Neuro 3000 Exam 2 OSU

Forward Genetics

Have a mutant animal with a known phenotype, want to identify the gene/mutated gene

Behavioral assay

Needed to screen for the mutant phenotype you are looking for

Goal of forward genetics

Saturation- identify all the genes contributing to a behavior and to molecularly identify them

Reverse Genetics

You have a known gene and want to find its function. Good example is a gene knockout.

Modern era human genetics methods

linkage analysis, GWAS, whole exome sequencing

transgenic organism

any organism in which foreign DNA is introduced and stably integrated into the genome

What is the transgene used for?

- Mark cells with a developmentally & functionally neutral enzyme from a cell- or tissue-specific promoter

- Overexpress a protein that should produce an effect when expressed

Main goal of transgenesis

Express a foreign protein in specific parts of the brain

Main goal of a gene knockout

To target a specific gene, eliminate its function, and determine what is wrong with the KO animal

How do transgenic mice and knockout mice contribute to neuroscience?

Contributes to the understanding of the molecular mechanisms underlying brain function and behavior

Founders of transgenic mouse technology

Brinster and Palmiter

Pronuclear injection

- Developed in the 1980s, DNA construct injected directly into fertilized mouse eggs (zygotes)

- DNA integrates into a random location in mouse genome

Gain-of-Function

Drive expression of a protein of interest in some cell type or region in the brain

DNA construct promoter

determines where the transgene will be expressed

DNA construct protein coding sequence

what you want to express in the cells of a living animal

Examples of transgenic mice and uses

- expression of neutral reporter, like GFP, to monitor patterns of gene expression & study living cells using live cell imaging

- expression of voltage- or Ca2+-sensitive fluorescent reporters to study cell physiology in living cells

- expression of a functional protein to disrupt development, or function

Problems with old way of making transgenic mice

- DNA integrates randomly in gene, intended expression gets perturbed by local DNA sequences

- Frequently, DNA would get integrated in multiple copies, causing variations in level/pattern of expression

- Hard to find good promoters that work as expected

ROSA26 gene

- widely expressed gene that allows expression of strong promoters that are inserted

- use knockout mouse technology involving ES cells

- Will destroys one copy of ROSA26 gene, other left in tact. Goal is not to knockout gene

When did knockout mice become relevant to the brain?

1990

How are KO mice different from transgenic mice?

In KO mice, a specific gene in the genome is being targeted for inactivation.

How are KO mice made?

- use embryonic stem cells and homologous recombination to introduce the DNA construct and target the gene of interest using.

Chimera

The first/founder animal created by mixing normal embryo cells with genetically modified ES cells

Homozygous knockout

The result after two breeding steps, has both copies of the inactivated gene

Early mouse development and chimerism

- Generated cells can be equipotential or pluripotential

- pluripotential can give rise to all tissues and organ systems

Embryonic stem cells

- pluripotential, cannot make extra embryonic cells

- can be manipulated like cell lines, can be transfected with DNAs

Homologous Recombination

- recombination vector using double selection

- vector introduced in ES cells, recombines with normal genes. Two recombination events are required

- distinct from standard transgenics where the DNA integrates randomly

Conditional Gene Targeting

- express Cre recombinase under the control of a cell type-specific promoter

- make a second mouse via knock-out to introduce loxP sites

- cross the two mouse lines to get a tissue-specific knock-out

- all cells in the body have the knocked-in loxP gene and the promoter-Cre expression construct

Knockout

when the gene is genetically inactivated

Knock-in

when the gene is modified, but not necessarily inactivated

When will conditional knockout occur?

Only in cells in which the Cre recombinase is expressed. Driven by the gene promoter that was chosen to link the Cre.

Inducible Cre (CreER)

similar to Cre, has been fused to the ligand binding domain of the estrogen receptor. Cannot move into the nucleus until the drug tamoxifen is provided. Ex: reversal of Rhett syndrome in mouse model

Tamoxifen

Analog of estrogen, either injected into animals or in cell culture medium to allow CreER to move into nucleus

Temporal Component

Important in neuroscience as it enables one to study a function of a gene and its encoded protein in adults. Delay

CRISPR/Cas9

- system of clustered regularly interspaced palindromic sequences (CRISPR) together with a nuclease enzyme called Cas9 that evolved in bacteria to destroy invading viruses.

- works by generating short RNA stretches that exactly match the viral DNA

- utilized for generating targeted mutations and for editing

- has potential to correct human diseases

Protospacer Adjacent Motif (PAM)

a three nucleotide NGG sequence at the end of the optimal 20 nucleotide stretch.

