2. Inquiries into the Biological Significance of TARP γ−8 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 613
muscle-relaxant properties without being a GABA receptor
antagonist. The development of this and other series of
compounds led to a number of compounds with potential for
neuroprotection, pain, epilepsy, drug dependence, and other
disorders of the CNS [6, 7]. However, it was not until 2012
that perampanel (Fycompa®
), a non-competitive AMPA
receptor antagonist, became the first FDA-approved
medication for the control of refractory partial seizures.
One issue associated with the pharmacological
modulation of AMPA receptors is the induction of side-
effects. For example, perampanel induces dizziness, vertigo,
problems with walking, and sleepiness [7, 8]. On the other
side of modulation are AMPA receptor potentiators which
had been discovered with the hope of cognitive enhancement
[9, 10] and more recently depression [11]. A known
drawback with some of these agents is the fact that supra
amplification of glutamatergic tone is associated with
neurotoxicity [12, 13].
AMPA receptors have been found to be associated with
transmembrane AMPA receptor regulatory proteins (TARPs)
[14-16] in addition to other proteins (e.g., cornichons) [17-
20]. TARPs are evolutionarily related to the claudin family
and calcium channel γ subunits of tight junction proteins.
TARPs are comprised of Type I and Type II variants based
upon structural similarity to one another. Group I TARPs
consist of γ−2 (also known as stargazin), γ−3, γ−4, and
γ−8(Cacng8); group II TARPs are comprised of γ5, and γ−7
[21-24]. It is established that TARPs are key regulators of
AMPA receptor pharmacology and function [12, 16]. For
example, TARPs control the translocation of AMPA
receptors to the synapse enabling normal AMPA receptor
function; in the absence of TARP γ−2, AMPA receptor
function is lost (stargazin mouse) [14, 25, 26]. Likewise,
TARPs can control the potency and selectivity of ligands
activating AMPA receptors. For example TARP γ−2 imbues
AMPA receptor flip and flop spice variants sensitive to
AMPA receptor potentiators as well as enhancing their
potencies [27]. However, it is unknown what biological
significance TARPs play in normal and pathophysiological
states in humans. One remarkable feature of TARPs which
likely plays a major modulatory role in the CNS is the fact
TARPS have highly specialized localization in the brain [15,
28]. For example, γ−8 TARPs have very high relative
densities in the hippocampus of rodent and human brains
[29, 30].
Since TARPs regulate AMPA receptor function and
pharmacological selectivity and potency, AMPA receptors
associated with γ−8 TARPs would likely have a high degree
of selective control over hippocampal function. A corollary
is that brain structures with AMPA receptors associated with
γ−2 TARPs are likely to have dominant control over
cerebellar function [14, 15, 28, 31]. Recently, it was
speculated, based on behavioral and electrophysiological
studies with perampanel, that an antagonist of AMPA
receptors which was selectively associated with γ−8 TARPs
might engender antiepileptic benefit without the motoric
side-effects associated with AMPA receptor blockade across
the entire CNS [32]. Earlier, it was suggested that an AMPA
receptor potentiator that selectively enhanced AMPA
receptor/γ−8 TARP complexes might be antidepressant since
AMPA receptor facilitation and hippocampal function in
general appear to be critical to antidepressant activity [11].
However, to date no selective ligands for the TARP γ−8
exist.
In the absence of selective pharmacological tools, one
approach toward the elucidation of biological function of
TARP γ−8 can be gained through research with TARP γ−8
null mice. γ−8 -/- mice have been genetically constructed and
have undergone some scrutiny. Compared to their wild type
controls, γ− 8 -/- mice, or knockout mice, are characterized
by the virtual absence of the γ−8 protein in the brain,
significantly fewer numbers of AMPA receptors in dendritic
synaptic, and extrasynaptic membranes [28, 33], and the lack
of long-term potentiation (LTP) with a mild decrease in the
synaptic AMPA receptor-mediated responses [33]. Menuz at
al. [28] suggested that very few physically observable traits
and behaviors distinguish γ−8 -/- mice from their wild type
(WT) littermates, hypothesizing that similar TARP family
members (e.g. γ−4) may compensate for the loss of γ−8.
However, γ−8 -/- mice have not been extensively studied for
phenotypic behavioral differences. Such investigations are
the primary aim of the present series of experiments.
MATERIALS AND METHODS
Mice
The TARP γ−8 -/- mice were created as described [33].
They were re-derived in a CD1 mouse line (Taconic Farms
Line 1834). Het x het breeding created WT and KO
littermate controls. Only male mice were used in the current
experiments as our housing vivarium did not accommodate
females and to avoid, in these studies, any gender factor. The
mice weighed 35-42 g at the- time of testing and were all
born and delivered within two weeks of each other. They
were grouped housed with ad libitum food and water in a
colony room which controlled heat, humidity, and light
(lights on 6am; off 6pm). Animals were maintained in the
colony room for at least 3 days before testing. They were
moved to a quiet room 1 hour prior to the start of a test. The
mice were used in these studies under the strict guidance and
approval of local animal care and use committees under
guidelines of the Institutional Animal Care and Use
Committee at Eli Lilly & Co. and in strict accordance with
the guidelines set forward by the National Institutes of
Health.
Compounds
d-Amphetamine SO4, phencyclidine HCl, imipramine
HCl, chlordiazepoxide HCl, pentylenetetrazole base and
NMDA base (Sigma Chemical Co., St. Louis, MO, USA)
were dissolved in 0.9% NaCl. LY392098 (Eli Lilly and Co.)
was suspended in 1% hydoxyethylcellulose/0.25%
Tween80/0.05% Dow antifoam. Kainic acid (Tocris
Bioscience, Bristol, United Kingdom) was dissolved in
water. Drug doses are expressed as the drug forms listed. All
compounds were prepared just prior to dosing and
administered i.p. or s.c. in a volume of 0.01 ml/g body
weight.
3. 614 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 Gleason et al.
Body Temperature
Animals (n=6 WT and n=6 γ−8 KO mice) were moved
from colony room into procedure room and allowed to
acclimate for 45-60 minutes. For basal body temperature,
animals were removed from home cage and put into holding
cages, 30 minutes after being put into holding cage, body
temperature was measured with a RET-3 rectal probe
attached to a Model BAT-12 (Physitemp Instruments,
Clifton, NJ, USA). Temperature was recorded in degrees
Celsius.
Locomotor Activity
Locomotor Arenas
Locomotor activity was measured with a 20 station
photobeam activity system (San Diego Instruments, San
Diego, CA, USA) with seven photocells per station.
Locomotor activity was recorded as the number of
ambulations, where ambulation was defined as the breaking
of adjacent photobeams. Animals were placed individually
into polypropylene cages (40.6 x 20.3 x 15.2 cm, no
bedding). Data were recorded for 90 minutes, animals were
removed weighed, injected sc with vehicle, 1, 3, or 10 mg/kg
d-amphetamine or 3 mg/kg PCP, and returned to the cage for
an additional 90 minute data collection period.
Circular Runways
In another set of studies, mice were tested for locomotor
activity in a circular runway with 4 pairs of photo-detectors
(RLC Products, Rockville, MD, USA) for 5 min. The
circular runway was 24 cm (outside diameter) x 14 cm
(inside diameter) x 4.5 cm wide x 5 cm high. The runway
was enclosed in a darkened enclosure. Total beam breaks,
clockwise rotations, and counterclockwise rotations were
recorded.
Marble-Burying
Mice were studied essentially per the method of Li et al.
[34]. Separate groups of mice were used in these
experiments and were conducted in a dimly lit testing room.
After 60 min acclimation to the experimental room, mice
were placed in a 17 x 28 x 12 cm high plastic tub with 5 mm
sawdust shavings (Harlan Sani-Chips, Harlan-Teklad,
Indianapolis, IN, USA) on the floor which was covered with
20 blue marbles (1.5 cm diameter) placed in the center. Mice
were left in the tub for 30 min. The number of marbles
buried (2/3 covered with sawdust) -was counted and
submitted to inter-observer reliability assessment. In one
experiment, the movement of mice in the marble-burying
arena was measured by a trained observer blind to treatment.
Every 5 min, each mouse was observed for 10 sec and rated
as being in motion (walking, rearing, climbing) or digging.
Thus, each mouse was observed on 6 occasions for the 30
min test period.
