Lecture 20- Axon guidance III

Question 1

How do dendrites differ from axons?

Answer 1

1. Only one axon but many dendrites
2. Axon is responsible for the output of a neuron; dendrite receive synaptic inputs
3. Different cytoskeletal components: axons contain Tau1 and dendrites contain MAP2 (different microtubule associated proteins= handy in study of the axons and dendrites)

Question 2

Do neurons have many different morphologies?

Answer 2

• yes
• most of the morphological differences in neurons are in the shape and direction of dendrites, can project in different places, layers

Question 3

What is special about amacrine cells?

Answer 3

-amacrine cells do not have axons, only dendrites= these use dendrites both for inputs and outputs -there are 16 types of these, differ in the dendritic morphology

Question 4

What is the functional significance of different dendritic morphologies?

Answer 4

-length and branching limits the number and type of inputs a neuron can receive -dendrite morphology influences how synaptic signals decay as they propagate towards the soma -the rate of signal decay depends on the diameter and branching of the dendrites = that is why it is critical for dendrites to develop properly

Question 5

What is a neurite?

Answer 5

-any process emanating from the cell body of a neuron that is not obviously an axon or a dendrite. Commonly used in developing neurons before the processes are specified or in injured neurons -most commonly used in cultured neurons as cannot tell if dendrite or an axon

Question 6

Which neurons are used in evidence for how neurites become an axon vs dendrite?

Answer 6

-hippocampal neurons

Question 7

What are the stages in formation

Answer 7

• they go through a number of stages
• initially symmetrical, have lamellipodia and filopodia
• then some filopodia extend and become neurites and at the end a growth cone, still symmetrical
• critical stage 3= one of the neurites takes off and grows quickly, the rest slow slowly
• 4 grow more
• 5- even more -starts as neurites and suddenly one of those grows really fast

Question 8

What is the evidence for how neurites become an axon vs a dendrite?

Answer 8

1. Evidence from cultured hippocampal neurons: a) Par polarity complex (intrinsic cue) b) extracellular cues 2. In vivo evidence

Question 9

What is the Par3/Par6/aPKC protein complex crucial needed for?

Answer 9

-crucial for A-P polarity of C- elegans at the single cell embryo stage

Question 10

What is the mPar6?

Answer 10

-mPar6 contains a Cdc42/Rac interactive binding (CRIB) domain that associates with the active (GTP-bound) form of Cdc42 and Rac1 -also binds aPKC

Question 11

What is the mPar3?

Answer 11

-mPar3 interacts with mPar6 and aPKC

Question 12

What does the function of the Par3/Par6/aPKC protein complex depends on?

Answer 12

• depends critically on its polarized sub-cellular distribution
• loss of any one member of the complex causes mislocation of the other 2 and results in defects in cell polarity
• if complex present in one part of the cell and not other, when division = different fate

Question 13

What is the evidence that the Par complex is involved in the formation of an axon by cultured hippocampal neurons?

Answer 13

-mPar3 localization becomes polarized at stage 3 -only present in the axon at stage 3 -ectopic overexpression of Par3 results in multiple long Tau 1 processes (instead of just one) -if block aPKC (part of the Par complex) blocks specification of axons

Question 14

How does the Par polarity complex influence axon specification?

Answer 14

-Affecting actin and/or microtubule dynamics -Local application of cytochalasin-D (inhibitor of actin polymerization onto a dendrite induces it to become an axon -polarized actin filament instability determines

Question 15

What can the hippocampal neurons grow on?

Answer 15

1. pily-L-lysine+ laminin 2. poly-L-lysine+NgCAM -substrates -when hippocampal neurons are grown on striped substrates, axons form predominantly on one susbstrate -axons are always the neurites that are grown on one substrate -so one thing that can regulate which neurite becomes the axon is the extracellular matrix

Question 16

Is there competition between neurites?

Answer 16

• there appears to be some sort of competition between the neurites
• when encounter laminin, there is a time when rate of growth is declines= like tug of war and one wins and takes off really fast
• suggests there is competition between the neurites
• when two or more processes contact LN at approximately the same time, axon formation is delayed
• A-E phase micrographs photographically lightened in regions corresponding to the LN stripes

Question 17

Do all neurons select an axon from several neurites?

Answer 17

-no -some neurons extend a single process that becomes an axon, and dendrites appear later (in vivo evidence)

Question 18

How does an axon develop in retinal ganglion cells?

Answer 18

-retinal ganglion cells do not have multipolar stage= no multiple neurons -apical= always dendrite -basal process always the axon

Question 19

How does an axon develop in the cerebral cortex?

Answer 19

-in cereberal cortex do have multipolar stage - the leading process becomes dendrite -the lateral process becomes the axon

Question 20

How do dendrites grow?

Answer 20

-very dynamic -new branches generated that then extend or retract -constant remodeling

Question 21

What are neurons surrounded by in vivo?

Answer 21

-surrounded by a heterogenous environment (extracellular matrices, secreted factors, various cell types) -these microenvironmental cues are finely coordinated and orchestrate intracellular signaling pathways (in vivo evidence)

Question 22

What are the 3 things controlling dendrite growth?

