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Bio 222 Cell/Molec Neuroscience > Exam 2 > Flashcards

Exam 2 Flashcards

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1
Q

Central Dogma

A

Crick

DNA –transcription–> RNA –translation–> protein

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2
Q

gene structure

A

promoter: initiates expression

5’UTR: regulates gene expression

open reading frame: RNA information (introns and axons)

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3
Q

mRNA: location and function

A

functions in nucleus, migrates to ribsomes

carries DNA sequence info to ribosomes

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4
Q

tRNA: location and function

A

functions in cytoplasm

provides linkage b/w mRNA and AAs, transfers AAs to ribosomes

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5
Q

ribosomal RNA/rRNA: location and function

A

functions in cytoplasm

structural component of ribosomes

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6
Q

post transcription regulation: how are eukaryotic genes segmented?

A

enzymes cut out introns

exons spliced together to make mRNA

more than 90% of pre-mRNA is destroyed (introns)

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7
Q

alternative splicing

A

single gene can code for multiple proteins by mixing and matching exons

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8
Q

mutations occurs as ____ and cause changes in the _____

A

random chance events, DNA sequence

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9
Q

if mutations occur in ____, then ____

A

gametes, they can be passed onto offspring

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10
Q

mutations may be caused by ____

A

exposure to toxins or radiation (mutagens)

others lead to variations that are good for organism to adapt to environment

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11
Q

two kinds of gene mutations

A
  • Gene mutations:
    • Single gene
    • Substitution, stop, inversion, insertion, and deletion
  • Chromosomal mutations:
    • Abnormal chromosome structure (affects multiple genes)
    • Substitution, stop, inversion, insertion, deletion, and translocation
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12
Q

down syndrome

A

trisomy 21 (extra 21 chromosome)

alters child’s phenotype - characteristic facial features, short stature

usually some mental retardation

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13
Q

major types of genetic disordesr

A
  • Autosomal
    • single genes
    • multiple genes
  • Sex-linked
  • Chromosome abnormalities (eg down syndrome)
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14
Q

Recessive traits vs dominant

A

recessive: normally loss-of-function mutation

dominant: normally gain-of-function mutation

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15
Q

autosomal genetic disorders + examples

A

caused by alleles on autosomes (chromosomes other than sex chromosomes)

most are recessive (need two alleles)

carriers ex (have 1 recessive) : CF, sickle cell

dominant ex: huntingtons (only need 1 allele)

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16
Q

sex linked genetic disorders

A

common Y-linked disorder: male infertility

X-linked recessive disorders (most common in males): hemophilia, color blindness, muscular dystrophy, fragile-X syndrome

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17
Q

2 ways of identifying disease mutations

A
  • Linkage analysis:
    • Data collected for family members
    • Good for rare disorders
  • Genome-wide association studies (GWAS)
    • Data colleced for unrelated individuals
    • Good for common diseases
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18
Q

autosomal dominant vs recessive, how to identify

A

dominant: affected parents have affected children

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19
Q

how to identify x linked dominant vs recessive

A
  • x-linked dominant:
    • affected mother: either son or daughter can be affected
    • affected father: only can pass to daughter, but not son
  • x linked recessive:
    • female carrier: only son can be affected
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20
Q

SNP vs microsatellite

A

single nucleotide polymorphyism: just one base changed at specific point

microsatellite: series of bases repeated several times

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21
Q

types of SNPs: figure

A

synonymous: single nucleotide change does not change AA sequence
missense: one AA change

