This document is a thesis presented by Sebastian Aguiar to the Keck Science Department in partial fulfillment of a Bachelor of Arts degree. It aims to characterize a novel monoclonal antibody targeting the AMPA receptor subunits GluA1, GluA2, and GluA3. The thesis provides background on AMPA receptor structure, function, trafficking, role in synaptic plasticity, involvement in neuropathology, and pharmacology. It then describes the research questions and methods used to visualize the antibody's binding in rat, monkey and human brain tissue using confocal and electron microscopy.

1. Characterizing a novel monoclonal AMPA
receptor 1/2/3 antibody in the hippocampus and
prefrontal cortex of rat, monkey, and human
A Thesis Presented
by
Sebastian Aguiar
To the Keck Science Department
Of Claremont McKenna, Pitzer, and Scripps Colleges
In partial fulfillment of
The degree of Bachelor of Arts
Senior Thesis in Human Biology
December 2013

2. Alan
Jones
Donald
McFarlane
Emily
Wiley
Thomas
Borowski

3. Characterizing a novel monoclonal AMPA receptor 1/2/3 antibody
in the hippocampus and prefrontal cortex of rat, monkey, and human
Sebastian Aguiar
Pitzer College!, W.M. Keck Science Department
!
John Morrison, PhD, Mentor
Dean of Basic Sciences and the Graduate School of Biomedical Sciences
Professor of Neuroscience, Geriatrics and Palliative Medicine
Friedman Brain Institute, Icahn School of Medicine at Mount Sinai
!
Alan Jones, PhD, First Reader
Professor of Neuroscience and Psychology, Pitzer College
Abstract
The excitatory, ionotropic glutamatergic AMPA receptor is the most common
membrane-bound receptor in the central nervous system. AMPARs and the
NMDA receptors are central to synaptic plasticity, memory, and mechanisms of
neurodegeneration. The AMPAR is an obligate heterotetramer, composed of
subunits GluA1-4. Subunit permutation determines ion conductance, trafficking
and other functional characteristics. Few available antibodies are subunit-specific,
disabling researchers from accurately visualizing differential AMPAR subunit
distribution in the nervous system. This study sought to visualize a novel
monoclonal GluA1/2/3 antibody with functional avidity for three of four receptor
subunits and to characterize the ultrastructural localization of these receptors using
confocal and electron microscopy.

4. Table of Contents
Acknowledgements
Introduction
Receptor Structure and Mechanics 1
Receptor Trafficking 3
Biogenesis 3
Synaptic Targeting 4
Exocytosis and Endocytosis 5
Synaptic Plasticity 7
Long-Term Potentiation 8
Long-Term Depression 10
Homeostatic Scaling 11
Neuropathology 11
Pharmacology 13
Allosteric Modulators 14
AMPAR Subunit Characterization 19
Research Questions 20
Methods and Materials
GluA1 Transformation 22
Transcardial Perfusion 23
Immunohistochemistry 23
Confocal and Electron Microscopy 24
Results
Anti-GluA Monoclonal Antibody 25
Single / Double Label Confocal Microscopy 28
PSD95, pAb AMPA, mAb Colocalization 30
Colocalization Plot 31
Immunogold Electron Microscopy 32
Discussion 33
References 34
Appendix 42

5. Table of Figures
Introduction
Figure 1: AMPAR Heterotetramer and Generic Subunit
Structure
Fig 2: Electron micrograph of GluA2 tetramer showing
dual N-terminal domains, ligand binding domains, and
the transmembrane domain
Fig 3: Proteins that associate with the C-terminus of the
AMPAR
Figure 4: Preliminary activity state models of the
AMPAR
Figure 5: TARP-mediated AMPAR trafficking from
endoplasmic reticulum to dendrite, followed by PSD95
binding
Figure 6: Schematic of AMPAR associated trafficking
proteins
Figure 7: LTP Recording Setup and Electrophysiology
Figure 8: Phosphorylation and Receptor Dynamics
Figure 9: Suspected Pathological Molecules in
Alzheimer’s Dementia
Fig 10: Perampanel and tezampanel
Figure 11: Locations of ligand binding for both AMPA
and kainate receptors
Figure 12: Effect of CX717 on Sleep Deprivation and
Cognition in DMS Task
Figure 13: Dose-Response Data
Fig 14: Glucose consumption PET scan
Figure 15: Dimer of dimer subunit ratios in rat Shaffer-
CA1 pyramidal neuron synapses

6. Results
Fig 1. Western blot
Figure 2. Non-antigen retrieved rat prefrontal cortex
stained with monoclonal GluA1/2/3 antibody
Figure 3: Non-antigen retrieved hippocampus stained
with monoclonal GluA1/2/3 antibody
Figure 4: Human Prefrontal Cortex with antigen
retrieval
Figure 5. Colocalization: antigen retrieved human
prefrontal cortex stain of DAPI, GluA1/2/3 and Post
Synaptic Density-95.
Figure 6. Double labeling with GluA1/2/3 and PSD-95
in human, monkey and rat hippocampus
Figure 7. Double labeling of rat CA1 hippocampus with
the GluA1/2/3 monoclonal antibody colocalized with the
polyclonal GluA2/3 antibody.
Figure 8. Colocalization plot
Figure 9. Electron microscopy
Figure 10. Dilution series

7. 1
Introduction
AMPA Receptors
The identification of the AMPA-type ionotropic glutamate receptor as the primary
regulator of fast excitatory transmission in the CNS constitutes one of the major
achievements of modern neuroscience. These ubiquitous receptors are critical for
synaptic plasticity, mediating memory-associated changes in dendritic morphology,
signaling the expression of neurotrophins, and are implicated in many neurological
disorders. 1
AMPA receptors (AMPARs) were named for the artificial
selective agonist and glutamate analogue !-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid by Tage Honore and colleagues
at the Royal Danish School of Pharmacy in Copenhagen, and
published in 1982 in the Journal of Neurochemistry. 2
Receptor Structure and Mechanics
AMPARs are obligate heterotetramers, composed of subunits GluA1-4, 3
with
each subunit possessing a binding pocket for the neurotransmitter glutamate. 4
From 1989
to 1992, the genes encoding the four subunits (GRIA1-4) were identified. 5
Different
combinations of subunits determine the pharmacological, functional and trafficking
characteristics of the channel. 6
Diversity can also come in the form of post-translational
modifications and splice variants (such as the Arg607 Q/R residue). 7
Figure 1: AMPAR Heterotetramer and Generic Subunit Structure. 8
AMPA

8. 2
Fig 2: Electron micrograph of GluA2 tetramer showing dual N-terminal domains, ligand binding domains,
and the transmembrane domain. 9
Receptor subunits are composed of an N-terminal extracellular domain (NTD)
with an intermediate ligand binding domain (LBD), four transmembrane domains (TMD),
and an intracellular C-terminal tail that interacts with a multitude of scaffolding proteins
involved in structure and signaling at the post-synaptic density. These include NSF, AP-2,
PICK1, GRIP, ABP, KIF5, PKC, PKA, SAP97, PSD95, and a family of Transmembrane
AMPAR Regulatory Proteins (TARPs) such as stargazin. 10, 11 12
Fig 3: Proteins that associate with the C-terminus of the AMPAR. 11
In adult excitatory hippocampal neurons, most AMPARs are composed of GluA1-
2 or GluA2-3 complexes. 13
All AMPARs are ligand-gated cation channels, allowing
sodium in and potassium ions out, tending toward an equilibrium potential of 0 mV
(about halfway between ENa+ and EK+). GluA2, however, is calcium impermeable. And
GluA2 trumps GluA1 in a heterotetramer. The majority of AMPARs in the hippocampus
(approximately 80%), however, possess GluA2 and are therefore calcium impermeable. 14

9. 3
As a result, AMPARs are “dependent” upon NMDA-mediated calcium influx in the
LTP/LTD response (see the section on Synaptic Plasticity below).
In 1998, Eric Gouaux’s laboratory reported the first structural assessment of the
isolated ligand-binding domain. They identified the N-terminal extracellular structure as
a “clamshell” that is closed by the binding of two glutamate molecules, leading to the
opening of the ion pore in a scissor-like motion. 15
Models of activation and
desensitization states have been developed using computational x-ray crystallography.
Figure 4: Preliminary activity state models of the AMPAR. Desensitization is characterized by reduced
response after prolonged exposure to agonist. This process is mediated by separation of the upper lobes of
the LBD dimer.
16
Receptor Trafficking
Biogenesis
AMPAR subunits are mostly synthesized in the somatic endoplasmic reticulum,
modified and assembled into tetramers in the Golgi apparatus and finally inserted into the
membrane where they laterally diffuse in and around the dendrite. AMPARs are
synthesized in response to neural activity, a process mediated by the metabotropic GluRs
1 and 5. 17
AMPARs are also synthesized within dendrites. The mRNA is packaged and
transported via microtubules in response to glutamatergic stimuli. Translational outposts
in the dendrites consist of polyribosomes and golgi structures. Interestingly, D1/D5
dopaminergic agonism is also capable of augmenting AMPAR synthesis and eliciting a

