www.sciencetranslationalmedicine.org/cgi/content/full/1/7/7ra17/DC1

Supplementary Materials for

Restoration of Norepinephrine-Modulated Contextual Memory in a Mouse Model of Down Syndrome

A. Salehi,* M. Faizi, D. Colas, J. Valletta, J. Laguna, R. Takimoto-Kimura, A.

Kleschevnikov, S. L. Wagner, P. Aisen, M. Shamloo, W. C. Mobley

*To whom correspondence should be addressed. E-mail: asalehi@stanford.edu

Published 18 November 2009, Sci. Transl. Med. 1, 7ra17 (2009) DOI: 10.1126/scitranslmed.3000258

This PDF file includes:

Materials and Methods References Fig. S1. Photographs showing nesting in 2N and Ts65Dn mice. Fig. S2. VMAT2 staining in the inner molecular layer of the dentate gyrus in 3- and 6-month-old mice. Fig. S3. Recorded responses from the granule cell layer after stimulation of the middle molecular layer. Fig. S4. The effects of releasing (10 µM) isoproterenol (ISO) in the middle molecular layer of dentate gyrus on changes in population spike amplitude of field EPSPs. Fig. S5. NE concentrations in 6-month-old L-DOPS–treated mice.

Supplementary Material Materials and Methods Immunocytochemistry Histological analyses were performed blind to genotype. LC neurons were identified by immunocytochemical staining for tyrosine hydroxylase (TH). TH is a rate-limiting enzyme converting L-tyrosine to dihyroxyphenylalanine (DOPA). TH is widely used as a marker for catecholaminergic neurons particularly NE-containing neurons in LC (1, 2). A polyclonal antibody (Protos Biotech Co, New York) was used to label TH-positive neurons. The brain of Ts65Dn, Ts1Cje, and 2N mice was examined at 3, 6, and 18 months of age. The cell bodies of LC were examined at each age. Mice were deeply anesthetized with sodium pentobarbital (200 mg/kg i.p., Fort Dodge Animal Health) and perfused for subsequent immunocytochemical detection of TH (1:2,000) in 70 μm free-floating coronal sections through brain stem and HC. For each group of mice, identical conditions were used. Unbiased stereological methods (Stereologer, Systems Planning & Analysis) were used to determine the number (optical fractionator method) of TH-positive neurons throughout the rostral-caudal extent of LC of each animal. The cross-sectional areas of TH-positive neurons randomly sampled throughout the rostral-caudal extent of LC was also determined using an Image Pro Plus (Bethesda, MD). To study monoaminergic terminals, a polyclonal antibody against mouse vesicular monoamine transporter (VMAT2, Phoenix Pharm. Inc) was used in the coronal sections through the HC. VMATs are members of a family of transmembrane carriers on synaptic vesicles. VMAT2 is an isoform of VMAT1, which is exclusively found in catecholaminergic neurons (3). Tissue preparation. Mice were deeply anesthetized using sodium pentobarbital (200 mg/kg i.p.) and brains were extracted immediately. The right hippocampal region dissected out on ice, weighed, and frozen shortly after. The rest of the right hemisphere was also weighed and collected. For homogenization, 1 ml of ice-cold 0.05 M phosphoric acid (Sigma) with 10 μl of 110 μg/ml of 3, 4-Dihydrxybenylamine hydrobromide (DHBA;Sigma) used as an internal standard. The tissue samples were sonicated for 3 seconds on ice. The resulting homogenates were centrifuged at 15,000g for 15 minutes at 4˚C. The resulting supernatant was transferred to a conical tube containing 1ml of 3M tris (pH: 8.6) with 90 mg acid-washed aluminum oxide (Sigma). The mixture was rotated at 4 ˚C overnight. The alumina was centrifuged for 10 seconds, and washed briefly with 6 mM tris (pH: 8.6) and 3 changes of water (HPLC grade). After final centrifugation, the resulting precipitate was mixed with 1.8 ml of 0.05M H3PO4 (Sigma, puriss >99%) for 30 minutes at 4˚C. The solution was filtered with a 0.2 μm filter and used for UV-based HPLC. HPLC method. A UV-HPLC machine (Varian) equipped with an auto sampler and 2 pumps was used for this study. The machine was connected to a Varian column (Pursuit PFP, 150 mm x 4.6 mm, 5μm, A3050150x046) optimized for catecholamine measurements in aqueous conditions. The mobile phase was consisted of 60% 0.1M citric acid (Sigma) and 40% 0.1M Na2HPO4 both in 0.3 mM HPLC-grade sodium octyl sulfate salt (Sigma) (44). The mobile phase was degassed using 0.2 μm filter and refreshed every week. The flow rate was set at 1.0 ml/min at room temperature and the detection was

