up to this point, the information packets from the dendrites and cell body travel primarily by electrotonic spread, with different packets of electrical activity coming together and summing in a graded (analog) manner. By contrast, the axon transmits information in a strictly digital fashion. If the combined electrogenic signal reaching the axon from the cell body is sufficiently strong, the axon will fire and pass the information along; otherwise, the signal stops and proceeds no farther in the circuit. If the decision is a “go,” the axon transforms the information into a self-propagating electrical wave known as an action potential that travels undiminished down the axon to its end. The action potential is an electrical signal that results from the coordinated functioning of sodium and potassium channels (see Chapter 49 for details), usually in collaboration with glial cells (see later).
Sensory Neurons
Mechanical Receptors
The simplest receptor cells of this type are used to receive information about touch and pain. As might be expected, most are located in the skin and other integuments. Receptors for light touch are illustrated in Figure 6-3. These receptor endings have different precisions and sensitivities and are specialized to receive different types of stimuli.3 Each receptor represents a specialized ending of a sensory ganglion axon. These include Ruffini’s endings or pacinian corpuscles (for deep receptors) and Merkel cells or Meissner’s corpuscles (for more superficial receptors). Sometimes the neurite of a mechanoreceptor is wrapped around the interfusal muscle fibers of one of the striated muscles. The cellular deformation associated with movement of the axonal membrane activates a series of stretch-sensitive ion channels. The ionic current through these channels initiates the electrical activity that signals a sensory stimulus to the organism.
A subset of mechanical receptors has evolved to serve the auditory and vestibular systems. The principal function of these cells is the same as for light touch receptors: deformation of a hair opens a number of specialized ion channels, which results in the generation of an electrical signal. The difference in the acousticovestibular system is that the “hairs” are actually cilia on the basal surface of the cell and hence are part of the receptor cell itself. Indeed, the ion channels that are opened in response to movement of the hair are located at the tip of the cilium. A diagram of this cell type is shown in Figure 6-4. In the auditory system, the vibration of sound waves is transduced into the vibration of fluid in the cochlea. Receptor cells at different positions in the cochlear spiral respond to different auditory frequencies and transmit both pitch and volume information to the auditory system. In the vestibular system, a morphologically similar configuration of receptors is found in the semicircular canals. Movement of the head in any of the three orthogonal planes leads to movement of fluid in the canals. This movement displaces the cilia of the vestibular receptors and initiates an electrical signal that is transmitted through the eighth nerve to the brain, where the vestibular system interprets the information to determine the orientation and movement of the organism in space. In each of these systems, however, the common feature of the receptor is that deflection of a hair leads to deformation of the cellular membrane, which in turn results in opening of a specific set of ion channels. The resulting change in conductance leads to an electrical “packet” of information that moves along the neuron to the rest of the brain.
Chemical Receptors
A second class of receptors responds directly to specific chemicals by generating an electrical response that can be propagated to other parts of the nervous system. Receptors in this group are found in the papilla of the tongue, where they respond to the presence of salt, sweet, bitter, and sour and project to the gustatory centers of the brain by way of the seventh and ninth cranial nerves. A more sophisticated and chemically diverse set of sensors in this class is found in the lining of the nasal epithelium.4 These receptors are responsible for endowing the organism with a sense of smell. This more elaborate mechanism of chemical reception is based on a large family of G protein–linked receptor molecules. Family members of this receptor class number in the thousands, each apparently encoded by a different gene. Each receptor recognizes a different chemical structure and responds to binding of the chemical by stimulating release of the bound G protein, which activates adenylate cyclase. This, in turn, leads to an elevation in cyclic adenosine monophosphate, which then opens a cyclic nucleotide–gated channel and results in the generation of an electrical signal that is transmitted along the neuron to the olfactory portions of the brain.
Physical Receptors
Certain nerve cells are sensitive to the physical properties of their environment. The temperature receptors of the skin are one example of this group of receptor cells; the light-sensitive cells of the eye are another. These latter cells are known as photoreceptors, and they respond to electromagnetic radiation in the visible spectrum. They are further subdivided into rods and cones, depending on their wavelength specificity. Cones are narrowly tuned to transmit information about color, whereas rods have a broad frequency range and are most useful in low-light situations. Both classes of photoreceptors contain many flat membrane sacks that are stacked like pancakes at one end of the cell (see Fig. 6-1B). These sacks contain the photosensitive pigment rhodopsin, which allows light energy to be transduced into an electrical signal. Reception of light in the photoreceptor evolved through the use of the same class of G protein–linked receptor molecules as in the olfactory system. When light strikes the rhodopsin molecule, a chemical cascade occurs that is nearly identical to that described for the olfactory receptors.
