Supplementary Figures Supplementary Table 1: Exercise regulated genes identified by custom microarray analysis.

Abbreviation Full Length Name Fold Upregulation

Standard Error

P value (t-test) Accession #

Growth Factor Signaling VGF VGF (nonacronymic) 3.60 0.36 6.11E-07 M60525 ERK2 extracellular signal-related kinase 2 1.86 0.24 0.00874547 M64300 MAPKP3 phosphatase MKP-3 1.76 0.32 0.05164833 X94185 GRB2 growth factor receptor bound protein 2 1.75 0.30 0.02940078 NM_030846

NTRK2 neurotrophic tyrosine kinase, receptor, type 2 1.75 0.39 0.06407797 NM_006180

CSF3G colony stimulating factor 3 1.74 0.27 0.00987398 NM_017104 MEK2 mitogen-activated, kinase 2 1.74 0.25 0.01779264 L14936 NEUR Neuritin 1.61 0.26 0.04207812 NM_008738 MAGED1 melanoma antigen, family D, 1 1.54 0.21 0.03320883 NM_053409

Transcription Factors & IEG NARP neuronal activity-regulated pentraxin 3.14 0.93 0.01175453 S82649 EGR1 early growth response 1 2.48 0.65 0.01038235 NM_012551 EGR2 early growth response 2 2.32 0.72 0.03859933 AB032420 EGR4 early growth response 4 2.10 0.51 0.03819028 NM_019137.1 ELK1 Ets-domain protein ELK1 2.04 0.30 0.00856434 NM_007922

Neurotransmitter/ Synaptic Signaling GRP78 glucose-regulated protein 78 2.10 0.28 0.0050497 S63521 SYNCAM synaptic cell adhesion molecule 1 2.05 0.41 0.01141024 AF539424 IL-16 interleukin 16 1.87 0.29 0.01614977 NM_010551

Gat1 sodium and chloride-dependent GABA transporter 1 1.79 0.37 0.04690756 NM_024371

GAD67 glutamic acid decarboxylase 67 1.74 0.34 0.03087761 M76177 LPHH1 R CL1BA; Latrophilin 1 GPCR 1.73 0.23 0.01251362 NM_022962

GIT1 G protein-coupled receptor kinase-interactor 1 1.60 0.26 0.04451577 NM_031814

GPCR37 G protein-coupled receptor 37-like 1 1.58 0.26 0.05251082 NM_134438 NPY neuropeptide Y 1.56 0.23 0.03851329 NM_012614 MFGE8 milk fat globule-EGF factor 8 1.55 0.24 0.04537911 NM_012811 Gabrr1 GABA receptor rho-1 subunit 1.50 0.21 0.04161858 NM_017291 SCG-2 secretogranin II 1.48 0.21 0.05073705 NM_022669 Syn-1 synapsin 1 1.47 0.21 0.04936608 X04655

Kinases, Phosphatases, & Enzymes Pkcl atypical protein kinase C lambda/iota 2.40 0.71 0.02662912 AB020615 STEP46 striatal enriched phosphatase 46 2.37 0.43 0.00290866 XM_193035 LDHA lactate dehydrogenase-A 1.90 0.38 0.03151884 NM_017025 E214K 14-kDa ubiquitin-conjugating enzyme 1.76 0.29 0.01027437 M62388 ODC1 ornithine decarboxylase 1.72 0.34 0.04952544 NM_012615

PEPCK phosphoenolpyruvate carboxykinase (GTP) 1.62 0.27 0.04460906 AH007109

Significantly regulated genes are shown, including abbreviations, full-length names, fold regulation, standard error, p values (t-test), and accession numbers. The genes arranged by functional group. Twenty seven of the 33 genes identified have not been previously reported to be regulated by exercise in brain.

Supplementary Table 2: Exercise-Regulated Gene Functional Information.

Abbreviation Function Growth Factor Signaling VGF+

Neuropeptide regulated by electroconvulsive shock 1 and neurotrophins (e.g. BDNF or NGF) 2-4 that functions in energy balance 5, learning and synaptic activity 6. Reported to be regulated by exercise in brain 7.

ERK2#

Serine/threonine kinase involved in growth factor signaling 8; Reported to be regulated by9 exercise in skeletal muscle 10 but not reported to be regulated by exercise in the hippocampus.

MAPKP3*

MAPKP3 (MKP3) is a dual specificity phosphatase (DSP) that selectively dephosphorylates phosphotyrosine and phosphothreonine residues within MAPK that are required for its activation11; MAPK family regulates gene expression, cell proliferation, differentiation, neuronal survival, and programmed cell death 12-19; MKP3 acts predominantly on ERK1 and ERK2 but not on JNK/SAPK or p38 MAP kinases 20-22; MKP3 is expressed in brain and has a cytosolic localization 23,24; it is also induced by nerve growth factor (NGF) in differentiating PC12 cells 25; cerebral hypoxia results in increased expression of MKP1 and MKP3 (MAPKP3) via a nitric oxide mechanism 26; MKP3 plays a role in regulating gluconeogenic gene expression and hepatic gluconeogenesis where altered expression of MKP3 and/or function in liver may contribute to the pathogenesis of insulin resistance and type II diabetes27. There are no reports on MAPKP3 being regulated by exercise in brain.

