ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2012

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 168

The Impact of GrowthHormone and Gamma-Hydroxybutyrate (GHB) onSystems Related to Cognition

JENNY JOHANSSON

ISSN 1651-6192ISBN 978-91-554-8552-8urn:nbn:se:uu:diva-185631

Dissertation presented at Uppsala University to be publicly examined in B21, BMC,Husargatan 3, Uppsala, Friday, January 18, 2013 at 09:15 for the degree of Doctor ofPhilosophy (Faculty of Pharmacy). The examination will be conducted in Swedish.

AbstractJohansson, J. 2012. The Impact of Growth Hormone and Gamma-Hydroxybutyrate (GHB)on Systems Related to Cognition. Acta Universitatis Upsaliensis. Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Pharmacy 168. 73 pp. Uppsala.ISBN 978-91-554-8552-8.

Drug dependence is a serious and increasing problem in our society, especially amongadolescents. The use of the large variety of substances available can result in a range ofphysiological and psychological adverse effects on individuals and negative consequences onthe society overall. Several different types of drugs induce neurotoxicological damages, which inturn can generate impairment in for example the reward system and affect cognitive parameters.

The drug gamma-hydroxybutyrate (GHB) is usually considered a harmless compound amongabusers, but has now shown to be highly addictive. Furthermore, GHB can cause memoryimpairments in both humans and animals. On the contrary, growth hormone (GH) and its mainmediator insulin-like growth factor 1 (IGF-1) have recently been suggested to improve memoryand learning in several studies. The hormones exhibit certain neuroprotective capabilities andhave also previously been demonstrated to reverse opioid induced apoptosis in hippocampalcells. These effects and the fact that GHB is shown to increase GH secretion, which attractedconsiderable attention among body builders, led us to initiate studies on GHB and its impact onrelevant systems in the central nervous system (CNS). Thus, the main purpose of the presentinvestigation was to elucidate some of the underlying mechanisms that could account for theeffects exerted by GH and GHB in the CNS.

We found that a) GH affects the density and functionality of GABAB-receptors and opioidreceptors in the male rat brain, b) GHB induces cognitive deficits and down-regulates GABAB-receptors, c) GHB treatment creates an imbalance between the endogenous opioids Met-enkaphalin-Arg6Phe7 (MEAP) and dynorphin B and increases the levels of MEAP in regions ofthe brain that are associated with drug dependence, and d) GHB affects the expression of IGF-1receptors but not the plasma levels of IGF-1. In conclusion, the present work demonstrates thatGH interacts with both opioid and GABAB-receptors in the male rat CNS and that GHB has animpact on brain regions associated with cognition and the development of dependence. Theseobservations may be of relevance in many aspects related to addiction and might be translatedinto humans.

Keywords: Growth Hormone, Gamma-Hydroxybutyrate, GABAB, Opioids, Insulin-likegrowth factor 1, Rats, Central Nervous System, Autoradiography, Radioimmunoassay,ELISA, Water Maze, Open Field

Jenny Johansson, Uppsala University, Department of Pharmaceutical Biosciences, Box 591,SE-751 24 Uppsala, Sweden.

© Jenny Johansson 2012

ISSN 1651-6192ISBN 978-91-554-8552-8urn:nbn:se:uu:diva-185631 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-185631)

Supervisor Associate professor Mathias Hallberg Co-supervisor Professor Fred Nyberg Faculty opponent Associate professor Linda Fryklund Members of the examining board Professor Lennart Dencker Associate professor Åsa Mackenzie Associate professor Anna Skottner

List of Papers

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

I Grönbladh, A., Johansson, J., Nyberg, F., Hallberg, M. (2012) Recombinant human growth hormone affects the density and func-tionallity of GABAB receptors in the male rat brain. Neuroendocri-nology, Epub ahead of press.

II Johansson, J., Grönbladh, A., Nyberg, F., Hallberg, M. (2013) Application of in vitro [35S]GTPγS autoradiography in studies of growth hormone effects on opioid receptors in the male rat brain. Brain Research Bulletin, 90, 100-106.

III Johansson, J., Grönbladh, A., Nyberg F., Hallberg, M. (2012) Gamma-hydroxybutyrate (GHB) induces cognitive deficits and af-fects GABAB receptors and IGF-1 receptors in male rats. In manu-script.

IV Johansson, J., Brolin, E., Grönbladh, A., Nyberg, F., Hallberg, M. (2012) Gamma-hydroxybutyrate (GHB) elevates Met-enkephalin-Arg6Phe7 (MEAP) levels in the frontal cortex of the male rat brain. In manuscript.

Paper I and II are published by kind permission of S. Karger AG, Basel, Switzerland and Elsevier, Oxford, United Kingdom.

Contents

Introduction ................................................................................................... 11  Gamma-hydroxybutyrate (GHB) .............................................................. 11  

History .................................................................................................. 11  Physiological and psychological effects of GHB ................................ 11  Synthesis and metabolism of GHB ...................................................... 12  Mechanisms of action .......................................................................... 14  

Growth Hormone (GH) and Insulin-like Growth factor-1 (IGF-1) .......... 15  History .................................................................................................. 15  The somatotrophic system ................................................................... 16  Pharmacodynamic aspects of GH and IGF-1 ....................................... 18  Physiological and psychological relevance of GH and IGF-1 ............. 20  

The brain reward system ........................................................................... 21  The opioid system ..................................................................................... 22  

Opioid peptides and receptors .............................................................. 22  Regulation of the reward system by opioid peptides ........................... 23  The GABAergic system ....................................................................... 24  Cognition; learning and memory ......................................................... 25  

The aims of the thesis .................................................................................... 27  Methods ......................................................................................................... 28  

Animal handling and drug treatment ........................................................ 28  Behavioural methods ................................................................................ 29  

The water maze test (WM) .................................................................. 29  The open field (OF) ............................................................................. 30  

Tissue and blood collection ...................................................................... 31  Autoradiographic methods ....................................................................... 31  

GABAB, mu and delta opioid receptor stimulated [35S]GTPγS autoradiography ................................................................................... 32  GABAB receptor autoradiography ....................................................... 33  IGF-1 receptor autoradiography .......................................................... 33  

IGF -1 Enzyme-linked immunosorbent assay (ELISA) ........................... 34  Peptide extraction and separation ............................................................. 34  Radioimmunoassay (RIA) ........................................................................ 35  Statistical analysis ..................................................................................... 35  

Results and discussion ................................................................................... 37  GABAB receptors ...................................................................................... 37  Mu and delta opioid receptors .................................................................. 40  Insulin-like growth factor-1 receptor ........................................................ 41  Insulin-like growth factor-1 in plasma ..................................................... 42  

Opioid peptides .................................................................................... 43  The water maze ......................................................................................... 44  The Open field .......................................................................................... 46  

Summarizing conclusion ............................................................................... 48  Populärvetenskaplig sammanfattning ........................................................... 49  Acknowledgements ....................................................................................... 50  References ..................................................................................................... 52  