Non-homologous end-joining (NHEJ)

- a way to generate short deletions or insertions

- engineer NGG sequence into a full length gRNA. gRNA attached to Cas9 will find matching sequence in genomic DNA of the cells, forming a RNA-DNA hybrid

- Cas9 nuclease will cut the DNA 3-4 nucleotides upstream of PAM, will then re-ligate (reconnect) the DNA

Homology directed repair (HDR)

- insert a much larger DNA sequence at the position of cleavage by Cas9

- need to inject a donor DNA into the cells along with Cas9 & gRNA

- donor DNA includes portion of the 20 nucleotide sequence at each end

- can also be used to "edit" a mutation by replacing it with a corrected sequence

RNA interference (RNAi)

reduces the level of RNA by degrading it, reducing the amount od protein that can be made

RNAi process

- express a short-hairpin RNA (shRNA) or double-strand RNA (dsRNA) that is designed to be complementary to a region of the mRNA that encodes the protein desired to be knoced-down

- shRNA is processed by the ribonuclease Dicer to make a short interfering (si) RNA. siRNA binds to the RISC complex, converting siRNA to a single-strand antisense RNA that is complementary to mRNA, mRNA is degraded

- main goal: reduce the level of specific protein to result in phenotype or knock down mutated proteins, knock down expression of protein.

RISC complex and Dicer

- part of normal system in cells that process microRNAs (miRNAs)

- miRNAs are encoded by genes, don't code for proteins. Regulates the level of selected proteins by causing degradation of their mRNAs

Antisense Oligonucleotides (ASOs)

- Designed, short, synthetic, single-stranded DNA molecules whose sequence complements a short stretch of sequence on the mRNA

- forms a double strand RNA-DNA hybrid

- mRNA is sense, ASO is antisense

- can be injected intrathecally (into CSF fluid in spinal cord) or intraventricularly (into CSF-filled ventricles in brain)

- therapy has shown no benefit in humans

Gene Expression Changes

Bulk Measures (no cellular resolution)

- Protein: Western blotting, ELISA

- mRNA: qRT-PCR, RNA-Seq

Cellular Level Measures

- Protein: Immunohistochemistry (IHC), Immuno EM

- mRNA: In situ hybridization, single cell RNA-Seq

Western Blotting

- homogenize tissue

- extract proteins: denature (completely uncoiled) using detergent (sodium dodecylsulfate)

- separate proteins based on size using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS places a uniform negative charge per unit length on unwound protein so they migrate towards positive pole at a rate determined by the length of the amino acid chain.

- transfer from gel onto blotting membrane, incubate membrane in primary antibody, antibody binds to protein of interest

- add secondary antibody that recognizes first, chemically modified to have compound that produces light, expose the membrane rto x-ray film

- quantify protein band by using a densitometer

Enzyme-Linked Immunosorbent Assay (ELISA)

- antigen/sample is added to plate

- blocking buffer is added to block remaining protien-binding sites

- a suitable primary antibody is added

- a suitable secondary antibody (HRPO conjugate) is added which recognizes and binds to the primary antibody

- TMB substrate is added and is converted by HRPO to detectable form

RNA expression

- roughly 10,000 genes expressed in any brain region, RNA extracted from tissue will be a complex mixture of all transcribed RNAs from expressed genes

- some genes highly expressed, some low, some in between

- level of expression is largely a reflection of the transcriptional rate of that gene

- a gene that is highly expressed makes up higher percentage of total RNA than weakly expressed, so the "signal" measured in any assay will be proportionally higher.

Reverse Transcriptase (RT)

- important exception to Central Dogma

- convert RNA to DNA

- use an enzyme from RNA viruses to make a DNA copy from an RNA template

Polymerase Chain Reaction (PCR) - Process

- for amplification of specific DNA sequences

- very sensitive

- carried out in computer-controlled heating block in 3-step cycle

- thermostable DNA polymerases are critical for PCR

Polymerase Chain Reaction (PCR) - Concept

- need primers to amplify a stretch of the sequence of interest

- Logarithmic amplification: each cycle doubles the DNA. 20 cycles = 1,000,000-fold amplification

qPCR/RealTime PCR

- powerful quantitative PCR method

- can only quantify one RNA at a time

- uses fluorescent dye that gives off light when bound to double stranded DNA. More dsDNA, more signal

- takes ~20 cycles to make baseline signal, then signal grows exponentially with each cycle

RNA-Sequencing

- measures changes in many RNAs at the same time

- purify RNA from cells or tissue; a bulk method, no cellular resolution

- use equal amount of RNA to generate short cDNA fragements

- Number of reads directly correlates with gene expression level

- high-throughput

Immunohistochemistry (IHC)

- a method to analyze protein expression at the single cell level

- can only analyze one protein at a time

- Quantifiable

In situ hybridization (ISH)

- a method to analyze RNA expression at the cellular level

- use RNA or DNA probe that is complementary to the RNA you want to detect

- study one RNA at a time

single cell RNA sequencing

- relatively new method that allows high throughput analysis of changes in gene expression in individual cell types

- cellular resolution of gene expression

use of light-gated channels from algae and other marine species.