GRB2*

Adaptor protein involved in ERK/MAPK pathway where following tyrosine kinase receptor activation GRB2 mediates the interaction between SHC and SOS (son of sevenless, guanylnucleotide exchange factor) leading to activation of RAS 28,29. Not regulated in skeletal muscle following exercise 30. No reports on GRB2 being regulated by exercise in brain.

NTRK2+

Preferred receptor for BDNF; mediates BDNF/NT-3 survival of fibroblasts 31; mediates the effects of BDNF on hippocampal LTP 32; modulates dendritic spine development 33; TrkB and TrkC signaling required for maturation and synaptogenesis of hippocampal connections 34; BDNF stimulation of NPY requires both TrkB phospholipase C gamma and Shc binding sites 35; mediates hippocampal plasticity by recruiting PLCgamma and phosphorylating CaMKIV and CREB 36; BDNF recruits full-length TrkB receptor into cholesterol-rich lipid rafts which may provide a mechanism for synaptic modulation 37. Reported to be regulated by exercise in brain 38.

CSF3G#

Haematopoietic growth factor that works by encouraging the bone marrow to produce more white blood cells; reported to be upregulated in blood of humans after exercise 39,40. Interestingly, there is a recent review stating that CSF3G is a potential novel neuroprotective factor 41. Also CSF3G reported recently to induce neurogenesis via VEGF 42 and angiogenesis 43. CSF3G provides trophic support to cholinergic neurons 44 which may be another mechanism where by exercise achieves its beneficial effects. There is also a report specifically discussing the potential for CSF3G as a novel neurotrophic factor that may have therapeutic value in treating neurodegenerative diseases 45. Reported to be regulated in blood in humans following exercise 46. Not reported to be regulated by exercise in the brain.

MEK2#

Kinase in MAPK kinase pathway involved in cell proliferation and differentiation; involved in growth factor signaling; MEK2/ERK involved in

growth arrest while MEK1/ERK activation preferentially provides proliferative signals at the G1/S boundary 47. Following resistance exercise a MEK inhibitor (PD098059) blocked exercise induced muscle protein synthesis 48. Exercise of human skeletal muscle activates the MAPK pathway (RSK2, MEK1, Raf-1), but not MEK2 in this study 49. Another report of exercise in human skeletal muscle does report increased phosphorylation of ERK 1&2 and MEK 1&2 phosphorylation) 50. There are no studies reporting MEK2 regulation by exercise in the brain.

NEUR*

Activity-regulated gene (immediate early gene) encoding a membrane-bound ligand that regulates dendritic and axonal arbor growth and synaptic maturation that is involved in synaptic plasticity 51-54. Promotes neuritogenesis and is regulated by neurotrophins (trkB mediated), glutamate, depolarization, and seizures 1,53-55 No reports of exercise regulation in brain.

MAGED1*

Novel member of the MAGE/necdin gene family whose members were shown to promote neuronal differentiation and cell arrest 56. Interacts with neurotrophin p75 receptor and facilitates NGF-dependent apoptosis. MAGED1 is able to overcome the anti-apoptotic effect of Bcl-2 57. MAGED1 was identified as a binding partner for the p75 neurotrophin receptor, the apoptosis inhibitory protein XIAP, and Dlx/MSX homeodomain proteins, where it is reported to block cell cycle progression and enhance apoptosis 58,59. No reports on being regulated by exercise in brain.

Transcription Factors & IEG NARP+

Immediate early gene that augments synaptic plasticity possibly via excitatory synaptogenesis where it clusters AMPA receptors 60,61; induced by synaptic activity and promotes neurite outgrowth 62; acute administration of methamphetamine induces NARP mRNA in PFC and reduces it in hippocampus 63; Referenced to be regulated by exercise in hippocampus 7.

EGR1+

Immediate early gene involved in neuronal plasticity induced by growth factors and synaptic activity 64-67; induced by BDNF 68; up regulated following classical conditioning 69 and plays a role in memory formation during a hippocampal spatial learning task 70; involved in late-phase long term potentiation, required for hippocampus-dependent long-term memory formation and for reconsolidation of memories via regulation of Arc 71. EGR1 activation via cAMP regulates synapsin 1 expression 72; up regulated following environmental enrichment in hippocampus 73; reported to be regulated by exercise in hippocampus 7,74.

EGR2*

Immediate early gene 66,75; regulates myelin protein zero (Mpz) gene which is critical for myelination of the peripheral nervous system 76; inhibition of EGR2 may contribute to glucocorticoid-induced osteoporosis 77; induction of EGR2 activates downstream transcription factors including c-fos, SRF, and c-myc which are involved in cell proliferation 78; role in PTEN-induce apoptotic pathway 79; Up regulated following environmental enrichment in hippocampus 73. No reports of being regulated by exercise in brain.

