Class Notes (786,821)
Canada (482,297)
55-101 (38)
Habetler (38)

Neurocytology I

46 Pages
Unlock Document

University of Windsor
Biological Sciences

Neuroscience Lecture Notes summary Neurocytology I - The structure of the neuron The variation in the dendritic spine length has lead to two general hypothesis about the function of spines: 1) They serve to isolate the spine electrically from the dendrite. This hypothesis suggests that spines would be capable of acting autonomically from the electrical events that travel up and down a dendritic shaft. Spines with long, thin neck would be particularly well isolated, whereas those with short, wide necks would act more like the shaft. 2) Spines provide for an isolated chemical environment in the spine head. This hypothesis is particularly relevant to the buildup of Ca+2 within the cytoplasm, an event associated with long-term changes in the excitability of a neuron’s membrane. Again, spines with long necks would be able to regulate Ca+2 conc locally where as those with short necks would follow a regulatory plan established for the dendrite as a whole. The axon hillock is devoid of ribosomes and RER. As a result, there is no means for synthesizing anything other than mitochondrial proteins along the length of an axon. The initial region of a axon is devoid of myelin. It is characterized by the presence along its membrane of a very high density of voltage-sensitive Na+ channels. Because of the insetion of these channels in its membrane, the initial segment is the sight for initiation of APs that travel down the axon. Along the length of axons covered with myelin are regularly spaced gaps in the myelin. As in the case for the initial segment, these myelin-free regions (nodes of ranvier) have membranes with high density of voltage sensitive Na+ channels. The nodes are sites at which the actively propogated AP is regenerated. Protein synthesis: All organelles for transcription, translation, post-translational processing and packing of proteins are present in the soma. Neuronal nuclei contain large amounts of heterochromatin, most likely a reflection of the fact that past a restricted period in development neuron’s cease mitotic activity and divide never again. Neuronal somata possess an extreme density of ribosomes, which are found in stacks of RER and are referred to as Nissl bodies. Ribosomal rosettes are frequently encountered in the cytoplasm of the soma. They are equally dense in the proximal dendrites, which permits these structures to show up in Nissl stains. AS the primary site of post translational processing the Golgi complex is well developed in the neuronal soma. Particularly important is glycosylation of proteins and their packaging into vesicles used for transport and secretion. Degredation of worn-out membrane and of material taken in by endocytosis if the function of the lysosome and the peroxisome. 1) lysosomes are characterized by the presence of acid hydrolases. Primary lysosomes are a produce of the golgi complex. They are filled with hydrolytic enxymes that work at low pH. Fusion of the primary lysosome with a phagosome produces a secondary lysosome. Material resistant to degradation by the acid hydrolases accumulates in what is referred to as residual bodies. In the CNS, some residual bodies contain a dark pigment and stacks of undigested membranes. These are lipofuscin granules, which accumulate throughout life and are particularly abundant in aged brains. Primary lysosomes (filled with acid hydrolases) + phagosome  secondary lysosome ( residual bodies with lipofuscin granules) 2) Peroxisomes are biochemically distinct membrane bound organelles responsible for the degradation of long chain fatty acids, among other membrane products. Peroxisomes for long chain fatty acid degradation, associated with sphingosine degradation disorders and adrenoleukodystrophy Microtubules are the primary determinant of neuronal morphology. Composition of microtubules includes: 1) α and β tubulin, which can be modified to fit the conditions of the individual neuron at a particular moment. Thru these modifications, microtubules can be quickly assembled and disassembled at the tips of dendrites and axons. 2) Microtubule associates proteins (MAPs), which play a prominent role in the stabilization of microtubules. Juvenile subunits of one MAP permits rapid and easy changes in the cross-linking of microtubules whereas the adult subunits serve to stabilize the structure. Neurofilaments are responsible for setting the diameter of an axon. 1) neurofilaments are neuronal intermediate filaments, composed of three proteins. Because the heavy neurofilament protein has a long side chain-arm that permits crosslinking with microtubules, the neurofilament dictates the spacing of cytoskeletal elements in the axon, and thus the caliber of the axon. 2) Neurofilaments aggregate in bundles, neurofibrils, that stain with silver ions. Axoplasmic transport moves vesicles and other organelles from the some to the tips of the neuron. Microtubules are the scaffold upon which all elements are transported. The individual tubulin molecules form polarized filaments that in axons are arranged with their plus ends toward the axon terminal and their minus ends towards the soma. Separate ATPases move vesicles and other elements in anterogrande (kinesin) or retrograde (dynein) directions. Actin filaments (microtubules) are a major constituent of the cytoskeleton along the inner surface of the plasma membrane. Actin are chemically heterogeneous, which permits some parts of the actin filament architecture to respond selectively to changes in its environment. Actin interacts with a variety of neuronal proteins, including dystrophin that is involved in Duchenne muscular dystrophy. Unusual means for interneuronal communication: 1) Gap junctions: assembled of connexons, gap junctions are a means for directly transferring the rapid changes in ion concentration across the plasma membrane from one neuron to another. 2) Extrasynaptic receptors: receptor proteins are inserted into membranes outside of anatomically recognizable synapse; these are frequently encountered on the membranes of synaptic terminals and are referred to as autoreceptors. 3) Signalling molecules that are gases rather than liquids can rapids diffuse thru plasma membranes and may directly influence that activity of neuronal targets without the necessity of binding to a membrane-bound receptor. Presynaptic events: Synaptic vesicles exocytosis Synaptic vesicles (SVs) are loaded with neurotransmitters (NTs) are docked at the active zone in the presynaptic plasma membrane. Upon Ca++ influx, docked SVs fuse with the plasma membrane to release their neurotransmitter across the synapse. SV proteins are then recovered from the cell surface in a process of endocytosis, allowing new SVs to be formed and filled with NTs. General functions of synaptic vesicle proteins: 1) Localizing SVs within the nerve terminal (e.g. synapsins – binds actin, rabs – binds GTP) 2) Transport across SV membrane (e.g. proton pump – acidifies interior of the cell to produce a gradient that can be exploited by other transporters to bring other things into the cell, ACh transporter, SV2) 3) Docking and fusion to plasma membrane (VAMP (a.k.a. synaptobrevin) – binds syntaxin, synaptotagmin – binds syntaxin, adaptin, neurexins) Four proteins were identified that form the 7S complex: 1) synaptotagmin – binds phospholipids in a calcium-dependant manner; a Ca+ sensor – unsure if the sensor acts as a brake or accelerator. bbb 2) VAMP – vesicle associated membrane protein 3) Syntaxin – a plasma membrane protein 4) SNAP-25 – synaptosomal associated protein of 25 kilodaltons. Three of these proteins form a core complex: VAMP, SNAP-25 and syntaxin. The core complex forms extraordinarily