10. 4
heightened frequency of spontaneous miniature excitatory postsynaptic currents
(mEPSCs) in the hippocampus. 18
Synaptic Targeting
Long-term potentiation, depression, and the attendant memory and learning are
mediated by rates of synthesis, membrane transport, and endocytosis of AMPA receptors.
Vesicular trafficking from the Golgi involves dyneins and kinesins, small molecular
motors that move cargo along microtubules. 19
Trafficking from the soma to the dendrites
is mediated by the TARP family of chaperone-like proteins.
Figure 5: TARP-mediated AMPAR trafficking from endoplasmic reticulum to dendrite, followed by
PSD95 binding. 11
A complex of GluR2, the PSD protein GRIP1, and kinesin can be
immunoprecipitated from brain lysates, and the expression of nonfunctional versions of
kinesin decreases synaptic abundance of AMPARs. 20
Within the dendritic spines, actin
and myosin have been implicated in clathrin-dependent endocytosis and local AMPAR
trafficking. 21
Stargazin, a calcium channel gamma-subunit homolog, plays a major role in
several points of the AMPAR secretory process. 22
Originally identified as the mutant
gene in the Stargazer mouse, which is afflicted with cerebellar ataxia and epilepsy, it has
since been shown to promote transport of AMPARs to the cell surface. Stargazer mouse
cerebellar granule cells consequently show very low AMPAR levels at the synapse. 23
Post-synaptic density protein 95 (PSD 95) is a structural protein critical to
AMPAR stability. Stargazin is complexed with both AMPARs and PSD95, which
anchors the group to the post synaptic density. Overexpression of PSD95 results in

11. 5
increased AMPAR-mediated synaptic currents, 24
and knockdown results in decreased
AMPARs at the synapse. 25
Exocytosis and Endocytosis
AMPARs have a metabolic half-life of approximately 30 hours. 26
Explaining
their synaptic dynamics, especially in light of their role in long-term memory, is a critical
area of research. For example, it remains controversial whether AMPARs are primarily
inserted into the synapse directly or into the adjacent extrasynaptic membrane and then
laterally diffuse to the synapse.
AMPAR insertion is dependent upon subunit composition. Insertion of long-tailed
C-terminus AMPARs GluA1/4 occurs slowly under basal conditions and is stimulated by
NDMA activation and neural activity. In contrast, receptors containing GluA2/3 tend to
be constitutively trafficked to the synapse under basal conditions and are not dependent
upon neural activity. 27
AMPA receptors are held in intracellular reserve pools and are fused with the
membrane as necessary. The administration of tetanus toxin cleaves SNAREs and blocks
AMPAR insertion at the synapse. 28
N-ethylmaleimide-sensitive fusion protein (NSF) has
been shown to bind with the C terminus of the GluA2 subunit, regulating rapid
exocytosis. AMPARs in the reserve pool complex with PICK1, a protein that is thought
to stabilize the reserve pool. When NSF binds with PICK1, the former dissociates from
AMPARs and enables them to fuse with the synapse. 29
Endocytosis of AMPARs is similar to that of G-protein coupled receptors
(GPCRs) because both processes feature clathrin-coated pits and require dynamin. 30
After being internalized, AMPARs are engulfed by early endosomes and sent either to a
specialized recycling endosome compartment that allows quick reinsertion to the surface
or to late endosomes and ultimately lysosomes for degradation. 31
The immediate early gene CPG2 mediates basal and activity-regulated AMPAR
internalization and localizes to the endocytic zone. CPG2 knockdown inhibited AMPAR
and NMDAR endocytosis. 32
Another immediate early gene, Arc, is induced by neural
activity and directly implicated in cognition and long-term memory. Arc mRNA is tightly
regulated and translated at activated synapses. Arc regulates AMPAR trafficking via

12. 6
interactions with the endocytic proteins dynamin and endophilin. 33
Finally, Tumor
Necrosis Factor Alpha (TNF-!) augments AMPAR insertion. 34
Figure 6: Schematic of AMPAR associated trafficking proteins. 1
Recycling and degradation may be coordinated, at least in part, by ubiquitination
signals and the ubiquitin-proteasome system (UPS). Monoubiquitination has been shown
in C. elegans to signal endocytosis, whereas polyubiquitination leads to proteasome

13. 7
degradation. 35
In mammalian neurons, targeted mutation of the ubiquitin peptide at
lysine 48 prevented AMPA-induced receptor internalization. 36
The role of ubiquitination
in AMPAR recycling remains an active area of study.
Synaptic Plasticity
Changes in synaptic strength are believed to underlie memory storage. 37
Hebbian
Learning postulates “neurons that fire together wire together,” and that neurons sensitize
or desensitize in response to repeated stimuli. In the terms of Neural Network Theory,
memory is encoded in the pattern of synaptic strength or “weights” distributed across the
probability space of possible connections. Likewise, forgetting is the loss or decay of this
pattern of synaptic weights.
Long-term potentiation (LTP) and long-term depression (LTD) are the two most
studied cellular models of synaptic plasticity. 38
AMPARs are the metaphorical “currency”
of LTP and LTD, and the NMDA receptor can be understood as the cashier – transacting
in AMPARs under various stimulus conditions. 39
Long-term memory formation
putatively involves cyclic AMP response element binding protein (CREB) and Mitogen-
activated protein kinases (MAPK). 40
Figure 7: A) LTP Recording Setup, B) Augmented AMPAR current and number of AMPARs at the
synapse in response to high frequency vs. low frequency stimulation. 7

14. 8
Long-Term Potentiation
Long-term potentiation can be defined as the long lasting enhancement of signal
transmission that results from synchronous stimulation. The phenomenon was first
demonstrated in the rabbit hippocampus by Terje Lømo and Tim Bliss in 1966, who
stimulated Schaffer collateral (pyramidal CA3) neurons and recorded from CA1
pyramidal cells. The effect has been shown to last for weeks in vivo, and to correlate with
performance on spatial memory tasks. 41
LTP also involves other regions of the brain
such as the limbic system and cortex. 42
Inducing LTP experimentally involves stimulating the presynaptic neuron with a
high frequency (typically 100 Hz for one second) tetanus, resulting in an excitatory post-
synaptic potential. The postsynaptic neuron will be sensitized and more likely to respond
with a higher amplitude output for an extended period of time. A single AMPAR-
mediated excitatory post-synaptic potential (EPSP) has a rise time-to-peak of
approximately 2–5 ms and a mean duration of 30 ms. If stimulated one hundred times a
second, the AMPAR will attempt to open every 10 ms (including the refractory period),
the EPSC will increase, and EPSPs will sum resulting in a moderate local depolarization,
triggering NMDARs and initiating a sequence that leads to increased synaptic weighting.
AMPARs are mostly found as GluA2-containing heterotetramers, which are
impermeable to calcium. For LTP to occur, NMDA (named for the selective agonist N-
methyl-D-aspartate) receptors must be activated. NMDARs are dual-ligand (glutamate
and D-serine or glycine) and voltage-gated due to magnesium ion blockade at resting
potential (-70 mV). AMPARs depolarize the membrane potential enough to dislodge the
Mg2+
ion (about 0 mV) and enable the NMDAR Ca2+
current to flow inward.
In short, the role of the AMPAR is to initiate a local membrane depolarization
sufficient to revoke the Mg2+
blockade, leading to the activation of the more “powerful”
NMDARs, which, in turn, instigate a cascade that results in the regulation of AMPAR
membrane insertion. Many excitatory synapses are actually thought to be
postsynaptically 'silent', possessing functional NMDARs but lacking AMPARs. The
acquisition of AMPARs at silent synapses may also be important in synaptic plasticity

15. 9
and neural development. 43
Kinase phosphorylation is crucial in the regulation of neural function, like in most
cell types. 44
AMPAR phosphorylation is also an integral part of LTP. 45
A substantial rise
in calcium concentration within the dendritic spine initiates LTP by activating kinases.
Low calcium influx activates phosphatases leading to long-term depression (LTD).
AMPARs are phosphorylated by Calcium/calmodulin-dependent kinase II
(CaMKII), which was discovered in the lab of Paul Greengard in 1978, another
monumental discovery in the molecular neuroscience of memory and learning. 46
CaMKIIA isoform knockout mice demonstrate a low frequency of LTP and fail to form
persistent, stable place cells in the hippocampus. 47
CaMKII itself is autophosphorylated
in the presence of NMDAR-mediated calcium influx. CaMKII does not appear to be
necessary for recruiting AMPARs to the synapse but phosphorylation Ser831 of GluA1
does indeed augment ion conductance. 48, 49, 50
Figure 8: Phorphorylation of AMPARs influences cation conductance, as
well as playing a role in dendritic spine trafficking and membrane
insertion. 45

