BMS236 Building Nervous Systems Flashcards
What ways are the PNS classified?
How they connect to CNS - Cranial and spinal nerves Direction of propagation - Afferent - Efferent Motorneurone's target effectors - Somatic and autonomic (sympathetic and parasympathetic)
What has research shown us about fish brains?
There is a tube that carries nerves from distal parts of body to a central point
- Unconscious and mechanical brain
What has research shown us about reptilian brains?
Nerves sorted into specialised modules e.g.. light sensitive = vision
- Mechanical and unconscious brain
What has research shown us about mammalian brains?
- Hypothalamus - reaction to stimuli
- Thalamus - vision smell and hearing to be used together
- Limbic system - emotions but unconscious
- Amygdala and hippocampus - crude memory
What has research shown us about human brains?
- Enlargement of areas associated with thinking, planning and communicating
- Larger cortex pushing cerebellum to current position
- Skull bones pushed outwards forming flat forehead
What are Brodmann’s areas of the brain?
Systematic map of brain based upon cell types
- Broca’s area (44)
- Wernike’s area (22)
- Motor cortex (4)
- Visual cortex (14)
What are Broca’s and Wernike’s areas of the brain?
- Broca’s - Produces speech by controlling muscles to speak
- Wernicke’s - has grammatical rules for language
What is anatomical modularity?
Connecting modules that work together to complete tasks
Why are some parts of the brain hard wired to become allocated t a specific task?
Evolution has made it essential that humans have vision, the ability to move precisely and speech
Why can humans do things that have no evolutionary advantage e.g. play music?
- Maybe to attract opposite sex and pass on genes
How does a musicians brain differ?
increased size
- caused by neurones sprouting new connections to allow for complex movements involved in musical performance
- Shows plasticity
What is brain plasticity?
Ability to produce new and destroy old connections between neurones in response to physical demands
Why is the brain so powerful?
Brain has 10^11 neurones and each has 1000-10000 synapses meaning 10^14 connections in human brain
How do sponges show signs of an early nervous system?
Water flow needs to be regulated by myocytes which are specialised muscle like cells which respond to stretch
What are the characteristics of primordial nervous systems?
- Appearance of neurones
- First neurones probably sensorimotor cells
- Began to differentiate down a nerve pathway instead of a skin pathway
What are the characteristics of a hydra nervous system?
Derivation of different types of neurones from ectoderm
- Motor neurones which receive inputs from the sensory neurones
- Interneurons - lie between sensory and motor neurones
How is a worms nervous system more complex than in hydra?
- Gangliation
- Cephalization - formation of brain - worms have difference in anterior/prosterior
- Bilateral symmetry
- Fasiculation - nerves beginning to bundle
- entire nervous system was internal
Give an example of how the segmented worms nervous system evolved?
- Fusion of longitudinal nerve cords
- Brain evolved to regulate feeding - sense food and ability to grab it
Give an example of how the C. Elegans nervous system has evolved?
- Nervous system mapped
- Ventral dorsal ad lateral nerve cords
- Most neurons derived from one cell
- Neurones share lineage with hypodermis (skin) - evidence that a decision is made whether to become skin or nervous system
Give an example of how the drosophila nervous system has evolved?
- Optic lobes
- Beginning of proper brains
- Formation of neuroblasts
Give an example of how vertebrates nervous system has evolved?
- have a common body plan
- early nervous system is similar across all families
- Nervous system forms from ectoderm that would otherwise be skin
- Nervous system is dorsal instead of ventral - somewhere in evolution head turned around so ventral things became dorsal
Give the common features of xenopus (vertebrate) and insects nervous systems
- Neurogenic region next to ectoderm
- Neurogenic region migrates downwards
- Gastrulation
Give the different features of xenopus (vertebrate) and insects nervous systems
- Neural cells do not delaminate
- Neural cells stay as a layer called the neuroepithelium (neural plate)
What is cell differentiation?
Process by which cells become different from each other and acquire specialised properties
Governed by gene expression which can be changed in response to morphogens or transcription factors
What is BMP?
A protein that will cause a cascade that will cause the differentiation of cells into skin ectoderm
- It has been conserved through time and evolution
What is chordin?
An antagonist to BMP (inhibits BMP signalling)
How does the neurogenic region arise?
BMP signalling is inhibited by chordin
What is the homologue of BMP and Chordin in insects?
BMP = dpp Chordin = sog
So sog inhibits DPP causing the cells to become the neurogenic region
Where in the cell does the neurogenic region develop?
Chordin and BMP diffuse from opposite ends so where they meet is where the neural tube develops
What is the pathway for ectoderm differentiating into epidermis?
- BMP binds to BMP receptor and a secondary signal (Smad) is phosphorylated
- Smad can then up regulate transcription factors like Msx1, GATA1 which leads to epidermal differentiation
What is the pathway for ectoderm differentiating into neural tissue?
- BMP inhibited
- No Smad phosphorylated so different transcription factors are up regulated (xlpou2, soxd)
- Neural differentiation pathway
What is neurulation?
When the neural plate rolls up to form the neural tube
What is the Spemann organiser?
A specialised part of the mesoderm that expresses transcriptional factors (Gsc) expressing antagonists of BMP eg. chordin which diffuse into the ectoderm and bind with BMP
- Forming neural plate in ectoderm neighbouring the organiser
What decides the location of the organiser in the mesoderm?
Low levels of the nodal protein leads to ventral mesoderm
High levels gives the organiser
What happens to the organiser cells in the mesoderm?
- Differentiate into anterior endoderm, prechordal mesoderm and notochord
- It involutes, intercalates and undergoes convergent extension
- Forms the midline of the neural plate
Give some experimental evidence for neural induction?
- A donor organiser was grafted from a donor onto a host newt
- found that a ‘twinned’ embryo developed with a secondary neural axis
- Secondary neural tube was host derived showing it was induced from the ectoderm in response to signals from the organiser
- The prechordal mesoderm ad notochord where donor derived showing they’re formed from the organiser
How were BMP inhibitors discovered?
Extracted all mRNA from organiser cells and reverse transcribed them to cDNA. This was tested to look for a protein that would mimic the organisers ability to induce a secondary neural plate
Give experimental evidence for the fate of the organiser (Henson’s node)
Cut out the node and culture
It develops into a long rod shape which is the notochord and at the anterior end the prechordal mesoderm develops
What is the activation transformation model?
The idea that neural inducing molecules such as BMP inhibitors (Chordin) and ant antagonists only remain in the prechordal mesoderm (part of organiser that is involuted first) not all of it. the later part of the node expresses Wnt, FGF and retanoic acid which posterialises anterior tissue
When establishing an A P regional identity what is present on the anterior side?
BMP and Wnt antagonists
When establishing an A P regional identity what is present on the posterior side?
Want, FGF and retanoic acid - promise posteriorisation and growth
What is the key concept when establishing a regional pattern?
having 2 antagonist molecules at each end of a forming structure
What are the two models for the use of concentration gradients by morphogens?
- Alan turing reaction diffusion model
- Lewis Wolpert’s French flag model
What is the French flag model?
The idea that different levels of a morphogen concentration leads to a different cell fate
What is a morphogen?
A diffusible molecule that causes a particular response from a cell depending on its concentration gradient
What are hox genes?
- Control segmentation and specify A P
- Massively conserved through time
What controls the patterns of hox?
Retanoic acid gradient
- high concs lead to neck
- low concs to tail
What is the neural plate border?
A specialised border that develops at the neural - ectoderm boundary (edge of the neural plate)
What are the border cells required for?
- Neural cress formation
- Roof plate formation and dorsal neural tube differentiation
What is responsible for the formation of the peripheral nervous system?
The neural crest
How is the neural crest formed?
- Border is established between neural plate and surface ectoderm which expresses transcription factors such as msx - induce at intermediates levels of BMP signalling
- Wnts, FGFs act together with msx1 - these transcription factors characterise neural plate border cell
- Wnt signals act together wot NPB transcription factors to up regulate more transcription factors (c-Myc, Id, Snail) that characterise neural crest cells
- Neural crest cell transplantation factors up regulate a further set of genes that promote epithelial mesenchymal cell transition.
- Neural crest cells delaminate from the border and migrate
What causes the early border cells to be established?
Intermediate levels of BMP signalling triggering transcription factors (msx)
What transcription factors are known to give cells ‘stem like” behaviours: proliferation and multipotency and what are there roles?
( c - MYC, Id, Sox9)
Role in formation of neural crest cells
They activate genes that control proliferation
Why are neural crest cells referred to as the ‘4th germ layer’?
Because it gives rise to high number of different types of cell
- Peripheral nervous sytem
- Facial cartilage
- Schwann cells
- dentine of teeth
- Enteric nervous system
How are neural crest cells fate determined?
- Partially by how genes
- time of generation of neural crest cells
- Migratory pathway - encounter different signals
Do all neural plate border cells form neural crest cells?
No
What happens to neural plate border cells that don’t develop into neural crest cells?
A few are retained at the border and form roof plate cells - it is not clear why some cells are retained are and others aren’t
Why are roof plate cells important?
Important in the final step of neurulation and in dorsal neural tube patterning
How do roof plate cells lead to neural tube progenitors to acquire dorsal identities?