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22
Q

GWAS has been applied to ___, and what does graph mean

A

alzheimers, autism, schizophrenia

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23
Q

which animal models easy to house large numbers

A

best (a): C. elegans (worm), fruit fly, zebra fish

also good (b): mice

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24
Q

which animals have large number of offspring

A

best (a): C. elegans, fruit fly, zebra fish

also good (b): mouse

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25
Q

animals that are good genetic tools

A

best (a): C elegans, fruit fly, mice

also good (b): zebra fish, monkey

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26
Q

which animals have short generation time

A

best (a): C elegans, fruit fly

also good (b): zebra fish, mice

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27
Q

which animals have transparancy

A

best (a): zebra fish

also good (b): C elegans, fruit fly

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28
Q

which aniamals have similarity in organization to human CNS

A

mice and monkeys

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29
Q

animals good for studying single neuron vs cognitive learning/memory

A

single neuron: C elegans

cognitive learning/memory: monkey

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30
Q

Morgan nobel prize (193)

A

studied sex limited inheritance in flies

discovered the role that chromosome in heredity

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31
Q

what are UAS-GAL4 and FRT-FLP systems used for

A

UAS-GAL4: used to manipulate gene expression

FRT-FLP: used to make mosaic clones

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32
Q

forward vs reverse genetics

A

forward genetics: identify interesting phenotype, then discover the genes defective in the mutants

reverse genetics: alter known gene and see how phenotype changes

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33
Q

forward genetics: how are random mutated genes introduced

A
  • Chemical: ethyl methyl sulfonate (EMS)
    • Point mutations, tedious mapping process
  • Radiation: X-ray or gamma rays
    • Chromosome deletions/rearrangement, inefficient
  • Transposons (P element)
    • trigger DNA insertion, easy mapping
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34
Q

classic F3 screen for recessive mutations

A
  • Diploid screen for dominant mutations (F1 screen)
  • Diploid screen for recessive mutations (F2 screen)
  • Diploid screen for recessive mutations (specific locus screen)
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35
Q

nobel prize in physiology or medicine, 1995

A

Lewis, Nusslein-Volhard, Wieschaus

nobel prize for discoveries on genetic control of early embryonic development

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36
Q

sonic hedgehog

A

can be loss of function (mutant embryo much smaller) or gain of function (double wing, double fingers, double head in snakes)

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37
Q

UAS-GAL4

A
  • used to manipulate gene expression in flies
  • derived from yeast
  • GAL4 = transciption factor
  • UAS = upstream activating sequence
  • when GAL4 bind to UAS, triggers gene expression
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38
Q

mosaic analyses definition

A

express mutation in only some daughter cells

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39
Q

FRT-FLP system

A
  • used to label cells via mosaic techniques
  • site-directed chromosomal recombination
  • FRT = specific DNA sequence
  • FLP = flippase or FLP recombinase
  • randomly label some daughter cell, but not all cells
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40
Q

general approach to doing reverse genetic manipulation

A
  1. Build a vector (brings foreign DNA into cell)
    1. Gene targeting vector for homologous recombination
  2. Use vector to transfect cells of interest and express foreign protein
    1. expression may be transient or stable
      1. transient expression may be inducible or repressible
  3. Assay structure or function
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41
Q

what is addgene

A

website that’s a database of vectors

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42
Q

reverse genetics: building an appropriate construct (what are the pieces)

GFP example

A

want to construct pcDNA3.1-GFP:

  1. CMV promotor (generic promoter to express genes in all cell types)
  2. GFP gene
  3. Stop sequence: bGH poly(A) signals
  4. pcDNA3.1-GFP for subcloning
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43
Q

reverse genetics: transfection involves what

GFP example

A
  • can transfect into cell lines/cultured neurons (in vitro):
    • calcium phosphate transfection: carries DNA into the nucleus, not high efficiency (not many neurons are transfected)
    • lipid transfection: higher efficiency
    • electroporation: makes pores in membrane for DNA to enter; highest efficiency
  • can also transfect in vivo:
    • electroporation in utero, then inject construct
    • low efficiency in vivo
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44
Q

transgeneic animals

A
  • integrate foreign DNA randomly
  • expression is controlled bc of endogenous sequences + inserted DNA has promoter sequence and coding sequence of gene of interest
  • possible in any animal
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45
Q

knockout or knockin animals

A
  • Replace endogenous gene w/ a version that cannot function (knockout, KO) or functions differently (knockin, KI)
  • Expression controlled by endogenous promoter
  • Works via homologous recombination
  • efficient only in flies and mice
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46
Q

how to make a transgenic mouse

A

make a transgenic vector containing promoter, gene of interest, and stop coding sequence; then integrate into genome