- Adapted as a technique for controlling neural firing via light

- Major advantages: selectivity and temporal precision

- excite or inhibit specific neurons in living animals

Electrical Synapses

- presence in cells helps to coordinate firing of cells or neurons

- only small molecules and ions can move through the gap junctions, like cAMP, ATP, Ca2+

Gap Junction

- six connexin subunits make up on connexon, two connexons make one gap junction channel, many gap junction channels needed for one gap junction.

- bidirectional, makes the cell electronically couples, enables faster communication

- Synchrony is important

- If connexin 36 is knocked out, lose synchrony

Otto Loewi

- in 1921, identified vagusstoff (acetylcholine) released as a chemical neurotransmitter.

- helped establish the concept of chemical transmission between neurons

Chemical Synapses

- have a presynaptic and postsynaptic side with a cleft

- cleft is not empty, but contains extracellular matrix proteins that help organize the synapse

- highly organized

- Presynaptic side: active zones, docked vesicles, secretory granules (large dense-core vesicles)

- Postsynaptic side: postsynaptic dendrites with receptors and associated proteins

CNS Synapses

- have different sizes and configurations

- synapses can occur between axons and dendrites, axons and cell bodies, axons and axons, and dendrites and dendrites

- astrocytes help regulate signaling

- two categories of CNS synaptic membrane types: Asymmetrical (Gray's Type I: usually excitatory) and symmetrical (Gray's Type II: usually inhibitory)

Neuromuscular Junction (NMJ)

- between motor neurons and muscles

- similar to CNS synapses, easier to study

- Fast, large, reliable

- Motor endplate and large number of active zones

- Active zones alligned with junctional folds on postsynaptic side, allow for more transmitter receptors

- Acetylcholine main neurotransmitter for NMJ (excitatory)

- An action potential in the motor neuron generates an endplate potential (EPP) that always results in a muscle action potential and contraction. ~40-50 mV

Steps of Synaptic Transmission

1. Synthesis of neurotransmitters

2. Loading into vesicles or secretory granules (dense-core vesicles)

3. Release of vesicles

4. Neurotransmitter binding to postsynaptic receptors

5. Electrical response through transmitter-gated channels or biochemical response through G protein-coupled receptors

6. Removal or destruction of neurotransmitter

Peptide Neurotransmitter Synthesis

synthesized in the soma like other proteins and transported in secretory granules via the fast axonal transport mechanism

Amine neurotransmitters Synthesis

synthesized at axon terminals and loaded into vesicles there by specific vesicular transporters

Glycine and glutamate Synthesis

present in all cells as basic amino acids used in protein synthesis, but are loaded into synaptic vesicles by specific vesicles vesicular transporters

GABA synthesis

synthesized from glutamate by glutamic acid decarboxylase and loaded into vesicles

Neurotransmitter release

the action potential depolarizes the axon terminus, opening voltage-gated Ca2+ channels, causing vesicle fusion and neurotransmitter release by exocytosis

Docking & Priming

- involve proteins that tether the vesicles to the active zone

- Ca2+ influx through voltage-gated Ca2+ channels results in vesicle membrane fusion at the active zone and release of vesicle contents into the synaptic cleft (exocytosis)

- diffuses across in 0.05 msec

Proteins involved with docking and priming

- Synaptobrevin is the v-SNARE, and SNAP-25, Syntaxin and Munc18-1 are the t-SNAREs. Together make up the SNARE complex which "snaresa" the vesicle to the active zone.

- Once Ca2+ enters the terminal, syntaxin in the active zone binds synaptotagmin on the vesicel and helps with vesicle fusion and subsequent release of transmitter

Synaptic vesicle recycling

- Three possible mechanisms: Kiss-and-run, clathrin-mediated endocytosis, ultrafast endocytosis and endosomal budding

- endocytosis = opposite of exocytosis

Neurotransmitter deactivation

- neurotransmitter must be deactivated after binding, could "lock up" a circuit or leak over to another synapse

- some neurotransmitters removed from synaptic cleft by reuptake transporters in presynaptic site, or by transporters in astrocytes

- can be degraded by enzymes

Inhibitors & Activators of neurotransmitter channels

- Sarin gas

- black widow spider venom

- curare (antagonist)

- nicotine (agonist)

Peptide neurotransmitters

- large dense-core vesicle containing neuropeptides are excluded from the active zone and located at greater distances from the synapse

- neuropeptides generally released in response to higher Ca2+ levels

- often released extrasynaptically and can be released from soma and dendrites

Neurotransmitter receptors

- neurotransmitters bind to receptors (specialized proteins) that match the molecular shape of the transmitter molecule (key in a lock). Ionotropic and metabotropic