EGR4*

Immediate early gene involved in cellular growth and differentiation 66,80; role in male fertility 81; in T cells EGR4 & EGR3 interact with NF-kappa B and control inflammatory cytokine gene transcription 82; induced by seizure and vibrissae stimulation 83; up regulated following environmental enrichment in hippocampus 73. No reports of being regulated by exercise in brain.

ELK1*

Transcription factor that can bind to purine-rich DNA sequences and can form a ternary complex with serum response factor (SRF) and the ETS and SRF motifs of the FOS serum response element 84,85; involved in hippocampal plasticity via

the MAPK pathway 86,87; signaling pathways of TrkB-ERK1/2-CREB/Elk-1 are highly activated in mossy fiber organization 88; thrombin activation of the Elk1 transcription factor leads to chemokine gene expression 89. No reports of being regulated by exercise in brain.

Neurotransmitter-Synaptic Signaling

GRP78#

Molecular chaperone; reduced by dietary restriction 90; promoter region of GRP78 responds to glucose deprivation 91. Reduction of glucose concentrations first leads to repression of GRP78 mRNA abundance, followed by induction of the mRNA only when glucose is nearly exhausted 92; localized in the ER and its expression is increased by environmental stressors where it has been demonstrated to play a neuroprotective function 93. In a running rat model, acute exercise activates the synthesis and accumulation of HSP72, GRP75 and GRP78 in liver cells 94. No reports on being regulated by exercise in brain.

SYNCAM*

Brain-specific, immunoglobulin domain-containing protein that binds to intracellular PDZ-domain proteins and functions as a homophilic cell adhesion molecule at the synapse; this synaptic cell adhesion molecule is critical for synapse formation and function 95,96. No reports on being regulated by exercise in brain.

IL-16*

Cytokine; natural ligand of CD4 molecules and induces chemotaxis in CD4-expressing cells such as T cells, eosinophils, dendritic cells and monocytes; generated by posttranscriptional cleavage by caspase-3 of two large precursor isoforms; smaller protein of 67 kDa (pro-IL-16) is expressed in cells of the immune system and contains three PDZ domains, where the larger 141-kDa neuronal variant (npro-IL-16) has two additional PDZ domains in its N-terminal extension that interact with neuronal ion channels; novel target of the MAPK pathway in T-lymphocytes 97; role in the disease process underlying rheumatoid arthritis and joint destruction 98 as well as in the exacerbation of chronic adult atopic dermatitis 99. No reports on being regulated by exercise in brain.

Gat1*

GABA transporter; Gat1 mRNA is expressed in GABAergic neurons and in non-GABAergic neurons and/or glial cells which are involved in GABAergic neurotransmission 100. GAT1 deficiency leads to enhanced extracellular GABA levels resulting in an over activation of GABAA receptors responsible for a postsynaptic tonic conductance. Chronically elevated GABA levels also down-regulate phasic GABA release and reduce presynaptic signaling via GABAB receptors thus causing an enhanced tonic and a diminished phasic inhibition 101. GAT1 over expression in mouse affected testis development 102. GAT1 over expressing mice also displayed cognitive deterioration in associative learning ability and new object recognition retention 103. No reports on being regulated by exercise in brain.

GAD67*

Rate-limiting enzyme in GABA synthesis; BDNF increases GABA content in striatal cultures by presumably increasing GAD67 and GAT1 mRNA 104. Interestingly, there is decreased BDNF, TrkB, and GAD67 mRNA in the PFC of schizophrenic patients 105. No reports on being regulated by exercise in brain.

LPHH1R*

Alpha-Latrotoxin (LTX) is the venom from the black widow spider and stimulates massive exocytosis of synaptic vesicles 106. LPHH1R is the G protein-coupled receptor (GPCR) for alpha-Latrotoxin and is expressed primarily in brain 107,108; No reports on being regulated by exercise in brain.

GIT1*

G protein-coupled receptor kinase-interacting protein (GIT1) is an adaptor protein that functions as a key regulator of spine morphology and synapse formation by targeting actin regulators and altering Rac (a GTPase involved in regulating actin cyctoskeleton dynamics) activity at synapses 109. GIT1 is suggested to also act as

a scaffolding protein to enhance c-Src-dependent activation of MEK1-ERK1/2 in response to GPCRs and tyrosine kinase receptors 110. No reports on being regulated by exercise in brain.

GPCR37*

G-protein coupled receptor 37 like 1 (GPCR37) also known as Endothelin B receptor-2 precursor (ETBRLP2) was cloned and shows 60% overall homology with the Endothelin type B receptor, however it is unable to bind endothelin. It is highly expressed in the human central nervous system 111. No reports on being regulated by exercise in brain.

NPY+

Up-regulated by exercise and BDNF 112 and has diverse functions in feeding, anxiety, memory processing, and seizure activity 113,114

MFGE8*

Milk fat globule-EGF factor 8 protein (MFGE8) is a secreted glycoprotein from macrophages that can bind to apoptotic cells and bring them to phagocytes for engulfment 115-117. MFGE8 also regulates the removal of apoptotic epithelial cells during mammary gland involution and absence of this glycoprotein leads to inflammation and abnormal mammary gland remodeling 118. No reports on being regulated by exercise.