tightly. The notion that the core complex represents a docked synaptic vesicle has gained considerable support because toxins that inhibit neurotransmission cleave VAMP, syntaxin, and SNAP-25. Tetanus toxin and the light chains of botulinum toxins B, D, F and G selectively cleave VAMP; bot toxins A and E cleave SNAP-25; and bot toxin C1 cleaves syntaxin. A toxin that depletes synaptic vesicles from nerve terminals (black widow spider venom, α- letrotoxin) acts indirectly thru synaptotagmin. SNARE hypothesis: proteins on the donor compartment or vesicle (v-SNARES such as VAMP) bind to proteins on the plasma membrane or target (t-SNARES such as syntaxin). According to the hypothesis, the specificity of vesicle trafficking may be guaranteed by the pairing of cognate SNARES. N-sec1 (Munc18) is a soluble protein that binds tightly to syntaxin. The two proteins are released upon phosphorylation of syntaxin by one of several kinases. N-sec1 has a role in controlling fusion pore expansion on the plasma membrane. Rab3 is a GTP-binding protein that is peripherally attached to synaptic vesicles. Members of this family are localized selectively to intracellular membrane compartments where they are thought to direct the vectorial transport of vesicles thru a cycle of GTP binding and hydrolysis. Rab3a dissociates from SVs upon exocytosis. A mouse rab3a KO releases NT normally upon stimulation, but shows defects upon repetitive stimulation. Thus, Rab3a may function in regulating the availability of SVs for NT release. Neurocytology II – Myelin and Glia Myelin and myelin-forming cells: oligodendrocytes (CNS) and Schwann cells (PNS). The myelin sheath has a high electrical resistance and a low capacitance, providing an effective insulator around the axon. Consequently, myelinated fibers conduct APs in a salutatory manner. In nonmyelinated fibers, conduction velocity is proportional to the square root of the diameter, whereas in myelinated fibers, conduction velocity is proportional to the diameter of the fiber itself. This in turn allows greater conduction velocity in a smaller space. There are many different Schwann cell phenotypes: • Schwann cells include the satellite cells around the sensory neurons in the dorsal root ganglia, around autonomic neurons and around the massive neuronal complex in the enteric nervous system, where the schwann cells have some distinctive properties and are often termed enteric glia. In each of these sites, the schwann cells cover nearly the whole surface of the nerve cell body and its processes, and have a basal lamina over their outer surface, isolating the neuron from the surrounding collagenous world. • Schwann cells of unmyelinated axons (ensheathing Schwann cells): most of the axons in he peripheral nervous system are non-myelinated, but are ensheathed by Schwann cells. Several axons may be ensheathed by a single Schwann cell, and the junctions between neighboring Schwann cells along a set of axons form a series of interdigitating processes. In unmyelinated axons, the propagation of impulses is graded, continuous and relatively slow, and ion channels appear to be evenly distributed along the surface of the axon. • The myelinating Schwann cell: the mulilamellated spiral that comprises the myelin sheath can be thought of as a lengthening and spiraling of the lips of the indentation in which the axon sits. Early in development Schwann cells typically ensheath communities of axons, rather than individual axons. With continued Schwann cell proliferation, some axon-Schwann cell units go on to form the normal mature ensheathing Schwann cell arrangements, with several axons ensheathed by individual schwann cells. In other fibers the Schwann cell all along the axon continue division until they have reached a 1:1 relationship with the axon. They then initiate the process of myelination. It is the axon that signals whether the Schwann cell is to make myelin or not. With the commitment to myelination, the Schwann cell changes in a variety of ways and adopts a series of new biochemical features. In addition, it undergoes a terminal differentiation: the myelinating Schwann cell never reenters the cell cycle in a normal nerve fiber and continues to support the myelin sheath that it forms throughout the normal life of the organism. Myelination proceeds by a process of continual lengthening of the inner lip of the Schwann cell. The Schwann cell nucleus rotates around the axon in the same direction as the inner loop; imagine it pushing the inner loop around. If the original myelin sheath is damaged or destroyed, or if the myelinated axon degenerates, the Schwann cell promptly reenters the cell cycle, abandons its myelinating biochemical characteristics, and amputates its old sheath, which is ultimately cleared. Oligodendrocyes: Oligos develop from specific glial progenitor cells and myelinate axons in the CNS. The process of myelin formation is in general similar to that in the PNS, with two important exceptions: 1) the oligo is capable of myelinating many axons (> 50). 2) the biochemical composition of CNS myelin is somewhat different than PNS myelin. Oligos are capable of proliferation and remyelination after demyelinating injuries, but they are much less effective in this role. Astrocytes: Astrocytes outnumber neurons 10:1. Functions of astrocytes: 1) Astrocyte basal lamina serves to isolate the nervous system from the collagenous world; all of the nervous system is within the basal laminae produced by the glial limitans and the cerebral blood vessels (contrast the isolation of individual nerve fibers by schwann cell basal lamina in the PNS that doesn’t surround the neuron completely, but surrounds the blood vessels). 2) The numerous gap-junctions b/t astrocytes imply that they provide a functional synctium for ions and small molecules. Astrocytic processes cover CNS nodes of ranvier in a similar fashion to the schwann cell microvilli in the PNS. 3) Nutritive support of neurons because of the capacity for vessel-neuron connection. Note that this connection might work in the opposite direction, clearing from the perineuronal space into the circulation unwanted substances (detoxification). 4) Astrocytes produce a variety of cytokines, chemokines, and growth factors that modulate the response of the CNS to injury. 5) Astrocytes appear necessary to induce tight junctions between endothelial cells. 6) Astrocytes may provide a supportive role in facilitating extension and guidance of axonal processes during development. 7) They proliferate and elaborate dramatic bundles of glial filaments, gliosis. Regions of gliosis have interdigitated astrocytic processes laced together by frequent desmosomal junctions, and are usually considered inimical to regeneration. Their importance may be that they are also inimical to CNS invasion by connective tissue from blood vessels or meninges. As such, they may function to keep the nervous system “neural.” Microglia are marrow derived macrophage lineage cells in the CNS. In early development, cells bearing monocytic markers invade the nervous system from the circulation and assume the distinctive stellate shapes typical of microglia. In this behavior, they resemble specialized cells of monocytic origin in other tissues. They then lose many of the markers of their ancestors, and in addition, unlike most circulating macrophages, they retain the capacity to proliferate locally. Whether the continue to be renewed from the circulation in the post-development period is uncertain. In the response to disease, microglia are known to: 1) proliferate locally 2) interact with white blood cells 3) differentiate into macrophages 4) Produce cytokines, substances that influence both immune cells and cells of the nervous system. Recently recognized has been the prominence of perivascular cells in both the CNS and PNC that lie just within or just without the basal lamina of the vessels. The perivascular cells are well suited for antigen presentation to trafficking T cells that enter the CNS. Conduction of Decremental and Regenerative Signals in Neurons 0. Fundamental Concepts + 1) Resting potential is –70 mV, close to E dKe to open K channels 2) Ion distribution gives rise to different equilibrium potentials via Nernst eqn - High Na outside (E =Na4 mV) , high K inside (E = -86 KV) - Very low Ca inside (E = Ca6 mV), high Cl outside (E = -78 mCl 3) The equilibrium potential for a given ion is the potential at which no net flux of that ion occurs across the membrane. 