Ionotropic receptors

- ligand- or transmitter-gated channel; fast acting

- 4-5 subunits

- not as selective as voltage-gated channels

- pore closed until ligand binds, ligand changes conformation and channel opens

- produces EPSPs or IPSPs

Metabotropic rececptors

G-protein coupled, not direct channel, but transmitter binding causes biochemical activation of effector pathways that may open a channel or cause other molecular changes; slow acting

Local Potentials

- action potential arriving in presynaptic terminal causes release of neurotransmitter

- if Na+ enters, depolarized

- if Cl- enters, hyperpolarized

Reversal Potential

the critical value of Vm at which the direction of current flow reverse = ~0 mV for the ACh receptor at the NMJ

EPSP summation

- a single action potential from a presynaptic axon in the CNS results in transmitter release and a local EPSP that is small and likely subthreshold

- Spatial and temporal summation can result in larger net effects on Vm when measured at soma

Dendritic cable properties

- determine the strength of any signal reaching the spike-initiation zone

- amount of depolarization falls of exponentially with distance from stimulation

- length constant (λ)

Shunting inhibition

- EPSP + no IPSP = small EPSP at soma

- EPSP + IPSP = negated EPSP

- anything that would reduce the leakage of depolarizing current and thus increasing membrane resistance would increase the length constant, increasing propagation of local potentials

G-protein coupled receptors

- activation does not directly evoke EPSPs or IPSPs, but modifies the effectiveness of EPSPs generated by other synapses with transmitter-gated channels

- this type of synaptic transmission is called modulation

Criteria for defining a chemical as a neurotransmitter

1. Presynaptic cell should manufacture, contain, & inactivate the chemical

2. Stimulation of cell should release chemical

3. Postsynaptic cell should have receptors for the chemical

4. The molecule, when experimentally applied, must produce a response in the postsynaptic cell that mimics the response produced by the release of neurotransmitter from the presynaptic neuron

5. Application of a receptor-specific antagonist should block the action of the chemical

Microintophoresis

- presumptive neurotransmitter can be applied to a target neuron with a pipette

- electrophysiological recording can be made

- response can be compared to that of the stimulated presynaptic axon

Agonist

any molecule, synthetic or natural, which binds to the receptor and causes the same action as the natural substance that normally binds to the receptor

Antagonist

any molecule, synthetic or natural, which binds to the receptor and blocks the action of an action of an agonist at that receptor

Dale's Principle

(1954) A given neuron contains and releases only one neurotransmitter and exerts the same functional effects at all its termination sites

(generally true)

Catecholamines

- subtype of monamines

- dopamine, norepinephrine, epinephrine (adrenaline)

Dopamine functions

executive functions (attention, inhibition, working memory, behavioral flexibility), motor control, motivation, arousal, reward (and addiction), and aversion

Norepinephrine (noradrenaline) functions

alertness, arousal, attention, readiness for action, feeding behavior, sleep, mood, learning & memory particularly related to fear conditioning and its extinction. Reduce background noise and increase responses to strong stimulation

Epinephrine functions

present in few neurons (role not fully understood). Important for fight-or-flight response. Regulates cardiac & respiratory function

Key enzymes in catecholamine synthesis

Tyrosine hydroxylase (TH)

Dopamine-β-hydroxylase

PNMT

Tyrosine hydroxylase (TH)

- rate limiting step in catecholamine synthesis; localizes in cytosol

- regulated at the protein and RNA levels

- found in ventral tegmental area, substania nigra, and locus coeruleus

Dopamine-β-hydroxylase

- present in NE neurons, localized in synaptic vesicles

- in locus coeruleus only

PNMT

- for synthesis of EPI in adrenergic (EPI) neurons; localized in cytosol

Catecholamine Vesicle Loading

- loaded into vesicle by vesicular monamine transporters (VMAT1 & VMAT2; V=vesicular; genes called SLC18A1 and SLC18A2)

Catecholamine transmitter recycling

- once released into the synaptic cleft, transported back into terminal by specific Na+ dependent transporters (DAT & NET; genes are SLC6A2 and SLC6A3)

Catecholamine Projection Pathways

dopaminergic and noradrenergic pathways

Dopaminergic pathways

Nigrostriatal: control of voluntary movement

Mesolimbic: control of reward and motivated behaviors (addiction, Nucleus accumbens - ventral striatum)

Mesocortical pathway: cognitive control, decision making

Noradrenergic pathways

- LC hypothalamic projections modulate sleep/wake

- LC amygdala and hippocampal projections modulate fear and escape behaviors, etc.

main neurotransmitter in the sympathetic chain

Norepinephrine

- NE neurons in the sympathetic ganglion chain send axons along spinal nerves to their organ targets