Elevated-Plus Maze
The maze (RLC Products, Rockville, MD, USA) consists
of four arms (two open without walls and two enclosed by
14 cm tall x 29.5 cm long walls). Each arm is 30 cm long
and 5 cm wide. At the junction of the arms, there is a 6x6 cm
open area. Each arm has two IR photobeam detectors
mounted 6.5 and 8.5 cm respectively (measured from center
of maze). Photobeam detectors are controlled by a custom
designed interface and connected to a computer for
recording. The entire apparatus was elevated 32 cm above
the surface it was placed on. Eight WT and TARP γ-8 KO
mice were studied in the maze for 5 min with one WT and
one KO mouse studied simultaneously in separate mazes.
The number of open and closed arm entries was measured
and the percentage of time in the open arms was also
calculated.
Forced-Swim Assay
Mice were placed in clear plastic cylinders (diameter 10
cm; height: 25 cm) filled to 6 cm with 22-25 °C water for six
min. The duration of immobility was recorded during the last
4 min of a six-minute trial. A mouse was regarded as
immobile when floating motionless or making only those
movements necessary to keep its head above the water. Data
were analyzed by post-hoc Dunnett’s test with alpha level set
at 0.05. The amount of time spent immobile was measured.
Seizure Testing
Mice were tested for the production of clonic or tonic
convulsive episodes after the administration of
chemoconvulsant agents. Clonus was defined as repetitive
moments of the forelimbs and or head and tonus was defined
as the full rigid extension of the fore-and hindlimbs. Minimal
numbers of mice were used in these assays to decrease the
number of mice which experienced convulsant events. Mice
were observed in plastic containers for 30 min post dosing of
the convulsant agent by an observer trained to detect clonic
and tonic convulsions in mice. The percentage of mice
exhibiting convulsions was recorded.
Neurochemistry
Analysis of Serotonin, Dopamine, and Norepinephrine
WT and γ−8 KO mice were brought into an experimental
room for one hour prior to decapitation. Brain areas were
dissected, weighed and immersed in 1 ml of cold 0.01 N HCl
containing an antioxidant (0.5 mg/ml L-cysteine) in 1.5 ml
centrifuge tubes. The tissues were sonicated,50 ul of 3 N
HClO4 (perchloric acid) was added, and tubes were shaken
by hand to precipitate proteins (final concentration is 0.15 N
perchloric acid). Samples were stored in refrigerator for
about an hour to cool before centrifuging. The samples were
then centrifuged at 12,000xg. About 200 ul of the
supernatant was transferred to a small plastic tube for
injection onto the HPLC system (injection volume was 10
ul).
Samples were run with an HPLC analytical method
capable of simultaneously detecting three monoamines (DA,
NE and 5-HT) and their metabolites DOPAC, 5-HIAA and
HVA. A BDS-Hypersil 5 µ C18 analytical column (4.6 x 150
mm from Keystone Scientific, Bellefonte, PA, USA) was
used. The mobile phase consisted of 75 mM sodium
phosphate monobasic, 350 mg/L 1-octanesulfonic acid
sodium salt, 0.5 mM EDTA, 0.8% tetrahydrofuran (HPLC
grade, inhibitor-free) and 8% acetonitrile at pH 3 (adjusted
4. Inquiries into the Biological Significance of TARP γ−8 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 615
with phosphoric acid). The flow rate was 1.20 ml/min. The
analytical column was maintained at 40o
C with a column
heater. An electrochemical detector (EG & G PARC,
Princeton, NJ, USA) with dual glassy carbon electrodes was
used (E = 700 mV, range = 10 nA). The data was collected
using an EZChrom chromatography data system (Scientific
Software, San Ramon, CA, USA) running on a Compaq
computer, which calculated peak heights and sample
concentrations. The sensitivity for monoamine and
metabolites was 0.1 pmol/ml.
Analysis of cAMP, Acetylcholine, Histamine, and
Histamine Metabolites
Mice were brought into an experimental room for one
hour prior to decapitation. The MAO inhibitor, pargyline
(Sigma Aldrich, Lot 077K1405), was used as a positive
control and prepared in 0.9% saline at 75 mg/kg, ip for 3
hours. Animals were pretreated with test compounds for 2
hours then sacrificed by decapitation and the striatum and
trunk blood collected. The mice were sacrificed using
microwave fixation (Thermax Thermatron, Louisville, KY,
USA) at setting 0.6 sec at high power. The mouse striatum
and cerebellum were collected in this study. Dissected
tissues were removed and placed immediately on a metal
sheet over dry ice. Trunk blood was collected in heparin-
lithium coated 1.5 mL Eppendorf tubes and spun in an
Eppendorf 5417R centrifuge at 12,600 rpm’s for 2 min at 4o
C and the plasma collected. Tissue weights were recorded
and both brain and plasma samples processed using probe
sonication in acetonitrile/0.1% formic acid using 4x tissue
weight or plasma volume. Processed samples were
centrifuged at 13,000 rpm’s for 15 min at 40
C. 100 uL of
each sample was assayed for the histamine metabolites, tele-
methylhistamine (t-MH) and tele-methylimidazoleacetic acid
(t-MIAA), using Agilent Technologies Inc. LC-MS/MS
(Santa Clara, CA, USA). The calibration standards, t-MH
(H137) and t-MIAA (M9265), were purchased from Sigma
Aldrich. Stocks were made at 1 pg/mL in acetonitrile/1% FA
and kept at 4o
C. The analysis of standards were carried out
using an Agilent 6410 series triple quad LC/MS/MS with
MassHunter data analysis software fitted with an
electrospray ion source and run in positive mode. The
chromatographic separation employed an Agilent Zorbax
RX-SIL Narrow-Bore 2.1 X 150mm 5-Micron Column. The
mobile phase consists of 3% acetonitrile in water with an
overall 0.1% formic acid content. Clearly delineated
chromatographic peaks with the retention time of authentic
standards and expected molecular weight were seen after
each injection of sample.
Plasma Lipids
The quantitation of lipids in plasma was accomplished
using liquid chromatography electrospray ionization-tandem
mass spectrometry. LC/ESI/MS/MS analysis of lipids was
performed using a TSQ Quantum Ultra-Triple quadrupole
mass spectrometer (Thermo Fisher, San Jose, CA, USA)
equipped with an ESI probe and interfaced with the Agilent
1290 infinity LC system (Agilent, Palo Alto, CA, USA). The
UPLC system consisted of an Agilent 1290 binary pump,
thermostat, TCC, and sampler. The injection volume was 10
µL for extracted sample. Lysophosphatidic acids and
lysophosphotidyl cholines were separated with a Zorbax
Eclipse Plus RRHD C8 column, 2.1x50 mm, 1.8 µm
(Agilent, Palo Alto, CA, USA). Mobile phase A was
water:25 µM EDTA:0.1% TEA. Mobile phase B was
methanol:25 µM EDTA:0.1% TEA. For ethanolamides and
n-acyl glycerols, the samples were separated via elution with
methanol:0.1% formic acid on an Eclipse Plus C18 RRHD,
2.1x50mm, 1.8 µm column (Agilent, Palo Alto, CA).
Sphingolipids were separated with a Poroshell 120 EC- C8
column, 2.1x50 mm, 2.7 µm,(Agilent, Palo Alto, CA).
Mobile phase A was water:methanol:formic acid
(45/55/0.4% by v/v). Mobile phase B was acetonitrile
:methanol:formic acid (50/50/0.4% by v/v). The valve,
sample loop, and needle were washed with
acetonitrile/methanol (50:50 % by vol) for 5 sec. Mass
spectrometric analyses were performed online using
electrospray ionization tandem mass spectrometry in the
positive and negative multiple reaction monitoring (MRM)
mode. Samples were prepared using Biomek FX (Beckman
Coulter, Brea, CA, USA). Small amount of plasma sample
was added to a 2-mL 96-well plate. Internal standard mixture
was added to the samples. Sphingolipids, lysophospholipids,
ethanolamides, and n-acyl glycerols were extracted using 1
phase extraction with methanol-dichloromethane, methanol,
and ethylacetate-hexane, respectively. Lipid levels were
quantified by the ratio of analyte and internal standard and
calibration curves obtained by serial dilution of a mixture of
lipid standards.
