Materials. B. Tsao-Nivaggioli (Avicena Group, Palo Alto, California, USA) provided the cGMP-grade cyclocreatine used for this study. All other chemicals were reagent grade.

Generation of Slc6a8–/y mice. A Cre-lox system (51) was used to generate a conditional Slc6a8 knockout in the brain. We first generated Slc6a8 floxed mice. Homologous recombination in C57BL/6N cells was carried out by transfecting a targeting vector containing a loxP site within intron 1 and a positive selection cassette containing the neomycin phosphotransferase gene (neo) flanked by Frt sites and the second loxP site within intron 4 (Figure 2A). Neomycin-resistant ES cell clones were screened for homologous recombination by PCR, followed by Southern blot analysis of ES cell genomic DNA using probes located outside the 5′ and 3′ homology arms. Correctly targeted ES cell clones were injected into MF-1 blastocysts. Male chimeras were crossed with C57BL/6J females to produce N1F0 offspring, which was confirmed by Southern blot and PCR analysis. The conditional allele was generated by breeding the heterozygous mice to a germline Flp deleter strain (Jackson Laboratory) to delete the neo cassette, leaving a single loxP and Frt sites in intron 4. The resulting mice were further bred with C57BL/6J females to be free of flpase. We then crossed Slc6a8fl/fl female mice with male mice (C57BL/6J) expressing a Cre recombinase driven by the CamkIIα promoter in the brain (52) to generate Slc6a8–/y mice and Slc6a8fl/y littermate controls. We chose this Cre construct because it drives expression throughout the mouse brain, but shows particularly dramatic expression in the hippocampus and cortex, areas controlling cognitive function, which is an important aspect of the human phenotype. It is also known that Slc6a8 is highly expressed in that area in mice (53). We did not observe problems with the floxed mice, so we used Slc6a8fl/y animals as control littermates. This minimizes the influence of genetic background on the observed phenotype. A total of 29 Slc6a8–/y mice and 28 male Slc6a8fl/y controls were used in this study. At 6 months of age, baseline assessment was performed for all mice. To assess the efficacy of cyclocreatine treatment, 17 of the 29 Slc6a8–/y mice and 15 of the 28 Slc6a8fl/y controls were further used for 9 weeks of treatment.

Experimental design. Mice were maintained on an ad libitum standard pelleted diet (Teklad irradiated standard diets; Harlan animal research laboratory) and on ad libitum water intake with 12-hour dark/light cycles in 70°F ± 2°F through the study. At the conclusion of the study, mice were analyzed for Slc6a8 mRNA levels. Other tests — such as baseline values for working and reference memory, novel object recognition, Morris water maze, body composition, home cage locomotor activity, rotarod, hanging wire grip, and beam walk tests — were done before and after treatment. At 12 months of age, Slc6a8–/y and Slc6a8fl/y mice were randomly assigned to groups and started on 1 of 3 treatments for 9 weeks: (a) cyclocreatine (n = 7 [Slc6a8–/y]; 5 [Slc6a8fl/y]), (b) creatine (n = 5 per group), and (c) maltodextrin as placebo (n = 5 per group). Each treatment compound was supplemented in the drinking water, and the concentration was adjusted to deliver 0.28 mg/g body weight/d. This is a standard creatine dose for human subjects (20 g/70 kg body weight/d), intended to induce maximum creatine or phosphocreatine concentrations inside the body. Body weight and water and food intake volumes were monitored every other day throughout the treatment period. After 9 weeks of treatment, each parameter was evaluated and compared with baseline values or between groups.

Genotyping. Genotyping of the conditional allele was performed by PCR with primers that flank the position of the loxP-Frt insertion with genomic DNA from mouse tail and brain templates (VS747 in intron 4, 5′-AGGTCCAGACAGTAACTACCCTTC-3′; VS584 in intron 4, 5′-TGGGTTTGCAGCTTGGTGTTATTGC-3′; Pnew in intron 1, 5′-TCCTACACCAATACCCCCATAAGC-3′) under the following conditions: 40 cycles of a reaction consisting of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 58°C, and 1 minute of elongation at 72°C, followed by a final extension for 10 minutes at 72°C. The expected product sizes were 422 bp for the WT allele, 548 bp for the floxed allele, and 346 bp for the knockout allele. PCR for Cre recombinase expression was performed with genomic DNA from mouse tail templates (Cam k1, 5′-GGTTCTCCGTTTGCACTCAGGA-3′; Cam k2, 5′-CCTGTTGTTCAGCTTGCACCAG-3′; Cam k5, 5′-CTGCATGCACGGGACAGCTCT-3′) under the following conditions: 30 cycles of a reaction consisting of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 67°C, and 1 minute of elongation at 72°C, followed by a final extension for 10 minutes at 72°C. The expected product sizes were 350 bp for Cre recombinase and 300 bp for the internal control. PCR products were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining; images were taken with a Universal Hood II (Bio-Rad Laboratories).

