Week 2:

 The dogma
o Stimulus leads to biological change (aka protein synthesis)
 How we respond to the environment
 DNA structure
o 5’ end has the phosphate while 3’ end doesn’t have it
o DNA strand is made up of many bases
 ATCG
 Know where the protein is interacting on the strand of DNA
 Transcription
o Process that requires sequence specific regulators = transcription factor (interacting with
DNA sequences)
 Specific DNA sequences are called motifs
 Interact with DNA sequences in response to cellular signals (APs that initiate
the transcription)
o Diagram
 Chromatin = unwinding the chromosome to expose strands of DNA for transcription
 The unwinding process is needed to read the DNA strand sequences
 RNA polymerase II
 What is needed for initiation of transcription
o Can be blocked, or be more efficient by various interactions
 Around RNA polymerase II, we need to build a transcription complex assembly
 To initiate transcription
 Made up of many different transcription factors (TF)
o Molecules that regulate gene transcription
 TFII
 Specific group related to polymerase II (for functioning properly)
 Main job
o Proteins that help position RNA polymerase II at the correct
promoter region
 Find polymerase, then put it into the right region and make
sure the unwinding of the DNA is done
 Activator molecules (co-activators) also work alongside transcription factor to
facilitate polymerase II
o Second diagram (initiation)
 (1) Starts with assembly of TFIID
 Recognizes an area of DNA strand that has TATA (called TATA box =area of
strand rich in Ts and As)
o Specifically, the TATA binding protein (portion of TFIID) recognizes
the TATA box
 (2) Causes the distortion of the DNA strand; signals all the other TF to come together
(and bind)
 Attracts TFIIA and TFIIB as well as the RNA polymerase (etc.)
o Each have unique functions
 TFIIH has the helicase to unwind the DNA
  (3) Together make the transcription initiation complex
 (4) Once ready to transcript, all of the TF are not required and disassembly
o Disassembly signifies transcription
 3’ to 5’
 Table
o Binding in a specific order
 Transcription
o Step 2: Elongation
o Step 3: Termination
 Capping
o A cap that stops anything from happening to it
 A m7G cap
 Splicing
o While introns are there, the mRNA is called pre-mRNA
o Diagram
 During splicing, there is removing of introns
 Now is a mature mRNA strand (what’s actually translated)
o Step 1
 U1 snRNP helps cleaves the 5’ end of the intron
o Step 2
 U2, U4 and U6 snRNP
 Brings 5’ close to the branch point
o There is a bend in the branch point as a result
 U5 brings the 3’ end close
 3’ end is cut and joined to 5’ end
 Left with a loop
 Called an RNA lariat
o What is removed from the mRNA
 Exons are now joined together
 Splicing diagram
o Lariat is removed later on
 Splicing video
o All processes happen all the same time (in multiple places)
 Not like one process happens in one time and in one position (at one period of times)
 Spliceosomes at different areas and RNA pol II transcribing
 Transcription factors
o Binds to parts of DNA called specific consensus sequences (DNA binding motifs)
 Important for supporting specific behavioural processes
o Distinct DNA sequences areas offer TFs patterns of hydrogen bonds (donor)
 TF can accept the hydrogen bonds and that’s how they are linked to the DNA strand
o TF bind to DNA binding motifs via structural motifs
 Actual part of the TF that binds DNA
 CREB (most important TF)
o CREB recognizes the CRE DNA sequence
o Diagram
  Linked to all forms of behaviour
 CREB is involved somehow to basically all forms of behaviour
o Diagram (structure)
 CREB has 3 main structural features
 (1) bZIP domain (at C terminus) facilitates dimerization (joining of 2 things)
between different family members
o Called the leucine zipper = bZIP side of the CREB molecule
o bZIP is the part of CREB that recognizes the CRE sites
 (2) KID
o A site for phosphorylation by PKA and others
 (3) Q2 domain
o Recruits the transcriptional machinery to the promoter
o Diagram
 A
 All pathways that lead to CREB activation
o Commonly activated
 B
 Leucine zipper or zipper region
 D
 Placed on a strand
 CREB
o Diagram (bottom)
 Idea that when CREB is activated, there is 2 of them
 They are phosphorylated which allows the attraction of CBP molecule
o That’s how RNA pol II is brought in (attracted RNA pol II so it
facilitates transcription)
o Diagram
 Actually, the HAT within the CBP molecule attracts the RNA pol II
 The domain that enables chromatin remodeling to allow RNA pol II to come
in
 Co-factors
o Other molecules that interact with TF
 Example
 HAT interacts with CREB process
o All co-factors
 Serve to enhance capabilities of the TF and its efficiency
 Example
o HAT
 Enhances unwinding of the chromatin so transcriptional
process takes place
 Does it by weakening the DNA histone binding
o Without HAT, not as effective at
transcription
o Compressors (opposite of co-factors)
 Example
  HDAC (enzyme)
o Makes the transcription less efficient
 By increasing DNA histone binding
Week 3:

 Chromatin
o Chromatin regulation is outside of the genetic code
 Controls genetic expression outside of being genetic code
o Euchromatic
 Organized in a structure that resembles beads on a string
o Nucleosome
 Each structured with a core particle that contains 147 bp
 Histone octamer
o Contains 2 copies of histones
 H2A. H2B, H3 and H4
 Nucleosomes are linked to H1 rod histone
o Heterochromatin
 Repressed and compaction of chromatin into 30 nanometer fiber
o Each core histone contains N-terminal tail that extends from the DNA histone core and can be
covalently modified
 The modifications include; methylation, phosphorylation, acetylation and more
 All modifications are reversible
 Histone modifications
o HATs
 Add acetyl groups to specific lysins
o HDAC
 Remove acetyl groups
 Methylation
o Diagram
 Cytosine ring that DMNT (DNA methyltransferases) add the methyl groups