List of abbreviations

1,4-BD 1,4-butanediol CNS Central nervous system DAMGO Tyr-D-Ala-Gly-NMe-Phe-Gly-ol DOP Delta opioid peptide DPDPE [D-Pen2-D-Pen5]-enkephalin Dyn B Dynorphin B ELISA Enzyme-linked immunosorbent assay GABA Gamma-aminobutyric acid GBL Gamma-butyrolacton GDP Guanosine diphosphate GH Growth hormone GHB Gamma-hydroxybutyrate GHD Growth hormone deficiency GHR Growth hormone receptor GTP Guanosine triphosphate GTPγS Guanosine 5’-[γ-thio] triphosphate IGF-1 Insulin-like growth factor-1 IGF-1R Insulin-like growth factor-1 receptor IU International units LTD Long-term depression LTP Long-term potentiation ME Met-enkephalin MEAP Met-enkephalin-Arg6Phe7 MOP Mu opioid peptide NMDA N-Methyl-D-aspartic acid OF Open field rhGH Recombinant human growth hormone RIA Radioimmunoassay s.c. Subcutaneous WM Water maze

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Introduction

Gamma-hydroxybutyrate (GHB) History GHB was initially synthesized in 1960 by the French surgeon Henri Laborit in an attempt to find a new GABA-like agent, which could easily penetrate the blood brain barrier. The drug appeared to have the ability to induce num-ber of pharmacological effects e.g. euphoria, sedation and relaxation and was later on clinically evaluated as a pre-surgeon anesthetic drug (Blumenfeld et al., 1962, Solway and Sadove, 1965). Due to the lack of analgesic properties, narrow therapeutically window and severe side effects GHB never became a general anesthetic agent (Vickers, 1968).

Researchers in the 1970s discovered an increase in REM sleep in associa-tion with GHB administration (Takahara et al., 1977) and GHB was later approved in several countries as a drug for narcolepsy and cataplexy (Fuller and Hornfeldt, 2003, Black and Houghton, 2006). Several studies have been performed regarding GHB and treatment of alcoholism (Gallimberti et al., 1992, Gallimberti et al., 2000) and GHB has also been proposed as a poten-tial therapeutic in the treatment of fibromyalgia (Scharf et al., 1998a, Staud, 2011) and opioid dependence (Rosen et al., 1997).

The use of GHB for non-medical purposes started in 1980-1990 in the body builder scene, since it increases growth hormone release (Takahara et al., 1977). Moreover, it was sold over the counter as a dietary supplement for weight control, but was later banned in most countries. The use of GHB as a recreational drug has spread over the two last decades, especially among adolescents. Unfortunately, these trends have been reported in several parts of the world e.g. Europe, Australia and the United States (Van Cauter et al., 1997, Nicholson and Balster, 2001, Degenhardt et al., 2002, Rodgers et al., 2004, Knudsen et al., 2008, Wood et al., 2009).

Physiological and psychological effects of GHB The use/misuse of GHB and its precursors γ -butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are increasing, although it is considered that relatively few people use the drug. Data collected on drug use from 36 European coun-tries report an overall lifetime use of GHB was approximately 1% (see re-

12

view and report (Bramness and Haugland, 2011, Hibell et al., 2011, van Amsterdam et al., 2012). However there is evidence pointing at a higher usage among some specifically geographic areas and sub-cultures e.g. on the rave scenes.

GHB is easily made at home by heating GBL or 1,4-BD with NaOH or KOH, followed by filtering and cooling. The mixture can be kept in a liquid form or dried to a powder form. The drug is among certain abusers consid-ered as harmless and is usually used in recreational settings, as it induces euphoria, relaxation, sociability and sexuality (Sumnall et al., 2008). To increase the effects, GHB is often combined with other drugs of abuse like alcohol, cannabis, cocaine, benzodiazepines, opioids and amphetamine, which may result in disastrous aversive effects (Louagie et al., 1997, Abanades et al., 2006, Anderson et al., 2006, Liechti et al., 2006, Knudsen et al., 2010)

The effects produced by GHB are biphasic, where the euphoric effects are pronounced at low concentrations, whereas the sedative effects emerge upon higher doses. As a consequence of the narrow therapeutic window, an over-dose can easily be administrated, resulting in sleep, convulsions, vomiting, confusion, respiratory depression and even death (Munir et al., 2008, Knudsen et al., 2010, Galicia et al., 2011). Moreover, in humans GHB is known to induce temporary amnesia and is therefore used as a so-called date-rape drug (ElSohly and Salamone, 1999, Schwartz et al., 2000, Varela et al., 2004). Interestingly, memory impairments have been confirmed in several animal studies (Sircar and Basak, 2004, Pedraza et al., 2009, Sircar et al., 2010).

In addition to the acute toxicity, GHB is highly addictive and is associated with severe withdrawal symptoms such as tremor, anxiety, hallucinations and sometimes delirium upon abrupt discontinuation of use (Galloway et al., 1997, Miotto et al., 2001, Perez et al., 2006)

Synthesis and metabolism of GHB As described above, GHB is a drug of abuse, but it is also an endogenous compound present in the discrete areas of the brain as well as in peripheral tissue (Doherty et al., 1978, Nelson et al., 1981). The main precursor of GHB in the brain is the inhibitory neurotransmitter GABA (figure 1). GABA is converted to succinic semialdehyde by GABA transaminase in the mito-chondria, which is subsequently released in to the cytosol and reduced by GHB dehydrogenase to GHB (Roth and Giarman, 1969, Gold and Roth, 1977). However, only 0.05% (in vitro) to 0.16% (in vivo) of the total amount GABA is converted to GHB leading to an equilibrium where the level of GHB corresponds to approximately 0.1% of the GABA concentration. The restricted populations of neurons where GHB is synthetized and the limited amount of succinic semialdehyde released to the cytosol from the mitochon-

13

dria may explain the reason for this very low conversion of GABA into GHB (Gold and Roth, 1977, Rumigny et al., 1981).

GHB is primary degraded by GHB dehydrogenase to succinic semialde-hyde and further to succinic acid by succinic semialdehyde dehydrogenase. Succinic then acid enters the citric cycle and is subsequently expired as car-bondioxide and water (Chambliss and Gibson, 1992). Alternatively GHB is metabolized by GHB dehydrogenase to succinic semialdehyde and further by GHB transaminase to GABA (Vayer et al., 1985). Less than 5% of the GHB remains unmetabolized and is leaving the body unchanged via urine (Thai et al., 2006).

As seen in figure 1, when the GHB analogues, GBL and 1,4-BD are in-gested, they rapidly degrade to GHB (Maxwell and Roth, 1972, Snead et al., 1982). GBL is metabolized by lactonase and the 1,4-BD by alcohol dehy-drogenase and aldehyde dehydrogenase to GHB.

Figure 1. An overview of the synthesis and metabolic pathways of GHB. Drawing based on ideas from (Schep et al., 2012, van Amsterdam et al., 2012).

