GLOBUS PALLIDUS NEUROCHEMICAL RESPONSES TO THE PARTIAL NMDA AGONIST GLYX-13 AMONG HD51CAG HUNTINGTON’S DISEASE MODEL RATS Antigone Wolfram-Aduan2, Aaron Antcliff2, Zach Semaan2, Jennifer Knirk2, John Wickwire2, Abby Knoll2, Becky Koepke2, Justin Gilardone2, Amanda Hartman2, Michael Mandziara2, Haley Tomlinson2, Dana Delaney2, Megan Altemus2 Andrew Crane2, Jessica Matyas3, Steven Lowrance, M.S.2, Naveen Jayaprakash, M.S.2, Joseph Moskal, Ph.D.6, Julien Rossignol, Ph.D.2,4,5, Gary Dunbar, Ph.D.1,2,3,5, and Michael Sandström, Ph.D.1,2,3 SandstromSandstrom Central Michigan University Dept. of Psychology1, Neuroscience2, Experimental Psychology3 Programs, and College of Medicine4, Mount Pleasant MI 48859 USA CentralCentral MichiganMichigan UniversityUniversity 5Field Neurosciences Institute, Saginaw MI 48604 USA LabLab NeuroscienceNeuroscience ProgramProgram 6Falk Center for Molecular Therapeutics, Dept. of Biomedical Engineering, Northwestern University, Evanston IL 60201 USA

HPLC RESULTS INTRODUCTION D. Median Baseline Huntington’s disease (HD) is a hereditary neurodegenerative disorder characterized by A. Glutamate B. Glutamine D. cognitive and behavioral dysfunctions (motor impairments) and striatal neurodegeneration 175 GLYX-13 150 GLYX-13 2500 7 in later stages. HD is caused by an expansion of repeated CAG codons for glutamine on Wild Type DialysateContent 150 125 6 the huntingtin gene, expanding the Huntingtin protein. It is hypothesized that abnormal Homozygote 2000 glutamate transmission may underlie the neuropathological changes seen in HD. In excess, 125 100 5 GABA(ng) glutamate can produce excitotoxicity in the brain, leading to neuronal cell death, and a 100 Wild Type 9 mo 75 1500 4 defect in glutamate reuptake by astrocytes may contribute to the phenotype exhibited. The 75 Homozygote 9 mo cell death observed in late-stage HD chiefly involves medium-sized spiny neurons (MSNs), 50 1000 3 90% of the striatum, and cortical neurons are also vulnerable. However, evidence indicates 50 25 neuronal dysfunction begins prior to the onset of symptoms or neurodegeneration. 25 2 GLU GLU GLN (ng) 500 Dysfunctions of basal ganglia circuitry become apparent before motoric deficit 0 0 Content Dialysate 1

expression and are quite prominent by the time movement disorders arise. The key % Glutamine Change % Glutamate Change -25 -25 structures affected by HD are represented by a reciprocal neuronal loop between the basal 0 0 ganglia and cortex. The striatum projects directly to the globus pallidus via GABAergic -50 -50 BSLN 5 6 7 8 9* 10* BSLN 5 6 7 8 9* 10* neurons. The rat globus pallidus similarly projects to the substantia nigra reticulata/ GLU GLN Collection # Collection # GABA entopeduncular nucleus via inhibitory GABAergic input (Fig 1). B. GABA This research aims to explore neurochemical system disruptions underlying HD along C. 75 GLYX-13 CONCLUSIONS with potential neurochemical and behavioral effects of the partial NMDA agonist GLYX-13, GABA Wild Type Aged (n=7) using a transgenic rat model of HD at two ages: Adult (nine months) and Aged (13-24 50 Homozygote Aged (n=9) months). GLYX-13 is a glycine-site specific NMDA receptor modulator that has previously FIGURE 1. (Above) Rat basal ganglia circuitry. GLU, GLN, and GABA concentrations were 75 GLYX-13 Wild Type 9 mo (n=8) Our original hypothesis was based on findings that in HD there is an abnormal basal been found to have a neuroprotective effect in vitro (Zhang, 2008). Concentrations of amino measured from the globus pallidus (GP) pre- and post- GLYX-13 administration. GLYX-13 was 25 Homozygote 9 mo (n=2) glutamatergic stimulation of striatal neurons. Theoretically, if this were the case, GABA acids glutamate (GLU), glutamine (GLN), and gamma-aminobutryic acid (GABA) were expected to have an affect on NMDA receptors located in the striatum. Subsequent effects of levels in the globus pallidus (GP, an efferent target of GABAergic striatal neurons, see 50 obtained via in vivo microdialysis from the globus pallidus of the aforementioned age groups GLYX-13 on the corticostriatal pathway are observed. Sources of GP GLU are corticopallidal and 0 Fig.1) would be initially heightened among HD subjects. Of course it is important to of wild-type and homozygote transgenic rats. Collections representing percent changes from the subthalamic nucleus. Sources of GP GABA are both striatopallidal and local pallidal. Figure 3 A, B, & C. recognize that an internal normalization is to some extent built in to GP GABA in that any from basal levels following systemic GLYX-13 administration were obtained and analyzed Microdialysis results pre– and post– GLYX-13 injection. GLN relates to astocytic reuptake and the GLU/GABA recycling physiology. 