Answer 22

1. extrinsic cues (neurotrophic factors:NT-3, BDNF, NGF; BMPs) 2. Intrinsic cues (regulators of actin dynamics eg. Rho family of GTPases; regulators of microtubule dynamics: par complex but not proven in vivo) 3. cell-cell interactions: if process grows out and encouters another= can affect fate of the neurite

Question 23

Are axon growth and dendrite growth related?

Answer 23

-many common molecules involved but -might be some specific extracellular cues and intracellular pathways involved in dendrite growth and branching -retinal ganglion cells seem to switch irreversibly from an axon to a dendrite growth cone -this is the example -specification of axon may be separated in time from specification of dendrite

Question 24

When do retinal ganglion cells grow axons and dendrites?

Answer 24

-seem to switch irreversibly from an axon to a dendrite growth mode -in dendrites the opposite of axons they grow after birth -axons grow before birth

Question 25

Do extrinsic factors have the same effect on all neurons?

Answer 25

-no -different effects of neurotrophic factors on different neurons -response of different neurons to different neurotrophic factors differs -e.g. cortical neurons layer 4 BDNF makes them the worng shape, layer 6 grow well with BDNF treatment

Question 26

What are the factors that influence the development of dendrites in pyramidal cells?

Answer 26

• multiple factors in the microenvironment, including cell-cell interactions -auto or paracrine action of neurotrophins
• neuron neuron interaction via TAG1
• gradient of TGF beta (regulates the way the axon will grow)

Question 27

What is tiling?

Answer 27

-complete but non-redundant innervation of a receptive area by dendrites of the same types of neuron -once they touch they don’t cross each other -tiling ensures that every part of the field is covered, usually only once by each type of neuron

Question 28

What is the mechanism of tiling?

Answer 28

-lesion of a patch of retinal ganglion cells results in neighbouring ganglion cells growing towards the area -suggests like repels like by contact mediated dendritic interaction or signaling via short range diffusible molecule -mechanism are likely to be neuron class specific -but recent evidence suggests that dendrite substrate interactions can also play a role by regulating the space in which they interact

Question 29

What is the role of Flamingo in dendritic fields in peripheral neurons in Drosophila?

Answer 29

-flamingo is an adhesion molecule related to cadherins -dendritic fields of some peripheral neurons overlap in flamingo mutants

Question 30

What is the role of integrins in Drosophila sensory neurons?

Answer 30

-da neurons tile the larval body wall -their dendrites grow in a 2D space on the ECM(extra-cellular matrix) secreted by the epidermis -removinh neuronal integrins or blocking epidermal laminin production causes dendrites to grow into the epidermis (instead of laying flat -once they didn’t go flat= lot of overlap)

Question 31

What is the summary of tiling?

Answer 31

-arise from contact-dependent repulsion and/or short range diffusible repulsive signals (not yet identified= the signals) -but for these mechanism to work, dendrites must encounter each other reliably: sometimes in a 2D space -thus substrate interactions that confine dendrites to particular locations are also essential

Question 32

What is the role of afferent input in dendrite growth?

Answer 32

-afferent input can influence dendritic maturation -most active phase of dendrite growth often correlates with the development of afferent innervation -exposure to an enriched environment or training on a motor learning task increases dendritic growth and branching of pyramidal neurons in the cerebral cortex

Question 33

What does the input regulates in Purkinje cells?

Answer 33

-input regulates development of Purkinje cell dendrites in the cerebellum -purkinje cell dendrites grow into the area occupied by parallel dibres to receive their inputs -disruption of inputs (by lesioning the granule cells) results in abnormal dendrites on the Purkinje cells

Question 34

What was the experiment that showed that activity regulates dendritic growth and morphology?

Answer 34

• rat littermates reared in 3 environments:
  a) individually in lab cages (IC)
  b) in pair in cased (SC)
  c) in a complex environment (EC)

Question 35

What is the effect of calcium on dendrite growth?

Answer 35

-calcium influx influences dendrite growth -when had neurons with more calcium = more dendrite growth -when you block the voltage sensitive Ca2+ channels that reduced the dendrite growth and have fewer dendrites

Question 36

What do developing neurons show?

Answer 36

-spontaneous Ca2+ transients

Question 37

What effects do CAM kinases have on dendrites?

Answer 37

-different CAM kinases have different effects -this is connected to the levels of Calcium (activated by that) -the kinases involved in the calcium response are specific -e.g. CaMK IV ca makes them grow, CaMKII ca doesn’t

Question 38

What does Ca2+ influx cause?

Answer 38

What does Ca2+ influx through voltage sensitive Ca2+ channels leads to activation of calclium/calmodulin dependent protein kinase IV (CaMKIV) -this is the mechanism that makes the calcium dependent effect happen

Question 39

Summary?

Answer 39

-dendrite development and growth is influences by intracellular signalling pathways (e.g. Par complex) cell adhesion molecules, interactions with substrates, extrinsic diffusible molecules, interactions with substrates, extrinsic diffusible factors (e.g. neurotrophic factors) and activity