Semiquantified RT-PCR. Total RNA was extracted from brain tissues of Slc6a8–/y and Slc6a8fl/y mice at 6 months of age using TRIzol reagent (Invitrogen) according to the manufacturer’s instruction and was quantified by determination of absorbance at A260. Then, total RNA was treated with RNase-free DNase (Ambion). RT priming with oligo (dT) primers (Invitrogen) was performed to generate cDNAs from 1 μg total RNA using Superscript II (Invitrogen) following the manufacturer’s instructions. Equal amounts of cDNA from all samples were subjected to PCR. PCR primer pairs were as follows: Slc6a8 forward, 5′-CCATGAAGACTGTGCCAATG-3′; Slc6a8 reverse, 5′-CCCCTTCCACACACAGAAGT-3′; Actb forward, 5′-GTGGGCCGCCCTAGGCACCAG-3′; Actb reverse, 5′-CTCTTTGATGTCACGCACGATTTC-3′. Reactions were performed in 25 μl total volume with 25 pM of each primer. Amplification conditions were as follows: 95°C for 10 minutes, followed by 28 or 25 cycles (for Slc6a8 or Actb, respectively) of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The expected product size was 202 bp. Amplified fragments were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. The intensity of each band was measured by Scion Image (Scion Corp.), and the intensity of Slc6a8 was expressed relative to that of Actb.

Cyclocreatine and creatine assay. Animals were deeply anesthetized with 1%–2% isoflurane delivered with oxygen. Whole blood was harvested with a 1-ml syringe and 27-gauge, 0.625-inch needle from the heart and centrifuged at 3,000 g for 20 minutes; serum was subsequently placed in Eppendorf tubes. Brain, liver, heart, kidney, lung, bladder, and soleus and gastrocnemius muscles were rapidly removed and frozen in liquid nitrogen. Urine and hair samples were also collected. The samples were each dropped into 250 μl boiling water and boiled for 20 minutes to remove protein and lyse cells. The total creatine content of the protein-free extract was assayed using the fluorometric method of Conn (54). Total cyclocreatine content of brain and hair was assayed using the method of Griffith (55). All chemicals were obtained from Sigma-Aldrich unless otherwise stated.

In vivo MRS. All data were collected on a Bruker BioSpec 7T system (Bruker BioSpec 70/30) equipped with 400 mT/m actively shielded gradients. In total, 6 mice were used at baseline assessment, and 18 mice after 9 weeks of treatment. Mice were anesthetized by 1%–2% isoflurane delivered in air, and the respiration rate was maintained at 60–100 breaths per minute. Core temperature was maintained at 37°C by warm air circulated through the magnet bore. All animals’ brains were scanned with a custom-built radio frequency coil. T2-weighted axial and sagittal localizer images were acquired with a fast-spin echo sequence. A proton double-spin echo sequence was used to shim on a voxel approximately 6 mm × 4 mm × 6 mm, covering most of the brain. The water proton line width averaged about 17 Hz. After shimming, 31P data were acquired with an ISIS sequence from the same voxel using a 4-second repetition time, 2,048 complex points, 8,000 Hz spectral width, and 4 repetitions of 480 averages each. Total phosphorus acquisition time was 2 hours. Individual metabolites were identified by chemical shift. Ratios of metabolites to total phosphorus were calculated by comparing the total peak amplitude of all phosphorylated metabolites with the individual peak heights. Metabolite peak heights were averaged and reported.

Morris water maze. Before and after 9 weeks of treatment, a total of 32 mice were subjected to Morris water maze testing, a hippocampal-dependent task of spatial learning and memory. The maze consisted of a circular fiberglass pool (122 cm diameter, 75 cm high; Rowland Fiberglass Inc.) filled with water (18 ± 1°C, 43 cm deep). A clear glass platform (10.5 cm × 10.5 cm square) was submerged 1 cm below the water surface. The pool was situated in a room containing extramaze cues (42-cm × 76-cm posters printed with contrasting patterns and shapes) that provide specific visual reference points for locating the submerged platform. A video camera mounted to the ceiling, directly above the center of the pool, was used for recording the probe trial. The recording was digitized by a computer and analyzed using CleverSystem Topscan software (Cleversys) for path analysis (distance traveled and percentage of time in the target platform area). Each mouse received 3 trials in the water maze on each of 5 days. The submerged platform remained in one quadrant of the pool throughout all trials, and latency to find the platform was recorded. If the mouse failed to reach the platform within 60 seconds, the trial was terminated, and the mouse was guided onto the platform for 5 seconds. On the sixth day, each mouse received a final 60-second probe trial in which the platform was removed from the pool.

