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Da steh ich nun, ich armer Tor! Und bin so klug, als wie zuvor.

”Faust - The First Part of the Tragedy” Johann Wolfgang von Goethe

Supervisor Docent Pierre Le Grevès

Co-supervisors Professor Fred Nyberg Docent Matthias Hallberg

Faculty opponent

Ph.D. Jan Kehr Members of the examining board

Professor Kjell Wikvall Professor Nina Mohell Docent Robert Fredriksson Ph.D. Åsa Wheelock Docent Mervi Vasänge

List of papers included in the thesis

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Rossbach, U.L., Steensland, P., Nyberg, F., Le Grevès, P.

(2007), Nandrolone-induced hippocampal phosphorylation of NMDA receptor subunits and ERKs. Biochem. Biophys. Res. Commun.,. 357(4):1028-1033

II Rossbach, U.L., Le Grevès, M., Nyberg, F., Zhou, Q., Le

Grevès, P. (2010), Acute 19-nortestosterone transiently suppresses hippocampal MAPK pathway and the phosphorylation of the NMDA receptor. Mol. Cell. Endocrinol.,. 314(1):143-149

III Rossbach, U.L., Flensburg, J., Nyberg, F., Le Grevès, P., A

quantitative phosphoproteomic study of rat hippocampal synaptoneurosomes following a single 19-nortestosterone administration. manuscript

IV Rossbach, U., Nilsson, A., Fälth, M., Kultima, K., Zhou, Q.,

Hallberg, M., Gordh, T., Andren, P.E., Nyberg, F. (2009), A quantitative peptidomic analysis of peptides related to the endogenous opioid and tachykinin systems in nucleus accumbens of rats following naloxone-precipitated morphine withdrawal. J.Proteome Res., 8(2):1091-1098.

Reprints are published with kind permission from Elsevier and American Chemical Society.

List of additional papers

Kultima, K., Nilsson, A., Scholz, B., Rossbach, U.L., Fälth, M., Andrén, P.E. (2009), Development and evaluation of normalization methods for label-free relative quantification of endogenous peptides. Mol. Cell. Proteomics, 8(10):2285-2295

Contents

Introduction...................................................................................................11 Anabolic androgenic steroids ...................................................................11

Androgens............................................................................................11 Anabolic androgenic steroids - psychological effects .........................14 Androgens - impact on the hippocampus ............................................14 Androgens and cognitive function.......................................................15

Signaling pathways and the hippocampus................................................16 Hippocampus .......................................................................................16 MAPK pathway ...................................................................................17 NMDA receptor ...................................................................................18 NMDA receptor and MAPK - functional interdependence in the hippocampus........................................................................................19 Anabolic androgenic steroids - a gateway to opioid dependence ........20

Opioids .....................................................................................................21 Mesolimbic system...................................................................................22 Neuropeptides...........................................................................................23

Opioid peptides....................................................................................23 Tachykinins .........................................................................................24

Protein analysis – methodological aspects ...............................................26 Antibody-based protein analysis..........................................................26 Mass spectrometry-based protein and peptide analysis .......................26

Aims..............................................................................................................29

Comments on methods..................................................................................30 Animal experiments and drug treatment ..................................................30 Sample preparation...................................................................................31 Protein analysis ........................................................................................32

SDS-PAGE and immunoblotting.........................................................32 Immunoprecipitation............................................................................33 Quantitative mass spectrometry-based proteomics..............................34

Quantitative peptidomics..........................................................................35 Quantitative real time PCR ......................................................................37 Statistics ...................................................................................................37

Results and discussion ..................................................................................38 The anabolic androgenic steroid nandrolone............................................39

Impact on phosphorylation of NMDA receptor and ERK...................39 Androgen receptor dependence of nandrolone-induced effects...........43 Impact on proteins involved in synaptic plasticity and neurogenesis..45 Phosphoproteomics..............................................................................47

Opioid withdrawal....................................................................................49 Effect on endogenous opioid and tachykinin peptides ........................49

Concluding remarks ......................................................................................52

Acknowledgements.......................................................................................53

References.....................................................................................................55

Abbreviations

19NT 19-nortestoterone (nandrolone) AAS anabolic androgenic steroid AMPA �-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate AR androgen receptor CA1, -2, -3 cornu ammonis 1, -2, -3 CaMKII calcium/calmodulin-dependent protein kinase II Cdk5 cyclin-dependent kinase 5 cDNA complementary DNA CNS central nervous system Da Dalton DHT 5�-dihydrotestosterone DOP delta (�) opioid peptide eIF4E eukaryotic translation initiation factor 4E ERK1/2 extracellular signal-regulated kinase 1, and -2 ESI electrospray ionization GABAA gamma-aminobutyric acid receptor subtype A GluN2A-D NMDA receptor subunit 2A-D KOP kappa (�) opioid peptide LC liquid chromatography LTP long-term potentiation LTQ linear ion trap MAPK mitogen-activated protein kinase MAPKAPK mitogen-activated protein kinase activated protein kinase MOP mu (μ) opioid peptide MS mass spectrometry MS/MS tandem mass spectrometry NAc nucleus accumbens NMDAR N-methyl-D-aspartate receptor p- phospho- PC prohormone convertase PKC protein kinase C PSD postsynaptic density Q-Tof quadrupole-time of flight RP reversed phase SCX strong cation exchange chromatography SP substance P

11

Introduction

Anabolic androgenic steroids Androgens The endogenous male sex hormone testosterone is the prototype of an anabolic (myotropic) and androgenic steroid. Testosterone is predominantly produced in the Leydig-cells of the testicle in males. To a lower extent, synthesis occurs in the ovary in females and in the adrenal cortex of both males and females. The release of testosterone is regulated by the hypothalamus and the pituitary. Testosterone in the systemic circulation is predominantly bound to the sex-hormone-binding globulin and only the free testosterone can reach its target [1]. Testosterone has further a half-life of 10-20 min due to fast inactivation in the liver. This was the reason for synthetic modifications of testosterone to obtain long-acting compounds [2]. Testosterone metabolites are quickly formed by phase I and phase II reactions in the liver to eliminate the compound from the body. Besides the vast number of metabolites, two are of special interest due to retained bioactivity. First, 5�-dihydrotestosterone (DHT), a reductive metabolite formed by 5�-reductases. DHT binds to the androgen receptor being more potent than testosterone (Fig. 1) [3]. Second, estradiol, an oxidative metabolite of testosterone, is formed by the aromatase enzyme complex (Fig._1). This enzyme is found in testes, ovaries, placenta and adrenals and catalyzes the aromatization of the A-ring by oxidative removal of C19 [4].

OH

OH

OH

OH

estradiol

testosterone

5α-dihydrotestosterone

O

OH

1

4 6

919

12

14 15

17

18

A B

C D

aromat

ase5α-reductase

Figure 1. Chemical structure of the gonadal steroid hormones testosterone, its reductive metabolite 5�-dihydrotestosterone and the oxidative metabolite estradiol.

12

The biological effects of testosterone are male phenotypic differentiation (masculinization) of the embryo. Moreover, it regulates the development of secondary sexual characteristics of the male body during puberty, e.g. promotion of muscle growth, voice deepening, growth of beard and body hair, regulation of the spermatozoa production, and the male libido [1].

Anabolic androgenic steroids Anabolic androgenic steroids (AAS) were originally synthesized to selectively improve the muscle growth properties of testosterone. It was believed that the anabolic activity can be fully separated from the androgenic activity but this goal was never accomplished. However, compounds with high anabolic/androgenic ratios are described [5,6]. The anabolic effect is explained by nitrogen-retention in the body mass, stimulated by androgens interaction with the androgen receptor. This is accomplished by either diminished protein degradation or increased protein synthesis [7].

Nandrolone (19-nortestosterone) has a diminished androgenic activity and a greater anabolic/androgenic ratio than testosterone [4,6,8]. The structure of nandrolone is identical to testosterone, except for the lacking methyl group at C19. Nandrolone, as well as testosterone, is available with ester modification at C17-hydroxyl group (Fig. 2). These esters are prodrugs, showing increased hydrophobicity and allow pharmaceutical formulation of oily solutions with long-term release from an intramuscular depot. Once the ester leaves the depot, esterases in the blood catalyze the hydrolyzation. Hereby, the rate limiting step is the release from the oily depot in the muscle while the hydrolysis occurs within an hour [9,10]. The free drug acts at its target protein or undergoes metabolization.

O

OH

19-nortestosterone (nandrolone) nandrolone decanoate

O

O

O

C9H19

Figure 2. Chemical structure of the AAS 19-nortestosterone (nandrolone) and its decanoate ester

The androgenic and anabolic (myotropic) properties of testosterone and AAS are used in a few clinical indications. Secondary hypogonadism can be treated with intramuscular injections of testosterone esters restoring physiological testosterone levels. Boys showing a delayed puberty and patients with hematologic disorders such as anemia can also benefit from androgen therapy [11]. A further indication for testosterone is the wasting syndrome associated with infections, e.g. human immunodeficiency virus,

13

where it augments the long-term survival [7]. Recommended therapeutic doses of intramuscular nandrolone decanoate administrations are 25-50 mg every 2-3 week. These can increase up to 200 mg/week for anemic patients [10].

Besides the clinical use of AAS, these drugs were taken first by bodybuilders and became later also popular among athletes having benefits from the muscle strengthening effects [12]. The performance-enhancing effects of AAS were so tempting that socialistic governments operated secret doping programs for their athletes to gain prestige from being successful in sports. Thousands of athletes were administered doping agents, predominantly anabolic steroids, e.g. nandrolone [13]. More recent studies have shown that the AAS are used as recreational drugs [14]. In 2006, a report about youth risk behavior in the US unveiled that 4% of the nationwide students had used steroids illegally at least once during their life time [15]. The drugs are obtained via the illegal market. Therefore, it is often difficult to determine the actually injected amount of steroid. Moreover, illicit users combine different AAS or administer injectable and oral steroid formulations parallel [16]. Treatment regimens exist corresponding to up to 100-times the physiological dose [16-18].

Various physiological side effects are reported for high-dose use of AAS. Gynecomastia in men occurs due to aromatase-mediated conversion of androgen to estrogens. Testicular atrophy as well as reduced sperm production is the consequence of the activated feedback cycle reducing the release of follicle-stimulating and luteinizing hormones. In addition, negative effects of AAS on the cardiovascular system were reported [19].

The androgen receptor The androgen receptor belongs to the steroid receptor, which is characterized by forming homodimers. The AR consists of a conserved C-terminal ligand-binding domain (LBD) and a DNA-binding domain (DBD). Upon binding of the steroid to the LBD in an allosteric fashion, the receptor undergoes conformational changes leading to a transcriptionally active state [20]. Therefore, this receptor can be seen as a ligand-activated gene regulatory protein.

The AR is found in the hippocampus showing accumulation in the CA1 and CA3 region, and absence in the dentate gyrus [21]. The intracellular distribution of the AR is still controversial. Studies, conducted on mammalian cell lines, revealed that the unoccupied AR can be predominantly nuclear, primarily cytoplasmic or evenly distributed. However, all studies found that the ligand-occupied AR is localized to the nucleus [22,23]. In hippocampal neurons, the AR is localized in the nucleus, and in the cytoplasm of axons and dendritic spines. Moreover, it was found that the AR was densely accumulated proximal to the postsynaptic density [24,25]. The role of this extranuclear localized AR is not clear,

14

although it can be speculated that the AR interacts with mitochondrial DNA. Moreover, a role in the initiation of signaling pathways has been discussed [24]. In addition, completely unrelated proteins have been suggested to mediate the rapid actions of steroid hormones. Membrane-bound androgen recognition moieties, coupled to G-proteins, were found to rapidly increase calcium influx in osteoblasts [26,27]. To date, G-protein-coupled steroid recognition sites situated in the CNS were only found for estrogens [28].

Anabolic androgenic steroids - psychological effects Besides the described physiological effects of AAS, studies were conducted to investigate the AAS effects on the psyche. Mood changes such as mania, hypomania or major depression were observed in some athletes when taking steroids but unaffected aggressive behavior [29,30]. Long-term treatment with high doses increased the occurrence of manic states and aggression in men. However, large inter-individual variations were shown [31,32]. In addition, aggression-altering behavior was assessed using animal studies [33]. Correlation of aggression to testosterone serum levels were observed in castrated adult rats receiving a sex steroid replacement therapy. However, serum levels above normal did not further enhance the aggressive behavior [34]. However, in another study, gonadally intact adult male rats receiving a long-term high testosterone dose treatment showed enhanced aggression patterns [35]. The impact of nandrolone on aggressive behavior showed ambiguous results [33]. Long-term AAS exposure to adolescent rats affected aggression long after cessation of the drug. This finding suggests that altered sex hormone levels during adolescence may lead to persistent changes of the nervous system, affecting aggression behaviors [36].

Studies investigated also the effect of androgens on anxiety in rodents. Reductions in anxiety-like behavior were found immediately after a testosterone injection [37]. Others report reduced anxiety after subchronic and chronic treatment with AAS [38,39]. However, opposing effects were reported in rats after chronic AAS treatment [40]. A study on humans was in agreement with animal studies, showing fear reduction after a single testosterone dose [41]. The anxiolytic effects of androgens could be inhibited by GABAA receptor antagonists suggesting a GABAA receptor modulation [37,42,43].

Androgens - impact on the hippocampus Besides other factors, gonadal steroids were found to have an impact on cellular and morphological changes in the central nervous system (CNS). In addition to the well-documented effects of estrogens on the hippocampal development, recent studies have been focused on the impact of male sex hormone [44-46]. Sex-dependent alterations of dentate gyrus morphology were

15

reported. The size of this hippocampal formation structure is larger, more asymmetric and contains more granule cells in male rodents than in the female counterparts [47,48]. CA3 pyramidal cell branching also differs with sex [49]. Decreased density of dendritic spine synapses were observed in male rats and male monkeys after castration [50,51]. Replacement therapy with testosterone and the non-aromatizable DHT reconstituted the spine synapse density to normal levels, but the administration of estrogen did not. This finding suggests that androgens but not estrogens affect the synapse density in males.

In female rats, changes of estrogen levels between proestrus and estrus correlate with the synapse density in the hippocampus. Estradiol replacement therapy of ovariectomized female rats restored the synapse densities in the hippocampal CA1 [52,53]. Interestingly, testosterone or dihydrotestosterone reconstitute the synapse density of ovariectomized females similar to estrogen. This argues for a general impact of androgens on the synapse formation independent from estrogen [54].

Androgens and cognitive function There is a vast amount of publications dealing with the question if gonadal hormones, i.e. androgens and estrogens, affect the cognitive function. Gender-related differences in cognitive function tests were found. Men perform better in spatial tasks while women have a greater capacity in verbal function. However, the effect of gender was found greater than the effect of current testosterone levels [55]. These effects might be explained by permanent morphological alterations of the brain due to exposure to sex hormones at critical time points in pre- and postnatal developmental stages [56]. In healthy adult men, positive correlations were found between endogenous testosterone levels and the performance in visuo-spatial tests, e.g. orientation, mental rotation of 3-dimentional drawings [57,58] while other experiments suggested an inverted U-shaped relationship between testosterone levels and performance [59,60]. Reports of prostate cancer patients, treated with the androgen receptor antagonists, showed marked declines in processing speed [61], spatial ability and visual memory [62]. However, there are studies, which failed to show relationships between androgen levels and cognitive skills. A review of Ulubaev concludes that there is evidence for an organizational role in the brain development but inconsistent findings regarding the activational effects of androgens on cognition [56]. Moreover, animal studies were conducted to investigate the impact of long-term high-dose treatments of AAS on cognitive functions. The results suggest an AAS-induced impairment, mediated via the AR [39,63].

16

Signaling pathways and the hippocampus The hippocampus has received much attention in the research for the understanding of how information is processed into memory. Lesions in the hippocampus impair consolidation of new memory. Further, the hippocampus is involved in working memory, in spatial memory and in processing of emotional information, e.g. anxiety [64]. The process of the conversion of information into memory requires changes of synaptic activity. These changes are mediated by long-term potentiation (LTP) and components of cell signaling pathways are critical for LTP [65].

