The Impact of Nandrolone Decanoate on Neuropeptidergic Mechanisms - DocsLib

sign in sign up

<<

Home , Aromatization, Dehydroandrosterone, Sex hormone

! " #$""%

""&'(&)* "+),-()(''.(,'-,( /0///1 (,.- ! "##$% &%"' ( ' ' )( (* ')(+,-( . / (,

0,##$,-(1 '2 2 ( 3 4 3. ,4 , %# ,"$ , ,1!2$567$%7""75"657%,

-( ' *44!+ ' ( . ( ''( ( ' ' ( ,44! '.(( '( ,) ( ( . 7 '' .( ( ! ) *!)+ ( ( 8 ( '' 9 ' ( ( , -( ( ( (( ( '( '' ,-( ' '' ( : '!) ( 4 ( !)*%75+ ;7 9 ( 74< ' , ' ( ( '' (9 ,-(. ' ( ' ( ' ( (( .( ,1 ( . :9' . . ,-( '( ( 44!7 ( '',

4 2 ! ) ( 4 0=) ) ( 3 4 ( .:)3

! " ! #$%& '()$&*+

>0 ##$

1!!2%<"%7<%$ 1!2$567$%7""75"657% & &&& 7%#56 *( &?? ,9,? @ A & &&& 7%#56 + Så finns du här så självklar, Linnea – strålande och fin Livets spirande blomma, du underbara dotter min

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 Magnusson, K., Hallberg, M., Kindlundh Högberg, AMS., Ny- berg, F. (2006) Administration of the anabolic androgenic steroid nandrolone decanoate affects substance P endopeptidase- like activity in the rat brain. Peptides, 27(1):114-21 II Magnusson, K., Hallberg, M., Bergquist, J., Nyberg, F. (2007) Enzymatic conversion of dynorphin A in the rat brain is affected by administration of nandrolone decanoate. Peptides, 28(4):851-8 III Magnusson, K., Birgner, C., Bergström, L., Nyberg, F., Hall- berg M. (2009) Nandrolone decanoate administration dose- dependently affects the density of kappa opioid peptide receptors in the rat brain determined by autoradiography. Neuropep- tides, 43(2):105-11 IV Magnusson, K., Hållner, A., Bazov, I., Clausen, F., Zhou, Q., Nyberg, F. Nandrolone decanoate administration elevates hippocampal prodynorphin mRNA expression and impairs Morris water maze performance in male rats. Submitted.

Reprints are published with kind permission from Elsevier Ldt./Inc.

List of additional Papers

Hallberg, M., Magnusson, K., Kindlundh, AMS., Steensland, P., Nyberg, F. (2005) The effect of anabolic androgenic steroids on calcitonin gene-related peptide (CGRP) levels in the rat brain. PharmacologyOnline, 1:178-196

Lindblom, J., Petrovska, R., Hallberg, M., Magnusson, K., Nyberg, F., Uhlén, S. (2005) Nandrolone treatment decreases the α1B-adrenoceptor mRNA level in rat kidney cells. Eur J Pharmacol., 19:527(1-3)

Sahlin, C., Lord, A., Magnusson, K., Englund, H., Almeida, CG., Greengard, P., Nyberg, F., Gouras, GK., Lannfelt, L., Nilsson, LN. (2007) The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase. J Neurochem., 101(3):854-62

Takahashi, K., Hallberg, M., Magnusson, K., Nyberg, F., Watanabe, Y., Långström, B., Bergström, M. (2007) Increase in [11C]vorozole binding to aromatase in the hypothalamus in rats treated with anabolic androgenic steroids. Neuroreport, 18(2):171-4

Contents

Introduction...... 11 Anabolic androgenic steroids ...... 11 Definition...... 11 Use in society...... 11 Testosterone...... 12 Nandrolone ...... 13 Administration patterns ...... 14 Physiological aspects...... 15 Psychological aspects ...... 15 Pharmacological aspects...... 17 The Substance P system ...... 18 The Dynorphinergic system ...... 19 The KOP receptor...... 20 Processing of neuropeptides...... 20 Enzymatic conversion of Substance P...... 21 Enzymatic conversion of Dynorphin A(1-17) ...... 22 Methodological aspects ...... 24 Radioimmunoassay...... 24 Autoradiography...... 24 Morris water maze ...... 25 TaqMan? real-time polymerase chain reaction ...... 25 Aims...... 26 Materials and methods ...... 27 Experimental animal procedures...... 27 Enzyme activities ...... 27 Tissue preparation...... 27 Assay for measurement of SPE-like activity ...... 27 Assay for measurement of DCE-like activity...... 28 HPLC characterization...... 28 Mass spectrometry characterization...... 28 Radioimmunoassay ...... 28 Autoradiography...... 29 Morris water maze...... 29 RNA isolation and cDNA synthesis...... 30

TaqMan? real-time polymerase chain reaction ...... 31 Statistics ...... 31 Results and discussion ...... 32 Effects on Substance P conversion (Paper I)...... 32 Effects on Dynorphin A(1-17) conversion (Paper II)...... 34 Effects on the kappa opioid peptide receptor (Paper III) ...... 35 Effects on Morris water maze performance (Paper IV) ...... 38 Effects on Prodynorphin gene transcription (Paper IV)...... 41 General considerations ...... 42 Nandrolone decanoate administration ...... 42 Summarizing conclusions ...... 43 Populärvetenskaplig sammanfattning ...... 45 Acknowledgements...... 47 References...... 48

Abbreviations

AAS Anabolic androgenic steroid ACE Angiotensin converting enzyme AD Alzheimer’s disease ANOVA Analysis of variance cAMP Cyclic adenosine 3’,5’ monophosphate cDNA Complementary deoxyribonucleic acid CNS Central nervous system CSF Cerebrospinal fluid Ct Threshold cycles DCE Dynorphin converting enzyme DHN Dihydronandrolone DHT Dihydrotestosterone DNA Deoxyribonucleic acid DOP Delta opioid peptide DSM IV Diagnostic and statistical manual of mental disorders, fourth edition Dyn A Dynorphin A(1-17) Dyn B Dynorphin B(1-13) EDTA Ethylenediaminetetraacetic acid FSH Follicle stimulating hormone GABA Gamma amino butyric acid hCG Human chorionic gonadotropin HDL High-density lipoprotein HPLC High performance liquid chromatography i.m. Intramuscular KOP Kappa opioid peptide LDL Low-density lipoprotein LH Luteinizing hormone MOP Mu opioid peptide mRNA Messenger ribonucleic acid MS Mass spectrometry MWM Morris water maze NEP Neutral endopeptidase

NK Neurokinin NMDA N-methyl-D-aspartate PAG Periaqueductal gray PC Prohormone convertase PCR Polymerase chain reaction PDYN Prodynorphin RIA Radioimmunoassay RNA Ribonucleic acid s.c. Subcutaneous SEM Standard error of the mean SP Substance P SPE Substance P endopeptidase VTA Ventral tegmental area

Introduction

Anabolic androgenic steroids Definition “Anabolic androgenic steroids” (AAS) is the designation of the endogenous male sex hormone testosterone and synthetic derivatives of the same. Com- mon effects of all AAS are enhanced tissue building, i.e. anabolic effects, and the development and maintenance of male sexual characteristics, i.e., androgenic effects [1].

Use in society The general belief in today’s society is that the use of AAS is a problem mainly connected to athletes and bodybuilders. However, there are reports, which indicate that the abuse of AAS is spreading among adolescents and young adults not connected to sports [2,3] and media constantly report cases of AAS-use in, for example, connection to violent acts, where the perpetrator has been under the influence of AAS. The prevalence of AAS-use in different countries has been investigated with a number of surveys. Among non-athletes, the estimated life-time prevalence typically ranges between 1-5% [4-9]. The prevalence is some- what higher among athletes [10]. The intake of AAS in order to enhance physical performance is however not a recent phenomenon; testosterone, as well as testosterone-derived substances, have been used for this purpose since the isolation and characterization of the male hormone as discussed more thoroughly below. As often the case with substances of abuse, the original intentions were to find pharmaceutical substances to treat different deceases and to improve health. There is still a clinical use of AAS in the treatment of for example hypogonadism, impotence, osteoporosis and anemia. Yet, the clinical use is minor compared with the illegal use.

11 Testosterone History In 1849, the German professor Berthold von Göttingen discovered that cas- tration of roosters resulted in altered sexual behavior and physical appear- ance [11]; the effects were reversed by the transplantation of testicular tissue into the abdominal cavity. This was thus an early experiment of replacement therapy that demonstrated the endocrine function of the testicles. In 1889, the French physiologist Brown-Séquard proclaimed that he had found a “re- juvenating elixir” and reported of increased strength, improved intellect, relief from constipation, and an increased arc of his urine [12]. He had injected himself with extracts from dog and guinea pig testicles and found the effects to be extraordinary. Several groups started the pursuit for the “male hormone” that resulted in the isolation of androsterone from male urine in 1931. A few years later, two independent research groups, led by Butenandt and Ruzicka, identified the primary male hormone testosterone [13]. For this work they were awarded the Nobel Prize in Chemistry in 1939. During the following years, researchers were dedicated to modify the structure of testosterone to find a substance with pure anabolic effects, avoiding the unwanted androgenic effects. However, to this date there are no scientific reports of the existence of such substances. Nevertheless, due to this quest, a number of testosterone derivatives are now available that together with testosterone comprise the group AAS. The use of AAS in sports commenced with the Russian weightlifters, spread to weightlifters in the USA and then further on to other sports [14]. As mentioned above, the use today also involves other subgroups in the society that are not connected to sports.

Biosynthesis and metabolism Testosterone is biosynthesized from its precursor cholesterol in the Leydig cells of the testicles in males and in the corpus luteum in females. Production also occurs in the adrenal cortex in both genders [15,16]. Testosterone un- dergoes both Phase I and Phase II metabolism, which leads to the formation of a number of metabolites. Phase I reactions generally involve oxidation, reduction or hydroxylation, yielding more polar molecules. Phase II metabolism results in glucuronides or sulfates, which enables elimination through the urine. Two bioactive testosterone metabolites have been of great interest; dihydrotestosterone (DHT) and estradiol, produced by the enzymes 5α- reductase and aromatase, respectively (Fig. 1). Interestingly, DHT displays a much more androgenic effect than that of testosterone. DHT is subsequently converted to either of the inactive metabolites androsterone or etio- cholanolone [16].

12

Figure 1. Testosterone and the metabolites dihydrotestosterone and estradiol generated by actions of the enzymes 5α-reductace (1) and aromatase (2).

Figure 2. Nandrolone decanoate; * marks the lack of a methyl group in C-19 position compared with testosterone.

Nandrolone Nandrolone (19-nortestosterone) was first synthesized in the year 1950 [17]. The steroid displays a reduced androgenic effect compared to testosterone, due to the lack of a methyl group in the 19-position (Fig. 2). Nandrolone is usually esterified with decanoic acid before administration, which makes it suitable as a depot drug for intramuscular (i.m.) injections (Fig. 2). As in the case of testosterone, 5α-reductase will reduce the C-4,5 double bond, yielding the bioactive metabolite dihydronandrolone (DHN) (Fig. 3). However, DHN is less androgenic than nandrolone as opposed to the relationship be-

13 tween DHT and testosterone. Also some aromatization of nandrolone to estrogens occur but to a lesser extent than that of testosterone [15,16]. Nan- drolone is one of the most popular AAS today due to relatively low androgenic effects combined with reduced potential for conversion to estrogens [18].

Figure 3. Nandrolone and the metabolite dihydronandrolone produced by the action of the enzyme 5α-reductase.

Administration patterns Bodybuilders and athletes usually administer AAS according to strict regimes. These regimes normally comprise 2-3 cycles of administration per year where each cycle lasts for 6-12 weeks. A resting period is usually included between the cycles with the intention to minimize adverse effects. In addition, doses are gradually increased, then decreased over the period of the cycle, called “pyramiding” to further reduce behavioral adverse effects caused by sudden withdrawal of the AAS. Furthermore, several different AAS are frequently administered within each cycle, also called “stacking” [19]. The theory behind stacking is that receptor down regulation is less likely to occur when several steroids are used instead of one single. In addition, some steroids are considered to work synergistically when combined. However, this has not been proven scientifically. Doses used within these regimes are often found to be up to 100 times greater than therapeutic levels [19,20]. With this in mind, it is not surprising that a number of physiological as well as psychological adverse effects usually develop after some time of abuse. It is therefore not unusual that a person abusing AAS administer a wide range of other drugs at the same time to reduce AAS-induced adverse effects or to enhance the anabolic effects. For instance, aromatase inhibitors are used to decrease the conversion to estrogens and thus reduce feminizing effects. In addition, since endogenous testosterone decline when exogenous androgens are administered, human chorionic gonadotropin (hCG) is used at the end of a cycle to increase endogenous testosterone production. The list of additional substances can be made long and AAS-users usually possess great

14 knowledge of what drugs to use in order to avoid or minimize unwanted effects as well as to reach the desired effects. The use of AAS is also associated with other drugs of abuse such as heavy alcohol drinking and the abuse of other illicit drugs [2,21-24]. This multi drug-abuse has become apparent among adolescents, even outside athletic and bodybuilding contexts, that take AAS to boost the self-esteem or to become intoxicated [2].

Physiological aspects The use of AAS frequently results in a number of effects besides the often- desired anabolic ones that are either caused by the administered steroid or metabolites thereof. Adverse effects are particularly common among users that administer very high doses of AAS. Typical physiological adverse effects are baldness and dermatological effects such as acne and striae [25]. Furthermore, the administration of high doses of exogenous androgens in men leads to decreased levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) through negative feedback mechanisms. This sequentially results in decreased endogenous testosterone production and may end up in decreased spermatogenesis and testicular atrophy [26]. The conversion of androgens to substrates with estrogenic activity may furthermore result in gynecomastia. In women, the use of AAS generates mascu- linazing effects such as deepening of the voice, characteristic male baldness and clitoromegaly [27]. More concerning physiological effects of AAS-abuse have been described in case reports that address cardiovascular complications, such as myocardial infarction, cardiac dysarythmia, cardiac hypertrophy and stroke [28-32]. Several studies demonstrate an increase in low-density lipoprotein (LDL) and cholesterol and a decrease in high-density lipoprotein (HDL), thus in- creasing the risk of atherosclerotic heart disease [32-35]. Furthermore, the orally active 17α-alkylated steroid derivatives are associated with jaundice and hepatic carcinoma [36,37].

