Exam 1 Flashcards
Antoine van Leeuwenhoek
Built the first compound microscope to achieve significant magnification and observed unicellular organisms and plant tissues
Antoine van Leeuwenhoek’s discoveries include:
Single-celled organisms from pond water, red blood cells, spermatozoa
Robert Hooke
A contemporary of Leeuwenhoek who coined the term cell from looking at plant tissues
Cell theory
Formalized in 1838 by Matthias Schleiden and Theodor Schwann
- All living organisms are composed of one or more cells
- The cell is the most basic unit of life
- All cells arise from pre-existing living cells
Camillo Golgi
Discovered a way to stain a subpopulation of neurons using the golgi stain– this stain completely stains a neuron at random via an unknown mechanism
What theory did Camillo Golgi propose?
“Reticular theory”– the idea that neurons are continuous rather than physically separate cells
Santiago Ramon y Cajal
Described neurons as being discrete– contiguous, not continuous
What is neuron doctrine?
The idea that the nervous system is made up of discrete individual cells
Theodor Meynert
A Viennese psychiatrist who noticed regional variations in structure of different parts of the gray matter in cerebral hemispheres– tried to link psychiatry with histology and is considered the founder of cerebral cytoarchitecture
How do we visualize neurons?
We can dye them randomly (golgi) or individually (dye injection); we can also dye entire populations of cells based on macromolecule specific dyes like Nissl or Cresyl Violet
What do Nissl and Cresyl Violet do?
Bind nucleic acids
What is the HRP enzyme?
An enzyme that uses H2O2 to oxidize a substrate, which then yields a color change.
What ways of classifying neurons are there?
Shape, what emerges from cell body, number or branching of dendrites, branching of axons
All neurons have ___ axon
1 primary (primary meaning emerging directly from cell body)
Bipolar neurons
2 processes extending from cell body
Pseudounipolar cells
2 processes capable of generating action potentials; one extends to spinal cord, other towards skin or muscle
Multipolar neurons
many processes from cell body, but only 1 is an axon
How do neurons differ from cells?
Big with complex morphology and lots of surface area
Excitable– must maintain ion gradient
Energetically demanding
Post-mitotic (non-dividing)
Must signal and store info on short and long timescales
Limit of resolution in Electron microscopy
r=0.61lambda/NA (shorter wavelength= smaller r so higher resolution
Electron microscopy provides _____ than visible light
higher resolution (due to shorter wavelength of EM radiation)
Transmission electron microscopy
High-resolution images of thin slices of the object- electron beams pass through the object, creating 2-d image
Scanning electron microscopy
Slightly lower resolution images of topogrophy of thicker object- 3D image and electrons bounce off of subject
Cytoskeleton types
- Actin filaments
- Intermediate filaments
- Microtubules
Actin filaments
Aka microfilaments, are two-stranded helical polymers made from actin. They are flexible, 5-9 nm in diameter, organized in bundles, 2d networks, and 3d gels– they are usually found mainly in the cortex just beneath the plasma membrane
Microtubules
Long, hollow cylinders made from tubulin, more rigid and 25 nm in diameter– alpha and beta subunits arranged into a linear protofilament (12 in animals)
Neurofilaments
10 nm in diameter and form the neuronal cytoskeleton along with microtubules and microfilaments (7 nm). Many monomers make coiled dimers, which join to make a terameric protofilament, making a protofibril which makes a filament
Microtubules
Hollow tubes with 13 columns of tubulin molecules, 25 nm with a 15 nm lumen, made of a and b tubulin– cell shape, motility, chromosome and orgamelle movement
microfilaments (actin)
two strands of actin, 7 nm, cell shape, contraction, cytoplasmic streaming, motility and cleavage furrow
intermediate filaments
neurofilaments– proteins coiled into cables, 8-12 nm, usually keratin based, cell shape, anchoring nucleus and organelles, nuclear lamina
Dendrites and axons have many
microtubules
Are microtubules polarized?
