Cellular neuroscience Flashcards
What does the nissl stain show and why?
Stains neurons NOT glia
Nissl binds to -ve charge of mRNA found mainly in soma of neurons
What does the golgi stain show and why?
Silver-chrome rxn
Randomly stains a few neurons
Can visualise entire neuron
What are the pros and cons of the nissl stain?
PROS
Stains majority of neurons
Good for looking at gross structure
CONS
Weakly stains glia
Can only see soma clearly
What are the pros and cons of the golgi stain?
PROS
Can visualise entire neurons
Easy to determine type of neuron due to this
CONS
Only a subset of neurons take up the stain
What are microtubules made of?
Hollow tubes of tubulin
Made of alpha and beta dimers
Dimers are added on at +ve end
What are neurofilaments made of?
Made of light, medium and heavy filaments and proteins
What are microfilaments made of?
Mainly made of actin and monomeric G-actin
What are the functions of microtubules?
They are used for motor proteins to carry their vesicles and proteins down the axon
Overall for movement of cellular components
What is rescue factor?
Stabilise and destabilises microtubules
What occurs at the +ve end of the microtubule?
Dimers are added and microtubule lengthens
What occurs at the -ve end of the microtubule?
Gamma-tubulin binds to GT complex and forms the gamma-tubulin ring complex which binds -ve end and is anchored to centrosome
What is kinesin?
Kinesin is a motor protein that walks vesicles toward the synapse
What is anterograde transport?
Done by kinesin - goes towards synapse
What is retrograde transport?
Done by dynein - goes back to soma with leftover stuff
What is dynein?
Dynein is a motor protein that walks vesicles toward the soma and away from synapse
How do motor proteins actually walk?
Uses ATP for every step
Phosphate broken everytime foot swings
What is the function of neurofilaments?
Predominant component of cytoskeleton
Huge mechanical strength
Most stable part of cytoskeleton
Very important in axonal integrity and diameter
What is the function of microfilaments?
Keep organelles in place and help cell movement
How does actin endocytosis work?
F-actin forms a scaffold and transports lyosomes filled w/ endocytosed proteins and retrograde transports them to microtubule highway
How does actin exocytosis work?
Myosin transfers vesicles along actin filaments until they reach synapse and are exocytosed
What is the central dogma of biology?
DNA –> RNA –> Protein
transcript. translate
Why do neurons need to make so many proteins?
Are highly dynamic
Receptors, vesicles etc
How does CaMKII phosphorylate AMPA receptors?
Ca2+ influx activates CaMKII
Phosphorylated tails of AMPA-bound TARP bind to PSD-95, anchors receptor to membrane
Phosphorylated AMPA tails cause a conf. change in the receptor allowing more Na+ into cell
What is direct IHC?
Using primary antibody to bind
Often immunofluorescence
What is indirect IHC?
Using secondary antibody to bind to primary antibody to allow visualisation
What are the advantages and disadvantages of direct IHC?
Simple
Fewer off-target effects
Expensive
Lower sensitivity
Antibody must be made specific to target
What are the advantages and disadvantages of indirect IHC?
Cheaper
Known and repeatable expression patterns
High sensitivity
More complex
Potential off target binding
What does MAP2 show?
Immunofluorescence
Shows blue, only in soma and dendritic tree
Binds to mRNA
What does ankG show?
Shows red
Only in initial segment of axon
How can we determine if a population is expression an RNA for a protein of interest? (FISH)
Fluorescence in situ hybridisation
Must know sequence for protein
Make complementary RNA sequence
Attach a fluorescent protein to manufactured sequence
If cell is expressing target RNA, fluorescence occurs
How can you identify the newly synthesised protein?
Add puromyocin to new protein
Make antibody against the new protein
If antibody for protein and puromyocin bind then fluorescence occurs
How are proteins made at the synapse?
DNA transcribed to mRNA and is either translated in soma or mRNA binds to a protein and translation is repressed
mRNA docks with kinesin
Synaptic activation causes mRNA to be unrepressed
Translated at synapse
What is the correlation of AD and microtubules?
Tau stabilises microtubules
Tau becomes phosphorylated and dissociates from microtubule
Causes neurofibrillary tangles
What is amyloidogenic process?
Cleavage by beta-secretase and then gamma-secretase
Causes amyloid beta
What are key differences between glial cells and neurons?
Glial:neuron = 50:1
Glia non-excitable
Glia undergo mitosis, neurons dont
Incl. oligodendrocytes, ependymal, schwann, microglia, astrocytes
What are some advantages of myelination?
Decreases energy consumption
Allows for rapid propagation down the axon
Which allows for predatory movement and long term integrity
What is contained in the juxtaparanode?
VG K+ channels
Is most lateral from node of ranvierW
What is contained in the paranode?
Has anchoring proteins
Immediately lateral to node of ranvier
What is contained in the node?
Node of ranvier
Contains VG Na+ channels
How is the node anchored to the axon?
Neurofascin-155, contactin, caspr
What is the process of protein synthesis in oligodendrocytes? (OG)
mRNA is transported down microtubules to the paranode
They are then translated locally
What is a key difference between OG and schwann cells?
OG myelinate multiple axons
Schwann cells myelinate one segment of an axon
What are the proteins of the juxtaparanode and internode?
MBP and PLP
Present only in CNS
Bind together to maintain lamellar structure of myelin sheath
What are monocarboxylate transporters (MCT)?
