《医学生理学 Medical Physiology》参考教材资料（PDF电子书籍）Neuroscience in Medicine, Third edition, 2008

8 Cohen and pfaff Fig4Electron micrograph of a hypothalamic neuron actively Fig. 5. Electron micrograph of a Golgi apparatus of a teins. Many cisternae of rough endoplasmic reticulum(er) apparatus on all sides. The cis face is located near the rough and associated dense-cored vesicles(arrowheads)are also visi- the trans Golgi network(TGN). Secretory material is detected ble(From Cohen RS. Pfaff Dw. Ultrastructure of neurons in within the peripheral portions of the cisternae(arrowheads)of he ventromedial nucleus of the hypothalamus with or without the tgN. a dense-cored vesicle(arrow)containing material of estrogen treatment. Cell Tissue Res 1981: 217: 463. similar density is seen at the periphery of the TGN reticulum vary and appear to depend on the prevail- The secretory material is later detected within the ing demands for secretory protein synthesis in a cisternae occupying the trans-Golgi network. Often, functional group of neurons In nerve cells that are at the periphery of the sacs, the membranes appear to quiescent in regard to the production of exportable constrict around a dense granule, which is the con proteins, cisternae of the rough endoplasmic reticu- centrated precursor protein(Fig. 5). The granule- lum appear as discrete sacs and seem to occupy only a containing membranous compartment is pinched small fraction of the cellular space. Neurons actively off, forming a dense-cored vesicle. Although the engaged in secretory protein synthesis contain large details of protein processing within the Golgi appa stacks of elongated cisternae that fill a considerable ratus have not yet been elucidated, its overall function portion of the perikaryon. is known: the preparation and packaging of the secre- 2.2.1.2. Packaging and Modification ot Exporta- to the axon terminal for storage or release<ransport tory protein, still in its precursor form, fo ble Neuropeptides in the Golgi Apparatus. The Golgi apparatus is composed of a series of flattened, smooth-surfaced, membranous sacs and a variety of 2.2.1.3. Formation of the Active Neuropeptide associated small vesicles, which encompass the Golgi Within the Neurosecretory Vesicle. The actual apparatus on all sides(Fig. 5). The Golgi cisternae enzymatic processing of the precursor and generation are arranged in a polarized fashion, reflecting their of individual biologically active peptides appear to morphology and function. The forming or cis face occur distal to the golgi apparatus as the secretory approaches the rough endoplasmic reticulum; the vesicle makes its way down the axon. Conditions, opposite side is designated as the maturing or trans such as pH, necessary for the maximal activity of face or trans-Golgi network. Vesicles bud off from the processing enzymes, are optimal. The relatively the smooth transitional portion of the rough endo- simple structure of the secretory vesicle-a dense core plasmic reticulum and transport their contents to surrounded by a single membrane-belies its dynamic Golgi sacs on the cis face. Here, they deliver the role in generating active neuropeptides. Various enzy- newly synthesized precursor protein by fusion of matic activities have been located in neurosecretory the vesicle membrane with the Golgi membrane. vesicles, and some have been purified and characterized

reticulum vary and appear to depend on the prevail￾ing demands for secretory protein synthesis in a functional group of neurons. In nerve cells that are quiescent in regard to the production of exportable proteins, cisternae of the rough endoplasmic reticu￾lum appear as discrete sacs and seem to occupy only a small fraction of the cellular space. Neurons actively engaged in secretory protein synthesis contain large stacks of elongated cisternae that fill a considerable portion of the perikaryon. 2.2.1.2. Packaging and Modification of Exporta￾ble Neuropeptides in the Golgi Apparatus. The Golgi apparatus is composed of a series of flattened, smooth-surfaced, membranous sacs and a variety of associated small vesicles, which encompass the Golgi apparatus on all sides (Fig. 5). The Golgi cisternae are arranged in a polarized fashion, reflecting their morphology and function. The forming or cis face approaches the rough endoplasmic reticulum; the opposite side is designated as the maturing or trans face or trans-Golgi network. Vesicles bud off from the smooth transitional portion of the rough endo￾plasmic reticulum and transport their contents to Golgi sacs on the cis face. Here, they deliver the newly synthesized precursor protein by fusion of the vesicle membrane with the Golgi membrane. The secretory material is later detected within the cisternae occupying the trans-Golgi network. Often, at the periphery of the sacs, the membranes appear to constrict around a dense granule, which is the con￾centrated precursor protein (Fig. 5). The granule￾containing membranous compartment is pinched off, forming a dense-cored vesicle. Although the details of protein processing within the Golgi appa￾ratus have not yet been elucidated, its overall function is known: the preparation and packaging of the secre￾tory protein, still in its precursor form, for transport to the axon terminal for storage or release. 