Hippocampus The hippocampal formation belongs to the limbic system and consists of

several cytoarchitectonically distinct regions: dentate gyrus, hippocampus, subiculum, presubiculum, parasubiculum, and entorhinal cortex. The hippocampus can be further subdivided into CA1, CA2, and CA3. The hippocampal formation is a functional unit, since the six regions are linked to each other by mostly unidirectional projections (Fig. 3). Starting with the perforant path, where neurons from the entorhinal cortex project onto granule cells in the dentate gyrus. The axons from the granule cells (mossy fibers) project towards the pyramidal cells of the CA3 region. The pyramidal projections (Schaffer collateral) innervate the pyramidal cells of the CA1 region. The unidirectional pattern connects the CA1 eventually with the entorhinal cortex via the subiculum. Informational processing is obtained by this unilateral circuit [66].

enthorinal cortex

subiculum

dentate gyrus

CA3

CA1

CA2

parasubiculum

presubiculum

A Brostral

caudal

Figure 3. (A) Horizontal section through the left hippocampus of a rat displaying the major regions of the hippocampal formation. The bold line in represents the granule cell layer of the dentate gyrus and the pyramidal cell layer in the CA1-3 regions. (B) Unilateral projections of the hippocampal circuit (adopted with changes from Amaral et al. [66])

17

MAPK pathway A signaling cascade is a network of consecutive molecules interacting as following: an extracellular signal interacts with its receptor and is converted into a primary cellular signal. This is further propagated via a series of signaling molecules within the cell to its target.

The mitogen-activated protein kinase (MAPK) pathway is a signaling cascade. It transmits extracellular signals from sites at the plasma membrane to target molecules within the cell. The linear downstream sequence architecture of the MAPK core cascade is conserved for all MAPK cascades and consists of MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK) and MAP kinase (MAPK).

MAPKKK is a substrate of an upstream signaling element transmitting the signal downstream to the MAPKK. From there, the signal is further propagated downstream onto the MAPK. To date, four different MAPK cascades are described and named according to the name of the MAPK: the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated kinase (p38) and big mitogen-activated protein kinase 1 (BMK1) [67].

Small GTPases

MAPKKK

MAPKK

MAPK

MAPKAPK

ERK1/2

MEK1/2

Raf-kinases

RSK1-4 Mnk

Ras

Figure 4. Schematic presentation of the ERK signaling cascade. Arrows indicate direct activation.

The ERK cascade has been found to be involved in the regulation of transcription, translation or cell proliferation. It is composed by a linear downstream sequence including Raf-kinase, MAPK/ERK kinase (MEKs) and ERK (Fig. 4).

The MEK isoforms MEK1 and MEK2 share the Ser-Xaa-Ala-Xaa-Ser motif in the activation loop, where MAPKKK-catalyzed serine phosphorylation occurs [67,68]. Upon activation, MEKs activate the downstream situated ERKs via dual phosphorylation of tyrosine and

18

threonine in the Thr-Xaa-Tyr motif [69]. ERKs are the only substrate of MEKs [70].

ERKs are proline-directed kinases leading to phosphorylation of serine or threonine neighbored by a proline [71]. Two main isoforms ERK1 (44 kDa) and ERK2 (42 kDa) exists beside several other. They share 90 percent identity [72], cellular localization and similar activation kinetics [67]. This argues for a functional redundancy. However, knock-out studies revealed that ERK1-deficient mice are born viable while ERK2-knockouts show embryonic lethality [73,74].

Examples of substrates of the ERK1 and ERK2 are the MAP kinase activated protein kinases (RSK, Mnk), transcription factors (c-fos, Elk), signaling components (PLA2, EGF receptor) and cytoskeleton proteins (MAP-2, tau) [75].

The cytoplasm is the locus for all members of the ERK cascade in resting cells. Upon an extracellular stimulus, Raf-molecules migrate to the plasma membrane interacting with activated GTPases, e.g. Ras. The following activation of MEKs, ERKs and RSKs releases them from cytoplasmic anchoring proteins, enabling migration into the nucleus or to other sites in the cell [67]. A target for the MAP kinase activated protein kinase Mnk1 is the eukaryotic translation initiation factor 4E (eIF4E), which play an important role in the initiation of cap-dependent translation of mRNA [76]. This factor is involved in synaptodendritic mRNA translation. ERK-dependent phosphorylation of eIF4E occurs in response to multiple forms of neuronal activity [77].

NMDA receptor The N-methyl-D-aspartate receptor (NMDAR) is one of three distinct ionotropic excitatory glutamate receptor types. The NMDAR is a non-selective cation channel with permeability for Ca2+, Na+ and K+ ions. In the resting state, when the membrane potential is hyperpolarized, the NMDAR is blocked by magnesium ions. When the postsynaptic cell is depolarized, the magnesium block is removed. Subsequent inward currents of ions occur upon binding of glutamate [78]. The NMDAR consists of four subunits. At least one is the NMDAR subunit 1 (GluN1) and the additional subunits are GluN2 subunits. There exist four different GluN2 subunits, named GluN2A/-B/-C/-D. Diheteromeric forms (e.g. GluN1/GluN2A, GluN1/GluN2B) are predominant although recent findings describe also triheteromeric NMDAR types (e.g. GluN1/GluN2A/GluN2B) [79].

Studies revealed an even distribution for GluN1 mRNA throughout the adult rat brain. A differential expression was observed for the GluN2A transcript predominantly found in hippocampus, cerebellum and cerebral cortex. GluN2B subunit mRNA appeared in the hippocampus and cerebral cortex. GluN2C transcripts were predominantly found in the cerebellum and

19

GluN2D in the brain stem [80]. Each subunit consists of an extracellular N-terminus, three transmembrane segments, an re-entrant loop, and an intracellular C-terminus [79]. The protruding C-terminal binds to the post-synaptic density (PSD) scaffold and forms the NMDAR signaling complex together with signal transducing enzymes [81]. Moreover, these tails posses sites for potential phosphorylation by protein kinases.

GluN2B subunit is phosphorylated by src family non-receptor tyrosine kinases. Studies on mice revealed prominent phosphorylation at tyrosine-1472 (Tyr1472), catalyzed by the src family member Fyn. Enhanced phospho-Tyr1472 levels were found after induction of long-term potentiation. Experiments with Fyn-deficient mice showed reduced phospho-Tyr1472 levels and impaired LTP, causing defect spatial learning [82,83]. Fyn-dependent Tyr1472 phosphorylation is associated with the retention of NMDAR at the synaptic scaffold. Phosphorylation prevents clathrin adaptor protein (AP-2) from binding to the GluN2B tail and internalization of GluN2B-containing NMDAR cannot occur [84].

The subunit GluN2A also possesses a long intracellular C-terminal with potential phosphorylation sites. Phosphorylation can occur at Ser1232, which was found to be catalyzed by the serine/threonine protein kinase Cdk5 [85]. Inhibition of Cdk5 prevented LTP induction in CA1 neurons. This shows the importance of Cdk5-mediated phosphorylation of the GluN2A subunit in synaptic plasticity [85,86]. Ser1232 phosphorylation by Cdk5 increased currents through recombinant NMDAR in HEK293 cells and enhanced synaptic NMDAR channel activity in CA1 neurons [85,87].

NMDA receptor and MAPK - functional interdependence in the hippocampus The MAPKs and the NMDAR have been found to be important key players in hippocampal synaptic plasticity. Synaptic plasticity can be defined as changes in the efficiency of the synaptic transmission between two neurons due to a coincident activity in both. This has been theorized first by Hebb[88]. Strengthening leads to a lasting association of the pre- and postsynapse and is named long-term-potentiation. On the contrary, long-term depression involves weakening of the association of pre- and postsynapses [89,90].

The NMDAR has been found to suit the conditions needed for plasticity. This ionotropic receptor is blocked by magnesium at resting potential, which is reversed by a postsynaptic depolarization evoked at other synapses of the same neuron. If a presynaptic glutamate release coincides with a postsynaptic action potential, calcium enters the neuron via the NMDAR. Therefore, the NMDAR can be seen as a coincidence detector [89]. Blockade of NMDAR was found to prevent induction of LTP. The increase in intracellular calcium affects signaling pathways. Targets for the elevated

20

calcium can be the protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII), Ras-specific guanine nucleotide releasing factor_1 (RasGFR1) or protein kinase A (PKA) [65,91,92]. These calcium sensors were described to reside in the post-synaptic density, which links regulatory molecules to their targets [93]. For PKC, an involvement in LTP could be shown, since activators of PKC induce LTP while PKC inhibitors prevent LTP [94]. Another calcium sensor in neurons is CaMKII�. It was found to be important for LTP induction, since the CaMKII� mutant mice exhibited severely reduced LTP [95]. Activation of the calcium sensors was hypothesized to be further conveyed onto the MAPKs leading to plasticity-associated gene induction or dendritic translation [91]. Studies have been conducted to elucidate whether ERK is important for synaptic plasticity. It was shown that ERK is activated during LTP and that inhibition of the ERK activation via MEK inhibitors prevents induction of LTP [96,97]. Recently, ERK activation has been associated with the regulation of GluN2B expression in the mouse hippocampus. Moreover, NMDAR phosphorylation was suggested to be mediated by ERK [98].

Anabolic androgenic steroids - a gateway to opioid dependence In 2000, Arvary and coworker conducted a study investigating whether human opioid addicts had a history of AAS use. It could be concluded that a certain proportion (9.3 percent) of these addicts were introduced to opioid drugs through their AAS dealers with the expectation to counteract AAS induced depression, insomnia and irritability [99]. A more recent study on 12th grade students in the United States of America investigated associations between the administration of AAS and other drugs of abuse. The results revealed a similar correlation between AAS use and the use of other drugs of abuse [100].

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Opioids Historically, opiates are natural products in juice of the unripe seed capsule from the opium poppy (Papaver somniferum). The juice contains many compounds, e.g. codeine, thebaine and morphine. The term “opioid” is limited to agonists and antagonists, which act at targets similar to morphine. It includes also the endogenous opioid peptides, i.e. enkephalins, dynorphins and endorphins.

The antinociceptive properties of opium extracts have been known for thousands of years. The first isolated alkaloids were morphine and codeine_[101] (Fig. 5). The first semi-synthetic opioid was diacetylmorphine (heroin) and later, fully synthetic opioids were introduced to the market e.g. pethidine and methadone. Opioids are used as analgesics, since they are the most potent drugs to relieve pain. Commonly prescribed compounds in this regard are morphine, fentanyl, mepiridine, and buprenorphine. Selection is based on the desired duration of pain relief and the dependence risk under long-term use. Codeine, and historically also heroin, has been used as an antitussive drug due to its cough suppressant properties. Unfortunately, all opioid receptor agonists posses an inherent addiction potential, which is still a reason for its restricted use, although careful choosing and dosing of appropriate opioids can avoid negative effects [102,103]. The reason for the drug misuse of opioids is their reinforcing and rewarding properties.

3

6

OON

O

HOHO

3

6

NO

OH

OH

3

6

O

N

O

O

O

O

3

6O

OH

ON

O H

morphine codeine diacetylmorphine naloxone(heroin)

Figure 5. Chemical structures of the opioid agonist morphine, codeine, heroin and the opioid receptor antagonist naloxone.

The opioid receptor With the availability of radiolabeled ligands, opioid binding site were detected in the mammalian brain [104-106]. Three different receptors were classified and named mu, kappa and sigma opioid receptor [107]. Later studies revealed a fourth type of opioid receptors, the delta opioid receptor [108]. However, studies in the early 1980s showed evidence that the sigma receptor was not an opioid receptor [109]. Opioid receptors are G-protein-coupled receptor of the Gi/o-type. Activation of this type inhibits the adenylate cyclase mediated formation of cyclic adenosine monophosphate (cAMP). This leads to an inhibition of neuron firing. The current nomenclature of the

22

opioid peptide receptors according to the IUPHAR is MOP-, KOP-, and DOP receptor. In the 1990s, the ORL1 (opioid-receptor like receptor) was found, showing a high structure homology to the known opioid receptors [110,111].

Mesolimbic system This system includes the ventral tegmental area (VTA), the nucleus accumbens (NAc), and the prefrontal cortex. Dopaminergic neurons from the VTA project towards the NAc releasing dopamine onto synapses of medium spiny neurons. This leads to an inhibition of the NAc neurons. Opioids exert an impact on the mesolimbic system via two distinct mechanisms. GABA-ergic neurons in the VTA control the dopaminergic neurons via inhibitory action. Activation of MOP receptors at GABA-ergic neurons inhibits the GABA release, eventually leading to a disinhibition of the dopamine release in the NAc. The second targets are opioid receptors on NAc neurons, where opioids posses inhibitory effects such as dopamine on D1- and D2- receptors [112].

Opioid tolerance is an adaptation due to repeated exposure to opioids. This leads to a diminished effectiveness of the given drug. Therefore, to obtain the same physiological responses a higher dose is needed. Tolerance to opioids is expressed as biochemical adaptations in the locus ceruleus and the mesolimbic system including the NAc. Chronic exposure to opioids leads to normalization of the drug-induced inhibition of firing rates in the locus ceruleus. In the NAc, elevated adenylate cyclase level were observed after chronic opioid exposure [113].

Cessation of administration of opioids leads to the withdrawal syndrome, which is characterized by physical and psychological disturbances [113]. Discontinuation in tolerant rats leads to elevated firing. Moreover, the time course of cAMP levels is paralleled with the time course of recovery from withdrawal [113,114]. A major role in withdrawal-induced behavior plays the locus ceruleus and the periaqueductal gray while the NAc plays a minor role [115]. In the past, studies examined the impact of opioid withdrawal on neuropeptides in the mesolimbic system. Met-enkephalin levels were increased in the striatum of Sprague-Dawley rats after naloxone-precipitated withdrawal from morphine [116]. Another study revealed similar results for Met-enkephalin, dynorphin A and Leu-enkephalin levels in striatum and NAc. In addition, dynorphin B levels were also found to be increased in the NAc [117]. Another investigation of neuropeptide levels in the NAc revealed strain-dependence, since basal neuropeptide levels were found to be different between rat strains [118]. However, studies conducted in the late 1970s have reported decreased or unchanged levels of enkephalins during opioid withdrawal [119-121].

23

Neuropeptides Neuropeptides are small proteins that act as neurotransmitters in the nervous system. A variety of neuropeptides is known e.g. glucagon, substance P (SP), follicle-stimulating hormone, gonadotropin-releasing hormone, �-endorphin and bradykinin. Some of these neuropeptides are known to be released into the blood circulation acting as hormones, while others show local action [122].

The neuropeptides are derived from large precursor proteins. Endoproteolytic cleavage occurs at basic amino acid pairs. This is catalyzed by prohormone convertases (PC) leading to intermediate and mature peptides. Subsequent removal of C-terminal basic amino acids is catalyzed by carboxypeptidase action. Some peptides need further modification to become bioactive, e.g. N-terminal acetylation, C-terminal amidation. To undergo �-amidation, which is important for the biological activity, a C-terminal glycine is required as an amino-donor [123]. Substance P and alpha-melanocyte-stimulating hormone (�-MSH) are C-terminal amidated. N-terminal acetylation of �-endorphin reduces the affinity for the MOP receptor. However, acetylation of �-MSH is associated with increased activity [124].

Opioid peptides The endogenous ligands of the MOP-, DOP-, and KOP receptors were discovered in the 1970s-1980s, and found to be peptides derived from precursor proteins. The three precursors, each of which is encoded by a separate gene, are posttranslational processed into the bioactive peptides (Tab. 1).

Pro-opiomelanocortin (POMC) is cleaved at basic amino acids by PC1 and PC2 [125]. The products are gamma-melanocyte-stimulating hormone (�-MSH), adrenocorticotropic hormone (ACTH), and �-lipotropin. Further processing of the ACTH gives rise to �-MSH and corticotropin-like intermediary peptide (CLIP), while processing of �-lipotropin produces �-lipotropin and �-endorphin [126]. �-endorphin is the only POMC-derived peptide acting at opioid receptors. Distribution of the POMC-derived peptides reveals medium levels in the NAc [127].

The prodynorphin gene encodes the prodynorphin precursor protein (PDYN). The endopeptidase-mediated (PC1) cleavage at pairs of basic amino acids leads to intermediates, which require further processing [128]. PC2 catalyses further cleavage to �-neoendorphin and the dynorphins (-A, -B, big-). Moreover, PC2 can cleave the precursor to most of the bioactive fragments although carboxypeptidase E is required for truncated dynorphins (DynA1-8, DynA10-17) and Leu-enkephalin [129]. In the rat, presence of

24

prodynorphin could be shown in the NAc and to a lesser extent also in the caudate putamen [130].