Extracellular membrane channels that trasnport lactate and pyruvate
MCT1,2,4 in CNS
What does the MCT-1 do?
Present in the OG plasma membrane
What does the MCT-2 do?
Present in axon plasma membrane, underneath the myelin
Transports lactate into axon
How do OG convert glucose to lactate?
Glucose enters via GLUT-1
Glycolysis turns it to pyruvate
Pyruvate –> Lactate via LDH
MCT-2 takes up lactate into axon
Lactate turned back to pyruvate and metabolised into ATP
How does the OG/SC know the axon needs energy?
NMDA on OG bind glutamate and allow Ca2+ into OG
Ca2+ signals increase transport of glucose
How is apoptosis triggered?
P38 MAP kinase, cytochrome C, ROS
All caused by excitotoxicity and excessive Ca2+ signalling
What are the main function of microglia?
Homeostasis, killing quiet synapses, phagocytosis, immunity
Where are microglia found?
Near synapses and in grey matter
Have large phenotypic diversity
What are the different morphologies of microglia?
Amoeboid = activated
Bushy = neurodegeneration and toxicity
Ramified = for surveillance
Under TM what do microglia look like?
Bean shaped w/ elongated nuclei
Scattered cisternae and rough ER + golgi at each pole
What is the function of microglia?
When resting its surveillant
Detects injury and responds to injury
by synthesis of chemokines
What is the role of the M2 phenotype?
Associated w/ cell growth and survival
Anti-inflam. IL4, 10, 13
What is the role of the M1 phenotype?
Associated with inflam. and destroying cells
Pro-inflam. IL6
What is the neuronal function of microglia?
Neurogenesis, apoptosis, induction, phagocytosis, maintenance of health
What is the immune function of microglia?
Pro and anti-inflam. response, cytokine and chemokine production, immuno-surveillance
How do TLRs work?
On the extracellular side of microbial products
On the cytoplasmic side:
Recruits signaling molecules
Alters kinase activation
Modifies gene expression
Function of resting microglia
Not moving
Ramified and branched
Immuno-surveillance
Function of activated microglia
Amoeboid
Moves freely
Phagocytoses debris
Prune cells and dendrites
What do ependymal cells do?
For CSF synthesis and circulation
Found in choroid plexus
Have apical microvilli and cilia
Regulates osmotic pressure
What happens when microglia become overactivated?
Produces cytotoxic factors
As AD progresses, microglia become overactivated
How are reactive oxygen species produced?
NADPH oxdiase activation
Activated in AD, PD and other neurological disorders
What is the basic shape of astrocytes?
Star-shaped, long branched processes
Most numerous in glia in grey matter
Non-excitable but communicable
How can astrocytes be distinguished?
Branching processes
Dominated by int. filaments, actin, mt. and microtubules
Large distal feet
What are the two types of astrocytes?
Fibrous: in white matter
Protoplasmic: in grey matter, majority of the astrocytes, envelop synapses
What are the general functions of astrocytes?
Structural integrity
BBB maintenance
Neuronal nutrition
Monitor neuron health
Modulate neuronal output
Tripartite synapse
Overall synchronisation
How do astrocytes maintain these functions?
Have K+ channels that move K+ from [high] –> [low]
What is the tripartite synapse?
When the astrocyte directly modulates ongoing communication between synapses
Uses Ca2+ as a way for astrocytes to communicate
How do mGluRs work?
Glutamate transmission increases intra. Ca2+ in astrocyte
Gliotransmitters released
Can directly affect pre. and post. signalling
Act on NMDA, GABA Rs etc
How do astrocytes influence numerous neurons?
Astrocytes make connections to numerous neurons
Eg: CB1 expressed on astrocytes and causes Ca2+ release
Can cause increased release of gliotransmitters
How does astrocytic synchronisation work?
Astrocyte contacts a number of neurons across 100uM
Results in neuronal synchronisation
How do astrocytes take up glucose?
Take glucose from blood at distal feet and convert to lactate
Neurons take lactate and feed to mt. to make ATP
How does neural activity lead to vasodilation?
Astrocytes detect energy demand thru GluRs
Ca2+ transient develops and travels to end feet
Release of vasodilators
Increase blood flow = more glucose for demands
How do astrocytes turn reactive?
In response to various symptoms of neuronal dysfunction
Two phenotypes A1 and A2
What does the A1 astrocytic phenotype do?
Cause cell death via inflammation
What does the A2 astrocytic phenotype do?
Repair of synapses and neuronal survival
What are the roles of astrocytes in PD?
Alpha-synuclein overproduced in dopaminergic cells
Astrocytes consider alpha-synuclein and insult and destory the dopaminergic neurons
How do knock ins and knock outs work?
Use enzymatic cutting to splice out a piece of specific DNA
OR
Encoding for a protein not usually expressed, DNA recombines w/ donor DNA and cell expresses new proteins
How does CRISPR work?
Viral DNA captured and integrated into bacteriums DNA
Viral DNA transcribed to RNA
RNA integrated into Cas as a guide
Guide RNA recognises viral sequence
Cas cuts viral DNA, disrupting it
How does optogenetics work?
Include channel rhodopsin for depolarisation and halorhodopsin for hyperpolarisation
Can selectively turn cells on/off
What does tetrode based electrophysiology allow us to measure?
Records APs fired from individual neurons
Can show place cell firing