- Roof plate cells up regulated BMPs and Wnts
- Secreted and diffuse into dorsal tube
- Indice expression of transcription factors (Pax6, Pac7, Pax3, Lim1) that cause neural tube progenitors to acquire dorsal identities
How is it thought that BMP’s induce a different dorsal cell type?
- It was thought that they act as morphogens
- But recent work shows that roof plates may express many different BMP’s which induce a particular dorsal cell type
What cell types differentiate dorsally?
Roof plate cells, neural crest cells, different classes of dorsal sensory relay interneurones
What cell type secrete a morphogen inducing a ventral fate?
Norochord
Floor plate cells located at the ventral midline
How was the role of floor plate cells tested?
- Ectopically implanting a donor notochord which induced an ectopic floor plate and saw it induced ventral neurones such as motor neurones
What causes the formation of the floor plate?
The notochord
What gene is responsible for the production of the protein that is secreted from the floor plate?
Hedgehog gene
What mRNA is expressed in the notochord and floor plate?
shh mRNA
- Only gets upregulated when notochord formed
Why does shh act as a morphogen?
Made and secreted from floor plate diffuses into neighbouring cells and establishes a concentration gradient resulting in ventralisation due to the induction of expression of transcription factors
What happens if shh soaked beads are implanted into a cell?
Secondary neural plate and notochord
How does Shh act as positivity feedback mechanism?
A high concentration of shh protein induces floor plate which unregulated shh gene leading to more shh protein
What is the role of shh in the developing nervous system?
shh acts at early stage to confer a DV pattern of transcription factor on progenitor cells
What are the morphogens that pattern the DV axis?
The opposing gradients of BMP and shh - act antagonistically to each other
shh does not act at the same early stages of BMP
What are the key stages in ventralisation?
- Notochord secretes shh
- shh diffuses through spinal cord - conc gradient (morphogen)
- It induces different patterns of transcription factors
- High concs of shh cause floor plate cells to develop which occupy ventral midline of neural tube - activates shh
- shh mRNA in both notochord and floor plate
- shh diffuses into neighbouring cells causing ventral fate
Why has the investigation into dorsal and ventralisation helped in drug discovery?
Knowing how processes work and how the nervous system forms allows us to push embryonic stem cells towards a defined fate which can be used in new drugs
What are radial glia?
Neural stem cells
How are radial glia formed?
- During early development, some cells continue to span the width of the neural tube
- Their nuclei migrate back/fourth at different stages in mitotic cycle
- These are radial glia
How do radial glial cells become a proliferating progenitors daughter cell?
Neural stem cells maintain a luminal contact and over time cell bodies come to occupy ventricular zone (ependymal zone)
- depending on the plane of diffusion they can give rise to two radial glia and proliferating daughter progenitor
- Proliferating progenitors move into the adjacent mantle zone and differentiated cells moves away into the outer marginal zone
What happens in a proneural and a neurogenic mutant?
Wild type drosophila
- Have cells that have proneural genes so are able to come neurones
Proneural mutant
- No cells become neurones
Neurogenic mutant (notch-/-)
- But in a neurogenic mutant more neurones are formed
Shows Notch required to prevent a cell becoming a neurone - acts locally on immediately to adjacent cells
What is lateral inhibition?
Involves transmission of an inhibitory signal between a pair or cluster of cells to prevent cells that receive the signal from adopting a specific fate
When the central cell in the pro neural cluster inhibits the surrounding cells and stops from becoming neuroblast
How does the neurectoderm become a neuroblast and epidermis cells through lateral inhibition?
- Cells express both Notch and Delta equally
- One cell starts to express more delta
- Other cells increase in notch in order to receive the amount of delta
- The increase in notch causes the inhibition of delta in those cells
- The difference in notch and delta increases leading to a neuroblast (with high delta) and epidermis (with high notch)
=> Lateral inhibition
Are all cells in a proneural cluster capable of becoming a neuroblast?
Yes the proneural glosser is an equivalence group
Notch -/- or Delta -/- causes all cells to become neuroblast - must be competent
What is a proneural cluster?
Groups of cells in the neurectoderm
One cell in the cluster will become a neuroblast and the others will become epidermis
What happens to progenitors that don’t differentiate?
They have a certain shape
Exist as radial glia (neural stem cells)
Provide a pool of undifferentiated cells that are used to build yo the nervous system over time in embryogenesis
What is the highest level in the motor system hierarchy?
Primary motor cortex
What is the middle level in the motor system hierarchy?
Brainstem
What is the lowest level in the motor system hierarchy?
Spinal cord
What is the role of the highest level in the motor system hierarchy?
Projects directly to spinal cord via corticospinal tract
Regulates the motor tracts that originate in brainstem
What is the role of the middle level in the motor system hierarchy?
Lateral descending system controls distal limbs is important for goal directed movements of hand and arms
What is the role of the lowest level in the motor system hierarchy?
Contains neuronal circuits that mediate reflexes autonomous such as walking
What does monosynaptic mean?
One sensory neurone and one motor neurone
- Simplest reflex
What does polysynaptic mean?
Many neurones and interneurones
What happens if you artificially stimulate the Motor cortex, brainstem or spinal cord?
No big movements at most twitches
What is the overall role of the basal ganglia and cerebellum in motor control?
- Monitor commands going down to muscles to make sure they are appropriate for the situation the person is in
- If commands are inappropriate then they calculate correction signals which they send back up to motor cortex for approval
What is the role of the basal ganglia in motor control?
Acts as a feedback loop and sends info back to primary motor cortex
- Helps initiate and terminate movements and establish a normal level of muscle tone
What is the role of the cerebellum in motor control?
Mainly feedback to motor cortex but can also send its error correcting signals straight down to the brainstem
When was the motor cortex discovered?
1870
What happens when you stimulate the frontal lobe?
Movements
How was the primary motor cortex discovered?
Different areas of the brain were electrically stimulated and ‘Brodmann’s area’ found to be the area that elected the most movement with the lowest intensity
- Primary motor cortex
Which parts of the body have the largest representation of control and why?
Hands fingers face - for fine movement
What is the role of the upper motor neurones?
Carry motor commands down through the brain, brainstem and to the spinal cord
Involved in planning initiating and directing movements
Output to lower motor neurones via interneurones
Where do upper motor neurones originate from?
Ancient motor centres of the brain stem
- Vestibular nuclei
- Superior colliculus
- Recticular formation
What are the two types of upper motor neurone pathways?
Direct and indirect motor pathways
What is the direct motor pathway?
Input to lower motor neurones from axons extending directly from cerebral cortex
What is the indirect motor cortex?
Input to lower motor neurones from basal ganglia, cerebellum and cortex
What is the final common pathway?
Leads to muscle contraction from lower motor neurones
Where are the basal ganglia found?
Causate, putamen, substantia nigra, sub thalamic nuclei
What is Gilles de la Tourettes syndrome?
Causes twitching, face movements, uncontrolled swearing (rare) and unable to terminate movements
- Caused by problem with basal ganglia
How does the cerebellum connect to the cortex?
via the thalamus and brainstem
What is required for muscle control?
Excitation of muscle by alpha - motor neurones
Continuous info from each muscle at each isntant
- Muscle length
- Muscle tension and rate of change of muscle tension development
- Lots of info relayed from these receptors to spinal cord
What is the role of muscle spindles?
Send muscle length information to the spinal cord and cerebellum
What are intrafusial and extrafusial fibres?
Intrafusial are inside the spindle and extrafusial are outside the spindle
Don’t actually contribute to muscle tension
Purely sensory role
What are afferent fibres in the muscle spindles?
Wraps around equator of dynamic bag, static bag fibres and long and short chain fibres
What is the role of the gamma efferent motor neurons?
innervate bag fibres and short chain fibres innervate bag fibres and short chain fibres in order to change they length and mechanical properties
Which terminal endings are wrapped around intrafusail muscle fibres?
Group Ia
What is the role of group Ia terminal endings in muscle control?
Wrapped around the equator of both bag and nuclear chain intrafusial fibres
Pulling apart these cooked initiates an action potential on the axon
What are the different functions in group I and II terminal endings?
I - dynamic stretches - sensory endings relay information on the ‘dynamic’ phase of muscle stretch
ie. as stretch is occurring
II - Static stretches - sensory endings relay information on the static phase of muscle stretch
ie. its final length
Muscle spindles are only useful under tension, how is tension maintained?
- Extrafusal fibres shorten around the spindles - loses tension
- gamma motor neurones are coactivated with alpha motor neurones to ensure that the spindle shortens with the extrafusal fibres in order to maintain tension
Why does the brain need to fine tune its muscles?
Because there are 635 muscle - it would cause a sensory overload
How does the brain fine tune its muscle?
- Serotonin increases gamma motor neurone activity ( action potential firing) - this causes intrafusal fibres can be slightly stiffer - stiffer materials transmit stretches with a greater fidelity
- Noradrenaline decreases gamma motor neurone activity - causing intrafusal fibres to be more elastic and floppy - elastic materials don’t transmit stretches well
What is the tendon jerk reflex?
