BIO304 - Midterm #1

Modules #1-5

What is the nervous system?

What is the nervous system?

Network of cells specialized to sense inputs & generate outputs

What is signaling in the nervous system?

Electrical impulses for information propagation + synapses for conveying information between neurons

What are 2 types of electrical impulses?

Graded potentials + action potentials

What are chemical synapses?

Convert electrical signals into chemical signals for postsynaptic cells

What barrier to electric signals do synapses overcome?

Hydrophobic phospholipid bilayer can’t conduct electrical signals

What are graded potentials?

Convey intensity of stimulus

What is the disadvantage of graded potentials?

Dissipate so no long distance

Why do graded potentials dissipate?

Quick depletion of ions/charges

What are graded potentials in the retina?

Photoreceptors of receptor cells to bipolar cells & then Ganglion cells both producing graded potentials

What is stimulus intensity in retinal graded potential?

Stronger light = more neurotransmitter secretion by receptor cell = stronger depolarization of bipolar cell

When does action potential arise in the retina?

Strong enough graded potential in excitable neurons like Ganglion cells

What is an excitatory potential?

Depolarizing (excitatory postsynaptic potential = EPSP)

What is an inhibitory potential?

Hyperpolarizing (inhibitory synaptic potential = IPSP)

What are action potentials?

All-or-nothing electrical impulse up to 120 m/s along axon

When are action potentials activated?

Graded potentials activate voltage-gated sodium (NaV) & potassium (Kv) channels

What are 2 advantages of action potential?

Long distance + don’t dissipate

How far apart are cells?

~10 microns

What are NaV channels?

Depolarize membrane

What are Kv channels?

Depolarize/hyperpolarize membrane

What are 2 functions of neuronal action potentials?

Transmit signals along axons + trigger presynaptic Ca2+ influx through CaV at nerve terminals

What is presynaptic Ca2+ influx at nerve terminals?

Regulated release of neurotransmitters by exocytosis

What is the function of graded/action potentials in muscle?

Contraction

What is the function of graded/action potentials in endocrine cells?

Secretion of hormones by exocytosis

What is the challenge of understanding the nervous system?

Integrating molecular biology with electrophysiology (physics)

What made the cell theory (1838) possible?

Combined observations of light microscopy

What are 3 tenets of the cell theory?

(1) All living things composed of cells, (2) cell is basic unit of organism structure & organization, (3) cells come from pre-existing cells

What is the reticular theory of the nervous system (1861)?

Made up of single contiguous network, not separate cells

Why was reticular theory preferred over cell theory?

No discrete cells under microscope of nervous tissue

Who were 2 proponents of reticular theory?

Camillo Golgi + J. von Gerlach

What are 2 alternate names of the Golgi stain?

La reazione nera + the black reaction

What is the Golgi stain?

Potassium dichromate & silver nitrate to dead fixed neurons to randomly label a subset of neurons black in their entirety for single cell tracing

What is the (ironic) significance of the Golgi stain?

Shows neurons as individual cells

Who was Santiago Ramon y Cajal?

Histologist studying nervous system tissues with Golgi stain that disproved reticular theory in favour of neuron theory

What is the neuron doctrine (theory)?

Nervous system is made up of separate cells

Who was Henrich Waldeyer?

Synthesized nervous system organization principles to propose the neuron theory

Who was Deiters?

Protoplasmic processes renamed dendrites by Waldeyer

Who was Kolliker?

Axis cylinders renamed as axons by Waldeyer

What were Cajal vs. Waldeyer’s contributions?

Cajal et al. generated data whereas Waldeyer only synthesized into neuron doctrine

• What are 4 tenets of the neuron doctrine?

(1) Neuron (cell) is functional unit of nervous system, (2) law of dynamic polarization, (3) neuron communication via synapses, (4) Dale’s law

What are 3 common structures of neurons?

Dendrites, soma, axons

What are nerve fibers?

Project from soma of single neurons

What is the neuron nucleus?

Nutritive center

What is the law of dynamic polarization (directionality)?

Dendrites & soma = receptor surfaces, single axon = effector

What is the direction of neuronal information?

Dendrites to axons

What was Cajal’s contribution to law of dynamic polarization?

Inferred directionality through diagrams of neurons (ex. retina)

What are synapses?

Regions of neuron-neuron contact

Who was Charles Scott Sherrington?

Proposed synaptic transmission through electron microscopy

What is Dale’s law?

Single neurons use single type of neurotransmitter (ex. glutamate = glutaminergic, glycine, GABA)

Which 3 scientists received Nobels for neuron doctrine?

Golgi, Cajal, Sherrington

• What are 4 issues with the neuron doctrine?

(1) Gap junctions resemble reticular theory, (2) reciprocal synapses, (3) action potential backpropagation, (4) Dale’s law exceptions

What are gap junctions?

Electrical rather than chemical synapses by connecting cytoplasms through pores at membranes

What are reciprocal synapses?

Axons can act as dendrites, dendrites can act as axons

What is action potential backpropagation?

Signals travelling against polarity from soma/axon to dendrites

What are exceptions to Dale’s law?

Some neurons secrete more than 1 neurotransmitter type

What were 3 early inroads to understanding the nervous system?