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performed at 270 nm. To achieve maximum performance, the mobile phase was pumped at 0.1ml/min flow the night before the experiment. The column was washed thoroughly with 50% methanol after each experiment. To calculate the amounts of catecholamines, the height of the peak generated by DHBA, NE, and L-DOPS were measured using Variant software. The following formula (4, 5) was used to calculate the amounts of NE per g tissue: ng NE per gram tissue = [(peak height NE/peak height DHBA)*(ng DHBA added to the tissue)*(peak height per ng DHBA/ peak height per ng NE)]/ tissue weight (grams). For standardizations, we used DHBA (Sigma), NE (hydrochloric salt, Sigma) and L-DOPS. Prior to these experiments, a very significant linear correlation between the DHBA, NE and L-DOPS peak height(s) (p<0.00001) and the concentration was established. Behavioral Studies Fear conditioning. Contextual and tone-cued fear conditioning tests were performed using the Fear Conditioning Video Tracking System (Med-Associates Inc.). To reduce the stress, each mouse was handled for 5 days. After that, the mice underwent three days of Training day, Tone-Cued in novel context testing day, and Contextual testing day. In the first day, mice went through the Training session. Prior to conditioning, each mice had 3 min to explore the Test Chamber (baseline activity and then they received five tone-shock pairings. The shock (0.5 mA, 50 Hz, 2 sec) delivered after 18 sec from the end of the tone (70 dB, 2 kHz, 20 sec). Therefore, an empty trace interval interposed between the tone and the shock in each conditional stimulus-unconditional stimulus pairing. On the second day (Tone-cued testing day), mice were placed in the novel context (new olfactory and visual cues) for 3 min and subsequently were presented three tone presentations (same as the Training Day) without any shocks. On the last day of the experiment, mice were placed in the context similar to the Training Day for 5 min without any tones or shocks. Effects of L-DOPS on fear conditioning. The effect of L-DOPS on fear conditioning was investigated by treating Ts65Dn mice and their controls 5 hours before the start of each test. The treated mice underwent three days of Training day, Cued testing day and Contextual testing day. L-DOPS was diluted (20mg/ml) in 0.2M HCL containing 2mg/ml ascorbic acid. The pH was neutralized with 10M NAOH just before the injection. To counter the peripheral effects of L-DOPS, S-(-) carbidopa was used. Carbidopa was diluted in the same solution (2.5mg/ml) as L-DOPS. Two groups (10 pairs each) of 5-6-month-old male Ts65Dn and 2N mice were either injected with Carbidopa (2.5mg/ml) or a combination of 20 mg/ml L-DOPS and 2.5 mg/ml Carbidopa (50 μl/g subcutaneously) five hours before the start in all 3 days of the experiment. Effects of Xam on fear conditioning. The effect of β1-partial agonist (Xam) on fear conditioning was investigated by treating Ts65Dn mice and their controls 1 hour before the start of each test. Xamoterol hemifumarate (Tocris Bioscience) was freshly diluted in saline. Two groups of 9-month-old (2N; n=19 and Ts65Dn; n=15) mice were subcutaneously injected either with xamoterol (3mg/kg) or saline (10ml/kg) exactly 1 hour before all 3 days of fear conditioning test.

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Statistical methods for behavioral testing The data tested using one-way analysis of variance (ANOVA) between the genotypes. The significance of genotype and treatment effects was confirmed by a non-parametric Mann-Whitney U-test. Chi square test was used to compare the frequency distribution of cell profile area between 2N and Ts65Dn mice. 2) Nesting Behavior Analysis The nesting behavior was analyzed in three different periods (sham, drug and no treatment): Sham Period Each mouse was placed in individual mouse cages for 4 days. On day 5, each mouse was injected subcutaneously at 1 PM with 50 μl/g of (2.5mg/ml) carbidopa (Sigma) and retuned to their individual cages. The next day, mice were injected the same amount of carbidopa as the day before at 1PM. At 6 PM, the mice were transferred to a rat cage and one nestlet was placed in each cage. Next morning, the nesting was scored by three investigators blind to the genotype (AS, JV, and DC). This was followed by weighing the remaining nestlets. Drug period Following the sham experiment, mice were retuned to their individual mouse cages without any nestlets for 6 days. On day 7, the mice were injected subcutaneously (50μl/g) of a combination of (20mg/ml) L-DOPS and carbidopa (2.5mg/ml) at 1PM and were returned to their individual mouse cages. Next day the injection was repeated at 1PM and at 6PM the mice were transferred to clean rat cage with one nestlet. The quality and quantity of nesting was scored the morning after. No Treatment (Washout) Period The mice were kept in individual cages for two weeks to minimize the effects of L-DOPS treatment on nesting. At the end of this period, each mouse was transferred to a rat cage and was exposed to one nestlet. The quality and quantity of nesting was scored the morning after. Recording of Evoked Field Potentials Slices were transferred into the recording chamber and superfused with ACSF at a constant rate of 2.5 ml/min at a temperature of 32°C (6). Recording electrodes were made of borosilicate glass capillaries (1B150F, World Precision Instruments, Sarasota, FL) and were filled with 2M NaCl (resistance 0.3-0.5 mΩ). Mono polar stimulating electrode was maid of Pt/Ir wires with diameter 25.4 µm (PTT0110)(World Precision Instruments) and had 100-µm-long exposed tip. The stimulating electrode was inserted under visual control perpendicular to the slice surface into the middle molecular layer, and the recording electrode into the granule cell layer of the DG upper blade. The distance between the electrodes was 250-300 µm. Testing stimuli evoked field responses with population spike amplitudes 65-75% of maximum. The amplitude of the population spike was measured as follows: (i) a line was drawn at the base of the population spike connecting the first and second peaks of the field response, (ii) a second line was drawn at the peak of the population spike (i.e., at the peak of the downward deflection), and (iii)