- Muscle stretched by hammer blow to its tendon
- Primary sensory endings are activated and sends action potentials along the limb to the spinal cord
- Muscle contracts in opposition to the stretch and the limb jerks as a consequence
What is jendrassiks manoeuvre?
(putting hands together and pull)
- causes excitation in upper segments of spinal cord
- Excitation spills over to rest of spinal cord - causes leg twitch to increase as more motor units are involved
What is the tonic vibration reflex?
- Group II afferents fire at about 50Hz when arm is straight
- Group III afferent fire at about 20Hz when fully flexed
- Vibrator applied to tendon drives the group II afferents in muscle so they discharge at 100Hz
- This makes the arm shorten as the brain must think it is really seethed as 50Hz is straight
What is the stretch reflex?
Polysynaptic reflex
- Spindle Ia makes excitatory connections on homonymous muscle (muscle the spindle is in) and are synergistic muscles (those that help the main muscle contraction)
- Ia’s also act through inhibitory interneurones that innervate antagonistic muscles
- When muscle is stretched the Ia firing rate increases
- This cause contraction f the homonymous muscle and relaxation of antagonistic muscle
- Reflex counteracts the stretch enhancing the springiness of the muscle
Where is the proprioreceptive info processed?
Spinal cord and somatosensory cortex
What is the central pathway in the somatosensory system known as?
Medial - lemniscal system
What are the steps in the dorsal medial lemniscal system?
- Central processes of dorsal root ganglia cells
synapse on neurones in gracile and cuneate nuclei
in lower medulla - Axons from these nuclei ascend in medial
lemniscus and synapse on neurones in ventral
posterior lateral nucleus of thalamus - Neurones of lateral nucleus send axons to
primary somatosensory cortex.
Where are the somatosensory cortices found?
Primary (s1)
The anterior parietal lobe and the posterior parietal cortex
Secondary (s11)
Deep within lateral sulcus
What would happen if there was a lesion in somatosensory cortices?
Proprioceptive defects (ability to discriminate size, texture and shape)
What is the morphology of neurones designed to detect?
Food
Predators
Mates
What is meant by the optic nerve being ‘information bottleneck’?
The optic nerve cat transmit all the information that is received by the retina as there is a limit to the thickness of it. So the retina also has to decide what is transmitted
What is the main function o the retina?
Image acquisition
What are the two main visual pathways?
Ventral stream - V1 cortex to inferior temporal cortex - Processes object identity Dorsal stream - V1 cortex to posterior parietal - Spatial location
What is the role of the pupil in the eye?
Regulate the amount of light
What is the role of the lens in the eye?
Focuses image on fovea
What is the fovea?
Fovea is part of the retina with the highest visual acuity
- Rest of the retina has Lowe acuity and contains primarily rods
What are muller cells?
Cells in the retina that light has to travel through before getting to the photoreceptors
What are horizontal and amacrine cells?
Interneurons between the layers of the retina
What are the three layers of the retina?
- Photoreceptors (rods and 3 types of cones)
- Bipolar cells
- Ganglion cells
What is the function of amacrine cells?
Inhibitory - inhibit bipolar cells and feed forward to ganglion cells - mostly gabaergic or gycinergic
Most diverse type of cell in retina (25 types) - showing importance of their function
Also have gap junctions between bipolar and ganglion cells
What is the function of the horizontal cells?
Inhibit photoreceptors nd feed forward to bipolar cells
What kind of light do rod and cone cells receive?
Rod - dim
Cone - active and bright - colour
What is a difference instructor between rod and cone cells?
rod - more disc and molecules because they need to be stimulated by smaller amounts of light
How does phototransduction occur?
In darkness
- Na+ and K+ channels open so the membrane is depolarised
- Activated by cGMP in the cytoplasm - keeping them open
In light
- ligand gated g coupled receptor is activated and the G protein breaks down into alpha and gamma subunits - activates phosphodiesterase - decreases cGMP in cytoplasm causing the Na+ K+ channel to close - hyper polarisation of photoreceptor
What is a ribbon synapse?
In retina
Constantly release glutamate - happens in darkness
Difference between on and off bipolar cells?
On cells - depolarise who light goes on them
Off cells - hyper polarise when light
Why do off bipolar cells hyper polarise in light?
Photoreceptors hyperpolarise in response to light so less glutamate and less activation of off bipolar cells
Why do on bipolar cells depolarise in response to light?
They don’t express ionotropic glutamate receptors (channel) like off cells
They express metabotropic glutamate receptors which are g coupled receptors
When metabotropic receptors are activated cGMP drops down in cytoplasm closing Na+ K+ channel - Hyper polarisation
If less glutamate, less activation of metabotropic channels then depolarisation
What is the difference between ionotropic and metabotropic glutamate receptors?
Ionotropic
- Are channels
Metabotropic
- G coupled
What is a receptive field in the retina?
An area of the retina which when illuminated activate a visual neurones
What is centre - surrounded organisation of the receptive field?
Stimulation of the centre of bipolar cells leads to depolarisation and the stimulation of the periphery is of opposite polarity (hyper polarises)
Why does stimulation of the centre and periphery of bipolar cells lead to responses of opposite polarities?
When stimulate centre of receptive field, that photoreceptor hyper polarises. It is directly synapsed with the bipolar cell so that also hyper polarises
When stimulate the periphery, those photoreceptors hyper polarise but they are synapses with horizontal cells - these hyper polarise which steps the hypopolarisating of photoreceptor which depolarises bipolar cells
How does the centre surround organisation of the ganglion cells differ to that of bipolar cells?
Illumination of the whole receptive field does not activate ganglion cells it appears that ganglion cells are designed to respond to differences in illumination that occur within the receptive field
What are the two classes of ganglion cells?
Paracellular - 80%
Magnocellular - 10%
Summerise the differences between paracellular and magnocellular ganglion cells?
Paracellular - Smaller with smaller dendrites - More densely packs - detect fine spatial details better - smaller receptive fields - Sustained response - Detects form/colour Magnocellular - Motion detection - High conduction velocity - More sensitive to light
How does the specific neuronal connectivity of the adult organism arise?
Two extreme hypothesis:
Weiss (1928) - Resonance theory - stochastic and diffuse neuronal outgrowth occurs to all targets followed by elimination of non functional connections
Sperry (1939) - Chemoaffinity hypothesis - Directed and specific outgrowth occurs through axons following “individual identification tags” carried by the “cells and fibers” of the embryo
What experiments evidence is there to prove Sperrys theories?
Sperry first did experiments in newts and then moved to frogs
- he cut the optic nerve and removed the temporal retina (allow just nasal axons to grow back)
- The axons grew back directly to the right place meaning sperry was right
How do we know that Weiss’s theory is incorrect by looking at embryos?
If Weiss were right, would expect to see random patterns of axons in embryos.
In fact see that patterns of axon outgrowth are highly organised, reproducible and stereotyped
Give some experimental evidence for axon guidance I chicks?
- Early stage embryo
- Cut and replace segment of neural tube before motor axons grow out
- Despite displacement, motor axons still found way to their correct target
- Suggests axon guidance
What are guidance cues?
Factors in the cells environment that axons use to find their correct target
What is Cajal’s growth cone?
The growing tip of the axon which Cajal proposed sensed cues in the environment
Why were insects used to identify locations of guidance cues?
- Relatively simple nervous systems
- Embryos easy to observe and manipulate
- In the large insects, individual cells could be ablated using lasers
Why are grasshoppers in particular good to use as a model organism when investigating guidance cues?
Detailed analysis resulted I the identification of almost every neuron in the embryonic nerve cord allowing a map of axon predictions to be made
How was the theory that guidance cues could be found on axons tested?
- Cells were ablated that were thought to be carrying potential cues
- this caused the axon to stop at the axon where it would usually turn as the cue was not present
- Shows evidence for growth cone and that axons can be directed in response to other axons
What is the labelled pathway hypothesis?
- Axons can selectively fasciculate with other axons
- Axon surface carry labels or cues
- Different growth cones express different sets of receptors for such cues
- Early axons (pioneers) form an axon scaffold on which later axons extend
- Established axon surfaces as one potential source of guidance cues
Give evidence for axon scaffold in vertebrate
- Ghosh and Shatz (1993)
Visual cortex - Ablated sybplate neurones from one area and innervation of lateral geniculate nucleus failed in that area - Evidence for scaffold
How do the pioneer axons find their way?
Growth cones appear to react at specific points
- For example, in the grasshopper embryo limb, the pioneer Ti1 (tibial1) growth cone makes a specific turn at the limb boundary, and then again as it approaches a specific cell, Cx1.
-Ablation of Cx1 causes the Ti1 growth cone to stall at the other side of the limb boundary.
- Neither Cx1 nor the limb boundary cells are distinguished by any obvious morphological features.
Cx1 and the other cells are sometimes referred to as ‘stepping stones’ or ‘guidepost cells’
Implies that there must be molecular differences in the environment
What are the three domains of the growth cone?
Central - Microtubules
Transitional - f actin
Peripheral - both
What are lamella in growth cones?
In peripheral domain
- The actin bundles are cross linked int a net
- Highly motile
What are filopodia in growth cones?
In peripheral domain
- The actin bundles are polarised to form larger bundles
- Highly motile
What are f actin treadmills in a resting growth cone?