Orally ingested, GHB is rapidly absorbed from the gastrointestinal tract and easily penetrates the blood brain barrier and distributes in the brain leading to a rapid onset of actions (Roth et al., 1966, Abanades et al., 2006, Bhattacharya and Boje, 2006). Furthermore, post mortem analysis reveals that GHB also distributes to several peripheral tissues and fluids, although the exact concentration is difficult to determine, due to post mortem conver-

Citric cycle

!-butyrolactone

!-hydroxybutyrate, GHB

Succinic semialdehyde Succinic acid

!-aminobutyric acid, GABA Glutamate

4-hydroxybutyraldehyde 1,4-butanediol

Lactonase

GHB dehydrogenase

GHB transaminase

Aldehyde dehydrogenase

Alcohol dehydrogenase

Succinic semialdehyde dehydrogenase

Glutamic acid decarboxylase decarboxylase

14

sions of GABA to GHB (Elliott et al., 2004, Mazarr-Proo and Kerrigan, 2005). The blood peak concentration occurs typically within 1 h after intake in humans (Ferrara et al., 1992, Palatini et al., 1993, Scharf et al., 1998b, Abanades et al., 2006). Generally, the effects experienced of the drug start within 10-30 min and last approximately 2-4 h. The drug is eliminated very quickly with an average half-life of approximately 27 min (20-53 min) (Ferrara et al., 1992, Palatini et al., 1993, Scharf et al., 1998b). As a conse-quence of the GHB’s short half-life, the compound is undetectable after ap-proximately 12 h (Verstraete, 2004).

Mechanisms of action The pharmacological mechanisms underlying the effects mediated by the compound is not fully clarified. Yet, evidence supports that endogenous GHB is stored in presynaptic vesicles and are released upon depolarization of neurons in a Ca2+-dependent manner (Maitre et al., 1983, Snead, 1987). The activation is terminated by active Na+-dependent reuptake (Benavides et al., 1982b, Hechler et al., 1985). GHB acts as a neuromodula-tor/neurotransmitter and mediates its pharmacological effects predominantly via GHB- and GABAB receptors, although a very recent study also suggest the involvement of GABAA receptors (Benavides et al., 1982a, Hechler et al., 1991, Absalom et al., 2012).

Belonging to the G-protein coupled family, GHB receptor binds GHB with a high affinity and is anatomically expressed in the limbic system, hip-pocampus, cortex, and regions associated with dopaminergic activity (Benavides et al., 1982a, Hechler et al., 1987, Hechler et al., 1989, Snead, 1996, Ratomponirina et al., 1998, Andriamampandry et al., 2003, Castelli et al., 2003, Andriamampandry et al., 2007). The receptors can be activated by low concentrations of GHB leading to dopamine release (Castelli et al., 2003, Brancucci et al., 2004, Molnar et al., 2006). On the contrary, higher concentrations of GHB, received by exogenously administration inhibits dopamine release, an effect that is predominantly mediated via low affinity GABAB receptors. GHB is therefore said to act in biphasic manner (Erhardt et al., 1998, Lingenhoehl et al., 1999). Activation of the G-protein coupled GABAB receptor induces neuronal hyperpolarization leading to a general inhibition of neurotransmitter release, including dopamine. The involvement of GABAA receptors has been discussed in the literature for a long time. For example an indirect activation of the GABAA receptors via GHB have been reported to induce an alteration of neurosteroids, e.g. allopregnanolone (Barbaccia et al., 2002). Recently Absalom and co-workers identified several high affinity binding sites for GHB on the GABAA receptor. The result demonstrated that the subunit combination α4δ seem to play a particular role in pharmacological response induced by GHB (Absalom et al., 2012). (More information about GABA receptors is found on page 23). In addition to di-

15

rect activation of the GABA receptors by GHB, as seen in figure 1, the com-pound is to some extent also metabolized to GABA, which in turn activates GABAA and GABAB receptors (Snead and Gibson, 2005, Carter et al., 2009).

As mentioned above, GHB alters dopaminergic activity in the brain, es-pecially in the mesolimbic and nigrostriatal pathways (Cheramy et al., 1977, Diana et al., 1991, Hechler et al., 1991). The increased release of dopamine have been demonstrated to be GHB receptor specific (Maitre et al., 1990). Increased dopamine release in particularly the mesolimbic pathways, are associated with reward and addiction. Thereby it has been postulated that some of the rewarding effects induced by GHB, may in fact be associated with GHB receptor stimulated dopamine release (Cruz et al., 2004). Fur-thermore it has been suggested that some of the motoric impairments, such as muscle relaxation and dystonia, may partly be explained by GHB’s inhibi-tory effect on dopaminergic neurons in regions implicated in motor control (Nissbrandt and Engberg, 1996, Schmidt-Mutter et al., 1999b, Itzhak and Ali, 2002). In addition, the alterations in dopamine release are also suggested to be indirectly modulated by endogenous opioids, and although the exact relationships between GHB and the opioid system are not clarified, this might explain why many of GHB related effects e.g. sedation and euphoria, are blocked by naloxone (Gobaille et al., 1994, Schmidt-Mutter et al., 1999a).

The GHB related memory impairments is explained by e.g. alterations seen in the cholinergic and glutamatergic systems. Decreased extracellular levels of acetylcholine, NMDA synaptic activity and NMDA receptor densi-ty in hippocampus, frontal cortex and striatum has been reported as a result of GHB administration (Sircar and Basak, 2004, Li et al., 2007). Further-more, GHB seem to alter glutamate levels in the hippocampus in a biphasic manner, where lower concentrations of GHB increase and high doses de-crease extracellular glutamate levels in the hippocampus. These effects are suggested to be mediated by GHB receptor and GABAB receptors respec-tively (Castelli et al., 2003). An increase in GH release has also been report-ed in several studies after GHB intake, a response that seems to involve cho-linergic mechanisms (Takahara et al., 1977, Takahara et al., 1980, Addolorato et al., 1999, Volpi et al., 2000).

Growth Hormone (GH) and Insulin-like Growth factor-1 (IGF-1) History In the beginning of the 20th century, several patients underwent pituitary surgery for acromegaly with successful results (Caton, 1893, Cushing, 1909)

16

and thereby the importance of pituitary gland and hypophysis for growth became well recognized. In 1922 Evans and Long (Evans and Long, 1922) discovered that pituitary extracts injected to rats exerted growth prompting effects. In 1956, GH was isolated from the human pituitary gland, and after further studies the first successfully GH treatment of a 17 year old hypopitui-tary man was reported in 1958 (Raben, 1957, 1958). Around that time a fam-ily of GH regulated factors, somatomedins was also discovered (Salmon and Daughaday, 1957). The circulating somatomedin C, later called IGF-1 has been demonstrated to exhibit important growth and differentiation properties (Van Wyk et al., 1974, Fryklund and Sievertsson, 1978, Skottner et al., 1987, LeRoith et al., 1995).

A method for extraction and purification of GH from homogenized pitui-tary glands by gel filtration was discovered by Swedish scientists in 1963 (Roos et al., 1963). Pituitary GH was used for treatment of growth hormone deficiency (GHD) patients for more than 20 years until 4 incidents of Creuzfeldt Jacob Disease was reported, and the product was ceased (see report (1985) and review (Ayyar, 2011)). After the biochemical structure of GH was recognized in the early 70s (Li et al., 1969, Niall et al., 1973), the work to develop recombinant DNA-derived human GH started. Around 1980, cDNA was first cloned and the first biosynthetic recombinant human GH (rhGH) was produced (Martial et al., 1979, Roskam and Rougeon, 1979, Fryklund et al., 1986).

We now know that human GH consists of a 191 amino acid (aa) single stranded protein, containing two disulfide bridges and the most common form weighs approximately 22 kDa, whilst IGF-1 is a smaller polypeptide consisting of only 70 aa, with a molecular weight about 7.5 kDa.

The somatotrophic system The somatotrophic axis consists of the hormones GH, IGF-1 and IGF-2, their protein binding carriers GH binding protein (GHBP) and IGF binding pro-teins (IGFBP) and their specific receptors GH receptor (GHR) and IGF-1 receptor (IGF-1R), respectively.