Neuron morphology, projection, (synaptic) connectivity

What facilitated early inroads to understanding the nervous system?

Technological advances like microscopy & staining techniques (ex. Golgi stain)

What was Cajal’s influence?

Understanding nervous system structure & connectivity = understanding information flow & directionality in nervous system

What is Cajal’s neural connectomics paradigm?

Significant ongoing research focused on new or improved neural imaging & tracing techniques for understanding NS function

What is a connectome?

Complete system of neural pathways in nervous system

What is the disadvantage of neural connectomics?

Studying electrical signalling necessary for NS function in biological context (mapping roadways in city not enough to understand traffic patterns during different times of day)

What are fluorescent dyes?

Inject into neuron to diffuse or transport throughout (some through gap junctions) for labelling live neurons

What is the advantage of fluorescent dyes over Golgi stain?

Label live neurons to track signals vs. dead fixed neuron

What is the mechanism of fluorescent dyes?

Electrons absorb light & emit fluorescence at different wavelength as they drop to lower energy orbitals (ex. Lucifer yellow emits yellow wavelength)

What is the relationship between excitation & emission wavelength?

Emission wavelength > excitation wavelength (emission energy < excitation energy)

What are 4 dyes labelling sub-cellular structures?

MitoTracker (mitochondria), LysoTracker (lysosomes), Hoechst & DAPI (nucleus)

What is dye conjugation?

Conjugate dyes to molecule for targeting specific cell part (ex. toxins, like phallacidin labels F-actin)

What is BODIPY FL Phallacidin?

Green fluorescent dye preventing depolymerization of F-actin for actin location in cell

What is protein and mRNA localization?

Genes to label & identify neurons

What are marker genes?

Expressed only in cells of interest

What is a marker gene for glutaminergic neurons?

VGLUT1, transporter for glutamate into vesicles

What is a marker for GABAergic neurons?

GAD1, synthesis of GABA

What is mRNA detection in cells/tissues?

In situ hybridization through complementary antisense RNA conjugated to fluorescent dye / other label as probe

What is the disadvantage of mRNA detection?

mRNAs mostly located in soma so often no location of translated protein

What is protein detection in cells/tissues?

In situ immunolabeling where protein epitopes generate antibodies that bind target proteins for detection

What are epitopes in protein detection?

Piece of protein injected into animal for antibody production, extraction & conjugation with label

What is the advantage of protein detection?

Determine function by location (ex. pre-synaptic protein)

What are fluorescent proteins?

Encoded by gene, absorb light & emit fluorescence like fluorescent dyes

What is green fluorescent protein (GFP)?

Cloned from jellyfish Aequorea victoria

What is the GFP chromodomain?

Absorbs & emits light

How is GFP genetically expressed in specific neurons?

Use cell-specific promoter for genome-inserted GFP expression in neurons of interest (ex. labelling tubulin structure during microtubule assembly)

What is the labelling capacity of GFP?

Labels entire neuron, like fluorescent dyes

What are fusion proteins of GFP?

Fuse GFP to protein of interest to visualize location in/out of cell

What is multicolour fluorescent protein labelling?

Mutated GFP & mRFP (monomeric red fluorescent protein) into many colours for simultaneous labelling

What is the nomenclature for GFP derivatives?

XFPs (ex. YFP = yellow fluorescent protein)

What is a brainbow?

In vivo transgenic technicolour (multicolour) Golgi stain developed by Jeff Lichtman at Harvard

What is the visual representation of neurons in brainbow?

Individual neurons = different coloured balls = different combo of fluorescent proteins

What is the approach of brainbow?

Randomly integrate 3 XFP gene copies (CFP, YFP, or RFP) into mouse genome so that neurons randomly express different combo of FPs for unique fluorescence profile

What is the promoter for brainbow?

Synthetic virus promoter inserted into mouse genome

What is Cre-mediated recombination in brainbow?

Cre recombinase cuts Lox sites between FP genes for recombination to control which FPs are expressed (ex. cutting RFP from RFP-YFP-CFP leads to either YFP-CFP or just CFP)

What are 3 examples of brainbow in other species?

Zebrafish, Drosophila, plants

What is the limitation of brainbow?

Ineffective at high magnification at synaptic connectivity due to tightly-packed neuron projections of CNS

Why is brainbow ineffective at high magnification?

Light microscopy resolution limit of >200 nm using photon beams vs. ~10s nm of synapses

What are static tracers of synaptically-connected neurons?

Injected into & transported only within cell in either anterograde or retrograde direction

What is the anterograde direction of neural circuit tracer?

Direction for law of dynamic polarization (ex. Phaseolus vulgaris-leucoagglutinin)

What is the retrograde direction of neural circuit tracer?

Backward direction for law of dynamic polarization (ex. Cholera toxin B)

What are adeno-associated viruses (AAVs) for neural circuit tracing?

Modified AAVs can label single neurons either anterograde or retrograde by promoting expression of detectable gene product

What are monosynaptic tracers?

Jump across single synapse

What are polysynaptic tracers?

Jump across series of synapses

What are trans-synaptic tracers?

Modified virus derivatives that can label neural circuits

What is an example of anterograde trans-synaptic tracers?

Modified herpes simplex virus