25 % GABA Change -25 increases in striatopallidal GABA may be offset by corresponding decreases in GABA while also monitoring locomotion changes. Shown as percent change from median baseline. release from local GP neurons. The partial NMDA agonist GLYX-13 acts at the glycine site -50 BSLN=last baseline. of NMDA receptors (NMDARs). Regarding pallidal GLU, there are two sources: one 0 BLSN 5 6 7 8 9* 10* directly from the cortex and the other from the subthalamic nucleus (STN). Vulnerable A. B. Collection # METHODS *Due to change in collection # taken across sessions, NMDA receptors are expressed within the striatum, cortex, GP, and STN, although % GABA Change -25 the last two data points were obtained from fewer aged expression on the GP is lower than the striatum and cortex. We initially expected GLYX-13 animals (7HD and 3WT). One collection was lost from related responses to appear in the form of genotype-specific GP GABA release. While an

-50 one HD animal in GABA (C). initial surge of GABA does seem to differ between WT and homozygote animals from the Male homozygous (HD) and wild-type (WT) littermates were bred in-house from BLSN 5 6 7 8 9* 10* younger adult group. This same younger HD group also seemed to exhibit higher GABA in heterozygote HD51CAG (currently known as TgHD) stock animals obtained from Hanover, Collection # Figure 3 D. (Far right) depicts initial median baseline response to GLYX-13, while the other subjects seemed to hug the baseline. However, the Germany. Genotypes for each animal were determined from a tail biopsy and PCR analysis. measurements of each compound with GABA showing young homozygote group was our lowest n due to other difficulties thus preventing solid All animals were treated according to guidelines of Central Michigan University’s against the right axis. conclusions. Regarding GLU measurements, we are of course intrigued by the fact that Institutional Animal Care and Use Committee (IACUC). the young WT group showed a significantly different distribution of GLU than the aged WT Microdialysis probes that measured 3.0mm in length were prepared in the laboratory as group (Mann Whitney U=53, p=0.004) but values were not statistically different for GLN described by Steffes and Sandstrom (2008). Only probes that yielded a percent recovery (Mann Whitney, U=22, p=0.487) or GABA (Mann Whitney, U=14, p=0.105). There was no greater than 5% based on a prior-performed vitro test were used for experimentation. statistically significant difference between the aged WT and aged homozygote animals on Three days prior to microdialysis, all rats underwent intracerebral guide cannula installation measures of GLU, GLN, or GABA (Mann Whitney, U=28, p=0.771;U= 30, p=0.874; U=15, surgery using sterile technique. Rats were anesthetized by isoflurane inhalation. Stereotaxic OPEN FIELD p=0.081 respectively). surgery was preceded by a subcutaneous injection of bupivacaine (0.125%), a longer- lasting local analgesic, and following a midline incision the cannula were inserted at FIGURE 2. (Above) A. Guide cannula placement surgical field. Isoflurane was provided via the Open Field Total Distance Traveled nose cone. Placement was very lateral, on a skull ridge, so only minimal muscle tissue needed A. Striatal neurons are activated by corticostriatal GLU bursts that occur far more robustly coordinates (from Bregma): a depth of 4.60mm from skull surface, -0.90mm posterior, and 150 Saline GLYX-13 during movement, which would have enhanced genotype distinctions in GP transmitter +4.50mm lateral, at a 20° angle lateral to the midline, providing a direct route to the globus to be displaced to expose the skull. B. Intracerebral guide cannula placement example in the globus pallidus: relative to Bregma: -0.90mm ANT, +4.50mm LAT, -4.60mm below skull surface. release if they had been present. Figure 4 indicates if anything movement generally pallidus and avoiding the ventricles (Fig 2A). On these rats a skull ridge for facial muscle 125 B. Open Field Basic Movements tapered with time without any apparent motor effects of the drug. It does seem clear that attachment resides close to the cannula hole so tissue was displaced conservatively so as C. Dialysis was performed in an open field environment. A syringe pump delivered aCSF at 2µl/ min through the microdialysis probe. Samples were collected in a vial attached to a swivel head something more than merely striatopallidal-generated release is influencing GABA levels in to maintain functionality while exposing the skull sufficiently. Cannula were held in place by 100 125 Saline the GP. At 9 months, when striatopallidal GABA might be expected to be higher in basal a head stage secured with small skull screws and dental cement, and