Gabrr1*

GABA is the major inhibitory neurotransmitter in the nervous system, and GABAC receptor, subunit rho 1 (Gabrr1) is a subunit of the GABAC receptor, which is a ligand-gated ion channel found in the retina, thalamus, hippocampus, pituitary, and gut 119-122. GABAC receptors may function in a diverse set of processes ranging from visual processing to regulation of sleep-wake rhythms to even pain perception, memory, and learning 122. Receptor antagonist studies of GABAC receptors have indicated altered sleep-waking behavior, inhibition of ammonia-induced apoptosis in hippocampal neurons by restoring pro-apoptotic BAD levels, and regulation of hormone release in the pituitary 120,123,124. No reports on being regulated by exercise.

SCG-2*

Secretogranin II (SCG-2) is an acidic secretory protein found in large dense core vesicles in endocrine, neuroendocrine, and neural tissues that is in the chromogranin family. Its promoter contains a CRE site 125 where it may be regulated by such transcription factors as CREB. SCG-2 is regulated by neurotrophic factors including nerve growth factor (NGF) and epidermal growth factor (EGF) 126,127 as well as second messengers such as calcium, protein kinase A, and protein kinase C 128,129. SCG-2 is processed into secreoneurin in the brain 130 and secreotneurin can elicit dopamine release from striatal slices 131. Secretoneurin may also function in processes such as chemotactic migration, inflammation, and angiogenesis 132-135. No reports on being regulated by exercise.

Syn-1+

Synapsin 1 (syn-1) is a synaptic-vesicle associated protein involved in vesicle pool formation and neurotransmitter release 136. Referenced to be regulated by exercise in brain 137.

Kinases, Phosphatases, & Enzymes Pkcl#

Atypical protein kinase C lamda/iota (Pkcl) is a kinase that resides in the cell nucleus found abundantly in lung, less abundantly in heart and skin, and very low expression in brain. It lacks a calcium regulatory domain and can be activated by phorbol esters 138,139. PKC activity has been implicated in insulin resistance and glucose transport 140,141. Following exercise in human skeletal muscle, atpical PKC (aPKC) activity was increased 142. No reports on being regulated by exercise in brain.

STEP46*

Striatal enriched phosphatase 46 (STEP46) is a cytoplasmic protein tyrosine phosphatase (PTPase) with a molecular weight of 46 kDa and is highly expressed in striatal neurons with dopamine D1-receptors. STEP can also dephosphorylate MEK and ERK. Over-expression of STEP46 induces neurite outgrowth via cAMP signaling 143-145.

LDHA#

Key enzyme in metabolism of lactate; regulatory elements in promoter region include an AP-1 binding site, 2 consensus Sp1 binding sites, and a CRE site 146; over expression perturbs glucose metabolism and insulin secretion in islet beta-cell types 147; PKC stabilizes LDHA mRNA and increases half life 148; up regulated by hypoxia via HIF-1 and CRE sites 149,150; induced by estrogen 151; regulated in the periphery by exercise 152,153. No reports to be regulated by exercise in brain.

E214K*

Up regulated following fasting in muscle, heart, liver, and kidney implicating a role of ubiquitin system in metabolic response to fasting 154; insulin and IGF1 reduce expression of E214K mRNA 155; reactive oxygen species (ROS) and mild oxidative stress increases protein degradation in skeletal muscle by up regulating E214K and other components in the ubiquitin-proteasome pathway9,156. No reports to be regulated by exercise.

ODC1#

Homodimer of 461 amino acids (in humans) that catalyzes the decarboxylation of ornithine producing, diamine putrescine which is the first and the rate limiting step in humans for the production of polyamines, compounds required for cell division. Polyamines accumulate following exercise in skeletal muscle and heart and lead to muscle adaptation to exercise 157-159. Polyamines also protect against oxidative stress 160; A beta-peptides induce ODC and subsequently polyamines which suggests a mechanism for repair from free radical damage 161. No reports of regulation by exercise in brain.

PEPCK#

Rate-limiting enzyme in gluconeogenesis in hepatic and renal cells 162; contains a CRE element in its promoter 163 which could mediate the induction of this gene by exercise in order to meet increased metabolic demands; role in maintaining blood glucose level where over expression results in symptoms of type II diabetes mellitus 164. No reference of regulation by exercise in brain.

(* ) novel exercise regulated genes, genes previous reported regulated by exercise in brain (+; 5 total) and peripheral tissues (#, 8 total). Functional information for each of the 33 exercise-regulated genes is shown above.

Supplementary Figure 1:

Immobilization stress decreases hippocampal BDNF and VGF. Sprague Dawley rats (300-350g) were subjected to 45 minutes of immobilization stress as described previously (Koo et al., 2003; Vollmayr et al., 2003) where (a) BDNF mRNA was decreased (38% DG and 20% CA3) and (b) VGF displayed a paralleled decrease (18% DG).