The proenkephalin precursor (PENK) is the translational product of the proenkephalin gene. PC1 cleavage leads to mainly larger immature enkephalin-containing peptides with minor amounts of mature enkephalins. PC2 mediated cleavage shows more complete processing [131-133]. Medium to high levels of enkephalins were found in the limbic system (NAc, bed nucleus of stria terminalis, CA3, central nucleus of amygdala) and in the dorsal horn. Low to medium levels were detected in the caudate putamen [127,134].

Enkephalins exert their effects mainly via DOP receptor interaction. Animal studies showed an enkephalin-mediated suppression of the withdrawal syndrome in mice [135]. Dynorphins act predominantly on the KOP receptor. Conditioned place aversion and attenuation of opioid withdrawal symptoms have been reported for dynorphins [136].

Table 1. The amino acid sequences of the endogenous opioid peptides. Selectivity for opioid receptors according to [137,138].

precursor peptide name sequence binding preference to

POMC β-endorphin YGGFMTSEKSQTPLVTLFKNAIVKNAHKKGQ MOP receptor

big-dynorphin YGGFLRRIRPKLKWDNQKRYGGFLRRQFKVVTdynorphin A YGGFLRRIRPKLKWDNQdynorphin B YGGFLRRQFKVVTLeu-enkephalin YGGFLα-neoendorphin YGGFLRKYPKβ-neoendorphin YGGFLRKYP

Leu-enkephalin YGGFLMet-Enkephalin YGGFMMet-Enkephalin-Arg-Gly-Leu YGGFMRGLMet-Enkephalin-Arg-Phe YGGFMRF

Endomorphin-1 YPWF-amideEndomorphin-2 YPFF-amide

PDYN

unknown

PENK

KOP receptor

DOP receptor

MOP receptor

Tachykinins The precursor proteins of the tachykinin family are encoded by three genes, preprotachykinin-A, -B and -C. Moreover, many splice variants are known. The precursor proteins undergo proteolytic processing releasing the bioactive tachykinin peptides, e.g. substance P, neurokinin A, neurokinin B and the neurokinin A-containing peptides neuropeptide K and neuropeptide_�. Apart from neurokinin B, the above listed peptides derive from the different splicing variants of the preprotachykinin-A gene. The members of the tachykinin peptide family share the C-terminal sequence

25

F-X-G-L-M-amide and bind to the G-protein coupled neurokinin receptors (NK) (Tab. 2). Substance_P shows the highest affinity to NK1, neurokinin A to NK2, and neurokinin B to NK3 receptors [139]. In the NAc, co-localization of preprotachykinin A with proenkephalins and prodynorphins is shown. Preprotachykinin B immunoreactivity was weak and patch-like distributed in the NAc [130].

After its release, substance P undergoes C-terminal �-amidation to become bioactive. Many studies have been conducted on substance P and its regulatory function. It is associated with pain. Release from sensory fibers into the dorsal horn was detected upon noxious stimuli and intrathecal injections of SP were found to produce hyperalgesia [140]. Moreover, interaction between opioids and SP was found in the nervous system of tolerant rats [141-143]. Increased SP levels were detected in the striatum of rats during opioid withdrawal [143].

Further processing by endopeptidases yields truncated forms of SP, e.g. SP1-7, SP1-8, SP8-11 and SP9-11 [144]. The biological effects of SP1-7 have been described being both similar and opposing to those of SP [145].

Table 2. The amino acid sequences of the tachykinin peptides are shown together with the corresponding receptors.

peptide name sequence selectivity towards neurokinin receptor

substance P RPKPQQFFGLM-amide NK1neurokinin A HKTDSFVGLM-amide NK2neurokinin B NMHNFFVGLM-amide NK3

neuropeptide γ DAGHGQISHKRHKTDSFVGLM-amide NK1, NK2neuropeptide K DADSSIEKQVALLKALYGHGQISHKRHKTDSFVGLM-amide NK2

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Protein analysis – methodological aspects A vast number of techniques for protein identification and quantitation have been developed, and to mention all would break the mold of this chapter. Therefore, the focus will be on a few methods, which were used or could have been used in this thesis.

The terms proteomics and peptidomics, such as “-omics” in general, imply that these methods aim to identify all proteins or peptides in the biological material, respectively. In addition, posttranslational modified proteins and peptides can also be studied.

Antibody-based protein analysis Antibody-based protein analyses utilize selective antibodies for identification and quantitation. A few examples are the well established semi-quantitative immunoblot detection, immunoassays and the protein microarrays. The immunoblot (Western blot) technique is a widely used protein detection method for identification and semi-quantitation of protein expression. Competitive binding immunoassays, e.g. radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), can be used to quantify a single or a small selection of proteins in many biological samples at a time_[146]. The recent protein microarray allows global protein expression profiling of biological samples.

The application of antibodies has advantages, e.g. high sensitivity and selectivity regarding the recognition of domains in proteins. Disadvantages can be limited availability of antibodies against certain posttranslational modified proteins, cross-reactivity, shell life and expenses.

Mass spectrometry-based protein and peptide analysis Mass spectrometry-based protein analyses utilize the mass spectrometer for identification. Besides identification, quantitation of the measured peptide ions can be achieved using mass spectrometry. To reduce the complexity of the biological material, sample separation is essential to detect the protein or peptide of interest. Low abundant proteins, e.g. peptide hormones, are often desired to be detected. However, highly abundant proteins, e.g. albumin, housekeeping proteins, might prevent their detection. Several approaches to enhance detection are described including gel-based methods [147,148], reversed phase chromatography or multidimensional chromatography [149].

Gel-based methods The 2-dimensional gel electrophoresis (2DGE) separates proteins first according to their isoelectric point (pI) and subsequently according to the molecular weight of the protein. After running the second dimension, the gel

27

can display up to several thousand spots, each representing a protein or posttranslational modification thereof. The quantitation of the protein abundances is carried out by measuring the optical density of the protein-containing spot. A more recent advantageous approach is the 2-dimensional differential gel electrophoresis (2D-DIGE) applying fluorescent dyes. This allows running of two samples plus an internal standard simultaneously on the same gel with higher sensitivity regarding quantitation [148]. Using the gel-based approach, identification of the proteins, situated in the gel, requires excision of the spot for subsequent protein digestion followed by mass spectrometry-based identification. Matrix-assisted laser desorption ionization (MALDI) is commonly used, but liquid-chromatography coupled electrospray ionization (ESI) would also be applicable [150].

Liquid chromatography-based methods Liquid chromatography, for sample separation, allows direct coupling to the mass spectrometer using electrospray ionization (LC-ESI-MS). For the hydrophobicity-based separation of peptides and proteins, reversed phase chromatography is well suited. The development of small diameter columns increases analyte concentration and enhances detection in the mass spectrometer. To increase the number of identified proteins, further reduction of the sample complexity is needed, which can be achieved by multidimensional LC [149,151,152]. These separations can be performed both off-line and directly coupled to the reversed phase LC. The general workflow for a mass spectrometry-based analysis consists of sample separation, ionization of the analyte, determination of the ion mass-to-charge ratios, measurement of relative ion abundance, selection of ions to be fragmented, and scanning the mass-to-charge ratios of the fragmented ions (MS/MS). For identification of the peptide sequence the MS/MS spectra data are correlated to amino acid sequences of virtually cleaved peptides stored in databases. Three distinct search algorithms have been described and are implemented into available search engines (SEQUEST [153], Mascot [154], X!Tandem [155]). Besides identification of the peptide sequence, also quantitation of the measured peptide ions can be achieved by the mass spectrometer. Different quantitation strategies exist, e.g. labeling the peptides with tags ((SILAC) [156], (ICAT) [157], (iTRAQ) [158])) or label-free techniques [159]. Labeling strategies allow simultaneous analysis of several samples per run. However, they possess drawbacks, e.g. increased complexity of the mass spectra, incomplete labeling and expenses. An alternative approach is the label-free quantitation based on the peptide peak area. This technique possesses no limitation regarding the number of samples to be analyzed. In contrast to the labeling-based methods, label-free approaches require individual sample analysis by LC-MS, and potential drifts in retention times require subsequent alignment. Both, alignment and quantitation, are performed in an automatic fashion using specific software

28

for large-scale proteomic analysis [159]. Large-scale mass spectrometry-based protein analysis is often conducted using the bottom-up strategy, also called shotgun proteomics. In this strategy, the biological sample is digested into small peptides either prior or subsequent to sample separation. Using ESI-MS/MS allows efficient identification and quantitation of the peptides eluted from reversed phase LC.

The distinction of the terms protein and peptide is somewhat arbitrarily and to separate the terms protein and peptide, a line must be drawn at a certain number of amino acid. In proteome research, the low molecular mass range (< 10kDa) is often classified as the peptidome, although this mass threshold is not absolute [160]. Peptidomic studies typically focus on the identification of bioactive peptides in biological samples [161]. These peptides might have undergone complex proteolytic processing mediated by various enzymes. Studying neuropeptide levels and their processing was commonly conducted using sensitive immunoassays, e.g. RIA. These techniques, requiring specific antibodies and were restricted to known peptides. Moreover, these assays are inherently sensitive to antibody cross-reactivity towards similar peptides. Moreover, application of antibody-based method to large-scale peptidomic studies is very labor-intensive. Peptide sizes below 10 kDa can be efficiently analyzed by LC-ESI-MS with moderate mass resolution (e.g. quadrupole-time of flight (Q-Tof)). Label-free quantitation approaches have the advantage not being depended on expensive and potential incomplete labeling due to posttranslational blocked residues. Therefore, mass spectrometry is a powerful tool for peptidome analysis.

29

Aims

The aims of the studies included in this thesis were:

• to investigate the in vivo effect of supratherapeutical doses of nandrolone decanoate (paper I) and the free nandrolone (paper II) on key elements of the signaling pathway in the rat hippocampus

• to elucidate whether supratherapeutical doses of nandrolone act

through a non-genomic rapid mechanism when regulating the phosphorylation of the NMDAR and ERK (paper II)

• to screen for AAS-induced alteration of phosphorylated proteins in rat hippocampal synaptoneurosomes following a single supratherapeutical 19-nortestosterone dose (paper III)

• to utilize LC-MS for the determination of neuropeptide levels of the endogenous opioid and tachykinin system in the rat nucleus accumbens following morphine withdrawal (paper IV)

30

Comments on methods

This thesis comprises several methods to investigate treatment-induced alterations of protein- and peptide levels in the rat brain. The methods chosen in paper I and II are employed to test hypotheses based on previous findings. Paper III extends the work done in paper I and II screening for the global effects of the AAS on the hippocampus. Paper IV uses the advantages of a recent mass spectrometry technique over the immunoassay to determine neuropeptide levels in the rat brain.

Animal experiments and drug treatment This thesis is based on animal experiments, which have been approved by the local experimental animal committee. All animals used in paper I - III were male Sprague-Dawley rats. In study IV, male rats from both Sprague-Dawley and Wistar were used.

Paper I-III The AAS doses of 15 mg/kg nandrolone decanoate (paper I) and 3 mg/kg free nandrolone (paper II and III) correspond to amounts taken by AAS abusers. Many studies investigating the effects of AAS misuse have chosen these doses [162-165]. The time points for decapitation of the animal were based on pharmacokinetic studies. Serum levels of nandrolone decanoate were reported to peak 10 to 43 hours after injection. Therefore sampling was conducted 24 h after administration of AAS. Subchronic treatment with AAS for 14 days leads to accumulation of the compound due to a depot half-life of 5 to 8 days [9,10,166].

Free nandrolone (19-nortestosterone) possesses a much shorter intramuscular depot half-life (0.6 h) [166], which may lead to serum peak levels within one hour. Therefore, the free nandrolone was chosen to study the acute effects on signaling pathways.

The decision for the dose regimen and duration of the flutamide pretreatment was based on affinity studies towards the AR and on pharmacokinetic data obtained from rats and humans. Flutamide is the prodrug for the bioactive 2-hydroxyflutamide. 2-hydroxyflutamide reaches maximum plasma concentrations in the rats 5 to 6 hours after oral flutamide administration. Studies in humans showed steady state concentrations after 4

31

days of treatment [167,168]. The 2-hydroxyflutamide binding affinity towards the AR was found to be approximately 100-fold lower than 19-nortestosterone [169,170]. To ensure total AR block a dose regimen of daily injection of 15 mg/kg of flutamide for 5 days was chosen. In humans, no symptoms of flutamide overdosing have been observed after a dose 20-fold higher than the therapeutic dose [171].

Paper IV The opioid receptor agonist morphine and the opioid receptor antagonist naloxone were used. Stepwise ascending doses ensured minimal respiratory depression of the rats in the beginning of morphine tolerance induction. In brief, the rats received subcutaneous injections of morphine hydrochloride, twice 2.5 mg/kg on day 1, twice 5 mg/kg on day 2, and twice 10 mg/kg morphine from day three onwards. After tolerance had been developed, naloxone hydrochloride (2 mg/kg) was injected subcutaneously to precipitate the withdrawal. The morphine withdrawal behavior was observed 30 min post induction and the animal was sacrificed after three hours later.

Sample preparation Sample preparation is a crucial step, since inappropriate handling of the material will have an impact on the results.

Paper I-III These studies were conducted on rat hippocampal synaptoneurosomes. This preparation consists of a subcellular compartment, enriched with pre- and postsynaptic membranes. Synaptoneurosomes are often used in studies of the regulation of proteins and signaling pathways in close proximity to the synapse. To minimize protein degradation, brain dissection was carried out on ice. The excised hippocampus was quickly frozen to -80°C. Preparation of the synaptoneurosomes was performed on ice. Both protease inhibitors (Complete Mini, Roche Diagnostics) and phosphatase inhibitors (Sigma) were used. The hippocampus was homogenized in ice-cold buffer. The crude homogenate was first filtered through an 80μm nylon mesh followed by a second filtration through a 5μm membrane filter. To pellet the synaptoneurosomes, the filtrate was centrifuged. Protein concentration was determined by use of commercial kits (2-D Quant-Kit; BCA)

Paper II Total RNA was isolated from hippocampus using TRIzol® reagent. To ensure quality of the extracted total RNA, the 28S/18S rRNA-ratio was estimated revealing equal quality between samples. RNA quantities were estimated by UV absorption. Complementary DNA was synthesized using a

32

reverse transcriptase kit (Invitrogen SuperScript® III). Absence of genomic DNA contamination could be excluded by a polymerase chain reaction (PCR) using primer for �-actin. Gel electrophoresis revealed bands of different sizes for the PCR product of the synthesized cDNA and genomic DNA.

Paper IV Sample preparation of biological material for peptidome analysis needs to be performed particularly careful in regard to time. Studies have shown that post mortem enzyme activity alters the quality of the sample [172]. Prolonged and uncontrolled post mortem enzyme activity will increase the number of peptides. This will distort the picture of composition and quantity of the endogenous peptides. Therefore, instant denaturation methods are needed. Exactly 90 second had elapsed between decapitation and freezing of the whole rat brains in -80ºC cold isopentane. Coronal slicing was performed at -10ºC to avoid thawing of the tissue. Subsequently, the slices were denaturized by rapid heating to +95ºC using the Stabilizor® [172]. Dissection of the NAc from the denatured brain slices was carried out at +4ºC. The peptides were extracted by microtip sonification in 0.25% acetic acid. Isolation of the peptide fraction was achieved using a centrifugal filter device with a nominal molecular weight limit of 10 kDa.

Protein analysis SDS-PAGE and immunoblotting Paper I and II SDS-PAGE and chemiluminescence-based detection of the immunoblot was chosen [173] (Fig. 6). Due to the complexity of biological samples, fractionation or separation is needed prior to immunoblotting. Electrophoretic separation in sodium dodecyl sulfate containing polyacrylamide gels (SDS-PAGE) can be used to separate proteins. Heating the protein mixtures in the presences of excessive sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) denatures the protein by giving it a strong negative charge and breaking the disulfide-bonds. The resulting protein mixture is than separated in a SDS-PAGE solely by the size of the protein. Depending on the protein size, a suitable polyacrylamide concentration of the gel needs to be chosen for optimal separation. For semi-quantitative studies, samples should be loaded in a randomized block fashion to minimize system inherent affection of the outcome. After the electrophoretic separation, the proteins are electro-transferred onto a nitrocellulose (NC) membrane. Blotting is necessary, since antibody penetration into the gels would be inefficient. Appropriate conditions for

33

blotting need to be found empirically, e.g. buffer composition, transfer current, and transfer time. Attention should be paid to evaluate the result of the blotting in regard to efficiency, completeness and evenness. This can be achieved by staining the membranes with reversible dyes, e.g. Ponceau-S, MemCode™.