 Covalent modification of DNA regulates memory formation
o Teaching fear (fear conditioning) to the mouse; specifically, auditory fear conditioning
 Accompany the tone with the shock
 Creating a fear response; freezing behaviour
 To show that they have learned the behaviour, we place the animal in the box and
play the same auditory sound but have no shock accompany it
 If it has learned, they will fear
o Dependent variable; freezing behaviour
 Percent of time frozen
o The addition of DMNT and then doing another testing, was to see if without DMNT inhibitor,
it changed the memory
 VEH = saline
o Saw that fear conditioning increased DMNT mRNA 30 minutes after training
 Thus, if we inhibited DMNT, we would not get the increase in mRNA
o Timing is important
  In order for long term memory to form, transcription must occur immediately after
learning a stimuli
 Acetylation
o Histone acetylation is regulated by opposing actions of 2 different classes of enzymes
 HATs and HDACs
 Balancing act that regulates transcription
 CBP Histone acetyltransferase activity is a critical component of memory consolidation
o Novel objection recognition
 Rodents are more likely to explore novel objects rather than familiar objects
 Must acclimatize to the environment (habituation phase)
o If the animal remembers what it explored previously, they will know which is familiar and
novel object
 Spend more time with novel object
o Graphs
 Found that mice with HAT domain of CBP eliminated, show deficit in object
recognition and spatial memory
 Go back to the familiar object (don’t prefer the novel object)
 But if their HAT activity is recovered (given treatment), then their memory deficits
are reversed back
o Ultimately, messing transcription means reducing translation
 Translation
o tRNA which also has 3 nucleotides
 Match the AA to the mRNA codons
 The matching set of codon from the tRNA strand is the anticodon
o On the tRNA molecule, there are enzymes called aminoacyl-tRNA synthetases
 The TRNA synthetases will attach the AA to the actual tRNA molecule thru a
covalent bond
 Initiation
o Initiation step is the rate limiting step
 Involves 12 protein factors and commonly know as eukaryotic initiation factors (eIF)
o Binding of tRNA to the AUG codon initiates translation
 (1) B4 that happens, there needs to be part of preinitiation complex
 The initiator tRNA binds to a small ribosomal unit with some of the eIFs
o Together forms the preinitiation complex
 The GTP attached to the eIF2 will find the initiator codon (AUG)
 (2) Next, the whole subunit binds to the 5’ cap mRNA and move forward to find the
AUG
 (3) The binding of large ribosomal subunit occurs and helps to grow the polypeptide
chain
o Why is initiation the rate limiting step?
 eIF2 phosphorylation by several kinases
 Leads to a decrease in general translation
o Biggest consequence of eIF2 phosphorylation is down regulation of
global translation and stimulation of translation of some mRNA
 Increases gene specific translation
 o Specifically of mRNAs that contain the uORF
 Example; CREB repressor
 Elongation
o Each ribosome has 3 binding sites for the tRNA
o Diagram
 Step 1
 tRNA binding to the A site
 Step 2
 Followed by forming the peptide bond in the P site
o Polypeptide chain is forming
 Once the peptide is added it moves onto the next tRNA molecule
 Step 3
 There is translocation of the large subunit (moves over) so that tRNA that has
pasted on its AA is in the E site
o The moving over of large subunits signals the bottom small subunit
to also move over
 Step 4
 The moving over ejects the tRNA molecule in the E site
o That gives the space to start all over again
 Termination
o Stop codons signals the end of translation
o Diagram
 Instead of a new tRNA molecule coming in, instead there is binding of a release
factor to the A site
 When that elongation step tries to continue, the protein is released
o This occurs via a chemical process whereby a water molecule is
added to the end of the peptidyl tRNA
 The water molecule frees the C terminal end of the
polypeptide chain from the tRNA
 This causes a whole dissociation of the entire complex
 Large ribosomal and small subunits will detach as well as the release factor
 IEGs
o In response to some environmental stimulus, these genes are the first to be transcribed
o Presence of IEGs = activity marker
 C-Fos
o C-Fos works alongside Jun
 Its transcription is stimulated by activation of CREB
o C-Fos the gene and Fos is the protein is produced by the translation of C-Fose gene
o Steps
 (1) The c-Fos promoter recruits SRF
 (2) SRF recognizes the SRE consensus sequence at the c-Fos promoter
 (3) This recruits CBP
 Removes the HDAC molecule
 (4) Initiates transcription at C-Fos site
Week 4:
 What is a NT?
o The specific amounts will determine the effect it will exert onto the postsynaptic neuron or
some specific target
o The NT never sits in the synaptic cleft forever
 Types of NT
o Neuroactive peptides
 Bigger sized NT = packed in larger dense-core vesicles
 Biogenic amines (produced and synthesized by the organism itself without any outside help) vs amino
acids (formed from carbohydrate substrates)
o NT can be classified as these 2 types
 Catecholamines (most important function is motor control)
o Step 1
 Tyrosine hydroxylase converts Tyrosine into L-DOPA
o Step 2
 L DOPA is decarboxylated by L-DOPA decarboxylase into dopamine
o Step 3
 DOPA beta-hydroxylase converts dopamine into norepinephrine
 The presence or absence of DOPA beta-hydroxylase determines whether a
neuron becomes dopaminergic or norepinephrine
o Determining enzyme
 Without DOPA beta-hydroxylase = dopamine neuron
o Step 4
 Phenyltethanolamine-N-Methyltransferase helps to make the norepinephrine into
epinephrine
 Regulation of TH
o There are different levels of catecholamines necessary for different type of behavioural
stimulus
o Stress causes rapid firing
 Rapid firing could cause a depletion of NT pool
o What regulates the catecholamines?