Yes, they have directionally aligned polarity (alpha and beta ends) which allows for directionality
Microtubule protein in axons
tau
microtubule protein in dendrites
map2
Cytoskeleton in neurons
the cytoskeleton provides routes for proteins, vesicles, and organelles to and from soma and distal regions of neuron
Axonal transport is important in
presynaptic terminal function (microtubules)
Steps in transport within neuron
- synthesis, export
- axonal transport
- neurotransmitter release, membtane recycling
- retrograde transport for degradation or reuse
Actin in neurons
supports cell surface, supports smaller processes like dendritic spines
Dendritic spine types
thin, stubby, mushroom
Endoplasmic reticulum
free and membrane bound polysomes translate mrna, which is transcrbed from dna and emerges into cytoplasm to form polyribosomes (complex with ribosomes) – secretory and membrane proteins translocate into rough er
What does the cytoskeleton do?
provide routes for proteins, vesicles, and organelles to traffic to and from soma and distal neuronal regions
What does active transport need?
atp motors walking along cytoskeleton
Anterograde
away from soma
retrograde
towards soma
Which organelles are unique to the cell body in neurons?
Nucleus
T or F: dendrites do not contain ER or golgi bodies
F
T or F: protein synthesis can occur distally to the soma
T
How does mRNA travel?
distally via the cytoskeleton
Dendrites and their organells
ribosomes to translate mRNA (allows for local modification of protein structure and function), ER, and Golgi, allowing for transmembrane and secreted protein translation
Role of ER in neuron
calcium source/buffer for signalling
Where are mitochondria found in neurons?
distributed everywhere, especially synapses- they provide energy for areas of high metabolic demand
Axons are myelinated by
oligodendrocytes (CNS) and schwann cells (PNS)– provide a sheath that insulated axons and allows quick conduction of electrical impulses
Astrocytes
make close contacts with synapses
Electron microscope
uses electrons with a wavelength of >.3 angstroms, uses fixed (killed) samples to see dine details, organelles, vesicles
light microscopy
uses visible light 350-700 nm on live or fixed samples, shows cell morphology, larger organelles, cell identity based on markers of proteins or nucleic acids
transmitted light microscopy
uses white light, contrast derives from the interaction of light through the specimen (diffraction); low contrast, so optics enhance contrast
how do cells affect visible light?
samples can lower the amplitude or make the lightwave go out of phase, or both
phase microscopy
the sample separates out the phase shifted light from unaffected light, separating the signal from the specimen from everything else
fluorescence microscopy
uses a specific wavelength of light for illumination, and collects specific lower-energy longer wavelength light. it relies on fluorophores (molecules with fluorescent priperties) and identifies cell morphology, organelles, cell types, and organelles
Stokes shift
the shift from a higher energy absoption to the emission of a lower energy (longer wavelength)
What are some advantages of fluorescence microscopy?
low background and high signal, making it easy to distinguish; it can also visualize specific cells, cell types, organelles, proteins/macromolecules, etc though macromolecules are at a lower resolution than light microscopy
DAPI stain
a fluorescent dye that binds dna, helping visualize the nucleus
mitotracker stain
fluorescent dye that accumulates in mitochondria
Immunofluorescence
antibodies have a variable region that binds an antigen; adding a fluorophore to the antibody causes immunofluorescence; you can either use primary or secondary antibodies
why might one use secondary instead of primary antibodies?
multiple secondary bind to a primary, creating signal amplification
GFAP
red immunofluorescent dye for astrocytes
map2
immunofluorescent blue or green dye for neurons
uses of immunofluorescence
specific cells, organelles, subcdellular structures
GM130 (green)
golgi apparatus
PSD-95
postsynaptic density labelling (immunofluorescence)
gfp
immunofluorescence to visualize neurons
synapsin
immunofluorescence for presynaptic terminal
if lots of psd-95 (red) and synapsin (blue/green) are present:
a synapse is likely there
steps to fluorescent tagging
fusion gene of fluorescent protein and promoter, targeting sequence, and or protein of interest, and introduce gene into cells or organisms
D1 dopamine receptor
drives td tomato expression
d2 dopamine receptor
drives egfp expression
advantages of fluorescent tagging
many colors, high brightness and specificity, helps see morphology and protein distribution, can use cell type promoters to drive fp expression, and fps can show enzyme activity, calcium dynamixs, protein interactions, etc
cells of the nervous system
Neurons, glia (astrocytes, microglia, oligodendrocytes)
astrocytes
metabolic and other support
microglia
immune cells
oligodendrocytes/schwann cells
myelination in cns/pns- insulate axon to speed up electrical conduction of impulses with regular breaks called nodes of ranvier; myelin extends from oligodendrocyte and weaps around multiple times
do regular immune cells enter brain
no so microglia scavenge brain for debris
Iba1 immunostaining
a specific microglia protein that stains microglia
astrocytes
structural, metabolic, and trophic support for neurons, and helps form the blood brain barrier; also plays roles in plasticity and other processes. for example, astrocytes clear extra glutamate from the synapse
how is cell membrane potential reached
separation of net charges across membtane (more positive ions outside)
how do we measure cell potential
insert microelectrode into nerve cell, keep extracellular reference electrode- resting potential is about -60 mn
why is there an electrical potential across the plasma membrane?