Supplementary Table 3: Elevated Plus Maze Infusion Data

Time in Open Arms (s)

Entries into Closed Arms

Entries into Open Arms

aCSF (n=7) 12.9±8.5 p=0.4 10.3±1.6 p=0.5 1.4±0.9 p=0.5 VGF (n=8) 15.5±6.4 p=0.4 10.3±1.0 p=0.5 1.4±0.5 p=0.5

% Open entries total # of entries time in center aCSF (n=7) 9.9±4.4 p=0.5 11.7±2.2 p=0.5 66.9±9.0 p=0.2 VGF (n=8) 9.5±3.4 p=0.5 11.6±1.5 p=0.5 73.3±3.8 p=0.2

Head dips stretch attend postures rears freezing

aCSF (n=7) 5.7±1.0 p=0.1 6.7±0.4 p=0.4 1.7±0.5 p=0.4 0.0VGF (n=8) 6.9±0.7 p=0.1 6.6±0.5 p=0.4 1.9±0.3 p=0.4 0.0

Elevated plus maze was performed following either infusion of vehicle or VGF peptide AQEE (1ug) where there was no significant difference in the behaviors measured.

Supplementary Table 4: Genotype Data –Elevated Plus Maze

Time in Open Arms (s)

Entries into Closed Arms

Entries into Open Arms

WT Sed (n=8) 6.1±4.5 18.1±2.1 2±1.2 VGF Sed (n=8) 8.6±4.2 15.1±2.5 2±0.8

% Open entries total # of entries time in center WT Sed (n=8) 8.1±4.1 20.1±2.6 100.1±10.4 VGF Sed (n=8) 10.7±4.7 17.1±2.9 97.9±13.9

head dips stretch attend postures rears freezing

WT Sed (n=8) 6.5±1.4 14.0±1.6 3.1±0.6 0.0VGF Sed (n=8) 5.5±1.4 11.3±1.4 2.9±0.9 0.0

EPM data is shown above for wild type and VGF+/- sedentary groups.

Supplementary Table 5: Genotype Data -Open Field Test

Center Center Entire Arena Rearing

Total duration (s) Total duration

(%) Distance moved

(cm) Frequency WT Sed

(n=8) 42.5±6.1 14.2±2.0 2865.3±270.9 24.5±6.8 VGF Sed

(n=8) 44.4±6.9 14.8±2.3 4135.3±895.7 21.8±3.6 Open field test data is shown above for wild type and VGF+/- sedentary groups.

Supplementary Figure 2: In Vivo Confirmation of VGF Induced Genes Following i.c.v. Infusion Using Real time PCR

Indicates in vivo confirmation of VGF induced genes. Isolated hippocampal RNA from mice is used to make cDNA for real time PCR confirmations.

Supplementary Table 6: Gene promoter analysis

Gene Regulation CRE Site TGACGTCA EGR 1 site GC[G/T]GGGCG VGF Ex only 8 of 8; TGACGTCA 7 of 8; GCGGG_CGggc EGR2 Ex+VGF 5 of 8; TGAtGctA 6 of 8; GtTGGGgCtgg GRB2 Ex+VGF 5 of 8; TGAtGTag 6 of 8; GCaGaGGCac Neuritin Ex+VGF 5 of 8; TGAtGTtg 6 of 8; CTGGGGGCtgg ODC1 Ex+VGF 6 of 8; TGACGaCg 5 of 8; GTGGGGGGCtgg Synapsin 1 Ex+VGF 6 of 8; TGACGaCg 6 of 8; GTGGGGGGCtgg Syncam 1 Ex+VGF 6 of 8; TGAacTCA 7 of 8; CTGGGGGCGtg Growth Factor Signaling ERK2 Ex only 7 of 8; TGACGTtA 6 of 8; GCaGGGGCagg MAPKP3 Ex only 6 of 8; TGggGTCA 6 of 8; GTGGGGaCGgg NTRK2 Ex only 6 of 8; cggTGtCGTCt 6 of 8; CTGGGCagc CSF3G Ex only 7 of 8; TGACGTCt 7 of 8; GCTGGGGCaac MEK2 Ex only 7 of 8; ttgTGACccTCA 7 of 8; GGGGaGGCGac MAGED1 Ex only 5 of 8; cccTGAgGcCc 7 of 8; CTGGGGCGac Transcription Factors & IEG NARP Ex only 5 of 8; TtAgcTCA 6 of 8; GTGGGGGGGGCG EGR1 Ex only 7 of 8; TGACGTgA 7 of 8; GCGGaGGCGgg EGR4 Ex only 6 of 8; gcgTGAtGTCc 5 of 8; GTGCaGGCagg ELK1 Ex only 6 of 8; accTGAgGTCt 6 of 8; GaGGGGGCaag Neurotransmitter - Synaptic Signaling GRP78 Ex only 6 of 8; gGAgGTCA little homology IL-16 Ex only 7 of 8; cgaTGAaGTCA 6 of 8; GTGGGGGGGGt Gat1 Ex only 6 of 8; TcACGgCA 6 of 8; GCaGGGGGaag GAD67 Ex only 6 of 8; ccaTGACacCA 7 of 8; GCGGGGGCagc LPHH1R Ex only 7 of 8; tgcTGACtTCA 6 of 8; CTGGGGGgagg GIT1 Ex only 6 of 8; ttcTGAtGTCt 6 of 8; GTGGGtGCtgg GPCR37 Ex only 4 of 8; TGAgGa 5 of 8; GTGGGCacagg NPY Ex only 5 of 8; gcTGAgGcCg 6 of 8; GTGGcGGCGtc MFGE8 Ex only 5 of 8; AgGTCA 6 of 8; CTGGGGGCcag Gabrr1 Ex only 6 of 8; atgTGACcTCt 5 of 8; GTGGGGgagac SCG-2 Ex only 8 of 8; gcaTGACGTCA 4 of 8; gaaGGGGGaag Kinases, Phosphatases, & Enzymes Pkcl Ex only 6 of 8; TGAaGaCA 5 of 8; GaaGaGGCaag STEP46 Ex only 6 of 8; cggTGAaGTCc 5 of 8; GTGGGGtCagg LDHA Ex only 6 of 8; ttgTGAtGTC 7 of 8; GCGGGCGgggc E214K Ex only 4 of 8; tgGTCA little homology PEPCK Ex only 7 of 8; TGACGTaA 5 of 8; GTGGGGGtcaa

