Neuro 3000 Unit 2 Exam

Lecture 7. Techniques 1
Transgenic mice, pronuclear injection – generally used when you want to express some
protein in a tissue/cell type of interest – gene promoter that targets expression to some
tissue or cell type + protein coding sequence of protein you want to express - could be a
neutral reporter protein that allows you to label some cells of interest for purposes of in
vivo imaging (like green fluorescent protein, aka GFP), or a protein or modified version
of a protein that you expect will have some effect when you express it, and you want to
explore its function or what happens (a toxin that kills or ablates cells, an inhibitor
protein that blocks some pathway, etc)
A knockout (KO) mouse is when you target a specific gene and inactivate it. A knock-in
(KI) is different because the goal may not be to inactivate a gene, but to change it in
some way to express a modified version. For example, switching a single amino acid to
study a known human mutation which does not inactivate the protein but changes its
function, knocking-in a coding sequence of a reporter (GFP, channel rhodopsin, etc) so
it can be driven by the promoter of the natural gene you knocked it in to, adding loxP
elements to a gene (“floxxing” it), etc. Generally your goal is to modify, not KO, a gene.
Know these terms: ES cells, homologous recombination, chimeras, global KO,
conditional KO’s using Cre-loxP, conditional and inducible KO’s using CreER-loxP,
etc...; don’t need to know all the details, just what they are used for – also
CRISPR/Cas9, NHEJ for targeted gene-inactivating small mutations (single nucleotide
insertion/deletion), and HDR for gene editing/repair or inserting large DNA fragments
Know idea behind RNAi, ASO, Huntington Holiday effect
Lecture 8. Techniques 2
Measurements of gene expression changes, bulk vs cellular resolution
Bulk for protein: western blotting, uses SDS-PAGE (gel electrophoresis) to separate
proteins, then an antibody to detect specific proteins; method is semi-quantitative –
ELISA’s are also bulk but more quantitative, more sensitive than westerns
Bulk for mRNA – qPCR or quantitative PCR – need to first convert mRNA to cDNA
using reverse transcriptase - PCR amplification is exponential, each cycle doubles the
amount of specific DNA target = 2x where x = number of cycles – 20 cycles produces
~1,000,000-fold amplification – RealTime PCR (reveals the exponential growth of DNA
amplification in ‘real time’) and Ct or threshold cycle, etc – curve deflection to left or right
indicates increased or decreased expression, respectively; 1-cyle = two-fold change, 2-
cycles = 4-fold, etc... – RNA-Seq is bulk but high-throughput because you can identify
changes in many mRNAs at the same time
In all the above methods you prepare protein or mRNA samples from tissues taken from
‘treated’ and ‘untreated’ animals or people, then use identical amounts of protein or
mRNA for the comparison – you are looking whether your protein or mRNA of interest
goes up or down (change in expression) after treatment – but you get no cell-type
resolution
Method for cell-type resolution for protein is IHC, and ISH for mRNA – scRNA-Seq for
mRNA is high-throughput and single cell resolution, typically can assess changes in
gene expression in ALL cell types at one time and for all mRNAs in all those cell types,

very powerful – do not need to understand the details of the scRNA-Seq method or the
clustering algorithm, but you should understand the key conclusions you can draw from
a Seurat cluster diagram like the figure in slide 34 that was also used for a TopHat
question: in this example the clusters indicate i) three types of astrocytes, and ii)
methadone significantly affects gene expression in all three types
Optogenetics, can be done in cell culture, or using transgenic or knock-in approaches in
whole animals – good way to study the function of specific cell types in a brain circuit
controlling a behavior in living animals – light-activated channels – channelrhodopsin
ChR2 is excitatory to excite neurons when you shine light on them – halorhodopsin
NpHR is inhibitory to do the opposite
Viral vectors for gene expression provides investigators control of where a protein of
interest gets expressed by injecting the virus directly into brain; different from
transgenics, because in transgenics every cell in the animal carries the transgene, but
with viral expression the gene is only in cells near where you inject the virus – the virus
is ‘engineered’ to carry the coding sequence of a protein you want to express in cells in
that brain region – the virus ‘infects’ those cells and expresses the protein - alternatively
can be used for gene therapy (intrathecal injection, for example)
Lecture 9. Synaptic transmission I
Electrical synapses - gap junctions, connexins, etc. - for electrical synchronization of
cells – important for function of selected brain circuits, like inferior olive in the brainstem
