PSYC 360: Neuroanatomy and Visualization Methods

How can GFP be used to view neurons in vivo?

- shave thinly the skull of an animal, or just cut out a window - light up the GFP cells - two-photon microscope can now view the GFP cells

what did the Television Test (Squire and Cohen 1979) show?

- showed that electroconvulsive therapy disrupts memory for recent, but not older, pilot TV shows - may be that some older memories are so weak that is no memory to disrupt

what Iba1?

- GFP promoter to stain microglia

What is the Thy1 promoter and what are its pros and cons?

- GFP version of the Golgi stain expressed in some brain cells, labels neurons pros: - allows cells to be monitored in time (as opposed to snapshots) b/c cells naturally express it - no interference from chemicals/injections/treatments - cells labeled all over the brain cons: - don't know exactly which cell types are using Thy1 promoter

What are the major characterizing features of neurons?

- action potentials - synapses

Theodule Ribot

- biology of memory - related memory to physiology - taught that memory is a side effect of physiology - experiences leave physiological traces (memory); think foam mattress

Give an example of a bipolar cell

- bipolar cell of retina

what shows that behavior has a physical basis (specifically in the brain)?

- brain dmg/manipulation leads to specific alterations in behavior - for every behavioral output, there is correlated activity in the brain

how are autistic individuals' interneurons abnormal?

- bridges between parvalbumin interneurons

How can you visualize doublecortin?

- can either be an antibody (immunohistochemistry) stain or used in GFP - can be used to label immature dentate gyrus neurons - b/c doublecortin used to extend axons to make connections with nearby cells, which is a process that is very active in immature cells, but not so much in mature cells

What is the key to visualizing neurons using genetics?

- choice of promoter

What is the CAG promoter and how is it used?

- expresses GFP in all cells - synthetic promoter used to express genes that are naturally expressed at high levels in mammals; inserted into genome so expressed in all genes - very active promoter - useful for tracking cells in transplanted tissue (cancer research)

Describe the stretch reflex

- doctor hits tendon with hammer, muscle stretches - sensory neuron senses stretch, signal goes to spinal cord, then through motor neuron to cause quads to contract, thus causing leg to kick up - at the same time, sensory neurons activate inhibitory interneuron that inhibits the motor neuron responsible for stretching the hamstring, to make sure the hamstring doesn't contract

Describe the hippocampal trisynaptic circuit

- cortical inputs to dentate gyrus: info from dorsal and ventral visual streams integrated in dentate gyrus - dentate gyrus granule cells -> CA3 pyramidal cells: CA3 receives info, feed back info to each other, so that all CA3 neurons wired together, so that when one CA3 is activated, all others fire too, and entire memory is retrieved; memory is stored/formed - CA3 -> CA1 pyramidal cells: CA1 required for understanding of temporal relationships of memories - CA1 -> out to cortex

What is the point of pruning synapses? What happens if they're not pruned?

- essential part of developmental plasticity and learning - microglia gets rid of associations that are no longer needed - lack of pruning in the PFC may underlie autism

How is electron microscopy used for neuroimaging?

- first label neuron with GFP - then label with antibodies - place neuron through chemical reaction to precipitate and make the labelled parts darker (once electron dense, then the microscope can detect the object) - cut object into thin films and place in resin - look with microscope (need to reassemble object from the thinly cut layers)

what are detonator synapses (mossy terminals)?

- found in CA3 - all or none, large impact - important for memory - synapses close to cell body - believed to be more likely to initiate action potential - in the dentate gyrus

Give an example of a pseudo-unipolar cell

- ganglion cell of the spinal cord

How can you tell through electron microscopy that a synapse is functional and connected to other neurons?

- if you see a post-synaptic terminal with a pre-synaptic terminal right beside it (very dark, electron dense strip is post-synaptic membrane) - little blobs inside the pre-synaptic terminal are the vesicles

what is calretinin?

- immunohistochemistry stain for inhibitory interneurons

Explain the function of inhibitory neurons in the hippocampus/dentate gyrus, and how they relate to seizures

- inhibitory neurons usually express GABA, which prevents neurons downstream from firing - axons carrying info from the cortex into the dentate gyrus pass across both the inhibitory neurons and the dentate gyrus neurons - inhibitory neuron "decides" whether to let the dentate gyrus neuron fire or not - both feedback and feed-forward inhibitory neurons are present - seizures are the result of failed inhibitors, or incorrect inhibitory/excitatory neuron balance

What is fluorescent dye filling of neuron? What is the main advantage?

- inject neurons with dye that is sensitive to a certain wavelength of light to see anatomy clearly - advantage: can record electrical property of cell (electrophysiology)

What are the four functional regions of a neuron?

- input: takes in info - integrative: integrates/combines info, allows neurons to do things conditionally - conductive: conducts info - output: releases peptides, contracts muscles, etc

what do neuroendocrine cells do?

- integrate information from endocrine system and nervous system

Give an example of a unipolar cell

- invertebrate neuron

What is DAPi?

- labels all cells by integrating into double-stranded DNA; used as counter stain - immunohistochemistry stain

What do endothelial cells do in the brain?

- line capillaries - form tight junctions that restrict passage of materials out of blood vessels into the brain

How do calcium indicators tell us that astrocytes respond to glutamate?

- load neuron up with calcium indicator, which emits fluorescence when the neuron is excited - when there is glutamate present, astrocyte emits waves of light

What does it mean if the stretch reflex does not occur?

- means that any synapse along the circuit could be broken

Ramon y Cajal

- neuronal basis of the brain - used Golgi stain on neurons - characterized diff. neurons throughout brains of small mammals

Can immunohistochemistry and light microscopy view individual synapses? Why?

- no - because of the limits of the wavelength of light

what is Ribot's Law?

