Module 2 - Visualizing the nervous system Flashcards
When was the cell theory introduced?
1838
Outline the cell theory
- All living organisms are composed of cells
- The cell is the basic unit of structure and organization in organisms
- All cells come from pre-existing cells
Did the reticular theory of the nervous system consider the cell theory?
No
What did the reticular theory of the nervous system believe?
The nervous system is made up of a single continuous network, not separate cells
When was the reticular theory introduced?
1861
Who were two strong proponents of the reticular theory?
Joseph von Gerlach and Camillo Golgi
What did Golgi (ironically) invent?
“La reazione nera ‘ or “the black reaction” AKA the Golgi stain
Outline the process of a Golgi stain.
Apply potassium dichromate and silver nitrate to fixed neurons
Randomly labels a subset of neurons in their entirety, permitting single cell tracing
Who was Santiago Ramon y Cajal?
A visionary histologist who studied a broad range of nervous system tissues.
Why Santiago Ramon y Cajal a significant figure in the understanding of the nervous system?
One of the first to see the great potential of the Golgi stain
Did seminal work that disproved the reticular theory in favour of the neuron theory (along with others)
Who introduced the neuron theory/doctrine?
Heinrich Waldeyer
Who generated the data that was used for formulating the neuron doctrine?
Ramon y Cajal and colleagues
Outline the tenets of the neuron doctrine (4)
- Neurons are the functional units of the nervous system
- Law of dynamic polarization
- Neurons communicate via synapses
- Dale’s Law
What is the law of dynamic polarization?
Nerve cells have a single axon that serves as an effector (i.e., passes information)
Dendrites and cell body serve as receptor surfaces
Information travels from dendrites to axons
Who put forward the concept of synaptic transmission?
Charles Scott Sherrington
What is Dale’s Law?
Single neurons utilize a single type of neurotransmitter
What are the 4 issues with the neuron doctrine?
Extensive gap junctions (electrical synapses) in the CNS encroach upon the reticular theory
Axons can acts as dendrites and vice versa
Signals can travel against polarity (e.g., from soma/axon to dendrites)
Some neurons can secrete more than one neurotransmitter type
What is a common theory about gaining insight about information flow in the nervous system?
Understanding nervous system structure and connectivity = understanding information flow in the nervous system
What is the analogy used to show why connectomics might not be adequate to understand information flow?
If you map the entire system of roadways in a city, would that be enough to understand all the traffic patterns that flow through it at different times of day?
How do fluorescent dyes work?
Have electrons that absorb light and emit fluorescence as they drop back down to lower energy orbitals
How do fluorescent dyes differ from the Golgi stain?
Can be used to label live neurons
Describe how labelling using fluorescent dyes works.
Inject into neuron
Diffuses or is transported throughout
Some dyes can travel through gap junctions, allowing us to track the flow of information
What is MitoTracker?
Fluorescent dye that labels mitochondria
What is LysoTracker?
Fluorescent dye that labels lysosomes
What is Hoechst and DAPI?
Fluorescent dyes that label nuclei
How can genes be used to label and identify neurons?
“Marker” genes are used, which are only expressed in cells of interest
What is in situ hybridization?
Detecting mRNAs in cells/tissues
How does in situ hybridization work?
Labelled antisense RNA that complements a target mRNA is used as a probe.
What is a drawback of using in situ hybridization?
mRNAs are mostly located in the soma, preventing us from seeing where the translated proteins end up
What is immuno-labelling?
Detecting proteins in situ
How does immuno-labelling work?
Protein epitopes (i.e., extracted portions of protein) are used to generate antibodies
Antibodies bind target proteins and can be detected
How do fluorescent proteins work?
Absorb light and emit fluorescence
What was green fluorescent protein (GFP) cloned from?
The jellyfish Aequorea victoria
What are the benefits of GFP?
Can genetically express GFP in specific neurons
Can create fusion proteins
How can GFP be expressed in specific neurons?
Use cell-specific promoters to drive expression in neurons of interest
Labels entire neuron
How are fusion proteins created with GFP?
GFP “sticks” to another protein of interest
What is a benefit of using GFP to create fusion proteins?
Visualize where the protein of interest spends its time inside/outside the cell
What is the Brainbow?
A technicolour Golgi stain
Who developed the Brainbow?
Jeff Lichtman and his team at Harvard
Describe the in vivo process of the Brainbow.
Randomly integrate XFP genes into the mouse genome, each capable of randomly recombining to express either CFP, YFP, or RFP.
Neurons will randomly express a different combination of fluorescent proteins, producing a unique fluorescence profile
What is the key limitation of the Brainbow?
Neuron projections in the CNS are tightly packed. Brainbow tracing is ineffective at high magnification where synaptic connectivity takes place
What are static tracers?
Static tracers are transported within the cell in either retrograde or anterograde direction, but are restricted to that cell
What are monosynaptic tracers?
Tracers that can jump across a single synapse
What are polysynaptic tracers?
Tracers that can jump across a series of synapses
What are trans-synaptic tracers generally derived from?
Modified viruses
Give examples of trans-synaptic tracers
Modified rabies and pseudorabies viruses can label neural circuits in a retrograde manner.
Modified herpes simplex viruses can label neural circuits in an anterograde manner.
What is serial EM reconstruction?
Serial sections made from fixed nervous system tissue
Sections are imaged with scanning electron microscopy
Computer algorithms identify separate cells and create a 3D reconstruction
Who reconstructed the full neural connectome for C. elegans and when?
Sydney Brenner and team in 1986
How many neurons are in the C. elegans connectome?
302
What was found after the neural connectome for C. elegans was reconstructed?
Useful to guide research, but does not explain nervous system function