The detection process of the antigen requires many steps. The optimal conditions have to be found empirically. This is the major disadvantage of this method, making it very labor-intense. The membrane is blocked by protein-mixtures to reduce non-specific binding of the antibody. The subsequent incubation with the primary antibody requires knowledge of the optimal antibody concentration. After several washing steps, the incubation of the secondary antibody is performed. Again optimal antibody concentration is crucial. To find the appropriate concentrations, laborious pre-experiments need to be conducted. The choice of the second antibody type determines the detection system and several are available, e.g. colorimetric, chemiluminescence, and fluorescent detection. For these studies, chemiluminescence visualization was chosen requiring a secondary antibody attached to horse-radish peroxidase (HRP). Chemiluminescence kits are offered, with detection levels of down to nanograms of antigen. Chemiluminescence has the advantage that it can be used with light-sensitive photographic films or charge-coupled device (CCD) imaging systems.

The disadvantage of the chemiluminescence detection method is, that only one antigen at a time can be examined. In the case, that proteins possess distinct molecular size, incubation with several antibodies for the respective antigens can be conducted. However, cross-reactivity needs to be excluded. Another remedy is cutting the membranes into pieces. Each molecular size range is incubated separately with the antibody against the protein of interest. Due to the number of steps, this method should be seen as semi-quantitative - yielding an approximation of the antigen content.

Immunoprecipitation Paper II This method has been used to enrich the low abundant androgen receptor from the synaptoneurosomal preparation prior to Western blot detection. The AR-antibody was attached to magnetic beads. Subsequent incubation with the AR-containing homogenate bound the AR onto the beads mediated by the AR-antibody. Using the magnetic properties of the beads eased separation of the AR from the homogenate. The AR was released by denaturation of the antibody followed by an SDS-PAGE separation and immunoblotting. Immunoblot detection was carried out using a different AR-recognizing antibody to improve selectivity.

34

synaptoneurosomes

immuno-based analysis MS-based analysis

protein analysis

SDS-PAGE

immunoblotting

antibody-based visualisation

identification and quantitation

protein digestion

strong cation exchange (SCX)

reversed phasechromatography

MS scan

quantitation data-dependent MS/MS

identification of the peptidesequences

normalization of quantitativedata

peptide ID and abundance

Figure 6. Flow chart describing the two different approaches chosen in this thesis to analyze proteins in the synaptoneurosomes.

Quantitative mass spectrometry-based proteomics Paper III Immunoblot-based screening for phosphorylated proteins, which are regulated by androgens, would be a long-lasting, elaborate procedure. The advantage of LC-MS-based methods is the higher throughput compared to the immunoblot technique. MS and computational tools for identification enable fast screening for proteins including modifications. This has the advantage, not being dependent on a specific hypothesis about the potential impact of the treatment. Moreover, the LC-MS method allows also relative quantitation of proteins.

A bottom-up phosphoproteomic study was conducted using multidimensional LC combined with ESI-MS based identification and quantitation (Fig. 6). The proteins in the synaptoneurosomes were digested using trypsin, and subsequent phosphopeptide enrichment was performed

35

using strong cation exchange chromatography. The obtained fractions were analyzed on a RP-nanoLC system coupled to a linear ion trap mass spectrometer (LTQ). A 120 min gradient was chosen to increase the detection due to reduction of the number of peptides eluted per second [174]. The peptides were analyzed without prior labeling, since label-free quantitative approaches could been shown rigorous and powerful in large-scale proteome studies [159]. The mass spectrometer was set to perform a full scan for quantitation and selection of ions to be fragmented (MS/MS). The selection of ions to be fragmented was carried out in a data-dependent fashion, where the software selects the three most intense ions with a dynamic exclusion of 30 seconds. An MS/MS/MS was conducted in case of the detection of a neutral loss of phosphoric acid. A prominent ion in the MS/MS spectra, showing a neutral mass loss of 98, 49 or 32.7 Da for the singly, doubly or triply charged ion, indicates a �-elimination reaction of phosphoric acid from serine or threonine residues of the peptide. In the case of tyrosine, a neutral loss of metaphosphoric acid (80, 40 or 26.7 Da) can be detected in a MS/MS scan [175].

To identify peptides, MS/MS data was searched against a non-redundant rat protein database using the search engine SEQUEST[153]. To validate the localization of the SEQUEST-determined phosphorylated residues, a recent probability-based approach was applied [176]. Quantitation, matching and normalization of the peptide ions intensities across all samples were conducted using DeCyder MS™. Normalization was conducted using the intensity-distribution mode of the software. Automated matching of the peptide identification data from SEQUEST to the intensity data was performed by DeCyder MS.

Quantitative peptidomics

Paper IV Opioid withdrawal-induced alterations in the neuropeptidome of the NAc were investigated. Male rats of two strains, Sprague-Dawley and Wistar, were treated either with saline or morphine (n = 7/group) until the morphine-treated had developed tolerance. Tolerance was assessed using the tail-flick-test. This test measures the latency of moving away the tail from a heat-inducing beam. On day 14, when tolerance was fully developed, the morphine- and saline-treated rats received a single injection of naloxone to induce withdrawal. Withdrawal-induced behavior was observed and the rats were sacrificed after three hours. The brains were immediately frozen and prepared as described above. For peptide analysis, an approach was chosen utilizing two distinct types of mass spectrometers (Fig. 7). The nanoLC-ESI-Q-Tof MS was applied for

36

measuring peak intensity areas with high mass accuracy, followed by nanoLC-ESI-LTQ MS/MS for identification of the peptides. The same liquid chromatography system was used in both measurements to minimize variations regarding the peptide retention times. For identification, MS/MS data were searched against three sequence collection from the SwePep database using X!Tandem[155]. The sequence collections consist of peptide sequences for all known matured peptides (SwePep peptide), all known precursors containing neuropeptides (SwePep precursor). The searches were conducted allowing unspecified cleavages, i.e. cleavage after each amino acid. The third SwePep database (SwePep prediction) contains in silico cleaved sequences of all possible peptides with dibasic cleavage sites. This database aims to identify novel peptides. Finally, a Mascot[154] search against the Swiss-Prot database using unspecified cleavage was conducted to find neuropeptides from precursors not annotated as neuropeptide-containing. For quantitation, ESI-Tof-MS data of all samples were time-aligned, and peptides were matched over all samples. Quantitation was performed using the DeCyder MS software. To correct for global intensity differences between the runs, a two step normalization was carried out [177]. The final step was to match the peptide masses of the identified peptide sequences with the peptides masses of the normalized intensities.

biological tissue

sample preparation

neuropeptides (<10kDa)

quantitation (ESI-Q-Tof-MS)

data-dependent MS/MS(ESI-LTQ-MS)

identification of the peptide sequences

(X!Tandem; Mascot)

matching the data between samples (DeCyder MS)

+normalization of quantities

peptidomics

matching the normalized abundance to peptide ID

Figure 7. Flow chart of the MS-based peptidomic analysis.

37

Quantitative real time PCR

Paper II Quantitative real time polymerase chain reaction (qRT-PCR) has been used to evaluate mRNA transcription of the NMDAR subunits. This method allows quantitation of mRNA, extracted from biological material. It is based on the simultaneous observation of the amount of PCR product being formed during amplification. Monitoring can be based on interaction of fluorescent dyes (SYBR™ Green, ethidium bromide) with double-stranded cDNA or using probes (TaqMan™ Gene expression assay) [178,179].

The quantitative evaluation of mRNA expression in this thesis utilizes the SYBR™ Green method. To assure amplification of a single product, melting point curves were recorded. To allow compensation for unequal cDNA load, the expression of house-keeping genes (HKG) was measured. The most stable set of HKG was used for the normalization of the expression of genes of interest [180].

Statistics

Paper I-IV Statistical analysis of the observed differences was computed using the paired and unpaired Student’s t-test (paper I and III). The analysis of variance (one-way ANOVA) followed by a Fisher’s PLSD post-hoc test was applied on the data obtained in paper II. In paper IV, a parametric one-way ANOVA was performed followed by Fisher’s PLSD post-hoc test for the behavioral data. The nominal data of behavior was analyzed by Fisher’s exact test (two-tailed) with Bonferroni’s p-value adjustment. Differences were considered significant when p < 0.05. For the peptide level data, moderated F- and t-statistics were used, based on the linear models and empirical Bayes method. P-values < 0.01 were considered to be significant.

38

Results and discussion

The main objective of the papers included in this thesis was to investigate the potential impact of drugs of abuse on the levels of peptides/proteins and their posttranslational modifications. Papers I to III are directed to potential impact of anabolic androgenic steroids on the rat hippocampus. Paper IV examines alterations of neuropeptide levels of rats during opioid withdrawal (Tab. 3).

Table 3. Overview of the experiments

paper I II III IV

project AAS AAS AAS opiate withdrawal

compound/ dose

nandrolone decanoate 15 mg/kg

nandrolone 3 mg/kg flutamide 15 mg/kg

nandrolone 3 mg/kg

morphine 10 mg/kg naloxone 2 mg/kg

species/ strain

Rattus norvegicus Sprague-Dawley



Rattus norvegicus Sprague-Dawley and Wistar

tissue hippocampus hippocampus hippocampus nucleus accumbens

preparation synapto-neurosomes

synapto-neurosomes

synapto-neurosomes

acidic extract of peptidome

analysis protein, and - phosphorylation (semi-quantitative)

protein, and - phosphorylation (semi-quantitative)

proteomics, phosphoproteomics (quantitative)

peptidomics (quantitative)

technique SDS-Page and Immunoblotting

SDS-Page and Immunoblotting

nanoLC-ESI-LTQ-MS/MS

nanoLC-ESI-Q-Tof-MS and nanoLC-ESI-LTQ-MS/MS

39

The anabolic androgenic steroid nandrolone Impact on phosphorylation of NMDA receptor and ERK Paper I The analysis was conducted to investigate, whether high nandrolone decanoate doses can affect synaptic signaling components reported to be involved in hippocampal plasticity. Synaptoneurosomes is a preparation enriched with pre- and postsynaptic membranes. It is often used in studies of the regulation of proteins and signaling pathways in close proximity to the synapse. Earlier studies have shown importance of the NMDAR, CaMKII�, and ERK activation for the induction of LTP in the hippocampus. LTP is a phenomenon of long-lasting strengthening of synaptic transmission between the pre- and postsynapse. LTP occurs at hippocampal CA3-CA1 synapses requiring NMDAR activation. The resulting calcium influx is converted into increased activation of ERKs via several potential signaling kinases [65,89,95-97].

Western blot analysis revealed that the NMDAR subunits GluN2A and GluN2B, which are present in the hippocampus, show increased phosphorylation 24 h after a single dose of nandrolone decanoate. The phosphorylation occurred at residues on the intracellular tails of the subunits, which anchor the receptor to the PSD scaffold of the synapse.

Figure 8. The effects of nandrolone decanoate on the phosphorylation of the NMDAR subunits (A) GluN2A at Ser1232 and (C) GluN2B at Tyr1472. Total levels of the NMDAR subunit (B) GluN2A and (D) GluN2B are shown. Representative immunoblots for each group of rats are shown (top). The values are means ± SEM. *P < 0.05; unpaired Student’s t-test; (with modification from [181]).

Phosphorylation of GluN2A is reported to occur at Ser1232, a site, which is phosphorylated by protein kinase Cdk5 [85]. Studies showed that inhibition of

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this protein kinase prevented LTP induction in CA1 neurons and reduced inward currents through the NMDAR [85,87]. As shown in paper I, a single supratherapeutical nandrolone decanoate dose led to increased Ser1232 phosphorylation of GluN2A (Fig. 8) suggesting increased Cdk5 activity and enhanced currents through the NMDAR.

In paper I, increased phosphorylation of the GluN2B subunit at Tyr1472 was observed 24 h after treatment with nandrolone decanoate (Fig. 8). Tyr1472 resides in the internalization motif, where clathrin adaptor protein (AP-2) binds. AP-2 binding is followed by internalization of GluN2B-containing synaptic NMDARs. Phosphorylation of Tyr1472 by Fyn prevents AP-2 binding, retaining the NMDAR at the synaptic scaffold [84]. Moreover, an association between Fyn, Tyr1472, and LTP was suggested, since phosphorylation of Tyr1472 was elevated by induction of LTP in the hippocampus [82,182]. Moreover, fyn-deficient mice showed impaired LTP and defect spatial learning [83].

Determination of the total GluN2B content in synaptoneurosomes did not reveal any changes (paper I), although enhanced retention of the NMDAR could be expected due to prevention of AP-2-induced internalization.

Multiple supratherapeutical administration of nandrolone decanoate over 14 days did not show any alterations regarding the phosphorylation and total amount of NMDAR subunits in synaptoneurosomes (Fig. 8).

Figure 9. The effects of nandrolone decanoate on the phosphorylation of (A) CaMKII�, (B) ERK1 and (C) ERK2. Representative immunoblots for each group of rats are shown. The values are means ± SEM of two independently conducted Western blot detections.**P < 0.01; ***P < 0.001 versus control; unpaired Student’s t-test; (with modification from [181]).

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An enhancement of currents through the calcium-permeable NMDAR may increase intracellular calcium levels. Elevated calcium levels interact with calcium sensors such as CaMKII, PKC, RasGRF1 and PKA. They reside in the post-synaptic density close to the NMDAR and are involved in the downstream propagation of the signal towards the ERK pathway [65,91-93]. Phosphorylation of the CaMKII� isoform is associated with increased activation of the protein kinase. In paper I, no alteration in CaMKII� was observed by any nandrolone decanoate treatment using immunoblotting (Fig. 9).

Parallel alteration of the phosphorylation state of both ERK1 and ERK2 was detected in paper I, suggesting a signal transduction towards the ERKs via other calcium sensors than CaMKII�. The phosphorylation of ERK1 and ERK2 could be shown to be increased by a single dose of nandrolone decanoate, while multiple dosing did not (Fig. 9). The effects of supratherapeutical nandrolone decanoate doses on the rat hippocampus (paper I) can be summarized as follows. A single dose of this AAS could be associated with parallel increases of NMDAR and ERK1/2 activation 24 h after the administration.

Paper II In paper II, the acute effects of nandrolone were investigated. Studying the acute effects of AAS required a formulation of nandrolone, which possesses a shorter depot half-life. The free nandrolone seemed suitable due to a half-life in muscle depot of only 0.6 h. Moreover, the androgen receptor antagonist flutamide was included to study whether the effects were AR-mediated. To ensure a complete block of the AR, a 5-day flutamide pretreatment was chosen. For the AAS treatment, short-term exposures (2 h and 6 h) to supratherapeutical free nandrolone were chosen.

Analysis of the same proteins as in paper I revealed a different picture (Fig. 10). Phosphorylation of the GluN2B subunit at Tyr1472 showed a significant decrease after 2 h while at 6 h the level was restored to normal. To test, whether this altered phosphorylation had an impact on clathrin-dependent internalization of the NMDAR, the total GluN2B level were measured. An analogous alteration was observed. Determination of the GluN2B subunit mRNA levels was performed to investigate potential alteration of transcriptional levels. The mRNA amounts were unchanged, arguing against transcriptional regulation of the NMDAR content.

The phosphorylation of the GluN2A subunit at Ser1232 was not significantly altered by free nandrolone at any time point (Fig. 10). However, immunoblotting revealed phosphorylation of ERK1/2, which matched the pattern of phospho-Tyr1472 GluN2B. Free nandrolone reduced the levels of p-ERK1/2 at 2 h although p-ERK2 did not reach significance

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levels. After 6 h, an elevation of the ERK phosphorylation was observed, restoring the p-ERK1/2 levels (Fig. 10).

Figure 10. The influence of time on the effects of 19-nortestosterone (19NT) on synaptoneurosomal p-ERK1 (A), p-ERK2 (B), p-GluN2BY1472 (C), GluN2B (D), p-GluN2AS1232 (E) and GluN2A (F) in male rat hippocampus. Representative immunoblots for each group of rats are shown on top of each diagram. The values are means ± S.E.M. of two independently conducted Western blot analyses. *P < 0.05, **P < 0.01 (one-way ANOVA followed by Fisher’s PLSD post-hoc test); (with modification from [183]).