Tubulin is dragged sporadically into the filopodia
Happens much more dramatically when the growth cone comes into contact with an attractive cue
What happens when a growth cone comes into contact with an attractive cue?
They reorganise
- F actin treadmilling slows and f actin accumulates
- This stabilises the filopodium and drags microtubules into the back of the filopodium
How does actin treadmilling control filopodial extension?
When encountered a promoting cue
- Molecular clutch is engaged and reward actin treadmilling slows - results in forward movement of the filopodium
- Actomyosin based actin tubules link pulls microtubules into the wake of extending filopodium
What is required for forward movement of the filopodium?
Attachment of the growth cone to a substrate is not enough to drive forward movement, need stimulus of cue to rearrange cytoskeleton.
How was it discovered that growth cones can be repelled as well as attracted?
Mixtures of neurone in culture were found to fasciculate only with their own kind
watching real growth cones showed that they were repulsed by each others axons and that contact led to their collapse
How does the collapse of a growth cone occur?
Destabilises f actin - the opposite effect of an attractive cue
What experiment was shown to cause collapse of the growth cone?
Treatment whir EphB1 protein
- Normally would have full growth cone collapse
- Would have localised collapse of filopodia contributing to growth cone reorganisation
What are semaphorins?
A family of inhibitory guidance cues
- Cause growth cones to turn
How were semaphorins discovered?
Biochemical purification of the factor from the retina responsible for causing the collapse of sensory axons
What firms can semaphorins come in?
Membrane bound
Secreted
What are the four forces?
Contact attraction
Contact repulsion
Chemoattraction
Chemorepulsion
What are permissive substrates?
Components that axons can attach and grow from
What is the relationship between strength of adhesion and the amount of axon growth?
No simple relationship
e.g. collagen is better than laminin for adhesion but laminin is better than collagen for outgrowth
Explain how laminin is permissive but not instructive
Laminin, a growth-promoting extracellular matrix protein, is localised in the optic nerve
Blockade of the receptors for laminin slows down the growth of retinal axons, but does not change their direction
Gradients of laminin in vitro do not direct axon growth.
What are permissive and non permissive substrates also known as?
Permissive substrates - Contact attractants
Non permissive factors - contact repellents
Axon growth is a balance between permissive and non permissive factors
What is the role of ephrins in axons guidance?
Non permissive factors used in early patterning and to guide axons - detected by receptor called ephs
What is the role of ephrins in the mammalian embryo?
Ephs and ephrins cause repulsion between cells
Early on, this helps to compartmentalise the embryo into discrete domains
- used t keep axons out of specific places
What is netrin?
A secreted protein similar to laminin which can associate with the extracellular matrix (in floor plate)
- Can turn commissural axons
What happens to a axons once they have encountered targets?
They reprogram
- Lose responsiveness to netrins once crossed midline
Why are lipophilic dyes useful when investigating axons?
- Hydrophobic end
- Can use them to trace axons, lineage and membrane trafficking
- Dil is an example
Give an example of how axons lose sensitivity to netrins during development?
Experiment
- On one side of the rodent hindbrain an ectopic floor plate is added so the commissural axons on that side sill have to cross it before the endogenous floor plate but other side doesn’t it
In the hindbrain
- axons exposed to ectopic floor plate before reaching midline turned
- Axons exposed to ectopic floor plate after crossing midline did not respond
How do commissural axons respond to repellants after crossing the floor plate?
They become sensitive to them
- Semaphotins and porteins called slit in the floor plate which repel them
- Also expressed in spinal cord - creating a channel for axons to grow though
How do growth cone sensitivities get reprogrammed?
As in the vertebrate floor plate, insect midline glial cells express diffusible attractants (netrins) and cell surface repellants (slits).
Levels of these determine
How do levels of slit receptor determine whether axons can cross the midline?
Roundabout gene, Robo, encodes a receptor for the inhibitory protein Slit. Robo protein is expressed at high levels on axons that don’t cross the midline. Commissural axons initially express low levels but high levels after they cross.
What gene is responsible for the inhibitory protein slit?
Roundabout gene, Robo
What happens in Robo mutants and Comm mutants?
In Robo mutants (no Robo protein made), Slit is no longer detected, so all axons go back and forth across the midline forming Roundabouts of axons
When Comm protein is missing (Comm mutant), Robo protein is expressed at hi levels in those cells that would normally cross the midline, and which now extend their axons longitudinally
What is Comm?
Comm is expressed only in those neurons that normally cross the midline and is switched off after they cross.
Is Robo or Comm dominant?
If Comm’s expression is forced in all neurons, Robo protein is lost everywhere resulting in a phenotype just like the Robo mutant. ie Comm controls Robo
How does Comm protein control Robo?
Comm encodes a “trafficking” protein that prevents Robo protein from reaching the cell surface so that the growth cone cannot receive Slit inhibitory signals before X-ing
Are comm and Robo expressed in vertebrates?
There is no comm homolog
Robo homolog
- Robo1 - expressed before and after crossing midline
- Rig1 - expressed only in pre crossing fibres and appears to block Robo1 signalling until the midline is crossed
What is controlling fasciculation?
How axons stay and get off the axons scaffold
- Involves homophillic binding by cell adhesion molecules
- e.g.. Fasciclin II
What are homophillic interactions?
Homophilic (like binds to like) interactions can bind two cell surfaces together
What do Fas II mutants show?
May defasciculated axons
What does over expression of Fas II cause?
Novel fasciculations
How cm axons get off the axon scaffold?
Interfere with adhesion by proteins that block
e.g. BEAT
Regulates FasII
How do axons select their targets?
- Discrete targets
- Topographic maps
Give an experiment used to show axons look for discrete targets
In Grasshopper and Drosophila, ablation of specific target muscles (by ablation of muscle precursor cells) leads to failure of relevant motor axons to leave main motor trunk at appropriate branch points
Suggests that axons are “looking” for specific “labels” on their targets
What happens if there is a loss of netrin in muscles?
the axons wander and do not make synapses (even though the muscle is there)
What happens if there is ectopic netrin in muscles?
leads to axons innervating the wrong muscles
What is Fas3?
The adhesion molecule Fas3 is expressed in specific muscles and in the motor axons that normally innervate them
What happens when here is ectopic expression of Fas3?
leads Fas3-expressing axons to innervate new targets
What was Sperrys theory regarding maintaining topology?
Neighbouring neurons send axons to neighbouring sites in their target to maintain the topology (order) in the target
Two possibilities how:
- Each axon has a unique label complementary to a unique label in the target
- That a co-ordinate system, encoded by gradients of signalling molecules, stamps a “latitude and longitude” onto cells of the target. This would be read by complementary gradients of receptors expressed in the retinal ganglion cells.
Why do we need colour?
Helps in object recognition
What are three types of cones?
Red
Green
Blue
Activate different types of bipolar cells
Where are green and blue cones located and why?
Green - top
Blue - bottom
Light from the top (blue sky) activates bottom of the retina and light from bottom (green grass) activates the top
What is the function of the superior colliculus?
Regulation of saccadic movements
Receives inputs from ganglion cells, auditory
system, somatosensory system
Integrates information from different sensory modalities
What is a retinotopic map?
organisation whereby neighboring cells in the retina feed information to neighboring places in their target structures (LGN, SC, cortex)
What type of organisation do command neurones have?
Retinotopic organisation
What is the role of the lateral geniculate nucleus?
LGN preprocesses the visual information
Give characteristics of lateral geniculate nucleus
6 layers
Monocular input
Layers alternate input from each eye
P and M ganglion cells remain segregated
Organised retinotopically
Ganglion cell axons make 1:1 connections with LGN projection neurons
What are the receptive fields of LGN like?
Receptive fields of LGN neurons are similar to the receptive fields of retinal ganglion cells
What Is the role of local interneurons in the LGN?
Important to process retina information
Outline an experiment into the understanding of object recognition
Subject looks at an image of a particular object.
Few neurons in higher cortical areas fire
Stimulation of the same neurons causes perception of the same object
What does object recognition allow us to do?
Recognise objects in an orientation not seen before
Recognise no matter what size
Effortless recognition of previously unseen objects
What is the hierarchical model of object recognition?
Detection of edges –> Detection of combination of edges and contours –> Detection of object parts —> Detection of objects from one point of view —> View invariant object detection –> categorisation
Each level is reached as the complexity of the stimulus increases
What is the reality of brain processing for object recognition?
Increase in complexity of responses of neurons along the ventral stream
Increase in the receptive field size of neurons along the ventral
What are the two key features of the cortical structure?
- Layering
- Columns (ocular dominance, orientation, direction and blobs)
How do the different layers in the cortex receive inputs?
Different layers receive inputs from different parts of the brain
Interneurons process this information - input between layers in the same cortex
What are the three types of columns in the cortex?
Ocular dominance column
Orientation (direction) columns
Blobs (colour)
Give a study into ocular dominance columns
Inject radioactive glucose in cortex and stimulate one eye with light
Shows that each column receives inputs from either ipsilateral or contralateral eye
What is a hypercolumn?
When all three types of columns come together
What are the three types of cells in the cortex?
Simple
Complex
Hypercomplex
Who was responsible for the discovering the cells in the cortex?