 Tyrosine and Tyrosine hydroxylase (TH) regulate its amount produced
 At baseline, the TH is completely saturated by tyrosine molecules
o If we were to increase the level of tyrosine, it will not change the rate
of TH activity
 Therefore, if we wanted to increase dopamine synthesis, and
give more tyrosine, it will not increase the amount of
dopamine produced
 Because TH is completely saturated
o Rate limiting step (bottleneck)
o TH is controlled in 3 ways (can actually change how much dopamine or epinephrine is
actually produced)
 (1) Phosphorylation of TH
 (2) End-product inhibition of TH
 Catecholamines can inhibit the activity of TH via competition of co-factors
that is needed
  (3) De novo TH synthesis (increased demand for catecholamine synthesis)
 This can be met by producing more TH molecules
 (1) De novo TH synthesis
o Diagram
 CREB TF can bind to the TH promoter to increase TH expression
 Activation causes a cascade of reactions that leads to phosphorylation of
CREB, leading to increased gene expression of TH
 (2) End-product inhibition
o BH4 is required for high TH activity (facilitates TH activity)
o Diagram
 When catecholamine binds to the flexible loop of TH, it causes a conformational
change
 Makes it less available for BH4 cofactor binding
o Competitive inhibitor
 (3) Phosphorylation
o Different phosphorylation sites cause different effects
 Diagram
 Generally, phosphorylation of the R domain of Th moves it from a position
of obstructing access to the active site to allowing the access site to be more
accessible
o However, its more complicated
 Depolarization (stimuli) causes increased in activity of PKC, PKA and CaM kinases
that affect TH in different types of phosphorylation
 Wherever within the flexible loop, the phosphorylation occurs, there is a
different effect
o Phosphorylation onto Ser40 by PKA
o Phosphorylation onto Ser31
o Phosphorylation onto Ser19 by CamKII
 Serotonin
o Here, the availability of tryptophan is the rate limiting step for serotonin (main difference
between catecholamines and serotonin)
 Other than that, the basic outline mechanism is similar to the catecholamines
 GABA
o Its not biogenic amine NT
 There needs to be a glucose metabolism
 Glucose is the #1 precursor
o GABA shunt (Diagram)
 Again, glucose is the precursor to GABA
 There are other precursors
 Step 1
 Transmission of GABA T
o Changes the glucose (alpha-ketoglutarate) into glutamate
 Step 2
 GAD catalyzes the decarboxylation of glutamate to get GABA
o The steps can be backwards as well
  GABA can be metabolized by GABA T to form succinic semialdehyde
 Recycling loop
o Since GAD is the final step
 Presence of GAD can indicate the presence of GABA
 Only appears in GABA neurons (specificity)
 Glutamate
o Glutamate can also be synthesized via glial cells
 Basically, 2 ways
 Conversion via GABA to glutamate and glial cells that convert glutamine
into glutamate
 ACH
o Involves only 1 enzymatic action
 Catalyzed by choline acetyltransferase (ChAT)
 ChAT determines whether there is ACh or not
o Rate limiting step
o ChAT combines acetyl CoA and choline into acetylcholine
 Acetyl CoA comes from our diet (glucose metabolism)
 Acetyl CoA comes from the mitochondria
o Thus, it must be transported into the cytoplasm to be used
 Rate limiting step as well
 Need enough availability
 Vesicular transporters
o Table
 VMAT is for catecholamines
 VaChT is for acetylcholine
 VGAT is for GABA
 VGLUT is for Glutamate
 VMAT
o Most transporters are stochiometric antiporters
 Involves charged particles and the electrical gradient
o For every 2 H+ ions going out of the vesicle, it leads to transportation of 1 NT into the vesicle
 This is maintained via the hydrogen ATPase
 Allows via energy consumption, lots of hydrogen to stay inside the vesicle to
encourage moving out
o Blocking or inducing the ATPase (increasing or decreasing)
 Effect = how much VMAT is able to bring in NT into the
vesicle
 Can be used to change the amount of NT moving into a vesicles
 2 methods of inactivation
o Diffusion
 Can drift away
 Can also be diluted by extracellular fluid whereby the concentration is non detectable
or is not useful in that condition
o Uptake
 Involves transporter molecules
  2 functions
o Either reuptake NT into storage vesicles
 Recycling method
o Or transporter causes enzymatic inactivation
 Breakdown of molecule into inactive components
 Reuptakes
o All based on the SLC transporter molecule
 Reuptake
o There is different transporter for specific and different NT that they are dealing with
 For monoamines, there’s SERT (serotonin), NET (norepinephrine) and DAT
(dopamine)
 For GABA/glycine, there’s GATs and GLYTs
 Location varies
 Reuptake - DAT
o Energy dependent
 Therefore, depends on the sodium co-transport and requires extracellular Cl-
 Diagram
o Sodium and dopamine go in at the same time (have to at the same
time)
 Causes a conformational change that will lead to dopamine
being transported inside
o General structure
 During splicing, there is different versions of N-terminals and C-terminals being
made
 Depending on how its spliced, it leads into different transporters for different
molecules
o In splicing is basically when we differentiate the different
transporters from each other
 DAT: regulation
o Reuptake is also closely regulated by all mechanisms we have discussed b4
 ERK pathways
 If activated by secondary messengers, it causes upregulation of DAT surface
levels
o Increasing transporter capacity = more reuptake
 PKC
 Causes reduced transport meaning PAC will not internalize dopamine back
o There is less reuptake, causing enhanced internalization (not open for
reuptake essentially)
 Enzymatic inactivation
o MAO
o COMT
 Diagram
o Use legend to look for Abcde (enzymes)
 Dopamine
  Dopamine can also be broken down by COMT and then subsequent enzymes
into homovanillic acid
o However, another way is that is can be first converted into
norepinephrine and then broken down by MAO
 Subsequent enzymes allow into homovanillic acid