GH and IGF-1 are regulated by a complex system. A simplified sketch of the regulation is presented in figure 2. GH synthesis and secretion in the pituitary is mainly determined by the balance between the stimulating GH-releasing hormone (GHRH) and the inhibiting somatostatin (SS) (Karin et al., 1990, McMahon et al., 2001, Goldenberg and Barkan, 2007, Gahete et al., 2009). Circulating GH can act directly on peripheral tissue where it mainly acts as a regulator for somatic growth as well as metabolic processes (Nilsson et al., 1986, Wong et al., 2008, Moller and Jorgensen, 2009). More-over, GH can stimulate IGF-1 release from the liver, which in turn is im-portant for stimulation of growth and is crucial for cell differentiation

17

(Daughaday and Trivedi, 1991, LeRoith and Roberts, 1991, Moller and Jorgensen, 2009).

Furthermore, the GH release is regulated with a negative feedback system where unbound GH and IGF-1 affect the hypothalamic hormones GHRH and SS, thus balancing the GH production and secretion from the pituitary (Daughaday, 2000, Le Roith et al., 2001a, Le Roith et al., 2001b). A short local feed-back system based on autocrine/paracrine mechanisms within the pituitary have also been reported (Fraser and Harvey, 1992, Asa et al., 2000). The levels of GHRH and SS are further affected by several other signal sys-tems, such as ghrelin, neuropeptides, steroid hormones and catecholamines (Kojima et al., 1999, Beleen et al., 2012, Kaminski and Smolinska, 2012). Moreover physiological parameters such as sleep and nutrition have also been suggested to play an important role in the regulation of the somato-tropic axis (Vicini et al., 1991, Muller et al., 1999). Extrapituitary production of GH has been reported, and is proposed to be regulated by autocrine and paracrine processes (Harvey and Hull, 1997).

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Figure 2. A simplified sketch over the regulation of the somatotropic system. GHRH released from the hypothalamus stimulates the secretion of GH from the anterior pituitary, whilst it is inhibited by SS. Upon release, GH exerts direct or indirect effects via IGF-1 on peripheral tissue. By a negative feed-back loop system, GH and IGF-1 control their own secretion. Stimulation and inhibition is indicated by (+) and (-) respectively.

Pharmacodynamic aspects of GH and IGF-1 Growth hormone receptor (GHR) The effects induced by GH are initiated by binding of the hormone to a spe-cific GHR on the cell surface. The GHR belongs to the cytokine receptor super family (Cosman et al., 1990, Kelly et al., 1993, Postel-Vinay et al., 1995) and is expressed in over 40 different tissues, including the brain (Lai et al., 1991, Nyberg, 2000, 2007). The receptor constitute of a single trans-membrane polypeptide chain, where the extracellular domain is composed of a N-terminal GH binding domain, and the intracellular domain is responsible for the regulation of the cytoplasmic effector system (Leung et al., 1987, Postel-Vinay, 1996). Binding of GH to the receptor results in a homodimeri-zation of two GHR (see figure 3) forming a GHR-GH-GHR complex (Cunningham et al., 1991, de Vos et al., 1992, Frank, 2002, Gent et al., 2002) The dimerization of the receptor is essential for proper intracellular

GH

GH GHRH

SS

IGF-1

SS -

-

- -

+

+

GHRH +

Pituitary

Hypothalamus IGF-1

IGF-1

Peripheral tissue

Liver

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response, involving activation of several kinases. Initially Janus kinas 2 (JAK2) is rapidly phosphorylated, which leads to interaction with other sig-naling molecules e.g. mitogenic-activated protein kinase (MAPK), signal transducers and activators of transcription (STAT) and mitogen-activated protein kinase (PKC) (Darnell et al., 1994, Herington, 1994, Horseman and Yu-Lee, 1994, Zhou and Waxman, 1999, Herrington and Carter-Su, 2001). As the concentration of GH increases, the GHR homodimers become more and more saturated leading to an increase in formation of monomeric recep-tor complexes (GHR-GH) (Figure 3), which in turn leads to an attenuated intracellular response. This scenario may lead to so-called bell-shaped dose-response effect (Fuh et al., 1992, Mustafa et al., 1997).

Besides the membrane bound receptor, a soluble form of the receptor called GHBP has been identified in many species (Amit et al., 1992, Talamantes and Ortiz, 2002). The GHBP correspond to the extracellular domain of the GHR and binds approximately 40-50 % of circulating GH. When GH is bound to GHBP it is less bioactive and is protected from degra-dation. Thus GHBP is important for the GH regulation (Postel-Vinay, 1996, Tzanela et al., 1997).

Figure 3. Hypothetical model of the GHR activation. JAK2; Janus kinase, STAT; Signal transducers and activators of transcription and PKC; Protein kinase C.

Insulin-like growth factor 1 receptor (IGF-1R) The IGF-1 receptor has been found in various regions in the brain (Bohannon et al., 1988, Laviola et al., 2007). It belongs to the large class of tyrosine kinase receptors and consists of two covalent bound " and two $ subunits (Figure 4). Binding of IGF-1 to the extracellularly " subunit induces a conformal change of the receptor leading to an autophosphorylation of the intracellular $ subunits (Kato et al., 1993). This leads to an intracellular sig-naling cascade including phosphorylation of other tyrosine containing sub-strates e.g. the Ras mitogen-activated protein kinase (MAPK) cascade (LeRoith and Roberts, 1991, Wine et al., 2009).

!!!!!!

GH GH

GH GH

GH

GH GH

JAK2 JAK2

STAT MAPK PKC

NO SIGNAL

Inactive GHR GHR homodimer Activated receptor

GHR monomer Inactive receptor

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Figure 4. Hypothetical model of IGF-1R. !; alpha subunit, "; beta subunit, P; phos-phorus and S; disulfide bridge.

Physiological and psychological relevance of GH and IGF-1 The hormones GH and IGF-1 are involved in many different processes in the body besides regulation of somatic growth and metabolism. Growing evi-dence verifies that GH and IGF-1 exert profound effects in the CNS includ-ing sleep, appetite, well-being, cognitive functions, neuroprotection and cell regeneration. The effects of the hormones on cognitive functions have also received attention during the last decades (Maruff and Falleti, 2005, Nyberg, 2009).

In many of these studies it has been suggested that both IGF-1 and GH can penetrate the blood cerebrospinal fluid barrier and blood brain barrier, either by diffusion or receptor mediated transport. A transport to the brain via the cerebrospinal fluid has also been proposed (Johansson et al., 1995, Burman et al., 1996, Coculescu, 1999, Pan et al., 2005, Nyberg, 2006).

There is a well-recognized relation between GHD and impaired cognitive functions (Deijen et al., 1996, Rollero et al., 1998). In fact, studies on GH replacement treatment show significant improvement in several cognitive parameters e.g. memory, attention, alertness and well-being in individuals suffering from GHD (Sartorio et al., 1995, Deijen et al., 1998, Falleti et al., 2006, Nilsson et al., 2007, Nieves-Martinez et al., 2010). These beneficial effects seem to be maintained several years after the treatment was intro-duced (Falleti et al., 2006, Laursen et al., 2008, van Nieuwpoort and Drent, 2008).