Neuronal Structure
Visualization of Neurons
Neurons, like most other cells, are pale, clear, and difficult to see in living tissue. As a consequence, many of the significant advances in the study of the nervous system were made possible by improved methods of visualizing the nerve cell and its processes. The basophilic dyes are the oldest and still most widely used method of staining nervous tissue. Common stains of this class include hematoxylin and cresyl violet. These dyes bind avidly to RNA and DNA and thus highlight the heterochromatin of the nucleus and the rough endoplasmic reticulum of the cytoplasm (Fig. 6-5A). Basophilic stains are most commonly used on sections with a thickness ranging from 2 to 20 µm. Examination of nervous tissue stained with such reagents results in a clear picture of the cell body and proximal dendrites; the axons and distal dendrites are usually invisible, so white matter and neuropil are generally clear of stain.
During the 1800s, silver salts were found to have a special avidity for nervous tissue. Because of their binding to neuron-specific classes of intermediate filaments, a variety of protocols were developed that revealed the neuronal axon with great clarity. Among this class of stains, the Bodian and Bielschowsky stains are still commonly used. A special class of silver stain is the Golgi impregnation method. In this procedure, pieces of tissue are incubated for many weeks in heavy metal salts. During this time, a small number of cells take up the salts, with their entire intracellular spaces being filled. The tissue is then embedded and sectioned. When the sections are “developed” in reducing agents, an opaque black precipitate is formed that fills the impregnated cells completely. For unknown reasons, only 1% to 2% of the cells react in this fashion (seemingly at random). The details of an individual cell can be seen against a clear background (Fig. 6-5B). Although the technique reveals the finest details of dendritic structure, axons are more resistant to filling and are commonly invisible in Golgi preparations. The technique is used to best advantage in sections ranging from 80 to 120 µm thick.
In the second half of the 20th century, new technologies dramatically expanded our ability to visualize and analyze the nerve cells of the brain. Beginning in the 1950s and 1960s, the transmission electron microscope led to a quantum leap in the ability to resolve the details of nerve cellular structure. In this method, small pieces of tissue (typically 2 to 3 mm wide) are embedded in plastic and cut with a glass or diamond blade in sections ranging in thickness from tens to hundreds of nanometers. Before embedding, the tissues are usually stained with uranyl acetate, lead citrate, and osmium tetroxide, lipophilic dyes that reveal membrane structure with a high degree of clarity. Phosphotungstic acid is a frequently used stain that has a particular affinity for synapses. The resolution afforded by the electron microscope allows the fine structure of the cell to be revealed, and the organelles of the cell body can be analyzed. The unique advantages of using electron microscopy to view the nervous system include the ability to resolve synaptic structures (see Fig. 6-2B and C) such as synaptic vesicles, details of the presynaptic and postsynaptic membranes, and the material of the synaptic cleft. Axon and dendrite morphology, with their unique collections and arrangements of filaments, can also be seen.
In the 1970s and 1980s, serum antibodies were developed as highly specific stains by using the techniques of immunocytochemistry. Lightly fixed tissue is exposed to an antiserum or monoclonal antibody raised against a particular neuronal epitope. The antibody selectively binds to the neural structure that contains that epitope. Most frequently, this primary antibody is revealed through the application of a secondary antibody that is derivatized to carry a detection molecule—either a fluorescent compound, such as fluorescein or rhodamine, or an enzyme, such as horseradish peroxidase or alkaline phosphatase. In the former case, the location of the antibody is determined by examining the tissue with a fluorescence microscope. In the latter instance, the marker enzyme is localized through the use of a specific substrate whose action deposits an insoluble, chromagenic product. Immunocytochemistry is most commonly used to reveal proteins (e.g., tyrosine hydroxylase, microtubule-associated protein 2 [MAP2], the α6 receptor of γ-aminobutyric acid [GABAA]), but the location of carbohydrates (e.g., polysialic acid, gangliosides, chondroitin sulfate proteoglycan) can also be determined. Immunocytochemistry can be applied at the electron microscopic level as well, where peroxidase or gold particles are used to reveal the location of the secondary antibody. Figure 6-5C presents an example of immunocytochemistry using an antibody against a neurofilament protein, one of the major cytoskeletal components of neuronal cytoplasm.
In the 1980s and 1990s, advances in molecular biology enabled the detection of messenger RNA for specific proteins through a technique known as in situ hybridization (see Chapter 3). Medium- to high-abundance messages are localized to the cell body transcribing them by exposing lightly fixed tissue to labeled nucleotide probes that are complementary to the message sequence. The labeled probe is detected either because of its radioactivity (35S-labeled nucleotide precursors are commonly used) or because of the presence of other derivatives (such as biotin or digoxigenin attached to nucleotides). In either case, the tissue is treated with proteinase to remove the bound proteins and then hybridized at temperatures that ensure the specificity of the probe-message interaction. If radioactivity is used, the location of the hybridized message is determined by apposing the section to x-ray film or by dipping the section in liquid photographic emulsion, which forms a thin coating and is subsequently fogged wherever the labeled nucleotide is found. If nonradioactive methods are used in the detection protocol, their location is revealed with a secondary probe (derivatized avidin in the case of biotin-labeled probes or antibody to digoxigenin). A digoxigenin-labeled probe hybridized to the message for the Purkinje cell–specific nuclear receptor RORα is shown in Figure 6-5D. An important aspect of the interpretation of such images is that the location of the message marks the cell body where the message is synthesized, not the place where the protein is found. For example, the image shown in Figure 6-5D could represent messenger RNA encoding RORα (a nuclear protein), the δ2-glutamate receptor (found predominantly in dendritic spines), or synaptophysin (found principally in the presynaptic axon terminal).