Hubel snd Weissel
Outline the response on simple cells to Hubel and Weissels experiment
When shined a light on centre of the receptive field - cells respond
Surrounding cells - respond on the dark - showing it has an inhibitory surround
- Change orientation of the bar and no response
- They concluded that ganglion cells were in a line down the middle
Outline the response on complex cells to Hubel and Weissels experiment
Differs from simple cell because responds to bar of light anywhere in the receptive field
Changes the orientation of the bar - cell doesn’t respond
Outline the response on hypercomplex to Hubel and Weissels experiment
Repsonds to bar of light anywhere in receptive field
If part of the bar is not over the receptive field then no response
If change the orientation of the part of the bar not over the receptive field then spikes again
Receptive fields downstream from V1 are complex so difficult to study, what methods are used?
Guessing
Computational models
How are computational models used to study receptive fields?
Record from a visual neuron Apply thousands of images Identify those that evoked spikes Average them or Build a computational model that predicts responses to all of the images
How is guessing used to study receptive fields?
Generate many images with defined geometrical properties (size, orientation, curvature, spatial frequency etc.)
Define orientation-, curvature-, frequency sensitivity
or
Make an educated guess of what should visual neuron respond to
What does probing the receptive fields in V2 show?
That cells in V2 cortex reposted to more complex stimuli then V1 - gets more complex
Give an experiment where face sensitive neurones in the temporal lobe are investigated?
Full face - cells respond
Partial face - not as many cells raspond
When characteristic features of a face are removed (e.g.. eyes, mouth, face shape) neurones don’t respond
- Neurones dont respond steadily - start to adapt
What is the Jennifer anniston neurone?
A specific neurone is fired when saw pictures of Jennifer anniston
- Could be at any scale and orientation - same response
Didn’t with other neurones
What are the main methods used to study vision?
Psychophysical methods and illusions
Lesions and other ways to silence neurons or parts of the brain
Anatomical studies and morphology (to study connections between neurons)
fMRI, electrophysiological recordings and imaging
Modeling and theoretical simulations
What model organisms do you use to study vision and what are the pros/cons of using them?
Humans - ethics, can get verbal feedback
Primates - ethics, similar model to humans
Lower mammals (cats, rodents) - Key organisation of the brain is the same
Lower vertebrates (zebrafish) - Brain different to humans, transparent embryo
Invertebrates (Drosophila) - not good to compare to humans
What is an example of the psychophysical experiment to study vision?
Investigate adaptation
- Illusions with colour
- Three pictures - George Bush and Barak Oboma
How do you investigate where vision is processed in the brain?
- Lesions - removing part of the brain and look at the damage or new behaviour
- Whole brain imaging techniques -
- Multi-electrode recordings in different brain areas
What types of lesions can be used to study vision?
Directed (animal models) - can’t get feedback from them
After strokes and other neurodegenerative diseases
What is fMRI?
Functional magnetic resonance imaging
How is fMRI used to study the brain?
Active neurones use a lot of oxygen and glucose - you can then see which parts of the brain uses these using functional magnetic resonance imaging
- Get a human subject and look at the images of the brain when looking at moving lights and stationary lights
- Can see cortex is much more active in movement showing its importance in perceiving motion
What methods in morphological studies can be used to study vision?
Fluorescence proteins
- Drive the expression of proteins in specific neurone subtypes so difference can be highlighted
Viruses
- Label one cell and pre synaptic neurones - can look at the circuitry - as the viruses will pass through the synapse as well
What is an advantage of using electrophysiological recordings to study vision?
Can be used in vivo
- Meaning it will have a sensory input as slices that have been cut out will have lost its innervation
Electrophysiological recordings can be used to study what kind of specimens?
In vivo
Slices
Cultured neurones (only for molecular mechanisms)
Give an example of a modern morphological study?
Enhancer traps
How do you validate an experiment?
Record activity of cell and neurones
Image the activity of a large population of neurones simultaneously
How do you validate experimentally by recording?
Patch clamp method
How do you validate experimentally by imaging?
GCaMP3
- Reports changes in neuronal calcium activity and therefore neuronal activity
- Brightness occurs when Intracellular Ca2+ is high
- By observing changes in brightness you can there foe feel when the neurone is active and how much is active
What are the advantages of validating experimentally by imaging?
Recording the activity of many neurons
Can record simultaneously the input and the output of a particular neuron (circuit reconstruction)
One can easily target particular neuronal types(e.g. Glu-ergic and GABA-ergic)
What are the disadvantages of validating experimentally by imaging?
Cant image as fast as electrophysiology
- Cant detect high frequency spikes
What is channelrhodopsin?
- Opens when yellow light
- Na+ enters
- Depolarisation of neurone occurs - spikes
What is halorhodopsin?
- Cl- channel
- Hyperpolarises with yellow light - doesn’t spike
- Depolarised with blue light - spikes
What is retinitis pigementosa?
Most abundant cause of blindness
- No treatments
- Caused by death of photoreceptors
How can you attempt to treat retinitis pigementosa and what are the problems?
Try to use an artificial retina
- But what part of the brain needs to be active as there are no photoreceptors
- What method would you use to activate it
Which parts of the brain would you stimulate to try and treat retinitis pigemtosa?
Retina (mostly for retinitis pigmentosa) Visual cortex (when the optic nerve didn’t develop or is destroyed)
Why is it important to activate as early (anatomically) as possible in the visual pathway when treating retinitis pigmentosa?
Retina performs complex computations
Different ganglion cells have different functions and project to different brain areas
Stimulation of retinal ganglion cells with simple stimuli is useless (e.g. motion sensitive RGC is active only when there is motion)
How could the you activate the retina cells in a patient with retinitis pigmentosa?
- Electrical stimulation
- Channel rhodopsin and halorhodopsin
What is the problem with activating an artificial retina?
Accessing bipolar cells or photoreceptors is difficult because ganglion layer is above so only accessible by electrode
What are neurotrophins?
A family of growth factors in the nervous system
Give an experiment conducted by Viktor Hamburger into neurotrophins?
Remove or add limb buds in chick embryo
- More/less innervation so more/less dorsal root ganglion neurones surviving
What is meant by having limbs having trophic effect?
Implies that limbs being innervated is giving an advantage for the organisms survival
What is the difference between tropism and trophism?
Trophism - relating to food or nutrition
Tropism - Meaning turning
What experiment into neurotrophins was conducted by Bueker in 1948?
He reasoned that fast growing muscle like cells might secrete survival factors (limb buds are fast growing)
- He therefore implanted sarcomas (tumours) into embryos
- It caused an increase in the size of dorsal root ganglion
- Provoked selective survival of non placodal sensory and sympathetic neurones
What followed Beukers experiment into neurotropins?
Demonstrated presence of diffusible growth factor
- Purified from snakes venom and mouse submaxillary gland
- Found antibodies to the purified growth factor that could block dorsal root ganglion growth in vivo and in vitro
What is the structure of the nerve growth factor (NGF)?
Activated from submaxillary gland Has subunits - alpha, beta, gamma Beta subunit - Dimer - Active component - alpha and gamma subunits only found in submax - storage
Give an experiment demonstrating how NGF’s work?
In a partition chamber
- DRG in centre - nerves grow out into outside chambers if NGF present in any chamber
- If NGF only present in centre compartment then no outgrow
- If NGF taken away from the axons that have outgrown then they die
Give evidence for NGFs being tropic and trophic?
Responsible for survival of axons - when taken away from experiment they die
Can also guide axons in vitro
What receptors for NGF’s are there?
TrkA - high affinity - it is a classic RTK (kinase) - they dimerise and phosphorylate - affects differentiation and growth P75 - NTR - low affinity - Promote cell death
Why is it difficult to identify another NGF in the nervous system?
Because there are low levels of them in the nervous system
What is the role of p75 in NGF’s?
Control whether or not cell death is occurring
Do all neurone types exhibit the same level of dependancy on neurotrophins?
No
- Placodal sensory ganglia e.g. nodose prefer BDNF over NT-3
- Crest- derived DRGs have subpopulations that respond to NGF, BDNF or NT3
- Sympathetics respond to NGF or NT3 but not BDNF
Is neurone dependancy on neurotrophins affected by time?
Yes neurones may have no dependancy
What is the role of NT3 in early development?
NT3 can support many neurone types early in development eg. on the way to targets
Arrival at the target often coincides with new expression of neurotropin by target
What do trigeminal neurones need during their development?
need BDNF and NT3 early
then NGF
then NGF or MSP
In animals that don’t have neurotophins, what do they have instead?
Dropsophilla and c.elegans - Other families Glia - derived neurotrophic factors e.g. GDNF supports midbrain dopaminergic neurons - Cytokines e.g. ciliary neurotrophic factor CNTF hepatocyte GF macrophage-stimulating protein (MSP) - Testosterone effects on motorneuron pools accounting for sex differences
Give an example of how neurotrophins effect dendritic morphology and connectivity?
Motor neurones innervating the triceps and pectoral muscles develop monosynaptic connections directly with PNS, whereas motor neurones innervating the cutaneous maximus (CM) and latissimus dorsi (LD) receive polysynaptic input from interneurons (IN).
