7. Synapse formation lecture.pdf

Synapse Formation Two phases of synapse formation 1. Target selection: An axon must switch from a motile axon guidance mode, to a mode where it recognizes its target. Involves adhesive and cell-cell recognition mechanisms that are generally independent of activity. Drugs like tetrodotoxin generally...

Synapse Formation Two phases of synapse formation 1. Target selection: An axon must switch from a motile axon guidance mode, to a mode where it recognizes its target. Involves adhesive and cell-cell recognition mechanisms that are generally independent of activity. Drugs like tetrodotoxin generally do not disrupt this first phase of synapse formation. This phase of synapse formation generates a roughly appropriate distribution of innervation. 2. Address selection: Involves major remodeling of the initial coarse distribution of synaptic connections. Synaptic contacts may be expanded or retracted. Generally an activity-dependent process that can continue into adult life in some regions of the adult mammalian brain. Nature vs Nurture - The neurobiological underpinning of Nature vs Nurture is based on activity-dependent vs activity-independent synapse formation. - Activity-independent synapse formation is programmed genetically (Nature) - Activity-dependent synapse formation is dependent on experience (Nurture) Activity-Dependent Synaptic Modifications - In most systems, activity-independent steps lead to an excess of synapses that are pruned in an activity-dependent manner - Critical periods regulate the plasticity in several of these systems - We will discuss two systems to exemplify activity-dependent synapse refinement: visual system and neuromuscular junction. Adapted from Lichtman and Colman, Neuron (2000) Kandel et al. Principles of Neural Science Fig 27-4 Why establish more synapses than needed? - Not enough genes to specify the quantity and quality of connections in each neural circuit; the formation of extra synapses may allow precise patterns of connectivity to be fine tuned by epigenetic mechanisms according to their use. - Provides a way to ensure that all parts of a target (i.e. each muscle fiber) is innervated by at least one axon. - Provides a way to ensure that all axons capture an appropriate number of target cells. Projections of the Visual System Retinal Ganglion Cells (RGCs) project axons to: 1. Lateral Geniculate Nucleus; main pre-cortical terminus for input to the visual cortex. 2. Superior Colliculus; control of saccadic eye movements. 3. Pretectum; control of pupillary reflexes Kandel et al. Principles of Neural Science Fig 27-4 Lateral Geniculate Nucleus (LGN) - Main relay for RGC projections - In primates, including humans, the LGN contains six layers of neurons. - Each individual layer receives input from one eye only. Kandel et al. Principles of Neural Science Fig 27-6 Kandel et al. Principles of Neural Science Fig 27-7 Ocular Dominance Columns in the Cortex - Neurons in the visual cortex have a columnar organization - Ocular Dominance Columns represent an orderly arrangement of cells that receive inputs only from the left or right eye. - Layer four of the visual area V1 (first stage from the lateral geniculate nucleus) receives information from both eyes, but most neurons are selective for input from one eye. - First described by Hubel and Wiesel in the 1960s; electrophysiological recordings showed that sensory input from two different eyes stimulated different cortical neurons. Cells with similar eye preference were grouped in columns. Kandel et al. Principles of Neural Science Fig 27-17 Formation of Ocular Dominance Columns - Classic experiment by Hubel and Wiesel demonstrated experience-dependence of ocular dominance columns and critical period. - Closing one eye of a juvenile cat leads to almost all of V1 responding only to the open eye; this paradigm leads to no change in an adult cat. - If not opened again during the critical period, the closed eye never regains space. Hubel Wiesel Nobel Prize 1981 Kandel et al. Principles of Neural Science Fig 56-3 Formation of Ocular Dominance Columns Is it experience-dependent? - Injection of tritiated proline in one eye of cats at different stages of development leads to transsynaptic labeling of geniculocortical projections - At stages of development before eye-opening, there is a lack of segregation of inputs. - After eye opening, visualization of ocular dominance columns - The lack of patterning in these experiments before eye opening was important evidence for a “tabula rasa” on which experience would then imprint. *** Caveat: spread of tritiated proline in the young cat could have been great due to a lack of extracellular matrix. Kandel et al. Principles of Neural Science Fig 56-4 Formation of Ocular Dominance Columns Is it experience-dependent? Injection into LGN is monocular - Larry Katz decided to reexamine the formation of ocular dominance columns using modern labeling techniques by injecting anterograde tracers into the LGN (already segregated for monocular targeting). Coincident with the arrival of LGN axons, you can already see segregation. 1956-2005 Crowley and Katz, Science 2000 Formation of Ocular Dominance Columns Injection into LGN Is it activity-dependent? - Larry Katz decided to reexamine the formation of ocular dominance columns using modern labeling techniques by injecting anterograde tracers into the LGN (already segregated for monocular targeting). - The segregation occurs even when both eyes are enucleated (completely activityindependent) ctrl Enucleated P18 Enucleated P0 Crowley and Katz, Nat. Neurosci. 