4) Due to very low capacitance of membrane (5 pF), very few ions most move to achieve equilibrium (Q = CV) 5) Membrance voltage (Vm) change produced by ionic movements is related to the number of charges moved (Q, in coulombs) divided by the membrane capacitance, Cm (in farads) Vm = Q/Cm 6) Current flow through ion channels can be approximated with Ohm’s Law Iion = Gion (Vm – Eion) where g is conductance (inverse of resistance) I. Passive current flow (aka cable property of dendrites and axons) A. Basic observations - At steady state, a point depolarization ΔV is graded: it tails off exponentially according to length scale λ, a measure of the relative resistance of the membrane and axoplasm - Due to resistance (ion channel) and capacitor (neuronal membrane) in parallel, upon current pulse voltage asymptotically increases to steady state value according to time constant τ, the product of resistance and capacitance of the membrane B. Consequences - Since λ goes as √r, resistance decreases with diameter of dendrite/axon - Spatio-temporal summation: depolarizations nearer axon hillock are more likely to cause action potential, input signal summation requires appropriate timing II. Active current flow (aka action potentials) - Small depolarizations do not result in action potentials due to outward K flow - Threshold depolarizations open voltage gated Na channels and initiate action potential - Na channels inactivate quickly, helping stop rise in membrane potential and give refractory period - Slower voltage gated K channels help repolarize cell - Experimental verification by Hodgkin and Huxley, using voltage clamp on squid giant axon, along with expts with selective ion channel blockage - Due to depolarization of nearby Na channels, action potential is propagated in all-or- none fashion; one-way due to refraction - Conduction velocity depends on passive current spread; myelination raises conduction velocity by increasing λ (bigger r) and decreasing τ (low membrane capacitance trumps higher resistance) Main points to take away from this lecture: 1) Conduction of signals in dendrites is graded and decremental. This allows for the integration of many incoming signals by spatio-temporal summation at the axon hillock and allows different inputs to be assigned different weights based on their point of contact in the dendritic arbor. 2) Conduction of signals in axons is regenerative and all-or-none. This derives from the presence of voltage-gated channels (particularly Na+) and allows for a high degree of fidelity in conduction of signals, sometimes over long distances. 3) The passive spread of current is a factor in the speed of both dendritic and axonal signal transmission. The major determinant of this is the caliber of the process. Synaptic Events: Neurotransmitter Release and Postsynaptic Potentials I. Ca couples action potential depolarization to neurotransmitter release - Whereas Na and K help propagate an action potential, Na and K current are not necessary at the presynaptic terminal for nxt release - Evidence: Depolarization with TTX and TEA blockage of Na and K channels still results in nxt release - Voltage-gated Ca channels and Ca current are necessary for nxt release - Evidence: 1) removal of external Ca or blockade of V-gated Ca channels during depolarization of the terminal inhibits NT release. 2) Ca influx into presynaptic terminals during NT release may be measured, either by recording the current flowing thru the Ca channels using an electrode inserted into the terminal or by loading the terminal with a fluorescent indicator that reports free Ca conc. 3) intraterminal injection of Ca thru a microelectrode or using Ca- containing liposomes can evoke or augment transmitter release. 4) intraterminal injection of a Ca chelator (e.g. BAPTA) will block transmitter release. - Ca promotes fusion of synaptic vesicles with presynaptic membrane - Candidate interactions: Ca may interact directly with structural proteins of the veicle or presynaptic membrane, via an effector such as calmodulin, or via a Ca-sensitive enzyme such as Ca-calmodulin dependant protein kinase or PK-C. Synaptotagmin, a Ca-binding protein present in the membranes of synaptic and other secretory vesicles, has been of particular interest recently. Injection into presynaptic terminals of synthetic peptides which interfere with the ability of synaptotagmin to bind Ca and phospholipids has been shown to block synaptic transmission in a manner that does not alter the amount of Ca that flows into the terminal during AP invasion. This suggests that synaptotagim has a role in vesicular release downstream from Ca entry. - Since nxt is stored in discrete vesicles of uniform size, nxt release is quantal and not graded. Synaptic vesicles are of uniform size and the fusion of a signle vesicle always causes the release of its entire contents. II. Neurotransmitters that affect ion channels cause predictable excitation or inhibition - Nxt bind postsynaptic receptorsstnd (1) open/close an ion channel or (2) modulate enzyme activity (focus on the 1 here) - ACh opens a channel equally permeable to Na and K whose reversal potential is ~ 0 mV (-11mV by Nernst eqn), therefore ACh depolarizes the cell - Nxt produce excitatory or inhibitory response if reversal potential of ion channel receptor (equil. potential if single ion) is above or below threshold, respectively - Consequence: opening of Na/Ca channels is excitatory, of K/Cl channels is inhibitory - Excitation and inhibition aren’t synonymous with de- and hyperpolarization, e.g. if depolarization is still inhibitory if reversal potential is below threshold III. Time course of synaptic transmission Presynaptic action potential  increased presynaptic Ca permeability; Ca influx  exocytosis of nxt vesicles  nxt binds postsynaptic receptors  activation of postsynaptic ion channels  postsynaptic action potential Summary of major points - Understand the role of Ca in nxt release - Be able to predict response to depolarization and hyperpolarization: is it excitatory or inhibitory? - Be able to describe the major events of synaptic transmission Astrocytes and astroglia 0. There are three types of glial cells 1) Astrocytes: maintain an appropriate extracellular environment for neuronal activity and provide metabolic support to neurons. They may also influence or regulate neuronal activity. 2) Oligodendrocytes: form myelin sheaths around axons (speeds action potential along axon). Analogous to Schwann cells. 