Pure synthetic standards of sphingolipids, lysophospho-
lipids, ethanolamides, and n-acyl glycerols were purchased
from Avanti Lipids and Cayman Chemical. Isotope labeling
and deuterated synthetic standards were synthesized
internally at Eli Lilly and Company.
Data Analysis
Differences between WT and KO mice were analyzed by
Student’s tests when only one group or dependent measure
was provided (body temperature). For all other dependent
variables except convulsions, two-way ANOVA were
conducted followed by post-hoc Dunnett’s test (locomotor
activity and forced-swim assays) or un-paired t-tests using
the Holm-Sidak correction for multiple comparisions. Clonic
and tonic convulsions were analyzed by Fisher’s Exact
probability test. For analysis of data on cAMP,
acetylcholine, and histamine and metabolites, data
exclusions were detected using the Grubb’s Test for Outliers
(also known as the ESD method or extreme studentized
deviate). For all analyses, the error acceptance rate was set a
priori at 5%.
RESULTS
Body Temperature
No significant differences in basal body temperatures
were observed between WT and γ−8 KO mice. The mean +
SEM body temperature of WT mice was 37.5 + 0.2 vs 37.6 +
0.1 for KO mice in degrees Celsius.
5. 616 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 Gleason et al.
Locomotor Activity
Basal levels of locomotion were higher in γ−8 KO mice
than in WT control mice. Activity was significantly higher in
the KO mice for the 90 min habituation period prior to
injection and then for the next 40 min post saline
administration (i.p.) (Fig. 1). These higher levels of basal
activity were maintained in two additional studies in separate
cohorts of WT and γ−8 KO pairs (Fig. 2). In the first study, a
dose-response curve of d-amphetamine was determined. For
the first 30 min post dosing, 10 mg/kg increased locomotion
in both genotypes; for the last 60 min of the study, 3 mg/kg
increased locomotion in the KO but not in the WT mice (Fig.
2, panel A). Phencylidine (3 mg/kg) also increased activity
in both WT and KO mice with significantly greater increases
occurring in the KO mice in the last 60 min of the study (Fig.
2, panel B).
In the circular runway, WT mice and KO mice did not
significantly differ in terms of overall photobeam breaks
(Fig. 3, top panel). However, γ−8 KO mice demonstrated
significantly less completed circular movements (counter-
clock wise or clockwise) than WT mice (Fig. 3, bottom
panel).
Marble Burying
Two experiments were conducting on separate groups of
WT and γ−8 KO mice and in one study separate observers
were employed. In both cases KO mice buried significantly
less marbles than their age-matched WT controls. The two
observers showed comparable records of marbles buried as
evidenced by the positive and significant correlation
coefficient across the 8 mice/genotype (r=0.92, p< 0.05). A
compilation of findings across the two studies is shown in
Fig. (4, top panel). The percentage of mice digging across
the six observation periods was markedly and significantly
reduced to near zero in the KO mice (Fig. 4, middle panel).
In contrast to these differences in burying and digging
behaviors, the percentage of mice in motion across the six
observation periods was not significantly different in WT vs
γ−8 KO mice (Fig. 4, bottom panel).
Elevated Plus Maze
Differences in anxiety-associated behaviors in the marble
burying test prompted independent evaluation in a separate
and distinct assay of detecting anxiety-associated
phenotypes. In the elevated plus maze, the WT and KO mice
did not display significant differences in behaviors as
measured either by the number of open arm entries or the
percentage of time spent in the open arms. The WT and KO
mice also did not differ significantly in the total number of
photobeam breaks (Fig. 5).
Forced-Swim Assay
AMPA receptor potentiators display antidepressant-like
effects in this assay (Li et al., 2003). We utilized this assay
to determine if γ−8 TARP affects the antidepressant-like
signature of an AMPA receptor potentiator. Compared to
Fig. (1). TARP γ−8 -/- mice (KO) have increased basal locomotor activity compared to wild-type controls (WT) which habituates over time.
Each point represents the mean of n=8, vertical bars represent ±SEM. Insert represents the mean locomotor activity over time. Pre:
Cumulative ambulations prior to dosing; Post: Cumulative ambulation post dosing. Black circles: WT; White circles: KO. F9,28=29.4, p<0.05.
*p<0.05 compared to WT (Dunnett's test). # p<0.05 compared to WT for respective dose (Dunnett's test).
Minutes
-100 -80 -60 -40 -20 0 20 40 60 80 100
Ambulations
0
200
400
600
800
1000
WT
KO
*
*
*
*
*
*
* * * * * * *
Dose
animals
WT KO
Ambulations
0
1000
2000
3000
4000
5000
Pre
Post
*
*
6. Inquiries into the Biological Significance of TARP γ−8 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 617
WT mice, γ−8 KO mice had a lower immobility time than
their matched WT controls (Fig. 6). Like genetic KO of γ−8,
the AMPA receptor potentiator LY392098 decreased
immobility time as did the tricyclic antidepressant
imipramine in WT mice (Fig. 6). However, in mice without
the γ−8 protein, LY392098 was without effect while the
antidepressant-like effect of imipramine was spared.
Seizure Testing
Pentylenetetrazole produced clonic convulsions which
increased in prevalence with dose. There were no significant
differences in the prevalence of convulsions in WT vs KO
mice (Table 1). NMDA was tested at only one dose that
engendered clonic and tonic convulsions and lethality. No
significant difference in convulsions was observed (Table 2).
Kainic acid produced dose-related increases in clonus until
60 mg/kg when tonic seizures were observed. Although there
were no significant differences in the percentage of mice
exhibiting clonic seizures across genotypes, γ−8 KO mice
showed a protection from the tonic convulsant effects of 60
mg/kg kainate and a corresponding reduction in lethality
(Table 3).
Neurochemistry
Compared to WT mice, γ−8 KO mice had significantly
reduced levels of norepinephrine in cerebellum (Fig. 7).
Although dopamine levels were not different across
genotypes in either nucleus accumbens or striatum, the
dopamine metabolite DOPAC was significantly lower in the
nucleus accumbens in the γ−8 KO mice and decreases in
dopamine turnover was observed in both brain areas (Fig. 8).
In contrast to the findings with norepinephrine and
dopamine, the monoamine serotonin and its metabolite 5-
HIAA were not significantly affected by deletion of the γ−8
protein (data not shown).
Levels of cAMP were measured in six brain areas;
significant reductions in levels were observed only in the
hippocampus of KO vs WT mice (Fig. 9). Significant
increases in levels of acetylcholine were seen in the
hypothalamus and prefrontal cortex of KO mice compared to
Fig. (2). Stimulatory effects of d-amphetamine (panel A) and of phencyclidine (Panel B) are enhanced in TARP γ−8 -/- mice compared to
wildtype control mice (WT). A) Each point represents the mean of n=8 male mice, vertical bars represent ± SEM. F7,56=8.55, p<0.05*p<0.05
compared to respective vehicle (WT or KO) (Dunnett's test). # p<0.05 compared to respective WT dose. B) Each bar represents the mean of
n=8 male mice, vertical bars represent ± SEM. Black circles: WT; White circles: KO. F3,27=10.4, p<0.05 *p<0.05 compared to respective
vehicle (WT or KO) (Dunnett's test). # p<0.05 compared to WT vehicle or PCP, respectively.
0-30 minutes
d-amphetamine (mg/kg,SC)
veh N.a.N. 1 3 10
Ambulations
0
2000
4000
6000
WT
KO
*
*
31-90 minutes
veh N.a.N. 1 3 10
-1000
0
1000
2000
3000
4000
5000
6000
WT
KO
d-amphetamine (mg/kg,SC)
Ambulations
*
# # #
#
#
*
*
*
0-30 minutes
WT N.a.N. KO
Ambulations
0
1000
2000
3000
4000
Vehicle
3 mg/kg, PCP
* *
#
31-90 Minutes
WT N.a.N. KO
Ambulations
0
1000
2000
3000
4000
Vehicle
3 mg/kg, PCP
*
* #
7. 618 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 Gleason et al.
Fig. (3). In a circular runway, wildtype (WT) mice and TARP γ−8
knockout (KO) mice did not significantly differ in terms of overall
photobeam breaks (top panel). However, γ−8 KO mice
demonstrated significantly less circular movements (counter-clock
wise or clockwise) than WT mice (bottom panel). Black bars: WT;
White bars: KO. *p<0.05 (Student’s t-test).