16. 10
Protein Kinase A (PKA) phosphorylation of GluA1 at Ser845 is also critical to
LTP. Intracellular perfusion of PKA into HEK 293 cells transfected with GluA1 led to a
40% potentiation of whole cell glutamate current peak amplitude, 51
likely by increasing
open-channel probability. 52
This potentiation effect was lost when Ser845 was mutated to
alanine. Site-directed mutagenesis and pharmacological studies indicate that this
phosphorylation is necessary but not sufficient for GluA1 membrane insertion during
LTP. 53
LTP was diminished in mice with mutated knock-in Ser381 and Ser845 sites that
resist phosphorylation. 54
Protein Kinase C (PKC) has also been shown to phosphorylate
Ser818 of GluA1, a modification that is implicated in synaptic trafficking during LTP. 55
Long-Term Depression
As downtrodden as this molecular process sounds, it is actually critical to synaptic
plasticity and rather something to be upbeat about. LTD is the means by which a
postsynaptic neuron becomes desensitized to input from the presynaptic neuron,
putatively by means of NMDAR-dependent AMPA receptor endocytosis. 56,57
The state is
induced by low frequency stimulus (1 Hz) over an extended period of time. Most studies
take place in hippocampal sections in vitro, so it remains a challenge determining how
the mechanism actually works in vivo. Heynen et al., however, have demonstrated that
hippocampal AMPAR postsynaptic membrane surface expression increased with LTP
and decreased with LTD in vivo. 58
Insulin plays a role in LTD, by activating phosphatidylinositol 3-kinase (PI3K)
and protein kinase B (PKB) via an NMDAR-dependent mechanism, 59
as well as by a
distinct AMPAR sorting pathway to specialized endosomes. 60
LTD is dependent upon
calcium influx and the activation of the phosphatase calcineurin. 61
For hippocampal LTD,
Ser845, the PKA site on GluA1, is dephosphorylated. Mice with this site mutated exhibit
deficits of LTD and NMDAR-induced AMPAR internalization. 62
In the cerebellum,
chemical activation of PKC was sufficient to phosphorylate Ser880 of GluA2 and induce
LTD and AMPAR internalization. 63
Interestingly, PKC does not appear to mediate the
phosphorylation of Ser880 in the hippocampus, indicating that this requirement is
satisfied by other kinases. 64

17. 11
Homeostatic Scaling
Chronic excitation or inhibition leads to compensatory mechanisms in cultured
neurons. Raising activity by blocking inhibitory synaptic transmission markedly
decreased the synaptic AMPAR population and reduced the EPSC. This also applied to
NMDARs. 65
Likewise, the chronic administration of AMPAR antagonists for hours to
days at a time increased the synaptic AMPAR population. 66
Both TNF-! and the immediate early gene Arc have been implicated in this
homeostatic process. By administering tetrodotoxin (TTX), a voltage-gated Na+
channel
blocker, glutamate release can be suppressed. This low level of glutamate causes glia to
release TNF-!, which upregulates AMPARs through an unknown mechanism. 67
Arc acts
as a sensor of neural activity, augmenting endocytosis when activity is elevated.
Conversely, blocking neural activity increases Arc levels and the AMPAR surface
population. Arc overexpression nullifies the gains in AMPARs that normally
accompanies TTX-induced activity reduction, and Arc hippocampal cell culture
knockouts exhibit no compensatory scaling in either direction. 68
This compensatory
mechanism poses an obstacle for the development of sustainable drug therapy. 69
Furthermore, any therapy must be precise enough to distinguish between constitutive and
regulated AMPA trafficking. The activity-independent constitutive pathway, typically of
GluA2/3 bearing receptors, maintains the total number of AMPARs and is thought to
replace damaged receptors and preserve newly formed memories. The regulated pathway
traffics predominantly GluA1-containing AMPARs, is inactive under basal conditions
and is activated by LTP. 70
Neuropathology
AMPARs are putatively involved in many diseases, including X-linked mental
retardation, Alzheimer’s disease, amyotrophic lateral sclerosis, limbic encephalitis,
epilepsy, ischemic brain injury, and Rasmussen’s encephalitis. 71, 72, 73, 74, 75
Given that Alzheimer’s dementia (AD) threatens to exacerbate the looming
demographic-healthcare crisis, the role of AMPAR dysfunction in AD deserves special
attention. An estimated 3.4 million people are affected by dementia in the United States.

18. 12
76
The elderly population (aged over 65) is expected to double by 2030, reaching 72
million, or 20% of the total U.S. population. 77
A similar shift awaits the developed world.
AD is characterized by the progressive loss of neural function, culminating in cell
death and impaired cognition. The fundamental etiology of AD remains controversial,
though there seem to be certain histological hallmarks including "-amyloid (A"42),
neurofibriliary tangles composed of hyperphosphorylated tau, inflammation, oxidative
stress, adrenergic and cholerinergic deficits. 78 79
A novel disease model even implicates
insulin resistance – the “type 3 diabetes” hypothesis. 80
Figure 9: Suspected Pathological Molecules in Alzheimer’s Dementia. A"42 can exist as intercellular
monomers, inhibiting the proteasome, as well as in the more familiar form of intracellular plaques. 81
Whether A" or tau are by-products of another underlying mechanism, or whatever
precise ratio of causal factors, AMPARs are intimately involved. A" secretion adversely
affects LTP and depresses both AMPAR and NMDAR currents in cultured slices. 82, 83
Furthermore, A" has proven capable of phosphorylating AMPARs at Ser880, signaling
LTD and endocytosis, indicating that A" may be responsible for synaptic depression,
AMPAR withdrawal, and dendritic spine loss. 84
The loss of spines and synaptic

19. 13
sensitivity presages cognitive impairment, and safe, effective methods of preserving
AMPAR function and neural integrity are certainly called for.
Pharmacology
Agonists
Each AMPAR has four glutamate binding sites, putatively formed by the
extracellular N-tail and the extracellular loop between transmembrane domains three and
four. The channel opens when two sites are bound, and as more sites are bound, more
current flows through the ionopore. 85
Full agonists are almost exclusively used to induce lesions for ablation
experiments. They include various AMPA analogues, williardiine analogues (found in
Mimosa bark), 86
domoic acid (culprit in amnesic shellfish poisoning and algal bloom
toxicity), 87
and quisqualic acid, an L-glutamic acid analogue found in Combretum
indicum (traditionally used as an antihelmintic). These few compounds that have been
characterized thus far are simply too potent for therapeutic use. (See Appendix for IC50
values).
Antagonists
Antagonists have demonstrated much more clinical relevance. One excessively
well-characterized compound, ethanol, partially acts as an AMPAR antagonist. 88
Upregulation of AMPARs in alcoholics is implicated in hyperactive excitation during the
withdrawal period. 89
The drug acamprosate (N-acetyl homotaurine) acts as an antagonist
at NMDARs and an agonist at GABAA receptors. The drug is of questionable efficacy, 90
but future compounds may be more effective if they target AMPAR regulation as well. 91
There is important emerging evidence for the involvement of AMPARs in addiction. 92, 93
Other antagonists include L-theanine (found at high levels in tea), a putative
cognitive enhancer 94
and neuroprotective agent; 95
kynurenic acid, an endogenous
tryptophan analogue implicated in schizophrenia 96
and cognitive impairment; 97
as well
as the experimental receptor blockers CNQX and NBQX. There is also reason to believe
that norepinephrine inhibits AMPAR currents in all layers of the rat temporal cortex. 98

20. 14
The AMPAR antagonists perampanel and tezampanel have found clinical
application in the treatment of epilepsy and as neuroprotective agents after stroke,
traumatic brain injury and the attendant excitotoxicity. 99
Furthermore, tezampanel
demonstrates analgesic and anxiolytic activity in animals. 100
Allosteric Modulators
Both positive and negative allosteric modulators (AMs) provide greater precision
of control or “fine tuning” of potential therapies. These drugs are also more selective for
receptor subtypes because they bind less conserved allosteric regions (rather than merely
binding the glutamate site of the LBD or blocking the ion pore with varying affinities). 101
Furthermore, AMs are believed to impact receptor dynamics less, leading to
reduced withdrawal and tolerance formation because these drugs work with pre-existing
receptor populations. AMs generally work by modifying the time course of deactivation
and/or desensitization. These drugs do, however, affect neurotrophin expression – a fact
that even further augments excitement about therapeutic application. 102
Negative AMs (NAMs) have not been studied much, but they include
extracellular protons (via a pH sensing region on the LBD), 103
neurosteroids (like
sulfated pregnenolone analogues), 104
and unsaturated fatty acids like arachidonic acid. 105
More research in this area is certainly warranted.
Positive AMs (PAMs) have been studied more extensively, and they work in at
least two different ways: some slow the rate of desensitization following repeated ligand
binding to the LBD, while others obstruct the exit of the agonist from the LBD.
There are presently three structural classes of PAM:
Fig 10: Perampanel and tezampanel