3) Microglial: play an important role in the brain’s response to injury and infection. Following injury they become highly motile and engulf dead cells and cellular debris. They may also be the brain’s immune cells. I. Astrocytes play a supportive role for neurons - Remove ions and neurotransmitters from extracellular space - Some processes ensheath synapses and have Glu transporters to remove Glu from extracellular space (which is toxic to neurons) - Potassium buffering hypothesis: astroctyes prevent accumulation of K in extracellular space (see physiology below) - Provide neurons with energy - Contain glycogen granules, perhaps to provide glucose or lactate to neurons - Ca waves in astrocytes may stimulate arteriole dilation and control brain microcirculation - Respond to neuronal activity or directly influence it - Express ion channels and nxt receptors, activated upon “spillout.” The functional significance of this neuron-astrocyte signaling is not known, but it may help to maintain the association of astrocytes with synapses. - Glu stimulates intracellular Ca oscillations, propagated as waves, and may excite nearby neurons via release of ATP and/or Glu. Recent studies also suggest that astrocyte Ca++ waves may be important for controlling the dilation of blood vessels in the brain, and thus brain microcirculation. - Secrete growth factors and guide neuronal migration - Enhance formation of synapses - True function of astrocytes hard to gauge because in situ observation is difficult II. Characteristics of astrocytes - Outnumber neurons 10:1 - Contact spacing (no overlap between astrocytes) - Express GFAP (Glial Fibrillary Acidic Protein; intermediate filament). GFAP- immunoreactive fibers are present in the primary processes of astrocytes, giving them their star shaped appearance. This protein may be important for providing structural support to brain tissue, as animals that lock this protein have an increased susceptibility to head trauma. Other immunocytochemical markers commonly used to identify astrocytes include S100β (a Ca_ binding protein), Gln synthetase (an enzyme that converts glutamate to glutamine), and GLAST/EAAT1 (a glutamate transporter). - The processes of astrocytes extend to make contact with capillaries. At the surface of the capillary, astrocyte processes expand to form an end feet specialization, which completely cover the capillary wall. - Astrocytes have conspicuous aggregations of glycogen granules in their cytoplasm suggesting they are a main site for energy storage in the brain. Glucose is transported into astrocytes from the blood at the endfeet by glucose transporters (GLUT-1) and used, released, or stored as glycogen. Glucose is provided to neurons either directly or after is has been converted to lactate. - Complex morphology with highly branching processes (may result in microdomains that are biochemically isolated from the rest of the cell) - The fine processes of astrocytes abut or ensheath synapses. These processes have a high density of glutamate transporters, which help to remove glutamate from the extraceullar space after it is released furing excitatory transmission. Without this astrocyte-dependant uptake, glutamate levels rise, tonic activation of receptors occurs and neurons eventually die as a result of excitotoxicity. A concern is that the elevation of extracellular K+ and decrease in extracellular Na+ that accompany brain ischemia may cause reversed cycling of glutamate transporters and contribute to neuronal death thru excitotic mechanisms. However, glutamate transporters are also expressed by neurons and it is not yet clear which transporters contribute to the increases in extracellular glutamate that occur in ischemia. - Physiology - Inexcitable – incapable of firing APs. - They have low membrane resistance due to their high resting K conductance, causing a very negative resting potential (~-90mV). - The ion channels responsible for the K+ conductance are enriched at the tips of astrocyte processes, the presumed end feet. - These features of the astrocyte membrane have led to the hypothesis that the high expression of K+ channels enables astrocytes to remove K+ that is released from neurons during activity and redistribute it thru the astrocyte network, or dump it into the circulation, preventing its accumulation in the extracellular space. This has been termed the K+ buffering or K+ siphoning hypothesis. - Forms syncytium via gap junctions, which are modulated by phosphorylation and often asymmetrical. The primary gap junction expressed by astrocytes is connexin 43 (Cx43). The function of this coupling is poorly understoof, but may be involved in ion buffering, metabolic homeostasis, regulation of proliferation, and/or astrocyte-astrocyte signaling. III. Astroglial cells - Astrocytes are absent in retina and cerebellum; instead astroglial cells do the job - In retina they are called Muller glial cells, in cerebellum Bergmann glial cells IV. Astrocytes in disease a) Alexander disease: a rare disorder of the nervous system that primarily affects children. Those afflicted exhibit macrocephaly, seizures, and mental retardation; the disease is typically fatal within the first decade of life. A characteristic feature of this disease is the presence of cytoplasmic inclusions in astrocytes, termed Rosenthal fibers. Immunocytochemical studies indicate that these inclusions are ubiquitinated protein aggregates composed of GFAP and small stress proteins, such as αβ-crystallin and HSP27. Analysis of DNA from patients indicate that they have mutations in the coding region of the GFAP gene. Interestingly, equivalent mutations in other intermediate filaments also produce disease. Thus, Alexander disease results from gain of function mutations in GFAP. b) Vasogenic edema is associated with a gain of water by the brain from the blood, resulting from a breakdown in the integrity of the BBB. This is often accompanied by cell swelling. However, cellular edema can occur without breakdown of the BBB and may occur during intense neuronal activity. Cellular edema often occurs during ischemia, hypoxia, or brain trauma and is associated with the swelling of astrocytes and neuronal dendrites. Cellular edema of brain: intense neuronal activity  increase uptake of ions, nxt by astrocytes  osmotic swelling. Swelling can induce the release of neuroactive substances (particularly glutamate) as well as ions that can have deleterious effects on neuronal signaling and survival. c) In many diseases the expression of glut transporters is decreased, perhaps as a result of alterations in neuron-astrocyte signaling. d) Excitotoxicity: extracellular high K and low Na  reversed Glu transporter  toxic to neurons e) Reactive astrocytosis: accompanies brain trauma; characterized by hypertrophy/hyperplasia of astrocytes, and the increased expression of GFAP. f) Cancer: astrocytomas (e.g. glioblastoma), multifocal as a result of the high migratory ability of these cells within the CNS. Astrocytomas are also associated with vascular proliferations perhaps because astrocytes in tumor secretes VEGF to stimulate angiogenesis. Development and Organization of the Brain – I Part 1 I) Development of neural tube and spinal cord: After the three germinal layers of the embryo are formed, some cells in the surface ectoderm migrate anteriorly thru a pore in the ectoderm (Henson’s node) and form the notochord, which lies in the mesodermal layer, immediately beneath the ectoderm. The ectoderm later forms both the skin and nervous system. i) Induction of neural plate – Neurulation: the CNS is derived from the surface ectorderm. Cells in the midline of the ectoderm proliferate and form a longitudinal strip of cells, the neural plate. These cells are located over the notochord and are induced to form the CNS while the more lateral ectoderm forms non-neural cells of skin. Due to increased cell proliferation along the midline, the edges of the neural plate become elevated to form bilateral folds, leaving a midline depression between them, the neural groove. The edges of the groove become elevated and form neural folds. The notochord releases trophic factors (e.g. SHH) that induces cell proliferation in the midline of the overlaying ectoderm. These dividing cell form the neural plate which becomes the CNS. SHH later induces formation of the floor plate in the ventral midline of the neural tube. Cells in the floor plate also express and release