Neurobiology 3

Nature

Lineage derived cues

Nurture

Environmentally-derived cues

Progenitor cells

Mitotic dividing cells, respond spatially or temporarilly variying cues that determine cell fate

Non-mitotic cells

Can no longer divide

Differentiating cells

Transcription factors from earlier phase induce the expression of transcription factors regulating specific aspects of differetiation (neurotransmitter, etc…)

Ontogeny

Developmental origin

sensory bristle 4 different types

2. Bristle cell

3. Socket shell

4. Sheath cell

SOP (sensory origin precurosor) cell

notch and numb determine which cell turns into what

Where does numb distribute to?

One daughter cell in SOP cell division

If you have no numb signaling..

-you are numb to life and senses because the hair cells won’t develop properly

-elevated socket and bristle cells= prevents neural development

If we get rid of notch

only sheathed and neuronal cells

there are many different transcription factor families that specify cell identity

interacts with other TFs to form hetereo or homo-dimers (by interacting with loops)(knitting) = subspecific cell fates

Mash 1 gone

no pan neuronal genes

Class I factors dominate

(minimal SHH)

Class II factors

(high SHH)(sonic hedgehog)

Interneuron types in spinal cord

all cell types generated through differential expression of transcription factors (don’t need to know types)

spinal cord

each layer has has their own transcription facotrs and gene encoding specific proteins

What genes do these TFs regulate?

gives us ideas whether the cells are excitatory or inhibitory, involved in both, and th phenotypes

Layer 1

intercortical axons and synapses

Layer 2/3

intercortical and callosal projections

L4

Neurons receiving thalamocortical projections

Layer 5/6

Layers of cerebral cortex

Different genes and TF’s in each layer

fezf2

“zinc finger” domains that bind DNA

Organoids

Cortical layers develop in organoids similar to fetal brains

Layer V CSMNs are missing in Fezf2 KO mice

CMSNs have large cell bodies- missing in the KO 

gain of function Fezf2 experiment

see expression in wrong region (l4) causes it to change the layer that it is within

which of the following belongs to the Zinc finger transcription factor family?

Fezf2

What genes does Fezf2 regulate?

RNA seek or microRNA libraries (tagged GFP on fezf2+ cells and then perform microarrays to determine genes-Ephb1 (epherin)

epherins are involved in..

axon guidance

In the absence of Fezf2 or ephb1…

CSMN axons project into the anterior commissure (AC) instead of projecting into the corticospinal tract (CST)

Fezf2 is highly expressed in which of the following layer during the development of the cortical plate?

Layer 5

what specifically entices a synapse to form?

we don’t know!

Weiss hypothesis

connections made during development are nonspecific and random

however, we commonly observe specificity wherein neurons make contacts with specfic partners and not others + different synapse types

synapse specificity

specific neurons make specfific types of synapses with different types of neurons

Roger Sperry

Optic nerve in frogs and connectivity. When optic nerve is turned and axons are regrown, the correct synapses are made.This formed a chemoaffinity hypothesis

Chemoaffinity “tag” hypothesis

1. Axons have different markers

2. Target cells have corresponding markers

3. Markers are the prouct of cellular differentiation

4. Axonal growth is actively directed by markers to establish specific connections

chemoaffinity: quantitative

Neurons being specified by molecular gradients of adhesive molecules. Based on these ideas, intensive efforts were made to isolate such gradient molecules
can help direct axons but cannot identify which neurons they synapse with

Chemoaffinity: proteins

Transmembrane cell adhesion molecules are uniquely suited to this role because they can interact with the surface of nearby neurons via extracellular. can act like a molecular glue (homophilic and heterophilic)

Neuroligin

Neurons grown over non-neuronal cells expressing neuroligin triggers presynaptic development

synapsin is a presynaptic marker

Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons

seen in SEM

Orchestra of Synaptic adhesion molecules

Multiple types not just neuroligin

How many genes in the human genome?

20,000 protein-coding genes > how can they encode for quadrillion proteins? → Alternative splicing

Alternative splicing

Different neurexins and their splice variants have dramatically different functions, suggesting that it is no longer possible to talk about neurexins as a homogeneous protein family
very few genes have this <1000 genes

Neuroligin triple knockouts have normal synapse numbers and morphology

neurologin triple knockouts have normal synapse numbers and morphology but altered functions

synaptic adhesion molecules

Unique combinations of cell adhesion molecules are necessary for forming synapses

Molecular gradients

Synapse number determined via gradients of cell recognition molecules acting through…

Glia

Astrocytes play a major role in synapse formation, whereas microglia contribute to synapse elimination
Glia also secrete powerful molecules how it is unclear

how are synapses established?

1. Synapses are established in a canonical process following partner choice

2. Synapses are established nonspecifically by default and partner choice is effected post hoc by eliminiation of noncognate synapses

3. Partner choice is part of diverse synapse establishment mechanisms mediated by distinct combinations of SAMs

Which of the following possibly contribute synapse formation?