Decrease levels of GHR mRNA, attenuated GH secretion and IGF-1 pro-duction with increased age have also been reported, effects that are suggest-ed to cause age-related impairment in cognitive functions (Lai et al., 1993, Zhai et al., 1994, Burman and Deijen, 1998, Gilchrist et al., 2002). However

!!!!P P

IGF-1

!

" !!

Phosphorylation of tyrosine containing

substrates

s

21

these symptoms may be reversed by GH treatment. Thus GH seems to have an overall positive effect on quality of life (Bengtsson et al., 1993, Burman et al., 1995, McGauley et al., 1996, Burman and Deijen, 1998, Gibney et al., 1999). In line with these findings GH has demonstrated to prevent neuronal loss in aged rat hippocampus (Azcoitia et al., 2005). Treatment with GH and IGF-1 also display a neuroprotective effect on damaged spinal cord and brain as well as cerebral hypoxic ischemia (Hanci et al., 1994, Sharma et al., 1997, Gustafson et al., 1999, Smith, 2003, Azcoitia et al., 2005, Reimunde et al., 2011).

Not only age but also different drugs of abuse can have a negative effect on cognition. For example extensive use or misuse of opiates impair the cognitive capability in human and animals (Spain and Newsom, 1991, Sjogren et al., 2000, Smith, 2000). In addition, opioids induce toxicity, in-hibit cell growth, stimulate apoptosis and impair cell regeneration of neurons (Hauser et al., 2000, Hu et al., 2002, Mao et al., 2002). These drug induced changes can probably also be reversed by GH. At least rhGH have been demonstrated to reverse opioid induced apoptosis in prenatal hippocampal neurons (Svensson et al., 2008).

The brain reward system The majority of drugs of abuse acts directly or indirectly upon the brain’s reward system, also referred to as the mesocorticolimbic dopamine pathway. As the mesocorticolimbic pathway is stimulated, dopamine is released and a sense of pleasure or “high” is experienced (Kalivas, 2004). Not all reward responses originate from addictive drugs, but the system is also stimulated by natural stimulants like food, sex and learning (Mitchell and Stewart, 1990, Avena et al., 2008, Wise, 2008).

One of the more established theories regarding the reward system de-scribes dopaminergic neurons projecting from the VTA to the nucleus ac-cumbens and the prefrontal cortex, (Figure 5) (Koob et al., 1998, Koob, 2006, Wise, 2008). These dopaminergic neurons are under the influence of other neurons containing different neurotransmitters, such as endogenous opioids (see page 23), glutamate, GABA, serotonin and noradrenaline, pro-jected from surrounding regions (the amygdala, thalamus, olfactory tubercle and ventral palladium) (see review (Belujon and Grace, 2011)). The dopa-mine neurons in the VTA are mainly regulated by glutamate and GABA. Upon stimulation, of the neurons, VTA release dopamine to the nucleus accumbens and the prefrontal cortex. The DA release in the mesocorticolim-bic pathway is further modulated by GABAergic medium-spiny projection neurons in the nucleus accumbens (Koob et al., 1998, Gardner, 2005). Inter-estingly, mechanisms of learning and synaptic plasticity related to drug ad-diction have also been reported to be associated with the dopaminergic

22

pathways described above (Berridge and Robinson, 1998, Kelley, 2004, Jones and Bonci, 2005, Kalivas, 2009).

Figure 5. A simplified model of the brain reward system, including dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NA) and the prefrontal cortex (PFC).

The opioid system Opioid peptides and receptors The opium extract, have been used as a recreational drug and for medical purposes for thousands of years. The existence of specific opioid binding sites were discovered in 1973 by three independent research groups (Pert and Snyder, 1973, Simon et al., 1973, Terenius, 1973) and was later referred as to mu-, delta- and kappa-opioid peptide receptors (MOP, DOP and KOP) (Martin et al., 1976, Lord et al., 1977, Chang and Cuatrecasas, 1979, Chang et al., 1980). The discovery of the receptors and the fact that they stimulated analgesia, which in turn was inhibited by the opioid antagonist naloxone (Akil et al., 1976) led to a search for endogenous opioids. The endogenous opioids, enkephalin, endorphin and dynorphin were also quickly found (Hughes et al., 1975, Pasternak et al., 1975, Terenius and Wahlstrom, 1975, Bradbury et al., 1976, Goldstein et al., 1979, Goldstein et al., 1981).

Each of the three classical endogenous opioid peptides i.e. enkephalin, endorphin and dynorphin are derived from different propeptides, namely proenkephalin, proopiomelanocortin (POMC) and prodynorphin. Moreover each propeptide is enzymatically cleaved, generating several smaller pep-tides. For example cleavage of proenkephalin results in Met-, Leu-Enkephalin, Met-enkephalin-Arg6Phe7 (MEAP) and Met-enkephalin-

VTA

NA

PFC

GABA neuron Dopamine neuron Dopamine

23

Arg6Gly7Leu8. POMC generates the peptides β-endorphin and β-lipotrophin, while prodynorphin serves as the precursor for dynorphin A and B (Dyn A and Dyn B) and neoendorphin. The N-terminal amino acid-sequence (Tyr-Gly-Gly-Phe-Met/Leu) is identical in all these opioid peptides and is fol-lowed by different C-terminal sequence resulting in 5-31 amino acid long peptides (Akil et al., 1998). Besides the classical opioids, there are also other opioid peptides that act on the opioid receptors but lacking the typical N-terminal e.g. endomorphin-1, endomorphin-2, hemorphins and β -casomorphin (Henschen et al., 1979, Brantl et al., 1986, Nyberg et al., 1997, Zadina et al., 1997).

Belonging to the Gi-protein coupled receptor family, all the opioid recep-tors inhibit adenylyl cyclase in response to activation, leading to neuronal inhibition. The MOP receptor predominantly binds endorphins and to some extent enkephalins. Enkephalins bind with a higher affinity to DOP receptors and the dynorphins prefer KOP receptors.

The opioid system is very complex and is associated with many different processes such as pain processing, stress, learning, memory, reward and reinforcement (For review see (Kieffer and Gaveriaux-Ruff, 2002, Bodnar, 2012).

Regulation of the reward system by opioid peptides It is well recognized that exogenous opioids, such as morphine and heroin stimulates the reward system, resulting in enhanced DA activity. This is also the case for the endogenous neuropeptides (Bozarth and Wise, 1984, Spanagel et al., 1992).

The opioid receptors are widely spread in the brain although with varying levels in different regions. Regarding the reward system, the MOP receptors are densely expressed in the ventral tegmental area VTA and KOP receptors in the nucleus accumbens, whereas DOP receptors are expressed in both above-mentioned regions (Mansour et al., 1988, Herz, 1997, Zhang et al., 2009). The endogenous MOP and DOP opioid receptor agonists indirectly stimulate DA activity in the mesolimbic system by acting on the receptors in the VTA and the nucleus accumbens, giving rise to a feeling of reward or ”high” (Figure 6). The MOP receptors in the VTA are situated on GABAer-gic interneurons, and by stimulation of these neurons, the DA neuron is indi-rectly disinhibited, resulting in enhanced DA activation (Margolis et al., 2012). On the contrary, the dynorphins create dysphoric effects by activation of KOP receptor on the terminals of the DA neurons, resulting in decreased levels of DA release (Spanagel et al., 1992, Herz, 1997, Shippenberg et al., 2009).