2.2.1.3. Formation of the Active Neuropeptide Within the Neurosecretory Vesicle. The actual enzymatic processing of the precursor and generation of individual biologically active peptides appear to occur distal to the Golgi apparatus as the secretory vesicle makes its way down the axon. Conditions, such as pH, necessary for the maximal activity of the processing enzymes, are optimal. The relatively simple structure of the secretory vesicle—a dense core surrounded by a single membrane—belies its dynamic role in generating active neuropeptides. Various enzy￾matic activities have been located in neurosecretory vesicles, and some have been purified and characterized. Fig. 4. Electron micrograph of a hypothalamic neuron actively engaged in the synthesis and packaging of exportable pro￾teins. Many cisternae of rough endoplasmic reticulum (er) are arranged in parallel stacks. The Golgi apparatus (GA) and associated dense-cored vesicles (arrowheads) are also visi￾ble. (From Cohen RS, Pfaff DW. Ultrastructure of neurons in the ventromedial nucleus of the hypothalamus with or without estrogen treatment. Cell Tissue Res 1981;217:463.) Fig. 5. Electron micrograph of a Golgi apparatus of a hypothalamic neuron. Small vesicles surround the Golgi apparatus on all sides. The cis face is located near the rough endoplasmic reticulum (er); the opposite side is designated as the trans Golgi network (TGN). Secretory material is detected within the peripheral portions of the cisternae (arrowheads) of the TGN. A dense-cored vesicle (arrow) containing material of similar density is seen at the periphery of the TGN. 8 Cohen and Pfaff

Chapter 1/Cytology and Organization of Cell Types 9 The vesicle membrane contains proteins that regulate cisternae of the rough endoplasmic reticulum. One of the internal vesicular environment. On the appropriate the most morphologically complex variations of these signal, active neuropeptides are released by exocytosis, structures resides in the dendritic spine, where paral which is fusion of the vesicle membrane with the plasma lel, smooth-surfaced cisternae alternate with electron- membrane at the presynaptic site. dense bands of unknown composition All of the diverse structures are thought to func- 2.2. 2. PROTEINS SYNTHESIZED FOR USE WITHIN tion in the release and sequestration of calcium THE NEURON within the neuron Calcium is central to most aspects The synthesis of exportable proteins represents of neuronal function, including membrane perme- only a portion of the total protein synthetic effort ability; mediation of the effects of neurotransmit- by the neuron. The elaboration of the cytoskeletal ters, hormones, and growth factors; cytoskeletal framework and the synthesis of other proteins includ- function; and vesicle release. In neurons,calcium mobilization is achieved, at least in part, by the tems, and other proteins destined for maintenance binding of inositol 1, 4, 5-triphosphate (IP)to intra and renewal of the cytoplasm and its organelles also cellular receptors located on the aforementioned depend on translation of messenger RNAS. These membranous cisternae. IP is a second messenger proteins are synthesized on free ribosomes that are generated on receptor-stimulated hydrolysis of plentiful in all nerve cells. Axons possess a highly phosphatidylinositol 4, 5-biphosphate by phospholi- organized transport system to convey proteins to pase C, an enzyme activated by signal transduction presynaptic terminals or back to the cell body. mechanisms Dendrites, their postsynaptic specializations, and dendritic spines also require proteins for growth, 2. 4. Mitochondria maintenance, and function. Although there is an 2.. MITOCHONDRIAL STRUCTURE IN NEURONS indication of molecular transport to these sites, the identities of the molecules and the nature of the Mitochondria from various brain regions and transport mechanism has just begun to be explored. neuronal, compartments(perikarya, dendrites, Moreover, evidence for local protein synthesis sug- brane architecture. Mitochondria in neurons con- gests that not all dendritic and postsynaptic proteins tain interconnected tubular and lamellar cristae, arrive from the cell body. The presence of polyribo tions and at the base of dendritic spines provides ipherally and the lamellar ones located more cen the machinery for local protein synthesis. The exis- tence of local protein synthetic mechanisms is further is unknown, although changes in cristae shape may supported by the presence of some messenger RNAs contribute to regulation of ATP production. The in distant dendritic arbors, suggesting that growth- outer mitochondrial membranes are also in close dependent and activity-dependent synaptic alterations association with membranes of the endoplasmic reti including changes in morphology, may be regulated culum, at sites of high calcium generation. Some partially by the local synthesis of key synaptic proteins. dria in the soma, the axon hillock, the nodes of Ranvier, and the nerve terminal; during neuronal development, mitochondria are also located in 2.3. Smooth Membrane Compartments in growth cones Neurons Serve as Reservoirs for Calcium In addition to the intramembranous compartments 2.4.2. MITOCHONDRIAL FUNCTION IN NEURONS that directly participate in the synthesis, packaging, Mitochondria are present in all cells and provide and transport of secretory peptides, other cisternal common functions irrespective of the particular cell and vesicular structures are evident within the various type. These functions include the generation of ATP domains of the neuron. The membranous sacs are as well as reactive oxygen species, intermediary visible as smooth-surfaced compartments in a variety metabolism, intracellular calcium signaling, and of configurations. Some appear as relatively short the regulation of apoptosis. However, the unique sacs, but others are longer and anastomose within compartmentalization of neurons into different neuronal processes. In the cell body, smooth mem- structural and functional domains and their high brane profiles are arranged in stacks or emanate from energy requirements necessitate an expanded role