The time-dependent effects of a supratherapeutical free nandrolone dose on the rat hippocampus (paper II) can be summarized as follows. Parallel alteration regarding phosphorylation of the NMDAR subunit GluN2B and ERK1/2 could be shown. Total GluN2B levels imitated the alterations of the phospho-Tyr1472 GluN2B. This is in accordance to earlier findings that Fyn mediated Tyr1472 phosphorylation prevents clathrin-dependent internalization retaining the NMDAR at the synapse [84].

The findings of paper I and II regarding the effects on ERK and NMDAR can be concluded as follows. The impact of nandrolone decanoate on GluN2B phosphorylation is comparable with ERK1/2 phosphorylation in the rat hippocampus. The effects seem to depend on the formulation of the drug and/or on the time points chosen, since the decanoate ester treatment induced a response distinct from free nandrolone.

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Why the decanoate ester yields opposing alterations compared to the free nandrolone can only be speculated on. One reason may be the different pharmacokinetics of the two compounds. High hydrophobicity of the ester causes a long depot half-life and late serum peak levels. Moreover, large deviation was reported regarding the time point of serum peak levels [9,10]. This fact made it difficult to known the amount of nandrolone circulating in the blood and in the brain. An interpretation may be that a single dose of nandrolone affects the hippocampal signaling in a biphasic fashion. Immediate effects might be inhibitory while later in time the signaling is activated. The effects observed after a single dose of nandrolone decanoate would fit this hypothesis, when the serum peak occurs earlier than 24 h after injection as it was determined by Wijand [10].

Another hypothesis is that the rapid effects of nandrolone are mediated by membrane-associated androgen receptors, while interaction of AAS with the classical AR, situated in the cytosol or nucleus, occurs later. Other report, that rendering DHT membrane-impermeable, had an inhibitory effect on ERK phosphorylation suggesting membrane-associated AR. In contrast, the membrane-permeable DHT acting via the classical receptor activated ERK [184]. If this is case, the effects mediated via the classical receptor needs to surpass the membrane-bound receptor-mediated effects, since nandrolone’s membrane-permeability allows also binding to the classical receptor. Different binding affinities, intrinsic activities of nandrolone at these androgen-binding targets, or adaptations of the cell signaling could be an explanation. To date, no study has been conducted examining the latter.

Androgen receptor dependence of nandrolone-induced effects Paper II Although effects on NMDAR and ERK phosphorylation were observed in animals treated with nandrolone, an involvement of the androgen receptor needed to be proved. To achieve this aim, rats received flutamide pretreatment followed by nandrolone or vehicle injections. The impact of flutamide on the nandrolone-induced effects was examined.

In paper II, flutamide pretreatment could be shown to restore the GluN2B phosphorylation to control levels while no impact of flutamide was observed on the phosphorylation of GluN2A. The alterations of total amounts of GluN2B appeared to be similar to those of phospho-GluN2B (Fig. 11). This is consistent with the reported findings that Tyr1472 phosphorylation controls the internalization of the GluN2B subunit containing NMDAR [84].

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Figure 11. The effect of subchronic (5 days) flutamide (Flu) and/or acute (2 h) 19-nortestosterone (19NT) treatment on ERK1 (A), p-ERK2 (B), p-GluN2BY1472 (C), GluN2B (D), p-GluN2AS1232 (E) and GluN2A (F) in rat hippocampal synaptoneurosomes. Representative immunoblots for each group of rats are shown on top of each diagram. Data are expressed as mean ± S.E.M. of two independently conducted Western blot analyses. #P = 0.06, *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Fisher’s PLSD post-hoc test). (with modification from [183]).

A slightly different picture was obtained in paper II, observing the results for flutamide-induced effect on the ERKs. Again the pattern looks similar to that of phospho-Tyr1472 GluN2B. The analysis revealed not only a restoration to control levels but a significant increase above it. Thus, the flutamide pretreatment seemed to exert a stimulatory effect on the ERK phosphorylation (Fig. 11). The published effects of the AR antagonist flutamide on signaling are inconsistent and somewhat unexpected. Flutamide showed an androgen-like induction of the ERK phosphorylation in prostate and breast cancer cell lines [185-187], but blocked nuclear AR-dependent ERK activation in glia cells [184]. Interestingly, both flutamide and DHT could be shown to increase the number of dendritic spine synapses in AR-intact and AR-deficient mice [188]. The mechanisms are still unclear, although it is speculated that extranuclear AR-mediated non-genomic action is involved. Such extranuclear AR could be shown to exist in axons and dendritic spines [24]. Studies were conducted showing that androgens induce ERK phosphorylation in cultured hippocampal neurons and in glia cells [184,189].

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Interestingly, Gatson and coworkers observed that membrane-impermeable DHT diminished the ERK phosphorylation in glia cells. It was suggested that the stimulatory effects of androgens are mediated via the nuclear AR, while membrane-associated ARs mediate the inhibitory effects. Flutamide was found to block nuclear AR-mediated actions but not those mediated by the membrane-associated AR [184].

The results, observed in paper II, could be interpreted, that rapid androgen effects, mediated via extranuclear membrane-associated ARs, lead to different responses compared to those mediated by the classical AR. This would be in accordance to earlier findings [184,189]. The subchronic pretreatment with flutamide increased the ERK phosphorylation, which is in agreement with observations made by many groups [185-187]. However, there is the opposing finding that flutamide inhibits androgen-dependent ERK induction [184]. The question why the free nandrolone dose was not able to reduce the flutamide-induced effects cannot be answered with full satisfaction. A possible explanation may be that the concentration of nandrolone was too low compared to flutamide concentrations achieved by the 5-day pretreatment.

Impact on proteins involved in synaptic plasticity and neurogenesis Paper I and II The effects of nandrolone on the key components of cell signaling were studied in paper I and II. In the past, others have studied the effect of androgens on in vivo LTP. Long-term androgen replacement therapy of castrated rats led to significantly diminished excitatory post-synaptic potentials after LTP stimuli [190].

The studies included in this thesis did not examine the impact of AAS on LTP. However, the here presented data on NMDAR and ERK phosphorylation suggests that synaptic plasticity might be affected by supratherapeutical doses of nandrolone. To examine this hypothesis, levels of marker proteins were examined.

The immediate-early gene product Arc/Arg3.1 is marker for neuronal activity. Upon synthesis, the protein is transported to dendrites and accumulates at active synapses. It is reported to be critical for LTD and LTP by interacting with AMPA receptors and F-actin [191]. Although the protein could be detected in the synaptoneurosomal preparation, no changes were observed (see paper I, Fig. 4).

The brain-derived neurotrophic factor (BDNF) has a regulatory function in hippocampal synaptic plasticity. It has been reported to activate ERK2, to induce LTP, and to be involved in the regulation of the cap-dependent translation of mRNA via eIF4E [192,193]. BDNF, acting via tyrosine kinase

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receptor (TrkB), leads to the release of eIF4E from its binding protein. Phosphorylation of released eIF4E at Ser209 via MAPKAPK activates the cap-dependent translation of mRNAs [192]. The factor eIF4E together with ERK is suggested as an important regulator of synaptodendritic protein synthesis [77]. The levels of BDNF, eIF4E, and phospho-eIF4E were studied for nandrolone induced alterations. Although a clear ERK2 activation was observed with nandrolone decanoate after 24 h, BDNF levels were not affected (see paper I, Fig. 4). The amount of eIF4E and phospho-eIF4E did not show alterations by any of the nandrolone and flutamide treatment regimen (see paper II, Fig. 4).

An additional protein associated with synaptic plasticity is the postsynaptic ephrinB2 in the hippocampus. Absence of this protein leads to a strong reduction of hippocampal LTP [194]. Previously, ephrinB2 was found to increase GluN2B Tyr1472 phosphorylation via binding to the ephrin-type B receptor, which in turn interacts with the src tyrosine kinase [98,195]. The ephrinB2 protein expression detected in paper II did not correlate with the NMDAR subunit phosphorylation (see paper II, Fig. 4). The altered levels of phospho-Tyr1472 GluN2B appeared not to be mediated via ephrinB2.

Subchronic supratherapeutical doses of nandrolone were reported to decrease neurogenesis and proliferation of neural stem cell in the dentate gyrus [196]. The post-mitotic markers TOAD/Ulip/CRMP (TUC-4) and �-tubulin III were used to evaluate potential neurogenesis and proliferation in the hippocampus [197,198]. However, immunoblot detection did not show any alterations due to nandrolone decanoate treatment (see paper I, Fig. 4). Since the dentate gyrus constitutes only a part of the hippocampus, analyzing a whole hippocampus preparation might dilute potential effects.

The results of the search for proteins affecting or being affected by the NMDAR and ERK activation can be summarized as follows. All of the described proteins could be detected in the synaptoneurosomes and an analysis for quantitative changes was performed. The nandrolone treatments did not reveal any changes of these proteins in hippocampal synaptoneurosomes. A reason could be that only subtle regulatory changes with transient character occur, which would require more sensitive quantitative investigation methods. Moreover, the inclusion of more time points considering the various on-sets of signaling components are recommended.

To conclude the findings of paper I and II: Interpreting the regulation of the phosphorylation of the NMDAR subunits and ERKs is difficult. The short-term AAS effects (2 h) are inhibitory. Six hours after nandrolone, levels similar to the control were found. After 24 h, enhanced phosphorylation was observed. However, multiple doses administered for 14 days did not have an impact on signaling key components. The underlying pharmacokinetic has not been fully studied allowing only estimation of the actual dose achieved

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by nandrolone decanoate or free nandrolone treatment. The hypothesis, that nandrolone affects synaptic plasticity and neurogenesis, is not supported by the results of the examination of marker proteins.

Phosphoproteomics Paper III The label-free multidimensional LC mass spectrometry was chosen to perform a search for additional AAS-affected phosphorylated proteins, not covered in paper I and II. To allow comparison with the findings in paper I and II, the impact of free nandrolone on protein phosphorylation in rat hippocampal synaptoneurosomes was studied 2 h after injection.

Protein identification revealed SCX enrichment of phosphopeptides in the investigated fractions (see paper III, Fig. 3). All SEQUEST-identified proteins (n = 1030) were evaluated by the DAVID bioinformatics resource database [199,200] in regard to their biological function. Functional cluster analysis revealed that the identified proteins were predominantly associated with the synapse and membrane fraction. This was in accordance with the earlier reported composition of the synaptoneurosomes, showing enrichment of the pre- and postsynaptic compartment of neurons [201]. Quantitative analysis could be performed for 86 identified phosphopeptides. A functional classification analysis of this subset revealed that these phosphopeptides belong to cytoskeleton-, synapse-, and membrane-related proteins (see paper III, Tab. 1). Of these 86 phosphopeptide, only a few have been reported earlier. A manual search in the databases UniProt and PHOSIDA [202] revealed that only 7 peptides contained residues known to be phosphorylated (Tab. 4). Of these 7 phosphopeptides, 4 carried the phosphorylation at database-reported residues. Moreover, SEQUEST’s site determination could be confirmed for these 4 peptides by Ascore calculation [176] (Tab. 4). The three remaining phosphopeptides were reported to be phosphorylated, although not at the residue determined by SEQUEST. However, Ascore calculation could not validate the determined site of phosphorylation. This implies that another residue in the sequence might carry the phosphorylation. However, the quality of the obtained spectra does not allow an accurate phosphorylation site determination for these three phosphopeptides.

The microtubule-associated protein tau derived phosphopeptide, carrying the predominantly occurring phosphorylation at Ser715, was previously reported to be present in fetal and adult rat brain [203]. The localization of the serine neighbored by proline is the target site for proline-directed protein kinases. ERKs are proline-directed protein kinases phosphorylating the MAPKAPK ribosomal protein S6 kinase (RSK). Cdk5 is also a proline-directed protein kinase known to phosphorylate proline-neighbored Ser1232

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[204]. The peptide M*S#RELHDVDLAEVK originates from N-myc downstream regulated gene 1 protein. It is strongly expressed in the developing postnatal hippocampal neurons and astrocytes coinciding with morphological differentiation [205]. The phosphopeptide ERNMS#PDLREDFNMMEQR, derived from gamma adducin, has been described as a calmodulin binding protein, and a substrate for protein kinase C (PKC) [206]. However, the PKC-mediated phosphorylation occurs not at the here observed Ser42 residue. The sequence NFS#AAKSLLKK originates from the delta isoform of the calcium/calmodulin-dependent protein kinase II. Long-lasting CaMKII activation has been observed after induction of long-term potentiation, which is essential for synaptic plasticity [207]. Different isoforms assemble the kinase and calcium/calmodulin binding leads to activation by autophosphorylation at Thr286. The position of the here observed phosphorylated Ser315, resides outside the calmodulin binding domain. No function of that particular phosphorylation site has been described so far. Table 4. Identified phosphopeptides with previously reported phosphorylation sites. a Posttranslational modifications (PTMs) were assigned by TurboSEQUEST. # indicates phosphorylation and * oxidation of the preceding residue, d Underlined sites of phosphorylation indicate a match between database reported and observed; h For each phosphorylated site in the phosphopeptide an Ascore was calculated, determining the certainty of the phosphorylated site. YES indicates >99% certainty. NO indicates no Ascore confirmation. SEQUEST indicates that each possible phosphosite was assigned by TurboSEQUEST search engine. protein name UniProt

accession no.

peptide sequence / position / PTM a SEQUEST score e

reported phospho-residues d

phosphosite confirmation by Ascore h

microtubule-associated protein tau P19332 707SPVVSGDTS#PR717 3.0 S707; S711;

S715; T714 YES

calcium/calmodulin-dependent protein kinase II delta P15791 313NFS#AAKSLLKK 323 2.2 S315; S319 YES

N-myc downstream regulated gene 1 protein Q6JE36 1M*S#RELHDVDLAEVK 14 2.1 S2 SEQUEST

adducin 3 (gamma) Q62847 38ERNMS#PDLREDFNMMEQR55 2.1 S42 SEQUEST

phosphofurin acidic cluster sorting protein 1 O88588 479T#PMKSSKADLQGS #ASPSK496 2.8 S493 NO

ELL associated factor 2 Q811X5 137T#SNLVQHSPSEDKLSPTSLM*DDIER 161 2.6 S146; S151; S154 NO

phosphoglycerate kinase 1 P16617 57S#VVLMSHLGRPDGVPM*PDKYSLEPVAAELK 86 3.1 Y76 NO

The SEQUEST search, together with Ascore confirmation of the phosphorylation site determination, revealed 44 peptides with unambiguous phosphorylation sites (see paper III, Suppl.-Tab. 1). The other phosphopeptides show ambiguous phosphorylation site-determining information. Although these peptide sequences fulfilled the requirement to be accepted as “identified”, a proper placement of the phosphorylation site cannot be secured.

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In addition to the screening, we aimed to elucidate potential androgen-induced alterations regarding phosphorylated proteins. Each group consisted of three biological replicates. Incomplete matching of peptide intensities across all samples and the minimum requirement of two samples per group for the quantitative comparison condensed the number of phosphopeptides to 44. Comparison of the abundances revealed a regulation of more than 30 percent for 9 identified phosphopeptide (see paper III, Tab. 2). Unfortunate, the regulations observed did not reach significant levels. This might be explained by the small number of biological replicates included in this study. Further studies need to include more biological replicates to allow appropriate quantitative comparisons. In summary, the rat hippocampal synaptoneurosomes were screened for potentially interesting phosphoproteins. A phosphopeptide enrichment followed by mass spectrometry was applied. The identification process led to over 80 phosphopeptides, where the majority has not yet been described to be phosphorylated. The proteins, where these phosphopeptides originated from, could be associated with the cytoskeleton, neuronal differentiation and synapse signaling. Four peptides, carrying the modification at earlier described amino acids, were detected and could be related to protein kinases involved in long-term potentiation and microtubule plasticity. Despite the successful identification, none of these phosphopeptides displayed AAS-affected regulation in the rat hippocampal synaptoneurosomes.

Opioid withdrawal Effect on endogenous opioid and tachykinin peptides Paper IV Studies conducted in the past, have shown partly inconsistent data regarding the regulation of opioid- and tachykinin peptides during opioid withdrawal [117-121]. Although the highly sensitive radioimmunoassay method was used throughout these studies, cross-reactivity of the antibodies between similar peptides could not be fully excluded. To date, the utilization of modern mass spectrometers with high resolution allows efficient analysis of neuropeptides. To investigate the neuropeptidome of two different rat strains, the recently described label-free LC-MS-based peptidomic approach was applied. Although the method allows whole peptidome studies, the included work (paper IV) was focused on endogenous peptides of the opioid- and tachykinin system.

