Twenty four (24) three (3) month old male Sprague-Dawley rats weighing approximately 250 - 350 g, were obtained following institutional ethical approval of

the protocol (UHWI/UWI/FMS Ethics Committee, AN02/2007/2008). Animals were housed in pairs, in a normal 12:12 hour light: dark cycle and fed food and water ad libitum.

Phase 1: Pre-conditioning, Selection and Group Assignments

Animals selected for inclusion in the experiment were allowed an initial twenty (20) minute exploration of the CPP apparatus, and scores of chamber entries and time spent in each chamber were recorded manually and correlated with the video recorded data. An entry was recorded when all four paws of the rat were placed in a chamber. Animals displaying at least 80% preference for the black CPP chamber during pre-conditioning, assessed as greater number of entries and time spent in the black chamber, were selected and randomly assigned to one of two treatment groups, AMP or SAL, each containing twelve (12) rats [9] [10] .

Phase 2: Conditioning

During the conditioning phase, rats assigned to the amphetamine treatment group (Group AMP) received intra-peritoneal injections (i.p.) of 5 mg/kg/ml of amphetamine sulfate (Sigma, USA) dissolved in physiological saline (0.9%). This dose has been shown to produce place preference in a number of studies [9] [10] . Rats in Group SAL were given only vehicle (1 ml 0.9% saline, i.p.).

Conditioning consisted of 8 days of daily, group-assigned i.p. injections, followed immediately by individual confinement to the white, “non-preferred” or black “preferred” chamber of the CPP box for thirty (30) minutes. On days 1, 3, 5 and 7, Group AMP received amphetamine sulfate (Sigma, USA) while non- drug exposed, vehicle treated rats received saline (Group SAL). Each rat in Groups AMP and SAL was then confined individually to the white “non-pre- ferred” chamber. On the four alternate days (i.e., Days 2, 4, 6, 8), all animals received an injection of saline (1 ml, i.p.) and were confined individually for 30 minutes in the black “preferred” chamber. Thus, Group AMP rats were conditioned to pair amphetamine with the white chamber and saline with the black chamber [9] [10] . On Day 9, rats were placed individually in the grey chamber, sliding doors then removed, and allowed to explore the CPP box. The number of entries and time spent in the white chamber were recorded over 20 minutes and the percentage entries and time spent calculated from the total. Data obtained revealed that all animals assigned to Group AMP exhibited a significant and positive CPP drug-seeking behavior for the white chamber (Figure 2).

Phase 3: Amphetamine Addiction and Drug Withdrawal

The addiction phase then ensued, with daily doses of amphetamine sulfate (5, 5, 6, 6, 7, 7 mg/kg/ml, i.p.) that increased in concentration over a 6-day period. Group SAL rats received only vehicle (i.p.) over the same period and represented the non-drug exposed controls for the addiction phase of the study.

Rats were subsequently taken through a period of three days without drug, vehicle or CPP exposure, and were fed standard rat chow with water ad libitum. On day 4, following drug withdrawal, each animal was individually placed in the grey start chamber and allowed a 20-minute period of exploration of the CPP box after the sliding doors were removed (Figure 2). The number of entries made into, and the time spent in each chamber, were recorded manually for all rats and the data compared with scores obtained from video recordings of exploration of the CPP box.

Phase 4: Oral Nutritional Supplementation

The orally administered, nutritional supplementation regimen used in this study consisted of three separate pharmaceutical grade components: 1) a mega vitamin with higher levels of calcium and magnesium; 2) omega 3 and 6 fish oils; and 3) a supplement capsule consisting of basic amino acids with higher levels of tyrosine (Manufacturer: All The Whey, Inc. Paoli PA, USA).

Nutritionally supplemented animals were proportionally fed a combination of the named component, at 0.123 g/day, 0.123 g/day and 0.0408 g/day, respectively, over a period of 8 weeks. These doses were calculated based on a 350 g rat, after proportions previously used in other studies for administration to a 75 kg man were scaled down [11] .

Preparation of the nutritional supplement for oral treatment involved crushing and weighing the megavitamins; whereas, the fish oil tablets were opened and drained into a calibrated beaker and the relevant volumes removed. Supplement capsules were also opened and the powder weighed for correct dosing. All animals were individually caged for the duration of the supplementation period.