The vesicle membrane contains proteins that regulate the internal vesicular environment. On the appropriate signal, active neuropeptides are released by exocytosis, which is fusion of the vesicle membrane with the plasma membrane at the presynaptic site. 2.2.2. PROTEINS SYNTHESIZED FOR USE WITHIN THE NEURON The synthesis of exportable proteins represents only a portion of the total protein synthetic effort by the neuron. The elaboration of the cytoskeletal framework and the synthesis of other proteins includ￾ing ion channels, receptors, second messenger sys￾tems, and other proteins destined for maintenance and renewal of the cytoplasm and its organelles also depend on translation of messenger RNAs. These proteins are synthesized on free ribosomes that are plentiful in all nerve cells. Axons possess a highly organized transport system to convey proteins to presynaptic terminals or back to the cell body. Dendrites, their postsynaptic specializations, and dendritic spines also require proteins for growth, maintenance, and function. Although there is an indication of molecular transport to these sites, the identities of the molecules and the nature of the transport mechanism has just begun to be explored. Moreover, evidence for local protein synthesis sug￾gests that not all dendritic and postsynaptic proteins arrive from the cell body. The presence of polyribo￾somes beneath postsynaptic membrane specializa￾tions and at the base of dendritic spines provides the machinery for local protein synthesis. The exis￾tence of local protein synthetic mechanisms is further supported by the presence of some messenger RNAs in distant dendritic arbors, suggesting that growth￾dependent and activity-dependent synaptic alterations, including changes in morphology, may be regulated partially by the local synthesis of key synaptic proteins. 2.3. Smooth Membrane Compartments in Neurons Serve as Reservoirs for Calcium In addition to the intramembranous compartments that directly participate in the synthesis, packaging, and transport of secretory peptides, other cisternal and vesicular structures are evident within the various domains of the neuron. The membranous sacs are visible as smooth-surfaced compartments in a variety of configurations. Some appear as relatively short sacs, but others are longer and anastomose within neuronal processes. In the cell body, smooth mem￾brane profiles are arranged in stacks or emanate from cisternae of the rough endoplasmic reticulum. One of the most morphologically complex variations of these structures resides in the dendritic spine, where paral￾lel, smooth-surfaced cisternae alternate with electron￾dense bands of unknown composition. All of the diverse structures are thought to func￾tion in the release and sequestration of calcium within the neuron. Calcium is central to most aspects of neuronal function, including membrane perme￾ability; mediation of the effects of neurotransmit￾ters, hormones, and growth factors; cytoskeletal function; and vesicle release. In neurons, calcium mobilization is achieved, at least in part, by the binding of inositol 1,4,5-triphosphate (IP) to intra￾cellular receptors located on the aforementioned membranous cisternae. IP is a second messenger generated on receptor-stimulated hydrolysis of phosphatidylinositol 4,5-biphosphate by phospholi￾pase C, an enzyme activated by signal transduction mechanisms. 2.4. Mitochondria 2.4.1. MITOCHONDRIAL STRUCTURE IN NEURONS Mitochondria from various brain regions and neuronal compartments (perikarya, dendrites, axons, and synapses) have essentially uniform mem￾brane architecture. Mitochondria in neurons con￾tain interconnected tubular and lamellar cristae, with the tubular-shaped cristae arranged more per￾ipherally and the lamellar ones located more cen￾trally. The functional significance of these features is unknown, although changes in cristae shape may contribute to regulation of ATP production. The outer mitochondrial membranes are also in close association with membranes of the endoplasmic reti￾culum, at sites of high calcium generation. Some neurons display a higher accumulation of mitochon￾dria in the soma, the axon hillock, the nodes of Ranvier, and the nerve terminal; during neuronal development, mitochondria are also located in growth cones. 2.4.2. MITOCHONDRIAL FUNCTION IN NEURONS Mitochondria are present in all cells and provide common functions irrespective of the particular cell type. These functions include the generation of ATP, as well as reactive oxygen species, intermediary metabolism, intracellular calcium signaling, and the regulation of apoptosis. However, the unique compartmentalization of neurons into different structural and functional domains and their high￾energy requirements necessitate an expanded role Chapter 1 / Cytology and Organization of Cell Types 9