SHH, which induces differentiation of motor neurons in the ventral part of the neural tube. II) Cell proliferation and pseudo-stratified epithelium: Surface ectoderm: the epithelial cells sit on a basal lamina and their apical and basal surfaces differ. The apical surface of cells has cilia that face dorsaly, toward the amniotic cavity. The basal surface faces down, resting on the basal lamina and facing the interior of the embryo. The basal lamina separates surface epithelial cells from mesoderm. Neural tube: the cells that form the walls of the neural tubes are called neuroectoderm. As cells proliferate, the ectoderm layer thickens. The nuclei appear to be at different levels, thus the term pseudo-stratified. Initially the apical surface of these cells faces the amniotic cavity. After the neural tube closes, the apical surfaces of the neuroepithelial cells face inward, toward the lumen of the neural tube. The basal surfaces of these cells face the outer surface of the neural tube and they rest on a basal lamina. This basal lamina later becomes the pia. III) Closure of the Neural tube: Apposition: the edges of the neural fold come together in the midline to form a tube. Fusion: dorsal margins of the neural folds fuse in the midline. Neural crest cells arise from the zone between neural tube and surface ectoderm. Surface ectoderm separates from the neural tube and fuses over the dorsal surface of the embryo (failure to fuse = spina bifida). IV) Differentiation of the neural tube wall: Initially the neural tube wall is a single layer of undifferentiated, proliferative cells. Due to proliferation and migration, these cells later form three concentric layers. Early stage: Neural tube is a single layer; all cells in the wall are neuroepithelial cells. Dorsal and ventral regions of the neural tube differ in structure and functions: The walls of neural tube have dorsal and ventral portions: Alar plate: dorsal, function: sensory, (afferent); dorsal horn of spinal cord. Basal plate: ventral, function: motor (efferent); ventral horn of spinal cord. The sulcus limitans divides alar and basal plates and this border is the location of autonomic neurons (preganglionic); forms the intermediolateral cell column in adults. Specialized midline structures: these zones are a single cell layer; no proliferation Roof plate: dorsal; roof of the ventricles and at some levels, the brain; also forms the choroid plexus which secretes CSF. Floor plate: ventral; the site where axons cross the midline from one side of the CNS to the other. At certain places in the CNS, the floor plate forms the commussures of the brain. V) Generation of cells in the CNS: Three concentric zones (ventricular, mantle and marginal) are seen in a cross section of the neural tube wall. • Ventricular Zone (vz) is a transient proliferative compartment that is innermost, close to lumen. In the spinal cord, all neuronal cells arise from vz – the germinal layer. The ventricular zone ultimately disappears leaving a single layer of ependymal cells that line the ventricular surface. • Mantle Zone (mnz): In the brain and spinal cord, neurals arise from cells in the vz. After proliferation, the cells migrate outward to form the mantle zone which contains neuronal cell bodies that form the grey matter of the adult cord. Mnz cells are differentiated neurons that no longer divide. • Marginal zone (mrz) contains neuronal processes, but very cell bodies. In the spinal cord, it forms white matter, which contains myelinated axons. VI) Neuronal Migration: • In the spinal cord, the first born neurons travel a short distance from the vz and ener the mnz. Later formed neurons migrae outward and displace the mnz cells, pushing them further out. This pattern produces an outside to inside gradient where the oldest neurons are located at the outer mnz. One result of this pattern is that the white matter is located at the outer border of the spinal cord in the place that was the mrz. • In cerebral cortex, a different mode of migration produces thickening of the neural tube wall. Beneath the marginal zone, a wide new zone appears (intermediate zone) which contains migrating neurons that travel a long distance. Newborn neurons from the vz migrate outward thru the intermediate zone and do not stop until they reach the marginal zone. These neurons form a dense layer of cell bodies termed the cortical plate situated beneath the mrz. Deep to the cortical plate is the intermediate zone, which later becones subcortical white matter when myelinated axons grow in. o In the cortical plate, successive waves of migration occur so that recently born neurons migrate outward past previously arrived neurons producing an inside-to-outside gradient. In cerebral cortex, the white matter is situated deep in the wall, beneath the cortical gray matter, whereas in spinal cord, the white matter forms the outermost later. Part 2: Development of brain – formation of brain vesicles (3-5) Three brain vesicles appear in the early fetus. Two of the vesicles later subdivide to form a total of five vesicles. All parts of the adult brain arise from the walls of these vesicles, each vesicle forming a particular brain region. The caudal neural tube forms the spinal cord. The rostral vesicles differentiate further and greatly enlarge during fetal life. The walls of the brain vesicles form the cellular tissue of the CNS. The lumen forms the ventricular system. Divisions of the fetal brain: Brain Vesicles Lumen Rhombencephalon – most caudal brain vesicle. Later in development the walls of the rhombencephalon form two divisions of the brainstem Myelencephalon – becomes the medulla and its roof plate IVth ventricle forms the this roof of the IVth ventricle. Metencephalon – consists of two brainstem structures: the base forms the pons, while cells in the dorsal portion proliferate to form a transient ridge (rhombic lip) that gives rise to the cerebellum. Mesencephalon – the dorsal portion forms the tectum which has 4 elevations on its dorsal surface, the superior and inferior colliculi. They participate in auditory and visual signaling. The base of the mesencephalon becomes the cerebral peduncles, which carry descending axons from higher to lower levels of the CNS. The central portion of the midbrain, the tegmentum, contains a network of grey matter and axonal bundles. Midbrain Cerebral aqueduct Prosencephalon- the most rostral vesicle of the embryonic brain; forms a single vesicle in the midline. It later expands into 3 portions, the diencephalons and two telecephalonic vesicles which grow extensively to become the cerebral hemispheres; the hemispheres give rise to the cerebral cortex and basal ganglia. The caudal part of the prosencephalon forms a single vesicle, the diencephalons. Its lumen is the IIIrd ventricle while its lateral walls become the thalamus and hypothalamus. Diencephalon IIIrd ventricle Telecephalon – forms as an evagination of the prosencephalon. Lateral ventricles (2) Consists of two parts: the dorsal wall is the pallium and forms the cerebral cortex; the ganglionic eminence forms the basal ganglia and produces GABA neurons that migrate to cortex. The background growth of the telencephalon produces a curve in the lumen that leads to the C-shape of the lateral ventricles. Brain flexures: • Brain stem formation: Cell proliferation in the walls of the neural tube causes the long axis of the CNS to bend and form flexures; the mesencephalic and pontine flexures being most prominent. o Pontine: a dorsal concavity in the rhombencephalon. Cells in the floor proliferate causing the ventral surface to expand, but neurons in the roof plate do not proliferate. Since the floor elongates more than the roof, the rhombencephalon bends, with the concave surface facing up (dorsalward) forming a U-shape. o Mesencephalic