Controlled by a neurotrophin secreted from CM and LD, which turns on the transcription factor (TF) Pea3 in the MNs.
What percentage of neurones are lost during development?
50%
Where does death occur in the developing nervous system?
Everywhere in the nervous system
Why can death occur even though tissue size is increasing?
Because neurones are refining
- Relationship between the size of the target and how many neurones are required
Give some examples of cell death in the developing nervous system
Somatosensory barrels - whiskers on rodents - In CNS cortex - refined Auditory system - refined - doesn't follow the correlation of size and neurones required - shows that other inputs are involved
How does cell death in the developing nervous system occur?
Apoptosis - programmed cell death
How do we know that apoptosis is an active process?
It is inhibited by actinomycin D or cycloheximide
How are cells which have undergone apoptosis cleaned up?
Macrophages
What is the intrinsic pathway (within a cell) for apoptosis?
DNA damage and p53 activation (tumour suppressor) —> Activation of mitochondria/ cytochrome c —-> activates initiator caspase 9 —> Effector cascade 3 —-> Apoptosis
What is the extrinsic pathway for apoptosis?
Death ligands —> activates death receptors —> activated initiator caspase 8 —> Effector cascade 3 —-> Apoptosis
What can pro and anti apoptotic signals do?
Enter the apoptosis pathway and activate apoptosis
Balance of the signals determine whether a cell lives or dies
Give an example an experiment into pro and anti apoptotic signals
C.elegans
- Knockout of Ced3 and Ced4
- Cells lived that weren’t suppose to
- Showing these pro apoptotic signals are required for cell death
What is the mammalian homologue for the anti apoptotic gene ced 9?
bcl-2
What was the idea of what promotes neurones survival?
That the target secretes a neurotrophic factor rewind for the presynaptic neurone to survive
How did NGF deprivation prove evidence for neurotrophins involvement in neurone survival in the developing nervous system?
Nerve growth factor (NGF - a type of neurotrophin)
- Deprived from sympathetic ganglion neurone in vitro
- SGNs have p75NTR, TRKA receptors
- Blocking NGF triggered apoptosis as the receptor for NGF is TRKA which is present
- Also shown in knockout mice in vivo
How can the withdrawal of NGF be rescued?
Adding anti-p75NTR blockers
- p75NTR is the other receptor present on the sympathetic nerve ganglion so even though TRKA isn’t activated by NGF, the other receptor stops the trigger of apoptosis
What is the role of neurotrophins in deciding an axons survival?
They provide pro and anti apoptotic signals
- An individual neurone needs both the correct neurotrophins and the absence of the wrong neurotrophins to survive
What are the roles of proneurotrophins in the control of cell death?
Pro NGF strongly activates p75NTR receptor which leads to apoptosis
- Levels of enzymes that cleave proneurotrophins are important - growth cones can release these enzymes
How is brain derived neurotrophic factor (BDNF) released?
ProBDNF is synthesis
- binds to carboxypeptide E
- Leads to secretion - triggered by depolarisation or ligand binding
- Cleaved to make proper form and release to axons
How are the axons in a neuromuscular junction refined?
Want to end with one axon innervating one muscle fibre but start with much more
- muscle fibre produces proBDNF which acts as a repellant
- the ‘winner’ axon converts proBDNF into BDNF allowing it to survive
- the ‘loser’ axon does not convert it - triggers retraction - if no other neuromuscular junction then apoptosis is triggered
How is synapse formation competitive?
Not all neurones make synapses - outcompeted
Synapse formation can dictate neuronal or target surivial
What is need to make a functional synapse?
Correct receptor being expressed
Synapse at the correct location
Correct part of the membrane to differentiate unto synapse
Receptors to make the target the tissue
The correct number of synapses made this can vary from 1 to 10000 per neuron
Outline synaptogensis at a neuromuscular junction
Motor axon differentiates to motor nerve terminal at contact point
Contact point is dictated by gap junctions made by Schwann cells - redistricts where the junction can occur
Muscle forms the post - synaptic apparatus
What morphological changes occur in synaptogensis in the spherical bushy cell (SBC)?
- Small vesicles at presynaptic membrane
- Narrow cleft between pre and post synaptic membranes - fill with special proteins
- Post synaptic membrane apparels thickened
What are the problems with the experiment conducting testing the morphological changes in synaptogensis in the spherical bushy cell (SBC)?
It was in vivo - so cant see what happens at the very early stages
What morphological changes occur when a growth cone twins into a pre synapse?
- Filopodia retraction - tight junction formation
- Membrane and cell gycloproteins are added
- Presynaptic vesicles, dense ECM and receptors accumulate at the cleft
When does synaptogensis occur?
When axons reach targets - highly variable
Give an example of when synaptogensis occurs?
Cat visual cortex
- Synapse density increased for one month after birth
What dictates where synaptic sites are?
Site availability may be restricted
- Astrocytes may cover cell body but dendrites are free
Post synaptic cells may have pre-prepared sites
How do synapse stick together?
Adhesion proteins
- Immunoglobulin domains that stick them together
How does receptor clustering occur in synaptogensis?
e.g.
Initially ACh receptors are present at moderate levels on myotubule surface
Innervated by AChRs cluster in post synaptic membrane
The clustering involves both redistribution of AChR proteins and localised synaptic synthesis of AChRs
Aggregation of neurotransmitter receptors is the principle feature of synaptogensis
What other receptors cluster during in synaptogensis?
Glycine, GABA, Glutamate
Give an experiment studying what evokes clustering of receptors in synaptogensis
Denatured and destroyed muscle to break neuromuscular junctions
- MMuscles regenerate and clustering occurs again
- Must be something in the basal lamina causing this
- The proteoglycan Agrin was discovered
What protein is responsible for receptor clustering in synaptogensis?
Agrin
Where is Agrin made?
Made in nerves and moves to basal lamia
What is the mechanism of action for Agrin?
Z+ agrin is released from the axon - has a high affinity for MuSK (a kinase)
- Agrin binds to MuSK protein and signals Raspyn
- Raspy clusters which have Ach receptors - end up with lots of Act receptors clustered
What occurs in MuSK knockout mice?
They are insensitive to agrin
How does the shape of the post synaptic apparatus change after maturation?
Junction changes from a simple oval plaque to a pretzel like set of branches
How does the topography of the post synaptic apparatus change after maturation?
Junctional membrane changes from a flat sheet to invaginated surface with gutters and folds - receptors cluster at the top
How does the extracellular membrane of the post synaptic apparatus change after maturation?
Changes to basal lamina over the ACh rich
membrane and cytoskeleton under it
How does the channel function of the post synaptic apparatus change after maturation?
Composition of AchR subunit changes
How does the molecular partitioning of the post synaptic apparatus change after maturation?
Ion and ligand gated channels segregate into discrete alternating domains
How does presynaptic division occur?
The growth cone has the receptors and the cell the ligands
- Growth cone - frizzled receptors
- Granule cells - express Wnt7a
Contact recruits vesicles and synaptic proteins to form presynaptic site
Explain climbing fibre loss as an example of refinement
Initially interact with lots of cells which is refined
Ina. mature cerebellum there is 1 climbing fibre per purine cell
In development, typically up to 4 climbing fibres per day
How does refinement occur in synaptogensis?
Synapse refinement is required to ensure the right number of synapses and that they are focused
Connection focusing
- Immature inputs are not segregated
- eg. Reorganise so cortex receives info from different parts
Transmitter choice
- Choice may be due to environment
- Not all hard wired in
How do excitatory synapses use activity to build a synapse?
Acquisition of presynaptic release machinery and postsynaptic NMDA receptors
If no additional input, they remain silent because they lack sufficient AMPA receptors
Activity recruits AMPA to activate synaptic activity
What are the types of memory?
Declarative (facts/events) and non declarative (unconsciously)
Long and short term
What neurotransmitters are present in presynaptic neurone for memory?
Excitatory - glutamate - leads to depolarisation
Inhibitory - GABA - hyperpolarisation
How can the sensitivity of the a synapse involved in memory be regulated?
- Regulated of voltage gated Ca2+ channel - activation released neurotransmitters
- Position of Ca2+ channels - closer it is to the vesicle containing neurotransmitters, the less depolarisation required for its release
What is the role of syntax, SNAP25 and synaptobrevin in memory in simple organisms?
They interact with each other and drive the synaptic vesicle to its release - releasing neurotransmitters
What is the role of synaptotagmin in memory synapses?
Calcium sensor
- without it the channel wouldn’t be calcium dependant
- Some require different concentration of calcium to activate it - change its sensitivity
What are the three types of vesicles in the presynaptic bouton?
Readily releasable poll - close to the membrane
Proximal pool - substitute readily
Reserve pool - resting pool
How can the types of vesicles available in the presynaptic vesicle lead to a type of plasticity?
The speed of release for readily releasable is quick but speed of conversion of proximal pool to readily releasable is slower - there will be a tie when no vesicles are ready to be released - lose sensitivity
What are the three types of glutamate receptors involved in post synaptic density in memory synapses?
NMDA receptor
AMPAR - Non NMDA receptor
mGlut receptor
What is the mechanism of action of mGlut receptors?
Bound to G protein - activation activates adenylate cyclase - phospholipase C - involved in increasing Ca2+ in cytoplasm
What is the mechanism of action of AMPAR receptors?