o Another way is being converted then into epinephrine and then by
MAO
 To get vanillylmandelic acid
o Memorize the end products and starting products as well where enzymes MAO and COMT
are
 No need for intermediaries
 ACh
o AChE (breaks down ACh)
 It promotes ACh hydrolysis by forming Acetyl-AChE intermediary which releases
the choline
 Thru hydrolysis; get back to initial products = Choline and acetyl-CoA
o Diagram
 ACh is first broken down and then it is recycled back into the cell
 ACh inactivation takes place extracellular
 Other enzymes can be first reuptake into the cell and then inactivated but
ACh is inactivated outside the terminal
Week 6:

 NT release depends on presynaptic depolarization

o Giant squids have really big neurons (no need to look under microscope)
 Just need electrodes
o Graph D
 Above 40 MV indicates that presynaptic terminal has reached threshold of
depolarization and that will cause release of NT
 Interestingly, the postsynaptic response is proportional to size of depolarization
 Change in voltage dictates the amount of NT being released
o The amplitude of EPSP dictates how much NT is released
o Graph B
 In terms of NT release, see NT release even if we block Na and K
 NT release is regulated by presynaptic Ca2+
o Depolarization occurs cause a change in calcium = results in change in postsynaptic potential
characterized by release of NT
o At base line, calcium concentrations are really small inside the cell
 Thus, any small fluctuations can cause large changes (triggers changes in NT)
 It is very controlled because so few ca ions are already at baseline inside the
cell that a few coming in can regulate how much NT is being released
 Ca2+ channels are concentrated in the active zones (close to presynaptic terminals)
o Microscope image
 Many calcium channels in presynaptic terminals
 No calcium channels in postsynaptic terminals
 Ca2+ channels
o Neuron contains 5 different classes of multi merit voltage gated calcium channels
 Each has specific properties and physiological functions
 4 types of calcium channels (L, PQ, N and R)
o Requires fairly strong depolarization to be activated (once threshold
is met)
 High voltage activated calcium channel
 What really causes the postsynaptic change = release
of NT
 Another type called T-type calcium channels
o Requires small depolarization (doesn’t have to be at the threshold)
 Help controls the excitability of the resting membrane
potential cause it doesn’t need to hit threshold to open
o How much calcium is needed to trigger NT release?
 Graph
 Non-linear relationship
o Requires 4 to 5 Ca ions to bind to trigger NT release
 NT release in Quantal
o Graph E
 Clustered around a peak of 8.3 amplitudes
 The greater the postsynaptic potential amplitude, the greater # of quanta
released
o But because they are clustered, each quantum releases a similar
amount
o Number of quanta determines the amount of NT released
 What determines the amount of NT release is the # of quanta
released; not the actual # of NT molecules because those are
the same within each quantum
o Why is transmission quantized?
 Neural circuits want to process stimuli quickly
 Sometimes info changes so fast that timing of events is important
o To increase the speed and control over the speed, why quantum
released
 Quanta will be enough to cause a robust enough signal to
cause postsynaptic change rather than a single NT release
each time
o Ca2+ and vesicle release
 The more Ca influx, the larger # of quanta released meaning the more NT is being
released
 Vesicle components
o B
 Many proteins involved
 Regulated by transcription and translation
 Vesicle restraint and mobilization
o How to we get the vesicles to the active zones?
  Regulated by synapsin proteins (located in the cytoplasm)
 Bind to ATP and actin which anchor the vesicles outside the active zones
o When there is an incoming signal, calcium is released
 Increase of Ca triggers phosphorylation of synapsin (occurs
via CAMK and PKA pathways)
 This will release the vesicle from the actin
o This de-anchoring allows it to go towards
the active zone
 Vesicle fusion
o Next step is how the vesicle fuses with the membrane at active zone for it to release the NT
 Since the membrane lipid bilayer is very stable, it requires a lot of energy to fuse the
vesicle
 Thus, neuron recruits a complex of proteins called the core or SNARE
complex
o Composed of 2 classes of SNARE proteins
 V SNARE (Synaptobrevin)
 Synaptobrevin is located in the vesicle
o Targets the T snares which are attached to
the membrane
 T SNARE (SNAP-25 and Syntaxin)
 Vesicle fusion
o Three SNARE proteins called Synaptobrevin, SNAP-25 and syntaxin
 Bind together to form a helix coil complex
 Allows the vesicle to get closer to the membrane
o Once the T and V SNARE proteins are closer together
 It makes the helix very stable and causes energy to be released
 Believed to draw the membrane negatively charged phospholipids into a way
that it can fuse together and cause prefusion
o Allows the – charged lipid bilayer to actually fuse together
o In reality, those 3 proteins are not enough for the fusion
 Munc18 (SM protein) is required to bind to synataxin to facilitate the snare assembly
 What actually binds the whole coil together
o Essentially, it operates as a clasp
 Controls the assembly of the helix
 So, when synaptobrevin and vesicle comes close, it
clamps it together to allow the attachment of the coil
 D
 Binds to the N-terminal of the syntaxin
o Because of that, when it opens up, it makes the space necessary for
the other proteins of the helix to come to go inside and then clamps it
 Like a clasp
o Now talk about opening the actual vesicle
 Synaptotagmin
 Binds to 5 Ca molecules (has to be 5) which allows it to then bind to the
whole SNARE complex
 o This will fuse the membranes together thru the synaptotagmin’s C2
domains
 The fusion promotes the pore opening for NT release from
the membrane
 Where does synaptotagmin get the calcium from?