21. 15
1. Pyrrolidinone and related piperidine compounds (e.g., aniracetam and CX614).
2. Benzothiadiazide compounds (e.g., cyclothiazide, diazoxide)
3. Biarylpropylsulfonamide compounds (e.g., PEPA, LY404,187).
Detailed crystallographic descriptions of these ligand-receptor interactions are
now being correlated with network circuitry and behavioral output. This literature is
quickly evolving and beyond the scope of this thesis. 106
What began with the -racetam family has expanded to encompass a host of other,
highly potent and selective compounds; for example, PEPA has one hundred times the
potency of aniracetam at the AMPAR in vitro. 107
PAMs are promising therapeutics for
dementia, major depression, ADHD, Parkinson’s, Huntington’s, ALS and other cognitive
disorders. Ailments characterized by neuron loss may be particularly amenable to PAM
treatment because several ampakines augment brain-derived neurotrophic factor (BDNF)

22. 16
expression in disease models. 108, 109, 110
Thus far, however, clinical trials for dementia
have been lackluster due to bioavailability and blood-brain barrier issues.
Figure 11: Locations of ligand binding for both AMPA and kainate receptors. 111
The ampakine farampator demonstrated an acute effect on short-term memory in
a preliminary clinical trial of healthy elderly. 112
One concern is that prolonged ampakine
use could downregulate synaptic AMPARs via compensatory homeostatic scaling.
Lauterborn et al found that continuous incubation of cultured hippocampal slices with
CX614 rapidly multiplied BDNF mRNA (over 3–12 h) but this was followed by a decline
to control values over the next 36 hours coupled with a comcomittant decline in
AMPARs at the synapse. 113
This finding portends a “crash.” Lauterborn, Gall, Lynch et
al addressed this concern, finding that CX614 can sustain increases in BDNF protein
without AMPAR downregulation when moderate doses were administered for three hour
intervals rather than continuous (24 hr) incubation of brain slices in the ampakine
solution (see data in appendix). 114
Deadwyler et al. demonstrated dramatic sleep-deprivation alleviating cognitive
effects of CX717 in non-human primates performing a delayed match-to-sample task.
Not only was performance augmented for 30-36 hour sleep-deprived monkeys (to the
point of removing impairment), but also CX717 significantly augmented performance of
well-rested monkeys in a dose dependent manner. Positron emission tomography
measures of regional cerebral metabolic rates for glucose (CMRglc) during the task
I

23. 17
revealed that activity in the PFC, dorsal striatum, and medial temporal lobe (including
hippocampus) was significantly enhanced by CX717. 115
As these data show, correct
matches were highly significantly increased (P < 0.001), and latency of response was
reduced by a similar magnitude.
Figure 12: Normally rested + vehicle vs. Normal + CX717 delayed match-to-
sample task. Animals treated with CX717 outperformed in both accuracy and
latency parameters. Sleep deprived animals administered CX717 outperformed
even those that were normally rested on both parameters. (N = 11) 115

24. 18
Figure 13: A) Dose-Response Data. CX717 is shown administered on consecutive sessions for nine
monkeys over three dose ranges (0.3–0.5 mg/kg, 0.8–1.0 mg/kg, and 1.5 mg/kg, IV). Each CX717 session
(C, arrows) was interspersed with a single normal vehicle (V) session. B) Monkeys were given 0.8 mg/kg
of CX717 midway of their sessions. C) Overall mean dose-effect relationship of CX717 on normal alert
DMS performance across monkeys (n = 9).
Fig 14: Glucose consumption PET scan. Brain Region
Abbreviations: Dorsal Prefrontal Cortex, Dorsal Striatum,
Thalamus, and Medial Temporal Lobe. 115

25. 19
AMPAR Subunit Characterization
Different permutations of subunits of the heterotetrameric AMPAR determine its
functional characteristics. There are efforts underway to develop antibodies that
selectively tag these receptors with subunit specificity with the expectation that novel
therapies will ultimately take advantage of differential subunit localization and activity in
disease states.
GluA2 receptors have been relatively easy to characterize because they exhibit a
signature inward rectifying current and are responsive to an array of polyamines and
toxins. The other three subtypes exhibit more subtle pharmacological and
electrophysiological signatures.
In Neuron, Lu et al in the lab of Roger Nicoll at UCSF published an innovative
single-cell knockout method that has yielded much greater insight into the precise subunit
composition at CA1-Shaffer pyramidal neuron synapses. 116
GluA4 is conspicuously
absent from their data, because although it is found in the brain, it has much lower
expression levels than the other three. 117
According to Lu et al, GluA4 does not play any
role in the AMPAR-mediated transmission in CA1 pyramidal neurons.
Figure 15: Dimer of dimer subunit ratios in rat Shaffer-CA1 pyramidal neuron synapses. GluA1/2
tetramers predominate both at the synapse (80%) and extrasynaptically (>90%), whereas GluA2/3 tetramers
compose only about 16% of synaptic AMPA receptors. 118
Subunit composition is no mere technical curiosity – it is a major mechanism by
which the brain regulates its level of excitation. For example, RNA editing replaces a
80%

26. 20
glutamine with an arginine at the TMD 607 residue of GluA2. This change accounts for
the calcium impermeability of GluA2 and its inwardly rectifying current properties
(letting more current in than out). Transgenic mice with impaired Q/R editing exhibit
epileptic seizures and die within two weeks after birth. 119, 120
Understanding this kind of
subtle change may yield profound dividends in human therapy.
Unaddressed Research Questions
Although great strides have been made, there are many questions that remain to be
answered with regard to AMPARs trafficking and cognition. Two prominent researchers
in the field, Jason Shepherd at the Picower Institute for Learning and Memory at MIT and
Richard Huganir at the Howard Hughes Medical Institute at Johns Hopkins, ask the
following questions in their comprehensive review: 1
“Many basic cell biological questions still remain to be addressed. What is
the role of locally synthesized receptors, and how are local translation and
mRNA trafficking regulated? How do receptors traffic in and out of the
entangled complex of proteins in the PSD? When does lateral diffusion
versus direct insertion/internalization occur at synapses?
In addition, a huge challenge remains to elucidate the role of AMPAR
trafficking in vivo, in terms of precise mechanisms, as well as determine
the role that these processes play in synaptic plasticity and behavior.
Information can be stored in the brain for years, yet AMPARs are highly
dynamic and have a metabolic half-life of only a couple of days. Therefore,
if AMPAR levels do determine synaptic strength, how can synaptic weights
be maintained for weeks, months, or years? Moreover, how do individual
synapses within a neuron know how many receptors it needs to maintain its
potentiated or depressed state?”
Research Question(s)
This experimental thesis set out to address the following questions: Does a newly
developed monoclonal antibody bind to AMPA receptors with subunit specificity and
under what conditions? If the antibody binds to the appropriate sites where AMPARs are
putatively found, and does so for numerous subunit permutations, then the antibody likely
demonstrates avidity for multiple AMPA receptor subtypes.

27. 21
The AMPAR 1/2/3 monoclonal antibody (mAb) functions as a sort of
experimental control – one can compare, for example, a GluA1-specific mAB to the
novel “pan-AMPA” (1/2/3) mAb. If, for whatever reason a researcher must visualize all
of the predominant AMPAR subunits of the CNS, this mAb would also serve that
purpose.
These questions were addressed using double-label immunohistochemistry,
confocal laser scanning microscopy and electron microscopy. These studies were
conducted in accordance with the methods of Morrison et al. 2005. 121

28. 22
Methods and Materials
Expression of GluA1 on a plasmid vector in transformed E. Coli.
GluA1 subunit with an N-terminally fused SUMO protein by the lab of Dr.
Thomas Moran, Director Microbiology Department, Icahn School of Medicine at Mount
Sinai. The entire GluR1 receptor subunit has a molecular weight of 106 kD (Milipore) to
101 kD (PhosphoSitePlus). Listed below is a portion of the extracellular N-terminal
domain putatively containing antigenic target portion of the GluR1, fused with a SUMO
peptide in bold. The molecular weight of this 376 peptide subsequence after the SUMO
protein has been cleaved is 43kD. Including the SUMO protein, it weighs 48 kD.
MGSSHHHHHHSSGLVPRGSHMASMSDSEVNQEAKPEVKPEVKPETHINLKV
SDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL
DMEDNDIIEAHREQIGGSNFPNNIQIGGLFPNQQSQEHAAFRFALSQLTEPPKLL
PQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSF
PVDTSNQFVLQLRPELQDALISIIDHYKWQKFVYIYDADRGLSVLQKVLDTAAEK
NWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIIKLEKNG
IGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPAKIMQQWKNSDARD
HTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVP
WGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNE
DDKFVPAATD
The clonal line of mAb producing B-cells was expanded with hybridoma
technology in the lab of Dr. Thomas Moran. The avidity of the 5 µg/mL GluA mAb was
verified using a Western blot by sodium dodecyl sulfate polyacrylamide gel

