Module 2 - Cellular Neuroscience Flashcards
L2.1 - Describe the morphology of neurons
Dendritic spines, dendrites, soma, axon hillocks (intermediate zone sometimes overlaps), axon (myelinated with nodes of Ranvier), axon terminals with boutons
L2.1 - Subdivide neurons into different classes
Unipolar (embryonic), bi-polar (retina), pseudo-unipolar (somato-sensory), multipolar (motor)
L2.1 - Describe the morphology of glial cells
Supporting cells, found in 1:4 in the cortex (less in cerebellum – 1:4) – different for CNS/PNS
They’re smaller than neurons but their morphology will differ depending on the glial type, and the morphology can also change. E.g. microglia can become reactive when pathogens are present, which changes its morphology.
Glial cells have a cell body and a number of processes
o Astrocytes have numerous processes giving them a star-like appearance
o Oligodendrocytes have fewer glial processes
o Microglial cells are smaller than other glia
L2.1 - Subdivide glial cells into different classes and describe their main functions
Oligodendocytes/Schwann cells: myelinating – oligodendocytes in CNS can have somewhat stem cell properties (have precursors to make new oligodendrocytes). Oligodendrocytes in the CNS can myelinate multiple neurons at once, but schwann cells can only wrap myelin around 1 neuron like a burrito blanket.
Oligodendocytes have the MBP marker
Astrocytes/satellite cells: support the chemical environment in synapses (take up glutamine and GABA), astrocytes support the BBB, form scar tissue after injury and has stem cell properties in the SVZ. Has the GFAP marker
Microglia: macrophage like cells, helps with the immune system – IBA1 marker
Ependymal cells are in the ventricular system and help CSF homeostasis
L2.1 - Describe basic neurohistological staining techniques
Nissl: cell bodies (purple) - nucleic acids
Klüwer barrera: myelin (blue)
Bodian: whole neurons (silver)
L2.2 - Explain the equilibrium potential
The EP is a process where there will be no net flux of different ions. Each ion has its own EP and will try to reach that while fighting the chemical and electric gradients.
Assuming full permeability, we can determine the equilibrium potential of an ion by Nernst equation: . This shows that the EP is all about the concentration, but these can change based on ATP pumps.
L2.2 - Explain the membrane potential
The weighted average of the permeable ions’ equilibrium potentials – we can determine it by the EP times the proportion of permeability. This permeability can be changed by ion channels opening and closing.
The membrane potential is the difference in charge inside and outside the neuron. The outside is always considered to be 0. The membrane potential is created by not having equal levels of ions on either side of the membrane, which often is further controlled by the ion permeability not allowing all types of ions to pass the membrane.
L2.2 - Explain the driving forces for directional movement of ions across the membrane
The driving forces are the electric force (wanting charges to be equal) and the chemical forces (wanting equal number of ions of each type on both sides of the membrane)
L2.2 - Describe the different ion channels and their gating mechanisms
There is ligand gated (will open for a NT), leak channels (always open), voltage gated (will open for a certain MP), TRP channels open for temps (V1 for capsaisin), K2P is PH sensitive, HCN senses protons
L2.2 - Explain the mechanism of ion channel voltage sensing
One of the transmembrane helixes have positive side chains (normally not charged), so when there is depolarization, the charges are pushed, so ions can go through – otherwise the channel is closed, but the “wings” being pushed causes a confirmational change to open the channel
L2.2 - Explain the mechanism of coupled transport
Coupled transport happens though co-transporters (2 things in or out) and antiporters (one in and one out) takes one thing up the electrochemical gradient and another down, so the energy “released” by the thing going down the gradient is used to lift the other up the gradient
L2.2 - Explain the mechanisms of ATP-driven ion pumps
The Na+/K+ pump takes up 50% of the brains energy – a phosphate group is separated from ATP to release the energy for the pump to move it’s open hinges from the inside to the outside, after which the 3 Na+ are released, 2 K+ goes in, and the pump opens on the inside of the cell again, where the 2 K+ are released
L2.4 - Distinguish between graded potentials and action potentials
Graded potentials will only lead to action potentials if they sum at the axon initial segment – the action potentials are all-or-nothing signals that will propagate down the axon. Graded potential can summate and be hyper or depolarizing (Aps can do neither). Graded potentials don’t travel well, but APs do.