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Figure 6. A simplified model of the dopaminergic reward system, including the regu-lation of endogenous opioids. VTA; ventral tegmental area.

The GABAergic system GABA is a neurotransmitter, which is wildly distributed in the mammalian brain and spinal cord. Acting as the main inhibitory neurotransmitter, it is involved in several psychological and physiological processes (McGinty, 2007).

There are three different classes of GABA receptors; GABAA, GABAB and GABAC. GABAA and GABAC receptors are rapid ionotropic receptors also called ligand-gated ion channels, whereas GABAB receptors are metabotropic receptors belonging to the G protein-coupled receptors family. The ionotropic GABAA receptor consists of a pentamer assembled from different combinations of subunits (", $, !, # and %), resulting in a large vari-ety of GABAA receptor types.

The metabotropic GABAB receptors, GABABR1 and GABABR2, were first characterized in 1980 (Bowery et al., 2002). They were later found to function as heterodimeric receptors (Jones et al., 1998, Kaupmann et al., 1998, White et al., 1998). Like GABAA receptors, they are widely distributed throughout the brain e.g. limbic system, and are located both pre- and postsynaptically. GABA serves as its own regulator on the presynaptic re-ceptors by depressing Ca2+ influx via voltage-activated Ca2+ channels. Such presynaptic stimulation is suggested to be involved in long-term potentiation (LTP), a phenomena underlying synaptic plasticity (Bliss and Gardner-Medwin, 1973, Bliss and Lomo, 1973, Mott et al., 1990). Further evidence for an involvement of GABAB receptors in cognitive processes has been proposed, although the results are somewhat contradictive. In several stud-ies, GABAB antagonists have been shown to improve learning and memory (Helm et al., 2005, Lasarge et al., 2009). However, in another study per-formed by Schuler et al. GABAB deficient mice performed poorer in a pas-sive avoidance test, indicating cognitive impairments (Schuler et al., 2001).

Dopamine neuron

NUCLEUS ACCUMBENS VTA

!-endorphin

Enkephalin!

EnkephalinEnkephalinDynorphin!

Dynorphin

GABA

neuron

Dopamine release!

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Cognition; learning and memory The formation of a memory is divided into three phases; immediate memory, short-term memory and long-term memory. The immediate memory corre-spond to a fraction of a second, e.g. remembering how an item looked like just after a glimpse. The short-term memory lasts for seconds to minutes without rehearsal, e.g. the everyday searching for lost objects. The more permanent storage of memories is called long-term memory and lasts for days, weeks or even a lifetime. The process of conversion immediate memory to long-term memory is called consolidation.

Information about the brain regions and mechanisms involved in the dif-ferent types of memory formations and amnesia are known thanks to clinical cases and animal models. For example, in a case where serious anterograde amnesia (loss of ability to create new memories) occurred after the hippo-campus and amygdala were removed (Scoville and Milner, 1957, Corkin et al., 1997, Scoville and Milner, 2000). In animals, bilateral lesions to the hip-pocampus have also been shown to induce similar effects e.g. retrograde amnesia and impaired spatial learning and memory in the WM (Squire et al., 2001, Clark et al., 2005).

We now know that one of the mechanisms behind the formations of new memories is LTP. LTP is a long lasting increase of signal transmission be-tween two neurons resulting in increased synaptic strength and plasticity (Bliss and Cooke, 2011). The molecular mechanism underlying LTP regula-tion of synaptic plasticity is not fully clarified, although it is well known that it involves excitatory NMDA and AMPA receptors (Morris et al., 1986, Derkach et al., 2007, Li and Tsien, 2009, Lee and Kirkwood, 2011). Since the first LTP studies were performed in the hippocampus, LTP based plastic-ity was assumed to be a process exclusively for learning and memory (Maren, 2005, Whitlock et al., 2006). However, today it is well established that different forms of plasticity occurs in most excitatory synapses (Griffiths et al., 2008, Luscher and Huber, 2010). It is therefore not surpris-ing that accumulating evidence demonstrates that drugs of addiction increase synaptic plasticity in brain regions involved in reinforcement and reward processes, thus triggering addiction (Nestler, 2001, Kauer and Malenka, 2007, Thomas et al., 2008). This drug induced pathological form of learning and memory seems to involve different systems such as glutamate transmis-sion in the VTA leading to LTP in dopamine cells (Ungless et al., 2001, Almodovar-Fabregas et al., 2002, Saal et al., 2003). Furthermore other stud-ies have demonstrated that blockade of the NMDA receptor prevents drug-induced behaviors in certain animal models (Schenk et al., 1993, Tzschentke and Schmidt, 1995, Harris and Aston-Jones, 2003, Harris et al., 2004, Kalivas, 2004).

As described earlier, several drugs of abuse exert deleterious effects on the brain resulting in cognitive dysfunction, including impairments in learn-

26

ing and memory. It is well established that use of different types of opioids and alcohol, causes brain damages and cognitive impairments in humans (Cipolli and Galliani, 1987, Hu et al., 2002, Harper and Matsumoto, 2005, Kameda et al., 2007). Furthermore, other drugs such as flunitrazepam and GHB induce e.g. short-term amnesia and disrupt working memory respec-tively (Schwartz et al., 2000, Gouzoulis-Mayfrank et al., 2003, Carter et al., 2006). In addition several similar outcomes have been demonstrated in ani-mal models for a variety of different drugs.

In this thesis we studied spatial working and reference memory in rats treated with GHB using the WM as our memory test model. The WM regime is further described under “Methods”.

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The aims of the thesis

The general aim of this thesis was to investigate biochemical and behavioral effects of rhGH and GHB in male Sprague Dawley rats. In these studies the main focus was devoted to cerebral systems associated to cognitive process-es and addiction.

More specifically, these were the aims;

• To study the effects of rhGH on GABAB receptor density and func-tionality in the male rat brain.

• To evaluate the impact of rhGH on the functionality of the opioid

peptide receptors, mu and delta, in the male rat brain.

• To investigate the effects of long-term GHB treatment on learning and memory using the WM model. In addition, to study the impact of GHB on IGF-1 and GABAB.

• To study the effects of GHB treatment on the IGF-1 levels in blood

plasma and the endogenous opioid peptides Dyn B and MEAP in discrete brain regions. Moreover, to study the effect of GHB on the explorative behavior and locomotor activity in rats using the open field (OF) test.

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Methods

Animal handling and drug treatment This thesis is based on animal studies, where male Sprague Dawley rats (Scanbur, Sollentuna, Sweden (paper I and II) and Taconic Farm Inc., Den-mark (paper III and IV)) have been used in the experiments. All the animals were housed in standard Macrolon 4 cages (59 x 38 x 20 cm) in air-conditioned, humidity-controlled (60%) rooms with constant temperature of 23 ± 1 oC. Food and water was provided ad libitum.

In the first two studies (paper I and II), the rats were housed in groups of 4 animals per cage on a normal dark-light cycle with the lights on at 0700. The animals were allowed to adapt to the new environment one week before any treatment was given. At the start of the experiment, the rats weighed 314-325 g and were divided into 3 different treatment groups, consisting of 8 animals in each group. Twice daily for 7 days, the animals were given s.c. injections of rhGH (0.07 IU/kg or 0.7 IU/kg) or saline.