  • Random insertion
  • Unknown copy number (may insert 1 or more copies)
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47
Q

how to make a transgenic mouse: method 1

A
  1. Microinjection of foreign DNA into fertilized oocytes (include promoter)
  2. Implant into foster mother
  3. Screen offspring by southern blotting or PCR
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48
Q

potential problems that can arise when using transgenic animals

A
  • lack of control over where DNA integrates
    • sometimes transgene is silenced by local elements)
    • sometimes transgene insertion can cause an unintended mutation
    • sometimes expression of phenotypes are not directly a result of the transgene
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49
Q

4 key steps to making KI and KO mice through homologous recombination

A

isolate and characterize gene of interest

generate a targeting vector

perform homologous recombination in embryonic stem cells

generate mice from the ES cells that express the modified gene

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50
Q

how to make a KO mouse: targeting and ES cell screening

A
  1. Design construct w/ stop codon to disable gene of interest
  2. Replace endogenous copy of gene by homologous recombination
  3. Transfect mouse ES cells (pluripotent) w/ KO construct
  4. Add positive selection marker (usually neomycin resistance) to select for cells that have taken up DNA (all cells w/o neo cassette will die)
  5. Add negative selection marker (construct w/ toxin; if randomly inserted, toxin will kill cells)
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51
Q

how to make a KO mouse: what do you do after developing your construct

A

inject ES cells into a mouse blastocyst

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52
Q

how to make a KO mouse: what do you do after you inject ES cells into a mouse blastocyst

A

transplant blastocyst into pseudopregnant mother

generates chimeric mice (usually use ES cells from a mouse line w/ different coat color than blastocyst to identify chimeras that have both colors)

cross best chimeric mice w/ WT mice to get germline transmission

identify gene targeting by southern blotting or PCR

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53
Q

summary steps of making KO mouse

A
  1. Make targeting construct
  2. ES cell transfection
  3. Positive (neo resistance) and negative (HSV-tk) screening
  4. Inject ES cells into blastocysts
  5. Implant blastocysts into foster mother
  6. Birth and breeding of chimeric mice
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54
Q

Nobel prize for creation of knockout mice

A

Capecchi, Evans, Smithies

developed principles for introducing gene modifications in mice by using ES cells

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55
Q

KI mice

A

directly overexpress GFP by replacing endogeneous genes

use positive (neo) andnegative (diptheria toxin, directly kills cells after expression) screen

express GFP w/ control of Gfib promoter

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56
Q

if ES cells are derived from mouse strain 129/SV with agouti coat and
the recipient blastocysts are derived from the mouse strain CD1 with white
coat, which offspring in the picture is the best chimeric mice?

A

mouse with most agouti (brown) coating

originates more from strain 129/SV (ES cells), so higher chance of having gene

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57
Q

common problems of making KO or KI mouse

A
  • many KOs have no phenotype
  • many transgenic, KO, or KO modifications are lethal during early development
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58
Q

name the different promotoers and where they are expressed

A
  • GFAP promoter: expression only in astrocytes
  • MBP promoter: oligodendrocytes
  • GAD67 promoter: GABA expression neurons
  • CAMKIIα promoter: glutamatergic neurons of forebrain (hippocampus)
  • ChAT promoter: motor neurons
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59
Q

example of how we can selectively knockin a gene in motor neurons

A

Conditional knockin mice:

  • Use ChAT promoter with Cre recombinase
  • Cross Lox-Stop-Lox transgenic mouse with ChAT Cre mouse
  • Cross triggers deletion of stop cassette only in cells w/ Cre, then promoter can drive transgene expression just in motor neurons
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60
Q

example of how we can selective knockout a gene in motor neurons

A

Conditional knockout mice:

  • Use ChAT promoter with Cre recombinase
  • Cross floxed mouse (gene sandwiched b/w two LoxP sites) with ChAT Cre mouse
  • Cross triggers deletion of gene only in cells w/ Cre
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61
Q

spatial control in fruit flies example (intersectional approach): GAL4 and FLP systems

A
  • GAL4 activates UAS system (but prevented by stop cassette flanked by FRT)
  • Use FLP to remove stop cassette to trigger gene expression
  • Use two different promoters for GAL4 and FLP; only when both promoters are active is the gene expressed (AND logic)
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62
Q

spatial control in fruit flies example (intersectional approach): GAL4 and GAL80 systems

A
  • GAL80 prevents GAL4 from interacting with UAS
  • only cells that express promoter A but not B can express the gene (NAND logic)
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63
Q

FRT-Flp system

A

Flp: catalyzes DNA recombination

FRT: 34bp DNA sequence

naturally occurring in yeast

  • if FRT oriented in same direction: delete DNA in b/w
  • if FRT oriented in different direction: invert DNA
  • if FRT on two diff homologous chromosomes: translocation of arms of chromosomes
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64
Q

Cre-Lox system

A

Cre: recognizes and cuts specific LoxP sites in DNA

orientation of LoxP sites determines how DNA is manipulated

LoxP is 34bp sequence as well

derived from bacteriophage

if LoxP in same direction: deletion

if LoxP in different direction :inversion

if LoxP on each homologous chromosomes: translocation of arms

65
Q

CreER

A

modified estrogen receptor (ER, fused to Cre recombinase) that; binds tamoxifen (TAM) and not endogenous estrogen

Cre sequestered in cytoplasm until TAM administrated, so no recombination can occur until desired

temporal control of recombination

66
Q

2008 nobel for discover of GFP

A

Tsien, Chalfie, Shimomura

67
Q

calcium indicators

A

GCaMP (genetically encoded calcium indicator) is a fusion of:

  • EGFP: fluoescence
  • Calmodulin (CaM): binds calcium
  • M13

can be used as a proxy for neuronal activity

68
Q

intersectional genetic techniques

A

use combinations of multiple genetic lines of mice to get specific expression of a certain gene

69
Q

chronic itch mouse model

A

3 separate mouse lines crossed together to selectively express human diphtheria toxin receptor

NPY-Cre + LPX1-FLPO + Tau reporter line

results in Tau-DTR (expresses DT receptor

70
Q

spatial control in mice example: Cre recombinase and lox orientations

A

if both LoxP sites are in same orientation: DNA b/w them deleted when Cre protein is present

if both LoxP sites are in opposite orientation: DNA b/w them inverted when Cre protein is present

71
Q

Flpo

A

mutant of Flp, higher recombination efficiency in mammalian cells

72
Q

DTR

A

diptheria toxin receptor

when diptheria toxin binds, inhibits protein synthesis and kills cells

73
Q

tet-on and tet-off system

A

based on a bacterial protein (tetR) that binds to specific sequences, dependent on tetracycline

tTA (tet transactivator) can only bind to DNA when doxycycline (Dox, Tet analog) is absent

rtTA (reverse tet transactivator) can only bind to DNA when it is bound to Tet or Dox

74
Q

limitations and challenges of current mouse KO technology

A

making and selecting ES cells, mouse breeding is time consuming

75
Q

nobel prize for discovery of RNA interference

A

Fire and Mello

76
Q

RNAi

A

dsRNA, rather single-stranded antisense RNA, is the interfering agent

highly specific and potent

knocks down, rather than eliminating (KO), expression

affects mRNA, unlike KO (genes)

77
Q

how does RNAi work

A

viral dsDNA integrates into eurkaryotic genome

Dicer recognizes and cuts shRNA

RISC generates siRNA

siRNA finds a complimentary sequence on mRNA, RISC cleaves and inactivates that mRNA