Naloxone-induced withdrawal behaviors of the rats were recorded during a 30 min period. The withdrawal behaviors wet-dog-shake, writhing, teeth chattering, grooming, ptosis, and diarrhea showed treatment-dependent occurrence after naloxone-induced withdrawal. Significantly higher

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occurrence of wet-dog-shake and writhing behavior was observed in Wistar rats.

The mass spectrometry-based analysis was conducted to identify and to quantify peptides in the NAc derived from proenkephalin- , prodynorphin- and protachykinin-1. From proenkephalin, the bioactive peptides Met-enkephalin, Met-enkephalin-Arg-Phe, and Met-enkephalin-Arg-Gly-Leu were detected. Moreover, several intermediate peptides and truncated forms thereof were also identified. The biological function of these neuropeptides is still unknown (Tab. 5).

From the prodynorphin precursor protein, five detected masses could be matched to the theoretical masses of dynorphin A, dynorphin B, Leu-enkephalin, and �-neoendorphin. However, the sequence of the C-terminal fragment SQENPNTYSEDLDV (PDYN235-248) could be confirmed by MS/MS. In addition, Leu-enkephalin, a bioactive peptide derived from both proenkephalin and prodynorphin could be detected. Unfortunately, MS/MS confirmation could not be obtained for this peptide (Tab. 5). The protachykinin-1 derived peptides substance P, substance P1-7, neurokinin A, the C-terminal flanking peptides ALNSVAYERSAMQNYE, and the intermediate peptide SDWSDSDQIK could be identified by MS/MS (Tab. 5).

Quantitation revealed strain-dependent alterations of the identified peptides (see paper IV, Tab. 2). Most of the neuropeptides showed differences comparing the basal levels of the rat strains. The analysis revealed that 6 of 27 identified peptides showed lower basal levels in the Sprague-Dawley strain compared to the Wistar strain. Two peptides, both derived from protachykinin-1, showed higher basal level in Sprague-Dawley rats. This supports earlier findings of strain-differences in regard to levels of opioid peptides [118].

The C-terminus of BAM-188-18, a proenkephalin-derived peptide, was found to be increased during opioid withdrawal in Sprague-Dawley rats. BAM-18, possessing affinity towards MOP- and KOP receptors, showed dose-dependent effects on pain [208,209]. However, for the detected C-terminus BAM-188-18 no biological function has been described so far. In Sprague-Dawley rats, the detected peptide levels of Met-enkephalin, Met-enkephalin-Arg-Phe and Met-enkephalin-Arg-Gly-Leu were increased by the treatment, although not reaching significance levels.

Increased levels of the prodynorphin-derived peptides �-neoendorphin and dynorphin B were found in Sprague-Dawley rats during naloxone-precipitated withdrawal. However, no change was observed for dynorphin A. Earlier reports, studying the neuropeptides during opioid withdrawal in Sprague-Dawley rats, found similar results for dynorphin B, but different for dynorphin A[117].

Comparable basal and withdrawal-altered amounts of the tachykinin substance P were observed for both Wistar and Sprague-Dawley rats. The

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withdrawal-induced elevation of substance P is in accordance to an earlier study, which was also conducted on Sprague-Dawley rats [143]. In contrast to other observed peptides in this study, the impact of withdrawal on the tachykinin substance P appeared to be strain-independent. However, the C-terminal truncated substance P1-7 showed a different pattern. The basal levels differ significantly between the Sprague-Dawley and Wistar strain. Opioid withdrawal seems to affect the two strains in opposite direction, although no statistical significance were observed. Neurokinin A and the C-terminal fragment peptide show different basal levels and a similar strain-dependent increase during withdrawal.

Table 5. Identified peptides derived from the proenkephalin-, prodynorphin-, and protachykinin-1 precursor. MS/MS confirmed peptide sequences are shown in bold font. Mass matched peptides are in lightface font. (with modifications from [210])

UniProt accession

no.:precursor name peptide name peptide sequence

and localization in precursortheoretical

mass

observed mass LTQ

Neoendorphin alpha 166YGGFLRKYPK175 1227.68 -Dynorphin B [1-13] 221YGGFLRRQFKVVT233 1569.88 -Dynorphin B-29 [16-29] 235SQENPNTYSEDLDV248 1609.67 1609.5Dynorphin A [1-17] 202YGGFLRRIRPKLKWDNQ218 2146.19 -

Leu-enkephalin YGGFL 555.27 -Met-enkephalin YGGFM 573.23 573.0Met-enkephalin-Arg-Phe 263YGGFMRF269 876.40 876.3Met-enkephalin-Arg-Gly-Leu 188YGGFMRGL195 899.43 -Met-enkephalin-Arg-Gly-Leu 188YGGFMRGL195 Oxidation (M) 915.43 915.2- 201LEDEAKEL208 945.47 945.3- 220GRPEWWM226 960.43 960.1- 202EDEAKELQ209 960.44 960.2- 201LEDEAKELQ209 1073.52 1073.3- 198SPQLEDEAKE207 1144.52 1144.3- 200QLEDEAKELQ209 1201.58 1201.3- 198SPQLEDEAKEL208 1257.61 1257.4- 198SPQLEDEAKELQ209 1385.67 1385.5- 120VEPEEEANGGEILA133 1455.67 1455.3- 85KDSSKQDESHLLA97 1456.72 1456.6BAM-18 [8-18] 219VGRPEWWMDYQ229 1465.64 1465.4- 118YPVEPEEEANGGEILA133 1715.79 1715.8- 117LYPVEPEEEANGGEILA133 1828.87 1828.7

Substance P 1-7 58RPKPQQF64 899.50 899.3Neurokinin A 83HKTDSFVGLM92 Amide (C-term) 1132.57 1132.4- 32SDWSDSDQIK41 1179.50 1179.2Substance P 58RPKPQQFFGLM68 Amide (C-term) 1346.73 1348.0C-terminal-flanking peptide 111ALNSVAYERSAMQNYE126 1844.84 1844.9

P06300

P04094

P06767

Proenkephalin A

Prodynorphin

Protachykinin-1

Summarizing the findings of the neuropeptidomic study indicates strain-differences regarding the neuropeptide levels. Comparisons of basal neuropeptide levels revealed the largest differences between the Wistar and Sprague-Dawley rats. Neuropeptide levels during morphine-withdrawal showed only minor differences between the strains. The observed strain-difference of the neuropeptides levels is in accordance with an earlier study [118]. The here presented MS-based quantitative neuropeptide analysis revealed results comparable to findings, obtained by earlier radioimmunoassay-utilizing studies. This shows the usefulness of this method. Besides the possibility to conduct studies focusing on a few peptides, this approach is well suited to study the whole peptidome.

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Concluding remarks

The outcomes of the studies included in this thesis are:

• A single dose of nandrolone decanoate led to increased phosphorylation of the NMDAR subunits GluN2A and GluN2B after 24 h in rat hippocampal synaptoneurosomes. Analogous increases were found for ERK1- and ERK2 phosphorylation.

• A single dose of free nandrolone led to a rapid and transient

decrease in the phosphorylation of the NMDAR subunit GluN2B paralleled by a decreased ERK phosphorylation.

• Nandrolone-induced alterations were abolished by the androgen

receptor antagonist flutamide. Unexpectedly, flutamide alone increased ERK phosphorylation.

• Neither nandrolone nor flutamide altered marker proteins related to

neuronal differentiation and synaptic plasticity.

• MS-based phosphoproteomics identified proteins with known and not yet described phosphorylated residues in the rat hippocampal synaptoneurosomes. However, quantitative nandrolone-induced alterations of these proteins were not observed.

• Quantitative MS-based peptidomics were successfully applied in

the investigation of neuropeptide levels during morphine-withdrawal. Strain-differences were observed regarding the levels of endogenous peptides of the opioid- and tachykinin system.

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Acknowledgements

The presented work was carried out during a 5-year period at the Division of Biological Research on Drug Dependence, Department of Pharmaceutical Biosciences, Faculty of Pharmacy, Uppsala University, Sweden. I would like to express my sincere gratitude to: Pierre Le Grevès, my supervisor, for supporting my wish to conduct my Ph.D.-studies at the Uppsala University. For introducing me to neuroscience, your guidance and support throughout my entire Ph.D.-student time. For all help and advice, and believing in me in good times as in bad. Professor Fred Nyberg, my co-supervisor, for giving me the opportunity to conduct my Ph.D.-studies in his lab, for the possibility to attend conferences and supporting my ideas. My co-supervisor, Matthias Hallberg, for discussions and funny Innebandy sessions. Collaborators and co-authors for contributions and sharing their expertise and techniques.

The mass spectrometry lab: Per Andrén, Anna Nilsson, Maria Fälth Savitski, Kim Kultima, Malin Andersson and Henrik Wadensten. John Flensburg from GE Healthcare, Uppsala Qin Zhou, Madeleine Le Grevès, Johan Alsiö, Torsten Gordh, Pia Steensland

Group members for:

Britt-Marie Johansson, the most valuable person in the lab and being a warm hearted person. Alfhild Grönblad, Anna Carlsson, Georgy Bakalkin, Hiroyuki Watanabe, Igor Bazov, Jenny Johansson, Kristina Magnusson, Milad Botros, Mumtaz Tariq, Nasim Ghasemzadeh, Richard Henriksson, Tatiana Iakovleva

54

The people involved in teaching:

Fredrik Jernerén, Sadia Oreland, Tomas Nilsson, Loudin Daoura, Inga Hoffmann, Marie Eketjäll

Erika Johansson, Agneta Bergström, Annika Häger, Raili Engdahl. Especially, Magnus Jansson for fast help, whenever the computer was in trouble. The prototype of a researcher, Professor Ernst Oliw, for advice. The past members of the group, Carolina, Tobias and Martin, for advice, for countless lunch times discussing things even beyond heaven and earth, and for joining parties. Birger, Henrik, Mats, Oskar and Måns, the guys from the Department of Toxicology for discussions, from highly scientific to hilariously trivial. For joining the SfN conference and beer degustations. Not to forget Ephi, Wolfi, Matze, Katrin, Elfi, Opel, Marten in Germany and elsewhere for keeping friendship. Danke sagen möchte ich: Mutter und Vater, für euer endloses Vertrauen und eure Unterstützung in all den Jahren. Vor allem für eure Entbehrungen das eigene Kind nicht in eurer Nähe zu wissen. Schwesterherz, dafür, dass ich dich zur Schwester habe. Den Omas und Opas, für eure Unterstützung. Ebenso Christel, Wolfgang und Matthias für eure Unterstützung. Vor allem aber Ulrike, für deine unendliche Liebe, deine Unterstützung und die Aufmunterungen in den Zeiten unseres Doktorandendaseins. Ohne Dich wäre mein Leben freudlos und leer. Pauline, für deine Freude bringende Unbekümmertheit, das Glück Dich aufwachsen zu sehen, und dein Papa sein zu dürfen. Funding was obtained from the Swedish Research council (Grant 9459), Uppsala Berzelii Center for Neurodiagnostics, K&A Wallenberg Foundation, G&JA Anérs Foundation and Åhléns Foundation. Travel grants were kindly provided by the Swedish Academy of Pharmaceutical Sciences.

55

References

1. Wilson, J.D. Androgens. in Goodman and Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (eds. Hardman J.G., Gilman A.G. & Limbird L.E.) 1441-1457 (McGraw-Hill, 1995).

2. Wilson, J.D. Androgen abuse by athletes. Endocr Rev 9, 181-199 (1988). 3. Brueggemeier, R.W. Male Sex Hormones, Analogs, and Antagonists. in Burger's

Medical Chemistry and Drug Discovery, Vol. Vol. 3 (ed. Wolff, M.E.) 445-510 (John Wiley & Sons, Inc., 1996).

4. Wolff, M.E. (ed.) Burger's medicial chemistry and drug discovery. Volume 3: Therapeutic agents., 458-460 (John Wiley & Sons, Inc., 1996).

5. Kochakian, C.D. Definition of androgens and protein anabolic steroids. Pharmacol Ther B 1, 149-177 (1975).

6. Edgren, R.A. A Comparative Study Of The Anabolic And Androgenic Effects Of Various Steroids. Acta Endocrinol (Copenh) 44, SUPPL87:81-21 (1963).

7. Evans, N.A. Current concepts in anabolic-androgenic steroids. Am J Sports Med 32, 534-542 (2004).

8. Sundaram, K., Kumar, N., Monder, C. & Bardin, C.W. Different patterns of metabolism determine the relative anabolic activity of 19-norandrogens. J Steroid Biochem Mol Biol 53, 253-257 (1995).

9. Minto, C.F., Howe, C., Wishart, S., Conway, A.J. & Handelsman, D.J. Pharmacokinetics and pharmacodynamics of nandrolone esters in oil vehicle: effects of ester, injection site and injection volume. J Pharmacol Exp Ther 281, 93-102 (1997).

10. Wijnand, H.P., Bosch, A.M. & Donker, C.W. Pharmacokinetic parameters of nandrolone (19-nortestosterone) after intramuscular administration of nandrolone decanoate (Deca-Durabolin) to healthy volunteers. Acta Endocrinol Suppl (Copenh) 271, 19-30 (1985).

11. Bagatell, C.J. & Bremner, W.J. Androgens in men--uses and abuses. N Engl J Med 334, 707-714 (1996).

12. Hoberman, J.M. & Yesalis, C.E. The history of synthetic testosterone. Sci Am 272, 76-81 (1995).

13. Franke, W.W. & Berendonk, B. Hormonal doping and androgenization of athletes: a secret program of the German Democratic Republic government. Clin Chem 43, 1262-1279 (1997).

14. Kindlundh, A.M., Hagekull, B., Isacson, D.G. & Nyberg, F. Adolescent use of anabolic-androgenic steroids and relations to self-reports of social, personality and health aspects. Eur J Public Health 11, 322-328 (2001).

15. Eaton, D.K., et al. Youth risk behavior surveillance--United States, 2005. MMWR Surveill Summ 55, 1-108 (2006).

56

16. Llewellyn, W. Anabolics 2002: Anabolic Steroid Reference Manual, (Molecular Nutrition Patchogue, NY, 2002).

17. Fudala, P.J., Weinrieb, R.M., Calarco, J.S., Kampman, K.M. & Boardman, C. An evaluation of anabolic-androgenic steroid abusers over a period of 1 year: seven case studies. Ann Clin Psychiatry 15, 121-130 (2003).

18. Pagonis, T.A., Angelopoulos, N.V., Koukoulis, G.N. & Hadjichristodoulou, C.S. Psychiatric side effects induced by supraphysiological doses of combinations of anabolic steroids correlate to the severity of abuse. Eur Psychiatry 21, 551-562 (2006).

19. Talih, F., Fattal, O. & Malone, D., Jr. Anabolic steroid abuse: psychiatric and physical costs. Cleve Clin J Med 74, 341-344, 346, 349-352 (2007).

20. Mangelsdorf, D.J., et al. The nuclear receptor superfamily: the second decade. Cell 83, 835-839 (1995).

21. Kerr, J.E., Allore, R.J., Beck, S.G. & Handa, R.J. Distribution and hormonal regulation of androgen receptor (AR) and AR messenger ribonucleic acid in the rat hippocampus. Endocrinology 136, 3213-3221 (1995).

22. Jenster, G., Trapman, J. & Brinkmann, A.O. Nuclear import of the human androgen receptor. Biochem J 293 ( Pt 3), 761-768 (1993).

23. Georget, V., et al. Trafficking of the androgen receptor in living cells with fused green fluorescent protein-androgen receptor. Mol Cell Endocrinol 129, 17-26 (1997).

24. Sarkey, S., Azcoitia, I., Garcia-Segura, L.M., Garcia-Ovejero, D. & DonCarlos, L.L. Classical androgen receptors in non-classical sites in the brain. Horm Behav 53, 753-764 (2008).

25. Tabori, N.E., et al. Ultrastructural evidence that androgen receptors are located at extranuclear sites in the rat hippocampal formation. Neuroscience 130, 151-163 (2005).