flexure and Isthmus: a ventral concavity. The dorsal surface of the midbrain elongates more than the ventral surface causing the midbrain to flex. This bend forms the mesencephalix flexure which is the only flexure present in the adult brain. Due to this flexure, a right angle is formed between brainstem and cerebral hemispheres. A constriction at the caudal end of the midbrain is the isthmus, which forms the caudal midbrain and marks the border between mesencephalon and pons (metacephalon). Rostrally, the midbrain merges with the diencephalons. Organization of the CNS: segmental and suprasegmental structures: Segmental structures: Spinal cord and brainstem Connect to peripheral structures Input-output functions Function: mediate reflexes, sensory-motor functions Suprasegmental structures: cerebral hemispheres + cerebellum No direct connections to peripheral structures Connect only to spinal cord and brainstem Function: modulate segmental parts of the CNS Integration Cerebral hemispheres: main functional connections: • Integration (cortex to cortex) o Corticocortical (direct) o  basal ganglia o  cerebellum • Hemisphere input via thalamus (sensation) • Hemisphere outputs o Somatic (skeletal muscle): Voluntary movement (direct – corticospinal) o Autonomic (visceral): indirect via hypothalamus • Function: heart rate, respiration, blood pressure, temperature regulation, GI tract, endocrine (hormones), food intake (appetite), reproduction Development II: Molecular Regulation of Neural Development I. Neural induction: the notochord plays a key role A) Quick review - During gastrulation, dorsal tissue migrates through blastopore lip to become mesoderm - Notochord forms from mesoderm, and induces ectodermal neural plate formation B) Neural induction - Default fate of ectodermal cells is neuronal; BMP4 induces epidermal fate, blocking neural fate - Notochord produces noggin, chordin to inhibit BMP4, thereby inducing neural diffn - However, if you plate out ectoderm, you get neural fate, suggesting an active mechanism. This is explained by the loss of BMPs during plating… so it is the lack of BMPs that induce neural differentiation/default state. II. Dorsal/ventral patterning: In addition to neural plate induction, the notochord is active in dorsal/ventral patterning. The notochord remains ventral to the spinal cord even after the neural plate has been induced and rolled up into the neural tube. Notochord produced SHH which in addition to its morphogenic signaling power, also induces the floor plate to produce more SHH. SHH a ventralizing morphogen that acts in a graded manner by inducing a pattern transcription factors with varying sensitivity to SHH concentration that combine to determine neuronal fate: high Shh (most ventral) induces motor neurons, intermediate induces interneurons, low produces commissural neurons. SHH signaling pathway: SHH binds to its receptor Patched (Ptc)  relieves Ptc’s default inhibition of Smoothened (Smo)  Smo acts of Gli genes (transcription factors)  Gli goes into nucleus to act as either activator or repressor, depending on processing of Gli. Repressor: Gli3 in dorsal spinal cord progenitor – Ptc inhibits Smo  Gli3 processed to Gli3 repressor  in nucleus, Gli3R inhibits SHH target genes since there is no SHH in the dorsal spinal cord progenitor, there is no need for the target genes. Activator: Gli3 in ventral spinal cord progenitor – Shh binds Ptc  relieves inhibition on Smo  blockage of Gli3 processing to Gli3 repressor, upregulation of Gli3 processing to Gli3 activator  relieves inhibition on and induces activation of SHH target genes which can now be expressed and are needed since SHH is present in the ventral side. Shh/Gli3 double mutants suggests that SHH normally antagonizes Gli3 and that positional information can come from other sources. Overlaying ectoderm produces BMP 4/7 which induces the roof plate to also produce BMP4, a dorsalizing morphogen. Roof plate ablation disrupts gene expression (e.g. Math1 and Neurogenin1 normally induced by roof plate; Mash1 normally repressed by roof plate) in the dorsal spinal cord. General principal: the transcription factors induced/repressed by SHH/BMPs have cross- reactive interactions that help to delineate and clarify boundaries between segments of transcription factor  gene activation. Neurons are produced by lateral walls of neural tube, NOT the roof or floor plate III. Neurocortical neurogenesis A) Ventricular zone (site of neurogenesis): expression of Notch1 (receptor) and Delta1 (ligand) in the mammalian telencephalic proliferative zone supports a role in cell fate specification. Most cells express Notch1, but only some express Delta1. Notch signaling can cause adjacent cells to acquire different fates through cell-cell interactions. CBF1 is the primary mediator of the Notch1 pathway. A cell that receives Notch stimulation is inhibited from differentiating and remains a progenitor stem cell type. Whereas a cell that expresses high levels of ligand, this cell will not be itself be seeing that much Notch1 signal, and that cell then will not have that repressive gene expression so it can continue on to undergo differentiation. This is a fundamental mechanism in generating neurons and maintaining progenitor pool for subsequent proliferation. In addition to maintaining the progenitor pool via inhibiting proliferation/differentiation, expression of a constitutively activated form of Notch in vivo promotes the maintenance of radial glial character. Radial glia serve as a migratory scaffold during neurogenesis but are also neural progenitors. Notch/Delta signaling in the developing neocortex maintains the radial glial progenitor scaffold during neurogenesis. Radial glia and some astrocytes may be lineally related neural stem cells: B) Molecular regulation that is important for establishing the proper lamination for the cortex: Reelin and scrambler: mutation can result in inversion of inside-out laminar organization. In the normal case, the first neurons formed form the pre-plate. The pre-plate then split to form the subplate and the Cajal-Retzius cells. The C-R cells express reelin protein. Then subsequent layers of neurons migrate inbetween the preplate and C-R cells, producing an inside- out layering with the earliest cells being closest to the preplate. In the reelin-mutant, the preplate never splits and as a result migrating neurons pile up below the subplate and don’t successfully migrate past their predecessors. As a result, you end up with inverted lamination with the earlier born neurons in the superficial layers. It should be noted that the neurons largely still make their correct connections despite their inverted position. Reelin influences radial neuronal migration thru signaling cascades involving LIS1 and DCX. These genes have been cloned because of their involvement in causing lissencephaly, improper lamination, gyri and sulci (“smooth brain”). Females with DCX mutations have a “double cortex,” or two grey matter areas separated by white matter, due to X-inactivation. Males with DCX mutations have lissencephaly, instead, because DCX is on the X chromosome. C) Nonradial migration: Non-radial cell migration during forebrain development: Most neocortical GABAergic interneurons are derived from ventral structure. Cells from the ganglionic eminences migrate to the neocortex; the homeobox transcription factor Dlx1 and Dlx2 are essential for this process. Leftover from Raymond’s notes: - Asymmetric divisions (parallel to base) give rise to neurons, whereas symmetric divisions (perpendicular to base) do not - Neurons are generated early and glial cells are generated later - Neuroblasts divide only 1-2 times before diffn into neurons - Glioblasts divide many times before diffn, ∴fewer glioblasts needed than neuroblasts - Signal