78
Q

sherrington

A

nobel prize winner

first to describe the gap between neurons, called synapse

79
Q

electrical synapses

A
  • Bidirectional transfer of info
  • Pre and postsynaptic cell membranes close to each other, connected by gap junctions that allow ion flow b/w cells
    • Faster than chemical synapses (<0.1 ms)
80
Q

Gap junction

A
  • Gap junction made up of two connexons (one in each membrane), each connexon made up of 6 connexins
  • An AP in presynaptic neuron produces response in postsyanptic neuron (bidrectional)
81
Q

Chemical synapse

A

Chemical messenger: NT or hormone

synaptic delay of 1-2 ms (time to open voltage gated Ca)

unidirectional

82
Q

types of CNS synapses

A
  • Axodendritic: axon to dendrite
  • Axosomatic: axon to cell body
  • Axoaxonic: axon to axon
  • Dendrodendritic: dendrite to dendrite
83
Q

nobel prize for discovery of chemical transmission of nerve impulses

A

Ach inhibitory in cardiac muscle: Loewi

Ach excitatory in skeletal muscle: Dale

84
Q

types of antagonists

A

competitive antagonist: binds to same site as agonist

non-competitive antagonist: binds elsewhere on the receptor

85
Q

positive modulator

A

ligand that has no effect on its own, but makes the agonist more effect

binds somewhere else than binding site

86
Q

4 criteria for identifying NTs

A
  1. Must be synthesized in neuron or be present in it
  2. When neuron is active, must be released and produce a response in a target
  3. Same response must be obtained when chemical is experimentally placed on target
  4. Mechanism must exist to remove for synapse
87
Q

Ach, Glutamate, GABA

excitatory or inhibitory

A

Ach: excitatory in skeletal muscle

Glutamate: dominant excitatory NT in brain

GABA: dominant inhibitory NT in brain

88
Q

what is the excitatory NT at arthropid NMJs

A

NOT Ach

Glutamate

89
Q

monoamine neurotransmitters can be….

A

secreted into extracellular space outside synapses to affect nearby cells (aka volume transmission)

90
Q

dorsal root ganglion neurons

A

have axons in spinal cord

primary sensory neurons sensing touch, temp, pain, itch

91
Q

Large dense-core vesicles (LDCVs)

A

75-100 nm

contain peptides and/or proteins

92
Q

Small clear-core vesicles (SCCVs)

A

classic synaptic vesicle

40-50 nm diameter

store/deliver small NTs

93
Q

ionotropic receptors

A

ligand gated ion channels

rapid communication across synapse

94
Q

metabotropic receptors

A

trigger intracellular signaling cascades to regulate ion channel conductance

exert function slowly

95
Q

3 types of ionotropic receptors

A
96
Q

examples of excitatory and inhibitory ionotropic receptors

A

excitatory: iGluR (ionotropic glutamate receptor)
inhibitory: GABA and glycine receptors

97
Q

all metabotropic neurotransmitter receptors…..

A

trigger G protein cascades (via GPCR)

98
Q

excitatory GPCR pathways

A
  1. cAMP is second messenger: activates Ca2+ channels causing depolarization
  2. diverse signaling pathways: alter membrane conductance and cause depolarization
99
Q

inhibitory GPCR pathways

A

activate K+ channels

inactivate Ca2+ channels

reduce cAMP level

100
Q

Katz

A

identified major mechanisms of synaptic function at the frog neuromuscular junction

101
Q

Katz’s standard recording set up

A

at NMJ: used glass electrode and ACh pipette

very close to nerve terminals, acetylcholinesterase inhibitor present, and TTX to block propagation of APs

102
Q

End-plate potential (EPP)

A

depolarization at the end-plate (where message is sent to muscle cells)

muscle specific name for EPSP

ACh evokes EPPs

103
Q

what did Fatt and Katz recordings of EPPs look like

A
  • at synapse: spontaneous mini EPPs w/o any nerve stimulation
  • not as noticeable 2mm from synapse
  • there is a delay in AP following nerve stimulation
104
Q

what are mEPPs cause by

A

release of multi-molecular packets of transmitter

105
Q

what leads to EPP failures

A

low [Ca2+]o or high [Mg2+]o reduces EPP size and leads to failures

106
Q

quantum hypothesis (Katz)