WT controls but not in the other four brain areas measured
(Fig. 9). Although histamine levels were not significantly
different in most brain areas studied (decrease in striatum) of
WT vs KO mice, the histamine metabolite tele-methyl-
histamine and tele-methyl-imidazole acetic acid were
significantly higher in multiple brain areas of KO vs WT
mice (Fig. 9). The turnover of histamine was increased in
multiple brain regions in KO vs WT mice (Fig. 10).
Plasma Lipids
Plasma from WT and γ−8 KO mice was evaluated for a
panel of lipids. Although several of these lipids appeared to
be differentially expressed in γ−8 KO mice compared to age-
matched controls (Table 4), statistical analysis revealed no
significant contrasts. However, two monoacylglycerols
(1OG and 2OG) were marginally non-significant (p=0.056)
with ratios of KO/WT of 0.44 and 0.23 for 1OG and 2OG
respectively.
DISCUSSION
We explored the biological significance of the TARP γ−8
protein using the same construct deletion used by Rouach et
al. [33]. We reveal for the first time a mild hyperactivity in
Fig. (4). Decreases in marble-burying (top panel) and digging
behavior (middle panel) but not general movement (bottom panel)
in TARP γ−8 -/- (KO) mice compared to their wildtype (WT)
controls. Each bar represents the mean of n=8 male mice, vertical
bars represent ± SEM. Black bars: WT; White bars: KO. *p<0.05
compared to WT controls by Student’s t-test.
Fig. (5). An anxiolytic phenotype of TARP γ−8 -/- mice (KO) is not
evidenced in the elevated- plus maze. Each bar represents the mean
of n=8 male mice. Vertical bars represent ± SEM. Black bars: WT;
White bars: KO. There were no significant differences in measures
across genotypes: F1, 42 = 2.208. However a significant interaction
of genotype x measures was detected: F2, 42 = 3.80, p<0.05.
W
T
K
O
0
100
200
300
Numberofmarblesburied
MiceDigging(%)
NumberorPercent
8. Inquiries into the Biological Significance of TARP γ−8 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 619
Fig. (6). TARP γ−8 -/- mice (KO) have reduced immobility in the
forced-swim assay and do not detect the effects of the AMPA
receptor potentiator LY392098 (1 mg/kg, i.p., 60 min prior) like
that of wildtype (WT) mice. Each bar represents the mean of n=7-8
male mice. Vertical bars represent ± SEM. Black bars: WT; White
bars: KO. Veh: vehicle injection; LY: LY392098 injection; IMI:
imipramine injection (15 mg/kg, ip, 30 min). prior). WT and KO
differed significantly from each other across drug treatments: F1, 41
= 14.37, p<0.05. Drug treatments also differed significantly: F2, 41 =
18.53, p<0.05, and the interaction of genotype and treatment was
also statistically significant: F2, 41 = 3.772, p<0.05. *p<0.05
compared to WT veh (Dunnett's test).
these mice. However, we also documented that increased
activity induced by γ−8 deletion is sensitive to the
environmental conditions under which it is studied. Thus, in
an open rectangular arena, γ−8 -/- mice are more active than
WT controls. However, in the elevated plus maze, in a
circular runway, or a smaller arena with sawdust and
marbles, there is no significant difference in the amount of
movement detected. The subtle environmental modulation of
increased movement indicates that hyperactivity in the
locomotor arena does not necessarily imply that
hyperactivity is a confounding behavioral response for the
interpretation of other behavioral effects. Thus, it might be
argued that if γ−8 KO mice are hyperactive, this increased
movement could render the antidepressant-like activity in the
forced-swim assay as a false positive. Although this remains
a possibility, proof of this idea cannot be argued without
further data. Further, if hyperactivity per se accounts for the
other behavioral effects of congenic γ−8 deletion, then
marble burying could have been predicted to have been
increased rather than decreased by the gene deletion; instead
the effect was the opposite.
Despite the mild hyperactivity of γ−8 KO mice in the
locomotor chambers, two psychomotor stimulants (d-
amphetamine and phencyclidine) had greater stimulant
effects in the KO mice than in WT controls. In
neurochemical studies, dopamine levels were not increased
in either nucleus accumbens or striatum that might account
for locomotor enhancements under basal conditions.
However, dopamine turnover was significantly reduced in
multiple brain areas. The increases in histamine metabolism
observed in the KO mice in the present study might have
relevance to these effects [35-37]. Future studies utilizing
specific pharmacological tools and lesioning studies will be
required to more fully appreciate the mechanism(s)
associated with these effects on locomotor activity.
Although AMPA receptors in general and hippocampal
AMPA receptors in particular have relevance to cognitive
Table 1. Effects of TARP γ−8 gene deletion (KO) on the convulsant effects of pentylenetetrazole (PTZ) compared to wildtype
control mice (WT) .1
Mouse Strain PTZ Dose (mg/kg sc) Clonus Tonus Lethality
WT 40 1/8 0/8 0/8
WT 50 1/6 0/6 0/6
WT 60 8/10 0/10 0/10
WT 70 8/10 0/10 0/10
KO 30 0/8 0/8 0/8
KO 40 4/6 0/6 0/6
KO 50 3/6 0/6 0/6
KO 60 5/6 0/6 0/6
KO 70 8/8 0/8 0/8
1
Values represent the number of mice exhibiting signs/number of mice tested.
Table 2. Effects of TARP γ−8 gene deletion (KO) on the convulsant effects of N-methyl-D-aspartate (NMDA) compared to
wildtype control mice (WT) .1
Mouse Strain Dose (mg/kg, sc) Clonus Tonus Lethality
WT 140 4/4 3/4 3/4
KO 140 4/4 4/4 4/4
1
Values represent the number of mice exhibiting signs/number of mice tested.
9. 620 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 Gleason et al.
Table 3. Effects of TARP γ−8 gene deletion (KO) on the convulsant effects of kainic acid (kainate) compared to wildtype control
mice (WT) .1
Mouse Strain Dose (mg/kg, sc) Clonus Tonus Lethality
WT 10 0/2 0/2 0/2
WT 15 0/4 0/4 0/4
WT 30 2/4 0/4 0/4
WT 60 6/6 6/6 6/6
KO 10 0/2 0/2 0/2
KO 15 0/4 0/4 0/4
KO 30 2/6 0/6 0/6
KO 60 6/6 2/6* 2/6*
1
Values represent the number of mice exhibiting signs/number of mice tested.
Fig. (7). Brain region-specific decreases in the brain norepinephrine (NE) in TARP γ−8 -/- mice (KO) compared to wildtype (WT) controls.
Each bar represents the mean of 4 (hypothalamus) or 8 mice and vertical bars represent ± SEM. Black bars: WT; White bars: KO. There was
not a significant difference between WT and KO mice across all brain regions: F1, 40 = 0.16. However, significant differences within regions
were detected. *P<0.05 by t-tests using the Holm-Sidak correction for multiple comparisons.
pmol/ml
pmol/ml
pmol/ml
pmol/ml
10. Inquiries into the Biological Significance of TARP γ−8 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 621
Fig. (8). Brain region-specific decreases in the brain dopamine (DA) metabolite, DOPAC, were observed in TARP γ−8 -/- mice (KO)
compared to wildtype (WT) controls. Each bar represents the mean of 4 (hypothalamus) or 8 mice and vertical bars represent ± SEM. Black
bars: WT; White bars: KO. No statistically-significant differences were obsereved for DA or DOPAC between WT and KO mice: F1, 16 =
0.96 and F1, 16 = 0.71, respectively. However, a significant trend for was detected for the brain concentration ratios of DOPAC/DA: F1, 16 =
3.64, p=0.075. *P<0.05 by t-tests using the Holm-Sidak correction for multiple comparisons.
11. 622 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 Gleason et al.
processing [9], the decrease in hippocampal AMPA receptor
populations through deletion of γ−8 [26, 31, 33, 38] could
result in decreased cognitive processing especially for
hippocampal dependent synaptic plasticity. NMDA-
dependent long-term potentiation is decreased in γ−8 -/-
mice [33]. γ−8 is necessary for the decrease in long-term
potentiation but is not due to the PDZ domain associated
with TARP γ−8 which appears to regulate synaptic
transmission independently [38]. It should also be noted that
cortical acetylcholine levels, a neurotransmitter also
implicated in cognitive processing, was enhanced, raising
now the opposite prediction of cognitive enhancement
observed with drugs that facilitate acetylcholine efflux [39].
