Lecture 1 Notes - Brain and Behaviour - PSYC 304B 2024

PSYC 304B: Brain and Behaviour September 12, 2024 **Lecture 1: 'Seeing' Neurons** **[Objectives]** 1. Appreciate that neurons and circuits are the foundational units of brain function. 2. Be able to label and differentiate between different types of neurons. 3. Be able to evaluate m...

PSYC 304B: Brain and Behaviour September 12, 2024 **Lecture 1: 'Seeing' Neurons** **[Objectives]** 1. Appreciate that neurons and circuits are the foundational units of brain function. 2. Be able to label and differentiate between different types of neurons. 3. Be able to evaluate methods for identifying neurons as anatomical building blocks, including Golgi, dye injection, genetically encoded fluorescent proteins, immunohistochemistry, electron microscopy and brain clearing 4. Understand research applications for different visualization techniques. **[Key Milestones in Neuroscience]** - Cell theory -- 1830's - Theodor Schwann and Matthias Jakob Schleiden - Idea that all living organisms are made up of cells - Reticular Theory -- 1873 - Camillo Golgi - Developed the silver stain/Golgi stain technique to visualize neurons - Idea that nervous system is a continuous network - Neuron Doctrine -- 1889 - Santiago Ramón Y Cajal - Neurons are independent units - Structurally and functionally distinct - Information is transmitted via synapses (gaps) - Electron Microscopy -- 1950's - Confirmed the neuron doctrine **[Neuron Anatomy]**  [Types of Neurons] page23image65001568  [Region and Functions] page24image47849344 - Shape of neuron is related to its specific function - More dendrites = integrates more information - Less dendrites = more specialized [Challenges of Visualizing Neurons and Circuits] - Connectome: the wiring/synaptic connectivity of all neurons - Allows us to infer the function of a neural circuit - Prediction of the flow of information - Understanding dysfunction + guide potential treatments - Visualizing a single neuron is simple, but mapping an entire circuit or connectome is not - There are so many neurons in the brain! - Mapping all of them at once information overload **[Methods of Visualization]** [Method 1: The Golgi Stain] - Chemical process that causes silver impregnation in neurons - Small % of neurons labelled - Unknown why some are labelled and not others - Can be used on dead tissue, including humans - Tried and true technique; relatively easy - Reveals changes in synaptic density - Result of synaptic pruning during early development - Excessive pruning schizophrenia (can be seen on Golgi stain with fewer dendritic spines) [Method 2: Dye Filling Neurons] - Dye injection - Dye spreads throughout the entire cell via diffusion allows us to see full structure - Difficult to pinpoint neuron + inject dye without damaging it - Can be done on live or dead tissue - Useful for studying specific neurons - Ex. Dye filling purkinje neurons - Large complex neurons critical for motor control - Studied in detail using dye injection to map their dendritic structure and connections [Method 3: Immunohistochemistry] - Targets/identifies any specific protein (antigen) -- biomarker localization - Uses antibodies to locate specific proteins (biomarkers) - Helps map out protein presence and function in neurons - Combines multiple antibodies - Multiple antibodies allow visualization of different proteins simultaneously - Different colours can be used for multiple proteins in one tissue sample. - Dead tissue, can be used on human tissue - Has been used to show that one of the first pathological signs of Alzheimer's is a loss of synaptic proteins - Reasonably cheap and feasible - Examples: [Method 4: Genetically-encoded fluorescent proteins] - All cells share same DNA, but differential transcription causes different genes to be expressed in different cells distinct neuron types, tissues, regions, etc. - Certain promoters are only active in specific cell types - Therefore, by artificially expressing genes under the control of a cell-specific promoter, you can get cell-specific gene expression - Green Fluorescent Protein (GFP) - Isolated from jellyfish - Can be mutated to have all colours of the rainbow - Can be done to other fluorescent proteins as well - Critical choice -- what promoter to use - Promoter determines specificity - Create genetic construct piece of DNA that contains the fluorescent gene - Transgenic animal lines - Modifying the DNA of an animal so all cells carry GFP gene - Viral transduction - Using a virus to deliver GFP gene into cells - Costly set up, cheap and efficient once you have the transgenic animal or virus - Can label genetically-identified cells - Promoters rely on cells' gene expression - Can verify successful gene manipulation - Choosing the right promoter: - CAG promoter - Active in all cells - Thy 1 promoter - Active in a fraction of all types of neurons - L7 promoter - Active in cerebellar Purkinje neurons - Doublecortin promoter - Active in immature neurons - Iba1 promoter - Active in microglia - GFAP promoter - Active in astrocytes [Disadvantages of Traditional Visualization Techniques] - Brain tissue must be cut to visualize neurons - Anything that is translucent scatters light - Thick brain tissue is challenging - Light gets scattered, reducing resolution and clarity - The solution?\... [CLARITY Technique -- Brain Clearing] - This preparation technique involves brain clearing - Lipids, fats, membranes removed so less light is scattered - Light can penetrate deeper, emitted light will be captured without scattering - Antibodies can also penetrate more easily (immunohistochemistry) - Allowing for thicker tissue samples - Allows imaging of FPs in larger blocks of tissue - Can track axons over long distances, identify networks/coarse neuroanatomy - Can only be done on dead tissue [Visualizing FPs with Confocal Microscopy] - Fluorescent microscope - All light from tissue is reflected to the eye piece - Confocal microscope - Pinhole eliminates out-of-focus light, allowing visualization of a single focal plane - Reconstruct images from different focal planes [2 Photon Microscopes] - Must image living tissue to see changes over time - 2 photon microscopes - Image deep into tissue through dura (100s um) - Dura -- brain's protective covering [Method 5: Electron Microscopy] - Can be used with immunohistochemistry, FPS, or on its own - Method: - Best resolution - Expensive! - Microscope itself is expensive, requires employing a technician - Requires stable environment - Needs to be in a vacuum - Time consuming - Took 12+ years to map connectivity of 302 neurons of the C-elgans (worm) NS