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Chapter 1:Neurons are discrete cells and not one continuum. They communicate via specialized
contacts.
Information passes through the neuron in a certain direction:
Dendrites -- > Soma -- > Axon.
Divergence: one neuron gives many outputs
Convergence: one neuron has many inputs
Neuropil: area between cell bodies where synaptic connections are
formed between dendrites, axon and glia cells.
Investigating connectivity:
- Staining: golgi, antibody, Nissle
- Lesions
- Dye: Anterograde & retrograde
- Reporter genes: GFP
- Calcium imaging
- Electrophysiology
- Brain imaging
Investigating connectivity: tracer studies
- Retrograde: injection into the axon terminals -- > diffusion to the cell
bodies.
- Anterograde: injection into the somata -- > diffusion to the nerve
endings.
Transgenice, KO and KI mice:
1. Isolate stem cells from a mouse with fur colour.
2. Introduce GFP-fusion construct in the stem cells.
3. Inject modified stem cells in a blastocyst from a white mouse donor.
4. Place the blastocyst back into a pregnant mouse.
5. Select mosaic/chimera mice.
6. Cross breed these mice with wild-type mice.
7. Cross breed heterozygous offspring with each other.
KO and KI mice:
1. Design a guide RNA to specifically bind your gene-of-interest.
2. Design a Homology directed Repair (HDR) template.
a. KO: leave out the 1st and/or 2nd exon.
b. KI: clone something (GFP, His-tag, …), in-frame after the start- or
before the stop-codon.
3. Introduce the guide RNA, the HDR template and the Cas9 protein into the
isolated stem cells (previous slide).
4. Cas9 has helicase and endonuclease activity=> double strand break.
5. HDR will use your template to repair.
6. Isogenize and sequence the cells.
Conditional genetics: Cre-Lox-P system
Only works in certain cells (with a cell specific promoter) and it only works in the
developmental stage (the promoter is active after this stage). Furthermore, it is
chemically infused with Tamoxifen.
Calcium imaging: This technique is used to
determine neuronal activity.
,Optogentics: Channel rhodopsins are used to influence the neuronal activity and this find
out the function of a neuron.
Brain imaging:
Chapter 2:
Membrane potential: difference of charge between the in- and outside.
Three different types of potentials:
1. Receptor potential: slow and small electrical change. This is a graded potential.
2. Synaptic potential: This is a graded potential.
3. Action potential: This is an all-or-nothing potential.
Passive response: when a currents comes into a cell which causes a little genes. But this
does not reach the threshold. If you put in a current which is higher than the threshold, it
will cause a action potential and be an active response.
Excitable membranes are equivalent to resister-capacitor (RC) circuits:
semi-permeable membranes like to store charges and also allow charges
to go through the membrane. The charge that is stored will be stored in
the membrane but the charge that are allowed to go through go through
the channels.
A capacitor (C) transiently stores charge. It determines the rate by which
a potential difference is established. The more current is added to the
membrane the higher the membrane resistance is. The membrane
resistance determines the amplitude of the potential change.
Ohm’s law: V = I x R
Passive currents decay with time and spine: You lose current across the leaky
membrane. The membrane is high resistance but the cytoplasm has a low
resistance (the inside of the axon).
Active currents boost passive ones and mediate long-distance signaling: The action
potential will go all the way and this will be maintained until the end.
Action potentials encode information in rate codes: the information of the amplitude is
encoded in the number of spikes one neuron gives. This action potential coding
represents logic circuits. AND = 2 neurons for actionpotential, NOT = 1 is bound, no
actionpotential, NOT AND = 2 bound but no actionpotential.
Summary:
- Passive electrical signals are dependent on capacitance and resistance of the cell
membrane, as well as the internal resistance of the cytoplasm.
- Passive properties shape the membrane potentials.
- Active currents, such as during action potentials, boost the spatial spread of
signals.
- Neurons compute and encode information via temporal and rate codes.
, Ion transporter: actively move selected ions against concentration gradient and create an
ion concentration gradient.
Ion channel: allows ions to diffuse down concentration gradients and are
selectively permeable to certain ions.
- Operate in open and closed states
- Highly selective for specific ions
- Ions flow down their concentration gradient and they flow fast.
- Responsible for changing membrane potentials
- Active signal generation
Ion pumps:
- Constitutively active
- Ions move against their concentration gradient and require
energy in the form of ATP
- Operate slow and maintain the resting conditions of cells.
When there is a higher concentration on one side compared to the other.
At some point a equilibrium will be reached with ions moving but the
electrical potential is set. This is called the equilibrium potential.
Calculating the equilibrium potential: to calculate at which potential there
is a equilibrium-- > Ex = (58/z)log([X]out/[X]in)
Actively changing the driving force: Changing the membrane potential away from
EK changes the directionality of the ion flux.
The membrane potential can be calculated (with the Goldman equeation) as the
sum of the equilibrium potentials and their relative permeability at this potential. At
rest Pna and Pa are often considered << Pk.
Kalium dominates the resting potential (slope van 58 mV) and natrium dominates
the action potential.
Chapter 3:
Ion channels:
- Leak (passive, K+ >> Na+;Ch2): resting potential.
- Ligand-gated (active, chemical, Ch6): neurotransmitter.
- Voltage-gated (active), channels mediating the action potential
-- >
- Light-gated (active, from algae)
- Mechanically gated (active, open up with an action)
- Temperature, pH, acidity (active ion channels).
Voltage-gated ion channels represented in Ohm’s law: The arrow means that an action is
needed to go into the open or closed state. To describe the flow of the current, you can
use Ohm’s law.
1. Ohm’s law: V = I x R
2. Conductance (G) is the inverse of resistance: G = 1 / R
3. A currents flow through a conducting ion channel
(x):
V is the membrane potential and E is the equilibrium potential.
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