Claires answer:
Graded potentials: This can be either a depolarization or a hyperpolarization. The amplitude varies in proportion to the size of the inputs generating them and multiple potentials can be summed. They are generally initiated by ligand gated ion channels. As they are small they can only travel a short distance before disappearing.
Action Potentials: these arise if a depolarizing graded potential reaches a specific thresholds activating the opening of a large number of voltage-gated sodium channels. The action potential is not graded- it is an all-or-none response of a fixed amplitude. Therefore, multiple action potentials cannot summate (see refractory periods below)
L2.4 - Explain the membrane length constant
L2.4 - Explain the membrane time constant
Time constant: The time it takes the Vm to rise to 63% of steady state value
The membrane time constant is the product of the input resistance and capacitance (low membrane resistance and capacitance for short time constant)
L2.4 - Explain the ionic basis (the Na+ and K+ permeability changes) of the different phases of the action potential
The initial phase (rising) of the AP is driven by Na channels opening – as it peaks, the Na channels close (inactivate) and the K channels open. As the signal decreases, K is still open and Na is inactivated. During the undershoot, the K channels are still open and Na is merely closed (not inactivated). This will allow another AP to be triggered, however, the signal has to be strong as we’re still in the relative refractory period.
L2.4 - Explain the absolute refractory period and relative refractory periods
In the absolute refractory period, the na channels are blocked (inactivated) through the ball and chain and no AP can happen. In the relative period, the Na channels are only closed and can be open, however, it will take more depolarization to reach the threshold as there is an undershoot
L2.4 - Explain how synaptic input can be coded as output
L2.4 - Identify the key structures responsible for action potential initiation and conduction
The AP is initiated by the axon initial segment (flexible in size and placement based on inputs) – conduction relies on the depolarization happening at the AIS. As the AP is generated, it proppegates down the axon and has to be refreshed at nodes of Ranvier for myelinated axons (saltatory conduction) or at the voltage gated channels spread over the axon for the unmyelinated ones.
The AP can’t move backwards due to Na+ channel inactivation.
Key factors for improving conduction is myelination (increases membrane resistance) or decreased internal resistance (larger axon diamater).
L2.4 - Explain the key determinates of conduction velocity in axons.
Conduction happens better in larger axons, as they have less resistance. Myelinated axons also have better conduction, as they incubate the signal.
L2.5 - Explain the difference between current clamp and voltage clamp techniques
In the current clamp, you set how much current that should be injected into the neuron and then look at the resulting membrane potential. We can measure the IPSP/EPSP.
For the voltage clamp, you clamp the voltage and look at the ionic changes (current changes) within the neuron. Both are a patch clamp technique where you attach an electrode to the neuron.
L2.5 - Describe different patch clamp techniques
Patch clamp: A technique to measure microscopic current flow though single channels. You use suction to get the pipet to fit tight around the membrane.
Whole cell: break the membrane and get access to the inside (here you can inject things through the electrode
Inside out: you break a part of the membrane off, so the inside you broke off gets access to the bathing medium and can react based on what you put into that
Outside-out: Allows you to study the influence of extracellular cues such as neurotransmitters as the outside membrane is exposed
L2.5 - Describe the in vivo and in vitro techniques that we can use to record from human neurons
Induced pluripotent Stem cells:
Advantages:
*Can make many different cell types
*Can take from patient groups to model specific diseases
*Can test pharmacology on the cells of the same species you want to treat. (important as ion channel or receptor expression may differ between species)
Disadvantages:
*Immature neurons and ion channels expression
*Lack native environment-crucial for excitability
Brain slices from epileptic patients taken at surgery
Advantages:
*From the adult human so mature cells
*Can last up to 48 hours
Disadvantages:
*Tissue normally from epilepsy patients and so likely to have abnormal excitability
*Only from certain areas
EMG and ENG – using surface (EMG) or needle (ENG) electrodes
Advantages:
*Relatively non-invasive
*Low risk
*Can make repeated measurements
*Relatively low cost
*Can use reflexes to probe spinal circuitry
*Can combine with non invasive brain stimulation to test motor pathways
This can be used to identify:
1. Demyelination
2. A focal compression or injury
3. Other problems with the peripheral nerve
L2.7 - Describe the early morphological development of the central nervous system
A zygote (week 1) becomes an embryoblast (week 2) -> gastrulation (week 3) forms the 3 germ layers -> part of the ectoderm thickens to the neural plate, which groves –> form the neural tube (lateral parts will be the neural crest). This develops into the primary brain vesicles (see below).