Psychological aspects In addition to the described physiological effects, there are a number of reports pointing at psychological effects as a result of AAS-abuse. However, there is extreme variability in psychological symptoms caused by AAS- abuse due to differences between the AAS, dosage, duration of use, person- ality of the abuser as well as previous or present use of other drugs. Further- more, the biochemical mechanisms behind these effects are less understood than those behind physical effects. A cocktail of steroids are also commonly used, making it even harder to distinguish and correlate psychological effects of a specific steroid.

15 Some of the more prominent psychological effects are manic-like states defined by irritability, aggressiveness, euphoria, grandiose beliefs, hyperac- tivity, and reckless or dangerous behavior [38-42]. It has been shown that AAS-abusers are involved in more fights, are more verbally aggressive, and more violent towards their significant others when using AAS than when not using AAS. The abuse of AAS is unfortunately associated with the term, “roid rage”, where individuals suddenly commit terrible acts of violence [19,41,43,44]. In fact, increased aggression is the most consistent behavioral effect of high-dose AAS exposure in surveys and prospective studies. Ag- gressive behavior as a result of chronic treatment with AAS is also reflected in animal models in rats and hamsters [45,46]. A number of studies report of AAS-dependence, where, in all cases, supraphysiological doses of AAS have been chronically administered [21,27,47-53]. As an example, results from a study conducted on 100 Aus- tralian competitive and recreational AAS-users showed that 23% met the diagnostic and statistical manual of mental disorders (DSM) IV criteria for substance dependence and 25% qualified for substance abuse [54]. Also a very recent study in Sweden reported of AAS-dependence and abuse according to the DSM IV criteria among 32 patients who were attending an addiction centre [21]. Furthermore, individuals who have abused supraphysiological doses of AAS for a long time period of time may display withdrawal-like effects such as depressive symptoms, impaired concentration, fatigue, and suicidal thoughts upon cessation of the abuse [38,40,48,55,56]. In addition, reinforcing properties of androgens was also shown in condition place preference-models in animals such as mice [57] and rats [58]. Other studies demonstrate that rats and hamsters self-administer testosterone when given orally as well as intravenously [59,60], thus further confirming the rewarding properties of androgens. It should however be noted that AAS are not addictive in the same manner as compounds such as amphetamine or heroine. Nevertheless, this does not imply that the steroids are harmless; especially considering the fact that AAS-dependence undoubtedly also is reliant on individual susceptibility. Other psychological features associated with administration of AAS are the development of depression as well as cognitive dysfunctions such as forgetfulness, distractibility, and confusion or delirious states [39,61]. How- ever, the literature describing the effects of AAS on cognition is limited. Only a few controlled studies in humans and in animals regarding cognitive effects of AAS existed when this thesis was initiated. Further, there was a lack of studies displaying biochemical effects in connection to altered cognitive behavior caused by AAS administration.

16 Pharmacological aspects Testosterone and other AAS are thought to exert their actions in the body in similar manners. Different mechanisms have been proposed and include, for example, direct interaction with the androgenic receptor, interference with the glucocorticoid receptor as well as non-genomic pathways mainly found in the central nervous system (CNS), as described more thoroughly in the recent review by Kicman [62]. It is likely that a range of pathways rather than a single constant mechanism conduct the cellular actions of steroids. The most explored mechanism of action for androgens involves the activation of androgenic receptors [63]. These intracellular receptors belong to the nuclear receptor superfamily and possess a ligand-binding domain as well as at least two transcriptional activation domains; this enables interac- tions with the DNA and modulation of transcription. In the absence of a steroid ligand, the androgenic receptor is found inactive in the cytoplasm of the cell, associated to a chaperone complex. Steroids are small, fatty molecules that are able to diffuse passively into the cell. Subsequent to passing the cell membrane, the steroid ligand binds to the receptor, which conveys conformational changes that result in activation of the receptor. The acti- vated receptor is dissociated from the chaperone complex and translocated into the nucleus of the cell where it forms homodimers with another receptor through cooperative interaction with the DNA [64]. This activates co- regulators, i.e., co-activators or co-repressors, which form a transcription complex that affects transcription. This will further affect protein translation and alteration in cell function, growth or differentiation [63,65]. Furthermore, AAS are clinically used as anticatabolic agents at catabolic conditions such as severe burns [66]. It has been suggested that AAS exert this anticatabolic effect by antagonizing the glucocorticoid receptor [67]. Indeed, a case report showed that a patient gave some response to testosterone treatment despite the occurrence of an amino-acid mutation in the androgen receptor DNA-binding domain, thus suggesting this effect to be exerted by mechanisms other than by the androgenic receptor [68]. However, AAS generally show a low binding affinity to the glucocorticoid receptor, which indicates a more complex explanation. Effects of steroids conducted via the classical genomic pathway outlined above, require comparatively long time, usually hours or even days. Interest- ingly, it has become obvious that steroids are able to activate also more rapid non-genomic pathways, since effects by steroids have been observed after a much shorter lag-time. As early as in 1963, a study showed cardiovascular effects of aldosterone in men after only 5 minutes [69]. Further on, effects of aldosterone were seen on sodium ion exchange in mammalian erythrocytes, known to be lacking a nucleus [70]. Much of the non-genomic actions of androgens are, however, yet to be elucidated, particularly with regard to effects in the CNS, which can be linked to behavioral mechanisms. Studies

17 on AAS-induced effects in the CNS that are independent of nuclear receptor signaling have been made. Chronic treatment with AAS, including nandrolone, has for example been shown to allosterically modulate the function of the GABAA receptor when given acutely [71-73]. Furthermore, AAS have been suggested to affect the serotonergic system [74-76], the dopaminergic system [77-79] as well as the glutamate system [80,81]. In addition, alterations within neuropeptidergic systems, such as the endogenous opioid systems [45,82-84] and the tachykinin system [85,86] have been displayed. Previous studies have in fact shown altered levels of the tachykinin Substance P (SP) as well as of dynorphinergic endogenous opioids as a result of nandrolone treatment in rats [83,85]. This may give rise to effects in terms of an altered receptor activation profile. Hence, the mechanisms generating altered peptide levels are of great interest in a behavioral context. The SP system and the dynorphinergic system are of relevance regarding AAS-abuse due to their association with regulation of behaviors such as aggression, reward, cognition, and depression; these behaviors are also reported to be affected during AAS-abuse. However, at the time this thesis was initiated, a lack of knowledge existed regarding mechanisms generating these altered peptide levels. Possible effects on enzymatic conversion of the peptides in specific brain areas are of particular interest.

The Substance P system SP was discovered by von Euler and Gaddum as early as 1931 [87]. Initial studies showed SP to be present predominantly in the gray matter of the CNS with the highest levels found in the hypothalamus and the substantia nigra. SP was also found in the spinal cord as well as in the peripheral nervous system [88]. In the early 1970s, SP was identified as an undecapeptide and was subsequently synthesized by Leeman and colleagues [89,90]. SP belongs to the tachykinin family, which also comprises for example neurokinin A and neurokinin B. Comparative analysis of these three peptides has led to the identification of three distinct neurokinin (NK) receptors, each with a favored ligand. SP binds to the so-called NK1 receptor whereas neurokinin A and neurokinin B predominantly bind to the NK2 and NK3 receptor, respectively [91]. SP derives from the preprotachykinin-A gene. Alternative RNA splicing of the gene transcript generates three mRNAs; α-, β-, and γ-preprotachykinin, which all encode for the SP precursor sequence [92,93]. SP is further released from its precursor by the actions of proteases and is subsequently amidated at the C-terminal by peptidyl-Gly-alfa-amidating monoxy- genase, yielding a more stabile peptide [94,95]. The SP system is extensively studied and is predominantly recognized to play an important role in modulating nociception at the spinal level. The

18 involvement of SP in inflammatory response has also attracted great interest [96-99]. Moreover, the implication of the SP-NK1 receptor system has been suggested in a number of behavioral mechanisms in the CNS. These include for example the regulation of mood and depression as well as aggressive behavior, which is of particular interest with regard to AAS-abuse [96,100- 102].

The Dynorphinergic system “Dynorphins” is the designation of opioid peptides that derive from the prodynorphin (PDYN) precursor. Early research suggested them to be one single peptide and it was named dynorphin (Dyn- from the Greek dynamis = power), due to its extraordinary potency, showed to be 700 times that of Leu-enkephalin in guinea pig ileum bioassays [103-105]. The PDYN gene contains four exons (1-4) in humans and in rodents where exons 3 and 4 contain the entire coding sequence [106]. After transcription and translation, the large inactive PDYN precursor is further processed into bioactive peptides by the prohormone convertases (PC), PC1 and PC2 and carboxypeptidase E [107]. Moderate to high PDYN mRNA levels have been found in e.g. amygdala, denate gyrus, nucleus accumbens, hypothalamus, caudate putamen, and hippocampus. Major naturally occurring dynorphins that have been identified are Dynorphin A(1-17), Dynorphin A(1-8), Dynorphin B(1-13), Dynorphin B(1-29), Big Dynorphin, α-neoendorphin, and β-neoendorphin, displayed in Table 1. Dynorphins exhibit binding affinity towards all three opioid peptide receptors, i.e., the kappa opioid peptide (KOP) receptor, the mu opioid peptide (MOP) receptor, and the delta opioid peptide (DOP) receptor. However, dynorphins show a clear preference for the KOP receptor [105,108,109]. The selectivity towards the KOP receptor is mediated by the C-terminal of the dynorphin peptides with Arg7 and Lys11 making the great- est contribution [110]. Dynorphins are involved in a number of physiological systems such as neuroendocrine regulation, pain regulation, motor activity, cardiovascular function, respiration, temperature regulation, feeding behavior, and stress responsivity [111]. Furthermore, dynorphins are suggested to mediate negative emotional states such as depression and dysphoria and to be involved in cognitive functions [112-115]. Activation of the KOP receptor can produce actions similar to MOP and DOP receptor activation. However, KOP receptor activation also produces effects that oppose the actions of MOP and DOP receptor activation. This can, for example, be seen in addiction-relevant brain areas where MOP and DOP receptor activation mediate conditioned place preference, whereas KOP receptor activation mediate conditioned place aversion in rats [113].

19 Table 1. Sequences of major naturally occurring dynorphins

Peptide Amino acid sequence

Dynorphin A(1-17) Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln

Dynorphin A(1-8) Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile

Dynorphin B(1-13) Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Gln- Dynorphin B(1-29) Glu-Asp-Pro-Asn-Ala-Tyr-Ser-Gly-Glu-Leu-Phe-Asp-Ala α-neoendorphin Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys

β-neoendorphin Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn- Big Dynorphin Gln-Lys-Arg- Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr

The KOP receptor PDYN-derived peptides show a clear binding-preference for the KOP receptor [109]. Several distinct KOP receptor binding sites have been postulated, i.e. KOP1, KOP2 and KOP3 receptor binding sites. Even though this discov- ery, which occurred during early behavioral and biochemical experiments, resulted in the proposal of existence of several KOP receptor subtypes [116- 118], only one KOP receptor has been cloned so far. The pharmacological properties of this cloned receptor mostly resemble those of KOP1 binding sites [119-121]. The KOP receptor is a G-protein coupled receptor and mediates effects such as inhibition of calcium channels and cAMP synthesis [122,123] as well as activation of potassium channels [124]. KOP receptors located presynaptically are shown to inhibit the release of dynorphins [125]. There appears to be a mismatch between dynorphin distribution and KOP-specific binding sites. Thus, later investigations suggest that the original idea of several KOP receptor subtypes therefore might be related to dynorphins interacting with non-opioid receptors, such as for example the NMDA receptor [126,127].

Processing of neuropeptides Neuropeptides are derived by enzymatic cleavage of larger prepropeptides, which are synthesized in the ribosomes and processed to yield propeptides. The propeptides will undergo enzymatic cleavage, which results in bioactive neuropeptides that can be released into the synaps. In contrast to classical neurotransmitters, which are often inactivated by reuptake processes, neuropeptides are inactivated or converted by proteases. Since no recycling oc-

20 curs, newly synthesized neuropeptides are constantly delivered to the nerve terminal from the cell body. The neuropeptides will after the release be de- graded by specific enzymes to either inactive or active peptide fragments, which will thus terminate, retain or convert the activity of the released peptide.

Enzymatic conversion of Substance P A number of enzymes in the CNS have been shown to possess the capacity to cleave SP, yielding smaller peptide fragments. This metabolism of SP generates peptide fragments with depleted, similar, or altered activities as compared to the mother peptide. Enzymes worth mentioning are for example the angiotensin converting enzyme (ACE), the neutral endopeptidase (NEP), the post-proline cleaving enzyme, and the prolyl endopeptidase. Cleaving sites of these enzymes can be seen in Figure 4. In addition, an enzyme called Substance P endopeptidase (SPE) has been shown to cleave SP, generating primarily the N-terminal fragment SP(1-7). This fragment has been suggested to maintain some of the activity attributed to SP. However, SP(1-7) has also been shown to exert opposing activities compared to SP and its C- terminal fragments. For example, SP(1-7) is able to reduce aversive behavior otherwise promoted by SP [128]. Furthermore, studies have shown that SP(1-7) is involved in a number of functions, including antinociception, opioid withdrawal, motor behavior, stress, learning, and memory functions [129-133].

Figure 4. The amino acid sequence of Substance P and a summary of main metabolizing enzymes and their cleaving sites.