10 Cohen and pfaff for mitochondria in these cell types, whereby mito- 2.4.3. MITOCHONDRIA AND NEURODEGENERATIVE chondria affect nerve transmission and vice versa. AND PSYCHIATRIC DISORDERS lion ng development, mitochondria appear to func- Mutations in mitochondrial DNA may result in as a determinant of neuronal polarity, in the the maternal transmission of neurologic disorders control of neurite outgrowth, and the differentiation These appear to include some cases of schizophrenia of neurons from precursor cells. They also play a some neuropathies, and retinitis pigmentosa. Mito- role in adult plasticity by influencing neurotransmit- chondrial dysfunction may also be involved in Alz ter release from presynaptic terminals, possibly via heimer's and parkinson's diseases. as well as stroke their role in calcium signaling On the postsynaptic In these cases, age-related changes in neuronal meta neurons for glutamate. Notably, environmental fac- tion may indicate prior age-related changes in tors, which may also influence plasticity, appear to calcium balance and interfere with synaptic plasticity affect mitochondria. Rats kept in an enriched envir- Moreover, accumulation of cytotoxic forms of cer onment, resulting in enhanced performance of a tain proteins implicated in Alzheimer's disease may spatial memory task, display an increase activity of result in mitochondrial dysfunction and/or displace- some mitochondrial proteins. That synaptic activity ment. For example, defects in the human presenilin 1 can alter mitochondrial biochemistry and function is gene, which is implicated in an aggressive form of evidenced by the upregulation of mitochondrial early-onset familial Alzheimer's disease, appear to genes by high-frequency stimulation of a tissue compromise kinesin-based axonal transport in neu- slice from the hippocampus rons Kinesin is a molecular motor important in ante- In terms of ATP many neuronal functions require rograde axonal transport. Neurons with the mutant ergy, including those functions associated with the form of presenilin I display reduced mitochondrial cytoskeletal proteins, as well as those involving phos- density in neuritic processes. In regard to other psy phorylation Calcium influx and its sequestration and chiatric disorders, patients with bipolar disorder release are also essential to neuronal function, and appear to shift their metabolism toward glycolytic- mitochondria are also key players here. The produc- based energy production, as opposed to one that tion of reactive oxygen species by this organelle may involves oxidative phosphorylation. Moreover, be involved in cell signaling, as well as membrane some patients with schizophrenia display fewer mito- lipid peroxidation, which, in turn, alters membrane chondria in specific brain regions, and neuroleptic protein function drugs appear to reverse this phenomenon Mitochondria play a role in apoptosis, a type of programmed cell death. Part of this process is mediated by Bcl-2 family members, which interact 3. CYTOSKELETON DETERMINATION OF with mitochondrial membranes to either increase or NEURONAL FORM decrease their permeability, resulting in apoptosis or, A singular feature of neurons is their overall extra alternatively, stabilize the membrane to check this ordinary length, enabling them to transmit signals process. During the process of apoptosis, mitochon- over great distances. This property is reflected in the drial membranes exhibit increased permeability and polarity of neuronal form and function, which is release cytochrome c Cytochrome c itself can activate governed by regional specialization of the plasma caspase-3, which, in turn, may cleave some protein membrane and by differences in the cytoskeletal com- substrates resulting in cell death. On the other hand, position of dendritic and axonal processes emerging when activated at sublethal levels, some caspases in from the cell body. Although the neuronal cytoskele synapses and dendrites may cleave specific glutamate ton provides a structural framework on which var receptor subunits, thereby modulating synaptic ious organelles and cellular events are organized, it is by no means a static configuration. Throughout the GTP binding protein-coupled receptors for neuro- neuron, molecular alterations in cytoskeletal proteins ransmitters and neuropeptides, glutamate, and neu- reverberate as microscopically visible changes in rotrophic factors can also affect mitochondria via movement of the cytoskeleton, its associated orga second-messenger pathways. These pathways target nelles, and the shape and extent of some of the pro- gene transcription factors, with the possibility of cesses. Although the cytoskeleton permits the general encoding proteins relevant to synaptic and neuronal pattern of individual neurons to remain constant and identifiable, alterations in cytoskeletal dynamics