In the 2 latter studies (paper III and IV) the animals were housed 3 indi-viduals per cage (paper III) and 4 per cage (paper IV). Since the experiments involved behavioural studies and rats are nocturnal, the animals were kept on a reversed 12 h dark/light cycle with the lights on at 1900. Prior to the exper-iments, the rats were allowed to adapt to the new environment for 19-20 days (paper III) and 14-19 days (paper IV). The rats, weighing 320-390 g (paper III) and 345-347 g (paper IV) at the start of the experiment were ran-domly divided in to 3 different groups and were treated with GHB (50 mg/kg or 300 mg/kg) or saline orally once daily for 16 days (paper III) or once dai-ly for 7 days (paper IV). Behaviour parameters such as spatial learning and memory, spontaneous explorative behaviour and locomotor activity were evaluated using the WM and OF paradigm respectively. To avoid direct ef-fects of intoxication on the parameters observed in the behavioural tests, the treatment was given 60 min prior to the WM and OF. For the same reason rats were administered 60 minutes before the decapitation. All the animals were weighed every third day and on the last day of the experiment.

All the animal procedures were performed according to a protocol ap-proved by the ethics committee in Uppsala and in accordance with the Swe-dish Animal Protection Legislation.

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Behavioural methods The water maze test (WM) The WM is one of the most widely used tests for studies of spatial learning and memory in rodents (Morris et al., 1982, Morris, 1984). In study III, the ability to learn the spatial location of a hidden platform situated in large cir-cular pool filled with water, was evaluated after GHB treatment. Located in a room exclusively used for animal behavioral experiments, the maze con-sisting of a dark pool (160 cm in diameter) was filled with tap water (22 ± 1 oC) and virtually divided in four equally large quadrants (Figure 7). A trans-parent platform (15 cm in diameter) was situated in the southwest quadrant, submerged 1.5 cm under the water surface in order to be invisible to the animals. All WM experiments were conducted on day 11 to day 15, during the dark phase, 60 min after treatment. Each session started by placing the rat into one of the quadrants of the pool facing the wall in a random order. The animals underwent a learning period (acquisition), for 4 consecutive days including 4 trials each day. The animals were allowed to search for the platform for a maximum of 90 s. If the platform was not found, rats were guided to the platform, where it stayed for 30 s to memorize the extra-maze cues. Behavioural parameters such as escape latency, latency to first visit in target quadrant, swim speed, swim distance and thigmotaxis were scored. On day 15 of the study, the platform was removed and a single 90 s long probe trial (retention) was performed. The number of times the animal crossed the area where the platform originally was positioned i.e. target zone crossings, number of visits and time spent in the different quadrants were analysed in addition to the parameters scored in the acquisition trials. The performance in the WM was recorded by a video camera situated above the pool and the behavioural parameters registered by computerized tracking system (Bi-observe Viewer, Bonn, Germany).

30

Figure 7. The water maze (WM) with a submerged platform situated in the middle one of the four quadrants.

The open field (OF)The OF test is a common test for evaluating locomotor function and sponta-neous explorative behavior and was used in study IV. The test was conduct-ed in a relatively light room, during the dark phase, 60 min after the last drug administration on day 6. The test was carried out as described elsewhere (Enhamre et al., 2012). Briefly, the OF consists of a dark circular arena (90 cm in diameter) with 35 cm high walls, divided in to 3 zones (Figure 8). These zones are referred as the central zone (30 cm in diameter), the middle zone (15 cm in width) and the outer zone (15 cm in width). Starting in the outer zone, the rats were allowed to freely explore the area for 10 min. Pa-rameters such as time to enter the middle and central zones, time spent in the different zones, number of visits to the respective zone, activity, velocity and track length were registered by using the Biobserve Viewer software system (Biobserve Viewer, Bonn, Germany).

!"!"!"!#$%$%#$%$&#

Computerized tracking system

Camera

Platform

!"!"!"!#$%$%#$%$&#

Computerized tracking system

CameraCamera

PlatformPlatformPlatformPlatform

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Figure 8. The open field (OF) divided into a central zone, middle zone and outer zone.

Tissue and blood collection After decapitation (study I, II and III), whole brains were rapidly removed and fresh-frozen in isopentane (-35 ± 5 °C) for 30 seconds and stored at -80 °C until further autoradiographic analysis.

In study IV, trunk blood was collected in tubes containing ice cold 1% EDTA in 0.9 % NaCl immediately after decapitation. After centrifugation for 10 min, 4 °C at 3000 rpm the plasma was removed and stored at -80 °C until further analysis of IGF-1. Using a brain matrix (Activational System, Warren, MI, USA), the brains were cut in coronal sections and selected brain regions were dissected according to the rat brain atlas of Paxinos and Wat-son (Paxinos and Watson, 1997). The posterior and anterior pituitary, hypo-thalamus, frontal cortex, caudate putamen, nucleus accumbens, hippocam-pus, amygdala and periaqueductal gray area (PAG) were collected and im-mediately put on dry ice, and stored in separate eppendorf tubes in -80 °C until further processing.

Autoradiographic methods Autoradiography is used to determine the localization of a radioactive sub-stance in tissue. In study I, II and III, two different types of autoradiography were performed on coronal brain sections taken from bregma +3.2, +1.6, -2.6 and -5.6. The density and location of receptors were determined using ligands labeled with a radioactive isotope, which are capable of binding to

Central zone

Middle zone

Outer zone

Central zone

Middle zone

Outer zone

32

specific receptors. Subsequently bound radioactive labeled substance is thereby detected using i.e. x-ray film or screens (Herkenham and Pert, 1982, Schmahl et al., 1996).

The second technique, [35S]GTP!S receptor autoradiography is a tech-nique which is used in studies on distribution and functionality of G-protein coupled receptors. It is based on a replacement of endogenous GTP to [35S]GTP!S upon receptor specific agonist activation. The activation of the receptor introduces conformational changes leading to an interaction be-tween the radiolabeled [35S]GTP!S and the G-proteins. The amount radioac-tive [35S]GTP!S incorporated can then be detected (Sim et al., 1995, Sovago et al., 2001).

Representative autoradiograms including the majority of the regions ana-lyzed in paper 1-III are presented below in figure 9.

Figure 9. Representative autoradiograms from male Sprague Dawley rat brain. Coronal sections from bregma +1.6, -2.6 and -5.6 are presented. Cg1; cingulate cortex 1, Cg2; cingulate cortex area 2, CPu; caudate putamen, AcbSh; accumbens nucleus shell, AcbC; accumbens nucleus core, CA 1-3; fields of hippocampus, LD; laterodorsal thalamic nucleus, VPM; ventral posteromedial thalamic nucleus, DMD; dorsomedial hypothalamus nucleus, Me; medial amygdaloid nucleus, VMH; ventromedial hypothalamic nucleus, DG; denate gyrus, VPL; ventral posterolateral thalamic nucleus, LGP; lateral globus pallidus, Ce; central amygdaloid nucleus, LH; lateral hypothalamic area, PV; paraventricular thalamic nucleus, PAG; peria-queductal gray, SNR; substantia nigra, MG; medial geniculate nucleus, VTA; ven-tral tegmental area, DEn; dorsal endopiriform nucleus.