Chemical synapses - Most synapses are chemical – Otto Loewi experiment on frog
heart, discovered ACh, slows down the heart – got Nobel Prize with Dale
Presynaptic – presynaptic axons can contact different parts of postsynaptic cells,
axosomatic, axo-spinous, etc... - presynaptic site has specializations called active
zones, where synaptic vesicles filled with neurotransmitter are found, can be just one
active zone or many active zones at a single synapse – vesicles are ‘docked’ at active
zones – large secretory granules (also called dense-core vesicles), usually some
distance from active zone, carry peptide neurotransmitters which are really
neuromodulators, are trafficked long-distance from the soma into axon
Postsynaptic specialization = postsynaptic density or PSD (where the neurotransmitter
receptors are located)
Grays Type I and II: respectively, pre-synaptic vs post-synaptic asymmetry = excitatory;
symmetry = inhibitory
NMJ – what are it’s unique features? – postsynaptic side called motor endplate (on the
muscle), many presynaptic active zones (on motor neuron axon terminals), fast, reliable
– active zones of motor neurons are aligned with junctional folds on postsynaptic side –
EPP (end-plate potential) always produced in muscle, when motor neuron fires an AP
Transmitter types – synthesis – peptides made in soma, transported in large dense core
vesicles, generally are further away from active zone than transmitter vesicles – amines
synthesized in terminals, and amines and amino acids loaded into vesicles using
vesicular transporters – docking/priming - voltage-gated Ca2+ channels, transmitter
release by exocytosis
Amount of transmitter released is dependent on frequency of AP

Docking/priming – v-SNARES (on vesicles) & t-SNARES (on the terminal membrane),
know the general idea of how they work – vesicles are docked and primed prior to
arrival of AP, just waiting – then AP leads to Ca2+ influx and syntaxin/synaptotagmin
coupling, leads to vesicle fusion, then exocytosis (transmitter dumped into synaptic
cleft)
Recycling of vesicle membrane is mainly by endocytosis (the opposite of exocytosis)
Transmitter removed from cleft by transporters on the presynaptic terminal membrane
and on astrocytic end-feet, or by degradation using enzymes (only for ACh, using
enzyme called ACh-esterase, which is secreted into synaptic cleft)
Lecture 10. Synaptic transmission II
Neurotransmitter (NT) receptors, ionotropic (fast) vs metabotropic (slow)
Ionotropic – not as selective as voltage-gated channel – often pass both Na+ and K+ -
receptor pore is closed until the ligand (NT) binds, then the channel opens to the ions
Local potentials in the postsynaptic cell are the main result of presynaptic NT release –
EPSPs (depolarizing) vs IPSPs (hyperpolarizing).
Even though ionotropic channels gate both Na+ and K+, they will typically depolarize a
cell which is at resting potential before the NT arrives – reversal potential determined by
Goldman equation with relative permeability to the two ions set at 1:1 – generally near
+8 mV in most cells, which is the maximum local potential that could be achieved under
sustained NT application, and which is well above the threshold potential of -55 mV –
such local potentials would be under the same constraints on propagation down the
dendrite that we discussed before, namely that they decline in strength with distance
from the source – whether the cell fires an AP depends on Vm at the spike initiation
zone.
I-V plot used to show the response of a NT-gated receptor – each receptor type will
have a unique plot depending on its unique ion preferences and permeability ratios –
the I-V plot uses a voltage clamp to artificially set the cell at desired Vm’s (x-axis), and
measure the resulting currents (y-axis; note that to do this kind of study you need to
prevent the cell from firing an AP which it would do above -55 mV, and which would
generate confounding currents – can block the AP using TTX; don’t need to know this
technical detail, although hopefully you find the information useful) – Vm above reversal
potential (> +8 mV) will generally result in net outflow of K+ current, below this (< +8
mV) will cause net inflow of Na+ current – remember that conductance for Na+ and K+
is equal, so any changes in current at different Vm values will be solely due to driving
force (I = g x driving force) - basically each ion is trying to reach its Eion, and at reversal
potential the two ions split the difference – even at reversal potential neither ion is truly
‘happy’, because it is still not at its Eion, but it is a ‘draw’, equal but opposite currents and
so no net current
One “quantum” of ACh is one vesicle being released – could measure this at the NMJ
by blocking voltage-gated Ca2+ channels (inhibits high-level transmitter release)
EPSP summation – NT stimulation generally results in graded potentials, different from