Radial maze. An 8-arm radial maze was used to test spatial working and reference memory at baseline with a total of 32 mice (n = 17 [Slc6a8–/y]; 15 [Slc6a8fl/y]). The maze consisted of an octagonal central platform (51.5 cm in diameter) with 8 radial arms (61 cm long, 12 cm wide, 10 cm high) extending outward (Lafayette Instrument Co.). The maze was elevated 70.5 cm above the floor in a room containing many extramaze visual cues. Mice must use these visual stimuli to navigate the maze, which lacked intramaze, nonspatial cues for navigation. First, a food deprivation schedule was carried out to reduce animals’ weight to 85% of baseline. During the entire training and test periods, water was available ad libitum. Next, an acclimation trial was conducted. On days 1 and 2, food pellets were placed near the end of all arms. From day 3, food pellets were located only in arms 3, 5, 7, and 8; the remaining 4 arms were empty. Mice received 1 or 2 training trials per day for 22 trials, and then were tested using reversed baited arms (food pellets in arms 1, 2, 4, and 6 only) for 28 trials, for a total of 50 trials. At the beginning of each trial, mice were placed in the central platform, then allowed to explore the maze. Trials were completed when the 4 food pellets were eaten or 15 minutes had elapsed. Arm entries for each mouse were recorded by an investigator. The total number of entries into unbaited arms was tabulated as an index of reference memory error, and the number of reentries into previously baited arms was used as an index of working memory error.

Novel object recognition. Before and after 9 weeks of treatment, the novel object recognition task was conducted in a Plexiglas open-field apparatus (60 cm × 50 cm × 21 cm high) to test short-term recognition. Mice were individually habituated to the open-field apparatus with no objects in the cage for 5 minutes at a time on 3 consecutive days. On the fourth day, 2 identical objects (50-ml falcon tubes, 11 cm high, 3.5 cm diameter, covered with yellow tape) were placed symmetrically 12 cm away from the wall. The mouse was placed near the wall at equal distance from both objects and observed for 5 minutes. A second 5-minute trial was done 3 hours later, in which one of the familiar objects was replaced by a novel one (tube 4 cm in diameter, covered with red tape). We defined exploration zones around the objects (10 cm diameter), and time spent inside the zones was used as an index of object exploration. The discrimination index was calculated by dividing the difference between exploration times of the new object and the familiar object by the total time spent in object zones.

Body composition analysis. Total lean tissue, fat tissue, and water of the 32 total Slc6a8–/y and Slc6a8fl/y mice was determined by MRI (56) before and after 9 weeks of treatment. After system calibration, an unanesthetized mouse was weighed, placed in the restraint tube, and inserted into the Echo MRI whole-body composition analyzer (EchoMedical Systems) for 45 seconds. Fat mass and lean body mass were determined.

Home cage locomotor activity. We measured the locomotor activity expressed by each animal in its home cage before and after 9 weeks of treatment. This allowed us to capture general locomotor activity, unrelated to the animals’ activity in a novel, anxiogenic environment. The cage rack frame (Lafayette Instrument Co.) equipped with infrared photobeams was placed around each animal’s standard shoebox home cage. Infrared photobeam interruption sensors (7X and 15Y) mounted in the frame detected movement, which was recorded and analyzed using HMM100 Motor Monitor software (Lafayette Instrument Co.). Vertical and horizontal activity within the home cage was recorded for 48 hours, and events were collapsed into 60-minute bins. In order to discern short- and long-term activity–related circadian rhythms, results were expressed as the average number of beam interruptions per group per hour.

Rotarod. The rotarod test of motor coordination and motor learning was performed before and after 9 weeks of treatment. On day 1, mice were placed on a stationary rod (3.2 cm diameter) of the apparatus (Type ENV-576M; Med associates) for 30 seconds. Daily training trials were then administered for the next 7 consecutive days: mice were placed on the rod at increasing speeds, from 16 to 32 rpm, for up to 2 minutes. On day 9, mice were placed on the stationary rod at maximum speed (32 rpm). The duration in seconds until the animal fell from the rod was recorded as a measure of motor coordination; cutoff time was 120 seconds.

Hanging wire grip. Before and after 9 weeks of treatment, the hanging wire grip test was performed by placing a mouse on a wire net (2.5-cm × 2.5-cm grid, 30 cm2 wire), then turning the net upside-down at approximately 50 cm above the cage floor to prevent the animal from easily climbing down. The elapsed time until the animal fell was recorded. Tests consisted of 3 trials with 30-second intervals; cutoff time was 540 seconds.

Beam walk. The beam walk is a test of complex motor coordination. Before and after 9 weeks of treatment, animals were trained to ambulate across the beam (1 m long, 9 mm diameter) to a 14-cm2 platform for 7 consecutive days. On day 8, mice were placed on the beam 50 cm from the platform, and latency to reach the platform was measured. Missed steps off of the beam were also recorded.

Statistics. Data are expressed as mean ± SEM. Statistical significance of mean differences for each parameter was determined by 2-tailed Student’s t test; by ANOVA followed by post-hoc Tukey test (treatment group as between-subject factor); or by repeated-measures ANOVA followed by post-hoc Bonferroni test for multiple comparisons (treatment group and treatment timing as between- and within-subject factors). A P value less than 0.05 was considered significant.

Study approval. The experimental procedures performed herein were in compliance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Cincinnati Institutional Animal Care and Use Committee.