the plasma membrane is selectively permeable to ions, and a concentration gradient is actively established across the plasma membrane by pumps; there is also an electrochemical gradient as ions run down concentration gradient and electrical potential
Na/K+ pump
uses atp to pump ions against concentration gradient, pumping Na out and allowing K in
Ion channels
passively allow ions to pass through membrane and are selectively permeable (opened/closed easily)
Resting Na/K gradient
Na tries to enter cell, K+ tries to exit but some are pulled back by the intracellular negative anions; cell is more permeable to sodium
Potassium, Na, Cl, and Ca inside vs outside the cell
more inside, more outside, more outside, more outside
there are more organic anions inside the cell
what is the simplified nernst equation
E(mV)= 58 log (ion outside/ion inside)
add a negative sign or flip when dealing with chlorine
How could a neuron make its membrane potential less negative?
Allowing sodium or calcium into the cell can cause depolarization
How can a neuron make its membrane potential more negative?
this is hyperpolarization; allowing K+ out or Cl- into the cell would allow this
Graph of membrane potential vs log [K out/Kin]
a positive slope of 58 mV for 10fold increase in K+ gradient
neurons have two types of changes in membrane potential:
graded depolarization (makes cell more excitable/positive)
action potential
graded hyperpolarization (makes inside more negative)
are action potentials all or none
yes; graded potentials do not always cause APs
threshold for an AP
-50 mV
graded or passive action potentials
occur in dendrites, soma, axon w/ different duration and amplitude; spreads from origin in all directions and reflects local change in membrane permeability, decays with distance
action potential
occurs only in axon with a defined duration and amplitude, travels away from soma, requires sequential change of membrane permeability (‘travels’ down axon)
does the nernst equation account for permeability?
no, but when compared to measured membrane potential may tell us about membrane permeability
Goldman equation
predicts Vm when membrane is permeable to multiple ions (Cl is negative so we flip it to in/out)
Goldman equation
Vm= 58mV log (PKout+PNaout+Pcl in/ PKin+PNain + P cl out)
usually membrane so impermeable to Cl that you drop it
earlier hypothesis of action potential
action potential is when membrane potential briefly goes to 0
hodgkin and huxley
measured membrane potential during an action potential
voltage clamp
used to measure ion currents in squid giant axons, hold voltage constant by injecting current, predicted ap changes from nernst and channel permeabilities
describe the setup of the voltage clamp
one internal electrode measures membrane potential and connects to the voltage clamp amplifier, which compares potential with the desired potential; if its different, it injects current through a second electrode, this current flowing back into the cell is measured
what did the voltage clamp experiment show
peak of ap is more positive than expected
current when hyperpolarization occured
capacitive current but nothing else
current when depolarization occured
capacitive current, transient inward, delayed outward
how does voltage clamp work with depolarization
the membrane is depolarized and membrane potential is held constant, so that the currents undelying the ap are triggered but an ap cannot happen because potential is constant
what happens to voltage clamp if no extracellular sodium
early current is outward instead of inward
what happens if k+ current is blocked?
inward current but no outward current
what did hodgkin and huxley find
29 mv is peak of ap, cell is selectively more permeable to Na than K
an early influx of sodium followed by delayed efflux of K+
using this permeability info we can calculate Vm
how does depolarization affect conductance
increases conductance of sodium and potassium
na vs k currents
at threshold, na and k are activated; na is fast and transient, k is slower and longer– quick depolarization to E na followed by return to Ek
how does the action potential travel down the axon?