Table illustrating CREB and EGR1 conserved promoter elements in VGF and exercise regulated genes.

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Supplementary Methods

Animals

Male C57BL/6J mice (21-26g, Jackson Laboratory) were obtained at 6-7 weeks of age and

allowed 1 week to acclimate before use in experiments. VGF+/– and wild type littermate

mice (30-40g) (backcrossed onto a homogenous C57Bl6 background) were acquired from

S. Salton (Mount Sinai School of Medicine). Male Sprague Dawley rats (260-330 g,

Charles River Labs) were used for studies of the behavioral actions of intracerebral

infusions and immobilization stress. All mice, sedentary, running, and cannulated, were

housed individually while cannulated rats were housed 2 per cage. All animals were housed

under standard lighting parameters (12 hr light/ dark cycle) with lights on at 7:00 am, and

were given free access to food and water. Animal use procedures were in accordance with

the National Institutes Guide for the Care and Use of Laboratory Animals and were

approved by the Yale University Animal Care and Use Committee.

Free wheel running

Running and sedentary mice were housed in standard plastic cages (12 x 30 x 13 cm) with

ad libitum access to food and water. Exercise cages are equipped with running wheels

connected to counters that record the number of wheel turns per minute. Exercise behavior

data is quantified with Clocklab software (Actimetrics, Evanston, IL).

Cannulation Surgery in Mouse and Rat

All surgeries were performed using aseptic conditions, and under anesthesia with a

ketamine-xylazine mixture. For the mouse, a unilateral cannula was placed into the right

lateral ventricle using a stereotaxic apparatus (from Bregma AP -0.6, ML -1.6; from skull,

DV -2.0). For rats, a bilateral cannula was placed into the hippocampal dentate gyrus (from

Bregma, AP -3.8, ML +/- 1.9; from skull, DV -3.3). After cannulation, rats were allowed

to recover for 1 week before training and testing. For both mouse and rat, animals were

allowed to recover for 1 week before infusions of vehicle or VGF and behavioral testing.

Immobilization Stress

Sprague Dawley rats (300-350g) were subjected to 45 minutes of immobilization stress as

described previously 1,2 and then placed back into home cages. Two hours later animals

were killed and brains harvested for VGF and BDNF in situ hybridization analysis.

Custom growth factor chip

A custom cDNA expression array containing primarily neurotrophic/growth factors and

related signal transduction genes, as well as transcription factors, G protein coupled

receptors, cAMP response element (CRE) regulated genes, and relevant neuropsychiatry

regulated genes was produced with the assistance of the Keck microarray facility at Yale.

Spotted genes are approximately 300 bp PCR products. For a full description of the custom

neurotrophic/growth factor microarray please see 3.

Microarray analysis of gene expression

Total hippocampal RNA from individual animals was isolated (RNA Aqueous, Ambion).