29. 23
electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and visualized
with anti-mouse secondary antibody conjugated with I125
.
Transcardial Perfusion and Brain Acquisition
The brains of six adult Sprague-Dawley rats and one macaque monkey (Macaca
fascicularis) were acquired by transcardial perfusion. The rats were were deeply
anesthetized with a lethal dose of chloral hydrate (300 mg/kg) and the monkeys were
deeply anesthetized with ketamine (25 mg/kg) and Nembutal (30 mg/kg). The animals
were transcardially perfused with cold 1% paraformaldehyde in PBS, followed with cold
4% paraformaldehyde and 0.125% glutaraldehyde in phosphate buffered solution (pH
7.4).
All experiments were performed according to the NIH guidelines for research on
vertebrate animals and the Institutional Animal Care and Use Committee (IACUC) at the
Mount Sinai School of Medicine approved all protocols. The brains were immediately
postfixed in 4% paraformaldehyde for an additional 6 h at 48° C. The brains were then
cut into 50 micron sections on a Vibratome and preserved in 1% sodium azide. The
human brain was accessed via Dr. Patrick Hof’s research program.
Immunohistochemistry
A subset of sections underwent antigen retrieval, a process that breaks aldehyde
crosslinks and renders the target antigens more “visible” to the antibody. The sections
were rinsed in 37 ° C 200 mL dH2O for 5 minutes followed by 6.5 minutes in 100 mL of
0.2M HCl and 1 mL pepsin. This was followed by three washes in RT phosphate
buffered solution.

30. 24
A blocking solution was used in order to reduce non-specific mAb binding. The
solution was made up of 5% bovine serum albumin, 0.2% cold water fish skin gelatin,
and 0.3% Triton-X100 permeabilizing agent in phosphate buffered solution. The same
protocol was followed for the primary (polyclonal GluA2/3 or monoclonal GluA1/2/3)
and secondary Ab (1:400 diluted anti-Mouse Alexa 488 and anti-Rabbit 555) double-
label incubations. The sections then were mounted on microscope slides, stained with
Vectashield and DAPI, dried and cover-slipped.
Confocal and Electron Microscopy
Immunofluorescence of anti-Mouse Alexa 488 and anti-Rabbit 555 secondary
fluorophores were analyzed and imaged using a Zeiss LSM 410 inverted laser scanning
confocal microscope equipped with a ArKr 488/568 laser and Zeiss Plan-Neofluar
objectives (Zeiss, Oberkochen, Germany). A colocalization correlation was generated
using built-in Zeiss analysis software.
Ultrastructural analysis was performed using a Hitachi 7000 (Tokyo, Japan)
electron microscope. We used 10 nanometer immunogold-conjugated secondary
antibodies (preparation credit to Rishi Puri). We also conducted a dilution series in order
to determine the appropriate concentration for secondary antibody, using 2.5, 5, and 10
µg/mL. All images were prepared using Adobe Photoshop 7.

31. 25
Results
Characterization of anti-GluA monoclonal antibody
Fig 1. Western blot analysis demonstrated immunoreactivity to GluA subunits 1, 2 and 3,
but not 4. The antigen binding motif region was recognized at approximately molecular
weight 48 kD. The control lane was using mock-transfected E. coli lysate supernatant.
Single Label Confocal Microscopy
Figure 2. Non-antigen retrieved rat prefrontal cortex stained with monoclonal GluA1/2/3
antibody. Roman numerals denote layers of the neocortex. Ab binding was nonspecific.

32. 26
Figure 3. Non-antigen retrieved rat
hippocampus and stained with
monoclonal GluA1/2/3. On the right
is a magnified image of CA1 and the
dentate gyrus.
26

33. 27
Figure 4. Human Prefrontal Cortex with antigen retrieval. The section was stained with
DAPI and GluA1/2/3. The binding pattern is puntate, indicating greater specificity.

34. 28
Double Label Confocal Microscopy
Figure 5. Antigen retrieved human prefrontal cortex stain of DAPI, GluA1/2/3 and Post
Synaptic Density-95. Yellow punctae denote the sum of GluA1/2/3 and PSD-95,
indicating a high degree of colocalization at the postsynaptic density.

35. Human Hippocampus
GluA1/2/3 + PSD-95
Monkey Hippocampus
GluA1/2/3 + PSD-95
Rat Hippocampus
GluA1/2/3 + PSD-95
Figure 6. Double labeling with GluA1/2/3 and PSD-95 in
human, monkey and rat hippocampus. Yellow denotes
colocalization.
29

36. 30
DAPI GluA1/2/3
Monoclonal
GluA2/3
Polyclonal
!
Figure 7. Double labeling of rat CA1 hippocampus with the GluA1/2/3
monoclonal antibody colocalized with the polyclonal GluA2/3 antibody.

37. 31
Figure 8. Colocalization plot of the monoclonal GluA1/2/3 and GluA2/3 showing
moderate colocalization. Generated using Zeiss Zen built-in colocalization correlation
function.

38. Figure 9. Electron microscopy of non-human primate (macaque) hippocampus labeled
with 10 nm immunogold spheres conjugated to GluA1/2/3, which was used at a
concentration of 10 µg/mL. The antibody was successfully localized to the post synaptic
density of the dentritic spines.
Figure 10. Dilution series of 2.5, 5, and 10 µg/mL immunogold nanospheres
conjugated to monoclonal GluA1/2/3 antibody.
32

39. 33
Discussion
The novel monoclonal antibody binds to the ionotropic glutamatergic AMPA
receptor subunits 1, 2, and 3. Confocal laser scanning microscopy showed that the
antibody forms discrete punctae in the neuropil alone and when colocalized with PSD-95,
validating expected synaptic presence. The electron microscopic studies display a precise
localization of gold particles at the postsynaptic density synapse, likely in synthetic pools
within spines as well as stabilized on the membrane. Antigen retrieval is strongly advised
to augment antigen specificity. The fact that the antibody does not bind GluA4 is
acceptable because this subunit does not appear to play any major role in AMPA receptor
trafficking.
The antibody does not colocalize with the polyclonal GluA 2/3 to a convincing
degree, this dysfunction may be due to time-dependent storage degradation of the
GluR2/3 antibody. A follow-up experiment is currently underway.
The monoclonal antibody is functional, particularly with antigen retrieval,
between 5 to 10 µg/mL, pending confirmation with more confocal and EM double-
labeling with different sized nanogold spheres. Upon further validation, this antibody will
be ready for the market and use by neuroscientists all over the world. Hopefully this
novel antibody will provide a higher degree of subunit-specific granularity for
experimentalists and will expedite the development of therapies that attenuate epilepsy,
dementia, amylotrophic lateral sclerosis and other neuropathologies associated with
deranged AMPA receptor dynamics.

40. 34
References
1
Shepherd JD, Huganir RL. The Cell Biology of Synaptic Plasticity: AMPA Receptor
Trafficking. Annu. Rev. Cell Dev. Biol. 2007;23:613–43
2
Honore T, Lauridsen J, Krogsgaard-Larsen P. The binding of [3H]AMPA, a structural analogue
of glutamic acid, to rat brain membranes. J. Neurochem. 1982;38(1): 173–178.
3
Collingridge GL, Olsen RW, Peters J., and Spedding M. A nomenclature for ligand-gated ion
channels. Neuropharmacology 2009;56, 2–5.
4
Wenthold RJ, Petralia RS, Blahos J, Niedzielski AS. Evidence for multiple AMPA receptor
complexes in hippocampal CA1/CA2 neurons. J Neurosci. 1996;16:1982–1989
5
Boulter J, Hollmann M, O’Shea-Greenfield A, Hartley M, Deneris E, Maron C, Heinemann S.
Molecular cloning and functional expression of glutamate receptor subunit genes. Science.
1990;249:1033–1037.
6
Shi S, Hayashi Y, Esteban JA, Malinow R. Subunit-specific rules governing AMPA receptor
trafficking to synapses in hippocampal pyramidal neurons. Cell. 2001;105:331–343.
7
Swanson GT, Kamboj SK, Cull-Candy SG. Single-channel properties of recombinant AMPA
receptors depend on RNA editing, splice variation, and subunit composition. J Neurosci.
1997;17:58–69
8
Fleming J and England P. AMPA Receptors and Synaptic Plasticity: A Chemist’s Perspective.
Nature Chemical Biology. 2010; 6(2):89-97.
9
Nakagawa T. The biochemistry, ultrastructure, and subunit assembly mechanism of AMPA
receptors. Mol Neurobiol. 2010;42(3):161–184.
10
Braithwaite SP, Meyer, S, Henley JM. Interactions between AMPA receptors and intracellular
proteins. Neuropharmacology. 2000; 39:919–930
11
Ziff EB. TARPs and the AMPA Receptor Trafficking Paradox. Neuron 2007; 53(5):627-633.
12
Collingridge GL, Isaac JTR, Wang YT. Receptor trafficking and synaptic plasticity. Nature
Reviews Neuroscience 2004; 5: 952-962.
13
Wenthold RJ, Petralia RS, Blahos J, Niedzielski AS. Evidence for multiple AMPA receptor
complexes in hippocampal CA1/CA2 neurons. J Neurosci 1996, 16:1982-1989.
14
Lu W, Shi Y, Jackson AC, Bjorgan K, During MJ, Sprengel R, Seeburg PH, and Nicoll RA.
Subunit Composition of Synaptic AMPA Receptors Revealed by a Single-Cell Genetic Approach.
Neuron 2009; 62: 254–268.
15
Armstrong N, Gouaux E. Mechanisms for activation and antagonism of an AMPA-sensitive
glutamate receptor: crystal structures of the GluR2 ligand binding core. Neuron 2000;28:165–181.