Similar enhancements in histamine turnover were observed
in the γ−8 KO mice and histamine has long been implicated
in arousal and potential cognitive enhancement (Witkin and
Nelson, 2004). These alternative possibilities were not tested
in the present study and remain an outstanding question for
which the γ−8 line provides an intriguing research tool.
Further, the mechanisms associated with the neurochemical
alterations observed with deletion of TARP γ−8 also need to
be studied in relation to the congenic changes engendered by
this protein obscuration.
Marble-burying behavior, which can detect antidepres-
sant and anxiolytic drugs [34], was dramatically reduced in
KO mice. If this effect was due to the anxiolytic effects
engendered by γ−8 removal, it would be predicted that
positive activity would be observed in another anxiety-
detecting assay. However, at least in one such assay, the
elevated plus maze, no differences were observed between
WT and KO mice that would reinforce the concept of an
anxiolytic phenotype. However, in the forced-swim assay
Fig. (10). Histmine turnover as assessed by the ratio of the
quantities of histamine metabolites tele-methylhistamine (t-MH) or
tele-methyl-imidazole acetic acid (t-MIAA) to the quantities of
histamine in discrete brain areas. Each bar represents the mean of 4
(hypothalamus) or 8 mice and vertical bars represent ± SEM. Black
bars: WT; White bars: KO. The ratio of t-MH/Histamine and t-
MIAA/Histamine brain levels were both statistically different
across genotypes: F1,75= 8.34, p<0.05 and F1,75 = 32.7, p<0.05,
respectively. *P<0.05 by t-tests using the Holm-Sidak correction
for multiple comparisions.
Fig. (9). Brain region-specific changes in acetylcholine levels and histamine metabolism were observed in TARP γ−8 -/- mice (KO)
compared to wildtype controls (KO). Each bar represents the mean of 7-8 or 4 (hypothalamus) animals and vertical bars represent ± SEM.
Black bars: WT; Striped bars: KO. Statistical evaluation of each analyte was conducted comparing WT and KO mice: cAMP; F1, 80 = 1.79;
Acetylcholine F 1, 76 = 5.24, p<0.05; Histamine: F1, 75 = 5.12, p<0.05; t-methyl-histamine: F1, 76 = 11.7, p<0.05; t-methyl-imidazole acetic
acid: F1, 76 = 30.7, p<0.05. *P<0.05 by t-tests using the Holm-Seidak correction for multiple comparisons.
H
ypothalam
us
Hippocam
pusCerebellum
PrefrontalC
ortex
Nucleus
A
ccum
bens
Striatum
H
ypothalam
us
Hippocam
pusC
erebellum
PrefrontalC
ortex
N
ucleus
A
ccum
bens
Striatum
H
ypothalam
us
H
ippocam
pusC
erebellum
PrefrontalC
ortex
N
ucleus
A
ccum
bens
Striatum
H
ypothalam
us
H
ippocam
pusC
erebellum
PrefrontalC
ortex
N
ucleus
Accum
bens
Striatum
H
ypothalam
us
H
ippocam
pusC
erebellum
PrefrontalC
ortex
N
ucleus
A
ccum
bens
Striatum
H
ypothalam
us
H
ippocam
pusC
erebellum
PrefrontalC
ortex
N
ucleus
A
ccum
bens
Striatum
13. 624 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 Gleason et al.
that detects multiple antidepressant mechanisms [40], KO
mice displayed decreases in immobility time like the
antidepressant imipramine; that is, an antidepressant-like
response. An understanding of this phenomenon is likely
important given the relevance of AMPA receptors to
antidepressant drug response and the role of hippocampal
AMPA receptors in particular [11, 41]. In this regard, the
antidepressant-like effect of the AMPA receptor potentiator,
LY392098, was fully prevented in γ−8 -/- mice. The fact that
comparable effects of imipramine could still be invoked in
γ−8 -/- mice attests to the specificity of this gene deletion
and its biological impact.
AMPA receptor antagonists are anticonvulsant in animals
and in humans [42]. If TARPγ−8-associated AMPA
receptors are initiators of convulsions, then it would be
predicted that the γ−8 KO mice would be more resistant to
convulsant stimuli than WT mice. This was not the case
since multiple and distinct mechanistic chemoconvulsants
engendered convulsions in the KO mice with equal potency
to the WT mice (pentylenetetrazole and NMDA, albeit only
one dose studied for NMDA). Thus, since AMPA receptor
antagonists are anticonvulsant, it can be concluded that the
TARP γ−8 protein-associated AMPA receptors are involved
in the control of seizure propagation or braking, not
initiation. In contrast to pentylenetetrazole and NMDA,
which act through GABAA and NMDA receptors,
respectfully, kainate, which can act upon AMPA receptors
[43], engendered convulsions that were strikingly attenuated
by TARP γ−8 deletion; tonic convulsions and associated
lethality were both significantly reduced in γ−8 KO mice.
Tomita and colleagues [12] likewise reported reductions in
cell death in hippocampus (CA1 and CA3 regions) after
kainate-induced seizures. Thus, consistent with their
predicted pharmacological actions, the convulsant effects of
kainate and the antidepressant-like effects of an AMPA
receptor potentiator (both acting upon AMPA receptors)
were reduced or absent in KO mice, a finding that re-affirms
the value of the TARP γ−8 KO mice for interrogation of the
functional significance of this transmembrane AMPA
receptor protein.
Since synaptic transmission (~20%) and LTP are
regionally reduced in the hippocampus of γ-8-/- mice [33], it
could be suggested that the biology of these mice might be
similar to mice with hippocampal lesions. Indeed, increases
in activity levels have been observed after hippocampal
lesions [44], whereas other behaviors such as climbing are
reduced [45]; burying behaviors and digging were reduced in
the present study. Likewise, marble-burying is markedly
reduced in mice with lesions of the hippocampus [45];
however, in that study mice were observed to dig but not
specifically bury marbles. Whether such an effect can be
related to effects on spatial memory processing remains an
open question. Other work has indeed demonstrated that
increases in movement after kainic acid lesions are
dependent upon the environmental context in which
movement is assessed [46] as observed in the present study.
In the forced-swim assay, hippocampal lesions have been
shown to decrease immobility, an effect dependent upon the
extent of lesion [47] and an effect consistent with the
decreased immobility times observed in the TARP γ−8 -/-
mice in the present study. However, kainic acid and NMDA
lesions of the dorsal hippocampus did not facilitate
methamphetamine-induced locomotion in mice [48].
Although TARP γ−8 deletion reduced tonic seizures and
lethality induced by kainic acid, lesions of hippocampus by
kainate increased seizure prevalence [49]. In contrast,
colchicine-induced hippocampal lesions [50] showed
reductions in kainate induced seizures in rats. Hippocampal
lesions have long been known to reduce kainate-induced
(Table 4) contd…..
Lipid Class Analyte Wildtype Gamma 8 KO
N-acylethanolamines
PEA (C16:0) 5.17 ± 2.22 4.02 ± 1.07
LEA (C18:2) 2.12 ± 0.61 1.82 ± 0.25
OEA (C18:1) 1.61 ± 0.32 1.08 ± 0.26
SEA (C18:0) 3.36 ± 3.25 2.55 ± 2.27
AEA (C20:4) 0.30 ± 0.06 0.25 ± 0.07
DEA (C22:6) 0.56 ± 0.20 0.35 ± 0.10
Mono acylglycerols
2PG (C16:0) 1113.2 ± 137.41 964.72 ± 111.97
1PG (C16:0) 24329.9 ± 1644.39 27681.3 ± 2117.23
2LG (C16:0) 1363.3 ± 678.29 480.91 ± 290.79
1LG (C16:0) 1369.2 ± 304.73 827.35 ± 268.92
2OG (C16:0) 1542.0 ± 767.44 357.47 ± 208.32
1OG (C16:0) 847.9 ± 170.4 374.2 ± 163.3
2SG (C16:0) 19262.4 ± 2262.20 17156.4 ± 1636.70
1SG (C16:0) 201078.8 ± 10234.39 217021.9 ± 14702.29
2AG (C16:0) 28.9 ± 10.4 11.9 ± 7.03
1AG (C16:0) 18.8 ± 6.37 17.0 ± 12.1
1
Data are in units of ng/mL. There were no significant differences between wild-type and TARP γ-8 knockout mice: F1,468 = 0.082.