for mitochondria in these cell types, whereby mito￾chondria affect nerve transmission and vice versa. During development, mitochondria appear to func￾tion as a determinant of neuronal polarity, in the control of neurite outgrowth, and the differentiation of neurons from precursor cells. They also play a role in adult plasticity by influencing neurotransmit￾ter release from presynaptic terminals, possibly via their role in calcium signaling. On the postsynaptic side, mitochondria appear to affect the sensitivity of neurons for glutamate. Notably, environmental fac￾tors, which may also influence plasticity, appear to affect mitochondria. Rats kept in an enriched envir￾onment, resulting in enhanced performance of a spatial memory task, display an increase activity of some mitochondrial proteins. That synaptic activity can alter mitochondrial biochemistry and function is evidenced by the upregulation of mitochondrial genes by high-frequency stimulation of a tissue slice from the hippocampus. In terms of ATP, many neuronal functions require energy, including those functions associated with the cytoskeletal proteins, as well as those involving phos￾phorylation. Calcium influx and its sequestration and release are also essential to neuronal function, and mitochondria are also key players here. The produc￾tion of reactive oxygen species by this organelle may be involved in cell signaling, as well as membrane lipid peroxidation, which, in turn, alters membrane protein function. Mitochondria play a role in apoptosis, a type of programmed cell death. Part of this process is mediated by Bcl-2 family members, which interact with mitochondrial membranes to either increase or decrease their permeability, resulting in apoptosis or, alternatively, stabilize the membrane to check this process. During the process of apoptosis, mitochon￾drial membranes exhibit increased permeability and release cytochrome c. Cytochrome c itself can activate caspase-3, which, in turn, may cleave some protein substrates resulting in cell death. On the other hand, when activated at sublethal levels, some caspases in synapses and dendrites may cleave specific glutamate receptor subunits, thereby modulating synaptic plasticity. GTP binding protein–coupled receptors for neuro￾transmitters and neuropeptides, glutamate, and neu￾rotrophic factors can also affect mitochondria via second-messenger pathways. These pathways target gene transcription factors, with the possibility of encoding proteins relevant to synaptic and neuronal plasticity. 2.4.3. MITOCHONDRIA AND NEURODEGENERATIVE AND PSYCHIATRIC DISORDERS Mutations in mitochondrial DNA may result in the maternal transmission of neurologic disorders. These appear to include some cases of schizophrenia, some neuropathies, and retinitis pigmentosa. Mito￾chondrial dysfunction may also be involved in Alz￾heimer’s and Parkinson’s diseases, as well as stroke. In these cases, age-related changes in neuronal meta￾bolism as a consequence of mitochondrial dysfunc￾tion may indicate prior age-related changes in calcium balance and interfere with synaptic plasticity. Moreover, accumulation of cytotoxic forms of cer￾tain proteins implicated in Alzheimer’s disease may result in mitochondrial dysfunction and/or displace￾ment. For example, defects in the human presenilin 1 gene, which is implicated in an aggressive form of early-onset familial Alzheimer’s disease, appear to compromise kinesin-based axonal transport in neu￾rons. Kinesin is a molecular motor important in ante￾rograde axonal transport. Neurons with the mutant form of presenilin 1 display reduced mitochondrial density in neuritic processes. In regard to other psy￾chiatric disorders, patients with bipolar disorder appear to shift their metabolism toward glycolytic￾based energy production, as opposed to one that involves oxidative phosphorylation. Moreover, some patients with schizophrenia display fewer mito￾chondria in specific brain regions, and neuroleptic drugs appear to reverse this phenomenon. 3. CYTOSKELETON DETERMINATION OF NEURONAL FORM A singular feature of neurons is their overall extra￾ordinary length, enabling them to transmit signals over great distances. This property is reflected in the polarity of neuronal form and function, which is governed by regional specialization of the plasma membrane and by differences in the cytoskeletal com￾position of dendritic and axonal processes emerging from the cell body. Although the neuronal cytoskele￾ton provides a structural framework on which var￾ious organelles and cellular events are organized, it is by no means a static configuration. Throughout the neuron, molecular alterations in cytoskeletal proteins reverberate as microscopically visible changes in movement of the cytoskeleton, its associated orga￾nelles, and the shape and extent of some of the pro￾cesses. Although the cytoskeleton permits the general pattern of individual neurons to remain constant and identifiable, alterations in cytoskeletal dynamics 10 Cohen and Pfaff

Chapter 1/Cytology and Organization of Cell Types enable the neuron to respond to environmental- compartments; only after sonication or extremely dependent or activity-dependent fluctuations. This acidic conditions do these tenacious structures dis- apparent contradiction is resolved by the inherent sociate into their component parts nature of cytoskeletal elements that exist in different structural and functional states of assembly and dis- 3. 