EPP in NMJ – graded potential can be summed – spatial vs temporal summation as we
discussed before
A graded potential that is trying to move towards the soma is affected by dendritic cable
properties - Length Constant, know what it is and its definition, but don’t need to study

equation – know what affects it, such as anything that opens K+ or Cl- channels would
tend to decrease it – can think of an open K+ channel as a ‘leak’ that decreases
depolarizing current (K+ out is hyperpolarizing, making Vm more negative) like a leaky
hose or think of it as decreased membrane resistance (allowing less current to flow
down the axon, and more current leaking out) – this kills off any spread of depolarization
– blocking K+ or Cl- channels would increase length constant, enhancing any
depolarizing potential and its movement towards the soma
Metabotropic receptors, GPCRs, mainly modulatory – activate G-proteins which can
directly activate or inhibit channels (slow compared to ionotropic receptors) or work
through 2nd messengers like cAMP to affect channels downstream (even more slow =
slowest) – showed example of NE  receptor, which can cause closure of K+ channel –
would increase length constant by plugging the leaky hose = greater propagation of a
depolarizing potential
Lecture 11. Neurotransmitter Systems I
Agonists (activate receptors) and antagonists (block receptors), help to define
transmitter receptor subtypes, and pharmacological properties of receptors – part of the
tool box of neuroscience
Dale’s Principle, mainly true for GABA and glutamate with respect to each other (GABA
never found in the same neurons with glutamate), but plenty of examples where
peptides or modulatory NT’s are co-transmitted or co-released with GABA, Glu or other
NT’s
Key synthesis steps of catecholamines – starts with tyrosine, TH needed for all, DBH
only for NE and E – enzymes in vesicles or cytoplasm?, study this (DBH only one in
vesicles, the rest are in cytoplasm near to active zones), and brain regions where found
– substantia nigra and VTA are DA-ergic; LC is NE-ergic
For the most part don’t need to study epinephrine (E) – focus on DA and NE
Catecholamine vesicle loading, VMAT vesicular transporters in SLC18 gene family
(VACh transporter also in this family; see next lecture) - plasma membrane transporters
used to uptake and recycle NT’s from cleft, plasma membrane transporters DAT and
NET in SLC6 gene family (GABA and Gly plasma membrane transporters in same
family, see table on slide 25)
Know main catecholamine projections and what they are for (nigrostriatal for voluntary
movement, etc.), but not details of targets of projections - know NE in sympathetic part
of autonomic nervous system (outside conscious control; ‘fight or flight’)
Serotonin aka 5-HT, chillness concept and HPA axis (but do not need to know details
about HPA) – synthesis of 5-HT, starts with tryptophan (different from catecholamines!,
which start with tyrosine), TPH is key enzyme; produced in raphe nuclei in midbrain and
brainstem
5-HT vesicle loading, VMAT in SLC18 gene family – plasma membrane transporter
used to uptake 5-HT from cleft, SERT in SLC6 gene family, SSRI’s (see slide 25)
DA, NE and 5-HT can be recycled back into vesicles, or any excess degraded by MAO
in mitochondria (not degraded in synaptic cleft, only ACh can do that)
Know volume transmission exists for the catecholamines – these transmitters can use
volume or synaptic transmission, varies from region to region in the brain

Autoreceptors respond to a cell’s own release of transmitter - 5-HT autoreceptors can
be presynaptic to control release, or postsynaptic to control the overall state of the
neuron
Lecture 12. Neurotransmitter Systems II
Amino acid neurotransmitter’s (NTs) and their main functions, general
GLU main excitatory NT, can be synthesized from glutamine, another amino acid;
GABA main inhibitory NT
GABA comes from glutamate, GAD key rate-limiting enzyme for this conversion, GAD is
a great marker for GABAergic neurons (for ISH or IHC)
GLU vesicle loading, VGLUT (vesicular transporter protein name) in SLC17 gene family
– plasma membrane uptake from cleft, EAAT (protein name) in SLC1 gene family (GLU
taken up by both presyn terminal and astrocytes)
GLU converted to glutamine in astrocytes (the cycle), transferred to neurons by
glutamine transporter – in contrast, GLU reloaded into vesicles in presynaptic terminal
GABA, Gly vesicle loading, VIATT (vesicular transporter protein name) in SLC32 gene
family, used for both GABA and Gly loading into vesicles – plasma membrane uptake,
by GAT and GLYT, both in SLC6 family (taken up by terminal and astrocytes)
GABA converted to glutamine in astrocytes, transferred to neurons by glutamine
transporter
Cotransport (plasma membrane transporters) vs countertransport (vesicular
transporters), know the examples of these two terms and the differences in their
mechanisms
ACh synthesis, rate-limiting step, and ChAT enzyme (great marker for cholinergic
neurons!)