through notch receptor inhibits neural development; this may be a way cells decide to diffn into neurons (cell with delta ligand becomes neuronal; cell with notch receptor remains nonneuronal) Intermediate zone: postmitotic neurons migrate through this layer - Neurons that migrate radially along the processes of radial glial cells to the CP to become excitatory neurons - Neurons that migrate tangentially (e.g. from ganglionic eminences) become inhibitory interneurons Cortical plate: where postmitotic neurons stme to rest - Cortical plate consists of 6 layers (1 is most superficial) - Neurons populate each layer sequentially starting with layer 6, ending w/ layer 1 - The laminar fate of a cell is decided between S and M phase Development III: Axon guidance and Neuronal regeneration 0. Key points • Long and short range guidance cues direct axons and dendrites to their appropriate targets. • The dynamic regulation of growth cone cytoskeleton components underlies attractive and repulsive guidance. • Signal transduction pathways, conserved between neuronal and non-neuronal cells and also across phylogeny are employed to regulate cytoskeleton dynamics and provide clinical entry points for promoting neuronal regeneration. • Guidance across the CNS midline provides a model for understanding how a guidance cue must both direct guidance events and locally modulate responses in order to facilitate the formation of complex axonal trajectories. • Regeneration of central and peripheral neuronal projections differs owing to inhibitory cues present in the CNS by not in the PNS. • CNS inhibitory cues have been identified and they appear to utilize some of the signaling mechanisms which mediate inhibition during neural development. • Blocking intrinsic and extrinsic repulsive signaling pathways can promote adult neuronal regeneration. Part 1: Cell biology of axon guidance I. Principles of axon guidance A) Historical perspective: early evidence of axonal guidance cues - Cajal and Harrison observe dynamic nature of axonal growth cone - Sperry demonstrates topographic mapping in retina-to-tectum - Spinal cord inversion does not affect how motor axons select peripheral pathways B) Sequence of events: pathfinding, target selection, synapse formation and refinement II. Neuronal growth cone organization: The neuronal growth cone contains precise distribution of polarized actin and microtubule polymers. - Actin: found in growth cone, undergoes treadmilling via myosin and actin polymerization; adhesive molecules and myosin mediate membrane protrusion; present in peripheral or cortical cytoplasm of axons and dendrites. - Microtubules: Present in axons in uniform ordered arrays (plus end pointing away from the cell body) as long tracks, and in dendrites as randomly oriented polymers; primarily axonal and rare in terminal endings such as growth cones and presynaptic terminals. Fills in voids in growth cone when actin is attached to membrane complexes in response to extracellular cues (this is the method of growth cone advance). Both actin and microtubule polymers are not static. - Intermediate Filaments: primarily axonal and rare in terminal endings such as growth cones and presynaptic terminals; function to stabilitze axon and determine process caliber. III) Growth cone advance proceeds via protrusion, adhesion, locomotion: - Growth cone has filopodia and lamellipodia -1) Mechanism of growth cone exploration: Lamellipodia and filopodia protrude and retract, leading to rapid changes in growth cone shape. Actin fill the lamellipodia and filopodia and are mostly oriented with their faster growing ends toward the membrane. Most microtubules are found in the growth cone center, but some extend depp into the lamellipodia. They also change their distribution rapidly. This redistribution occurs by polymerization, movement and bending. - 2) Protrusion of lamella by inhibiting retrograde actin flow: a) retrograde flow is likely driven by myosin-type motors, which are hypothesized to bind to the membrane. Actin- assembly occurs at the membrane, and actin disassembly occurs toward the center of the growth cone. B) When ligands engage extracellular matric receptors, it is proposed that a protein complex can then bind to actin, retarding retrograde flow relative to substrate. Continued assembly of actin and the continued action of myosin lead to forward protrusion of membrane. 3) Site selection and site-stabilization in growth cone turning: During site selection, actin accumulates at the point where the growth cone contacts the guidance cue and is depleted in the zone adjacent to it. The growth cone remains spread in other directions. Microtubules extend toward the contact site. At this stage, microtubules may occupy other regions of growth cone as well. During site stabilization, microtubules have coalesced toward the contact site and form a bundle in the growth cone. The growth cone membrane has begun to collapse around it. - Individual filopodia can steer entire growth cone via mechanism above Experimental evidence for this model: • cytochalasin B, which inhibits actin polymerization, leads to cessation of filopedial protrusion and retraction of cytoplasmic networks away from leading edge. • Myosin 1 inhibitors lead to a cessation of retrograde flow, but not filopodial protrusion. • Vinblastine (blocks MT dynamic instability) blocks directed outgrowth, but not filopodial protrusion. IV) Conserved signaling pathways regulate actin dynamics: Know one way to promote actin assembly and one way to promote actin disassembly V) Rho GTPases are key regulators of neuronal growth cone cytoskeleton: Rho GTPases: a small Ras-related GTPases, which, like Ras, cycle between a GTP- bound active form and a GDP-bound inactive form. GDPGDP modulated by guanine nucleotide exchange factors (GEFs) and GTPGDP modulated by GTPase-activating proteins (GAPs). • In fibroblasts, activated forms of three Rho family members, CDC42, RAC, and RHO, have specific effects on cytoskeleton organization with respect to structureal requirements for motility. They appear to act sequentially: CDC42 induces filopedia, lamellipodia, and stress fibers and local adhesions. The later structures are dependant on Rac and Rho. Several kinases are targets of these GTPases, and Rho can regulate phosphoinositide production, which in turn can modulate ABPs, including profiling and gelsolin. Cdc42  Rac  Rho Filopodia Lamellipodia Stress fibers, focal adhesions • In neurons Rho family members function to regulate neuronal morphology. Dros Ras effects neurite outgrowth. In mice, constitutively active Rac in Purkinje cells  reduction in axon terminals, and reduction of dendritic spine size with a concomitant increase in the number of spines. • General principals: Axonal Function Dendritic Function Rho Limits growth or initiation Limits growth or initiation Rac, Cdc42 Promotes growth Promotes initiation Part 2: Axon guidance: Cellular and molecular mechanisms: I) General guidance principals: • Sequence of events: Pathfinding, target selection, initial synapse formation, synapse refinement. • The growth cone detects and responds to axon guidance cues. The lamellipodia contain cross-linked F-actin filaments. The filopodia extend and retract thru regulation of the rates of actin polymerization and depolymerization at the plus (+) and minus (-) ends of actin filaments, respectively, and of F-actin retrograde flow. Repulsive and attractive cues influence growth cone morphology by regulating these processes. II) Guidance as a paradigm dynamic growth cone guidance: • Spinal commissural axons extend toward the floor plate at the base of the spinal cord, cross the floor plate, then on the contralateral side of the floor plate extend rostrally without any extendion back toward the floor plate.  