Exam 2 Flashcards
(159 cards)
1
Q
Central Dogma
A
Crick
DNA –transcription–> RNA –translation–> protein
2
Q
gene structure
A
promoter: initiates expression
5’UTR: regulates gene expression
open reading frame: RNA information (introns and axons)
3
Q
mRNA: location and function
A
functions in nucleus, migrates to ribsomes
carries DNA sequence info to ribosomes
4
Q
tRNA: location and function
A
functions in cytoplasm
provides linkage b/w mRNA and AAs, transfers AAs to ribosomes
5
Q
ribosomal RNA/rRNA: location and function
A
functions in cytoplasm
structural component of ribosomes
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)
7
Q
alternative splicing
A
single gene can code for multiple proteins by mixing and matching exons
8
Q
mutations occurs as ____ and cause changes in the _____
A
random chance events, DNA sequence
9
Q
if mutations occur in ____, then ____
A
gametes, they can be passed onto offspring
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
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
12
Q
down syndrome
A
trisomy 21 (extra 21 chromosome)
alters child’s phenotype - characteristic facial features, short stature
usually some mental retardation
13
Q
major types of genetic disordesr
A
- Autosomal
- single genes
- multiple genes
- Sex-linked
- Chromosome abnormalities (eg down syndrome)
14
Q
Recessive traits vs dominant
A
recessive: normally loss-of-function mutation
dominant: normally gain-of-function mutation
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)
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
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
18
Q
autosomal dominant vs recessive, how to identify
A
dominant: affected parents have affected children
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
20
Q
SNP vs microsatellite
A
single nucleotide polymorphyism: just one base changed at specific point
microsatellite: series of bases repeated several times
21
Q
types of SNPs: figure
A
synonymous: single nucleotide change does not change AA sequence
missense: one AA change
22
Q
GWAS has been applied to ___, and what does graph mean
A
alzheimers, autism, schizophrenia
23
Q
which animal models easy to house large numbers
A
best (a): C. elegans (worm), fruit fly, zebra fish
also good (b): mice
24
Q
which animals have large number of offspring
A
best (a): C. elegans, fruit fly, zebra fish
also good (b): mouse
25
animals that are **good genetic tools**
**best (a):** C elegans, fruit fly, mice
**also good (b):** zebra fish, monkey
26
which animals have **short generation time**
**best (a):** C elegans, fruit fly
**also good (b):** zebra fish, mice
27
which animals have **transparancy**
**best (a):** zebra fish
**also good (b):** C elegans, fruit fly
28
which aniamals have **similarity in organization to human CNS**
mice and monkeys
29
animals good for studying single neuron vs cognitive learning/memory
single neuron: C elegans
cognitive learning/memory: monkey
30
Morgan nobel prize (193)
studied sex limited inheritance in flies
discovered the role that chromosome in heredity
31
what are UAS-GAL4 and FRT-FLP systems used for
UAS-GAL4: used to manipulate gene expression
FRT-FLP: used to make mosaic clones
32
forward vs reverse genetics
**forward genetics:** identify interesting phenotype, then discover the genes defective in the mutants
**reverse genetics:** alter known gene and see how phenotype changes
33
forward genetics: how are random mutated genes introduced
* 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
34
classic F3 screen for recessive mutations
* Diploid screen for dominant mutations (F1 screen)
* Diploid screen for recessive mutations (F2 screen)
* Diploid screen for recessive mutations (specific locus screen)
35
nobel prize in physiology or medicine, 1995
Lewis, Nusslein-Volhard, Wieschaus
nobel prize for discoveries on genetic control of early embryonic development
36
sonic hedgehog
can be loss of function (mutant embryo much smaller) or gain of function (double wing, double fingers, double head in snakes)
37
UAS-GAL4
* 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
38
mosaic analyses definition
express mutation in only some daughter cells
39
FRT-FLP system
* 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
40
general approach to doing reverse genetic manipulation
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
41
what is addgene
website that's a database of vectors
42
reverse genetics: building an appropriate construct (what are the pieces)
GFP example
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
43
reverse genetics: transfection involves what
GFP example
* 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**
44
transgeneic animals
* 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
45
knockout or knockin animals
* 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
46
how to make a transgenic mouse
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)
47
how to make a transgenic mouse: method 1
1. Microinjection of foreign DNA into fertilized oocytes (include promoter)
2. Implant into foster mother
3. Screen offspring by southern blotting or PCR
48
potential problems that can arise when using transgenic animals
* 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
49
4 key steps to making KI and KO mice through homologous recombination
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
50
how to make a KO mouse: targeting and ES cell screening
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)
51
how to make a KO mouse: what do you do after developing your construct
inject ES cells into a mouse blastocyst
52
how to make a KO mouse: what do you do after you inject ES cells into a mouse blastocyst
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
53
summary steps of making KO mouse
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
54
Nobel prize for creation of knockout mice
Capecchi, Evans, Smithies
developed principles for introducing gene modifications in mice by using ES cells
55
KI mice
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
56
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?