41. 35
16
Kumar J and Mayer ML. Functional Insights from Glutamate Receptor Ion Channel Structures.
Annu. Rev. Physiol. 2013. 75:313–37
17
Xiao MY, Zhou Q, Nicoll RA. Metabotropic glutamate receptor activation causes a rapid
redistribution of AMPA receptors. Neuropharmacology 2001; 41:664–71
18
Smith WB, Starck SR, Roberts RW, Schuman EM. Dopaminergic stimulation of local protein
synthesis enhances surface expression of GluR1 and synaptic transmission in hippocampal
neurons. Neuron 2005; 45:765–79
19
Hirokawa N, Takemura R. Molecular motors and mechanisms of directional transport in
neurons. Nat. Rev. Neurosci. 2005; 6:201–14
20
Setou M, Seog DH, Tanaka Y, Kanai Y, Takei Y. Glutamate-receptor-interacting protein
GRIP1 directly steers kinesin to dendrites. Nature 2002; 417:83–87
21
Osterweil E, Wells DG, Mooseker MS. A role for myosin VI in postsynaptic structure and
glutamate receptor endocytosis. J. Cell Biol. 2005; 168:329–38
22
Nicoll RA, Tomita S, Bredt DS. Auxiliary subunits assist AMPA-type glutamate receptors.
Science 2006; 311:1253–56
23
Chen L, Chetkovich DM, Petralia RS, Sweeney NT, Kawasaki Y, et al. Stargazin regulates
synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 2000; 408:936–43
24
Beique JC, Andrade R. PSD-95 regulates synaptic transmission and plasticity in rat cerebral
cortex. J. Physiol. 2003; 546:859–67
25
El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS. PSD-95 involvement in
maturation of excitatory synapses. Science 2000; 290:1364–68
26
Archibald K, Perry MJ, Molnár E, Henley JM. Surface expression and metabolic half-life of
AMPA receptors in cultured rat cerebellar granule cells. Neuropharmacology. 1998;37(10-
11):1345-53.
27
Hayashi Y, Shi SH, Esteban JA, Piccini A, Poncer JC, Malinow R. Driving AMPA receptors
into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science
2000; 287:2262–67
28
Lu W, Man H, Ju W, Trimble WS, MacDonald JF, Wang YT. Activation of synaptic NMDA
receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal
neurons. Neuron 2001; 29:243–54
29
Hanley JG, Khatri L, Hanson PI, Ziff EB. NSF ATPase and alpha-/beta-SNAPs disassemble the
AMPA receptor-PICK1 complex. Neuron 2002; 34:53–67
30
Wang YT, Linden DJ. Expression of cerebellar long-term depression requires postsynaptic
clathrin-mediated endocytosis. Neuron 2000; 25:635–47

42. 36
31
Ehlers MD. Reinsertion or degradation of AMPA receptors determined by activity-dependent
endocytic sorting. Neuron 2000; 28:511–25
32
Cottrell JR, Borok E, Horvath TL, Nedivi E. CPG2: a brain- and synapse-specific
protein that regulates the endocytosis of glutamate receptors. Neuron 2004; 44:677–90
33
Steward O, Worley PF. Selective targeting of newly synthesized Arc mRNA to active synapses
requires NMDA receptor activation. Neuron 2001; 30:227–40
34
Yin HZ, Hsu CI, Yu S, Rao SD, Sorkin LS, Weiss JH. TNF-! triggers rapid membrane
insertion of Ca2+
permeable AMPA receptors into adult motor neurons and enhances their
susceptibility to slow excitotoxic injury. Exp Neurol. 2012;238(2):93-102.
35
Burbea M, Dreier L, Dittman JS, Grunwald ME, Kaplan JM. Ubiquitin and AP180 regulate the
abundance of GLR-1 glutamate receptors at postsynaptic elements in C. elegans. Neuron 2002;
35:107–20
36
Patrick GN, Bingol B, Weld HA, Schuman EM. Ubiquitin-mediated proteasome activity is
required for agonist-induced endocytosis of GluRs. Curr. Biol. 2003; 13:2073–81
37
Martin SJ, Grimwood PD, Morris RG. Synaptic plasticity and memory: an evaluation of the
hypothesis. Annu. Rev. Neurosci. 2000; 23:649–711.
38
Malenka RC, Bear MF. LTP and LTD: an embarrassment of riches. Neuron 2004; 44:5–21
39
Turrigiano, G. G. AMPA receptors unbound: membrane cycling and synaptic plasticity. Neuron
2000; 26: 5-8.
40
Perkinton, MS; Sihra TS, Willams RJ. Ca2
+
-permeable AMPA receptors induce
phosphorylation of cAMP response element-binding protein through a phosphatidylinositol 3-
kinase-dependent stimulation of the mitogen-activated protein kinase signaling cascade in
neurons. Journal of Neuroscience 1999; 19 (14): 5861–5874.
41
Barnes CA. Memory deficits associated with senescence: a neurophysiological and behavioral
study in the rat. J Comp Physiol Psychol; 1979. 93(1):74-104.
42
Maroun M. Stress reverses plasticity in the pathway projecting from the ventromedial
prefrontal cortex to the basolateral amygdala. Eur J Neurosci. 2006; 24(10):2917-22.
43
Liao R, Scannevin RH, and Huganir R. Activation of Silent Synapses by Rapid Activity-
Dependent Synaptic Recruitment of AMPA Receptors. J Neurosci 2001; 21(16):6008–6017.
44
Greengard P. The neurobiology of slow synaptic transmission. Science 2001; 294:1024–30
45
Thomas GM, Huganir RL. MAPK cascade signaling and synaptic plasticity. Nat. Rev. Neurosci.
2004; 5:173–83
46
Schulman H, Greengard P. Stimulation of brain membrane protein phosphorylation by calcium
and an endogenous heat-stable protein. Nature 1978; 271: 478-479.

43. 37
47
Soderling, T. CaM-kinases: modulators of synaptic plasticity. Current Opinion in Neurobiology
2000; 10 (3): 375–80
48
Barria A, Derkach V, Soderling T. Identification of the Ca2+/calmodulin-dependent protein
kinase II regulatory phosphorylation site in the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-
propionate-type glutamate receptor. J. Biol. Chem. 1997; 272:32727–30
49
Benke TA, Luthi A, Isaac JT, Collingridge GL. Modulation of AMPA receptor unitary
conductance by synaptic activity. Nature 1998; 393:793–97
50
Huganir RL and Song I. Regulation of AMPA Receptors During Synaptic Plasticity. Trends
Neurosci. 2002; 25(11):578-88
51
Roche KW, O’Brien RJ, Mammen AL, Bernhardt J, Huganir RL. Characterization of multiple
phosphorylation sites on the AMPA receptor GluR1 subunit. Neuron 1996; 16:1179–88
52
Banke TG, Bowie D, Lee H, Huganir RL, Schousboe A, Traynelis SF. Control of GluR1
AMPA receptor function by cAMP-dependent protein kinase. J. Neurosci. 2000; 20:89–102
53
Malinow R. AMPA receptor trafficking and long-term potentiation. Philos. Trans. R. Soc.
London Ser. B 2003; 358:707–14.
54
Lee HK, Barbarosie M, Kameyama K, Bear MF, Huganir RL. Regulation of dis-
tinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity. Nature 2000;
405:955–59.
55
Boehm J, Kang MG, Johnson RC, Esteban J, Huganir RL, Malinow R. Synaptic incorporation
of AMPA receptors during LTP is controlled by a PKC phosphorylation site on GluR1. Neuron
2006;51 (2): 213–25.
56
Carroll RC, Lissin DV, von Zastrow M, Nicoll RA, Malenka RC. Rapid redistribution of
glutamate receptors contributes to long-term depression in hippocampal cultures. Nat. Neurosci.
1999; 2:454–60
57
EC, Carroll RC, Yu X, Morishita W, Yasuda H, et al. Regulation of AMPA
receptor endocytosis by a signaling mechanism shared with LTD. Nat. Neurosci. 2000; 3:1291–
300
58
Heynen AJ, Quinlan EM, Bae DC, Bear MF. Bidirectional, activity-dependent regulation of
glutamate receptors in the adult hippocampus in vivo. Neuron 2000; 28:527–36
59
van der Heide LP, Kamal A, Artola A, Gispen WH, Ramakers GM. Insulin modulates
hippocampal activity-dependent synaptic plasticity in a N-methyl-d-aspartate receptor and
phosphatidyl-inositol-3-kinase-dependent manner. J Neurochem. 2005;94(4):1158-66.
60
Lin JW, Ju W, Foster K, Lee SH, Ahmadian G, et al. Distinct molecular mechanisms and
divergent endocytotic pathways of AMPA receptor internalization. Nat. Neurosci. 2000; 3:1282–
90