Adhesive molecules, glia, specific neuron identity, all of the above!!

chemical synapses

-neurotransmitters, uni=directional, found acorss the brain in all brain regions, slower

electrical synapses

• Directly connects cytoplasms, bi-directional flow, transfer ions and small molcules, faster

• Connexins

Ephb1 KO and Fezf2 KO cause the same defects in axon guidance

In the absence of Fez or Eph CSMN axons project into the anterior commissure (AC) instead of projecting into the corticospinal tract (CST)

Development of a neuron

1. round

2. Neurite

3. opposite neurites

4. Multipolar stage

Acquisition of neuronal morphologies: neurons are polarized cells

Axons and dendrites

axons

One per cell

microtubule + end at terminal

major MAP is tau

Action potentials

May be myelinated

dendrites

Many per cell

Microtubules polarity is mixed

No myelinated or APs

Major MAP is MAP-2Dev

Development of neural polarity

Lamellopodia

Motor processes

Axonal outgrowth***

Dendritic outgrowth

Maturation

Severing the axon leads to a replaced axon being recruited

regrew after 24 hours/ the stump retracted slightly from the point of lesion, but was not reabsorbed

Disociated culture “stripe” essay

If neurons are put into PLL Laminin, the first neuron crossed to NG-CAM becomes that axon

-neurites respond to a different culture and not the specific protein presence for this experience

semaphorin

Repulsive molecule. Repels axo growth (like ephrin but an entirely independent signal)

three cytoskeletal systems

Microtubules, neurofilaments, and microfilament

Microtubule polymerization and depolymerization

-The cell can alter local conditions to favor polymerization or depolarization

MAPs can make MTs more stable (reduce probability of depolymerization) in the axons(?)

grow outwards

structural MAPs

-Microtubule-associated proteins

-MAP-2 and Tau in axons

-help stabilize

-Crosslinked microtubules add stability

Motor MAPs

kinesins and dyneins

Moving out and moving towards soma

what is labeled for in green?

Tau proteins → axon

Neurofilament

-polymers

-Smaller and less crosslinking, found only in neurons

-good for long distances

-highly enriched in AXONS and principal support. Increse the number increases the diameter of the axon and its conduction velocity

How much faster is axonal transport than simple diffusion?

300x faster

In axons, all MTs are oriented so hat the plus ends are away from the cell body

In dendrites, MTs are randomly oriented

Axon guidance mechanisms

Innate guidance of an axon refers to the inherent ability of a growing axon to regulate and reach its target. Growth cones as primary

Pioneer growth cone

come from non-neuronal (vascular, glial cells, etc..) accomplished by contact medium or contract chemoacttractant. Allow other axons to join it

Pioneer cell

TI cells. Lay path from developing limb to ganglion. Phillopedia and moves growth cones around guide cell until finding the second one. Distance is small and can reach it very quickly.
Guide cells = stepping stones

semaphorin is a…

repulsive cue + in a gradient

no csx1=

no boundary, crosses semaphorin line (?)

Growth cone guidance by fasciculation with earlier or pioneer axons

Connected by cadherins/cAMPS and follow path of pioneer axon
positive mediated guidance term
Pioneer axon attached to laminin molecules on extracellular matrix

what happens if we ablate the pioneer neurons?

harder to navigate where to go and go the wrong direction + make multiple axons (misrouting during development)

Elli was performing an experiment to understand the mechanism of a growth cone guidance in a chemoattractant model. She blocked TRPC channel for 10 minutes and what did she observe from this experiment?

Decrease in calcium signal, turning of growth cone towards an attractant prevented, neurite growth unaffected

contact mediated attraction

fasciculation occurs as the growth cone of a follower axon contacts the shaft of a pre-existing pioneer axon, adheres to it and uses it

-Defasciculation occurs through detachment

Fasll

-CAM

-growth cone guidance by fasciculation with earlier axons

-normal=normal fasciculation

-loss of function= defasciculation

-gain of function= refasciculation and altered fasciculation (combined pathways)

adhesivity guidance cue

Growing axons have preferences

-Growth cones elongated on the palladium-shadowed areas vs areas lacking Pd deposits depending on the relative adhesivity of the growth cones to the substrate

-in polyornithine-coated dishes they elongate for great distances on the pd-free areas

repulsive cues trigger cessation of growth cone motitilityy before restarting

when movement restarts it will be in a random direction

-if that movement is away from the repulsive cue it will continue

-otherwise, growth cones continues collapsing

excitatory synapses

-glutamate synapse

-Depolarizing

-Promote firing

-Glutamate, actylcholine, serotonin, histamine, catecholamines

Inhibitory synapses

-

- GABA, Glycine

active zone

area where in the presynaptic zone right before entering the synaptic cleft

PDZ domain proteins

-very small part of a protein that is found in many molecules
-Domains are named after the prototypical family members

-domains bind to consensus sites in many proteins

-found in multipile pre and post-synaptic scaffold proteins

PSD-95

-three PDZ zones, has three scaffolding PDZ domains (like beads)

Gephyrin

Gephyrin forms a hexogonal scaffold (add more)

What about PSD95 knockout?

Each MAGUK has some unique and overlapping roles in synaptic structure, function, and plasticity, particularly regarding anchoring glutamate receptors
-triple MAGUK knockdown reduces activity, area, and length of the PSD, and number of receptors

Mun18

Key regulator of vescile exocytosis
-interacts with different binding molecules
knockout abolishes neurotransmitter release, but does not prevent normal brain development or synapse formation

80% of excitatory synapses are on…

Dendritic spines (mammals)

Filopodium

-Transient, none have postsynaptic densities

Protospine

Semistable, 82% have postsynaptic densitities

Dendritic spine

100% have postsynaptic densities

Welcome packets

Mobile, prepared transport packets of “assembeled components” are captured at new contact sites

Presynaptic differentiation prcedes postsynaptic differentiation

stubby form

-Intermediate between thin and mushroom of the postsynaptic receptors

We are observing a phenotype where dendrites aren’t making synaptic contacts. What type of spine are we likely seeing on the dendrites?

Filopodium

synapses that are rarely used are targeted for elimination via synaptic pruning

Microglia proccesses dynamically interact with developing synapses and can detect local changes in neuronal activity

Autism spectrum disorders result in synaptic pruning loss and overexcitation

This overabundance of connections …(WRITE MORE)

Initial synapse formation is thought to be:

activity independent