1999 *** Hence, formation of ocular dominance columns does not require activity The Critical Period and Eye Disease Treatments - Strabismus is an abnormality in which children cannot align their eyes properly or fuse the images from the two eyes. These children typically stop using one eye which leads to sensory deprivation to one eye. - In the 1970’s, strabismus often led to incurable visual impairment in one eye. - Because diagnosing and treating strabismus is more difficult in infants,doctors typically would delay treatment until the children were four or five, inadvertently increasing the degree of vision lost. - Studies on the critical period of visual system development led to ealier treatments, well before the age of four, when vision can be restored. www.eyesite.ca/7modules/Module1/ html/fr_Mod1Sec6.html Sorting of LGN is activity-dependent - The sorting of LGN into eye-specific layers occurs before eye-opening, but is blocked by TTX infusions into the eye. - Requires spontaneous activity in retinal ganglion cells; these result in waves of activity going through cells and thus nearest neighbours firing close together. - This gives an opportunity for Hebbs rule (i.e. neurons that assist in firing the postsynaptic cell should be strengthened) to lead to activity-dependent tuning without requiring visual input - Waves use both gap junctions and nicotinic signaling, but underlying mechanism is still somewhat unclear. Kandel et al. Principles of Neural Science Fig 56.9 Proposed model for activity-dependent fine-tuning of synaptic connections in the visual system - Axon from the left eye fires alone leading to a small depolarization of the target cell. This is not sufficient to activate NMDA receptors and the small amounts of neurotrophic factors available do not sustain the axon. - Axons from the right eye fire synchronously leading to a larger depolarization of the target cell. This causes the target cell to release increased amounts of neurotrophic factor which is taken up by the active presynaptic terminals from the right eye during endocytic retrieval of the plasma membrane. - The inactive axons from the left eye, which has not taken up neurotrophic factors, retracts. - Axon branches from the right eye, stimulated by the neurotrophic factor, sprout and occupy the vacated target sites Kandel et al. Principles of Neural Science Fig 56-12 Activity-dependent synapse remodeling Visual system: Ocular dominance columns: activity not required for formation of columns but appears to play a role in their maintenance during the critical period. The main role of visual experience during the critical period might be to reinforce and augment an already appropriately situated basic set of connections. Lateral Geniculate Nucleus: spontaneous activity but not experience is required for sorting of eye-specific projections in the LGN. Neuromuscular Junction (NMJ) - In the mature motor system, each fiber of a muscle is innervated by a single motor neuron. - Neuromuscular synapses are established before birth but the transition from multiple to single axonal innervation takes place in the first few postnatal weeks. - Is branch retraction an active process? Adapted from Lichtman and Colman, Neuron (2000) Kandel et al. Principles of Neural Science Fig 55-11 Elimination of synapses at the NMJ - Competition between axons (each fiber is an independent asynchronous competition; i.e. one neuron can win some and lose others) - Winner is not just based on synaptic space, often the axon starting with only 30% of space can eventually win; receptors may not normally go away as winning axon takes over space of losing axon. Lichtman and Colman, Neuron (2000) Walsh and Lichtman, Neuron (2003) Elimination of synapses at the NMJ Is it activity-dependent? - Complete block of activity leaves synapses intact - Imbalance in activity at a NMJ leads to elimination of synapses - Hence synapse elimination depends less on the presence of activity per se than on the differential activity by competing inputs (true competition). Kandel et al. Principles of Neural Science Fig 55-11 Elimination of synapses at the NMJ Is it activity-dependent? - Asynchronous activity within a NMJ triggers competition while synchronous activity slows down competition. If all the motor neurons innervating a single muscle fiber were firing at the same time, the muscle fiber would not be able to discriminate among inputs. no diferential activity/ advantage for any of 1. 2. 3. them Wyatt and Balice-Gordon, 2003 1. At time of birth, motor neuron action potential activity is temporally correlated. This may be as a result of prevalent GAP junctional coupling. 2. Several days after birth, correlated activity between neurons start to disappear. Loss of correlated activity may trigger synaptic loss at NMJs. 3. Weeks after birth, each muscle fiber is innervated by a single motor neuron and motor neuron activity is no longer correlated. Proposed Mechanism of synapse elimination at the NMJ Synapse factor Adapted from Lichtman and Colman, Neuron (2000) 1. Activation of postsynaptic cell by release of neurotransmitters generates intracellular signals in the postsynaptic cell. 2. At the site of receptor activation, a local signal (BDNF?) protects the active synaptic sites from the damaging effects of activity. 3. A destabilization signal (inter-synaptic) is generated affecting surrounding, asynchronously firing, synapses that do not have protection. 