o Ca2+ microdomains trigger exocytosis
 B
 We know that opening the calcium channels in the
active zone is what leads to influx of calcium
o Pink areas called microdomains of calcium
 Where calcium is abundant
 Since the vesicle dock near the microdomains of
calcium, this is what triggers the vesicle to bind to
the membrane
o After fusion of complex, the whole SNARE complex dissociates (needs help tho)
 Via help of NSF and SNAP proteins
 Thru ATP hydrolysis; it allows the vesicle to be released from the membrane
and go inside
 Vesicle fusion
o Summary slide
 With all the steps
o Now go back in each steps and add more information
 Vesicle life cycle
o Decide which vesicle is the one to fuse with the membrane
 Docking step
 When we decide which vesicle is used
o Docking describes the phenomenon of where synaptic vesicles are
considered to be close enough to the membrane to fuse
 When vesicles are starting to fuse, we are breaking it down
into stages
 Priming stage I
 Need 3 proteins
o Rim protein, Munc18 and Mun13
 These are involved in the SNARE complex
 (a)
o RIM and Munc13
 They open up the whole SNARE complex (grabs on to the
Munc18 of the complex)
 Guides it inward
o Gives the space for the vesicle to dock
 Priming stage II
 Complexin protein activates the SNARE complex b4 synaptotagmin (comes
into play)
o Comes in and goes onto of the complex
  It clamps down the SNARE complex to keep it into place
until there is enough Ca ions to bind to the synaptotagmin to
trigger the fusion of the pore
 Fusion-pore opening
 Again; important to know that calcium binding to the C2 domain of
synaptotagmin is essential as it allows binding of phospholipids together
o Displacing the Complexin clamp from the SNARE complex
 Fusion completion
 NSF and SNAP dissociates the whole SNARE complex
 Endocytosis and recycling
o Now we need to un-fuse the membrane, so cell membrane doesn’t grow endlessly
 If vesicles fuse, it will enlarge more and more
o 3 ways to do so
 Fastest way
 Reversible fusion pore (reverses the opening and closing)
o Vesicle collapse back into the membrane
 Kiss and run approach
 Used mostly in low and normal frequency rates
o Not once threshold is met (when APs occur)
 Classic pathway
 Uses endocytosis via clathrin coated pits to recycle vesicles
o Takes some time (not as fast)
 Once AP occurs
 Bulk retrieval
 Uses a different protein
 Classic pathway (used the most when AP is happening)
o Need to recruit the claritin coat
 It coats the whole area and pulls the membrane back into the cell thru scission
o (1) Binding and recruitment
 How do we know which bit of the membrane should be endocytosed?
 Via AP2 protein
o Its high affinity for synaptotagmin will find where synaptotagmin
was and it will trigger claritin to be recruited to that area
o (2) Invagination
 Folding that part of the membrane back in, forming a cavity
 Via F-BAR domain
o (3) Maturation
 Dynamin and BAR domain protein
 Compress the membrane (pull it together)
o (4) Scission
 Envelope the whole thing and pull the vesicle inwards (back in)
Week 7:

 Main receptor classes

o Ionotropic
  Fast synaptic actions
 Happen in ms and last for few ms
o Metabotropic
 Slower to be activated
 Actions are a lot slower (minutes)
o The slower reactions modulate behaviour
 Don’t directly change the behaviour or cause a response
 Do so by altering the excitability of neurons,
strength of synaptic plasticity, and length of AP
 Ionotropic receptor families
o Relatively large
 Typically composed of 3-5 subunits
 When combined forms ion permeable pore that crosses the membrane
o 3 main families
 ACh, GABA, and glycine receptors
 All are pentamers (composed of 5 subunits)
o There are several types of subunits (characterized based on the
subunits)
 Alpha, beta, gamma, or delta subunits
 Each different subunits has a membrane domain and
4 different membrane spanning helices
 Glutamate receptors
 Tetramers (composed of 4 subunits)
o M2 membrane spanning helix forms a loop at the bottom and isn’t
transmembrane
 Dips in and out of the cytoplasm; regulates the channel being
able to open and accept ions
 ATP receptors
 Trimers (3 subunits)
o Mainly; M1 and M2
 Nicotinic ACh receptor (nAChR)
o Made up of 5 subunits; 2 alpha, 1 beta, 1 gamma, and 1 delta
 Alpha subunits are most important because that’s where the bonding sites for ACh are
 Binding sites aren’t on the top of receptors but midway
o Between the alpha subunits and whatever is next to it
 May be between alpha and gamma or beta
o Once 2 ACh molecules bind in the inside part; that’s what opens the channel and allows ions
to flow thru it
 nACh receptor
o The way the hydrophobic regions are arranged such that it forms a barrel (with a hole in the
middle)
 Where the negatively charged amino acids on each subunit are faced inwards; so it
can repel anions and makes it selective to cations
 Specifically allows sodium and potassium ion in only
o No negatively charged ions like chloride
  Importantly, M2 region is always facing inwards and creates the ring of negatively
charged AA
o Once ACh accesses the binding sites, it changes its conformation which allows the opening of
channel
 It’s attracted to the charge of inner pore
o What happens if the whole receptor was phosphorylated?
 Three kinases are involved:
 PKA phosphorylates the delta and gamma subunits
 PKC phosphorylates the delta subunit
 Tyrosine kinase phosphorylates the beta, gamma, and delta subunits
 Thru phosphorylation, it causes the receptors to enter the desensitized state
 Which is a state in which receptor remains closed even in presence of ACh
 5-HT3 receptor
o Mainly a metabotropic receptor but we will focus on this specific ionotropic type
 Acts like a ACh receptors
o Homomeric complex (its repeating the same subunits)
 Most are alpha 7 subtype which is normally found in nAChRs
o Not widely found in the brain and central nervous system as the other serotonin receptors
 Importance
 This receptor is target of many psychotropic medications that effect
behaviours
o Drug antagonists for this receptors = to treat nausea, psychosis and
so forth
 GABAA
o Again, composed of 5 subunits
 Usually, 2 alpha, 2 beta and one of either delta or gamma subunit
o Activated by binding of 2 GABA molecules in the cleft which are formed between the alpha
and beta subunits
 Glycine
o Similar to GABAA
o More defined in its subunits
 Know exactly which one of subunits each time
o Because of 3 alpha subunits = can require up to 3 bindings of molecules to open the receptors
 # of NT binding depends on # of alpha subunits
 Glycine and GABAA selectivity
o In this case, the M2 domain is facing middle part but it’s a positively charged part to allow
attraction of anions (selective to negatively charged ions)
 Glutamate
o In terms of ionotropic glutamate receptors
 Theres the 3 families NMDA, AMPA and kainate
 However, classified as non-NMDA or NMDA receptors
o Why?