21 Substance P endopeptidase SPE was first characterized in human cerebrospinal fluid (CSF), but has also been found in rat CSF, human and rat spinal cord and in different brain tissues of the rat [134-138]. This enzyme cleaves SP at the Phe7-Phe8 and Phe8- Gly9 bonds, and is thus capable of generating the N-terminal fragments SP(1-7) and SP(1-8) along with the corresponding C-terminal fragments SP(8-11) and SP(9-11). Substrate selectivity is indicated since other tachykinins, such as neurokinin A and neurokinin B, which lack the double- Phe residues, are not converted by SPE; enkephalins, β-casomorphin or SP(1-8) are also not affected by SPE. The enzyme is suggested to be a thiol- sensitive endoprotease, inhibited by EDTA and dithiothreitol but not by captopril and phosphoramidon [138-140].

Figure 5. The amino acid sequence of Substance P and its N-terminal fragment Substance P(1-7) and a summary of some of their proposed effects.

Enzymatic conversion of Dynorphin A(1-17) Several enzymes in the CNS acting on Dynorphin A(1-17) (Dyn A) have been described [141-143]. The earlier mentioned enzyme NEP cleaves peptides on the N-terminal side of hydrophobic residues. Thus, NEP cleaves Dyn A mainly between Gly3-Phe4 but minor hydrolyzation by NEP between Arg7-Ile8 and Lys11-Leu12 has also been reported [144,145]. The Tyr1-Gly2 bond in Dyn A is cleaved by aminopeptidases and an enzyme called dynorphin converting enzyme (DCE), further addressed below, has been shown to cleave Dyn A at the Arg6-Arg7 bond. Cleaving sites of main acting enzymes on Dyn A as well as on the bioactive fragment Leu-enkephalin-Arg6 (Leu- enk-Arg6) are shown in Figure 6. [141,142].

22

Figure 6. The amino acid sequence of Dynorphin A (top) and the fragment Leu-enkephalin- Arg6 (bottom) and a summary of main metabolizing enzymes and their cleaving sites.

Dynorphin converting enzyme Characterization of DCE has previously been performed in the CSF of rats [146]. The enzymatic actions in the CNS are likely to be reflected in the CSF and, in fact, DCE was also characterized in rat spinal cord [147]. DCE has further been reported in human CSF, pituitary and spinal cord [148-152]. DCE in human CSF is suggested to be a serine protease whereas the rat CSF enzyme seems to be a cysteine protease [139,146]. In addition, a negative correlation between the concentration of PDYN-derived peptides and the activity of DCE in human CSF has been observed [153]. The enzyme displays a high specificity for Dyn A, Dynorphin B(1-13) (Dyn B) and α- neoendorphin [146]. DCE has been shown to cleave Dyn A at the Arg6-Arg7 bond, thus releas- ing the DOP/MOP receptor preferring hexapeptide Leu-enk-Arg6 [146]. Hence, cleavage at this site produces a shift in the opioid peptide receptor activation profile (Fig. 7).

Figure 7. Amino acid sequence, mechanism of action, and some of the proposed effects of Dynorphin A(1-17) and its N-terminal fragment Leu-enkephalin-Arg6.

23 Methodological aspects Radioimmunoassay Quantitative measurements of the often low concentrations of biological active substances in the body can be a problem for researchers. New but quite expensive methods exist. However, the relatively non-expensive radioimmunoassay (RIA) technique has long been used for this purpose and is still suitable owing to its sensitivity and specificity [154]. These qualities are obtained based on its immunological approach. RIA is hence well suited for measurements of peptides at low concentrations in large sets of samples. It is in fact possible to determine peptide concentrations as low as in the femto- mole range with this method. The principle of RIA involves competition between an unlabeled antigen (the analyte of interest) and the corresponding labeled antigen for a limited number of antibody binding sites. A fixed amount of antibody and a relative excess of antigen are often used. After equilibrium is reached, the antigen will remain free to some extent, but a portion will be bound in an antigen- antibody complex. As the amount of unlabeled antigen increases, the per- centage of bound labeled antigen will decrease. Separation of the bound antigen from the unbound followed by measurement of the radioactivity in either fractions enables calculation of the proportion of the antigen bound to the antibody. The concentration of unlabeled antigen is obtained using a standard curve for each assay-run. Several separation methods of the bound antigen from the unbound exist. Separation based on the removal of free antigen can be obtained by adsorption to dextran-coated charcoal. This is based on the higher affinity of the free antigen to the dextran-coated charcoal than that of the antigen-antibody complex. Centrifugation leaves the antigen-antibody complex in the supernatant, which is measured for radioactivity. Another separation method is based on double-antibody precipitation. This involves a second antibody directed to the initial antigen-antibody- complex. Hence, the difference in weight and size between the complex and the free fraction is increased. After reincubation, separation can be made by centrifugation whereafter the radioactivity in the precipitate is counted.

Autoradiography Autoradiography is suitable for the analysis of effects on receptor expression in the brain and has been used by many researchers for this purpose. The method enables visualization of the binding sites of a studied substance through incubation of experimental tissues such as brain sections with a ra- dioactive labeled ligand. The binding sites can be visualized by development on appropriate films where the distribution of the radioactivity will be dis-

24 played. Specific binding can be calculated by subtracting the non-specific binding obtained by concurrent incubation with an antagonist of the ligand.

Morris water maze The Morris water maze (MWM) performance test evaluates spatial learning and memory of animals. The method uses escape from water to motivate learning and the principle of the test is based on the ability of animals to learn the spatial location of a hidden platform in a pool filled with water. The platform itself offers no visible cues to guide the animal. Instead, visual cues are kept constant around the pool. In principle, the rat could find the platform by swimming randomly in the pool. However, normal rats learn very quickly to swim towards the hidden platform from any starting point. Comparison of the performance of vehicle- versus nadrolone-treated rats does thus offer valuable information regarding effects of AAS on learning and memory.

? TaqMan real-time polymerase chain reaction TaqMan? quantitative real-time reverse transcriptase polymerase chain reaction (PCR) is a reliable and sensitive method compared with some other PCR methods commonly used for quantification. The principle of the method is based on the cleavage of a TaqMan? probe by the Taq polymerase. The TaqMan? probe is dual-labeled, with a reporter dye at the 5’ end of the probe and a quencher dye at the 3’ end. When the probe is intact, the quencher dye suppresses the reporter fluorescence resulting in no fluorescence at all. During the PCR, the probe specifically anneals between the forward and reverse primer sites. Subsequently, the 5’-3’-nucleolytic activity of the Taq polymerase will cleave the probe between the reporter- and quencher dyes. However, this will only occur if the probe is hybridized to the target. The separation of the two dyes results in increased fluorescence of the reporter dye. Hence, the accumulation of PCR product can be detected directly by monitoring the increase in fluorescence. Since the increase in the fluorescence signal is detected only if the target sequence is complementary to the probe and amplified during the PCR, no non-specific amplification is detected. This makes the method specific and is thus one of the advantages with TaqMan? PCR.

25 Aims

The general aim of this thesis was to investigate the effects of the anabolic androgenic steroid nandrolone decanoate on neuropeptidergic systems in the male rat brain.

The specific aims of this thesis were:

To study the effect of nandrolone decanoate administration on the conversion of Substance P to the bioactive fragment Substance P (1-7) in the male rat brain.

To study the effect of nandrolone decanoate administration on the conversion of Dynorphin A(1-17) to the bioactive fragment Leu- enkephalin-Arg6 in the male rat brain.

To study the effect of nandrolone decanoate administration on the kappa opioid receptor density in the male rat brain.

To study the effect of nandrolone decanoate administration on spatial learning and memory.

To study the effect of nandrolone decanoate administration on the expression of the gene transcript of prodynorphin in the hippocampus of male rats.

26 Materials and methods

Experimental animal procedures Adult male Sprague-Dawley rats were used in all studies. The rats were randomly divided into two groups (Papers I, II and IV) or three groups (Paper III) and allowed to adapt to the laboratory environment for one week before the experiments started. During the following 14 days, the animals received i.m. injections in the hind legs (Paper I and II) or subcutaneous (s.c.) injections in the neck of either nandrolone decanoate or oil vehicle (sterile ara- chidis oleum). The dose of administered nandrolone decanoate was 15 mg/kg daily (Paper I and II) or every third day (Paper IV). In Paper III, one group of animals received a daily dose of 15 mg/kg whereas another group received a daily lower dose of 3 mg/kg. In Papers I, II and IV, the brains were rapidly removed after decapitation and dissected using a rat brain ma- trix. Brain areas of interest were immediately put on dry ice and thereafter stored separately at -80°C until further use. In the study presented in Paper III, the brains were immediately removed and quickly frozen in 2-methyl- butan (-25°C) and subsequently stored at -80°C. All procedures described in this thesis were approved by the Uppsala County Animal Ethics board and follow the rules and regulations of the Swedish Animal Welfare Agency.

Enzyme activities Tissue preparation In the studies presented in Papers I and II, the frozen brain tissues were thawed on ice and homogenized in 20 mM Tris buffer (pH 7.8). The ho- mogenates were centrifuged at 8000 x g for 20 min whereafter the supernatants were recentrifuged at 10000 x g for another 20 min. The new supernatants were collected and analyzed for enzyme activity.

Assay for measurement of SPE-like activity The SPE-like activity was investigated by measuring the conversion of SP to its N-terminal fragment SP(1-7) in the different brain regions. Briefly, the

27 supernatants were preincubated in Tris buffer (pH 7.4) at 37°C together with the enzyme inhibitors captopril and phosforamidon, which inhibit ACE and NEP, respectively. The substrate SP was subsequently added, and the incu- bations proceeded. Fractions were withdrawn after 20 and 40 min and the enzymatic activity was terminated by the addition of ice-cold methanol. The samples were dried in a Speed Vac centrifuge, re-dissolved in methanol-HCl and analyzed by RIA for the generated fragment SP(1-7). Optimizations regarding dilution of tissue preparations were made for each of the studied brain structures.

Assay for measurement of DCE-like activity DCE-like activity was measured by the conversion of Dyn A to the C- terminal fragment Leu-enk-Arg6. The supernatants were preincubated in Tris buffer (pH 7.4) at 37°C together with the enzyme inhibitors captopril, phosforamidon and amastatin to prevent further degradation of the product. Dyn A was thereafter incubated in the mixture for 20 and 40 min after which the reactions were terminated in the same manner as described for SPE-like activity measurements.

HPLC characterization In order to study generated fragments from SP (Paper I), evaporated incubates were redissolved and analyzed by high performance liquid chromatography (HPLC) using a Pharmacia SMART system. The column used was a μRPC C2/C18, SC 2.1/10 column and elution was carried out with an ace- tonitrile gradient. For more detailed information, se Paper I.

Mass spectrometry characterization Verification of the metabolite Leu-enk-Arg6 from Dyn A (Paper II) was carried out by mass spectrometry. Pooled incubates were analyzed by an Ul- traflex II TOF/TOF, which was equipped with a SmartBeamTM laser. Fur- thermore, the target of choice for the MALDI approach was an Anchor- ChipTM target. For more detailed information, se Paper II.

Radioimmunoassay In Paper I, the fragment SP(1-7) was measured by the charcoal adsorption technique. Briefly, triplicates of samples or standards were dissolved in methanol-HCl and incubated together with antibodies and the [125I]-labeled analogue of the peptide. Incubation was carried out for 24 hours at 4°C and was subsequently terminated by the addition of active charcoal buffer after

28 which the samples were centrifuged for one min to separate the free and bound peptide. The supernatant was collected and the radioactivity was determined in a gamma-counter. In Paper II, the fragment Leu-enk-Arg6 was measured based on the double antibody precipitation technique. Briefly, dissolved samples and standards were incubated together with [125I]-Leu-enk-Arg6 and antibodies against the peptide for 24 hours at 4°C before the addition of anti-rabbit antiserum. In- cubation proceeded for one additional hour and the samples were subsequently centrifuged. The supernatants were discarded and the radioactivity in the precipitate was determined using a gamma-counter. The detection limit for SP(1-7) as well as for Leu-enk-Arg6 was 2 fmol/tube and tracer replacement was reached at approximately 10 fmol/tube. The cross reactivity in the RIAs with other related peptides was shown to be minor and is described elsewhere [152,155].

Autoradiography In Paper III, rat brains were taken out immediately after decapitation, frozen in 2-methyl-butane (-25°C), cut in 14 μm coronal sections in a cryostat and thaw-mounted on polysin glass. The slides were incubated with the ligand [3H]-977, specific for the KOP receptor. To be able to calculate specific binding of the ligand, non-specific binding was determined using the opioid antagonist naloxone. Following incubation and washing of the slides, they were exposed to [3H]-sensitive hyperfilm together with [3H]-micro-scales with known amounts of 3H. The films were developed manually after 10 weeks and digitalized using an Epson scanner. Optical densities were converted to fmol/mg using NIH Image software and brain regions were identified using a rat brain atlas [156]. Hence, the density of [3H]-977-binding sites could be determined in various brain regions.

Morris water maze In Paper IV, the rats were carefully placed in the water, positioned to face the wall of the pool and allowed to locate the hidden platform, which was submerged 2 cm below the surface of the water. Four training trials were conducted each day for four consecutive days. At the beginning of each trial, an animal was started at one of four different starting points, in random order (designated north, east, south and west). On experimental day 7, the platform was removed and the animals underwent a probe trial to determine the extent to which they remembered the location of the platform. The amount of time the rats spent in each of the four quadrants was observed.

29 The pool was located in a room exclusively used for behavioral studies with a constant environment and lamps positioned so as to provide a dim light without reflections in the pool. A video camera connected to a tracking system (HVS Image Ltd.Buckingham, U.K.) was mounted above the pool to allow thorough analysis of each trial. Analysis of the time taken to find the platform, the distance swum, and the swimming speed provided information regarding to what degree that the rats learned the spatial position of the platform and escaped the water. The study setup in Paper IV is displayed in Fig- ure 8.