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at the peak of the spike, a line was drawn vertically between these two lines, thus giving the amplitude of the population spike. The magnitude of the fEPSP was measured as the initial slope of the linear part of the fEPSP, which occurred 0.1-1 ms after the presynaptic volley. After stabilization of evoked responses and at least 30 min of stable recordings, an agonist of the adrenergic β1 and β2 receptors isoproterenol (1 µM) was bath applied for 10 min. Supplemental References 1) Y. Liu, M.-J. Yoo, A. Savonenko, W. Stirling, D. L. Price, D. R. Borchelt, L.

Mamounas, W. E. Lyons, M. E. BlueM. K. Lee, Amyloid pathology is associated with progressive monoaminergic neurodegeneration in a transgenic mouse model of Alzheimer's disease. J. Neurosci. 28:13805-13814 (2008)

2) J. N. O'neil, P. R. Mouton, Y. Tizabi, M. A. Ottinger, D. L. Lei, D. K. IngramK. F. Manaye. Catecholaminergic neuronal loss in locus coeruleus of aged female dtg APP/PS1 mice. Journal of chemical neuroanatomy 34:102-107 (2007)

3) E. Weihe, C. Depboylu, B. Schutz, M. K. SchaferL. E. Eiden. Three types of tyrosine hydroxylase-positive CNS neurons distinguished by dopa decarboxylase and VMAT2 co-expression. Cellular and molecular neurobiology 26:659-678 (2006).

4) T. Kumai, K. Asoh, T. Tateishi, M. Tanaka, M. Watanabe, H. Shimizu S. Kobayashi Involvement of tyrosine hydroxylase up regulation in dexamethasone-induced hypertension of rats. Life sciences 67:1993-1999 (2000).

5) W. J. Pearce, S. P. DucklesJ. Buchholz Effects of maturation on adrenergic neurotransmission in ovine cerebral arteries. The American journal of physiology 277:R931-937 (1999).

6) J. R. Moyer, Jr.T. H. Brown. Methods for whole-cell recording from visually preselected neurons of perirhinal cortex in brain slices from young and aging rats. Journal of neuroscience methods 86:35-54 (1998).

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Supplemental Figures Fig. S1. Photographs showing nesting in 2N and Ts65Dn mice. The amount of nesting material used by each mouse is indicated. 2N mice used a larger percentage of their nesting material compared with Ts65Dn mice. Fig. S2. VMAT2 staining in the inner molecular layer of the dentate gyrus in 3 and 6 month-old mice. (A) The optical density was measured in the inner ML and normalized to the optical density of the corpus callosum. At 3 months of age, there was a significant shift to higher values in the distribution of VMAT2 density values in Ts65Dn mice (3 χ2=21.8, P<0.01, 2N=5, blue line, Ts65Dn=5, red line). However, at 6 months of age, there was a significant shift to the lower values in Ts65Dn mice (χ2=29.8, P<0.005, 2N=3, Ts65Dn=3). (B) Surface imaging of VMAT2 optical density in the inner molecular layer of DG of a 6-month-old Ts65Dn mouse and its 2N control. The y axis indicates VMAT2 optical density and x axis depicts the anatomical localization starting from dentate granule cell layer toward the pyramidal cell layer of the hippocampus. Fig. S3. Recorded responses from the granule cell layer after stimulation of the middle molecular layer before (red) and 30 min after (black line) application of ISO (10 μM, 10 min) in slices of a Ts65Dn (6-month old) and its age-matched 2N control. ISO generated a much stronger response in Ts65Dn than 2N mouse. Fig. S4. The effects of releasing (10 μM) ISO in the middle molecular layer of dentate gyrus on changes in population spike amplitude of fEPSPs in 3-and 6-months-old Ts65Dn mice and their 2N controls. In both 2N and Ts65Dn mice and both age groups, ISO generated a significant increase in population spike amplitude of fEPSPs. Application of ISO enhanced the population spike amplitude for at least 30 min in 3-month-old 2N mice. However, in 6 months old mice, the increase lasted more then one hour.. Fig. S5. NE levels in 6-month-old L-DOPS-treated mice. A) No significant differences were found in NE levels in the hippocampus between 2N and Ts65Dn mice (P=0.4726, Ts65Dn; n=3, 2N; n=8). B) Similarly, no significant differences were found in the amount of L-DOPS in the hippocampus of 2N and Ts65Dn mice (P=0.5628, n=3, 2N; n=8).

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