 AMPA and kainite is blocked by the same drug (CNQX)
 Have the same mechanisms they operate and similar
effects
  But NMDA is only blocked by APV
o AMPA receptors
 Have 3 domains on the subunits
 Amino terminal domain = for attracting the receptor
 Ligand binding domain = where it actually binds to
 Transmembrane domain (TM or M)
o M1, M2 and M3
o Subunit composition
 AMPA receptors are considered tetrameric structures
 Each have combo of different subunits
o GluA1, GluA2, GluA3, and GluA4
 Most AMPA receptors are gluA2 in the brain
 Having GluA2 renders the receptors impermeable to Ca2+ ions
o Only permeable to Na+ ions
 A lot of our plasticity, learning and memory (behavioural
functioning) depends on involvement of calcium
 In order to turn GluA2 impermeable to calcium…
 There has to be RNA editing
o One glutamine molecule is replaced with an arginine molecule
(single nucleotide modification)
 This modification leads to gluA2
 Glutamate
o NMDA receptors
 Special qualities
 Permeable to Ca (as well as to Na and K)
 Opening of the channel requires extracellular glycine as a cofactor
 Its both ligand-gated and voltage-dependent
 At rest…
 Magnesium blocks the entrance of pores and stays there unless the cell is
already depolarized
 Once depolarized, it allows the magnesium block to pop off and allow entry
for sodium and calcium
 Voltage dependence aspect
 Graph
o In normal conditions, Mg block is present
o At +30 to +60 (reaching depolarization); magnesium block is
removed
 Due to conformation change
o If we remove the block experimentally, it will be open all the time
 No matter the voltage
o NMDA receptor structure
 Similar to the AMPA receptors
 Even have the M2 domain that isn’t transmembrane
 Main receptor classes revisited
o Structural differences between metabotropic and ionotropic receptors
  Table (see table)
 Physiological action
o Ionotropic receptors regulate channel that are basically on and off
switches
 Make the neuron fire AP or inhibit it from firing AP
o Metabotropic receptors are its longer and sometimes, molecules are
diffusing instead of acting quickly across the synapse
 They have modulating properties (not mediating)
 Effect of NT binding
o In ionotropic receptors, it always leads to increase in opening of
ionotropic receptors
o Activation of metabotropic receptors; may or may not open
 Effects are varied and much tightly regulated
o Metabotropic receptors regulate a variety of channel types
 Its slow action are normally insufficient on their own to cause a cell to fire an AP
 Thus, gaol is to influence the existing electrophysiological properties of that
neuron (modulating activities)
o Many ways
 Keep the cell more excitable or less excitable
 Make it more positive or negative
 They can operate in different parts of the cell (since activity is diffuse; doesn’t have
to be close to synapse to affect)
 Diagram
o Act on the cell body by acting on resting and voltage gated channels
 Alter the resting potential of neurons
 Changes the threshold of potential or potential’s duration of
AP
 Change repetitive firing of AP
o Acts on channels on the terminal (postsynaptic membrane)
 Modulate NT release
 Target opening of other ionotropic receptors
 How long its opened or closed
o Acts on presynaptic cell
 Activate the second messengers that can modulate
 N and K channels
 How NT is released
o Docking of NT
 How many NT is docking
 GPCRs
o They consist of a single polypeptide
 7 membrane-spanning helical segments (instead of 5 like the other ones)
 N-terminus
o Extends on the top into the extracellular space
 C-terminus
o Resides in the cytoplasm
 o With exception of GABA B and glutamate metabotropic receptors
 All look similar to the beta adrenergic receptor
 All have aspartate in the TM3 (third transmembrane subunit)
o Regulates glutamate binding
 It can reduce binding by over 100 folds
 If they have a serine, it reduces binding by 10,000 folds
 Importance; regulation of what’s coming in or being passed thru depends on what is
on the transmembrane subunits
 GPCR structure
o The composition of AA in specific positions is what determines the stability of the ligand
binding as well as what type of ligand can bind to a receptor
 Diagram B
 Variations in the 5 AA and their positions specify the type of GPCR (what’s
facing inwards)
o Allows what it can bind to
 Differing between the beta adrenergic and mACh receptor
 GPCR activation logic
o Not only is the general structure of GPCRs fairly conserved, but also the sequence of events
following their activation is conserved
o Diagram
 General cascade when first messenger (NT) binds to the receptor
 For all GPCRs, it leads to transducer and primary effector which then lead to
second messenger
o Sometimes, a secondary effector is involved
o Specific second messenger effector enzymes change depending on receptor type
 What differentiates between different receptor types
 But overall cascade stays the same
o In the brain there are 20 different types of subunits
 Called alpha subunits because it contains alpha and beta subunits
 There are 12 different types of gamma subunits
 Depending on different subunits, it differentiates different classes and which
effectors as well as what effects are ultimately happening in the brain
o Example
 Beta adrenergic Rs
 Activates the adenylyl cyclase by acting on Gs proteins
o Leading to activation of calcium channels
 Muscarinic ACh Rs
 Inhibits adenylyl cyclase by acting on Gi proteins
 GPCR activation logic
o Understand how ligand binding leads to activation of G proteins
o Proposed models of its activation
 Assumes the GPC receptor can spontaneously isomerize between inactive and active
states
 At equilibrium; without presence of agonist, its in an inactive state
o No G protein activation is happening
  When ligand binds; a conformation change happens
o Shifts to active form where it begins the cascade activation
o Antagonist
 (1) bind to inactive state so it can’t transition anymore into an active state
 (2) bind to both inactive and active states
 Freezes them in place
 Presynaptic mechanism
o Activation of G (alpha)
 In presynaptic terminal, GPCR activation of G alpha triggers a cascade
 Leading to activation of PKA and PKC
o Go on to phosphorylate proteins involved in vesicle recruitment,
docking fusion
 Then can cause inhibition or facilitation of synaptic
transmission
o Modulation of NT release by PKA and PKC targets
 Snap25; PKA and PKC phosphorylate the serine molecule which influences the
activity of SNAP-25
 Overall, fine tuning the NT release
o PKC phosphorylation of SNAP-25
 How does PKC phosphorylate part of SNAP 25?