A

transmitter is released in multimolecular packets, or quanta

packets are released spontaneously at low frequency (mEPP is 1 packet released)

arrival of AP increases frequency of release

107
Q

transmitter release is a ______ event

A

probabilistic

108
Q

binomial distribution to predict quantal content: what do variables stand for

A

p = probability of success per trial

probability of getting k successes in n trials

109
Q

when can Poisson distribution be used to predict quantal content

A

when p<<1 and n → ∞

probability of success (release) is very low and number of trials is very high

m is the average number of release

at NMJ, n is large; with low calcium, p is low

110
Q

is the poisson distribution relevant when synapses operate at physiological calcium

A

NO

p is no longer small

for most synapses in CNS and physiological calcium, binomial distribution should be used

111
Q

vesicle hypothesis

A

a quantum of transmitter is that amount stored in a synaptic vesicle

release occurs via exocytosis

112
Q

calcium ___ rises in presynaptic terminals following APs

A

rises

113
Q

each quantum is about _____ at the frog NMJ

A

10,000 ACh

114
Q

how to measure Calcium current at a synapse

A
  • voltage clamp presynaptic and postsynaptic side of synapse
    • Add TTX (block Na channels) and TEA (block K)
  • in presynaptic terminals, there are only voltage gated Ca currents (measure this)
  • Repeat at multiple voltages to get peak current vs voltage curve, then divide by driving force to get g-V curve
115
Q

conclusions of voltage clamp ca current experiment

A

synaptic delay of ca release results from sluggish opening of calcium channels

all steps after calcium entry are very rapid

116
Q

key steps in excitatory synaptic transmission: presynaptic to postsynaptic

A
  • AP travels down axon
  • Voltage gated Na channels open (depolarization)
  • Ca channels open and cytosolic Ca at release sites increases
  • Ca triggers synaptic vesicle fusion and NT release
    • NT content of one vesicle = quantum of NT
  • Opening of NT gated ion channels postsynaptic
  • Opening of voltage gated Na channels, new AP starts
117
Q

key steps in inhibitory synaptic transmission: presynaptic to postsynaptic

A
  • transmitters: GABA or glycine
  • opening of GABAA or glycine receptors
    • trigger hyperpolarization of membrane
118
Q

synaptic function is measure electrophysiologically by …..

A
  • excitatory or inhibitory postsynaptic currents (EPSCs or IPSCs)
  • or excitatory or inhibitory postsynaptic potentials (EPSPs or IPSPs)
119
Q

synaptic transmission is _____

key step is _____

A

rapid (1-5 ms)

neurotransmitter release

120
Q

synaptic process that govern NT release

A
  • synaptic vesicle fusion
  • Ca triggering of fusion
    • very fast (0.1 ms)
    • cooperative (5 ca ions)
    • localized ca influx
121
Q

nobel winners for research on vesicle transport in cells

A

Rothman, Schekman, Sudhof

122
Q

Scheller and Sudhof

A
  • biochemically purified synapic vesicles, identified as many proteins as possible, and try to figure out what they do
    • synaptobrevin (VAMP) was first protein they isolated from synaptic vesicles
      • also found synaptotagmin (another vesicle protein) and syntaxin (plasma membrane protein)
123
Q

Rothman

A
  • looked for proteins that bind SNAP
  • identified 3 proteins that form a complex that tightly bind SNAP (called SNARE proteins)
    • Synaptobrevin (previously found)
    • Syntaxin (previously found)
    • SNAP-25 (plasma membrane protein)
124
Q

vesicle transport: function of proton pump (V-ATPase)

A

establishes a pH and potential gradient that the vesicular neurotransmitter transporters use

there are separate transporters to load ACh, GABA, glutamate, etc.