mouse with most agouti (brown) coating
originates more from strain 129/SV (ES cells), so higher chance of having gene
57
common problems of making KO or KI mouse
* many KOs have no phenotype
* many transgenic, KO, or KO modifications are lethal during early development
58
name the different promotoers and where they are expressed
* 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
59
example of how we can selectively **knockin** a gene in motor neurons
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
60
example of how we can selective **knockout** a gene in motor neurons
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
61
spatial control in fruit flies example (intersectional approach): GAL4 and FLP systems
* 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**)
62
spatial control in fruit flies example (intersectional approach): GAL4 and GAL80 systems
* GAL80 prevents GAL4 from interacting with UAS
* only cells that express promoter A but not B can express the gene (**NAND logic**)
63
FRT-Flp system
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
64
Cre-Lox system
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
CreER
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
2008 nobel for discover of GFP
Tsien, Chalfie, Shimomura
67
calcium indicators
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
intersectional genetic techniques
use combinations of multiple genetic lines of mice to get specific expression of a certain gene
69
chronic itch mouse model
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
spatial control in mice example: Cre recombinase and lox orientations
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
Flpo
mutant of Flp, higher recombination efficiency in mammalian cells
72
DTR
diptheria toxin receptor
when diptheria toxin binds, inhibits protein synthesis and kills cells
73
tet-on and tet-off system
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
limitations and challenges of current mouse KO technology
making and selecting ES cells, mouse breeding is time consuming
75
nobel prize for discovery of RNA interference
Fire and Mello
76
RNAi
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
how does RNAi work
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
sherrington
nobel prize winner
first to describe the gap between neurons, called synapse
79
electrical synapses
* 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
Gap junction
* 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
Chemical synapse
Chemical messenger: NT or hormone
synaptic delay of 1-2 ms (time to open voltage gated Ca)
unidirectional
82
types of CNS synapses
* **Axodendritic**: axon to dendrite
* **Axosomatic**: axon to cell body
* **Axoaxonic**: axon to axon
* **Dendrodendritic:** dendrite to dendrite
83
nobel prize for discovery of chemical transmission of nerve impulses
Ach inhibitory in cardiac muscle: **Loewi**
Ach excitatory in skeletal muscle: **Dale**
84
types of antagonists
competitive antagonist: binds to same site as agonist
non-competitive antagonist: binds elsewhere on the receptor
85
positive modulator
ligand that has no effect on its own, but makes the agonist more effect
binds somewhere else than binding site
86
4 criteria for identifying NTs
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
Ach, Glutamate, GABA
excitatory or inhibitory
Ach: excitatory in skeletal muscle
Glutamate: dominant excitatory NT in brain
GABA: dominant inhibitory NT in brain
88
what is the excitatory NT at arthropid NMJs
NOT Ach
## Footnote
**Glutamate**
89
monoamine neurotransmitters can be....
secreted into extracellular space outside synapses to affect nearby cells (aka **volume transmission**)
90
dorsal root ganglion neurons
have axons in spinal cord
primary sensory neurons sensing touch, temp, pain, itch
91
Large dense-core vesicles (LDCVs)
75-100 nm
contain peptides and/or proteins
92
Small clear-core vesicles (SCCVs)
classic synaptic vesicle
40-50 nm diameter
store/deliver small NTs
93
ionotropic receptors
ligand gated ion channels
rapid communication across synapse
94
metabotropic receptors
trigger intracellular signaling cascades to regulate ion channel conductance
exert function slowly
95
3 types of ionotropic receptors
96
examples of excitatory and inhibitory ionotropic receptors
excitatory: iGluR (ionotropic glutamate receptor)
inhibitory: GABA and glycine receptors
97
all metabotropic neurotransmitter receptors.....
trigger G protein cascades (via GPCR)
98
excitatory GPCR pathways
1. cAMP is second messenger: activates Ca2+ channels causing depolarization
2. diverse signaling pathways: alter membrane conductance and cause depolarization
99
inhibitory GPCR pathways
activate K+ channels
inactivate Ca2+ channels
reduce cAMP level
100
Katz
identified major mechanisms of synaptic function at the frog neuromuscular junction
101
Katz's standard recording set up
at NMJ: used glass electrode and ACh pipette
very close to nerve terminals, acetylcholinesterase inhibitor present, and TTX to block propagation of APs
102
End-plate potential (EPP)
depolarization at the end-plate (where message is sent to muscle cells)
muscle specific name for EPSP
ACh evokes EPPs
103
what did Fatt and Katz recordings of EPPs look like
- 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
what are mEPPs cause by
release of multi-molecular packets of transmitter
105
what leads to EPP failures
low [Ca2+]o or high [Mg2+]o reduces EPP size and leads to failures
106
quantum hypothesis (Katz)
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
transmitter release is a ______ event
probabilistic
108
binomial distribution to predict quantal content: what do variables stand for
p = probability of success per trial
probability of getting k successes in n trials
109
when can Poisson distribution be used to predict quantal content
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
is the poisson distribution relevant when synapses operate at physiological calcium
NO
p is no longer small
for most synapses in CNS and physiological calcium, binomial distribution should be used
111
vesicle hypothesis
a quantum of transmitter is that amount stored in a synaptic vesicle
release occurs via exocytosis
112
calcium ___ rises in presynaptic terminals following APs
rises
113
each quantum is about _____ at the frog NMJ
10,000 ACh
114
how to measure Calcium current at a synapse
* 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
conclusions of voltage clamp ca current experiment
synaptic delay of ca release results from sluggish opening of calcium channels
all steps after calcium entry are very rapid
116
key steps in **excitatory** synaptic transmission: presynaptic to postsynaptic
* 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
key steps in **inhibitory** synaptic transmission: presynaptic to postsynaptic
* transmitters: GABA or glycine
* opening of GABAA or glycine receptors
* trigger hyperpolarization of membrane
118
synaptic function is measure electrophysiologically by .....