26. Lieberherr, M. & Grosse, B. Androgens increase intracellular calcium concentration and inositol 1,4,5-trisphosphate and diacylglycerol formation via a pertussis toxin-sensitive G-protein. J Biol Chem 269, 7217-7223 (1994).

27. Foradori, C.D., Weiser, M.J. & Handa, R.J. Non-genomic actions of androgens. Front Neuroendocrinol 29, 169-181 (2008).

28. Brailoiu, E., et al. Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system. J Endocrinol 193, 311-321 (2007).

29. Pope, H.G., Jr. & Katz, D.L. Psychiatric and medical effects of anabolic-androgenic steroid use. A controlled study of 160 athletes. Arch Gen Psychiatry 51, 375-382 (1994).

30. Malone, D.A., Jr., Dimeff, R.J., Lombardo, J.A. & Sample, R.H. Psychiatric effects and psychoactive substance use in anabolic-androgenic steroid users. Clin J Sport Med 5, 25-31 (1995).

31. Pope, H.G., Jr., Kouri, E.M. & Hudson, J.I. Effects of supraphysiologic doses of testosterone on mood and aggression in normal men: a randomized controlled trial. Arch Gen Psychiatry 57, 133-140; discussion 155-136 (2000).

32. Kouri, E.M., Lukas, S.E., Pope, H.G., Jr. & Oliva, P.S. Increased aggressive responding in male volunteers following the administration of gradually increasing doses of testosterone cypionate. Drug Alcohol Depend 40, 73-79 (1995).

57

33. McGinnis, M.Y. Anabolic androgenic steroids and aggression: studies using animal models. Ann N Y Acad Sci 1036, 399-415 (2004).

34. Albert, D.J., Jonik, R.H., Watson, N.V., Gorzalka, B.B. & Walsh, M.L. Hormone-dependent aggression in male rats is proportional to serum testosterone concentration but sexual behavior is not. Physiol Behav 48, 409-416 (1990).

35. Lumia, A.R., Thorner, K.M. & McGinnis, M.Y. Effects of chronically high doses of the anabolic androgenic steroid, testosterone, on intermale aggression and sexual behavior in male rats. Physiol Behav 55, 331-335 (1994).

36. Farrell, S.F. & McGinnis, M.Y. Long-term effects of pubertal anabolic-androgenic steroid exposure on reproductive and aggressive behaviors in male rats. Horm Behav 46, 193-203 (2004).

37. Aikey, J.L., Nyby, J.G., Anmuth, D.M. & James, P.J. Testosterone rapidly reduces anxiety in male house mice (Mus musculus). Horm Behav 42, 448-460 (2002).

38. Bitran, D., Kellogg, C.K. & Hilvers, R.J. Treatment with an anabolic-androgenic steroid affects anxiety-related behavior and alters the sensitivity of cortical GABAA receptors in the rat. Horm Behav 27, 568-583 (1993).

39. Kouvelas, D., et al. Nandrolone abuse decreases anxiety and impairs memory in rats via central androgenic receptors. Int J Neuropsychopharmacol 11, 925-934 (2008).

40. Minkin, D.M., Meyer, M.E. & van Haaren, F. Behavioral effects of long-term administration of an anabolic steroid in intact and castrated male Wistar rats. Pharmacol Biochem Behav 44, 959-963 (1993).

41. Hermans, E.J., Putman, P., Baas, J.M., Koppeschaar, H.P. & van Honk, J. A single administration of testosterone reduces fear-potentiated startle in humans. Biol Psychiatry 59, 872-874 (2006).

42. Jorge-Rivera, J.C., McIntyre, K.L. & Henderson, L.P. Anabolic steroids induce region- and subunit-specific rapid modulation of GABA(A) receptor-mediated currents in the rat forebrain. J Neurophysiol 83, 3299-3309 (2000).

43. Masonis, A.E. & McCarthy, M.P. Effects of the androgenic/anabolic steroid stanozolol on GABAA receptor function: GABA-stimulated 36Cl- influx and [35S] TBPS binding. J Pharmacol Exp Ther 279, 186-193 (1996).

44. Mukai, H., et al. Modulation of synaptic plasticity by brain estrogen in the hippocampus. Biochim Biophys Acta (2009).

45. Parducz, A., et al. Synaptic remodeling induced by gonadal hormones: neuronal plasticity as a mediator of neuroendocrine and behavioral responses to steroids. Neuroscience 138, 977-985 (2006).

46. MacLusky, N.J., Hajszan, T., Prange-Kiel, J. & Leranth, C. Androgen modulation of hippocampal synaptic plasticity. Neuroscience 138, 957-965 (2006).

47. Roof, R.L. & Havens, M.D. Testosterone improves maze performance and induces development of a male hippocampus in females. Brain Res 572, 310-313 (1992).

48. Wimer, C.C. & Wimer, R.E. On the sources of strain and sex differences in granule cell number in the dentate area of house mice. Brain Res Dev Brain Res 48, 167-176 (1989).

49. Parducz, A. & Garcia-Segura, L.M. Sexual differences in the synaptic connectivity in the rat dentate gyrus. Neurosci Lett 161, 53-56 (1993).

50. Leranth, C., Petnehazy, O. & MacLusky, N.J. Gonadal hormones affect spine synaptic density in the CA1 hippocampal subfield of male rats. J Neurosci 23, 1588-1592 (2003).

58

51. Leranth, C., Prange-Kiel, J., Frick, K.M. & Horvath, T.L. Low CA1 spine synapse density is further reduced by castration in male non-human primates. Cereb Cortex 14, 503-510 (2004).

52. Woolley, C.S. & McEwen, B.S. Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat. J Neurosci 12, 2549-2554 (1992).

53. Woolley, C.S., Gould, E., Frankfurt, M. & McEwen, B.S. Naturally occurring fluctuation in dendritic spine density on adult hippocampal pyramidal neurons. J Neurosci 10, 4035-4039 (1990).

54. Leranth, C., Hajszan, T. & MacLusky, N.J. Androgens increase spine synapse density in the CA1 hippocampal subfield of ovariectomized female rats. J Neurosci 24, 495-499 (2004).

55. Falter, C.M., Arroyo, M. & Davis, G.J. Testosterone: activation or organization of spatial cognition? Biol Psychol 73, 132-140 (2006).

56. Ulubaev, A., Lee, D.M., Purandare, N., Pendleton, N. & Wu, F.C. Activational effects of sex hormones on cognition in men. Clin Endocrinol (Oxf) 71, 607-623 (2009).

57. Silverman, I., Kastuk, D., Choi, J. & Phillips, K. Testosterone levels and spatial ability in men. Psychoneuroendocrinology 24, 813-822 (1999).

58. Driscoll, I., Hamilton, D.A., Yeo, R.A., Brooks, W.M. & Sutherland, R.J. Virtual navigation in humans: the impact of age, sex, and hormones on place learning. Horm Behav 47, 326-335 (2005).

59. Moffat, S.D. & Hampson, E. A curvilinear relationship between testosterone and spatial cognition in humans: possible influence of hand preference. Psychoneuroendocrinology 21, 323-337 (1996).

60. O'Connor, D.B., Archer, J., Hair, W.M. & Wu, F.C. Activational effects of testosterone on cognitive function in men. Neuropsychologia 39, 1385-1394 (2001).

61. Salminen, E.K., Portin, R.I., Koskinen, A., Helenius, H. & Nurmi, M. Associations between serum testosterone fall and cognitive function in prostate cancer patients. Clin Cancer Res 10, 7575-7582 (2004).

62. Cherrier, M.M., Rose, A.L. & Higano, C. The effects of combined androgen blockade on cognitive function during the first cycle of intermittent androgen suppression in patients with prostate cancer. J Urol 170, 1808-1811 (2003).

63. Magnusson, K., et al. Nandrolone decanoate administration elevates hippocampal prodynorphin mRNA expression and impairs Morris water maze performance in male rats. Neurosci Lett 467, 189-193 (2009).

64. Holscher, C. Time, space and hippocampal functions. Rev Neurosci 14, 253-284 (2003).

65. Adams, J.P. & Sweatt, J.D. Molecular psychology: roles for the ERK MAP kinase cascade in memory. Annu Rev Pharmacol Toxicol 42, 135-163 (2002).

66. Amaral, D.G. & Ritter, M.P. Hippocampal formation. in The rat nervous system (ed. Paxinos, G.) 443-493 (Academic Press, Inc., San Diego, 1995).

67. Shaul, Y.D. & Seger, R. The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim Biophys Acta 1773, 1213-1226 (2007).

68. Alessi, D.R., et al. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. Embo J 13, 1610-1619 (1994).

59

69. Payne, D.M., et al. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). Embo J 10, 885-892 (1991).

70. Seger, R., et al. Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. J Biol Chem 267, 14373-14381 (1992).

71. Seger, R. & Krebs, E.G. The MAPK signaling cascade. Faseb J 9, 726-735 (1995). 72. Boulton, T.G., et al. ERKs: a family of protein-serine/threonine kinases that are

activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65, 663-675 (1991).

73. Saba-El-Leil, M.K., et al. An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development. EMBO Rep 4, 964-968 (2003).

74. Pages, G., et al. Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Science 286, 1374-1377 (1999).

75. Lewis, T.S., Shapiro, P.S. & Ahn, N.G. Signal transduction through MAP kinase cascades. Adv Cancer Res 74, 49-139 (1998).

76. Ueda, T., Watanabe-Fukunaga, R., Fukuyama, H., Nagata, S. & Fukunaga, R. Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development. Mol Cell Biol 24, 6539-6549 (2004).

77. Kelleher, R.J., 3rd, Govindarajan, A., Jung, H.Y., Kang, H. & Tonegawa, S. Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell 116, 467-479 (2004).

78. Mayer, M.L., Westbrook, G.L. & Guthrie, P.B. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 309, 261-263 (1984).

79. Paoletti, P. & Neyton, J. NMDA receptor subunits: function and pharmacology. Curr Opin Pharmacol 7, 39-47 (2007).

80. Monyer, H., Burnashev, N., Laurie, D.J., Sakmann, B. & Seeburg, P.H. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12, 529-540 (1994).

81. Kennedy, M.B. Signal-processing machines at the postsynaptic density. Science 290, 750-754 (2000).

82. Nakazawa, T., et al. Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N-methyl-D-aspartate receptor. J Biol Chem 276, 693-699 (2001).

83. Grant, S.G., et al. Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science 258, 1903-1910 (1992).

84. Prybylowski, K., et al. The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2. Neuron 47, 845-857 (2005).

85. Li, B.S., et al. Regulation of NMDA receptors by cyclin-dependent kinase-5. Proc Natl Acad Sci U S A 98, 12742-12747 (2001).

86. Angelo, M., Plattner, F. & Giese, K.P. Cyclin-dependent kinase 5 in synaptic plasticity, learning and memory. J Neurochem 99, 353-370 (2006).

87. Wang, J., Liu, S., Fu, Y., Wang, J.H. & Lu, Y. Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors. Nat Neurosci 6, 1039-1047 (2003).

60

88. Morris, R.G. D.O. Hebb: The Organization of Behavior, Wiley: New York; 1949. Brain Res Bull 50, 437 (1999).

89. Lamprecht, R. & LeDoux, J. Structural plasticity and memory. Nat Rev Neurosci 5, 45-54 (2004).

90. Lisman, J.E. & McIntyre, C.C. Synaptic plasticity: a molecular memory switch. Curr Biol 11, R788-791 (2001).

91. Waltereit, R. & Weller, M. Signaling from cAMP/PKA to MAPK and synaptic plasticity. Mol Neurobiol 27, 99-106 (2003).

92. Krapivinsky, G., et al. The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1. Neuron 40, 775-784 (2003).

93. Yamauchi, T. Molecular constituents and phosphorylation-dependent regulation of the post-synaptic density. Mass Spectrom Rev 21, 266-286 (2002).

94. Tanaka, C. & Nishizuka, Y. The protein kinase C family for neuronal signaling. Annu Rev Neurosci 17, 551-567 (1994).

95. Silva, A.J., Stevens, C.F., Tonegawa, S. & Wang, Y. Deficient hippocampal long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice. Science 257, 201-206 (1992).

96. English, J.D. & Sweatt, J.D. Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem 271, 24329-24332 (1996).

97. English, J.D. & Sweatt, J.D. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem 272, 19103-19106 (1997).

98. Nateri, A.S., et al. ERK activation causes epilepsy by stimulating NMDA receptor activity. Embo J 26, 4891-4901 (2007).

99. Arvary, D. & Pope, H.G., Jr. Anabolic-androgenic steroids as a gateway to opioid dependence. N Engl J Med 342, 1532 (2000).

100. Denham, B.E. Association between narcotic use and anabolic-androgenic steroid use among American adolescents. Subst Use Misuse 44, 2043-2061 (2009).

101. Koob, G.F., Le Moal, M. Neurobiology of addiction. , (Academic Press Elsevier: San Diego, CA, USA, 2006).

102. O'Brien, C.P. Chapter 23. Drug Addiction and Drug Abuse. in Goodman & Gilman's The Pharmacological Basis of Therapeutics, (eds. Brunton, L.L., Lazo, J.S. & Parker, K.L.) (McGraw-Hill, 2005).

103. Gutstein, H.B. & H., A. Chapter 21. Opioid Analgesics. in Goodman & Gilman's The Pharmacological Basis of Therapeutics, (eds. Brunton, L.L., Lazo, J.S. & Parker, K.L.) (McGraw-Hill, 2005).

104. Pert, C.B. & Snyder, S.H. Opiate receptor: demonstration in nervous tissue. Science 179, 1011-1014 (1973).

105. Simon, E.J., Hiller, J.M. & Edelman, I. Stereospecific binding of the potent narcotic analgesic (3H) Etorphine to rat-brain homogenate. Proc Natl Acad Sci U S A 70, 1947-1949 (1973).

106. Terenius, L. Stereospecific interaction between narcotic analgesics and a synaptic plasm a membrane fraction of rat cerebral cortex. Acta Pharmacol Toxicol (Copenh) 32, 317-320 (1973).

107. Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E. & Gilbert, P.E. The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog. J Pharmacol Exp Ther 197, 517-532 (1976).

61

108. Lord, J.A., Waterfield, A.A., Hughes, J. & Kosterlitz, H.W. Endogenous opioid peptides: multiple agonists and receptors. Nature 267, 495-499 (1977).

109. Walker, J.M., et al. Sigma receptors: biology and function. Pharmacol Rev 42, 355-402 (1990).

110. Bunzow, J.R., et al. Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, delta or kappa opioid receptor type. FEBS Lett 347, 284-288 (1994).

111. Mollereau, C., et al. ORL1, a novel member of the opioid receptor family. Cloning, functional expression and localization. FEBS Lett 341, 33-38 (1994).

112. Wise, R.A. Brain reward circuitry: insights from unsensed incentives. Neuron 36, 229-240 (2002).

113. Nestler, E.J., Hope, B.T. & Widnell, K.L. Drug addiction: a model for the molecular basis of neural plasticity. Neuron 11, 995-1006 (1993).

114. Rasmussen, K., Beitner-Johnson, D.B., Krystal, J.H., Aghajanian, G.K. & Nestler, E.J. Opiate withdrawal and the rat locus coeruleus: behavioral, electrophysiological, and biochemical correlates. J Neurosci 10, 2308-2317 (1990).

115. Maldonado, R., Stinus, L., Gold, L.H. & Koob, G.F. Role of different brain structures in the expression of the physical morphine withdrawal syndrome. J Pharmacol Exp Ther 261, 669-677 (1992).

116. Gudehithlu, K.P., Tejwani, G.A. & Bhargava, H.N. Beta-endorphin and methionine-enkephalin levels in discrete brain regions, spinal cord, pituitary gland and plasma of morphine tolerant-dependent and abstinent rats. Brain Res 553, 284-290 (1991).

117. Nylander, I., Vlaskovska, M. & Terenius, L. The effects of morphine treatment and morphine withdrawal on the dynorphin and enkephalin systems in Sprague-Dawley rats. Psychopharmacology (Berl) 118, 391-400 (1995).

118. Nylander, I., Vlaskovska, M. & Terenius, L. Brain dynorphin and enkephalin systems in Fischer and Lewis rats: effects of morphine tolerance and withdrawal. Brain Res 683, 25-35 (1995).

119. Bergstrom, L. & Terenius, L. Enkephalin levels decrease in rat striatum during morphine abstinence. Eur J Pharmacol 60, 349-352 (1979).