Remember: ‘cholinergic’ refers to ACh – ‘catecholamine’ refers to dopamine and NE
Cholinergic neurons much more sparse in the brain compared to GABAergic and
GLUtamatergic – mainly brain stem and basal forebrain
ACh vesicle loading, VAChT (vesicular transporter protein name) in SLC32 gene family
ACh degraded to choline + acetic acid in cleft by AChEsterase (AChE) (the only NT
degraded in cleft)
Special transporter for choline uptake for recycling choline to make more ACh, in axon
terminal (there is no specific plasma membrane ACh transporter)
Two main cholinergic projection pathways in the brain: basal forebrain (cognition,
attention,addiction) and brainstem (facial motor, sustained attention, REM sleep)
Parasympathetic – rest and digest – uses ACh (for example, damps down heart rate,
due to M2 ACh receptors which activates a K+ channel, hyperpolarizing the heart
muscle)
Other NT’s – neuropeptides, important examples are endogenous opioids and HPA axis
peptides, SST and VIP used for circuit studies in cerebral cortex (don’t need to know
details for any of these) – purines, ATP, excitatory – adenosine inhibitory – NO, not in
vesicles, released by postsynaptic neurons, retrograde signal affects transmitter release
- endocannabinoids, not in vesicles, retrogradely activate CB1 and CB2 GPCR
receptors on presynaptic terminal, acts to damp down NT release
Ionotropic nACh receptor, subunit structure = pentamer, each subunit has 4 TM’s, binds
ACh only on alpha subunits, 2 bound ACh molecules opens the channel (so seems a

minimum effective number of alpha subunits is 2) – CHRN gene family encodes the
subunits, CHRNA for alpha subunits, CHRNB for beta, etc – 10 alpha subunit genes,
and 4 beta subunit genes – different subunits expressed in different brain regions, a key
concept is that there is huge diversity of nACh receptor subtypes across the brain based
on which neurons and brain regions express each specific subunit gene; we will find the
same kind of thing for glutamate and GABA ionotropic receptors
Lecture 13. Neurotransmitter Systems III
Ionotropic glutamate (GLU) receptors – 3 different types based on selective agonists,
subunits encoded in separate gene families called GRIA, GRIN and GRIK, all are
tetramers – in addition, all subunits have 4 trans-membrane (TM) domains, but unlike
for all the other ionotropic receptors, TM2 is only partial for GLU receptors, which is a
unique structural element – AMPA, NMDA and Kainate are all permeable to Na+ and
K+, but NMDA also permeable to Ca2+ - other unique NMDA features are voltage-
dependency, and has Mg2+ block which is removed at depolarizing voltage of -30 mV –
NMDA receptors and Ca2+ are linked to mechanisms of learning and memory
GABAA receptor, ionotropic, similar to nACh receptor, pentameric, GABA binds at alpha
subunit, and just like ACh need at least two alpha subunits and two molecules of GABA
to open channel - forms a Cl- channel – modulated by Valium and phenobarbital in
same direction, but by distinct mechanisms
Glycine receptor, also a Cl- channel, similar in structure to AChR and GABAA receptors
(pentamer, alpha subunit for Gly binding, etc) – strychnine is an antagonist
Metabotropic G-protein coupled receptors, 7-TM is iconic structural motif – know basic
mode of operation of G-proteins
G proteins are heterotrimers – Galpha (G) and Gbeta (G) and Ggamma (G). G and
G are two separate arms that act on different targets
Some G-proteins can activate K+ channels (hyperpolarizing), for example direct
interactions with activated G, like M2-ACh receptors in the heart - which btw, in the
brain, would tend to reduce propagation of depolarizing local potentials (decreasing the
length constant)
Others can inhibit K+ channels (depolarizing), like  NE receptor, through activation of
adenylyl cyclase and PKA; which btw, in the brain, would tend to facilitate propagation
of depolarizing local potentials (increased length constant)
Both of the above examples of metabotropic receptors are slower than ionotropic
receptor channels; and mACh would be faster than  NE, since for mACh receptors,
G directly acts on the K+ channel and does not need a second messenger.
NE and 2 NE receptors have opposite effects on adenylyl cyclase – NE is
stimulatory through Gs (also called Gs), and 2 NE is inhibitory through Gi (also
called Gi) – thus, opposite effects on cAMP levels
Main advantage of GPCRs is amplification
However, another difference from ionotropic receptors is that metabotropic responses
may be more sustained; in comparison, ionotropic receptors are fast, but are terminated
immediately – we did not really discuss this though in the lecture, so don’t worry about it