Optical density values (260/280) were consistently at or over 1.9. Five micrograms of total

RNA from 1 week exercise and sedentary mice (n=4 each group) was reverse-transcribed

into cDNA and indirectly labeled using a sensitive fluorescent labeling procedure

(Genisphere). A two-step hybridization and labeling protocol was used where the chip was

hybridized to cDNA overnight, washed stringently to remove nonspecifically bound probe,

and then poststained with fluorescent dendrimers. After posthybridization and washes,

slides were scanned using a GenePix scanner (Axon Instruments). Image analysis was

performed using GenePix Pro 4.0 software. Resulting files from GenePix 4.0 (Axon

Instruments) analysis were imported into Genespring 5.0 (Silicon Genetics) for additional

visualization and data mining. Hybridization spots were considered positive only if there

was a signal intensity of twice the background or more in at least one channel of half of the

replicates. Per-chip normalization was performed by dividing the expressed genes by the

median of two housekeeping control genes, � -tubulin and cyclophilin, that were not

regulated. Gene regulation was determined by taking the log ratio of the median

experimental (running) channel signal to the median control (sedentary) channel signal. Up-

regulated genes were defined as having an average expression ratio of >1.3, and the down-

regulated genes were defined as having an average expression ratio of <0.7. These values

were determined by performing homotypic hybridizations where the same sample is

hybridized in both channels (cy3 & cy5). These cutoff levels are also consistent with

expected levels of gene regulation in brain tissue relative to cultured cell reports by others

4,5. Statistical analysis was performed by an unpaired t test using the cross-gene pooled

error method in Genespring software. Significance was set at p < 0.05. Significantly

regulate genes were then classified into relevant functional categories.

VGF Treated PC12 Cell Conditions.

VGF responsiveness of undifferentiated PC12 cells was performed by plating PC12 cells

on collagen IV-coated 6 well dishes. Medium was changed to 2.5 mL DMEM with 20%

“spent medium” (i.e. medium removed from cells and then 4 mL of this is added to 16 mL

of DMEM without serum, then 2.5mL of this mix is put back in each well) in the morning.

Three hours later VGF C-terminal peptide (AQEE; 1ug/ml) was added or vehicle (HBSS

alone) and left for 6 hours. Cells were then washed in ice-cold PBS, solubilized with

RNAaqueous (Ambion), briefly sonicated, and then frozen at -80 degrees Celsius until

microarray analysis.

In Situ Hybridization

In situ hybridization was conducted according to standard procedures used in this lab 6.

Briefly, coronal brain sections (14 �m) were fixed in 4% paraformaldehyde solution,

washed, delipidated, and dehydrated. The sections were then incubated overnight with

hybridization buffer containing 35S-labeled riboprobes as indicated, followed by washing

and RNase treatment. The sections were dried and exposed to Hyperfilm (Amersham,

Arlington Hts, IL, USA) for 4-7 days. The specific RNA signal was then quantified by

NIH image.

Immunohistochemistry

Immunohistochemical techniques entail using fresh frozen cryostat cut coronal brain

sections (14 µm). Sections were fixed and rinsed followed by incubation in

blocking solution (2.5% BSA in PBS), rinsing in PBS, and then incubation overnight at

4°C in primary antibody solution (0.1% Triton-X 100, 1%BSA). The antibodies used were

rabbit anti-VGF (1:1000, Dr. Stephen Salton), mouse anti-NeuN (1:200, Chemicon,

Temecula, CA), rabbit anti-BDNF (1:1000, Chemicon), mouse anti-synaptophysin

(1:1000, Chemicion), mouse anti-PSA-NCAM (1:100, Chemicon). Sections were then

washed in PBS and incubated with fluorescent secondary antibodies: anti-rabbit Alexa

Fluor 546 (1:250) or anti-mouse Alexa Fluor 488 (1:250) both from Molecular Probes

(Eugene, Oregon), Cy3 AffiniPure donkey anti-mouse (1:125, Jackson Immuno Research,

West Grove, PA). Sections were then washed in PBS, dried and mounted with Vectashield

Hard Set Mounting Medium with DAPI (VGF and BDNF); or with biomeda corp

Gel/Mount aqueous mounting medium with anti-fading agents for the double-

immunohistochemical studies (VGF/Neu-N, VGF/synaptophysin, VGF/PSA-NCAM) 6.

Equipment and Settings

Acquiring immunohistochemistry images was performed on Zeiss Axioskop 2 microscope

using AxioVision 3.1 software where images for each protein were taken under the same

exposure conditions.

Forced Swim Test and Locomotor Activity in Mice

A 2-day forced swim test procedure was used to assess the effect of VGF peptide

infusions on immobility. Mice were given a 15 minute pre-swim on day 1 (19 cm diameter

glass cylinder filled to 10 cm with 23-25 ºC water). On the following day, mice received

unilateral infusions (lateral ventricle) of vehicle (aCSF) or VGF (0.2, 1.0, 5.0 µg) in a total

volume of 2.0 � l over a 2 minute period, and the injection syringe was left in place for an

additional 3 minutes. The VGF peptide used is a 30 amino acid peptide (AQEE-30, 588–

617). Four hours following injections mice were placed in the swimming chambers for 15

minutes and the behavior was videotaped. The recorded behavior was scored by an

observer. Immobility was defined as the absence of all movement except motions required

to keep the animals head above the water. A single forced swim test session was conducted

for VGF+/- and wild type mice.

For the cannulated mice, a second unilateral infusion was given (as described

above) one week following the forced swim test and locomotor activity was quantified 4

hours later. Locomotor activity was monitored in standard mouse cages (dimensions) using

a video tracking system (EthoVision pro, Noldus Inc., Leesburg, VA). Ethovision

software was used to calculate the distance traveled by each animal over a 10 minute test

period.