* excitatory or inhibitory postsynaptic currents (EPSCs or IPSCs)
* or excitatory or inhibitory postsynaptic potentials (EPSPs or IPSPs)
119
synaptic transmission is \_\_\_\_\_
key step is \_\_\_\_\_
rapid (1-5 ms)
neurotransmitter release
120
synaptic process that govern NT release
* synaptic vesicle fusion
* Ca triggering of fusion
* very fast (0.1 ms)
* cooperative (5 ca ions)
* localized ca influx
121
nobel winners for research on vesicle transport in cells
Rothman, Schekman, Sudhof
122
Scheller and Sudhof
* 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
Rothman
* 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
vesicle transport: function of **proton pump (V-ATPase)**
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
vesicle transport: function of **synapsin, synaptobrevin, synaptotagmin, and Rab3**
**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
synaptic vesicles undergo recycling: 2 modes
* Clathrin-mediated endocytosis (vesicle fuses w/ membrane)
* Ultrafast endocytosis: kiss and run (transient form of fusion)
127
SNARE hypothesis for vesicle fusion
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
vesicle fusion: how many helices
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
fusion mechanism: water and SNAREs
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
where does calcium bind on vesicle
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
model of the calcium activated membrane fusion complex
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
additional molecules that regulate fusion
complexin, Munc18, Rab3 and Rim
**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
summary: proteins involved in vesicle fusion
v-SNARE: synaptobrevin (VAMP)
t-SNARE: SNAP-25, syntaxin
complex drives vesicle fusion
Munc18 regulates this complex
134
summary: proteins involved in Ca triggerings
synaptotagmin (ca sensor) and complexin
135
summary: proteins involved in ca channel tethering
Rab3, RIM, Munc13
complex drives vesicle close to calcium channel, then fuses and NT release
136
kiss and run model of transmitter release
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
neuromuscular junction and nicotinic ACh: what are the parts in presynapse and postsynapse
presynapse: synaptic vesicles (ACh), voltage gated Ca channels
postsyanpse: acetylcholinesterase, AChR (receptor), voltage gated Na channels
138
Cys-loop superfamily: cation and anion channels
cation channels: nAChR, 5-HT3R
anion channels: GABAAR, GABACR, GlyR
139
how was muscle AChR protein purified
based on binding to α-bungarotoxin
140
nicotinic AChR structure (nAChR)
**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
how can you draw an I-V curve based on voltage step graph
reversal potential
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
at reversal potential
Na influx equals K efflux
143
dendritic spines vs dendritic shaft
**excitatory** synapses target **dendritic spines** (contain postsynaptic density)
**inhibitory** synapses target **dendritic shaft** (lack postsynaptic thickening)
144
excitatory and inhibitory synapses contain....
unique set of channels, scaffolding proteins, and other molecules
145
excitatory synaptic transmitters: ionotropic and metabotropic **glutamate receptors**
* 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
glutamate receptor structure
**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
GABAA receptors
* 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
competitive antagonist of GABAA receptors
bicuculline
binds at GABA binding site
149
non-competitive antagonist of GABAA receptors
Picrotoxin
binds at different site than GABA
blocks the pore
150
positive allosteric modulators of GABAA receptors
* **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
why is there lethal synergism b/w valium and alcohol
* 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
how can we detect postive allosteric modulation of GABAA receptors
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
mechanisms by which postive modulators could produce larger currents
must prolong activation
* make channel open more frequently
* make bursts (channel openings) longer
154
benzodiazepines _____ while barbituates \_\_\_\_
**benzodiazepines:** promote channel opening
**barbituates:** lengthen burst duration
155
chloride and GABAA
GABAA (chloride channel) regulates intracellular chloride concentrations
* **low intracellular chloride:** hyperpolarization, inward current
* **high intracellular chloride:** depolarization, outward current
156
NKCC1 and KCC2
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
somatic EPSPs from dendritic inputs in a neuron: an EPSP must spread to....
an EPSP must spread to the the spike-initiation zone, and this zone must be depolarized beyond threshold to generate an AP
158
the effectiveness of an excitatory synapse (on dendrite) for triggering AP depends on
how far the synapse is from the spike initiation zone
synapses closer to soma result in larger depolarization
159
spatial vs temporal integration
**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
