Investigations of TARP gamma-8 Null Mice

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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. TARP γ−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: [email protected] §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

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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.

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


with phosphoric acid). The flow rate was 1.20 ml/min. The analytical column was maintained at 40oC 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 40C. 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 4oC. 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.


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).

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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.

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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.

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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 1Values 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 1Values represent the number of mice exhibiting signs/number of mice tested.


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* 1Values 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


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.


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.

Hypothala

mus

Hippocampus

Cerebell

um

Prefro

ntal C

ortex

Nucleus A

ccumben

s

Striatu

m

Hypothala

mus

Hippocampus

Cerebell

um

Prefro

ntal C

ortex

Nucleus A

ccumben

s

Striatu

m

Hypothala

mus

Hippocampus

Cerebell

um

Prefro

ntal C

ortex

Nucleus A

ccumben

s

Striatu

m

Hypothala

mus

Hippocampus

Cerebell

um

Prefro

ntal C

ortex

Nucleus A

ccumben

s

Striatu

m

Hypothala

mus

Hippocampus

Cerebell

um

Prefro

ntal C

ortex

Nucleus A

ccumben

s

Striatu

m

Hypothala

mus

Hippocampus

Cerebell

um

Prefro

ntal C

ortex

Nucleus A

ccumben

s

Striatu

m


Table 4. Effects of TARP γ−8 gene deletion (KO) on the levels of lipids in plasma compared to wildtype control mice1.

Lipid Class Analyte Wildtype Gamma 8 KO

Lysophosphatidic acids

LPA (C16:0) 11.4 ± 3.37 15.2 ± 7.85

LPA (C18:0) 14.6 ± 3.17 10.6 ± 1.94

LPA (C18:1) 15.3 ± 3.06 14.6 ± 1.11

LPA (C18:2) 31.2 ± 5.55 35.8 ± 10.9

LPA (C20:4) 22.9 ± 3.98 21.2 ± 6.31

Lysophosphatidyl Cholines

LPC (C14:0) 340.6 ± 135.3 259.7 ± 98.53

LPC (C16:0) 96532.7 ± 26412.8 108236 ± 23914.5

LPC (C16:1) 1631.8 ± 566.01 1203.6 ± 422.19

LPC (C18:0) 54914.9 ± 16043.3 73825.8 ± 4718.60

LPC (C18:1) 45727.3 ± 13376.4 35262.2 ± 9258.05

LPC (C18:2) 42078.3 ± 14899.6 45531.9 ± 8752.88

LPC (C18:3) 640.8 ± 178.4 753.9 ± 230.4

LPC (C20:1) 468.6 ± 84.63 518.9 ± 123.7

LPC (C20:2) 524.3 ± 134.6 407.2 ± 140.3

LPC (C20:3) 4772.8 ± 1593.8 2897.6 ± 1347.0

LPC (C20:4) 12184.0 ± 3892.92 10803.4 ± 4102.98

LPC (C20:5) 426.6 ± 155.6 281.6 ± 106.0

LPC (C22:6) 7949.4 ± 2431.4 6027.5 ± 1439.1

Sphingosine Sphingosine-1-phosphate Sphinganine-1-phosphate

Dihydroceramides

Sph 14.3 ± 1.51 24.6 ± 12.2

S1P 332.4 ± 46.92 439.7 ± 107.6

Sa1P 139.9 ± 32.88 144.7 ± 34.99

DHCer (C22:0) 18.9 ± 13.9 39.6 ± 15.7

DHCer (C24:1) 10.0 ± 7.51 25.1 ± 25.3

DHCer (C24:0) 7.75 ± 2.73 35.1 ± 28.7

Total DHCers 36.7 ± 17.9 99.9 ± 65.3

Ceramides

Cer (C16:0) 50.4 ± 14.8 72.2 ± 37.7

Cer (C18:0) 17.2 ± 2.01 13.6 ± 4.49

Cer (C20:0) 27.6 ± 8.15 19.0 ± 8.18

Cer (C22:0) 553.9 ± 121.8 540.5 ± 228.2

Cer (C23:0) 174.6 ± 33.00 167.6 ± 78.13

Cer (C24:1) 454.6 ± 104.0 502.8 ± 142.0

Cer (C24:0) 620.1 ± 120.4 828.3 ± 482.4

Total Cers 1898.6 ± 329.10 2144.4 ± 757.42

Hexocyl Ceramides

HexCer (C16:0) 277.5 ± 88.34 937.5 ± 1013.2

HexCer (C18:0) 25.1 ± 14.8 39.5 ± 29.1

HexCer (C20:0) 2146.6 ± 87.79 173.8 ± 61.27

HexCer (C22:0) 2166.3 ± 898.08 2021.3 ± 825.71

HexCer (C24:1) 2101.0 ± 707.47 3093.8 ± 1732.8

HexCer (C24:0) 666.3 ± 291.6 910.8 ± 477.3

Total HexCers 5451.0 ± 2025.3 7176.9 ± 2897.0


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 TARP γ−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 1Data are in units of ng/mL. There were no significant differences between wild-type and TARP γ-8 knockout mice: F1,468 = 0.082.


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.

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Received: August 16, 2014 Revised: January 30, 2015 Accepted: February 1, 2015

PMID: 25921737

Investigations of TARP gamma-8 Null Mice

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