After behavioral tests, animals were killed by decapitation. Brains were removed

and stored at -80 ºC until use. To assess the cannula placement, sagittal sections were made

and then staining with cresyl violet. Animals with improper cannula placement were not

included in the final analysis. 21 mice out of 97 were not included in the final analysis due

to improper cannula placement and/or infusion complications.

The results of the forced swim test and locomotor activity are presented as percent

of control (vehicle) or wild type to account for variation in baseline activity between

experiments, which were conducted over several months. For vehicle controls the average

immobility time in the forced swim test (15min duration) was 301 ± 23.5 sec, and the

average locomotor activity distance (assessed for10 min duration) was 2322 ± 146 cm

(mean ± S.E.M.). For VGF wild type littermates the average immobility time in the forced

swim test (15min duration) was 238 ± 41.5 sec, and the average locomotor activity distance

(assessed for 10 minutes) was 3564 ± 171 cm (mean ± S.E.M.).

Tail Suspension Test in Mice

The tail suspension test consists of individually taping the mouse tail to a piece of Tygon

tubing and suspending it (60 cm) above the floor. The behavior is videotaped and the

duration of immobility, defined as the absence of all movement except for those required

for respiration, was measured for 6 minutes. Unilateral infusions (i.c.v.) of vehicle (aCSF)

or VGF peptide (0.2, 1.0, 5.0 µg) were conducted as described for the forced swim test in a

total volume of 2.0 µl. Mice were tested in the tail suspension test four hours following

infusions. The VGF+/- and wild type mice were subjected to the same tail suspension test

for 6 minutes. The results are presented as percent of control (vehicle) or wild type as

discussed for the forced swim test. For the vehicle control the average immobility time was

177 ± 11.8 sec and for the VGF wild type littermates the average was 80.5 ± 28.2 sec

(mean ± S.E.M.).

Elevated Plus Maze & Open Field Test in Mice

For the elevated plus maze, mice that had not been previously tested were administered

VGF (1.0 µg) infusions (i.c.v.) as described above and were tested for 5 minutes on the

elevated plus maze. This apparatus consists of a center platform (5 cm x 5 cm) 36.5 cm off

the ground with four branching arms (30 cm x 5 cm) where two of the arms are open and

the other two arms are enclosed by plastic walls (16 cm high). Testing occurred during light

phase in a dimly lit room (40 lux). Animals were placed on the center platform and scored

for arm entries and time spent in open arms. The open field test was conducted two days

following the elevated plus maze. Mice again received infusions of vehicle (aCSF) or VGF

(1.0 µg) (i.c.v.) and four hours later were placed into the center of a Plexiglas box (50 cm x

50 cm x 40.5 cm) in a brightly lit room. The distance moved and time spent for either the

entire open field or for a 25 cm2 center area were recorded using the EthoVision pro video

tracking system and software (Noldus Inc,. Leesburg, VA).

Novelty Induced Hypophagia

This paradigm consists of three days of habituation where mice are exposed to sweetened

milk in their home cage. On the forth day latency to drink from the sweetened milk bottles

is scored in their home cage. On the fifth day infusions of either vehicle or VGF (1.0 µg),

as described previously, were performed followed by measuring latencies to drink the

sweetened milk in a novel cage.

Forced Swim Test and Locomotor Activity in Rat

In these studies both locomotor activity and forced swim test were assessed in the same

animals after vehicle or VGF infusions. On the first day of a two day forced swim test, the

animals were placed in a plexiglass tank (30 cm diameter) filled with water (23-25 ºC) to a

height of 50-60 cm for 15 minutes. On the following day (testing), rats received bilateral

infusions of vehicle (aCSF) or VGF (1.0 or 2.5 µg) in a total volume of 1.0 µl over a

period of 10 minutes. The injection syringe was left in place for an additional 5 minutes to

allow for diffusion. Four hours following infusions locomotor activity was accessed by

measuring the total number of beam breaks in a 20 minute session (cage size: 24cm X

45cm) (Micropro version 1.30; AccuScan Instruments; Columbus, OH). The results are

presented as percent of control number of beam breaks during this time. Immediately

following locomotor activity assessment, the forced swim test was performed where rats

were placed in a water tank and videotaped for 10 minutes. Animals were scored for

immobility, swimming, and climbing by using a sampling technique to rate the predominant

behavior over a 5 second interval (therefore 120 total counts over 10 minutes) 7. Immobility

is defined as absence of all movement except motions required to keep the head above the

water. Climbing is defined as thrashing movements along the sides of the water tank while

swimming behavior consists of horizontal motion moving from one quadrant of the water

tank to another. After behavioral tests, animals were killed by decapitation. Brains were

removed and stored at -80 ºC until use. Cannula placement was assessed by analysis of

cresyl violet stained sagittal sections. For these rat surgeries, no animals had to be removed

because of improper cannula placement.

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