14. Inquiries into the Biological Significance of TARP γ−8 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 625
seizures [51]. Other seizure inducing agents are also altered
by hippocampal lesions. For example, kainate-induced
lesions of hippocampus increased the acute and chronic
dosing effects of pentylenetetrazole in rats [52]. Some of the
differences in effects of lesions of hippocampus in the
literature will be dependent upon the specific damage to the
hippocampus and the extent of damage that are dependent
upon the lesion methodology.
In summary, congenic deletion of the TARP γ−8 protein
results in a mild hyperactivity under some conditions, an
enhancement of psychomotor stimulant-induced locomotion,
and a reduction in marble-burying and immobility in the
forced-swim assay. Convulsions can be engendered by
multiple chemical agents in the knockout mice. Deletion of
TARP γ−8 attenuates AMPA-mediated events such as
kainate-induced tonic seizures and lethality, and the
antidepressant-related phenotype induced by an AMPA
receptor potentiator. The present study measured only a
small subset of dependent measures. A noted absence of data
in the current report is that of spatial navigation and learning.
These studies will be important in the future given the high
density localization of the TARP γ−8 protein in the spatially-
guiding hippocampus. Mice without TARP γ−8 will continue
to provide great utility in understanding the biological
significance of TARP γ−8 and the role of hippocampal
AMPA receptors in normal and pathophysiological states.
The mice also provide an additional inroad into interrogating
questions of selective activation or blockade of specific and
brain localizable AMPA receptors.
LIST OF ABBREVIATIONS
5-HIAA = 5-Hydroxy-Indoleacetic Acid
AMPA = α-Amino-3-Hydroxy-5-Methyl-4-
Isoxazolepropionic Acid
cAMP = Adenosine 3’,5’-Cyclic Monophosphate
DA = Dopamine
DOPAC = 3,4-Dihydroxyphenylacetic Acid
GYKI 52466 = 4-(8-Methyl-9H-1,3-Dioxolo[4,5-h][2,3]
Benzodiazepin-5-yl)-Benzenamine
LTP = Long-Term Potentiation
LY392098 = N-2-(4-(3-Thienyl)Phenyl)Propyl 2-
Propanesulfonamide
NE = Norepinephrine
NMDA = N-Methyl-D-Aspartate
TMH = Tele-Methyl-Histamine
TMIAA = Tele-Methyl-Imidazole Acetic Acid
CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.
ACKNOWLEDGEMENTS
We thank Drs. Kjell Svensson and David L. McKinzie
for their kind support of some of these experiments.
REFERENCES
[1] Takagaki G. The dawn of excitatory amino acid research in Japan.
The pioneering work by Professor Takashi Hayashi. Neurochem Int
1996; 29(3): 225-9.
[2] Curtis RR, Phillis JW, Watkins JC. The depression of spinal
neurones by gamma-amino-n-butyric acid and beta-alanine. J
Physiol 1959; 146: 185-203.
[3] Schoepp DD, Conn PJ. Metabotropic glutamate receptors in brain
function and pathology. Trend Pharmacol Sci 1993; 14: 13-20.
[4] Willard SS, Koochekpour S. Glutamate, glutamate receptors, and
downstream signaling pathways. Int J Biol Sci 2013; 9: 948-59.
[5] Tarnawa I, Farkas S, Berzenvi P, Pataki A, Andrasi F.
Electrophysiological studies with a 2: 3-benzodiazepine muscle
relaxant: GYKI 52466. Eur J Pharmacol 1989; 167: 193-9.
[6] Mellor IR. The AMPA receptor as a therapeutic target: current
perspectives and emerging possibilities. Fut Med Chem 2010; 2:
877-91.
[7] Zaccara G, Giovannelli F, Cincotta M, Iudice A. AMPA receptor
inhibitors for the treatment of epilepsy: the role of perampanel.
Expert Rev Neurother 2013; 13: 647-655.
[8] Kramer LD, Satlin A, Krauss GL, et al. Perampanel for adjunctive
treatment of partial-onset seizures: a pooled dose-response analysis
of phase III studies. Epilepsia 2014; 55: 423-31.
[9] Lynch G. Memory and the brain: unexpected chemistries and a new
pharmacology. Neurobiol Learn Mem 1998; 70: 82-100.
[10] O'Neill MJ, Dix S. AMPA receptor potentiators as cognitive
enhancers. IDrugs 2007; 10: 185-92.
[11] Alt A, Nisenbaum ES, Bleakman D, Witkin JM. A role for AMPA
receptors in mood disorders. Biochem Pharmacol 2006; 71: 1273-
88.
[12] Tomita S, Byrd RK, Rouach N, et al. AMPA receptors and
stargazin-like transmembrane AMPA receptor-regulatory proteins
mediate hippocampal kainate neurotoxicity. Proc Natl Acad Sci
USA 2007; 104: 18784-8.
[13] Lau A, Tymianski M. Glutamate receptors, neurotoxicity and
neurodegeneration. Pflugers Arch 2010; 460: 525-42.
[14] Chen L, Chetkovich DM, Petralia RS, et al. Stargazin regulates
synaptic targeting of AMPA receptors by two distinct mechanisms.
Nature 2000; 408: 936-43.
[15] Tomita S, Chen L, Kawasaki Y, et al. Functional studies and
distribution define a family of transmembrane AMPA receptor
regulatory proteins. J Cell Biol 2003; 161: 805-16.
[16] Kott S, Werner M, Körber C, Hollmann M. Electrophysiological
properties of AMPA receptors are differentially modulated
depending on the associated member of the TARP family. J
Neurosci 2007; 27: 3780-9.
[17] Schwenk J, Harmel N, Zolles G, et al. Functional proteomics
identify cornichon proteins as auxiliary subunits of AMPA
receptors. Science 2009; 323: 1313-9.
[18] Schwenk J, Harmel N, Brechet A, et al. High-resolution proteomics
unravel architecture and molecular diversity of native AMPA
receptor complexes. Neuron 2012; 74: 621-33.
[19] Kalashnikova E, Lorca RA, Kaur I, et al. SynDIG1: an activity-
regulated, AMPA- receptor-interacting transmembrane protein that
regulates excitatory synapse development. Neuron 2010; 65: 80-93.
[20] von Engelhardt J, Mack V, Sprengel R, et al. CKAMP44: a brain-
specific protein attenuating short-term synaptic plasticity in the
dentate gyrus. Science 2010; 327: 1518-22.
[21] Kato AS, Zhou W, Milstein AD, et al. New transmembrane AMPA
receptor regulatory protein isoform, gamma-7: differentially
regulates AMPA receptors. J Neurosci 2007; 27: 4969-77.
[22] Kato AS, Siuda ER, Nisenbaum ES, Bredt DS. AMPA receptor
subunit-specific regulation by a distinct family of type II TARPs.
Neuron 2008; 59: 986-96.
[23] Kato AS, Gill MB, Yu H, Nisenbaum ES, Bredt DS, TARPs
differentially decorate AMPA receptors to specify
neuropharmacology. Trends Neurosci 2010; 33: 241-8.
[24] Kato AS, Gill MB, Ho MT, et al. Hippocampal AMPA receptor
gating controlled by both TARP and cornichon proteins. Neuron
2010; 68: 1082-96.
[25] Tomita S, Adesnik H, Sekiguchi M, et al. Stargazin modulates
AMPA receptor gating and trafficking by distinct domains. Nature
2005; 435: 1052-8.
15. 626 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 Gleason et al.
[26] Menuz K, O'Brien JL, Karmizadegan S, Bredt DS, Nicoll RA.
TARP redundancy is critical for maintaining AMPA receptor
function. J Neurosci 2008; 28: 8740-6.
[27] Tomita S, Sekiguchi M, Wada K, Nicoll RA, Bredt DS. Stargazin
controls the pharmacology of AMPA receptor potentiators. Proc
Natl Acad Sci USA 2006; 103: 10064-7.