The initial Groups AMP and SAL were further equally divided, assigned a numerical code and randomly assigned to either the supplemented diet or standard rat chow group, as indicated below:

・ Group AMP-S: Amphetamine treated rats, fed with the nutritional supplement after the addiction phase

・ Group AMP-N: Amphetamine treated rats, fed with standard rat chow throughout the experiment

・ Group SAL-S: Saline treated rats, fed with the nutritional supplement after addiction phase

・ Group SAL-N: Saline treated rats, fed with standard rat chow throughout the experiment

Rats in Groups AMP-S and SAL-S were individually housed in cages and received the nutritionally supplemented chow as first feed daily for the 8-week treatment period. Non-supplemented, standard rat chow was administered for the rest of the day, only after chow with supplement was fully consumed. The non-supplemented groups (i.e., Groups AMP-N and SAL-N) were administered food and water ad libitum throughout the day. Following this 8-week period, animals were randomly and individually placed in the CPP box and their performance in a final 20-minute exploration of the paradigm was assessed from video-recordings. Figure 2 is a graphical summary flow chart which explains the phases of the experiment. Data on time spent and entries in the drug-paired white chamber were collated for the different groups after aligning scores obtained to the numerical code assigned to the rats.

The results of this study show for the first time, that complete reversal of amphetamine drug-seeking behavior in stimulant addicted animals can be demonstrated solely after application of a nutritional intervention.

The widely accepted criteria for acquisition of addiction using the conditioned place preference paradigm were evident in our baseline results. At baseline, all of the animals spent significantly less time in the white chamber, than they did in the black, naturally preferred chamber. Amphetamine administration paired with the white, non-preferred CPP chamber significantly increased the percent entries into, and percent time spent in the otherwise aversive white chamber during withdrawal (p < 0.05). Eight weeks of rat chow re-constituted to incorporate the formulated nutritional supplement resulted in the behavioral transition to significantly fewer entries into, and time spent in the drug-paired white chamber (p < 0.05). Amphetamine addicted, nutritionally supplemented animals performed similarly to the saline-treated, drug-naïve animals in white chamber time and entries. The saline treated controls maintained “baseline” white chamber aversion throughout the experiment.

The results of this study have the potential to profoundly revolutionize therapeutic management of drug addiction. The multi-nutrient supplement regimen, which previously anecdotally diminished withdrawal symptoms in human alcoholics and heroin addicts, has been shown in this study to eliminate drug-seeking behavior by amphetamine addicted rodents in the CPP paradigm, a valid, reliable and frequently used animal model of drug addiction.

These results complement those of an extensive, 50-year review of the literature related to nutrition as therapy in drug abuse recovery done previously [12] . They corroborate evidence that vitamin, mineral and amino acid therapy can reduce withdrawal symptoms, increase treatment retention and quality of life and contribute to higher abstinence rates in stimulant addicted subjects [12] . The rehabilitative role of amino acid supplementation with tyrosine and tryptophan, aimed at restoration of catecholamine balance in creating a foundation for complete recovery, has also been previously discussed [13] .

A cutting-edge review of the neurobiology of addiction recognizes three stages of the disease, and affirms that each stage influences and is fed by disordered circuitry in different brain regions. In the binge/intoxication stage, previously neutral stimuli become associated with drug availability, promoting habit formation that fosters excessive drug seeking via increases in dopamine and glutamate neurotransmission. Those authors associate this first stage with diminished reward function via dopamine and opioid peptide deficits (2). The animals in this study were likely confined to that initial stage of the continuum of drug addiction, as we employed a relatively short time line. The human chronic drug addict, however, would also likely have derangements in executive function via the dysregulation of glutamatergic, GABAergic, and dopaminergic neuronal networks in the prefrontal cortex (2).

Bearing in mind the gut and neuronal derangements associated with stimulant addiction, it is likely that the balance of fatty acids, minerals and vitamins in this supplement may potentially replenish catecholamine neurotransmitters [14] . It may also contribute to the restoration of gut health and neuronal cellular function in order to improve CNS regulatory mechanisms and restore normal decisions to behave as if there had been no drug exposure.

A nutritional supplement that contained fish oil (5.6 grams a day); a multiple vitamin; a brain supplement with ginkgo and vinpocetine; acetylcholine (acetyl- l-carnitine and huperzine A), and antioxidant (alpha-lipoic acid and n-acetyl- cysteine) was found to play a significant role in substance abuse rehabilitation and brain injury reversal [15] . These authors reported on improvements in cognitive functions of attention, memory, reasoning and speed of information processing. Brain SPECT scans provided information on brain perfusion showing improvements in cerebral blood flow also. Our results complement those findings, but we have demonstrated more effectively the ability of our supplement to significantly decrease drug seeking behaviour.

Our work demonstrated unequivocally that rats became amphetamine addicts, as we employed the international gold standard for addiction studies. Our method of including the nutritional supplement in the first feed of each day enabled the results to be more uniform, regardless of how much each animal consumed. The limitations are that we can only interpret cue-directed animal behavior in terms of positive CPP or drug-seeking behavior; and negative CPP or drug aversion. The physiological derangements and other qualitative and emotional aspects of human addiction may not have been modeled in our study.

This study opens the door to continued behavioral analysis of nutrient therapy for drug addiction. Much scope exists for inclusion of this supplement in stimulant drug abuse rehabilitation programs.