The isthmus is important in development, as it divides the expression of many factors that will decide the division between the mesencephalon and the metencephalon
Neurons will be formed from radial glia, and the Sulcus limitans will divide the caudal neural tube into the alar plate (sensory) and basal plate (motor) –> this will grow into the central canal
The diencephalon develops from the middle of the telencephalon and the retina also develops from here
Telencephalon develops as a lateral evagination from the prosencephalon -> pallium forms the cortex and corpus striatum the basal ganglia
Please refer to https://virmik3.sund.ku.dk/content.aspx for further divisions
L2.8 - Explain the basic principles involved in neural tube development and regionalization
The process of the neural tube being formed is called neurulation, which is controlled by the notochord. The neural tube form from the neural plate (an ectoderm structure), and starts grooving inwards. This creates the roof and floor plate, the roofplate being closely associated with the neural crest, which is a diverse cell type that can form bone, muscle, neurons, Schwann cells and so forth. In development, the neural tube forms first the primary brain vesicles and then the secondary ones (see above). The regionization depends on transcription factors, and different ones will be present anterior to (Otx2), posterior to (Gbx2) or on (fgf8) the mid/hind-brain boundary (the isthmic).
HOX genes will control anterior/posterior fate and are relevant for the hindbrain and spinal cord
L2.8 - Describe the concept of rostro-caudal and dorso-ventral neural patterning
The rosto/caudal patterning is based on concentrations of wnt (more wnt caudal). Ventral patterning is controlled by concentration of Sonic Hedge hack (more shh – more ventral) – secreted from the notocord. Dorsal patterning is controlled by BMP, which is secreted from the overlying ectoderm. Later on, this becomes more complex and Retinoic Acid starts playing a larger role in the rostral/caudal patterning. For the spinal cord, more RA is present in the upper spinal cord and more FHF8 in the lower. Note that the gradient of transcription factors don’t lead to gradient of cell types, but actual divisions that possess different markers.
L2.8 - Describe the organization of the ventricular zone and the characteristics of radial glial cells
As the primary and secondary brain vesicles develop from the neural tube, the inside of the tube is hollow and forms on the border of the tube the ventricular zone. This is where the radial glia is placed. The radial glia work as stem cells, which divide and allow neuronal cells to migrate up their processes into a new brain layer (interkinetic nuclear migration). As the neurons always migrate outwards, the most superficial brain layer is the youngest cells. From the ventricular zone (where progenitor cells are), the intermediate zone comes (early postmitotic cells) and then the mantel zone (mature cells) as we more outwards.
The brain holds 6 layers in addition to the ventricular and subventricular zone. Primates furthermore has an Outer Subventricular Zone.
Interneurons are not only able to migrate up the radial glia processes, but also across (tangential migration) them to find their final destination (non-interneurons don’t migrate).
L2.8 - Understand how developmental biology can be applied to differentiate neurons from stem cells for transplantation therapy
In order to create progenitor cells for e.g., midbrain dopaminergic cells for PD or retinal pigment epithelia cells (RPE), we need to know exactly what concentration of factors that needs to be applied (and for how long) to create the right patterning. Finding the correct recipe can take many years, but the more it’s perfected, the higher concentration of the cell type that we’re interested in you will get. Without the correct recipe, we can’t guide the iPSCs (which are like embryonic cells) to differentiate into the correct cell type and they will therefore be useless. This is an example of why understanding which factors that become relevant (and when) in the nervous system is so important in transplantational therapy.
iPSCs are reprogrammed in the first place using Yamanakas 4 factors and then later differentiated into different cell types –> better than hESCs as there are less ethical concerns
Other examples of translational applications could be to retinal pigment epithelia cells for macular degeneration or inhibitory interneurons for epilepsy
L2 (EXTRA) - Calculate the equilibrium potential for K+/Na+/Cl- and explain how they impact the membrane potential
K+ in: 140 mM – out: 5 mM
Na+ in: 10 mM – out 145 mM
Cl+ in: 5 mM – out 110 mM
Nernst equation: 58/(charge) * log(out/in) - assuming 20 degrees
-84 for K+
67 for Na+
-78 for Cl-