 It increases the association between snap 25 and syntaxin
o Leads to more of the SNARE complex to form and facilitates
exocytosis (NT release)
o PKA phosphorylation of Syntaphilin
 PKA phosphorylates syntaphilin which competes with SNAP 25 for syntaxin binding
 If attached to syntaxin, SNAP 25 can’t attach to syntaxin meaning it cant join
together to make the SNARE complex
 PKA phosphorylation pop off the syntaxin from the syntaphilin
 Leading to SNARE complex and more NT release
 Postsynaptic mechanisms
o GPCR activation can modulate excitability thru PKA mediated closure of K channels
 Graphs
 Adding serotonin which activates the GPCR thru cAMP which leads to
increase in PKA
o Leading to closure of K channels
 Importance
o Serotonin is important in regulating depolarization thru the activity
of cAMP and PKA
o Also increase activation postsynaptically thru long term changes in synaptic transmission
 Since PKA activity leads to increase in transcription and chromatin structure (via
PKA activation of CREB)
 Metabotropic glutamate receptors (mGluRs)
o mGluRs exist as dimers meaning it requires binding of 2 glutamate molecules
o Interestingly
 Its ECD consists of
  VFT
o Traps the glutamate
o Also has CRD which attaches to 7TM domain subunit
 Muscarinic ACh receptors (mAChRs)
o Muscarine binds to these acetylcholine metabotropic receptors (named as such)
o Present pre and post synaptically
 See that it can activate the same cascade but cause different effects
 Presynaptically
o Leads to more NT release
 Postsynaptically
o Slows depolarization to increase excitability of neuron
 More likely to fire another AP
o Divided into 2 classes based on different TM makeup
 Ones that contain M1, M3, M5
 Bind mostly to Gq
o Causes an excitatory cascade
 Ones that contain M2, M4
 Active Gi or Go
o Causing an inhibitory cascade
o M2 and M4
 Coupled thru Gi and Go pathway
 Decreases cAMP (by blocking it)
o Leading to suppression of NT
 Decreasing amount of ACh and glut being released
 Dampens excitability
 Dopamine receptors
o Found both pre and post synaptically
o Structure is similar to other catecholamines
o We know they have 5 subtypes
 Not sure of their functions of each subtypes
 Thus; differentiate between D1-like or D2-like receptors
o Other subtypes follow similar functions as one of those 2 categories
o Main distinction between D1 and D2 like types
 How it acts on adenylyl cyclase?
 D1-like receptors activate adenylyl cyclase thru interactions with Gs proteins
 D2-like receptors inhibit adenylyl cyclase via acting on Gi or G0 proteins
o Can see metabotropic receptors can be the same class using same NT
but depending on where they are located and how they are activated
 Can cause different cascade of reactions to increase or
decrease excitability
Week 8:

 Implicit vs explicit memory

o Breakdown long term memory into 2 types of memory
 Implicit memory = Autonomic movements (not accurate but good enough)
  Procedural memory
o Riding a bike
 Non associative learning
o Habituation and sensitization
 Aplysia
o Giant sea snail
 Useful because we know of its nervous system
 Nerve cells are very big
 Easy to do electrophysiology studies and stimulate neurons
 Nobel prize-winning research
o Habituation in Aplysia
 Respiratory organs; gill (in yellow) and siphon (used to expel seawater and waste)
 Together part of defense reflex
o Withdraw the gill and siphon away from danger
 Mild touching the siphon; causes it to reflex withdraw
immediately
 Withdraws both the gill and siphon
o If repeated touching, the withdrawal reflex
is habituated
 Stops all together or becomes not as
strong
 Gill withdrawal reflex circuit
o Touching the siphon excites the population of mechanoreceptor sensory neurons; these
innervate the siphon
 Release glutamate
 Generates an EPSP onto an interneuron and motor neuron
o Both these are temporally and spatially summated
 Which causes the motor neurons to discharge strongly
 Fires an AP and lots of NT release
o Leads to very strong withdrawal
o In habituation
 If we repeat the stimulus; the EPSPs produced by the sensory neuron from the
interneurons onto the motor neurons start to decrease overtime = less gill withdrawal
 In addition, decrease the overall synaptic transmission
o Graph C
 Initial touch doesn’t change but the sensory neurons’ EPSPs onto interneuron (caused
by decreased release of NT) is less, and withdrawal decreases over time
o Because the whole circuit uses only one main synapse
 It’s called a homosynaptic depression
o Habituation is short lived
 If waited one hr, the reflex goes back to baseline
 Can this habituation be long term?
o Yes, it can be long term
 Repeated sessions in a single day can produce long term
habituation
  Withdrawal reflex will last weeks
o Graph B
 At control see connections are reduced after habituation for one day, for one week but
starts to recover after 3 weeks
 Long term habitations is characterized by a change in the # of synaptic
connections but in short term habituation, its characterized by change in NT
release
o So how do we convert the short term change into long term change?