2. The Components of the Neuronal sembly. Moreover, these structures may be stabilized Cytoskeleton Include Microtubules, and destabilized, providing yet another dimension to Neurofilaments, and Microfilaments he number of possible conformations of cytoskeletal and Their Associated Proteins form The interactions of various cytoskeletal elements The dual nature of the neuronal cytoskeleton, with their associated proteins or with each other con- reflected in its rigidity and plasticity, is a function of tribute to the unique structural and functional iden- three filament types: microtubules, neurofilaments, tity of axons and dendrites and their associated and microfilaments or actin filaments. Each cytoske- dendritic spines. The cytoskeleton also interacts letal element acts in conjunction with a specific set of with the neuronal plasma membrane at specific associated or binding proteins. Some of these cross- sites, including the initial segment of the axon, special link the filaments to each other, the plasma mem- loci along the axon called nodes of Ranvier, and brane, and other intracellular organelles and are complex submembrane filamentous arrays. Such sistency of the cytoskeleton. Other associated and membranous-cytoskeletal associations may restrict binding proteins affect the rate and extent of filament the movement of important membrane proteins, polymerization, providing a mechanism for localized such as receptors, at that site or communicate events plastic changes occurring at the membrane to underlying areas. In Microfilaments consisting of the protein actin are this section, we describe components nts of the neuronal 6 nm in diameter and are prominent in cortical cytoskeleton and how they contribute to the architec- regions, particularly in the highly specialized sub ture of the neuron and confer specificity to each membrane filamentous structures, such as the presy- structural domain haptic and postsynaptic membrane specializations Microtubules are long, tubular structures that are 3. 1. The Neuronal Cytoskeleton Provides 25 nm in diameter and form tracks for the transport arious organelles and molecules, although the Internal Support microtubules are themselves also capable of move- Axons and dendrites emerge from the perikaryon ment(Fig. 6). The microtubules and actin consist of as delicate strands. Axons may be as much as a mil- globular subunits that can assemble and disassemble lion times longer than they are wide. Consequently, with relative ease. Neurofilaments that are 10 nm in hese fragile processes require internal support. The diameter are a subdivision of the ubiquitous class of igidity of the cytoskeletal network is apparent after intermediate filaments found in all cells(Fig. 6) removal of the neuronal membrane with detergents Mammalian neurofilaments consist of three fibrous hat selectively extract membrane lipids and proteins. subunits that have a very high affinity for each other, In experiments using detergent-treated cultured nerve and polymers composed of these subunits are very cells, isolated neuronal processes, and isolated sub- stable. Neurofilament subunits are synthesized and membranous cytoskeletal patches, the cytoskeleton assembled in the cell body and then directed down the remains intact, and its shape is virtually identical to axon, where they contribute to its resiliency and its its original conformation. The cylindrical form of the caliber Neurofilaments are degraded at the entrance axonal cytoskeleton is so cohesive that investigators, to the nerve terminal by Ca-activated proteases using the classic model of the squid giant axon to located at that site. study axonal transport mechanisms, equate the extru- sion of its contents, the axoplasm, to that of tooth- 3.2.1. ACTIN AND TUBULIN POLYMERS paste being squeezed out of its tube. Even isolated Subunits of actin, a 43-k Da globular protein, and submembrane filamentous arrays, such as those microtubules, a heterodimer of two 50-kDa globular found beneath the postsynaptic membrane, appear proteins called a-tubulin and B-tubulin, assemble to retain their curvature after the rigorous processes into polymers that bind to identical subunits at each of homogenization of brain and centrifugation, end of a preexisting polymer. The lengths of the poly lysis, and detergent treatment of isolated synaptic mer are determined by cellular mechanisms that

enable the neuron to respond to environmental￾dependent or activity-dependent fluctuations. This apparent contradiction is resolved by the inherent nature of cytoskeletal elements that exist in different structural and functional states of assembly and dis￾assembly. Moreover, these structures may be stabilized and destabilized, providing yet another dimension to the number of possible conformations of cytoskeletal form. The interactions of various cytoskeletal elements with their associated proteins or with each other con￾tribute to the unique structural and functional iden￾tity of axons and dendrites and their associated dendritic spines. The cytoskeleton also interacts with the neuronal plasma membrane at specific sites, including the initial segment of the axon, special loci along the axon called nodes of Ranvier, and presynaptic and postsynaptic membranes, forming complex submembrane filamentous arrays. Such membranous-cytoskeletal associations may restrict the movement of important membrane proteins, such as receptors, at that site or communicate events occurring at the membrane to underlying areas. In this section, we describe components of the neuronal cytoskeleton and how they contribute to the architec￾ture of the neuron and confer specificity to each structural domain. 