Figure 8. Scheme of the experimental design used for nandrolone- or vehicle-treated male rats. Down arrow: weighing; Injections: treatment with nandrolone decanoate (15 mg/kg) or vehicle every third day; MWM: Morris water maze task; T: training trial; P: probe trial; D: decapitation

RNA isolation and cDNA synthesis In order to extract total RNA from the frozen brain tissues in Paper IV, the RNeasy Lipid Tissue Mini Kit (QIAGEN, Maryland, USA) was used. Tis- sues were not thawed before the preparation and the manufacturer provided an appropriate protocol for RNA extraction. Briefly, tissue samples were homogenized in QIAzol? Lysis reagent, chloroform was added and the ho- mogenate was centrifuged. The aqueous supernatant was transferred to a new tube, where after ethanol (70%) was added and the RNA was eluted using RNeasy MiniSpin Columns. To obtain total RNA concentration as well as a preliminary quality control, the RNA samples were measured in a NanoDrop® ND-1000 Spectrophotometer. For a more thorough assessment regarding the quality of the RNA, the samples were analyzed on an Expe- rion™ System for RNA analysis (Bio-Rad Instruments, Hercules, CA). Sam- pled displaying clear ribosomal RNA, 18S and 28S, were subsequently used in cDNA synthesis using the High Capacity cDNA Archive Kit. Random hexamer primers were used and the absence of genomic DNA was controlled in a reaction lacking the reversed transcriptase. For a more detailed description, se Paper IV.

30 ? TaqMan real-time polymerase chain reaction In Paper IV, TaqMan? real-time quantitative PCR was used to detect the levels of PDYN mRNA. The TaqMan? Gene Expression Assay (Applied Biosystems, Foster City, CA) included a TaqMan? probe, which was dual- labeled, with a reporter dye, FAM (6-carboxyfluorescein), at the 5’ end of the probe and a quencher dye at the 3’ end. Reactions containing cDNA template, primers, probes and TaqMan? Universal PCR Master Mix were carried out in 96-well plates. The iCycler Real-Time PCR System (Bio-Rad Instruments, Hercules, CA) was used to monitor the increase in fluorescence from the reporter dye and the threshold cycles (Ct) were obtained. The Ct, defined as the number of PCR cycles required to reach a preset fluorescence threshold, is proportional to the initial amount of cDNA. Cycling parameters were: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, and 60°C for 1 min. Further, mRNA expression was calculated by relative quantification using a normalization factor, the geometric mean of two reference genes. The qBASEplus program was used for data analysis, including inter- nal and external calibration. For a more detailed description, se Paper IV.

Statistics Statistical analyses of differences between treated and non-treated groups were conducted using the unpaired Student’s t-test in studies presented in Paper I and II. In Paper III, statistical evaluation was performed using one- way ANOVA and Fisher’s post hoc test. In Paper IV, statistical analysis of data from the Morris water maze test was conducted using the non- parametric Mann-Whitney U test whereas the Student’s t-test was used for evaluation of PDYN transcription. P-values below 0.05 were considered significant. Data from all studies were tested for normal distribution using the Shapiro-Wilk’s W-test.

31 Results and discussion

Effects on Substance P conversion (Paper I) The generation of SP(1-7) from synthetic SP was detected in all rat brain regions examined, i.e. the amygdala, caudate putamen, frontal cortex, hippocampus, hypothalamus, nucleus accumbens, periaqueductal gray (PAG), substantia nigra and the ventral tegmental area (VTA). Two weeks of i.m. treatment with nandrolone decanoate (15 mg/kg) significantly decreased the conversion of SP compared with controls in the caudate putamen (44%), hypothalamus (43%), substantia nigra (32%) and the VTA (27%). Some of the results are displayed in Figure 9. Since the enzyme SPE was not characterized in the present study, the conversion of SP to SP(1-7) is herein referred to as SPE-like activity. As can be seen, the most pronounced effect was observed in the caudate putamen. This region is known to be associated with, for example, regulation of motor activity, adverse behavioral effects, and stimulus-response learning [157-159]. An earlier study showed that nandrolone treatment caused increased SP levels while SP(1-7) levels decreased in this region [85]. The decreased SPE-like activity observed in Paper I is thus in congruence with these previous findings. Since the amount of the metabolite SP(1-7) is decreased while its mother peptide SP is increased, it is tempting to speculate that decreased SPE-like activity in this region is a main contributor for these alterations and could thus put the SP system out of balance. Another interesting aspect is the fact that other enzymes such as the NEP and the ACE have been reported to display high activities in this region. This indicates that caudate putamen is an active region with regard to neuropeptide conversion [160,161]. In the hypothalamus, SPE-like activity was decreased by 43% as a result of nandrolone treatment. This area is known to be involved in e.g. aggressive and defensive behavior [102]. Furthermore, hypothalamus is an important region regarding integration of autonomic, endocrine, and somatomotor functions. Earlier studies have shown elevated SP levels following nandrolone administration in this region [85]. Hence, enhanced SP levels could be a result of decreased SP conversion and/or increased biosynthesis. In line with this, the density of the NK1 receptor has previously been shown to attenuate in hypothalamus as a result of nandrolone administration [86]. The observed alterations in this region are of special interest considering the reports of increased aggressiveness and violent acts associated with AAS-abuse.

32 The VTA of the brain is an important region with regard to drug rewarding effects. SPE-like activity was decreased by 27% in this area. Considering that SP(1-7) is suggested to decrease glutaminergic transmission and the fact that activation of the NMDA receptor has been postulated to generate rewarding properties, decreased SPE-like activity might cause a sense of reward as a result of declined SP(1-7) levels. SP is additionally suggested to enhance dopamine transmission, which further supports this theory. How- ever, one cannot compare the rewarding effects induced by AAS with effects obtained by drugs such as for example heroine or amphetamine. Neverthe- less, AAS are suggested to serve as a gateway to the use of other drugs [23]. The results clearly demonstrate that nandrolone treatment affects the SPE- like activity by reducing the conversion of SP to SP(1-7) in four of the brain regions examined. The mechanisms behind this decline in enzymatic activity are however not yet clarified and one could speculate in different alterna- tives. One explanation could be an effect by nandrolone on gene regulation, resulting in decreased biosynthesis of SPE. Alternatively, stimulation by nandrolone on increased biosynthesis of other peptides that act as competing substrates or antagonists to the enzyme could also occur.

Figure 9. Generated SP(1-7) (pmol/min/mg tissue), in six regions of the male rat brain, after daily nandrolone decanoate (ND) (15 mg/kg) or vehicle (Control) administration for 14 days; *P<0.05, **P< 0.01 versus controls, analyzed using the Student’s t-test. Data are given as means and SEM, n=8.

33 Effects on Dynorphin A(1-17) conversion (Paper II) The formation of Leu-enk-Arg6 from synthetic Dyn A was measured, and referred to as DCE-like activity. The results are summarized in Figure 10 and, as can be seen, the enzymatic activity was found to be highest in the caudate putamen and the lowest in the hypothalamus. Daily i.m. treatment during two weeks with nandrolone decanoate (15 mg/kg) significantly induced alterations compared with controls in four of the investigated regions, i.e., the caudate putamen, hypothalamus, nucleus accumbens and the PAG. Notably, a decrease in DCE-like activity by 40%, 34% and 36% in caudate putamen, hypothalamus and the PAG, respectively, was observed whereas an increase by 44% was encountered in the nucleus accumbens. The presence of Leu-enk-Arg6 in the incubate was confirmed by MALDI-TOF/TOF mass spectrometry. Previous results have shown increased Dyn B immunoreactivity in the hypothalamus, striatum as well as in the PAG in male Sprague-Dawley rats following nandrolone treatment [83]. The peptides Dyn A and Dyn B derive from the same precursor and are both cleaved by DCE. An attenuated DCE- like activity might give rise to increased levels of dynorphins and hence, the decreased DCE-like activities encountered in these regions in Paper II are in congruence with the earlier results [83]. The opposite effect was previously encountered in the nucleus accumbens of Wistar rats where an nandrolone-induced reduction in Dyn B immunoreactivity was reported [45]. Interestingly, this was the only region where DCE-like activity in Paper II was found to be increased. Further investigations on the effect of nandrolone treatment on PDYN mRNA expression in the nucleus accumbens displayed no significant changes (data not published). Hence, this indicates that the decreased dynorphinergic peptide levels in the nucleus accumbens are more likely caused by increased peptide degradation rather than an increased peptide biosynthesis. The altered dynorphin transformation may cause a change in the endogenous opioid receptor activation profile, provided that the observed enzymatic activity reflects the neurotransmission by Dyn A as well as the formed enkephalin product. Thus, nandrolone administration may give rise to an im- balance in the activation profile of the opioid peptide receptors and thereby cause psychological effects. In fact, some of the psychological and behavioral adverse effects attributed to AAS-abuse such as aggression, anxiety, irritability, cognitive dysfunction, and altered drug abuse pattern, are known to be partly controlled by the endogenous opioids.

34 The explanation as to why DCE-like activity increases or decreases in different brain regions is not yet known since the mechanisms, as in the case of SPE, are not fully understood. One might speculate in altered biosynthesis of the enzyme, release of unknown factors affecting the enzymatic activity or the scenario where altered DCE-like activity is a compensatory mechanism due to changed levels of the substrate.

Figure 10. Generated Leu-enkephalin-Arg6 (pmol/min/mg protein), in five regions of the male rat brain, after daily administration of nandrolone decanoate (ND) (15 mg/kg) or vehicle (Control) for 14 days; *P<0.05, **P< 0.01, ***P< 0.001 versus controls, analyzed using the Student’s t-test. Data are given as means and SEM, n=8.

Effects on the kappa opioid peptide receptor (Paper III) The density of the KOP receptor, measured by specific binding of the ligand [3H]Cl-977, was examined in various regions of the male rat brain at bregma 1.60 and -2.80. The highest binding was observed in the shell of nucleus accumbens. Nandrolone-induced effects were examined after daily administration of two different doses (3 mg/kg versus 15 mg/kg) of nandrolone decanoate during two weeks. Altered density of the KOP receptor was encountered in several brain regions. A significant decease in specific binding of [3H]Cl-977 was observed in the shell of nucleus accumbens, lateral hypothalamic area, ventromedial and dorsomedial hypothalamic nucleus, central

35 amygdaloid nucleus, lateral globus pallidus, and in the stria terminalis after treatment with the higher dose (Fig. 11). Interestingly, a significant increase in specific binding of [3H]Cl-977 was on the contrary detected in the caudate putamen and the dorsal endopiriform nucleus. The results from rats treated with the lower dose showed tendencies that are in congruence with the significant differences observed after administration of the higher dose, which indicates a dose-dependent cause of action. KOP receptors are found in a vast number of brain regions, with a dense distribution in e.g. the amygdala, caudate putamen, endopiriform nucleus, hypothalamus, and the stria terminalis. These receptors have been suggested to be involved in for example the modulation of drug seeking behavior, stress response, mood alterations, as well as in cognitive functions [162- 164]. To further address one of the regions where an alteration was displayed, a significant down-regulation of KOP receptors was observed in most areas of the hypothalamus after treatment with the higher dose of nandrolone decanoate. In the same region, a significant decrease in Dyn A conversion was shown following nandrolone administration (Paper II). It is thus tempting to suggest that attenuated biotransformation account for the higher levels of dynorphinergic peptides that are able to activate the KOP receptors; the down regulation of KOP receptors in hypothalamus would, hence, reflect a neuroadaptive response due to increased ligand levels. In fact, earlier studies show nandrolone-induced enhanced Dyn B levels in hypothalamus (Table 2) [83]. Moreover, a dose dependent reduction in KOP receptor binding was observed in the shell of nucleus accumbens. In this region, KOP receptor ligands are suggested to act predominantly presynaptically, regulating the release of dynorphins and other neurotransmitters [165]. An elevated biotransformation of Dyn A is described in Paper II. These results together with our previous report of attenuated Dyn B levels in this region as a result of nandrolone treatment [45], provides additional support that AAS administration has a pronounced impact on the biochemistry in the nucleus accumbens, a region with strong association with addictive behavior. In fact, the same study also verified correlations between alterations in PDYN-derived peptides and addictive behavior, i.e. voluntary alcohol intake, as a result of nandrolone administration in rats [45]. This region is of special relevance considering the reports of AAS-dependence as well as altered patterns of abuse regarding other illicit drugs [21,48,54]. Further, other studies have shown connections between high-dose AAS administration and behaviors known to be associated with the dynorphinergic system, such as anxiety and cognition [166-168]. Interestingly, another region where dose-dependent effects were observed was the amygdala. This region is suggested to influence neural activity and memory storage in the limbic system, since activation of amygdala improved caudate- and hippo-

36 campal dependent learning tasks [169]. Injections of testosterone enanthate in this region in rats resulted in impaired spatial memory and learning in the MWM task [166]. The observed effects on KOP receptor density could be a result of direct effects exerted by nandrolone or be attributed to compensatory mechanisms. Thus, nandrolone administration may result in cellular adaptations, such as altered KOP receptor density, and/or affinity for ligands in order to maintain synaptic homeostasis. The discussed effects caused by nandrolone decanoate administration on the dynorphinergic system in four of the brain regions investigated are summarized in Table 2.

Figure 11. Specific binding of [3H]Cl-977 (fmol/mg) in four brain regions of the male rat brain after 14 days of daily administration of 3 respective 15 mg/kg nandrolone decanoate (ND) or vehicle (Control); *P<0.05, **P< 0.01, ***P< 0.001 versus controls, analyzed using one-way ANOVA and Fisher’s post hoc test. Data are given as means and SEM, n=8.