44. 38
61
Zhou Q, Xiao M, Nicoll RA. Contribution of cytoskeleton to the internalization of AMPA
receptors. Proc. Natl. Acad. Sci. 2001; 98:1261–66
62
Lee HK, Takamiya K, Han JS, Man H, Kim CH, et al. Phosphorylation of the AMPAreceptor
GluR1 subunit is required for synaptic plasticity and retention of spatial memory. Cell 2003;
112:631–43
63
Chung HJ, Steinberg JP, Huganir RL, Linden DJ. Requirement of AMPA receptor GluR2
phosphorylation for cerebellar long-term depression. Science 2003; 300:1751–55
64
Kim CH, Chung HJ, Lee HK, Huganir RL. Interaction of the AMPA receptor subunit GluR2/3
with PDZ domains regulates hippocampal long-term depression. Proc. Natl. Acad. Sci. 2001;
98:11725–30
65
Lissin DV, Gomperts SN, Carroll RC, Christine CW, Kalman D, et al. Activity differen- tially
regulates the surface expression of synaptic AMPA and NMDA glutamate receptors. Proc. Natl.
Acad. Sci. 1998; 95:7097–102
66
Liao D, Zhang X, O’Brien R, Ehlers MD, Huganir RL. Regulation of morphological
postsynaptic silent synapses in developing hippocampal neurons. Nat. Neurosci. 1999; 2:37– 43
67
Stellwagen D, Malenka RC. Synaptic scaling mediated by glial TNF-!. Nature 2006;
440:1054–59
68
Shepherd JD, Rumbaugh G, Wu J, Chowdhury S, Plath N, et al. Arc/Arg3.1 mediates
homeostatic synaptic scaling of AMPA receptors. Neuron 2006; 52:475–84
69
Turrigiano G, Nelson S. Thinking Globally, Acting Locally: AMPA Receptor Turnover and
Synaptic Strength. Neuron 1998; 21(5):933–935.
70
Malinow, R, Mainen, ZF, Hayashi, Y. LTP Mechanisms: from silence to four-lane traffic. Curr.
Opp. Neurobio. 2000; 10:352-357.
71
Hsieh H, Boehm J, Sato C, Iwatsubo T, Tomita T, Sisodia S, Malinow R. AMPAR removal
underlies Abeta-induced synaptic depression and dendritic spine loss. Neuron 2006;52:831–843.
72
Lai M, Hughes EG, Peng X, Zhou L, Gleichman AJ, Shu H, Mata S, Kremens D, Vitaliani R,
Geschwind MD, et al. AMPA receptor antibodies in limbic encephalitis alter synaptic receptor
location. Ann Neurol. 2009; 65:424–434.
73
Rogers SW, Andrews PI, Gahring LC, Whisenand T, Cauley K, Crain B, Hughes TE,
Heinemann SF, McNamara JO. Autoantibodies to glutamate receptor GluR3 in Rasmussen’s
encephalitis. Science. 1994; 265:648–651.
74
Soundarapandian MM, Tu WH, Peng PL, Zervos AS, Lu Y. AMPA receptor subunit GluR2
gates injurious signals in ischemic stroke. Mol Neurobiol. 2005; 32:145–155.

45. 39
75
Wu Y, Arai AC, Rumbaugh G, Srivastava AK, Turner G, Hayashi T, Suzuki E, Jiang Y, Zhang
L, Rodriguez J, et al. Mutations in ionotropic AMPA receptor three alter channel properties and
are associated with moderate cognitive impairment in humans. Proc Natl Acad Sci 2007;
104:18163–18168.
76
Plassman BL, Langa KM, Fisher GG, Heeringa SG, Weir DR, Ofstedal MB, Burke JR, Hurd
MD, Potter GG, Rodgers WL, Steffens DC, Willis RJ, Wallace RB. Prevalence of dementia in the
United States: The aging, demographics, and memory study. Neuroepidemiology 2007; 29:125–
132.
77
Federal Interagency Forum on Aging-Related Statistics. Older Americans 2012: Key Indicators
of Wellbeing.
78
Mudher A, Lovestone S. Alzheimer's disease-do tauists and baptists finally shake hands?
Trends Neurosci.. 2002; 25(1):22–26.
79
Heneka MT, Nadrigny F, Regen T, Martinez-Hernandez A, Dumitrescu-Ozimek L, Terwel D,
Jardanhazi-Kurutz D, Walter J, Kirchhoff F, Hanisch UK, Kummer MP. . Locus ceruleus controls
Alzheimer's disease pathology by modulating microglial functions through norepinephrine. Proc
Natl Acad Sci 2010; 107:6058–6063
80
de la Monte S, Wands J. Alzheimer’s disease is type 3 diabetes–evidence reviewed. J Diabetes
Sci Technol 2008; 2: 1101–1113.
81
LaFerla FM, Green, KN, Oddo, S. Intracellular amyloid-! in Alzheimer’s disease. Nature
Reviews Neuroscience 2007; 8:499-509.
82
Cirrito JR, Yamada KA, Finn MB, Sloviter RS, Bales KR, et al. Synaptic activity regulates
interstitial fluid amyloid-beta levels in vivo. Neuron 2005; 48:913–22
83
Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, et al. Regulation of NMDA
receptor trafficking by amyloid-!. Nat. Neurosci. 2005; 8:1051–58
84
Hsieh H, Boehm J, Sato C, Iwatsubo T, Tomita T, et al. AMPAR removal underlies A!-
induced synaptic depression and dendritic spine loss. Neuron 2006; 52:831–43
85
Armstrong N, Sun Y, Chen GQ, Gouaux E. Structure of a glutamate-receptor ligand-binding
core in complex with kainate. Nature 1998; 395 (6705): 913–7.
86
Patneau DK, Mayer ML, Jane DE, Watkins JC. Activation and desensitization of
AMPA/kainate receptors by novel derivatives of willardiine. J Neurosci. 1992; 12(2):595-606.
87
Hogberg HT and Bal-Price AK. Domoic Acid-Induced Neurotoxicity Is Mainly Mediated by
the AMPA/KA Receptor: Comparison between Immature and Mature Primary Cultures of
Neurons and Glial Cells from Rat Cerebellum. J. Toxicology 2011; [Epub].
88
Moykkynen T, Korpi E, Lovinger D. Ethanol Inhibits "-Amino-3-hydyroxy-5-methyl-4-
isoxazolepropionic Acid (AMPA) Receptor Function in Central Nervous System Neurons by
Stabilizing Desensitization. JPET 2003; 306(2): 546-555