Evidence that commissural axons are indeed attracted to the floor plate by a long-range attractant. Identification of floor plate attractant, Netrin: Is expressed in the floor plate and is similar to a portion of the ECM component Laminin (ECM cues: a form of short range guidance. Laminin in ECM are permissive to pathfinding (binds integrin receptors)). It may, therefore, be incorporated in the ECM following secretion. Netrin-1 mutant mice have severe defects in commissural axons extension toward the floor plate.  COS cells expressing netrin attract commissural axons. • Guidence cues are often bi-functional, acting as both attractants and repellents: Trochlear motor axons normally extend away from the CNS midline. They are repelled, not attracted, by netrin. • Netrin-1 leads to activation of RhoGTPases to promote axon extension, and utilizes specific receptors for attractive and repulsive guidance.  The netrins are secreted proteins that bind to transmembrane proteins of the DCC family. The composition of netrin receptor complexes dictates the growth cone response to netrin. Binding of netrin to DCC homodimers induces association of DCC cytoplasmic domains, which leads to growth cone attraction. The adapter protein Nck is involved in facilitating this attractive response. In contrast, binding of netrin to UNC-5-DCC heterodimers leads to growth cone repulsion. It is the cytoplasmic domains that encode attraction or repulsion. cAMP also can regulate netrin attraction: high cAMP promotes netrin attraction, low cAMP turns netrin into a repellant. • The floor plate is a transitional zone for commissural axon guidance responses: Slit (receptor complex that binds netrin) is a secreted cue expressed at the floor plate, and it signals repulsion thru Robo. • Receptor mediated silencing allows for growth cones to block netrin attraction. In combination with slit-mediated repulsion this gives commissurual axons the ability to not turn back toward the floor plate even though netrin is still to be found here.  Ligand gated interations between either netrin or slit and a Robo/DCC heteromultimer silences these guidance receptors. This silenceing is mediated by interactions between the cytosolic domains of these receptors. • Sensory afferent patterning: Ventral spinal cord secretes semaphorins, a signal inhibitory to NGF-responsive afferents (these terminate in dorsal spinal cord) but not to NT3- responsive afferents (these reach ventral motoneurons) III) Neuronal Regeneration: - CNS environment is not permissive to axonal growth/regeneration - CNS myelin (oligodendrocytes) contains Nogo which suppresses axon growth - Blocking Nogo could be key in axonal regeneration therapy - Peripheral nerve grafts can overcome CNS inhibition) - NgR is the Nogo receptor on axons; Nogo and NgR are associated with other proteins - In addition to Nogo, MAG and OMgp, two inhibitory components of CNS myelin also appear to use the NgR to signal repulsion. Further, the NGF receptor p75 may be the common signal transducer. Activation of p75 can lead to activation of Rho and as discussed above the promotion of inhibition. - MAG inhibition of DRG outgrowth is developmentally regulated: MAG acts as a repellent for older neurons, however until early postnatal time points MAG actually acts as an attractant. MAG, like several guidance cues, is therefore bifunctional. - Conditioning lesions in peripherery promotes central regeneration of DRG axons and neurons isolated from DRG following a peripheral lesion are not inhibited by MAG. Therefore, intrinsic state of pre-injured neurons somehow overcomes MAG inhibition. - Many cues are in fact bifunctional, and cyclin nucleotides appear able to modulate attractive and repulsive events – this can be demonstrated at the level of single growth cones in culture. MAG can be shifted from repulsion to attraction by elevation of cAMP levels or PKA activation. Remember that high cAMP levels also support netrin-mediated attraction. While the role of endogenous cyclic nucleotide signaling in mediating normal guidance events remains undetermined, these observations suggest clear strategies for overcoming MAG-mediated repulsion. These include raising cAMP levels as a way of overcoming CNS inhibition. In rat this appears to works. Elevation of camp prior to dorsal column lesion results in enhanced regeneration. Development IV: Cell Death During Development and Synapse Formation I. Function and mode of cell death in nervous system development - 4 functions of cell death: systems matching (a means to match neuron and target cell populations), error correction, removal of cells to generate sexual dimorphism in nervous system, removal of cells that have transient function. - Neuron death generally occurs during the time in which neurons innervate their target tissue due to lack of trophic support from target tissue. - Neurotrophic hypothesis: competition over limited supply of neurotrophic factor from target tissue leads to death of incorrectly targeted neurons - Many cells provide trophic support, but the major ones are target-derived - Glial cells, oligodendrocytes also die during development, perhaps requiring trophic support from axons II. Cell biology of apoptosis (general) - Apoptosis is ATP-dependent and is clean and controlled; necrosis is not - C. elegans worm is a classis system to study apoptosis; its genes are highly homologous to vertebrate apoptotic genes: Cytochrome C exits from mitochondia (pore activated by BH3/Bax; inactivated by Bcl-2)  binds to Apaf-1 adaptor  2 CytC/Apaf1 complexes bind to homophilic CARD domains  results in cleavage of procaspase-9 into active caspase 9  promotes apoptosis. III. Nerve Growth Factor (NGF) is the prototypical neurotrophic growth factor - There are many families of growth factors that affect neurons; NGF will be our focus - NGF functions at least in part as a target-derived neurotrophic growth factor for certain populations of peripheral neurons. Evidence for this comes from: 1) NGF rescues neurons that would otherwise die during development. 2) Transgenic mice overexpressing NGF in skin have an increased number of sympathetic neurons. 3) Administration of anti-NGF antibodies result in almost complete loss of sympathetic neurons and many neural crest-derived sensory neurons. 4) Pure cultures of sympathetic and sensory neurons are completely dependant of NGF for survival. 5) Death of sensory and sympathetic neurons following axotomy can be prevented by systemic administration of NGF. 6) NGF is expressed in target tissues of sympathetic and some sensory neurons. - The NGF receptor is a receptor tyrosine kinase (TrkA). • Signaling via Trk receptors: NGF induces dimerization and transphosphorlyation of TrkA on multiple tyrosine residues. Autophos on tyrosine residues has two functions: 1) it increases the catalytic activity of the receptor and 2) it provides docking sites for PID-and SH-2 domain containing proteins that interact with the phosphorylated receptor, thereby recruiting signal transducing molecules to inner surface of membrane. • NGF signaling (inhibits apoptosis): NGF binds to TrkA  activates Bcl-2  inhibits Apaf-1  caspase inactive  cell survival. - Expression of receptor subtypes and responsiveness to growth factors are restricted to particular populations of neuronal and non-neuronal cells IV. Synapse formation and elimination - Presynaptic diffn: not much is known - Postsynaptic diffn: in case of motoneurons, two methods of AChR clustering, translocation and transcriptional regulation of receptors: 1) nerve-induced clustering at synapse, regulated by agrin. The RTK MUSK mediates agrin signaling within muscle cells. 2) increased AChR txn by subsynaptic nuclei, regulated by neure
More Less

Related notes for 55-101

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.