3.1. The Neuronal Cytoskeleton Provides Internal Support Axons and dendrites emerge from the perikaryon as delicate strands. Axons may be as much as a mil￾lion times longer than they are wide. Consequently, these fragile processes require internal support. The rigidity of the cytoskeletal network is apparent after removal of the neuronal membrane with detergents that selectively extract membrane lipids and proteins. In experiments using detergent-treated cultured nerve cells, isolated neuronal processes, and isolated sub￾membranous cytoskeletal patches, the cytoskeleton remains intact, and its shape is virtually identical to its original conformation. The cylindrical form of the axonal cytoskeleton is so cohesive that investigators, using the classic model of the squid giant axon to study axonal transport mechanisms, equate the extru￾sion of its contents, the axoplasm, to that of tooth￾paste being squeezed out of its tube. Even isolated submembrane filamentous arrays, such as those found beneath the postsynaptic membrane, appear to retain their curvature after the rigorous processes of homogenization of brain and centrifugation, lysis, and detergent treatment of isolated synaptic compartments; only after sonication or extremely acidic conditions do these tenacious structures dis￾sociate into their component parts. 3.2. The Components of the Neuronal Cytoskeleton Include Microtubules, Neurofilaments, and Microfilaments and Their Associated Proteins The dual nature of the neuronal cytoskeleton, reflected in its rigidity and plasticity, is a function of three filament types: microtubules, neurofilaments, and microfilaments or actin filaments. Each cytoske￾letal element acts in conjunction with a specific set of associated or binding proteins. Some of these cross￾link the filaments to each other, the plasma mem￾brane, and other intracellular organelles and are responsible for the gelatinous and relatively stiff con￾sistency of the cytoskeleton. Other associated and binding proteins affect the rate and extent of filament polymerization, providing a mechanism for localized plastic changes. Microfilaments consisting of the protein actin are 6 nm in diameter and are prominent in cortical regions, particularly in the highly specialized sub￾membrane filamentous structures, such as the presy￾naptic and postsynaptic membrane specializations. Microtubules are long, tubular structures that are 25 nm in diameter and form tracks for the transport of various organelles and molecules, although the microtubules are themselves also capable of move￾ment (Fig. 6). The microtubules and actin consist of globular subunits that can assemble and disassemble with relative ease. Neurofilaments that are 10 nm in diameter are a subdivision of the ubiquitous class of intermediate filaments found in all cells (Fig. 6). Mammalian neurofilaments consist of three fibrous subunits that have a very high affinity for each other, and polymers composed of these subunits are very stable. Neurofilament subunits are synthesized and assembled in the cell body and then directed down the axon, where they contribute to its resiliency and its caliber. Neurofilaments are degraded at the entrance to the nerve terminal by Ca2+-activated proteases located at that site. 3.2.1. ACTIN AND TUBULIN POLYMERS Subunits of actin, a 43-kDa globular protein, and microtubules, a heterodimer of two 50-kDa globular proteins called a-tubulin and b-tubulin, assemble into polymers that bind to identical subunits at each end of a preexisting polymer. The lengths of the poly￾mer are determined by cellular mechanisms that Chapter 1 / Cytology and Organization of Cell Types 11

Cohen and pfaff microtubule-associated proteins (MAPs)and tau proteins. These proteins induce the assembly and stabilization of microtubules by binding to them. The tau proteins facilitate polymerization by bind ing to more than one tubulin dimer at the same time mAPs have two domains. one of which binds to the microtubule and the other to an adjacent MAP molecule, filament, or cell organelle. MAPs provide the neuron with a mechanism for struc tural plasticity and variability. About 10 kinds of MAPs have been identified, and they appear to be differentially expressed during brain development Specific MAPs appear to be restricted to different neuronal processes. MAP 2, for example, is expressed in dendrites but not axons(Fig. 7);con- versely, MAP 3 is present in axons but not in dendrites. Although microtubules appear in parallel array, actin filaments in neurons are usually visible as a tangled meshwork, The network sometimes appears as a dense submembranous array, as in the postsynaptic density(PSd) immediately beneath Fig6 Electron micrograph of the axonal cytoskeleton. The two the postsynaptic membrane. However, the network prominent cytoskeletal elements in this region are microtubules may be less dense, as in the subsynaptic web imme- (m)and neurofilaments(n). The microtubules are 25 nm in diately beneath the PSD and extending throughout diameter and form tracks for the transport of various orga- the dendritic spine. Actin filaments are also asso- nelles, such as vesicles(arrowheads ) The neurofilaments belong ciated with a group of accessory proteins, the may contribute to the resilience relatively stable polymers that actin-binding proteins, which bundle them or in diameter. Neurofilaments y and caliber of axons cross-link them to form a gel. Some binding pro- teins join the filament ends to obstruct further polymerization, and others link actin to the mem- control the rates of association and disassociation at brane. Actin-binding proteins are regulated the ends of each rod. In some polymers, there is a by second messengers, such as calcium or cyclic constant flux of monomers at each end. In more nucleotides stable polymers, dissociation of the subunits at each end is slow or does not occur at all. Stability can be 3. 