 Via sensitization (different type of learning and memory)
 Enhancing gill withdrawal
o When any animal repeatedly encounters a harmless stimulus, its responsiveness habituates
(learn to ignore)
o However, for harmful stimuli, animal learns fear
 Releases a forceful response to the stimuli (responds vigorously to any other stimulus
happening at the same time)
 In defensive situation, our escape/ withdrawal behaviour becomes heightened
(sensitized)
o Learned fear response
o Graph B
 Fearful stimuli; shocking the tail = for aversive stimuli (light blue)
 Fast withdrawal but goes back to baseline
 After 4 tail shocks; the duration of withdrawal increases and last longer
 Short term learning
 After 4 sessions of tail shock in 4 days; even greater withdrawal response and lasts
much longer
o Sensitization is achieved via an increase in synaptic transmission at several connections
(including the same connection we talked about b4 by sensory and motor neurons +
interneuron)
 Circuit is bi-directional response
 It can sensitize and increase transmission
 It can habituate and decrease transmission
 Enhancement in synaptic transmission at multiple levels
o Not homosynaptic transmission
 Heterosynaptic potentiation
 Depends on additional modulatory interneuron
o Is activated by the tail shock (not involved in habituation)
o Graph
 Tail shock is the same
 In sensitization
 However, increased EPSP in motor neuron (withdrawal effect is stronger)
 In sensory neuron, there is broadening of AP
o Lasting a bit longer b4 returning to base line
 Involves calcium influx thru voltage gated calcium channels
 Which in turn enhances NT release
o Image
  3 groups of modulatory interneurons involved in sensitization process
 Best studied is the one that uses serotonin as a NT
o Serotonin interneuron which forms a synapse on a region of sensory
neuron (axoaxonic synapse)
 Serotonin is released from interneuron after tail shock
 Binds to receptors on the sensory neuron
o Coupled to G proteins and activate adenyl
cyclase
 Produces 2nd messengers for cAMP
pathways; which in turn activates
cAMP dependent protein called
PKA
 Also, actives second type of GPCR that leads to
hydrolysis of phospholipids and activation of PKC
o Essentially, phosphorylation mediated by PKA and PKC enhances
release of NT from sensory neuron
 Since there are 2 proteins; 2 different mechanism are
involved
 (1) PKA phosphorylates K channels causing them to close
 Broads the AP
o Potassium stays in the cell instead of leaving the cell
 Allowing the AP to take longer to come back to baseline
o Also, it enhances calcium influx thru voltage gated calcium channels
 This in turn leads to more NT release at the active zones
 (2) Phosphorylation thru PKC
 Enhances the functioning of NT release directly
o Serotonin receptor activates G protein coupled receptors
 Causes activation of PKC and it goes to facilitate NT release
at active zones
 Again remember; PKC phosphorylates SNAP 25
which causes it to facilitate the SNARE complex
formation
o So presynaptic facilitation in response to a release by serotonin caused by the tail shock lasts
for period of minutes (short time)
 Repeated stimuli = strengthening of synapse = long term
 Looking at that via classical conditioning
 Pairing stimuli with response
o Classical conditioning = more complex form of learning
 How animals associate one stimuli with another
 Siphon touch with the tail shock
o Like Pavlov’s dog
o In our case; it’s the gill withdrawal reflex in response to a weak touch of the siphon
(condition stimulus)
 We classically condition a gill withdrawal reflex, we are causing a sensitization
 Sensitize to the siphon touch
o (1) paired stimulation
  Siphon touching is happening almost immediately b4 the tail shock (in 4 minutes
apart)
 Becoming associated
o After training, sensitization is occurring to the siphon touch
 The unconditioned stimulus must be paired super close to allow the prediction so
when siphon touch occurs, you learn that it predicts the scary shock that follows
 Order matters; siphon touch (predictor) b4 the tail shock
o Paired pathway
 Broadening the AP because you are converging both signals at the same time
 Molecular mechanism
o Convergence of 2 mechanisms happening at the same time = presynaptic facilitation
 Cultured neurons (now study in cultured neurons)
o When doing in culture, can do 5 spaced training sessions per hour
 Leads to one or more days of long term sensitization
 If continued same pattern of training = lasts weeks and weeks
o Since there is no tail shock; we are artificially activating the modulatory serotonin circuit thru
a puff of serotonin (via pipette)
 Mimicking the interneuron circuit
 Long-term facilitation requires de novo protein synthesis
o If u added a protein synthesis inhibitor (like azinomycin), you abolish the long term
facilitation
 True for RNA synthesis inhibitors as well
 Molecular mechanism
o Know long term sensitization involved genes and protein expression
o 1-2
 Called a memory suppressor gene because it blocks CREB-1 from acting on
transcription and translation
o 4
 The ubiquitin hydrolase that is synthesized by the activation of its IEG; is a a
regulatory subunit of PKA
 When ubiquitin hydrolase joins with PKA
o It leads to persistent phosphorylation of the substrate proteins of
PKA
 More NT release
 Since PKA is more active; interacts with vesicle
docking mechanisms
o 5
 CAAT box enhancer (TF) that forms a dimer with itself and also a heterodimer with
another TF called AF (activating factor)
 Together these factors travel downstream and trigger the growth of synaptic
connections
o Which support long term memory
o For long term memory
 Need persistent PKA activation for strengthening NT release and C/EBP traveling
with AF to support the growth of new synaptic connections
o Graph
 In sensitization; explosion of more synapse’s connections