37 Table 2. The impact of nandrolone decanoate administration on KOP receptor density, DCE- like activity, Dyn B concentration, and PDYN mRNA level in four of the brain regions investigated. Data on Dyn B concentrations are from previous studies [45,83]. KOP receptor DCE-like activity Dyn B concentra- PDYN mRNA

density tion level ↓ ⎯ Amygdala ↑ ↓ ↑ Caudate putamen ↓ ↓ ↑ Hypothalamus ↓ ↑ ↓ ⎯ Nucleus accumbens

Effects on Morris water maze performance (Paper IV) Administration of nandrolone decanoate every third day during two weeks prior to the MWM test resulted in prolonged latencies before the rats found the hidden platform. On the second day of trial, the latencies were significantly longer in the nandrolone-treated rats compared with controls, as can be seen in Figure 12. Average latencies on the second trial-day were 13 seconds in the control group and 18 seconds in the nandrolone-treated group. The difference in performance was, however, not statistically significant on trial-days three and four. The latencies in the control-group were consistent with previous studies using the same rat strain and lab facilities [170]. In addition, the probe trial, which was performed three days after the last trial day, displayed that nandrolone-treated animals spent significantly less time (P< 0.01) in the quadrant where the platform was previously positioned compared with the controls. Control animals spent 42% of the time whereas nandrolone-treated rats spent only 30% of the time in the target quadrant. There were no differences regarding swim speed between or within the groups on different days (Fig. 13). Thus, no motoric effects as a result of nandrolone-treatment were observed. Hormones such as testosterone are shown to be associated with forms of cognitive processes [171]. For example, a study on aging mice with a loss of testosterone exhibited spatial learning deficits, which were reversed by administration of therapeutical levels of testosterone [172]. Further, endogenous testosterone concentrations have been suggested to be related to sex differences in spatial cognitive ability also in humans [173]. Nevertheless, investigations of the effects of endogenous testosterone concentrations on spatial cognitive ability have produced conflicting results, with researchers reporting either a positive correlation between increased testosterone and improved spatial ability, the opposite pattern or no effect at all [173-176]. Varying study designs and different steroids may cause discrepancies in the results. It should also be noted that the cognitive effects of supraphysiological levels of androgens might differ from the effects of endogenous levels.

38 The results presented in Paper IV indicate impaired spatial learning and memory in male rats as a result of nandrolone decanoate administration. A number of studies have previously examined the effects of exogenous testosterone or other AAS on the performance of male rats in reference memory tasks such as the MWM task. For example, one study showed impairments in probe trial performance of young and middle-aged rats in the MWM task following long-term treatment with testosterone [177]. In addition, microinjections of testosterone directly into the CA1 region of the hippocampus of male rats also showed impaired performance in the MWM task [178,179]. Similar effects of testosterone were also shown following injections directed at the basolateral amygdala [166]. A resent study indicates negative effects of high-dose nandrolone on social memory in the olfactory social memory test. The authors suggest it to be effects via activation of central androgen receptors, since co-administration of the androgen antagonist flutamide eliminated the effects [180]. The mechanisms behind this impairment are not fully understood. How- ever, one might speculate in some explanations: Apart from effects in the CNS via the intracellular androgenic receptor, steroid hormones have been shown to modulate memory processes by the regulation of acetylcholine- transferase and acetylcholinesterase activities, affecting the levels of acetylcholine [181]. Another mechanism of interest is the possible interference with neurosteroids, which have been implicated in both memory acquisition and memory loss in rodents. AAS may alter the neurosteroidogenesis and/or interfere with neurosteroid-binding sites on for example GABAA-, NMDA- and σ1-receptors [182-186]. Injections of nandrolone have also been shown to alter glutaminergic transmission; which is reported to affect recognition in visual and olfactory memory tasks [187,188]. Moreover, endogenous levels of testosterone decline when high-doses of exogenous androgens are administered due to a negative feed-back mechanism. Low levels of endogenous testosterone have been shown to give rise to cognitive impairments and could be a contributing factor in this matter. In addition, significant effects on PDYN mRNA expression levels were observed in the memory-impaired rats studied in Paper IV. These effects are further addressed below. Even though one should be careful with transferring results from animal studies to effects in humans, the fact still remains that nandrolone decanoate administration significantly impaired spatial learning and memory in rats and that indications exist of similar effects in human high-dose AAS-abusers [43,61].

39

Figure 12. Results from the Morris water maze behavioral task from day 1 to day 4 of training following nandrolone decanoate (ND) or vehicle (Control) administration in male rats. One block of four training trials was given on each day. The escape latency (time to reach the platform in seconds) is shown. *P<0.05 versus controls, analyzed using the Mann-Whitney U test. Data are given as means and SEM, n=24.

Figure 13. Results from the Morris water maze behavioral task from day 1 to day 4 of training following nandrolone decanoate (ND) or vehicle (Control) administration in male rats. One block of four training trials was given on each day. Swim speed (m/s) is shown. Data are given as means and SEM, n=24.

40 Effects on Prodynorphin gene transcription (Paper IV) Quantitative real-time PCR analysis showed significantly elevated gene transcription levels of PDYN in the brain region hippocampus in the nandrolone- treated animals compared with controls (Paper IV). Normalized relative levels of mRNA in the two treatment-groups showed that the transcription levels of PDYN were significantly elevated by 28% in the nandrolone-treated animals compared with controls (*P<0.05). Dynorphins have been suggested to play a modulatory role on cognitive acquisition and appear to enhance memory retention in negative reinforcement-based tasks [189,190]. On the contrary, they have an inhibitory effect on memory and learning in tasks based on positive or neutral stimuli. For example, studies on mice have revealed that aged PDYN knockout mice performed better in the MWM task than similarly aged wild-type mice [114,115]. Studies on rats showed that learning deficient rats display an increased abundance of PDYN mRNA as well as higher hippocampal dynorphin A(1-8) levels than control animals [114]. Furthermore, microinjections of a KOP receptor agonist into the CA3 region of the rat hippocampus sup- pressed learning and memory performance in the MWM task [191]. A recent study showed that prolonged stress exposure impairs learning and memory performance. Since the impairment of novel object recognition was abol- ished by antagonism of the KOP receptor or PDYN gene disruption, the authors suggested this effect to be mediated by the activation of KOP receptors [192]. Association between the dynorphinergic system and decreased memory function has not only been observed in rodents, but also in humans and a recent study suggests a role of PDYN gene polymorphism in episodic memory and verbal fluency in healthy elderly humans [193]. In congruence with this, and of particular relevance to human disease, patients with Alz- heimer’s disease (AD) displayed markedly elevated Dyn A levels in the frontal cortex and a correlation with neuritic plaque density was also observed [194]. Moreover, increased KOP receptor levels have been reported in brains of AD patients [195], and cognitive performance in AD patients can be improved by administration of the non-selective opioid antagonist naloxone [196]. Provided that the observed increase in PDYN mRNA expression reflects an increase in PDYN protein translation and biological active dynorphinergic peptides, this could be one of the mechanisms behind the cognitive impairments observed in the same animals. However, calculation with Spearman’s rank correlations between the MWM latency and the expression of PDYN mRNA did not reveal any significant correlation. This may be indicative of the involvement of other factors, in addition to those derived from the dynorphin system.

41 General considerations Nandrolone decanoate administration The administration schedules in this thesis were chosen to mimic the nandrolone decanoate intake of AAS-abusers, which often display doses up to 100- fold the therapeutic one. The steroid was injected i.m. in Papers I and II and III, whereas s.c. in Paper IV. The steroid was administered as a decanoate, yielding a depot effect. Plasma concentrations of nandrolone were measured by RIA in the rats from the study presented in Paper IV and showed an average concentration of 30 nM still remaining in the blood-stream one week after the last injection (unpublished data). This aspect of nandrolone decanoate administration is well known among AAS-abusers, referring to nandrolone decanoate as a long-acting steroid.

42 Summarizing conclusions

The results presented in this thesis display significant effects of nandrolone decanoate administration in male rats on neuropeptidergic mechanisms related to for example cognition, aggression, reward, and dependence. Two weeks of i.m. treatment with nandrolone decanoate (15 mg/kg) significantly decreased the conversion of synthetic SP into the bioactive metabolite SP(1-7) in the brain regions caudate putamen, hypothalamus, substantia nigra, and the VTA. The results may thus reflect nandrolone-induced neurochemical alterations in the SP-system in these brain regions, that also are in congruence with previous studies on altered SP and SP(1-7) levels. The formation of Leu-enk-Arg6 from synthetic Dyn A was measured following daily i.m. treatment during two weeks with nandrolone decanoate (15 mg/kg). The results show significantly decreased conversion of Dyn A compared with controls in the caudate putamen, hypothalamus and the PAG, whereas an increase was encountered in the nucleus accumbens. The observed effects may be a sign of an altered enzymatic conversion of Dyn A possibly resulting in an altered opioid peptide receptor activation profile in these regions. Further, the density of the KOP receptor measured by the specific binding of [3H]Cl-977 was examined in various regions of the male rat brain. The results showed that daily administration of two different doses (3 mg/kg versus 15 mg/kg) nandrolone decanoate during two weeks induced dose- dependent alterations in several brain regions. A significant decease was observed in the nucleus accumbens shell, lateral hypothalamic area, ventromedial and dorsomedial hypothalamic nucleus, central amygdaloid nucleus, lateral globus pallidus, and in the stria terminalis whereas a significant increase was found in the caudate putamen and in the dorsal endopiriform nucleus. The results indicate neuraoadaptive changes in dynorphinergic transmission caused by nandrolone administration. Investigation of MWM task performance following two weeks of s.c. nandrolone decanoate administration (15mg/kg every third day) revealed impaired spatial learning and memory in terms of prolonged latencies before the rats found the hidden platform. On the second day of the trial, the latencies were significantly longer in the nandrolone-treated rats compared with controls. In addition, the probe trial displayed that nandrolone-treated animals spent significantly less time in the quadrant where the platform was previously positioned compared with the controls. Further, the memory-

43 impaired rats also displayed increased PDYN mRNA levels, measured by TaqMan? reversed transcriptase real-time PCR, in the brain region hippocampus, a region associated with cognitive processes. This may be one of the explanations to the observed impaired spatial memory and learning. The results in this thesis provide further information regarding neuroadaptive changes caused by administration of high doses of nandrolone decanoate. The observed effects on neoropeptidergic mechanisms are of relevance in the pursuit of a greater understanding concerning the mechanisms related to nandrolone-induced effects on for example cognition, aggression, reward, and dependence.

44 Populärvetenskaplig sammanfattning

Definitionen av anabola androgena steroider (AAS) inkluderar det manliga könshormonet testosteron samt ämnen som liknar testosteron i strukturen. Alla idag kända AAS har både muskeluppbyggande och maskuliniserande effekter. Tidigare har användandet av AAS främst förekommit bland atleter och kroppsbyggare som tar dessa i syfte att öka sina fysiska prestationer. I dagens samhälle används dock AAS av personer även utanför den idrottsliga sfären och med andra syften, som t.ex. för att öka självförtroendet. Utöver de fysiska effekter som kan uppstå vid AAS-missbruk föreligger det även risker för psykiska effekter såsom ökad aggression, humörsvängningar, depression och minnesstörningar. Dessutom rapporteras det om AAS-beroende hos personer som tagit AAS i höga doser under en längre tid. Psykiska effekter uppstår som en följd av påverkan på hjärnans kommu- nikationssystem. Hjärnans celler kommunicerar med varandra via ämnen, s.k. transmittorer, som frisätts från en cell och sedan binder till mottagaren- heter, s.k. receptorer, på nästa cell. I hjärnan finns det många olika transmittorsystem med olika funktioner. Två av dessa transmittorer är Substans P (SP) och Dynorfin A (Dyn A). Frisättning av SP är bl.a. kopplat till aggres- sivt beteende och Dyn A till kognitiva processer och beroendemekanismer i specifika hjärnregioner. Dessa transmittorer är därför av relevans vid studier på effekter av AAS. Både SP och Dyn A kan brytas ner till mindre transmit- torsubstanser, vilka fortfarande är biologiskt aktiva men som binder till andra receptorer än SP och Dyn A och därför kan ge upphov till helt andra effekter. I avhandlingen undersöks effekter av behandling med nandrolon, som är en vanlig AAS, hos hanråttor. Resultaten visar tydliga effekter på nedbryt- ning av både SP och Dyn A i specifika hjärnregioner. Vidare visas effekter på den receptor som Dyn A binder till, KOP receptorn. De påvisade föränd- ringarna i SP- och dynorfin-systemen kan vara några av förklaringarna bak- om de beteendeförändringar som rapporterats hos AAS-missbrukare. Resultaten från ett beteendeförsök visar också på ett tydligt försämrat rumsligt minne och inlärningsförmåga efter nandrolonintag. Dessa djur upp- visade även förändringar i dynorfin-systemet i hippocampus, ett hjärnområde som är starkt kopplat till minnesfunktioner. Det bör tilläggas att det inte rakt av går att översätta resultat från studier på djur till effekter hos människa. Trots detta ger resultaten ändå en finger- visning om möjliga effekter som också skulle kunna uppstå hos mänskliga

45 AAS-missbrukare. Resultaten från samtliga studier i avhandlingen bidrar därmed till ökad kunskap kring de förändringar som kan uppstå i hjärnans transmittorsystem och kan ge upphov till beteenderubbningar som en följd av AAS-missbruk.

46 Acknowledgements

The studies presented in this thesis were carried out at the Department of Pharma- ceutical Biosciences, Faculty of Pharmacy, Uppsala University, Sweden.

I would like to express my sincere appreciation to all the people who have played a part in the completion of this thesis, especially:

My main supervisor, Professor Fred Nyberg, for providing a PhD education with the possibility of free-thinking. Thank you also for always believing in my abilities and for being the caring person that you are! My co-supervisors, co-authors and all present and past colleagues, none mentioned but none forgotten, at the Division of Biological Research on Drug dependence. My co-authors and colleagues at the Division of Pharmaceutical Pharmacology for a valuable teaching experience that also taught me a lot. All people at the Department of Neuroscience, Division of Neurosurgery, where parts of the study in Paper IV was conducted. Thank you, co-authors Dr Fredrik Clausen and Anders Hållner, for your help and Polly-influence. Magnus Jansson for always helping and not laughing too much at me whenever my computer “got the flue”. Jakob, my brother in law, for structuring my molecules. Not skabbigt . Sadia for making me only the second most stressed PhD student this summer and Jenny for your constant laughter. All the rest of my friends for brightening up my life! Nelly, my “big little sister”! Thank you for all your help with this thesis and for your incredible friendship! What would I have done without you? My sisters Margareta, Cecilia and Anette. You are invaluable to me! My parents Karin and Stig for your love and endurance! Patrik for your support and for being the best father Linnea could possibly wish for! Last but not least, sweet Linnea, you are the sunshine of my life!