- older memories are more resistant to disruption

Donald Hebb

- physiological basis of behavior - proposed idea of "circuits" that supported a process that resulted in behavior

what are the Calyx of Held?

- presynaptic terminals found in the brainstem - used to localize sound in space - fires really quickly (600Hz) to calculate difference in sound reaching each ear - completely cover cell body of post-synaptic cell

what is GFAP?

- promoter for GFP to stain astrocytes

What is the L7 promoter, and its pros and cons?

- promoter that expresses GFP only in cerebellar Purkinje neurons pros: - locate one specific type of cell cons: can't distinguish between subtypes of a cell

How does the blood-brain barrier affect the brain?

- protects brain from infections because since brain is in the rigid skull, cannot become inflamed and swell up (because then neurons would not functions properly due to the pressure) - BBB also creates challenge for delivery of therapeutics to the brain

What are the functions of astrocytes?

- regulate synaptic function because they have processes at synapses, so can strengthen neurons - regulate blood flow from blood to brain (BBB permeability) - maintain extracellular milieu by absorbing/cleaning up excessive neurotransmitters like glutamate

What are the functions of microglia?

- respond to injury; found everywhere in brain but migrates quickly to site of injury; can fix ruptured blood vessels - engulf debris/dying cells - constantly grows and retracts to sample environment for damage or dangerous neurons - prunes/eat synapses through endocytosis of synaptic proteins - contain BBB disruptions

what are two examples of Hebb's circuits?

- some neurons in circuits summed up/integrated the stimuli of other neurons - some circuits cycle/undergo reverberating activity in order to maintain a behavior (activity)

What is Clarity? What advantages does it offer over immunohistochemistry?

- technique that clears fats from brain to enable deep imaging and increase antibody access - allows microscopes to see deep into brain w/o light being refracted by brain matter - eliminates need to cut brain into thin slices before viewing (b/c brain matter in the way of microscope); prevent, thus eliminating need to reassemble slices together to form whole object

Which taxa have multipolar cells? Give three examples

- unique to mammals - motor neuron - pyramidal cells - Purkinje cells

What is immunohistochemistry? Pros and cons?

- use antibodies labelled with fluorescent tags to target specific proteins pros: - doesn't require much to use technique (no breeding, etc) - incredibly versatile - can pinpoint exact location of a protein on a cell (vs. doublecortin only indicating presence of a protein on a cell, but not exact location) - can be used in human samples cons: - antibody very big, cannot cross through membranes by itself (need to break membrane)

How can viruses be used to label neurons? Pros and cons?

- virus manipulated to carry labeling protein - inject virus into a cell, cell starts replicating viral DNA (which is labelled) - can be used to precisely determine age of cell, because can use retrovirus which only infects dividing cells; can know that a cell was born when the virus was injected pros: - spatially isolated; only inject where needed - do not require transgenic animals - precision about age of cell cons: - performing surgery to inject virus = pain in the butt

What are five ways to visualize neurons?

1) Golgi stain 2) dye injection 3) fluorescent protein labels controlled by genetic expression (GFP) 4) immunohistochemistry 5) Electron microscopy

why do gaps exist in biopsychology?

1) no computer can accomplish what the brain does 2) no other organs compare in function or organization 3) neuron identity + function is a product of genetics AND experience (as opposed to only the former)

What are the four kinds of glial cells?

1) oligodendrocytes 2) Schwann cells 3) microglia 4) astrocytes

what are the five steps in getting approval to use animals in research at UBC?

1) submit scientific proposal to funding agency, and have independent review panel make sure that the research will make significant contributions 2) UBC Animal Care Committee reviews entire project. If approved, a four-year compliance certificate that must be reviewed annually is issued 3) Researcher must pass training in animal handling protocol 4) Animal care staff help refine procedures, train personnel, and care for animals. Independent vet monitors and make sure regulations are obeyed 5) Animal Care Committee reviews project annually (back to step 2)

what are the three Rs of animal research?

Reduce: (number of animals used); look at other studies to see if info you need is already there Refine: (experiments to reduce suffering) - environmental, physical, and social enrichment of the rat (influences rat brain structure/function so might need to redo older experiments) Replace: replace with dummies, blue brain project to computationally model the entire human brain; limitations: fMRI cannot replace animal research because can't see molecular or synaptic level

What are the functions of oligodendrocytes and Schwann cells?

oligodendrocytes: myelinate CNS neurons Schwann cells: myelinate PNS neurons

Pros and cons of electron microscopy?

pros: - best resolution cons: - expensive - ultra thin sections; time consuming to reconstruct (can't see much by looking at just one slice) - methodically vigorous, challenging/even vibrations can **** up the process

Pros and cons of of genetic expression of GFP?

pros: - can be less invasive to cell - quick and easy to use if cell line exists - can use virus to induce GFP in normal, non-transgenic animals; some viruses can give added specificity - promoters can be exploited to determine specificity of expression cons: - expensive and timing consuming to set up if no transgenic line of animals exist

Pros and cons of immunohistochemistry?

pros: - can target and localize specific proteins - extremely versatile because can use multiple antibodies - can be used on humans cons: - tissue must be dead

Pros and cons of dye injection?

pros: - cheap, easy - selective (to some extent; can't select for cell type) - know that the fluorescence will expose the entire cell - combine with electrophysiology cons: - surgery is a pain - ethical concerns - can't visualize over multiple days, limited to hours

Pros and cons of Golgi staining?

pros: - sparse labeling (can see labelled cells more clearly) - old, optimized technology - cheaper and easier to perform - fewer animals needed (relative to transgenic animals) - can be used to study human brain cons: - not as high resolution as EM - selectivity is unclear, taken to be random - cells are dead - can't relate form to function