H+ flows out, NT flows in

125
Q

vesicle transport: function of synapsin, synaptobrevin, synaptotagmin, and Rab3

A

synapsin: regulates location of vesicles

synaptobrevin (VAMP): component of core fusion complex, binds to partners on plasma membrane

synaptotagmin: key calcium sensory

Rab 3: regulates fusion

126
Q

synaptic vesicles undergo recycling: 2 modes

A
  • Clathrin-mediated endocytosis (vesicle fuses w/ membrane)
  • Ultrafast endocytosis: kiss and run (transient form of fusion)
127
Q

SNARE hypothesis for vesicle fusion

A

3 major proteins involved in vesicle fusion:

  • vesicle associated SNAP receptor (v-SNARE) in neurons is synaptobrevin (VAMP)
  • target sites have corresponding t-SNARE (syntaxin and SNAP-25)

v-SNARE and t-SNARE interact, causing vesicle fusion

128
Q

vesicle fusion: how many helices

A

4 helices involved:

1 helix from vesicle (VAMP/synaptobrevin, v-SNARE)

3 from plasma membrane (t-SNARE complex, 2 from SNAP-25, 1 from syntaxin)

129
Q

fusion mechanism: water and SNAREs

A

cytoplasmic domains of vesicular and target SNAREs strongly bind in zipper-like fashion, pushing out H2O

force generated by binding brings vesicle and target membranes together, causing them to fuse

130
Q

where does calcium bind on vesicle

A

synaptotagmin-1 is the calcium sensor

binding ca allows it to bind to membranes

KO of syt1 impairs ca triggered release (but miniEPSCs, spontaneous release w.o AP, still occur w/ presence of sucrose)

131
Q

model of the calcium activated membrane fusion complex

A

synaptotagmin has 2 calcium binding domains

binding loops of syt insert into bilayer when calcium is present

PIP2 helps 2 domains insert into bilayer

helps SNAREs to interact

132
Q

additional molecules that regulate fusion

complexin, Munc18, Rab3 and Rim

A

complexin: co-activator of synaptotagmin-1

Munc18: regulate assembly of SNARE complex and interaction w/ synaptotagmin

Rab3 and Rim: holds vesicles near the calcium channels

133
Q

summary: proteins involved in vesicle fusion

A

v-SNARE: synaptobrevin (VAMP)

t-SNARE: SNAP-25, syntaxin

complex drives vesicle fusion

Munc18 regulates this complex

134
Q

summary: proteins involved in Ca triggerings

A

synaptotagmin (ca sensor) and complexin

135
Q

summary: proteins involved in ca channel tethering

A

Rab3, RIM, Munc13

complex drives vesicle close to calcium channel, then fuses and NT release

136
Q

kiss and run model of transmitter release

A

opening of narrow fusion pore, followed by rapid pore closure

results in rapid vesicle recycling

full collapse fusion occurs in most transmitter release events

high calcium concentration shifts the mode of full collapse model to kiss and run mechanism

137
Q

neuromuscular junction and nicotinic ACh: what are the parts in presynapse and postsynapse

A

presynapse: synaptic vesicles (ACh), voltage gated Ca channels
postsyanpse: acetylcholinesterase, AChR (receptor), voltage gated Na channels

138
Q

Cys-loop superfamily: cation and anion channels

A

cation channels: nAChR, 5-HT3R

anion channels: GABAAR, GABACR, GlyR

139
Q

how was muscle AChR protein purified

A

based on binding to α-bungarotoxin

140
Q

nicotinic AChR structure (nAChR)

A

pentamer made up of four types of subunits

in torpedo and embryonic mammels: α2βγδ

in adult mammals: α2βεδ

each receptor must bind 2 ACh to open (binds at α-δ and α-γ/α-ε interface)

141
Q

how can you draw an I-V curve based on voltage step graph

reversal potential

A

look at peak current at each voltage step

reversal potential (when I=0) is the membrane potential at which current flow changes from inward to outward flow

142
Q

at reversal potential

A

Na influx equals K efflux

143
Q

dendritic spines vs dendritic shaft

A

excitatory synapses target dendritic spines (contain postsynaptic density)

inhibitory synapses target dendritic shaft (lack postsynaptic thickening)

144
Q

excitatory and inhibitory synapses contain….