47 References

[1] Marshall E. The drug of champions. Science 1988;242 (4876):183-4. [2] Kindlundh AM, Isacson DG, Berglund L, Nyberg F. Factors associated with adoles- cent use of doping agents: anabolic-androgenic steroids. Addiction 1999;94 (4):543- 53. [3] Sjoqvist F, Garle M, Rane A. Use of doping agents, particularly anabolic steroids, in sports and society. Lancet 2008;371 (9627):1872-82. [4] Wanjek B, Rosendahl J, Strauss B, Gabriel HH. Doping, drugs and drug abuse among adolescents in the State of Thuringia (Germany): prevalence, knowledge and attitudes. Int J Sports Med 2007;28 (4):346-53. [5] Tahtamouni LH, Mustafa NH, Alfaouri AA, Hassan IM, Abdalla MY, Yasin SR. Prevalence and risk factors for anabolic-androgenic steroid abuse among Jordanian collegiate students and athletes. Eur J Public Health 2008. [6] Nilsson S, Baigi A, Marklund B, Fridlund B. The prevalence of the use of androgenic anabolic steroids by adolescents in a county of Sweden. Eur J Public Health 2001;11 (2):195-7. [7] Pallesen S, Josendal O, Johnsen BH, Larsen S, Molde H. Anabolic steroid use in high school students. Subst Use Misuse 2006;41 (13):1705-17. [8] Irving LM, Wall M, Neumark-Sztainer D, Story M. Steroid use among adolescents: findings from Project EAT. J Adolesc Health 2002;30 (4):243-52. [9] Kindlundh AM, Isacson DG, Berglund L, Nyberg F. Doping among high school students in Uppsala, Sweden: A presentation of the attitudes, distribution, side effects, and extent of use. Scand J Soc Med 1998;26 (1):71-4. [10] Stilger VG, Yesalis CE. Anabolic-androgenic steroid use among high school foot- ball players. J Community Health 1999;24 (2):131-45. [11] Berthold AA. Transplantation des Hoden [Transplantation of Testis]. Archives Anatomic Physiologie Wissenshaftliche 1849;16:42-6. [12] Brown-Sequard E. The effects produced on a man by subcutaneous injections of a liquid obtained from the testicles of animals. Lancet 1889;2:105-7. [13] Butenandt A, Hanish G. Umwandlung des dehydroandrosterone in Androstendiol and Testosterone, ein weg zur darstellung des testosterons aus cholesterin. Hoppe Seylers Z Physiol Chem 1935;237:89-97. [14] Wade N. Anabolic Steroids: Doctors Denounce Them, but Athletes Aren't Listen- ing. Science 1972;176 (4042):1399-403. [15] Mottram DR, George AJ. Anabolic steroids. Baillieres Best Pract Res Clin Endocri- nol Metab 2000;14 (1):55-69. [16] Schanzer W. Metabolism of anabolic androgenic steroids. Clin Chem 1996;42 (7):1001-20. [17] Birch JA. Hydroaromatic steroid hormones. I 10-nortestosterone. J Chem Soc 1950:367-8.

48 [18] Eklof AC, Thurelius AM, Garle M, Rane A, Sjoqvist F. The anti-doping hot-line, a means to capture the abuse of doping agents in the Swedish society and a new ser- vice function in clinical pharmacology. Eur J Clin Pharmacol 2003;59 (8-9):571-7. [19] Pagonis TA, Angelopoulos NV, Koukoulis GN, Hadjichristodoulou CS. Psychiatric side effects induced by supraphysiological doses of combinations of anabolic steroids correlate to the severity of abuse. Eur Psychiatry 2006;21 (8):551-62. [20] Rogol AD, Yesalis CE, 3rd. Clinical review 31: Anabolic-androgenic steroids and athletes: what are the issues? J Clin Endocrinol Metab 1992;74 (3):465-9. [21] Skarberg K, Nyberg F, Engstrom I. Multisubstance use as a feature of addiction to anabolic-androgenic steroids. Eur Addict Res 2009;15 (2):99-106. [22] Meilman PW, Crace RK, Presley CA, Lyerla R. Beyond performance enhancement: polypharmacy among collegiate users of steroids. J Am Coll Health 1995;44 (3):98- 104. [23] Arvary D, Pope HG, Jr. Anabolic-androgenic steroids as a gateway to opioid dependence. N Engl J Med 2000;342 (20):1532. [24] Kanayama G, Cohane GH, Weiss RD, Pope HG. Past anabolic-androgenic steroid use among men admitted for substance abuse treatment: an underrecognized problem? J Clin Psychiatry 2003;64 (2):156-60. [25] Scott MJ, 3rd, Scott AM. Effects of anabolic-androgenic steroids on the piloseba- ceous unit. Cutis 1992;50 (2):113-6. [26] Dohle GR, Smit M, Weber RF. Androgens and male fertility. World J Urol 2003;21 (5):341-5. [27] Gruber AJ, Pope HG, Jr. Psychiatric and medical effects of anabolic-androgenic steroid use in women. Psychother Psychosom 2000;69 (1):19-26. [28] Akhter J, Hyder S, Ahmed M. Cerebrovascular accident associated with anabolic steroid use in a young man. Neurology 1994;44 (12):2405-6. [29] Kennedy MC, Corrigan AB, Pilbeam ST. Myocardial infarction and cerebral haem- orrhage in a young body builder taking anabolic steroids. Aust N Z J Med 1993;23 (6):713. [30] Kennedy MC, Lawrence C. Anabolic steroid abuse and cardiac death. Med J Aust 1993;158 (5):346-8. [31] Kennedy C. Myocardial infarction in association with misuse of anabolic steroids. Ulster Med J 1993;62 (2):174-6. [32] Dickerman RD, Schaller F, Prather I, McConathy WJ. Sudden cardiac death in a 20- year-old bodybuilder using anabolic steroids. Cardiology 1995;86 (2):172-3. [33] Hartgens F, Rietjens G, Keizer HA, Kuipers H, Wolffenbuttel BH. Effects of androgenic-anabolic steroids on apolipoproteins and lipoprotein (a). Br J Sports Med 2004;38 (3):253-9. [34] Cheever K, House MA. Cardiovascular implications of anabolic steroid abuse. J Cardiovasc Nurs 1992;6 (2):19-30. [35] Bagatell CJ, Heiman JR, Matsumoto AM, Rivier JE, Bremner WJ. Metabolic and behavioral effects of high-dose, exogenous testosterone in healthy men. J Clin En- docrinol Metab 1994;79 (2):561-7. [36] Velazquez I, Alter BP. Androgens and liver tumors: Fanconi's anemia and non- Fanconi's conditions. Am J Hematol 2004;77 (3):257-67. [37] Creagh TM, Rubin A, Evans DJ. Hepatic tumours induced by anabolic steroids in an athlete. J Clin Pathol 1988;41 (4):441-3.

49 [38] Pope HG, Jr., Katz DL. Psychiatric and medical effects of anabolic-androgenic steroid use. A controlled study of 160 athletes. Arch Gen Psychiatry 1994;51 (5):375-82. [39] Daly RC, Su TP, Schmidt PJ, Pagliaro M, Pickar D, Rubinow DR. Neuroendocrine and behavioral effects of high-dose anabolic steroid administration in male normal volunteers. Psychoneuroendocrinology 2003;28 (3):317-31. [40] Papazisis G, Kouvelas D, Mastrogianni A, Karastergiou A. Anabolic androgenic steroid abuse and mood disorder: a case report. Int J Neuropsychopharmacol 2007;10 (2):291-3. [41] Pope HG, Jr., Kouri EM, Hudson JI. Effects of supraphysiologic doses of testosterone on mood and aggression in normal men: a randomized controlled trial. Arch Gen Psychiatry 2000;57 (2):133-40; discussion 55-6. [42] Perry PJ, Kutscher EC, Lund BC, Yates WR, Holman TL, Demers L. Measures of aggression and mood changes in male weightlifters with and without androgenic anabolic steroid use. J Forensic Sci 2003;48 (3):646-51. [43] Porcerelli JH, Sandler BA. Anabolic-androgenic steroid abuse and psychopa- thology. Psychiatr Clin North Am 1998;21 (4):829-33. [44] Choi PY, Pope HG, Jr. Violence toward women and illicit androgenic-anabolic steroid use. Ann Clin Psychiatry 1994;6 (1):21-5. [45] Johansson P, Lindqvist A, Nyberg F, Fahlke C. Anabolic androgenic steroids affects alcohol intake, defensive behaviors and brain opioid peptides in the rat. Phar- macol Biochem Behav 2000;67 (2):271-9. [46] Harrison RJ, Connor DF, Nowak C, Nash K, Melloni RH, Jr. Chronic anabolic- androgenic steroid treatment during adolescence increases anterior hypothalamic vasopressin and aggression in intact hamsters. Psychoneuroendocrinology 2000;25 (4):317-38. [47] Brower KJ, Blow FC, Young JP, Hill EM. Symptoms and correlates of anabolic- androgenic steroid dependence. Br J Addict 1991;86 (6):759-68. [48] Brower KJ, Blow FC, Beresford TP, Fuelling C. Anabolic-androgenic steroid dependence. J Clin Psychiatry 1989;50 (1):31-3. [49] Yesalis CE, Vicary JR, Buckley WE, Streit AL, Katz DL, Wright JE. Indications of psychological dependence among anabolic-androgenic steroid abusers. NIDA Res Monogr 1990;102:196-214. [50] Bahrke MS, Yesalis CE, 3rd, Wright JE. Psychological and behavioural effects of endogenous testosterone levels and anabolic-androgenic steroids among males. A review. Sports Med 1990;10 (5):303-37. [51] Hays LR, Littleton S, Stillner V. Anabolic steroid dependence. Am J Psychiatry 1990;147 (1):122. [52] Kashkin KB, Kleber HD. Hooked on hormones? An anabolic steroid addiction hypothesis. Jama 1989;262 (22):3166-70. [53] Brower KJ. Anabolic steroid abuse and dependence. Curr Psychiatry Rep 2002;4 (5):377-87. [54] Copeland J, Peters R, Dillon P. Anabolic-androgenic steroid use disorders among a sample of Australian competitive and recreational users. Drug Alcohol Depend 2000;60 (1):91-6. [55] Pope HG, Jr., Katz DL. Affective and psychotic symptoms associated with anabolic steroid use. Am J Psychiatry 1988;145 (4):487-90.

50 [56] Malone DA, Jr., Dimeff RJ, Lombardo JA, Sample RH. Psychiatric effects and psychoactive substance use in anabolic-androgenic steroid users. Clin J Sport Med 1995;5 (1):25-31. [57] Arnedo MT, Salvador A, Martinez-Sanchis S, Pellicer O. Similar rewarding effects of testosterone in mice rated as short and long attack latency individuals. Addict Biol 2002;7 (4):373-9. [58] Alexander GM, Packard MG, Hines M. Testosterone has rewarding affective properties in male rats: implications for the biological basis of sexual motivation. Behav Neurosci 1994;108 (2):424-8. [59] Johnson LR, Wood RI. Oral testosterone self-administration in male hamsters. Neuroendocrinology 2001;73 (4):285-92. [60] Wood RI, Johnson LR, Chu L, Schad C, Self DW. Testosterone reinforcement: intravenous and intracerebroventricular self-administration in male rats and hamsters. Psychopharmacology (Berl) 2004;171 (3):298-305. [61] Su TP, Pagliaro M, Schmidt PJ, Pickar D, Wolkowitz O, Rubinow DR. Neuropsy- chiatric effects of anabolic steroids in male normal volunteers. Jama 1993;269 (21):2760-4. [62] Kicman AT. Pharmacology of anabolic steroids. Br J Pharmacol 2008;154 (3):502- 21. [63] Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blum- berg B, Kastner P, Mark M, Chambon P, Evans RM. The nuclear receptor superfamily: the second decade. Cell 1995;83 (6):835-9. [64] Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988;240 (4854):889-95. [65] Perissi V, Rosenfeld MG. Controlling nuclear receptors: the circular logic of cofac- tor cycles. Nat Rev Mol Cell Biol 2005;6 (7):542-54. [66] Demling RH, Orgill DP. The anticatabolic and wound healing effects of the testosterone analog oxandrolone after severe burn injury. J Crit Care 2000;15 (1):12-7. [67] Hickson RC, Czerwinski SM, Falduto MT, Young AP. Glucocorticoid antagonism by exercise and androgenic-anabolic steroids. Med Sci Sports Exerc 1990;22 (3):331-40. [68] Tincello DG, Saunders PT, Hodgins MB, Simpson NB, Edwards CR, Hargreaves TB, Wu FC. Correlation of clinical, endocrine and molecular abnormalities with in vivo responses to high-dose testosterone in patients with partial androgen insensitiv- ity syndrome. Clin Endocrinol (Oxf) 1997;46 (4):497-506. [69] Klein K, Henk W. [Clinical experimental studies on the influence of aldosterone on hemodynamics and blod coagulation.]. Z Kreislaufforsch 1963;52:40-53. [70] Spach C, Streeten DH. Retardation of Sodium Exchange in Dog Erythrocytes by Physiological Concentrations of Aldosterone, in Vitro. J Clin Invest 1964;43:217- 27. [71] Yang P, Jones BL, Henderson LP. Mechanisms of anabolic androgenic steroid modulation of alpha(1)beta(3)gamma(2L) GABA(A) receptors. Neuropharmacol- ogy 2002;43 (4):619-33. [72] Bitran D, Kellogg CK, Hilvers RJ. Treatment with an anabolic-androgenic steroid affects anxiety-related behavior and alters the sensitivity of cortical GABAA receptors in the rat. Horm Behav 1993;27 (4):568-83.