2.3. MoLECULAR MOTORS achieved by blocking the dissociation reaction at Other proteins associated with the cytoskeleton either end. both tubulin and actin monomers are asymmetric so that they can only link up with each are the molecular motors, which harness energy to other in a specific orientation. Consequently, the propel themselves along filaments. These proteins are enzymes that hydrolyze ATP and GTP and use resultant polymer is polarized and has plus and the liberated energy to move themselves along the minus ends, a feature permitting polymers to grow in a directed manner polymer. Motion is achieved because each of the steps of nucleotide binding, hydrolysis, and release 3.2.2. CYTOSKELETAL-ASSOCIATED PROTEINS of ADP or gdp plus phosphate causes a concomi tant change in the conformation of the motor pro- Cytoskeletal-associated proteins regulate cyto- tein such that it is directed forward. Myosin skeletal structure and function and characterize spe- motors walk along actin filaments, which are cific neuronal domains. Purified tubulin monomers pulled along in the process. This action is impor- can spontaneously assemble into microtubules in tant in the motility of growth cones, the pioneering the presence of GTP. However, polymerization is tip of developing nerve cell processes. Motor pro greatly enhanced in impure preparations. The teins are also associated with microtubules. where impurities are actually a group of accessory pro- they are involved in organelle transport in neuro- teins that are subdivided into two categories: nal processes

control the rates of association and disassociation at the ends of each rod. In some polymers, there is a constant flux of monomers at each end. In more stable polymers, dissociation of the subunits at each end is slow or does not occur at all. Stability can be achieved by blocking the dissociation reaction at either end. Both tubulin and actin monomers are asymmetric so that they can only link up with each other in a specific orientation. Consequently, the resultant polymer is polarized and has plus and minus ends, a feature permitting polymers to grow in a directed manner. 3.2.2. CYTOSKELETAL-ASSOCIATED PROTEINS Cytoskeletal-associated proteins regulate cyto￾skeletal structure and function and characterize spe￾cific neuronal domains. Purified tubulin monomers can spontaneously assemble into microtubules in the presence of GTP. However, polymerization is greatly enhanced in impure preparations. The impurities are actually a group of accessory pro￾teins that are subdivided into two categories: microtubule-associated proteins (MAPs) and tau proteins. These proteins induce the assembly and stabilization of microtubules by binding to them. The tau proteins facilitate polymerization by bind￾ing to more than one tubulin dimer at the same time. MAPs have two domains, one of which binds to the microtubule and the other to an adjacent MAP molecule, filament, or cell organelle. MAPs provide the neuron with a mechanism for struc￾tural plasticity and variability. About 10 kinds of MAPs have been identified, and they appear to be differentially expressed during brain development. Specific MAPs appear to be restricted to different neuronal processes. MAP 2, for example, is expressed in dendrites but not axons (Fig. 7); con￾versely, MAP 3 is present in axons but not in dendrites. Although microtubules appear in parallel array, actin filaments in neurons are usually visible as a tangled meshwork. The network sometimes appears as a dense submembranous array, as in the postsynaptic density (PSD) immediately beneath the postsynaptic membrane. However, the network may be less dense, as in the subsynaptic web imme￾diately beneath the PSD and extending throughout the dendritic spine. Actin filaments are also asso￾ciated with a group of accessory proteins, the actin-binding proteins, which bundle them or cross-link them to form a gel. Some binding pro￾teins join the filament ends to obstruct further polymerization, and others link actin to the mem￾brane. Actin-binding proteins are regulated by second messengers, such as calcium or cyclic nucleotides. 3.2.3. MOLECULAR MOTORS Other proteins associated with the cytoskeleton are the molecular motors, which harness energy to propel themselves along filaments. These proteins are enzymes that hydrolyze ATP and GTP and use the liberated energy to move themselves along the polymer. Motion is achieved because each of the steps of nucleotide binding, hydrolysis, and release of ADP or GDP plus phosphate causes a concomi￾tant change in the conformation of the motor pro￾tein such that it is directed forward. Myosin motors walk along actin filaments, which are pulled along in the process. This action is impor￾tant in the motility of growth cones, the pioneering tip of developing nerve cell processes. Motor pro￾teins are also associated with microtubules, where they are involved in organelle transport in neuro￾nal processes. Fig. 6. Electron micrograph of the axonal cytoskeleton. The two prominent cytoskeletal elements in this region are microtubules (m) and neurofilaments (n). The microtubules are 25 nm in diameter and form tracks for the transport of various orga￾nelles, such as vesicles (arrowheads). The neurofilaments belong to the ubiquitous class of intermediate filaments and are 10 nm in diameter. Neurofilaments are relatively stable polymers that may contribute to the resiliency and caliber of axons. 12 Cohen and Pfaff