4. Effect on “Synapse factor” leads to disassembly of postsynaptic site and axon withdrawal What is the “Synapse factor”? Synaptotrophins or Synaptotoxins? Balancing competition at the NMJ - Motor pools are all around the same size (i.e. each motor neuron wins approximately the same number of competitions). - Competitions between two individual motor neurons are all the same; the winner is always the neuron with less connections already. - Synaptic strength appears to be spread out evenly between all synapses; the more synapses, the weaker each individual synapse. Summary Activity-dependent synapse remodeling Visual system: Ocular dominance columns: activity not required for formation of columns but appears to play a role in their maintenance during the critical period. Lateral Geniculate Nucleus: spontaneous activity but not experience is required for sorting of eye-specific projections in the LGN. Neuromuscular Junction: Asynchronous activity is required for synapse elimination during development. 1. During the activity-dependent phase of synaptic competition, what is the effect of applying the acetylcholine receptor blocker bungarotoxin to all the synapses. What would be the effect of applying an acetylcholine agonist to all the synapses. The elimination of synapses at the neuromuscular junction is dependent on the differential activity of synaptic inputs. If you apply bungarotoxin to all synapses, you block the activity in all synapses, thereby eliminating the difference in activity between synapses. Hence all synapses will remain intact. A similar phenomenon will be observed if an agonist of the acetylcholine receptor is added at the neuromuscular junction since all synapses will be activated and there will be loss of differential activity between synapses. Elimination of synapses at the NMJ Is it activity-dependent? - Complete block of activity leaves synapses intact - Imbalance in activity at a NMJ leads to elimination of synapses - Hence synapse elimination depends less on the presence of activity per se than on the differential activity by competing inputs (true competition). Kandel et al. Principles of Neural Science Fig 55-11 Long Answer Question (10 points): 3. You are given a mutant mouse with retinal ganglion cell axonal pathfinding defects that include the formation of an ectopic chiasm and wandering axons; however, Slit-1 and Slit-2 expression are found to be normal. A) Design and describe one in vivo experiment that you would perform on the mutant mouse strain to demonstrate that it has retinal ganglion cell axonal defects. B) Based on your knowledge of Slit signaling propose two molecules that could be disrupted in the mutant mouse. C) Design and describe one in vitro experiments that you would perform with this mutant mouse strain to test the involvement of Slit signaling in this guidance phenotype. A) An in vivo experiment would involve injecting a dye inside the retina of one eye. The dye would be transported along the axons and you would be able to visualize the optic chiasm in the mutant mouse and compare its morphology to the optic chiasm of a wild-type mouse. A) B Since the phenotype observed in this mouse is similar to the phenotype observed in Slit mutant mice, it is likely that Slit signaling is disrupted. One molecule which may be disrupted is Robo which acts as a receptor for Slit-mediated repulsion. Another possibility would be disruption of sr-GAP which is a GTPase activating protein that controls the activity of Rac GTPases downstream of the Robo receptor. A) C An in vitro experiment would involve explants of the retina from wild-type and knock-out animals and evaluate the responsiveness of retinal ganglion cell axons to a source of slit. You could put the retinal explants next to fibroblast-like cells that secreted Slit. You would expect axons from RGCs isolated from wild-type mice to be repelled by the Slit but not axons from RGCs isolated from mutant mice. Another option would be to isolate RGCs from wild-type and mutant mice and use the in vitro turning assay. You would evaluate the ability of RGC axons to turn away from slit molecules released from a pipette. The axons from wild-type RGCs would turn away but the axons from mutant RGCs would not be repelled Slits in RGC Axon Guidance - Slit-1 and Slit-2 are expressed at the optic chiasm - Ablation of Slit-1 and Slit-2 expression leads to axonal pathfinding defects that include the formation of an ectopic chiasm, and wandering axons. All figures from Plump et al. (2002) Neuron, 17:219-232. Slits and their Receptors - Three vertebrate Slits (1-2-3) - Secreted chemorepellents - Slits regulate several biological processes including migration, axon pathfinding, and axonal and dendritic branching - Robo family of transmembrane proteins are Slit receptors Huber et al. (2003) Ann. Rev. Neurosci. 26; 509-563 A direct assay for axon guidance was established in the laboratory of M.-M.Poo in 1997, using primary neurons from the spinal cord of embryonic Xenopus that were cultured individually in vitro.A micropipette loaded with a guidance cue is positioned 45 degrees to the direction of a growing axon and about 100 µm from its tip.Time-lapse microscopy allows one to study precisely the effect of a putative cue on the axon. It can establish roles of putative cues in attraction and repulsion, or their effects on motility.Typical results are shown in panel a. Guan K-L. and Rao Y. (2003) Signalling mechanisms mediating neuronal responses to guidance cues. Nat Rev Neurosci. 4(12):941-56.