120. Childers, S.R., Simantov, R. & Snyder, S.H. Enkephalin: radioimmunoassay and radioreceptor assay in morphine dependent rats. Eur J Pharmacol 46, 289-293 (1977).

121. Wesche, D., Hollt, V. & Herz, A. Radioimmunoassay of enkephalins. Regional distribution in rat brain after morphine treatment and hypophysectomy. Naunyn Schmiedebergs Arch Pharmacol 301, 79-82 (1977).

122. Nestler, E.J., Hyman, S.E. & Malenka, R.C. Molecular Neuropharmacology: a foundation for clinical neuroscience, (McGraw-Hill, New York, 2001).

123. Bradbury, A.F., Finnie, M.D. & Smyth, D.G. Mechanism of C-terminal amide formation by pituitary enzymes. Nature 298, 686-688 (1982).

124. Smyth, D.G., Massey, D.E., Zakarian, S. & Finnie, M.D. Endorphins are stored in biologically active and inactive forms: isolation of alpha-N-acetyl peptides. Nature 279, 252-254 (1979).

125. Benjannet, S., Rondeau, N., Day, R., Chretien, M. & Seidah, N.G. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci U S A 88, 3564-3568 (1991).

62

126. Bicknell, A.B. The tissue-specific processing of pro-opiomelanocortin. J Neuroendocrinol 20, 692-699 (2008).

127. Stengaard-Pedersen, K. & Larsson, L.I. Comparative immunocytochemical localization of putative opioid ligands in the central nervous system. Histochemistry 73, 89-114 (1981).

128. Dupuy, A., et al. Processing of prodynorphin by the prohormone convertase PC1 results in high molecular weight intermediate forms. Cleavage at a single arginine residue. FEBS Lett 337, 60-65 (1994).

129. Day, R., et al. Prodynorphin processing by proprotein convertase 2. Cleavage at single basic residues and enhanced processing in the presence of carboxypeptidase activity. J Biol Chem 273, 829-836 (1998).

130. Furuta, T., Zhou, L. & Kaneko, T. Preprodynorphin-, preproenkephalin-, preprotachykinin A- and preprotachykinin B-immunoreactive neurons in the accumbens nucleus and olfactory tubercle: double-immunofluorescence analysis. Neuroscience 114, 611-627 (2002).

131. Breslin, M.B., et al. Differential processing of proenkephalin by prohormone convertases 1(3) and 2 and furin. J Biol Chem 268, 27084-27093 (1993).

132. Johanning, K., Mathis, J.P. & Lindberg, I. Role of PC2 in proenkephalin processing: antisense and overexpression studies. J Neurochem 66, 898-907 (1996).

133. Mathis, J.P. & Lindberg, I. Posttranslational processing of proenkephalin in AtT-20 cells: evidence for cleavage at a Lys-Lys site. Endocrinology 131, 2287-2296 (1992).

134. Miller, R.J. & Pickel, V.M. The distribution and functions of the enkephalins. J Histochem Cytochem 28, 903-917 (1980).

135. Fukunaga, Y. & Kishioka, S. Enkephalinergic neurons in the periaqueductal gray and morphine withdrawal. Jpn J Pharmacol 82, 175-180 (2000).

136. Bruijnzeel, A.W. kappa-Opioid receptor signaling and brain reward function. Brain Res Rev 62, 127-146 (2009).

137. Cox, B.M., et al. Opioid receptors. in IUPHAR database (Last modified on 2009-10-29.).

138. Mansour, A., Hoversten, M.T., Taylor, L.P., Watson, S.J. & Akil, H. The cloned mu, delta and kappa receptors and their endogenous ligands: evidence for two opioid peptide recognition cores. Brain Res 700, 89-98 (1995).

139. Conlon, J.M. The tachykininpeptide family, with particular emphasis on mammalian tachykinins and tachykinin receptor agonists. in Tachykinins, Vol. 164 (ed. Holzer, P.) 25-61 (Springer-Verlag, Berlin, 2004).

140. Boyce, S. & Hill, R.G. Substance P (NK1) receptor antagonist -analgesics or not? in Tachykinins, Vol. 164 (ed. Holzer, P.) 441-457 (Springer-Verlag, Berlin, 2004).

141. Nylander, I., Sakurada, T., Le Greves, P. & Terenius, L. Levels of dynorphin peptides, substance P and CGRP in the spinal cord after subchronic administration of morphine in the rat. Neuropharmacology 30, 1219-1223 (1991).

142. Tiong, G.K., Pierce, T.L. & Olley, J.E. Sub-chronic exposure to opiates in the rat: effects on brain levels of substance P and calcitonin gene-related peptide during dependence and withdrawal. J Neurosci Res 32, 569-575 (1992).

143. Bergstrom, L., Sakurada, T. & Terenius, L. Substance P levels in various regions of the rat central nervous system after acute and chronic morphine treatment. Life Sci 35, 2375-2382 (1984).

63

144. Nyberg, F., Le Greves, P., Sundqvist, C. & Terenius, L. Characterization of substance P(1-7) and (1-8) generating enzyme in human cerebrospinal fluid. Biochem Biophys Res Commun 125, 244-250 (1984).

145. Hallberg, M. & Nyberg, F. Neuropeptide conversion to bioactive fragments--an important pathway in neuromodulation. Curr Protein Pept Sci 4, 31-44 (2003).

146. Paulie, S., Perlmann, H. & Perlmann, P. Enzyme-linked Immunosorbent Assay. Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester. DOI: 10.1038/npg.els.0004021 (2005).

147. Ong, S.-E. & Liang, C.M.Y.R. Two-dimensional Gel Electrophoresis. Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester. DOI: 10.1038/npg.els.0006191 (2006).

148. Granlund, I., Hall, M. & Schroder, W.P. Difference Gel Electrophoresis (DIGE). Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0021881 (2009).

149. Delahunty, C. & Yates, R.J. Proteomics: A Shotgun Approach without Two-dimensional Gels. Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester. DOI: 10.1038/npg.els.0006197 (2006).

150. Roepstorff, P. Mass Spectrometry Instrumentation in Proteomics. Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester. DOI: 10.1038/npg.els.0006194 (2006).

151. Link, A.J., et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 17, 676-682 (1999).

152. Thingholm, T.E. & Jensen, O.N. Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry. Methods Mol Biol 527, 47-56, xi (2009).

153. Eng, J.K., McCormack, A.L. & Yates 3rd, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry 5, 976-989 (1994).

154. Perkins, D.N., Pappin, D.J., Creasy, D.M. & Cottrell, J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551-3567 (1999).

155. Fenyo, D. & Beavis, R.C. A method for assessing the statistical significance of mass spectrometry-based protein identifications using general scoring schemes. Anal Chem 75, 768-774 (2003).

156. Ong, S.E., et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1, 376-386 (2002).

157. Gygi, S.P., et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17, 994-999 (1999).

158. Ross, P.L., et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3, 1154-1169 (2004).

159. Zhu, W., Smith, J.W. & Huang, C.M. Mass spectrometry-based label-free quantitative proteomics. J Biomed Biotechnol 2010, 840518.

160. Baggerman, G., et al. Peptidomics. J Chromatogr B Analyt Technol Biomed Life Sci 803, 3-16 (2004).

64

161. Fricker, L.D., Lim, J., Pan, H. & Che, F.Y. Peptidomics: identification and quantification of endogenous peptides in neuroendocrine tissues. Mass Spectrom Rev 25, 327-344 (2006).

162. Tirassa, P., et al. High-dose anabolic androgenic steroids modulate concentrations of nerve growth factor and expression of its low affinity receptor (p75-NGFr) in male rat brain. J Neurosci Res 47, 198-207 (1997).

163. Lindblom, J., Kindlundh, A.M., Nyberg, F., Bergstrom, L. & Wikberg, J.E. Anabolic androgenic steroid nandrolone decanoate reduces hypothalamic proopiomelanocortin mRNA levels. Brain Res 986, 139-147 (2003).

164. Lindqvist, A.S. & Fahlke, C. Nandrolone decanoate has long-term effects on dominance in a competitive situation in male rats. Physiol Behav 84, 45-51 (2005).

165. Alsio, J., et al. Impact of nandrolone decanoate on gene expression in endocrine systems related to the adverse effects of anabolic androgenic steroids. Basic Clin Pharmacol Toxicol 105, 307-314 (2009).

166. van der Vies, J. Implications of basic pharmacology in the therapy with esters of nandrolone. Acta Endocrinol Suppl (Copenh) 271, 38-44 (1985).

167. Xu, C.J. & Li, D. Pharmacokinetics of flutamide and its metabolite 2-hydroxyflutamide in normal and hepatic injury rats. Zhongguo Yao Li Xue Bao 19, 39-43 (1998).

168. Schulz, M., Schmoldt, A., Donn, F. & Becker, H. The pharmacokinetics of flutamide and its major metabolites after a single oral dose and during chronic treatment. Eur J Clin Pharmacol 34, 633-636 (1988).

169. Simard, J., Luthy, I., Guay, J., Belanger, A. & Labrie, F. Characteristics of interaction of the antiandrogen flutamide with the androgen receptor in various target tissues. Mol Cell Endocrinol 44, 261-270 (1986).

170. Kumar, N., et al. 7alpha-methyl-19-nortestosterone, a synthetic androgen with high potency: structure-activity comparisons with other androgens. J Steroid Biochem Mol Biol 71, 213-222 (1999).

171. Läkemedelsfakta Eulexin (Schering-Plough), (Läkemedelsindustriföreningen (LIF), Stockholm, 2007).

172. Skold, K., et al. The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: stathmin 2-20 and peptides as sample quality indicators. Proteomics 7, 4445-4456 (2007).

173. Bjerrum, O.J. & Heegaard, N.H.H. Western Blotting: Immunoblotting Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0000995.pub2 (2009).

174. Jaffe, H., Veeranna, Shetty, K.T. & Pant, H.C. Characterization of the phosphorylation sites of human high molecular weight neurofilament protein by electrospray ionization tandem mass spectrometry and database searching. Biochemistry 37, 3931-3940 (1998).

175. Boersema, P.J., Mohammed, S. & Heck, A.J. Phosphopeptide fragmentation and analysis by mass spectrometry. J Mass Spectrom 44, 861-878 (2009).

176. Beausoleil, S.A., Villen, J., Gerber, S.A., Rush, J. & Gygi, S.P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24, 1285-1292 (2006).

65

177. Kultima, K., et al. Development and evaluation of normalization methods for label-free relative quantification of endogenous peptides. Mol Cell Proteomics 8, 2285-2295 (2009).

178. Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M. Real time quantitative PCR. Genome Res 6, 986-994 (1996).

179. Wong, M.L. & Medrano, J.F. Real-time PCR for mRNA quantitation. Biotechniques 39, 75-85 (2005).

180. Vandesompele, J., et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, RESEARCH0034 (2002).

181. Rossbach, U.L., Steensland, P., Nyberg, F. & Le Greves, P. Nandrolone-induced hippocampal phosphorylation of NMDA receptor subunits and ERKs. Biochem Biophys Res Commun 357, 1028-1033 (2007).

182. Rosenblum, K., Dudai, Y. & Richter-Levin, G. Long-term potentiation increases tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit 2B in rat dentate gyrus in vivo. Proc Natl Acad Sci U S A 93, 10457-10460 (1996).

183. Rossbach, U.L., Le Greves, M., Nyberg, F., Zhou, Q. & Le Greves, P. Acute 19-nortestosterone transiently suppresses hippocampal MAPK pathway and the phosphorylation of the NMDA receptor. Mol Cell Endocrinol 314, 143-149.

184. Gatson, J.W., Kaur, P. & Singh, M. Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells. Endocrinology 147, 2028-2034 (2006).

185. Peterziel, H., et al. Rapid signalling by androgen receptor in prostate cancer cells. Oncogene 18, 6322-6329 (1999).

186. Zhu, X., Li, H., Liu, J.P. & Funder, J.W. Androgen stimulates mitogen-activated protein kinase in human breast cancer cells. Mol Cell Endocrinol 152, 199-206 (1999).

187. Lee, Y.F., et al. Activation of mitogen-activated protein kinase pathway by the antiandrogen hydroxyflutamide in androgen receptor-negative prostate cancer cells. Cancer Res 62, 6039-6044 (2002).

188. MacLusky, N.J., Hajszan, T., Johansen, J.A., Jordan, C.L. & Leranth, C. Androgen effects on hippocampal CA1 spine synapse numbers are retained in Tfm male rats with defective androgen receptors. Endocrinology 147, 2392-2398 (2006).

189. Nguyen, T.V., Yao, M. & Pike, C.J. Androgens activate mitogen-activated protein kinase signaling: role in neuroprotection. J Neurochem 94, 1639-1651 (2005).

190. Harley, C.W., Malsbury, C.W., Squires, A. & Brown, R.A. Testosterone decreases CA1 plasticity in vivo in gonadectomized male rats. Hippocampus 10, 693-697 (2000).

191. Bramham, C.R., Worley, P.F., Moore, M.J. & Guzowski, J.F. The immediate early gene arc/arg3.1: regulation, mechanisms, and function. J Neurosci 28, 11760-11767 (2008).

192. Soule, J., Messaoudi, E. & Bramham, C.R. Brain-derived neurotrophic factor and control of synaptic consolidation in the adult brain. Biochem Soc Trans 34, 600-604 (2006).

66

193. Ying, S.W., et al. Brain-derived neurotrophic factor induces long-term potentiation in intact adult hippocampus: requirement for ERK activation coupled to CREB and upregulation of Arc synthesis. J Neurosci 22, 1532-1540 (2002).

194. Grunwald, I.C., et al. Hippocampal plasticity requires postsynaptic ephrinBs. Nat Neurosci 7, 33-40 (2004).

195. Takasu, M.A., Dalva, M.B., Zigmond, R.E. & Greenberg, M.E. Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors. Science 295, 491-495 (2002).

196. Brannvall, K., Bogdanovic, N., Korhonen, L. & Lindholm, D. 19-Nortestosterone influences neural stem cell proliferation and neurogenesis in the rat brain. Eur J Neurosci 21, 871-878 (2005).

197. Quinn, C.C., Gray, G.E. & Hockfield, S. A family of proteins implicated in axon guidance and outgrowth. J Neurobiol 41, 158-164 (1999).

198. Jiang, Y.Q. & Oblinger, M.M. Differential regulation of beta III and other tubulin genes during peripheral and central neuron development. J Cell Sci 103 ( Pt 3), 643-651 (1992).

199. Huang da, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, 44-57 (2009).

200. Dennis, G., Jr., et al. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4, P3 (2003).

201. Hollingsworth, E.B., et al. Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3':5'-monophosphate-generating systems, receptors, and enzymes. J Neurosci 5, 2240-2253 (1985).

202. Gnad, F., et al. PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol 8, R250 (2007).

203. Watanabe, A., et al. In vivo phosphorylation sites in fetal and adult rat tau. J Biol Chem 268, 25712-25717 (1993).

204. Kesavapany, S., et al. Neuronal cyclin-dependent kinase 5: role in nervous system function and its specific inhibition by the Cdk5 inhibitory peptide. Biochim Biophys Acta 1697, 143-153 (2004).

205. Wakisaka, Y., et al. Cellular distribution of NDRG1 protein in the rat kidney and brain during normal postnatal development. J Histochem Cytochem 51, 1515-1525 (2003).

206. Matsuoka, Y., Hughes, C.A. & Bennett, V. Adducin regulation. Definition of the calmodulin-binding domain and sites of phosphorylation by protein kinases A and C. J Biol Chem 271, 25157-25166 (1996).

207. Fukunaga, K., Stoppini, L., Miyamoto, E. & Muller, D. Long-term potentiation is associated with an increased activity of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 268, 7863-7867 (1993).

208. White, J.D., Gall, C.M. & McKelvy, J.F. Proenkephalin is processed in a projection-specific manner in the rat central nervous system. Proc Natl Acad Sci U S A 83, 7099-7103 (1986).

67

209. Hurlbut, D.E., Evans, C.J., Barchas, J.D. & Leslie, F.M. Pharmacological properties of a proenkephalin A-derived opioid peptide: BAM 18. Eur J Pharmacol 138, 359-366 (1987).

210. Rossbach, U., et al. A quantitative peptidomic analysis of peptides related to the endogenous opioid and tachykinin systems in nucleus accumbens of rats following naloxone-precipitated morphine withdrawal. J Proteome Res 8, 1091-1098 (2009).

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