51 [73] Jorge-Rivera JC, McIntyre KL, Henderson LP. Anabolic steroids induce region- and subunit-specific rapid modulation of GABA(A) receptor-mediated currents in the rat forebrain. J Neurophysiol 2000;83 (6):3299-309. [74] Bonson KR, Johnson RG, Fiorella D, Rabin RA, Winter JC. Serotonergic control of androgen-induced dominance. Pharmacol Biochem Behav 1994;49 (2):313-22. [75] Kindlundh AM, Lindblom J, Bergstrom L, Nyberg F. The anabolic-androgenic steroid nandrolone induces alterations in the density of serotonergic 5HT1B and 5HT2 receptors in the male rat brain. Neuroscience 2003;119 (1):113-20. [76] Martinez-Conde E, Leret ML, Diaz S. The influence of testosterone in the brain of the male rat on levels of serotonin (5-HT) and hydroxyindole-acetic acid (5-HIAA). Comp Biochem Physiol C 1985;80 (2):411-4. [77] Kindlundh AM, Lindblom J, Bergstrom L, Wikberg JE, Nyberg F. The anabolic- androgenic steroid nandrolone decanoate affects the density of dopamine receptors in the male rat brain. Eur J Neurosci 2001;13 (2):291-6. [78] Birgner C, Kindlundh-Hogberg AM, Alsio J, Lindblom J, Schioth HB, Bergstrom L. The anabolic androgenic steroid nandrolone decanoate affects mRNA expression of dopaminergic but not serotonergic receptors. Brain Res 2008;1240:221-8. [79] Birgner C, Kindlundh-Hogberg AM, Nyberg F, Bergstrom L. Altered extracellular levels of DOPAC and HVA in the rat nucleus accumbens shell in response to sub- chronic nandrolone administration and a subsequent amphetamine challenge. Neu- rosci Lett 2007;412 (2):168-72. [80] Le Greves P, Huang W, Johansson P, Thornwall M, Zhou Q, Nyberg F. Effects of an anabolic-androgenic steroid on the regulation of the NMDA receptor NR1, NR2A and NR2B subunit mRNAs in brain regions of the male rat. Neurosci Lett 1997;226 (1):61-4. [81] Le Greves P, Zhou Q, Huang W, Nyberg F. Effect of combined treatment with nandrolone and cocaine on the NMDA receptor gene expression in the rat nucleus accumbens and periaqueductal gray. Acta Psychiatr Scand Suppl 2002 (412):129- 32. [82] Johansson P, Ray A, Zhou Q, Huang W, Karlsson K, Nyberg F. Anabolic androgenic steroids increase beta-endorphin levels in the ventral tegmental area in the male rat brain. Neurosci Res 1997;27 (2):185-9. [83] Johansson P, Hallberg M, Kindlundh A, Nyberg F. The effect on opioid peptides in the rat brain, after chronic treatment with the anabolic androgenic steroid, nandrolone decanoate. Brain Res Bull 2000;51 (5):413-8. [84] Guarino G, Spampinato S. Nandrolone decreases mu opioid receptor expression in SH-SY5Y human neuroblastoma cells. Neuroreport 2008;19 (11):1131-5. [85] Hallberg M, Johansson P, Kindlundh AM, Nyberg F. Anabolic-androgenic steroids affect the content of substance P and substance P(1-7) in the rat brain. Peptides 2000;21 (6):845-52. [86] Hallberg M, Kindlundh A, Nyberg F. The impact of chronic nandrolone decanoate administration on the NK1 receptor density in rat brain as determined by autoradiography. Peptides 2005;In press. [87] US VE, Gaddum JH. An unidentified depressor substance in certain tissue extracts. J Physiol 1931;72 (1):74-87. [88] Pernow B. Distribution of substance P in the central and peripheral nervous system. Nature 1953;171 (4356):746.

52 [89] Tregear GW, Niall HD, Potts JT, Jr., Leeman SE, Chang MM. Synthesis of substance P. Nat New Biol 1971;232 (29):87-9. [90] Chang MM, Leeman SE, Niall HD. Amino-acid sequence of substance P. Nat New Biol 1971;232 (29):86-7. [91] Teichberg VI, Cohen S, Blumberg S. Distinct classes of substance P receptors revealed by a comparison of the activities of substance P and some of its segments. Regul Pept 1981;1 (5):327-33. [92] Carter MS, Krause JE. Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma. J Neurosci 1990;10 (7):2203-14. [93] Krause JE, Chirgwin JM, Carter MS, Xu ZS, Hershey AD. Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A. Proc Natl Acad Sci U S A 1987;84 (3):881-5. [94] Eipper BA, Stoffers DA, Mains RE. The biosynthesis of neuropeptides: peptide alpha-amidation. Annu Rev Neurosci 1992;15:57-85. [95] Harmar A, Schofield JG, Keen P. Cycloheximide-sensitive synthesis of substance P by isolated dorsal root ganglia. Nature 1980;284 (5753):267-9. [96] De Felipe C, Herrero JF, O'Brien JA, Palmer JA, Doyle CA, Smith AJ, Laird JM, Belmonte C, Cervero F, Hunt SP. Altered nociception, analgesia and aggression in mice lacking the receptor for substance P. Nature 1998;392 (6674):394-7. [97] Otsuka M, Konishi S. Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat. Nature 1976;264 (5581):83-4. [98] Goldin E, Karmeli F, Selinger Z, Rachmilewitz D. Colonic substance P levels are increased in ulcerative colitis and decreased in chronic severe constipation. Dig Dis Sci 1989;34 (5):754-7. [99] Lembeck F, Holzer P. Substance P as neurogenic mediator of antidromic vasodila- tion and neurogenic plasma extravasation. Naunyn Schmiedebergs Arch Pharmacol 1979;310 (2):175-83. [100] Rupniak NM, Carlson EC, Harrison T, Oates B, Seward E, Owen S, de Felipe C, Hunt S, Wheeldon A. Pharmacological blockade or genetic deletion of substance P (NK(1)) receptors attenuates neonatal vocalisation in guinea-pigs and mice. Neuro- pharmacology 2000;39 (8):1413-21. [101] Kramer MS, Cutler N, Feighner J, Shrivastava R, Carman J, Sramek JJ, Reines SA, Liu G, Snavely D, Wyatt-Knowles E, Hale JJ, Mills SG, MacCoss M, Swain CJ, Harrison T, Hill RG, Hefti F, Scolnick EM, Cascieri MA, Chicchi GG, Sadowski S, Williams AR, Hewson L, Smith D, Carlson EJ, Hargreaves RJ, Rupniak NM. Dis- tinct mechanism for antidepressant activity by blockade of central substance P receptors. Science 1998;281 (5383):1640-5. [102] Shaikh MB, Steinberg A, Siegel A. Evidence that substance P is utilized in medial amygdaloid facilitation of defensive rage behavior in the cat. Brain Res 1993;625 (2):283-94. [103] Cox BM, Opheim KE, Teschemacher H, Goldstein A. A peptide-like substance from pituitary that acts like morphine. 2. Purification and properties. Life Sci 1975;16 (12):1777-82. [104] Goldstein A, Tachibana S, Lowney LI, Hunkapiller M, Hood L. Dynorphin-(1-13), an extraordinarily potent opioid peptide. Proc Natl Acad Sci U S A 1979;76 (12):6666-70.

53 [105] Goldstein A, Fischli W, Lowney LI, Hunkapiller M, Hood L. Porcine pituitary dynorphin: complete amino acid sequence of the biologically active heptadecapeptide. Proc Natl Acad Sci U S A 1981;78 (11):7219-23. [106] Horikawa S, Takai T, Toyosato M, Takahashi H, Noda M, Kakidani H, Kubo T, Hirose T, Inayama S, Hayashida H, et al. Isolation and structural organization of the human preproenkephalin B gene. Nature 1983;306 (5943):611-4. [107] Day R, Lazure C, Basak A, Boudreault A, Limperis P, Dong W, Lindberg I. Pro- dynorphin processing by proprotein convertase 2. Cleavage at single basic residues and enhanced processing in the presence of carboxypeptidase activity. J Biol Chem 1998;273 (2):829-36. [108] Corbett AD, Paterson SJ, McKnight AT, Magnan J, Kosterlitz HW. Dynorphin and dynorphin are ligands for the kappa-subtype of opiate receptor. Nature 1982;299 (5878):79-81. [109] Chavkin C, James IF, Goldstein A. Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science 1982;215 (4531):413-5. [110] Chavkin C, Goldstein A. Specific receptor for the opioid peptide dynorphin: structure--activity relationships. Proc Natl Acad Sci U S A 1981;78 (10):6543-7. [111] Fallon JH, Leslie FM. Distribution of dynorphin and enkephalin peptides in the rat brain. J Comp Neurol 1986;249 (3):293-336. [112] Pfeiffer A, Brantl V, Herz A, Emrich HM. Psychotomimesis mediated by kappa opiate receptors. Science 1986;233 (4765):774-6. [113] Bals-Kubik R, Ableitner A, Herz A, Shippenberg TS. Neuroanatomical sites medi- ating the motivational effects of opioids as mapped by the conditioned place preference paradigm in rats. J Pharmacol Exp Ther 1993;264 (1):489-95. [114] Jiang HK, Owyang VV, Hong JS, Gallagher M. Elevated dynorphin in the hippocampal formation of aged rats: relation to cognitive impairment on a spatial learning task. Proc Natl Acad Sci U S A 1989;86 (8):2948-51. [115] Nguyen XV, Masse J, Kumar A, Vijitruth R, Kulik C, Liu M, Choi DY, Foster TC, Usynin I, Bakalkin G, Bing G. Prodynorphin knockout mice demonstrate dimin- ished age-associated impairment in spatial water maze performance. Behav Brain Res 2005;161 (2):254-62. [116] Martin WR. History and development of mixed opioid agonists, partial agonists and antagonists. Br J Clin Pharmacol 1979;7 Suppl 3:273S-9S. [117] Chang KJ, Hazum E, Cuatrecasas P. Novel opiate binding sites selective for ben- zomorphan drugs. Proc Natl Acad Sci U S A 1981;78 (7):4141-5. [118] Kosterlitz HW, Paterson SJ, Robson LE. Characterization of the kappa-subtype of the opiate receptor in the guinea-pig brain. Br J Pharmacol 1981;73 (4):939-49. [119] Meng F, Xie GX, Thompson RC, Mansour A, Goldstein A, Watson SJ, Akil H. Cloning and pharmacological characterization of a rat kappa opioid receptor. Proc Natl Acad Sci U S A 1993;90 (21):9954-8. [120] Yasuda K, Raynor K, Kong H, Breder CD, Takeda J, Reisine T, Bell GI. Cloning and functional comparison of kappa and delta opioid receptors from mouse brain. Proc Natl Acad Sci U S A 1993;90 (14):6736-40. [121] Mansson E, Bare L, Yang D. Isolation of a human kappa opioid receptor cDNA from placenta. Biochem Biophys Res Commun 1994;202 (3):1431-7. [122] Sharma SK, Klee WA, Nirenberg M. Opiate-dependent modulation of adenylate cyclase. Proc Natl Acad Sci U S A 1977;74 (8):3365-9.

54 [123] Surprenant A, Shen KZ, North RA, Tatsumi H. Inhibition of calcium currents by noradrenaline, somatostatin and opioids in guinea-pig submucosal neurones. J Physiol 1990;431:585-608. [124] North RA, Williams JT, Surprenant A, Christie MJ. Mu and delta receptors belong to a family of receptors that are coupled to potassium channels. Proc Natl Acad Sci U S A 1987;84 (15):5487-91. [125] Nikolarakis KE, Almeida OF, Yassouridis A, Herz A. Presynaptic auto- and allelo- receptor regulation of hypothalamic opioid peptide release. Neuroscience 1989;31 (1):269-73. [126] Dubner R, Ruda MA. Activity-dependent neuronal plasticity following tissue injury and inflammation. Trends Neurosci 1992;15 (3):96-103. [127] Caudle RM, Dubner R. Ifenprodil blocks the excitatory effects of the opioid peptide dynorphin 1-17 on NMDA receptor-mediated currents in the CA3 region of the guinea pig hippocampus. Neuropeptides 1998;32 (1):87-95. [128] De Araujo JE, Huston JP, Brandao ML. Opposite effects of substance P fragments C (anxiogenic) and N (anxiolytic) injected into dorsal periaqueductal gray. Eur J Pharmacol 2001;432 (1):43-51. [129] Tomaz C, Silva AC, Nogueira PJ. Long-lasting mnemotropic effect of substance P and its N-terminal fragment (SP1-7) on avoidance learning. Braz J Med Biol Res 1997;30 (2):231-3. [130] Roske I, Oehme P, Hecht K, Nieber K, Hilse H, Wachtel E. [The role of the N- terminal of the substance P molecule in its action in stress-related behavioral and blood pressure disorders]. Pharmazie 1986;41 (11):799-805. [131] Reid MS, Herrera-Marschitz M, Terenius L, Ungerstedt U. Intranigral substance P modulation of striatal dopamine: interaction with N-terminal and C-terminal substance P fragments. Brain Res 1990;526 (2):228-34. [132] Kreeger JS, Larson AA. Substance P-(1-7), a substance P metabolite, inhibits withdrawal jumping in morphine-dependent mice. Eur J Pharmacol 1993;238 (1):111-5. [133] Stewart JM, Hall ME, Harkins J, Frederickson RC, Terenius L, Hokfelt T, Krivoy WA. A fragment of substance P with specific central activity: SP(1-7). Peptides 1982;3 (5):851-7. [134] Zhou Q, Karlsson K, Liu Z, Johansson P, Le Greves M, Kiuru A, Nyberg F. Sub- stance P endopeptidase-like activity is altered in various regions of the rat central nervous system during morphine tolerance and withdrawal. Neuropharmacology 2001;41 (2):246-53. [135] Karlsson K, Nyberg F. Purification of substance P endopeptidase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fluid and with chymotrypsin. J Mol Recognit 1998;11 (1-6):266-9. [136] Karlsson K, Eriksson U, Andren P, Nyberg F. Purification and characterization of substance P endopeptidase activities in the rat spinal cord. Prep Biochem Biotech- nol 1997;27 (1):59-78. [137] Persson S, Le Greves P, Thornwall M, Eriksson U, Silberring J, Nyberg F. Neu- ropeptide converting and processing enzymes in the spinal cord and cerebrospinal fluid. Prog Brain Res 1995;104:111-30. [138] 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 1984;125 (1):244-50.