46. 40
89
Haugbøl SR, Ebert B, Ulrichsen J. Upregulation of glutamate receptor subtypes during alcohol
withdrawal in rats. Alcohol 2005; 40(2):89-95.
90
Mason, BJ. Treatment of alcohol-dependent outpatients with acamprosate: a clinical review.
The Journal of Clinical Psychiatry 2001; 62(20): 42–8.
91
Sanchis-Segura C, Borchardt T, Vengeliene V, Zghoul T, Bachteler D, Gass P, Sprengel R,
Spanagel R. Involvement of the AMPA receptor GluR-C subunit in alcohol-seeking behavior and
relapse. J Neurosci. 2006;26(4):1231-8.
92
Good CH, Lupica CR. Afferent-specific AMPA receptor subunit composition and regulation of
synaptic plasticity in midbrain dopamine neurons by abused drugs. J Neurosci. 2010;
30(23):7900-9.
93
Bowers MS, Chen BT, Bonci A. AMPA receptor synaptic plasticity induced by
psychostimulants: the past, present, and therapeutic future. Neuron. 2010; 67(1):11-24
94
Nathan PJ, Lu K, Gray M, Oliver C. The neuropharmacology of L-theanine (N-ethyl-L-
glutamine): a possible neuroprotective and cognitive enhancing agent. J Herb Pharmacother.
2006; 6(2):21-30
95
Kakuda T. Neuroprotective effects of theanine and its preventive effects on cognitive
dysfunction. Pharmacol Res. 2011; 64(2):162-8
96
Erhardt S, Schwieler L, Nilsson L, Linderholm K, Engberg G. The kynurenic acid hypothesis
of schizophrenia. Physiol. Behav. 2007; 92 (1–2): 203–9
97
Schwarcz R, Elmer GI, Bergeron R; Albuquerque E,; Guidetti P, Wu H. Reduction of
Endogenous Kynurenic Acid Formation Enhances Extracellular Glutamate, Hippocampal
Plasticity, and Cognitive Behavior. Neuropsychopharmacology 2010; 35 (8): 1734–1742.
98
Dinh, L, Nguyen T, Salgado H, Atzori M. Norepinephrine homogeneously inhibits alpha-
amino-3-hydroxyl-5-methyl-4-isoxazole-propionate- (AMPAR-) mediated currents in all layers
of the temporal cortex of the rat. Neurochem Res 2009; 34 (11): 1896–906.
99
French, JA et al. Adjunctive perampanel for refractory partial-onset seizures: Randomized
phase III study 304. Neurology 2012; 79(6): 589–596.
100
Alt A, Weiss B, Ogden AM, Li X, Gleason SD, Calligaro DO, Bleakman D, Witkin JM. In
vitro and in vivo studies in rats with LY293558 suggest AMPA/kainate receptor blockade as a
novel potential mechanism for the therapeutic treatment of anxiety disorders.
Psychopharmacology. 2006; 185(2):240-7.
101
Sobolevsky AI, Rosconi MP, and Gouaux E. X-ray structure, symmetry and mechanism of an
AMPA-subtype glutamate receptor. Nature 2009; 462:745–756.

47. 41
102
Lauterborn JC, Lynch G, Vanderklish P, Arai A, and Gall CM. Positive modulation of AMPA
receptors increases neurotrophin expression by hippocampal and cortical neurons. J Neurosci
2000; 20:8–21.
103
Lei S, Orser BA, Thatcher GR, Reynolds JN, and MacDonald JF. Positive allosteric
modulators of AMPA receptors reduce proton-induced receptor desensitization in rat
hippocampal neurons. J Neurophysiol 2001; 85:2030–2038.
104
Yaghoubi N, Malayev A, Russek SJ, Gibbs TT, and Farb DH. Neurosteroid modulation of
recombinant ionotropic glutamate receptors. Brain Res 1998; 803:153– 160.
105
Kovalchuk Y, Miller B, Sarantis M, and Attwell D. Arachidonic acid depresses non-NMDA
receptor currents. Brain Res 1994; 643:287–295.
106
Arai AC, Kessler M. Pharmacology of ampakine modulators: from AMPA receptors to
synapses and behavior. Curr Drug Targets. 2007;8(5):583-602.
107
Sekiguchi, M; Fleck, MW; Mayer, ML; Takeo, J; Chiba, Y; Yamashita, S; Wada, K. A novel
allosteric potentiator of AMPA receptors: 4-2-(phenylsulfonylamino)ethylthio-2,6-difluoro-
phenoxyacetamide. Journal of Neuroscience 1997; 17 (15): 5760–71.
108
Simmons DA, Rex CS, Palmer L, Pandyarajan V, Fedulov V, Gall CM, and Lynch G Up-
regulating BDNF with an ampakine rescues synaptic plasticity and memory in Huntington’s
disease knockin mice. Proc Natl Acad Sci; 2009. 106:4906 – 4911.
109
Destot-Wong KD et al. The AMPA receptor positive allosteric modulator, S18986, is
neuroprotective against neonatal excitotoxic and inflammatory brain damage through BDNF
synthesis. Neuropharmacology 2009; 57: 277–286.
110
Jourdi H, Hsu YT, Zhou M, Qin Q, Bi X, Baudry M. Positive AMPA receptor modulation
rapidly stimulates BDNF release and increases dendritic mRNA translation. J Neurosci. 2009; 29
(27): 8688–8697.
111
Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB,
Yuan H, Myers SJ, and Dingledine R. Glutamate Receptor Ion Channels: Structure, Regulation,
and Function. Pharmacol Rev 2010; 62:405–496.
112
Wezenberg E, Verkes RJ, Ruigt GS, Hulstijn W, and Sabbe BG. Acute effects of the ampakine
farampator on memory and information processing in healthy elderly volunteers.
Neuropsychopharmacology 2007; 32:1272–1283.
113
Lauterborn JC, Troung G, Baudry M, Bi X, Lynch G, Gall C. Chronic elevation of brain-
derived neurotrophic factor by ampakines. J Pharmacol Exp Ther 2003;307:297–305
114
Lauterborn JC, Pineda E, Chen LY, Ramirez EA, Lynch G, Gall CM. Ampakines cause
sustained increases in BDNF signaling at excitatory synapses without changes in AMPA receptor
subunit expression. Neuroscience 2009; 159 (1): 283–295.

48. 42
115
Porrino LJ, Daunais JB, Rogers GA, Hampson RE, Deadwyler SA. Facilitation of Task
Performance and Removal of the Effects of Sleep Deprivation by an Ampakine (CX717) in
Nonhuman Primates. PLoS Biol 2005; 3(9): e299.
116
Lu W, Shi Y, Jackson AC, Bjorgan K, During MJ, Sprengel R, Seeburg PH, Nicoll RA.
Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach.
Neuron. 2009;62(2):254-68.
117
Petralia RS and Wenthold RJ. Light and electron immunocytochemical localization of AMPA-
selective glutamate receptors in the rat brain. J Comp Neurol 1992; 318:329 –354.
118
Beique J and Huganir R. AMPA Receptor Subunits Get Their Share of the Pie. Neuron 2009;
62: 165-67.
119
Brusa R, Zimmermann F, Koh DS, Feldmeyer D, Gass P, et al. Early-onset epilepsy and
postnatal lethality associated with an editing-deficient GluR-B allele in mice. Science 1995;
270:1677–80
120
Kim DY, Kim SH, Choi HB, Min C, Gwag BJ. High abundance of GluR1 mRNA and reduced
Q/R editing of GluR2 mRNA in individual NADPH-diaphorase neurons. Mol. Cell. Neurosci.
2001; 17 (6): 1025–33.
121
Janssen W, Vissavajjhala P, Andrews G, Moran T, Hof PR, and Morrison JH. Cellular and
synaptic distribution of NR2A and NR2B in macaque monkey and rat hippocampus as visualized
with subunit-specific monoclonal antibodies. Experimental Neurology 2005; 101: S28–S44.

49. 43
Appendix
Appendix Figure 1
Adapted From Lauterborn et al. 114
: “Spaced ampakine treatments sustain elevated BDNF protein content
without down-regulating AMPAR expression.
A) Hippocampal slices were treated with CX614 for varied intervals (1 – 6 hr) and harvested 24 h after
treatment onset.
B) Bar graphs show group mean ± S.E.M. in situ hybridization labeling densities for GluR1 mRNA in str.
granulosum and CA1 str. pyramidale. With CX614 at 50 µM (light bars), labeling was unaffected through 3
h but was lower than control values after 5 h treatments (*p < 0.05, **p < 0.01, ***p < 0.001 vs. con, SNK).
CX614 at 25 µM (dark bars) did not affect GluR1 mRNA levels.
C) Slices were treated with 50 µM CX614 for 3 h on four successive days; slices were collected daily (i) at
the end of treatment for GluR1 mRNA analysis (D) or (ii) at the end of the 24 h period for ELISA measures
of BDNF protein (E).
D) Bar graph shows that GluR1 mRNA levels in str. granulosum (SG) remained at control values through
treatment day 4 [at 50 µM]
E) Plot of protein measures shows that total slice BDNF protein content was elevated at the end of the first
day of treatment and remained elevated through treatment day 4 (**p < 0.01 vs. Con group) whereas GluR1
protein levels were unchanged throughout treatment. Mean ± S.E.M. values shown for n ! 8/group.”

50. 44
The following tables are adapted from Traynelis et al. 111
:
Carboxyl-terminal protein binding partners for AMPAR subunits
Post-Translational Modifications of C-terminal Domains of GluA

51. 45
AMPAR Subunit Agonists at Micromolar Concentrations
The Douglas Adams Equation and Secret to Universe Cube Matrix (Sum 3 x 3).
(Congratulations for Finishing the Paper)