there is a passive spread of charge in membrane potential— the change in membrane potential decreases with distance, and current injected follows the path of least resistance to the extracellular space
what is the change in vM mathematically
decays exponentially with distance from the site of current injection
what does lambda length constant mean
the distance at which Vm is 37% of its value at the point of current injection
describe the propogaction of an acrtion potential
an AP going from right to left causes a difference in potential in 2 adjacent regions; this difference creates a current that facilitates the passive spread of current so current spreads ahead and behind the AP
however, since more K exits the cell following the AP, the previous area is hyperpolarized and the sodium coming in isn’t enough to depolarize
why does the action potential travel only one way down the axon?
depolarization goes in both directions but the inactivation of VGNa+C prevents depolarization from reactivating the AP
closed vs inactive voltage Na
in the resting state, the activation gate is closed and inactivation gate is opened
when activated Na flows in
then the inactivation gate closes and after repolarization the inactivation gate is open but the activation gate is closed
what does it mean when they say propagation of an action potential is both passive and active?
passive because local current flow spreads to adjacent areas, causing depolarization
active because this causes the opening of voltage gated channels- the action potential mechanism
nodes of ranvier
nodes in a myelinated axon that facilitate saltatory conduction
what does myelin do
increase action potential conduction speed
squid giant axon
no myelin, 25 m/s, 500 um
motor axons
a alpha and a gamma, both myelinated
80-120 m/s, 13-20 um
4-24 m/s, 5-8 um
sensory axons
alpha, beta, delta all myelinated (delta is thin), c is unmyelinated
autonomic nerves
preganglionic b type is myelinated, postganglionic c type is not
the majority of macromolecules in the cell are
proteins
carbohydrates
energy storage, protein modification
lipids
membrane and energy storage
nucleic acids
dna and rna
proteins
everything else
how are amino acids added on
to the c terminus, the n terminus is 1
protein shorthand
1 letter code, then the nth amino acid in a protein
proteins are joined by
polypeptide bonds
protein structure levels
1- amino acid sequence
2- substructures like alpha helices and beta sheets
3- full 3d structure
4- multiple proteins
monomeric
no quarternary structure
monomer
1 protein, may or may not participate in 4 structure
multimeric
a protein complex with 4 structure
subunit
1 protein in a multimer
both pumps and ion channels are
multimers
describe the process of x-ray crystallography
purified, concentrated protein solution yields crystals of the protein; an x-ray beam is shot through to get a diffraction pattern of electron density since the beam is scattered and scattered waves reinforce eachother now and then
this diffraction pattern helps produce an atomic model along with the sequence
what do molecular pumps do
set membrane potential, transport ions and molecules directionally, and require energy from ATP or another gradient
describe the structure of the na k pump
it has domains for nucleotide binding, phosphorylation, an actuator domain that acts as a dephosphorylase
adp binds to the nb domain and 2 k in, 3 na out
atp phosphorylates the pump which causes the na pump to turn outward and decrease affinity for na, after which a k binds , phosphate is released and pump goes to original conformation
structure of the serca Ca2+ pump
areas for nucleotide binding, phosphorylation, ion translocation activity
what does the ca pump do
2 ca out, 2-3 h in
ATPase pumps
hydrolyze atp into adp, pump ion across membrane gradient
domain of sodium potassium pump
ouabain inhibits na k pump
3 na ions bind
2 k ions bind
ATPase domain
pump itself gets phosphorylated
alpha subunit of Na/K pump
alpha subunit has phophorylation site, atp site, ouabain site, and na/k site
beta subunit of Na/K pump
helps with maturation and structure
what changes are associated with the conformational change of the na k pump
changes in location pf phosphorylation, nucleotide binding, and activator domains
pattern of na efflux
na efflux is reduced when extracellular k is removed, k+ restores recovery, efflux is again inhibited by metabolic inhibitors like dinitrophenol which blocks atp synthesis, recovery when atp is restored
what role does the na k pump play in setting vm
sets up gradient for resting membrane potential and action potential– but ONLY -1 MV
how much do pumps and leak channels contribute to resting membrane potential
K+ leak current provides about -64 mv to resting membrane potential
ion exchangers
use energy from existing gradient to pump another ion/molecule AGAINST its gradient (antiport since in different directions)
co transporters
use energy from an existing gradeint to pump another ion against its gradeint but in the SAME DIRECTION