Na+ channel
- Bind glutamate - Na+ influx - depolarisation
What is the mechanism of action of NMDAR receptors?
Selective for Ca2+
- Has a Mg2+ bound to the pore region - needs to depolarise membrane to move it - just binding of glutamate is not sufficient
What are the advantages of studying memory in invertebrates?
Large neurones
Circuit complexity
Temperature- dependance
Genetically mapped
What are the two forms of simple memory?
Habituation - Responds less after repeat stimuli
Sensitisation - Nervous system responds to stimulus more and more
Give some examples of habituation in humans?
Habituation of eye blink reflex
Habituation of relative non harmful stimulus - e.g. living on a busy road
What Is the gill and siphon reflex?
Reflex in aplysia - snails
2 organs - Responsible for withdrawal reflex requires 2 neurones - sensory neurone activated with touching the siphon - glutamate - motor neurone contraction to remove
How was the cellular basis of habituation researched in aplysia and the gill siphon reflex?
Test which neurone becomes desensitised by recording from each neurone
- From sensory neurone - same response overtime - terminals are not desensitised
- Record from motor neurone - same
- Record from both and you can see response getting smaller with repeated stimulus
- Concluded that it is the synapse that desensitises
Why does the response get smaller in habituation?
Synapse desensitises
- Less than 50% of neurotransmitters are released
How can sensitisation lead to increased gill withdrawal reflex?
Addition of painful stimulus just after
- Sensory neurone activated by touching siphon
- L29 neurone stimulated by a shock
- L29 activation leads to the release of serotonin - activates receptors and G proteins - adenylate cyclase converts ATP and CAMP - activates protein kinase A which inactivated K+ channels in synapse
- Prolongs depolarisation - more vesicles released during one depolarisation
- Bigger contraction of muscle
- Sensitisation
How can aplysia show pavlovian conditioning?
Weak siphon touch paired with strong shock
- Paired stimulus - withdrawal reflex is very large - lasts long
- Timing is critical
What is the simple bear model?
Ca2+ influx due to depolarisation
Serotonin release
What was the key message shown from the Lymnaea - Kemenes 2006 research paper?
In addition to synaptic plasticity, other mechanisms can directly involve the synapse
What was the experimental model for the Lymnaea - Kemenes 2006 research paper?
- Single trail associative learning
- Present attractive feeding stimulus stimulus (sucrose) - snails will eat it because its sweet
- Also a neutral stimulus present (amyl acetate)
- Pair them
- Then snail shows feeding behaviour to amyl acetate alone - association
What is the role of serotonergic cerebral giant cells?
Serotonergic cerebral giant cells contact many cell types and permit feeding but not involved in feeding behaviour - if it fires feeding is possible if It doesn’t its not
How does association of the two stimulus occur in the Lymnaea - Kemenes 2006 research paper?
24 hours after the trial the resting potential of the serotonegic cerebral giant cells had raised by 10mv
- Only change seen
- The depolarisation lasted for weeks
- Depolarisation occurs after the behavioural change
What were the conclusions of the Lymnaea - Kemenes 2006 research paper?
Depolarisation needed for long term memory but not short term
Long term memory at least partially encoded at a site distant to the neurones
How could we test that depolarisation is necessary and sufficient for memory?
Necessary: Hyperpolarise neurones and train the animals
Sufficient: Depolarise neurones on untrained animals
Results showed it was sufficient
What are the three types of neurones found in the hippocampus?
Dentate gyrus
CA1
CA3
What synapse in the hippocampus was studied for long term potentiation?
CA1 and CA3 synapse
What does tetanic stimulation of CA3 neurone in a CA1 CA3 synapse lead to?
High frequency stimulation - causes summation of EPSP’s
Causes long term potentiation
What is the main input to the hippocampus?
The entorhinal cortex
What neurone produces the longest lasting potentiation in the hippocampus after stimulation?
CA3
How is associative memory formed?
Two pathways converging on the same target can both be strengthened if they fire together as long as membrane is depolarised
Is tetanic stimulation required for long term potentiation?
No stimulus just needs to depolarise the membrane
Does long term potentiation occur pre or post synaptically?
Evidence suggests post
What are the two glutamate receptors involved in long term potentiation?
AMPA - depolarise post synaptic neurone
NMDA - Selective for Ca2+
Give the main characteristic of on NMDA receptor?
Mg2+ ion binds to the pore of the receptor and blocks the channel from glutamate
In order to remove glutamate, the membrane of the post synaptic neurone must be depolarised
This is a key mechanism for long term potentiation
Give the mechanism of removal of the Mg2+ block on NMDA receptors?
Tetanic stimulation —> glutamate release —> AMPA receptor activation —> Depolarisation —> Mg2+ removal —> NMDA activation
What are the two stages of long term potentiation?
Early - Does not require protein synthesis
Late - requires protein synthesis
Give the mechanism of early phase long term potentiation
NMDAR mediated
- Ca2+ released and activated calmodulin kinase II - CaM
- NMDA receptor is opened and autophosphorylated so that it remains open even after Ca2+ is gone
- Prolongs the activity of calmodulin kinase II
Give an experiment investigating how phosphorylation of NMDA receptors enhance AMPA currents
Apply glutamate for short term and wash it out
Trigger long term potentiation
Apply glutamate again
Saw that AMPA currents increases
What is the advantage and disadvantage of using inhibitors to research enzymes involved in long term potentiation?
- They allow the animal to develop properly first and then add them
- Not 100% specific - usually inactivate something else as well
What is the advantage and disadvantage of using knockout mice to research enzymes involved in long term potentiation?
Specific
Don’t develop properly as effected many processes
What are the two ways to increase the activity of AMPA receptors?
- Change the open probability of the AMPA receptors - open more
- May involve amplification - increased number of AMPA receptors on the post synaptic site
When does late phase long term potentiation occur?
1 hour after stimulation
What is the role of cAMP signalling in late phase long term potentiation?
Important in the regulation of gene expression
- Activates protein CREB-2 (calcium response element) - blocks expression of gene
- CREB1- Replaces CREB 2 and activates gene expression and is phosphorylated by protein kinase A
What is the key mechanism of late phase long term potentiation?
Glutamate activates AMPA
Depolarises post synaptic membrane
Removes Mg2+ block
Ca2+ moves in through NDMA
Activates calcium calmodulin
Activates adenylate cyclase and calcium calmodulin kinase
Activates protein kinase A - gene expression —> proteins go to synapse
Calmodulin kinase 2 phosphorylates AMPA receptors –> increased numbers due to exocytosis
How would you test if long term potentiation is memory?
Behavioural test
- Animal model had to learn something
- Then inhibit LTP by inhibiting NDMA receptor
- Check if animals memorise less
- Inject inhibitor in hippocampus
- Could also over express something required in LTP and see if animals learn better
What are the problems with studying long term potentiation?
There are different types of synapses - some don’t use NMDA receptors, some don’t use CamK11
- LTP varies in non hippocampal areas
Give an experiment linking LTP to memory
Hidden platform in water for mice - First time they look for it, second time they know where it is
Use knockout mice of CaMKII or NMDA receptors - cant find it as quick
Is long term potentiation sufficient for memory formation?
No
It is only necessary for some memory formation
What is long term depression?
Active evoked long-lasting reduction in synaptic efficacy
What are the two main types of long term depression?
Depotentiation - Removal of previous potentiation - Reversal of long term potentiation Long term depression de novo - No previous potentiation
What are the general mechanisms for long term depression?
- Often requires NMDA receptors but not always
- Often evoked by low frequency stimulation but not always
- Often requires Ca2+ influx and activation of serine, threonine phosphotases
- Often involves glutamate but also diffusible transmitters eg. 5-HT
Explain the connections in the cerebellum
The cerebellum receives in pt from two fibres
- Mossy fibres - synapse with granula cells
- climbing fibres which synapse with one purkinje cell
Why is there a strong depolarisation in the cerebellum?
Because there are many synapses to one purkinje cell
- Main output of cerebellum is purkinje cell
Explain what happens when you stimulate a climbing fibre and parallel fire together?
If stimulate just parallel - ESPS
If stimulate both - the amplitude of ESPS in purkinje cells is much smaller - Long term depression
What is the Albus Marr model?
Climbing fibre input indicates a motor error and therefore weakens the parallel fibres so synapse between parallel fibre and purkinje cell is weakened
What receptors are involved in cerebellar long term depression mechanisms?
metabotropic glutamate receptors and voltage gated Ca2+ channels are involved
What is the mechanism of cerebellar long term depression?
Climbing fibres activated - release glutamate depolarisation of purkinje cells - activation of post synaptic Ca2+ cells - Ca2+ influx
Parallel fibres activated - GPCR cascade activated phospholipase C - DAG - activates protein kinase C
The two pathways interact with each other - increases protein kinase C more
Protein kinase C then phosphorylates AMPA receptor on a different side to the LTP
Leads to endocytosis of AMPA and reduction in AMPA receptor current
How is hippocampal long term depression thought to occur?
If the synapse of the cell is active but the rest of the cell is not depolarised then it becomes weakened
What decides whether long term depression or long term potentiation is going to occur in the hippocampus?