[28] Fukaya M, Tsujita M, Yamazaki M, et al. Abundant distribution of
TARP gamma-8 in synaptic and extrasynaptic surface of
hippocampal neurons and its major role in AMPA receptor
expression on spines and dendrites. Eur J Neurosci 2006; 24: 2177-
90.
[29] Lein ES, Hawrylycz MJ, Ao N, et al. Genome-wide atlas of gene
expression in the adult mouse brain. Nature 2007; 445: 168-76.
[30] Hawrylycz MJ, Lein ES, Guillozet-Bongaarts AL, et al. An
anatomically comprehensive atlas of the adult human brain
transcriptome. Nature 2012; 489: 391-9.
[31] Hashimoto K, Fukaya M, Qiao X, Sakimura K, Watanabe M, Kano
M. Impairment of AMPA receptor function in cerebellar granule
cells of ataxic mutant mouse stargazer. J Neurosci 1999; 19: 6027-
36.
[32] Zwart R, Sher E, Ping X, et al. Perampanel, an Antagonist of α-
amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA)
Receptors for the Treatment of Epilepsy: Studies in Human
Epileptic Brain, Non-Epileptic Brain, and in Rodent Models. J
Pharmacol Exp Ther 2014; 351: 124-33.
[33] Rouach N, Byrd K, Petralia RS, et al. TARP gamma-8 controls
hippocampal AMPA receptor number, distribution and synaptic
plasticity. Nat Neurosci 2005; 8: 1525-33.
[34] Li X, Morrow D, Witkin JM. Decreases in nestlet shredding of
mice by serotonin uptake inhibitors: comparison with marble
burying. Life Sci 2006; 78: 1933-9.
[35] Brabant C, Alleva L, Grisar T, et al. Effects of the H3 receptor
inverse agonist thioperamide on cocaine-induced locomotion in
mice: role of the histaminergic system and potential
pharmacokinetic interactions. Psychopharmacology 2009; 202:
673-87.
[36] Toyota H, Dugovic C, Koehl M, et al. Behavioral characterization
of mice lacking histamine H(3) receptors. Mol Pharmacol 2002; 62:
389-97.
[37] Okuda T, Zhang D, Shao H, et al. Methamphetamine- and 3: 4-
methylenedioxymethamphetamine-induced behavioral changes in
histamine H3-receptor knockout mice. J Pharmacol Sci 2009; 111:
167-74.
[38] Sumioka A, Brown TE, Kato AS, Bredt DS, Kauer JA, Tomita S.
PDZ binding of TARPγ-8 controls synaptic transmission but not
synaptic plasticity. Nat Neurosci 2011; 14: 1410-2.
[39] Tzavara ET, Bymaster FP, Overshiner CD, et al. Procholinergic
and memory enhancing properties of the selective norepinephrine
uptake inhibitor atomoxetine (Strattera), a novel non-stimulant
treatment for ADHD. Mol Psychiatry 2006; 11: 187-95.
[40] Cryan JF, Markou A, Lucki I. Assessing antidepressant activity in
rodents: recent developments and future needs. Trends Pharmacol
Sci 2002; 23: 238-45.
[41] Santarelli L, Saxe M, Gross C, et al. Requirement of hippocampal
neurogenesis for the behavioral effects of antidepressants. Science
2003; 301: 805-9.
[42] Chappell AS, Sander JW, Brodie MJ, et al. A crossover, add-on
trial of talampanel in patient with refractory partial
seizures.crossover, add-on trial of talampanel in patient with
refractory partial seizures Neurology 2002; 58: 1680-2.
[43] Bleakman D, Lodge D. Neuropharmacology of AMPA and kainate
receptors. Neuropharmacology 1998; 37: 1187-204.
[44] Reinstein DK, Hannigan JH, Jr, Isaacson RL. Time course of
certain behavioral changes after hippocampal damage and their
alteration by dopaminergic intervention into nucleus accumbens.
Pharmacol Biochem Behav 1982; 17: 193-202.
[45] Deacon RMJ, Rawlins NP. Hippocampal lesions, species-typical
behaviours and anxiety in mice. Behav Brain Res 2005; 156: 241-9.
[46] Gröticke I, Hoffmann K, Löscher W. Behavioral alterations in a
mouse model of temporal lobe epilepsy induced by
intrahippocampal injection of kainate. Exp Neurol 2008; 213: 71-
83.
[47] Arushanyan EB, Beier EV. Relationships between the epiphysis
and hippocampus during formation of a response. Neurosci Behav
Physiol 1998; 28: 608-12.
[48] Han W, Wang F, Qi J, et al. NMDA receptors in the medial
prefrontal cortex and the dorsal hippocampus regulate
methamphetamine-induced hyperactivity and extracellular amino
acid release in mice. Behav Brain Res 2012; 232: 44-52.
[49] Bouilleret V, Ridoux V, Depaulis A, et al. Recurrent seizures and
hippocampal sclerosis following intrahippocampal kainate injection
in adult mice: electroencephalography, histopathology and synaptic
reorganization similar to mesial temporal lobe epilepsy.
Neuroscience 1999; 89: 717-29.
[50] Maeda T, Hashizume K, Sako K, Tanaka T. The effect of
hippocampal dentate granule cell lesions upon the limbic seizure
model of rats. No To Shinkei 1998; 50: 643-9.
[51] Okazaki MM, Nadler JV. Protective effects of mossy fiber
degeneration.lesions against kainic acid-induced seizures and
neuronal degeneration. Neuroscience 1988; 26: 763-81.
[52] Czuczwar SJ, Turski L, Turski W, Kleinrok Z. Convulsant action
of pentetrazol in rats with selective lesions of the hippocampal
pyramidal cells with intracerebroventricular kainic acid. Methods
Find Exp Clin Pharmacol 1982; 4: 293-8.
Received: August 16, 2014 Revised: January 30, 2015 Accepted: February 1, 2015
PMID: 25921737
![Send Orders for Reprints to reprints@benthamscience.ae
612 CNS & Neurological Disorders - Drug Targets, 2015, 14, 612-626
Inquiries into the Biological Significance of Transmembrane AMPA
Receptor Regulatory Protein (TARP) γ−8 Through Investigations of
TARP γ−8 Null Mice§
Scott D. Gleason, Akihiko Kato, Hai H. Bui, Linda K. Thompson, Sabrina N. Valli, Patrick V. Stutz,
Ming-Shang Kuo, Julie F. Falcone, Wesley H. Anderson, Xia Li and Jeffrey M. Witkin*
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
Abstract: Transmembrane AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor
regulatory protein (TARP) γ−8 is an auxiliary protein associated with some AMPA receptors. Most
strikingly, AMPA receptors associated with this TARP have a relatively high localization in the
hippocampus. TARPγ−8 also modifies the pharmacology and trafficking of AMPA receptors.
However, to date there is little understanding of the biological significance of this auxiliary protein. In
the present set of studies we provide a characterization of the differential pharmacology and
behavioral consequences of deletion of TARP γ−8 by comparing the wild type (WT) and γ−8 -/-
(knock-out, KO) mouse. KO mice were mildly hyperactive in a locomotor arena but not in other
environments compared to WT mice. Additionally, the KO mice demonstrated enhanced locomotor stimulatory effects of
both d-amphetamine and phencyclidine. Marble-burying and digging behaviors were dramatically reduced in KO mice. In
another assay that can detect anxiety-like phenotypes, the elevated plus maze, no differences were observed in overall
movement or open arm entries. In the forced-swim assay, KO mice displayed decreases in immobility time like the
antidepressant imipramine and the AMPA receptor potentiator, LY392098. In KO mice, the antidepressant-like effects of
LY392098 were prevented whereas the effects of imipramine were unaffected. Convulsions were induced by
pentylenetetrazole, N-methyl-D-aspartate, and by kainic acid. However, in KO mice, kainic acid produced less tonic
convulsions and lethality. KO mice had reduced levels of norepinephrine in hippocampus and cerebellum but not in
hypothalamus or prefrontal cortex, decreased levels of cAMP in hippocampus, and increased levels of acetylcholine in the
hypothalamus and prefrontal cortex. KO mice displayed decreased turnover of dopamine and increased histamine turnover
in multiple brain areas In contrast, serotonin and its metabolites were not significantly affected by deletion of the γ−8
protein. Of a large panel of plasma lipids, only two monoacylglycerols (1OG and 2OG) were marginally but non-
significantly altered in WT vs KO mice. Overall, the data suggest genetic inactivation of this specific population of
AMPA receptors results in modest changes in behavior characterized by a mild hyperactivity which is condition
dependent and a marked reduction in digging and burying behaviors. Despite deletion of TARP γ−8, chemoconvulsants
were still active. Consistent with their predicted pharmacological actions, the convulsant effects of kainate and the
antidepressant-like effects of an AMPA receptor potentiator (both acting upon AMPA receptors) were reduced or absent
in KO mice.