A

unique set of channels, scaffolding proteins, and other molecules

145
Q

excitatory synaptic transmitters: ionotropic and metabotropic glutamate receptors

A
  • ionotropic receptors:
    • NMDA receptors (NMDAR): ligand is Glu, NMDA; Ca and K permeable
    • AMPA receptors (AMPAR): ligand is Glu, AMPA; Na permeable
    • Kalnate receptors (KAR): ligand is Glu, KA; Na permeable
  • metabotropic Glu receptors (mGluR): ligand is Glu
146
Q

glutamate receptor structure

A

four subunits: can be homotetramers or heterotetramers

NMDAR is usually heterotetramer

each subunit has 3 transmembrane helices

LBD: region that binds agonists

ATD: region that binds multiple modulators

147
Q

GABAA receptors

A
  • Cys loop family
  • Are chloride selective channels
  • Each subunit has large extracellular N terminal domain (cys loop) and large intracellular loop b/w 3rd and 4th transmembrane helices
  • 5 subunits
148
Q

competitive antagonist of GABAA receptors

A

bicuculline

binds at GABA binding site

149
Q

non-competitive antagonist of GABAA receptors

A

Picrotoxin

binds at different site than GABA

blocks the pore

150
Q

positive allosteric modulators of GABAA receptors

A
  • Barbituates: postive allosteric modulator of most
  • Diazepam (valium): works on some subunit combinations, not all
  • Ethanol: works on some

have no action when bound to receptor alone, but when agonist binds, changes probability that channel opens

151
Q

why is there lethal synergism b/w valium and alcohol

A
  • lethality due to inhibition of neurons that need to be active to drive respiration
  • Valium and ethanol both increase activity of GABAA receptors, increasing total amount of inhibition to lethal levels
152
Q

how can we detect postive allosteric modulation of GABAA receptors

A

keep [GABA] constant and vary [modulator]: no effect modulator alone

→ keep [modulator] constant and vary [GABA]: when diazepam used, less GABA required to achieve a response

153
Q

mechanisms by which postive modulators could produce larger currents

A

must prolong activation

  • make channel open more frequently
  • make bursts (channel openings) longer
154
Q

benzodiazepines _____ while barbituates ____

A

benzodiazepines: promote channel opening

barbituates: lengthen burst duration

155
Q

chloride and GABAA

A

GABAA (chloride channel) regulates intracellular chloride concentrations

  • low intracellular chloride: hyperpolarization, inward current
  • high intracellular chloride: depolarization, outward current
156
Q

NKCC1 and KCC2

A

early expression of NKCC1 and late expression of KCC2 determines developmental changes in intracellular chloride concentration

  • NKCC1: expressed early in life; Na-K-Cl cotransporter; high [Cl-]i; activation of GABAR exerts excitatory effect
  • KCC2: expressed in adults; K-Cl cotransporter; low [Cl-]i; activation of GABAR exerts inhibitory effect
157
Q

somatic EPSPs from dendritic inputs in a neuron: an EPSP must spread to….

A

an EPSP must spread to the the spike-initiation zone, and this zone must be depolarized beyond threshold to generate an AP

158
Q

the effectiveness of an excitatory synapse (on dendrite) for triggering AP depends on

A

how far the synapse is from the spike initiation zone

synapses closer to soma result in larger depolarization

159
Q

spatial vs temporal integration

A

spatial: two EPSPs at different spatial locations summate when they converge along their path to the soma

temporal: two EPSPs produced at the same synapse summate since they arrive at the same time

Bio 222 Cell/Molec Neuroscience (7 decks)

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