The degree of NMDA receptor activation
If activated a small amount then LTD and if more than LTP
- because LTD has mainly phosphatsaes which are triggered by NMDA receptors response to a small increase in Ca2+
- LTP has mainly kinases - large increases of Ca2+ lead to more kinases to increase AMPA efficacy
Give an experiment investigating whether long term potentiation and depression are memory?
On group of marmosets are in enriched environments and another not
- Looked at morphology of neurones
- Counted numbers and density of post synaptic boutons
- Increased number of dendritic spines
- Not only change strength of synapses but change shape and number of them too
- So memory is not just LTD and LTP but it is involved
What is the difference between anterograde and retrograde amnesia?
Retrograde - previous memories not intact
Anterograde - inability to form new memories
What is needed to transmit signals?
Transmitters eg. glutamate or GABA Receptors ECM - 'glues' synapse other neurones Glial cells - control the extracellular environment eg.ion concentrations
Why is a negative membrane potential required? for action potential?
So that Na+ channels can open when membrane potential increases and to keep them closed when needed
What is required for a resting membrane potential to be generated?
Pumps/transporters - to regulate ionic gradients
Na+, Ca2+ channels - depolarisation
K+ channels - to terminate action potential
How can the complexity of neurones excitability vary?
By changing what channels and transporters are present in the membrane of the neurone
How can the resting potential of neurones change during development?
By changing the channels present in the membrane
Also by glial cells
- Astrocytes increase causing extracellular [K+]to decrease from 35mM to 3mM
Does the ability for a neurone to produce an action potential occur at the same time during development?
No action potentials are seen early as well as neurones. develop at different stages
What is input resistance?
The measure of the ability of the cells for current to flow across the membrane - can be changed due to channels being inserted into the membrane
What is the membrane time constant?
How quickly the neurone can respond to a cell
What does the membrane time constant depend on?
Resistance and the capacity (ability to store charge) of the neurone
Why does the membrane time constant of neurones decrease during development?
Because neurones get bigger
What occurs to input resistance and membrane time constant when the resting membrane potential becomes more negative?
They both decrease
What are the two principle routes for action potential development?
Long Ca2+ dependant then short Na+ dependant action potentials
Short Na+ dependant
What are Rohon-Beard cells?
They are a transit population of mechanosensory neurones found in the spinal cord
What are the properties of an early action potential in a Rohon-Beard cell?
Looks like a cardiac action potential because it is Ca2+ driven
- Calcium is let in and the channels don’t shut quickly
- Not many K+ channels present to terminate so action potential appears elongated
What are the properties of an action potential that has started to mature in a Rohon-Beard cell?
Delay rectifier K+ channels are inserted into the membrane - open after a delay after activation
So after Ca2+ open, K+ channels ope after a delay so action potential is still a bit elongated
What are the properties of an action potential in a mature Rohon-Beard cell?
Normal action potential as Na+ channels have been inserted
More sensitive
Fast firing
- Occurs due to Na+ channels will snap shut quickly after it depolarises and also allows K+ channels to open to allow it to repolarise
What are delayed rectifiers?
Channel that allows current through one way
A delayed rectifier is a channel that opens some time after its voltage threshold is reached
Outward delayed K+ rectifiers allow positive charge out of neurones after an action potential
Appearance of such channels shortens the action potential
Apart from the addition of channels into the membrane, how else is complexity of excitability achieved?
Mouse cortex
- Different spiking activity as neurones develop
- fast spiking cells increase Kv3.1 expression the mature allowing to depolarise quicker
Purkinje cells bursting pattern
- Regulated by KCa channels increased expression
- Opens in response to influx of Ca2+
- Blocking these K channels stops the mature bursting pattern
What is the role of T current calcium channels?
Give the action potentials in early development
What is the role of N current calcium channels?
found in axon terminals ad control neurotransmitter release
What is the role of L current calcium channels?
Controls transcription
What are the spontaneous Ca2+ waves caused by calcium channel used fro in development?
Process growth
Involved in differentiation
What changes occur in receptors in neuronal development?
Neuronal development
- Populations
- Subunits
- Localisation
- Efficacy
How can some GABA be excitatory in developing neurones?
There is high intracellular [Cl-] in immature neurones
- Cl- channels on membrane have changed meaning that it pumps Cl- out instead of in
- GABA still activates these receptors meaning it has an excitatory role
Why is regeneration in the mature nervous system important?
Traumatic injuries in PNS, spinal cord and brain
Stroke
Loss of neurone in disease eg. Alzheimers
What is regeneration in the newt like?
Can regenerate whole limb with or without the presence of a nerve
What is regeneration in the xenopus like?
Tail regeneration is dependant on BMP signalling
Has a critical window of response
- At 47 hours of development - ail will not regrow
What is regeneration in the starfish like?
Needs the radial nerve to be intact for limb to regrow
How are nerve injuries classified?
Neuropraxia - Mildest - No physical peak but block of conduction Axonotmesis - Sectioning of axon but preservation of nerve itself Neurotmesis - Severe - Whole nerve is sectioned
How does the position of the nerve sectioning affect the likely head for cell survival?
If it is close to the cell soma - likely for cell death
Away from cell soma - cells might survive but will undergo changes such as reorganisation and re expression of the immature features eg. tubulins
What is Wallerian degeneration?
Discovered in 1840 and is the first state of regeneration in mature nervous system
- Part of the damaged axon furthest away from the soma (distal end) degenerated by macrophages trying to remove dying Schwann cells
- The part closest to the soma remains the same
- Occurs quite quickly so a new one can be made
What happens if a muscle becomes denerved?
Muscle atrophy
- AChR start to change to a immature form
- Muscle specific kinases increase
- If external electrical input is added it my help to prevent atrophy
How does regeneration of nerves that have minor damage happen in the PNS?
Wallarian regeneration
Mitosis of Schwann cells
- To cover area to help axon grow
- Also form bands of bunger to guide the new axon
Sprouting may also occur
- Another nerve tries to innervated the muscle to prevent loss of function
How do compression and cut injuries affect nerves differently?
Compression - Basal lamina damaged and ECM unaffected Cut - Disrupts both basal lamina and ECM - May not regrow accurately
What happens when the spinal cord is damaged?
Sprouting may be attempted but the fibres cannot find where they need to be so - failed regeneration
Formation of cysts and glial scars
Outline an experiment showing how regeneration in the spinal cord is difficult due to its environment?
Damaged axons from CNS where put in the environment of the PNS
They were able to regenerate unlike in the CNS
Suggesting environmental factor and not intrinsic
Give evidence for myelin inhibiting CNS regeneration
CNS neurones were trying to avoid oligodendrocytes
Removing myelin/oligodendrocytes improved regeneration
If you auto-immunise a micr to myelin proteins - enhances regeneration
What is present in CNS myelin which inhibits regeneration?
NOGO -a (protein)
What types of NOGO are there?
NOGO - a - large and donut in oligodendrocytes
NOGO b - Many cells
NOGO - c - muscle
Give evidence for the role of NOGO- a in inhibiting regeneration in the CNS
NOGO a is not found in the fish and salamanders or in human PNS which can regenerate
How does NOGO-a block regeneration in the CNS?
Works on P75 receptors interfering with the roles of neurotrophins which support the development of new fibres
What objections are there to NOGO a being responsible for the inhibition of regeneration in the CNS?
Knockout mice showed variable results
No correlation between NOGO receptor level and regenerative capacity
Transplanted hippocampal neurones grow axons
Much myelin is removed by macrophages after damage
How are scarred glia though to inhibit regeneration in the CNS?
When astrocytes are damaged they change in other to contain the inflammation
This causes a scar which fibres can not grow through
How are sectioning of nerves in both CNS and PNS surgically treated?
Gap in nerve that is too big
Scaffold tube inserted which connected the nerve and allows fibres to grow inside it
- Can be synthetic or biological
- If synthetic it wold have to be filled with ECM
What diseases have transplanted foetal cells been used to treat?
Parkinsons and Huntingtons
- Little success
What research into regeneration of nerves currently undergoing?
Transplanted foetal cells
Transplanted human embryonic stem cells
Transplant umbilical cells
Transplant autologous NS cells
Give evidence to suggest that neurogenesis can occur in the adult brain normally
Canaries
- In their high vocal centre, new neurones are formed as the canary learns to sing
- Suggests that neurogenesis can occur in mature animals
Mammals
- In dentate gyrus (in hippocampus) and olfactory bulb, neurogenesis can occur naturally
If there are parts of the adult brain that can undergo neurogenesis naturally, what is a new idea that can be researched for regeneration in the adult nervous system?
if there are stem cells capable of neurogenesis then they could be used for spontaneous regeneration under the right conditions
What are olfactory ensheathing cells?
Supporting cells
- Support and encourage regeneration
When studying commissural axons what is the problem with knocking out Shh and how can it be overcome?
Without Shh, mouse wouldn’t live long enough to reach the stage where commissural axons cross the midline
Cre recombinase can be used to knockout shh just from the tissue being studied so that the rest of the development is normal
How do BMPs and Shh guide commissural axons?
BMPs are released from roof plate and BMP is a repellant
Shh is released from floor plate and Shh is an attractant