55 [139] Nyberg F. Peptidases in human cerebrospinal fluid converting dynorphin and substance P to bioactive fragments. Adv Biosci 1987;65:65-72. [140] Nyberg F, Sundqvist C, Le Greves P. Substance P converting endopeptidase in human cerebrospinal fluid. Protides Biol Fluids 1986;34:181-5. [141] Malfroy B, Kado-Fong H, Gros C, Giros B, Schwartz JC, Hellmiss R. Molecular cloning and amino acid sequence of rat kidney aminopeptidase M: a member of a super family of zinc-metallohydrolases. Biochem Biophys Res Commun 1989;161 (1):236-41. [142] Benuck M, Berg MJ, Marks N. Membrane-bound enzymes and their role in processing of the dynorphins and of the proenkephalin octapeptide Metenkephalin-Arg- Gly-Leu. Neurochem Res 1984;9 (6):733-49. [143] Inaoka Y, Tamaoki H. Purification and characterization of enkephalinase B from rat brain membrane. Biochim Biophys Acta 1987;925 (1):27-35. [144] Hersh LB. Reaction of opioid peptides with neutral endopeptidase ("enkephalinase"). J Neurochem 1984;43 (2):487-93. [145] Matsas R, Kenny AJ, Turner AJ. The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11. Biochem J 1984;223 (2):433-40. [146] Nyberg F, Nordstrom K, Terenius L. Endopeptidase in human cerebrospinal fluid which cleaves proenkephalin B opioid peptides at consecutive basic amino acids. Biochem Biophys Res Commun 1985;131 (3):1069-74. [147] Silberring J, Sakurada T, Nyberg F. Dynorphin converting enzyme in the rat spinal cord. Decreased activities during acute phase of adjuvant induced arthritis. Life Sci 1992;50 (12):839-47. [148] Nyberg F, Silberring J. Conversion of the dynorphins to Leu-enkephalin in human spinal cord. Prog Clin Biol Res 1990;328:261-5. [149] Persson S, Post C, Alari L, Nyberg F, Terenius L. Increased neuropeptide- converting enzyme activities in cerebrospinal fluid of opiate-tolerant rats. Neurosci Lett 1989;107 (1-3):318-22. [150] Silberring J, Brostedt P, Thornwall M, Nyberg F. Approach to studying proteinase specificity by continuous-flow fast atom bombardment mass spectrometry and high- performance liquid chromatography combined with photodiode-array ultraviolet detection. J Chromatogr 1991;554 (1-2):83-90. [151] Silberring J, Castello ME, Nyberg F. Characterization of dynorphin A-converting enzyme in human spinal cord. An endoprotease related to a distinct conversion pathway for the opioid heptadecapeptide? J Biol Chem 1992;267 (30):21324-8. [152] Nyberg F, Nylander I, Terenius L. Enkephalin-containing polypeptides in human cerebrospinal fluid. Brain Res 1986;371 (2):278-86. [153] Thornwall M, Schwarzmayr S, Almay B, Post C, Vaeroy H, Nyberg F. Dynorphin converting enzyme in human cerebrospinal fluid. Regul Peptides 1994;53 (suppl. 1):155-6. [154] Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin in man. J. clin. Invest. 1960;39:1157. [155] Eriksson U, Andren PE, Caprioli RM, Nyberg F. Reversed-phase high-performance liquid chromatography combined with tandem mass spectrometry in studies of a substance P-converting enzyme from human cerebrospinal fluid. J Chromatogr A 1996;743 (1):213-20.

56 [156] Paxinos G, Watson C. The rat brain stereotaxic coordinates. San Diego, CA, USA: Academic Press, 1997. [157] Compton DM. Behavior strategy learning in rat: effects of lesions of the dorsal striatum or dorsal hippocampus. Behav Processes 2004;67 (3):335-42. [158] Loonam TM, Noailles PA, Yu J, Zhu JP, Angulo JA. Substance P and chole- cystokinin regulate neurochemical responses to cocaine and methamphetamine in the striatum. Life Sci 2003;73 (6):727-39. [159] McDonald RJ, White NM. A triple dissociation of memory systems: hippocampus, amygdala, and dorsal striatum. Behav Neurosci 1993;107 (1):3-22. [160] Waksman G, Bouboutou R, Devin J, Besselievre R, Fournie-Zaluski MC, Roques BP. Binding of the bidentate inhibitor [3H]HACBO-Gly to the rat brain neutral endopeptidase "enkephalinase". Biochem Biophys Res Commun 1985;131 (1):262- 8. [161] Strittmatter SM, Lo MM, Javitch JA, Snyder SH. Autoradiographic visualization of angiotensin-converting enzyme in rat brain with [3H]captopril: localization to a striatonigral pathway. Proc Natl Acad Sci U S A 1984;81 (5):1599-603. [162] Carlezon WA, Jr., Beguin C, DiNieri JA, Baumann MH, Richards MR, Todtenkopf MS, Rothman RB, Ma Z, Lee DY, Cohen BM. Depressive-like effects of the kappa- opioid receptor agonist salvinorin A on behavior and neurochemistry in rats. J Pharmacol Exp Ther 2006;316 (1):440-7. [163] McLaughlin JP, Li S, Valdez J, Chavkin TA, Chavkin C. Social defeat stress- induced behavioral responses are mediated by the endogenous kappa opioid system. Neuropsychopharmacology 2006;31 (6):1241-8. [164] Zhang Y, Butelman ER, Schlussman SD, Ho A, Kreek MJ. Effect of the endogenous kappa opioid agonist dynorphin A(1-17) on cocaine-evoked increases in striatal dopamine levels and cocaine-induced place preference in C57BL/6J mice. Psy- chopharmacology (Berl) 2004;172 (4):422-9. [165] Svingos AL, Colago EE, Pickel VM. Cellular sites for dynorphin activation of kappa-opioid receptors in the rat nucleus accumbens shell. J Neurosci 1999;19 (5):1804-13. [166] Naghdi N, Oryan S, Etemadi R. The study of spatial memory in adult male rats with injection of testosterone enanthate and flutamide into the basolateral nucleus of the amygdala in Morris water maze. Brain Res 2003;972 (1-2):1-8. [167] Wittmann W, Schunk E, Rosskothen I, Gaburro S, Singewald N, Herzog H, Schwarzer C. Prodynorphin-derived peptides are critical modulators of anxiety and regulate neurochemistry and corticosterone. Neuropsychopharmacology 2009;34 (3):775-85. [168] Rocha VM, Calil CM, Ferreira R, Moura MJ, Marcondes FK. Influence of anabolic steroid on anxiety levels in sedentary male rats. Stress 2007;10 (4):326-31. [169] McGaugh JL, Cahill L. Interaction of neuromodulatory systems in modulating memory storage. Behav Brain Res 1997;83 (1-2):31-8. [170] Marklund N, Clausen F, Lewen A, Hovda DA, Olsson Y, Hillered L. alpha-Phenyl- tert-N-butyl nitrone (PBN) improves functional and morphological outcome after cortical contusion injury in the rat. Acta Neurochir (Wien) 2001;143 (1):73-81. [171] Galea LA, Kavaliers M, Ossenkopp KP, Hampson E. Gonadal hormone levels and spatial learning performance in the Morris water maze in male and female meadow voles, Microtus pennsylvanicus. Horm Behav 1995;29 (1):106-25.

57 [172] Flood JF, Farr SA, Kaiser FE, Morley JE. Age-related impairment in learning but not memory in SAMP8 female mice. Pharmacol Biochem Behav 1995;50 (4):661-4. [173] O'Connor DB, Archer J, Hair WM, Wu FC. Activational effects of testosterone on cognitive function in men. Neuropsychologia 2001;39 (13):1385-94. [174] Liben LS, Susman EJ, Finkelstein JW, Chinchilli VM, Kunselman S, Schwab J, Dubas JS, Demers LM, Lookingbill G, Darcangelo MR, Krogh HR, Kulin HE. The effects of sex steroids on spatial performance: a review and an experimental clinical investigation. Dev Psychol 2002;38 (2):236-53. [175] Silverman I, Kastuk D, Choi J, Phillips K. Testosterone levels and spatial ability in men. Psychoneuroendocrinology 1999;24 (8):813-22. [176] Clark AS, Mitre MC, Brinck-Johnsen T. Anabolic-androgenic steroid and adrenal steroid effects on hippocampal plasticity. Brain Res 1995;679 (1):64-71. [177] Goudsmit E, Van de Poll NE, Swaab DF. Testosterone fails to reverse spatial memory decline in aged rats and impairs retention in young and middle-aged animals. Behav Neural Biol 1990;53 (1):6-20. [178] Naghdi N, Majlessi N, Bozorgmehr T. The effect of intrahippocampal injection of testosterone enanthate (an androgen receptor agonist) and anisomycin (protein synthesis inhibitor) on spatial learning and memory in adult, male rats. Behav Brain Res 2005;156 (2):263-8. [179] Naghdi N, Nafisy N, Majlessi N. The effects of intrahippocampal testosterone and flutamide on spatial localization in the Morris water maze. Brain Res 2001;897 (1- 2):44-51. [180] Kouvelas D, Pourzitaki C, Papazisis G, Dagklis T, Dimou K, Kraus MM. Nandro- lone abuse decreases anxiety and impairs memory in rats via central androgenic receptors. Int J Neuropsychopharmacol 2008;11 (7):925-34. [181] McEwen BS, Biegon A, Davis PG, Krey LC, Luine VN, McGinnis MY, Paden CM, Parsons B, Rainbow TC. Steroid hormones: humoral signals which alter brain cell properties and functions. Recent Prog Horm Res 1982;38:41-92. [182] Chen L, Dai XN, Sokabe M. Chronic administration of dehydroepiandrosterone sulfate (DHEAS) primes for facilitated induction of long-term potentiation via sigma 1 (sigma1) receptor: optical imaging study in rat hippocampal slices. Neuro- pharmacology 2006;50 (3):380-92. [183] Barbaccia ML, Scaccianoce S, Del Bianco P, Campolongo P, Trezza V, Tattoli M, Cuomo V, Steardo L. Cognitive impairment and increased brain neurosteroids in adult rats perinatally exposed to low millimolar blood alcohol concentrations. Psy- choneuroendocrinology 2007;32 (8-10):931-42. [184] Mellon SH. Neurosteroids: biochemistry, modes of action, and clinical relevance. J Clin Endocrinol Metab 1994;78 (5):1003-8. [185] Vallee M, Mayo W, Le Moal M. Role of pregnenolone, dehydroepiandrosterone and their sulfate esters on learning and memory in cognitive aging. Brain Res Brain Res Rev 2001;37 (1-3):301-12. [186] Schumacher M, Weill-Engerer S, Liere P, Robert F, Franklin RJ, Garcia-Segura LM, Lambert JJ, Mayo W, Melcangi RC, Parducz A, Suter U, Carelli C, Baulieu EE, Akwa Y. Steroid hormones and neurosteroids in normal and pathological aging of the nervous system. Prog Neurobiol 2003;71 (1):3-29. [187] Hlinak Z, Gandalovicova D, Krejci I. Behavioral deficits in adult rats treated neona- tally with glutamate. Neurotoxicol Teratol 2005;27 (3):465-73.

58 [188] Rossbach UL, Steensland P, Nyberg F, Le Greves P. Nandrolone-induced hippocampal phosphorylation of NMDA receptor subunits and ERKs. Biochem Biophys Res Commun 2007;357 (4):1028-33. [189] Kuzmin A, Madjid N, Terenius L, Ogren SO, Bakalkin G. Big dynorphin, a prodynorphin-derived peptide produces NMDA receptor-mediated effects on memory, anxiolytic-like and locomotor behavior in mice. Neuropsychopharmacology 2006;31 (9):1928-37. [190] Wall PM, Messier C. Concurrent modulation of anxiety and memory. Behav Brain Res 2000;109 (2):229-41. [191] Daumas S, Betourne A, Halley H, Wolfer DP, Lipp HP, Lassalle JM, Frances B. Transient activation of the CA3 Kappa opioid system in the dorsal hippocampus modulates complex memory processing in mice. Neurobiol Learn Mem 2007;88 (1):94-103. [192] Carey AN, Lyons AM, Shay CF, Dunton O, McLaughlin JP. Endogenous kappa opioid activation mediates stress-induced deficits in learning and memory. J Neuro- sci 2009;29 (13):4293-300. [193] Kolsch H, Wagner M, Bilkei-Gorzo A, Toliat MR, Pentzek M, Fuchs A, Kadusz- kiewicz H, van den Bussche H, Riedel-Heller SG, Angermeyer MC, Weyerer S, Werle J, Bickel H, Mosch E, Wiese B, Daerr M, Jessen F, Maier W, Dichgans M. Gene polymorphisms in prodynorphin (PDYN) are associated with episodic memory in the elderly. J Neural Transm 2009. [194] Yakovleva T, Marinova Z, Kuzmin A, Seidah NG, Haroutunian V, Terenius L, Bakalkin G. Dysregulation of dynorphins in Alzheimer disease. Neurobiol Aging 2007;28 (11):1700-8. [195] Hiller JM, Itzhak Y, Simon EJ. Selective changes in mu, delta and kappa opioid receptor binding in certain limbic regions of the brain in Alzheimer's disease patients. Brain Res 1987;406 (1-2):17-23. [196] Reisberg B, Ferris SH, Anand R, Mir P, Geibel V, De Leon MJ, Roberts E. Effects of naloxone in senile dementia: a double-blind trial. N Engl J Med 1983;308 (12):721-2.

59 $2 $ 3 4 5 5

4 5 5$ $25 5 5 6 7 8" 5 5 2 " $ 4 5 569: 5*' ; 2" $ 4 5 5<6=

66 2 (,.-

Web Analytics