Dendritic Structure
The dendrite represents a smooth, tapered extension of the neuronal cell body.5 The main tapered dendritic shafts have many of the same organelles as the cell body, including mitochondria, microtubules, neurofilaments, smooth endoplasmic reticulum, and ribosomes. The existence of ribosomes is correlated with clear evidence of local protein synthesis, and studies have illustrated that a unique subset of the total population of messenger RNA is transported into the dendrites6 based on specific sequences contained within the message. Despite this rough similarity to the cell body, the dendritic domain has several unique features that make it identifiable with both light and electron microscopy. Biochemically, the dendrite contains several proteins that are located nowhere else in the nerve cell. These proteins include MAP2, as well as several specific receptor species and channel proteins that differ from cell to cell.
As outlined earlier, the most typical site of contact between a postsynaptic cell and a presynaptic axon is a dendritic spine (see Fig. 6-2B and C). Spines can be found on the cell body (although this is rare in adults) or on the main dendritic branches, but they are most common on the fine terminal branches of the dendrites. The development of a dendritic spine is heavily influenced by the presence of a presynaptic afferent but does not require it. Studies of several pathologic conditions have shown that well-proportioned spines and their associated ultrastructure can be maintained (and possibly developed) in the absence of any presynaptic element. In this case, the structure is usually ensheathed by a glial cell.
Cell Body Structure
Neurons are synthetically active cells. Although the brain typically makes up only 2% of the weight of the human body, it consumes as much as 25% of the oxygen used by the organism. It has been estimated that more than 50% of human genes are either highly enriched or unique to the nervous system. Alternative splicing of transcripts is also more prevalent in the brain than in any other tissue.7 The ultrastructure of the nerve cell body reflects a high level of protein translation. The Golgi apparatus and rough endoplasmic reticulum are prominent features of the cell body. When there is a main apical dendrite, the Golgi apparatus and rough endoplasmic reticulum are often located between the nucleus and the emanation point of the dendrite. The concentration of rough endoplasmic reticulum is sufficiently great that the term Nissl substance is used to describe its prominent, dark floccular appearance on light microscopic images of basophilic dye preparations. Mitochondria are also in abundance, as might be expected given the high aerobic activity of the neuron. Primary and secondary lysosomes are present, and with aging, these organelles tend to accumulate large quantities of a waxy substance known as lipofuscin.8 The nucleus of most neurons is oval or spherical, with relatively clear nucleoplasm. In most large neurons there is a single prominent nucleolus, whereas in small neurons, such as the granule cells of the olfactory bulb, hippocampus, and cerebral or cerebellar cortex, there are scattered clumps of heterochromatic material with no nucleolus.
Axonal Structure
Although it is less prominent than in dendrites, recent work has shown that protein synthesis can also be detected in the axon.9 Even with this local synthetic source, the topology of the nerve cell presents a significant maintenance problem to neurons when the synaptic terminal is more than a meter away from the cell body. To achieve effective translocation between the protein synthetic machinery in the perikaryon and the axon terminal, the axon uses several mechanisms of transport. In the orthograde direction (cell body to axon terminal), bulk materials tend to flow at a pace of about 0.5 mm/day. Organelles and some proteins, however, are transported by rapid axonal transport, which can achieve rates of 400 mm/day. Material also moves from the axon terminal to the cell body, in the retrograde direction, at half the speed of fast orthograde transport.
Synaptic Structure
The physiology of synaptic function is covered in detail in Chapter 49. Underlying the details of ionic fluxes, however, are a number of crucial elements in the cell biologic structure of the neuron. On the presynaptic side, the axon terminates in a highly specialized presynaptic structure (see Fig. 6-2B and C). The shaft of the axon broadens in diameter and assumes a shallow cup-like form. Although microtubules are a prominent feature of the central axon domain, few extend into the terminal area. Instead, the major structural elements of the presynaptic terminal include neurofilaments and actin filaments. Mitochondria are more abundant than in the axon shaft, and a collection of small vesicles appears near the synaptic cleft itself. These vesicles contain the neurotransmitter substances that will be released on invasion of the terminal by an action potential. The vesicles are polymorphic in appearance. Those at excitatory synapses tend to be round, whereas those at inhibitory synapses are more ovoid or flattened. Synapses that release peptides contain larger vesicles with electron-dense material in their centers. These dense-core vesicles are typical of neurosecretory cells. Well in excess of a dozen proteins have been identified as being crucial to the process, which involves filling of the vesicle with transmitter, docking, fusion of the vesicle with cellular membranes, release, and finally, recycling.