contained a dummy- hanging above the free-moving rat in the open field testing environment below. Collections were analyzed via HPLC for concentrations of glutamate (GLU), glutamine (GLN), and GLYX-13 levels due to greater drive (see Fig. 3D), GLYX-13 seemed to induce a more rapid enclosure to keep it debris-free until the time of experimentation. Post surgery, following 75 100 increase in GP GABA release. This is of interest because if only the striatopallidal was fluid replacement, each animal was closely monitored until normal motor function returned. gamma-aminobutyric acid (GABA), using pre-column OPA/βME derivitization across a C18 column with a phosphate buffer mobile phase. influenced by GLYX-13 a decrease in GABA release would be expected. Post-surgery rats monitored daily for signs of pain/distress (none found) and habituated to 50 their head stages in single housing for a minimum of 48 hours before testing. cm/min Mean 75 It is difficult to interpret our results in terms of the direct influence of GLYX-13 with the Freely-moving microdialysis was performed in a novel testing area. Following careful 25 lower-than-expected subject numbers and multiple transmitter sources. To tease out the insertion of the dialysis probe, the rat was placed into a movement-monitoring open field 50 various sources of transmitters responding to GLYX-13 it may be necessary for future environment (Kinder) for the duration of the session (Fig 2C). There was a 1 hour discard C. 0 experiments to deliver this drug via direct infusion into the striatum, although such a 1 2 3 4 5 6 7 8 9* 10* period in which artificial cerebrospinal fluid (aCSF) was delivered at a rate of 2.0 µl/min via 25 delivery would not be practical for therapeutic efforts. The one aspect that seemed to

a syringe pump, to avoid collecting trauma-related neurochemical release resulting from Collection # movement/min Mean correspond to our original expectations was that basal GP GABA seemed be lower, probe insertion. Rats were closely monitored for any abnormal behavior indicative of brain C. approaching statistical significance, among HD than WT in the aged group and higher trauma. After the discard period, an intraperitoneal (IP) injection of isotonic sodium chloride Open Field Fine Movements 0 among HD than WT in the younger group. At present, it is difficult to determine if the 1 2 3 4 5 6 7 8 9* 10* solution was administered as an injection control. After 20 minutes, four 10 minute dialysis 75 Saline GLYX-13 GLYX-13 would normalize GABA release in the HD animals as hypothesized. collections were taken to establish baseline neurochemical levels (20µl each). Each Collection # collection was capped and frozen on dry ice at 20µl. The rat then received a 5 mg/kg GLYX-13 IP injection. After 20 minutes, four to six 10 minute collections were taken. At REFERENCES session end, all collections were frozen at -80°C until later analyzed for concentrations of 50 glutamate, glutamine, and GABA, using high performance liquid chromatography (HPLC). Steffes, S.K. and Sandstrom, M.I. (2008) Constructing Inexpensive, Flexible, and Versatile Microdialysis Following dialysis, rats were anesthetized with pentobarbital and transcardially perfused Figure 4. Results of open field movement analysis Probes in an Undergraduate Microdialysis Research Lab, The Journal of Undergraduate Neuroscience with a 4% paraformaldehyde solution. Brains were harvested and fixed with 10% pre– and post– GLYX-13 administration. Each data Education (JUNE), 7(1):A33-A47. point is an average of ten minute intervals. paraformaldehyde and sunk in a cryoprotective 30% sucrose solution. Tissue was sectioned 25 Zhang XL, Sullivan JA, Moskal JR, & Stanton PK. (2008). A NMDA receptor glycine site partial agonist, GLYX- into 40µm coronal slices via cryostat. Sections were mounted on slides and subsequently Efforts were made to correlate the movement intervals with the collections measured. 13, simultaneously enhances LTP and reduces LTD at Schaffer collateral-CA1 synapses in hippocampus. stained with Cresyl violet and neutral red, for verification of probe placement in the globus Neuropharmacology. 55(7), 1238-50. pallidus. Data from rats with placement outside of globus pallidus were not analyzed. movement/min Mean A. Total distance traveled. 0 B. Basic movements. ACKNOWLEDGEMENTS 1 2 3 4 5 6 7 8 9* 10* C. Fine movements. Collection # Special thanks to Dr. Ming Lu, Travis Hulse, and Andrew Crane who helped with our genotyping efforts. Also, thank you to Dr. Joseph Moskal from the Falk Center for Molecular Therapeutics at Northwestern University for his generous gift of GLYX-13. This research was supported by the College of Humanities & Social & Behavioral Sciences.