Keywords: AMPA receptors, convulsions, d-amphetamine, elevated plus maze, forced swim test, locomotor activity,
LY392098, marble burying, mice, phencyclidine, TARP gamma 8.
INTRODUCTION
Glutamate was identified as a neurotransmitter in the
central nervous system (CNS) only about 35 years ago.
Before that time, some important data were acquired by bold
investigations into its potential physiological roles. Hayashi
provided as early as 1944 that glutamate engendered seizures
[1] and Curtis and colleagues reported that iontophoretically-
applied glutamate onto cat spinal neurons induced
depolarization and firing [2]. Since then, glutamate has been
*Address correspondence to this author at the Lilly Research Laboratories,
Lilly Corporate Center, Indianapolis, IN 46285-0501, USA; Tel: 317 277-
4470; Fax: 317 276-7600; E-mail: jwitkin@lilly.com
§
This paper is dedicated to the memory of Dr. J. David Leander, a great drug
hunter and mentor who died too early.
widely accepted as the primary neurotransmitter regulating
excitatory neurotransmission in the mammalian CNS.
Glutamate initiates its biological actions via interaction with
ionotropic (AMPA, NMDA, kainate) and metabotropic
receptors (mGlu1-8) that are classified based upon sequence
homology and pharmacology [3, 4].
AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid) receptors are ion channels in the central nervous system
(CNS) of mammals which regulate fast-synaptic
transmission of glutamate. Their importance in the biology
of the CNS is well-known and continues to be the source of
intense investigation. In addition, AMPA receptors have
been a valued protein target for drug discovery over the past
several years. General interest in therapeutics was
additionally heightened by the discovery of the first AMPA
receptor antagonist [5]; GYKI 52466 was reported to have
1996-3181/15 $58.00+.00 © 2015 Bentham Science Publishers](https://image.slidesharecdn.com/b7ec38ff-b945-4320-81f7-52f91d76291d-160201170946/85/investigations-of-tarp-gamma8-null-mice-1-320.jpg)
![Inquiries into the Biological Significance of TARP γ−8 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 5 613
muscle-relaxant properties without being a GABA receptor
antagonist. The development of this and other series of
compounds led to a number of compounds with potential for
neuroprotection, pain, epilepsy, drug dependence, and other
disorders of the CNS [6, 7]. However, it was not until 2012
that perampanel (Fycompa®
), a non-competitive AMPA
receptor antagonist, became the first FDA-approved
medication for the control of refractory partial seizures.
One issue associated with the pharmacological
modulation of AMPA receptors is the induction of side-
effects. For example, perampanel induces dizziness, vertigo,
problems with walking, and sleepiness [7, 8]. On the other
side of modulation are AMPA receptor potentiators which
had been discovered with the hope of cognitive enhancement
[9, 10] and more recently depression [11]. A known
drawback with some of these agents is the fact that supra
amplification of glutamatergic tone is associated with
neurotoxicity [12, 13].
AMPA receptors have been found to be associated with
transmembrane AMPA receptor regulatory proteins (TARPs)
[14-16] in addition to other proteins (e.g., cornichons) [17-
20]. TARPs are evolutionarily related to the claudin family
and calcium channel γ subunits of tight junction proteins.
TARPs are comprised of Type I and Type II variants based
upon structural similarity to one another. Group I TARPs
consist of γ−2 (also known as stargazin), γ−3, γ−4, and
γ−8(Cacng8); group II TARPs are comprised of γ5, and γ−7
[21-24]. It is established that TARPs are key regulators of
AMPA receptor pharmacology and function [12, 16]. For
example, TARPs control the translocation of AMPA
receptors to the synapse enabling normal AMPA receptor
function; in the absence of TARP γ−2, AMPA receptor
function is lost (stargazin mouse) [14, 25, 26]. Likewise,
TARPs can control the potency and selectivity of ligands
activating AMPA receptors. For example TARP γ−2 imbues
AMPA receptor flip and flop spice variants sensitive to
AMPA receptor potentiators as well as enhancing their
potencies [27]. However, it is unknown what biological
significance TARPs play in normal and pathophysiological
states in humans. One remarkable feature of TARPs which
likely plays a major modulatory role in the CNS is the fact
TARPS have highly specialized localization in the brain [15,
28]. For example, γ−8 TARPs have very high relative
densities in the hippocampus of rodent and human brains
[29, 30].
Since TARPs regulate AMPA receptor function and
pharmacological selectivity and potency, AMPA receptors
associated with γ−8 TARPs would likely have a high degree
of selective control over hippocampal function. A corollary
is that brain structures with AMPA receptors associated with
γ−2 TARPs are likely to have dominant control over
cerebellar function [14, 15, 28, 31]. Recently, it was
speculated, based on behavioral and electrophysiological
studies with perampanel, that an antagonist of AMPA
receptors which was selectively associated with γ−8 TARPs
might engender antiepileptic benefit without the motoric
side-effects associated with AMPA receptor blockade across
the entire CNS [32]. Earlier, it was suggested that an AMPA
receptor potentiator that selectively enhanced AMPA
receptor/γ−8 TARP complexes might be antidepressant since
AMPA receptor facilitation and hippocampal function in
general appear to be critical to antidepressant activity [11].
However, to date no selective ligands for the TARP γ−8
exist.
In the absence of selective pharmacological tools, one
approach toward the elucidation of biological function of
TARP γ−8 can be gained through research with TARP γ−8
null mice. γ−8 -/- mice have been genetically constructed and
have undergone some scrutiny. Compared to their wild type
controls, γ− 8 -/- mice, or knockout mice, are characterized
by the virtual absence of the γ−8 protein in the brain,
significantly fewer numbers of AMPA receptors in dendritic
synaptic, and extrasynaptic membranes [28, 33], and the lack
of long-term potentiation (LTP) with a mild decrease in the
synaptic AMPA receptor-mediated responses [33]. Menuz at
al. [28] suggested that very few physically observable traits
and behaviors distinguish γ−8 -/- mice from their wild type
(WT) littermates, hypothesizing that similar TARP family
members (e.g. γ−4) may compensate for the loss of γ−8.
However, γ−8 -/- mice have not been extensively studied for
phenotypic behavioral differences. Such investigations are
the primary aim of the present series of experiments.
MATERIALS AND METHODS
Mice
The TARP γ−8 -/- mice were created as described [33].
They were re-derived in a CD1 mouse line (Taconic Farms
Line 1834). Het x het breeding created WT and KO
littermate controls. Only male mice were used in the current
experiments as our housing vivarium did not accommodate
females and to avoid, in these studies, any gender factor. The
mice weighed 35-42 g at the- time of testing and were all
born and delivered within two weeks of each other. They
were grouped housed with ad libitum food and water in a
colony room which controlled heat, humidity, and light
(lights on 6am; off 6pm). Animals were maintained in the
colony room for at least 3 days before testing. They were
moved to a quiet room 1 hour prior to the start of a test. The
mice were used in these studies under the strict guidance and
approval of local animal care and use committees under
guidelines of the Institutional Animal Care and Use
Committee at Eli Lilly & Co. and in strict accordance with
the guidelines set forward by the National Institutes of
Health.
Compounds
d-Amphetamine SO4, phencyclidine HCl, imipramine
HCl, chlordiazepoxide HCl, pentylenetetrazole base and
NMDA base (Sigma Chemical Co., St. Louis, MO, USA)
were dissolved in 0.9% NaCl. LY392098 (Eli Lilly and Co.)
was suspended in 1% hydoxyethylcellulose/0.25%
Tween80/0.05% Dow antifoam. Kainic acid (Tocris
Bioscience, Bristol, United Kingdom) was dissolved in
water. Drug doses are expressed as the drug forms listed. All
compounds were prepared just prior to dosing and
administered i.p. or s.c. in a volume of 0.01 ml/g body
weight.](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)