GABAB, mu and delta opioid receptor stimulated [35S]GTP!S autoradiography In paper I, II and III, the functionality of GABAB receptors and the opioid peptide receptors mu (MOP) and delta (DOP), were determined after treat-ment with GH and GHB, respectively. The method was performed as de-scribed elsewhere (Sim et al., 1995), although slightly modified. Fresh fro-zen 20 &m thick coronal brains sections thaw mounted on gelatin coated glass slides were equilibrated in a assay buffer containing 50 mM Tris-HCl, 4 mM MgCl2, 0.3 mM EGTA and 100 mM NaCl, pH 7.4 for 10 min in room temperature. Afterwards, the slides were placed in humid chambers and pre-incubation with assay buffer containing 10 mU/ml adenosine deaminase (ADA) and 2 mM GDP was conducted for 15 min in room temperature. The agonist-stimulated functionality was achieved in the presence of selective

AcbC

CPu

AcbSh

Cg1 Cg2 CA 1-3

VPM

Me PV

DG

Ce

VPL LGP

LD

VMH

DMD LH

PAG

VTA SNR DEn

MG

AcbC AcbC

CPu

AcbSh

Cg1 Cg1 Cg2

AcbC AcbC AcbSh

CA 1-3

VPM

MePV

DG

Ce

VPL LGP

LD

VMH

DMD LH

PAG

VTASNR

VTA

DEnDEn

MG

33

agonists, 100 µM (R)-baclofen (GABAB), 10 µM DAMGO (MOP) and 10 µM DPDPE (DOP) in assay-buffer containing 10 mU/ml ADA, 2 mM GDP and 0.04 nM [35S]GTPγS for 2 h in room temperature. Under the same con-ditions the specificity of respectively G-protein activated receptor was de-termined by adding the antagonists 100 µM CGP 35348 (GABAB) and 1 µM naloxone (MOP and DOP) to subsequent slides. Furthermore non-specific binding was assessed in the presence of unlabeled 10 µM GTPγS and the basal levels in absence of agonist. Following the last incubation step, slides were washed in cold 50 mM Tris-HCl, pH 7.4 (2x2 min), and distilled water for 30 sec and were exposed to Kodak BioMax MR-1 films. After 3-10 days the films were developed and analyzed and quantified according to Sims et al., in an Image J computer system (National Institutes of Health, Bethesda MD, USA) (Sim et al., 1995).

GABAB receptor autoradiography In vitro receptor autoradiography was performed to determine the density of GABAB receptors in cerebral brain sections after treatment with GH (paper I) and GHB (paper III). Slightly modified, the assay was performed according to Cremer et al., (Cremer et al., 2009). After being thawed for approximately 45 min, slides were pre-incubated for 15 min at 4 °C in a buffer consisting of 50 mM Tris-HCl, 2.5 mM CaCl2 (pH 7.4). Total binding was assessed by incubation with the same buffer containing 2 nM [3H]CGP54626 and non-specific binding was achieved with addition of 100 µM (R)-baclofen for 1 h in 4 °C. After being washed 3 x 30 s in ice-cold 50 mM Tris-HCl (pH 7.4) and rinsed in distilled water, the slides were dried over night. Together with a [3H]-microscales, the slides were exposed to a [3H] sensitive phosphor imager screens BAS-TR2040 (FUJI Science Imaging) for 14-20 days, and developed in a FUJI BAS 2500 scanner (Fuji Medical Systems). The recep-tor density was quantified and converted into nCi/mg using the microscales using the Image J computerized system (National Institutes of Health, Be-thesda, MD). Specific binding was calculated by subtracting non-specific binding from total binding.

IGF-1 receptor autoradiography In order to study the impact on IGF-1 receptors after long-term treatment with GHB, IGF-1 receptor autoradiography was performed (paper III) as previously described (Dore et al., 1997, Kar et al., 1997). Coronal sections (12 µm), cut in a cryostat and mounted on gelatine coated glass slides were pre-incubated in 50 mM Tris-HCl (pH 7.4) containing 10 mM MgCl2, 0.1% BSA (w/v) and 1 mg/ml bacitracin in room temperature for 15 min. The total binding was determined by incubating the slides in 50 pM [125I]rhIGF-1 and non-specific binding in 50 pM [125I]rhIGF-1 in addition with 100 nM rhIGF-

34

1, in the pre-incubation buffer for 24 h in 4°C. Subsequently, the slides were washed in ice-cold 50 mM Tris-HCl (pH 7.4) for 2 min and rinsed for 30 sec in distillated water. The slides were dried in room temperature over night. All sections, including the [125I]-microscale were exposed to Kodak Biomax MR-1 film, for 4 days. Developed films were analysed densitometrically and quantified using Image J software (National Institutes of Health, Bethesda, MD). Specific binding was achieved by subtraction of the non-specific bind-ing from total binding.

IGF -1 Enzyme-linked immunosorbent assay (ELISA) The concentration of IGF-1 in plasma was studied in paper IV after 7 days of GHB administration. The assay was performed using a commercial ELISA kit suitable for detecting IGF-1 in rat plasma (mouse/rat IGF-1 REF E25, Mediagnost, Reutlinger, Germany). The assay was conducted according to the manufactures instructions. Thawed plasma samples were first diluted 1:500 in sample buffer and analyzed in duplicates using a micro plate reader POLARstar OPTIMA (BMG LABTECH GmbH, Ortenberg, Germany).

Peptide extraction and separation As described previously (Zhou et al., 1998, Nyberg and Hallberg, 2011) peptides were extracted from discrete brain regions collected in study IV. Briefly, each tissue sample was heated in 1 M acetic acid, 95 °C for 5 min, cooled on ice and homogenized using a Branson Sonifier (Branson Ultrason-ics Corporation, Danbury, CT, USA). The samples were again cooled on ice and reheated for 5 min at 95 °C and then centrifuged (13 400 × g, 4 °C) for 15 min. The supernatants were collected and diluted with 1 mL Buffer I (0.1 M formic acid/0.018 M pyridine, pH 3.0). The peptide separation was per-formed using ion-exchange chromatography on a column containing SP-Sephadex C-25 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Buffer containing increasing concentrations of formic acid and pyridine were used for stepwise extraction of the opioid peptides (details presented elsewhere (Nyberg and Hallberg, 2011)). The fraction were dried in a vacuum centri-fuge (SpeedVac SC210A, Savant Instruments, Holbrook, NY, USA) and stored at -80 °C until further radioimmunoassay.

35

Radioimmunoassay (RIA) The radioimmunoassay technique was one of the first immunoassay tech-niques developed to determine nanomolar to picomolar concentrations of peptides in biological fluids and tissues. The low detection limit makes the technique suitable for analyzing levels of the opioid peptides. MEAP and Dyn B were detected and quantified in various brain regions after daily treatment of GHB (paper IV). The method we used is previously described elsewhere (Johansson et al., 2000). Antisera specified for each peptide were generated in rabbits by injections of thyroglobuline-conjugated Dyn B and oxidized MEAP (Glamsta et al., 1993). The MEAP antisera used in the as-says were diluted to a final concentration of 1:80 000 in gelatin buffer (0.15 NaCl, 0.025 M EDTA, 0.1% gelatin and 0.1 % BSA in a 0.05 M sodium phosphate buffer) and the antisera against Dyn B to a concentration of 1:180 000 in another gelatin buffer (0.15 NaCl, 0.02 % Na-azid, 0.1% gelatin, 0.1 % Triton X-100 and 0.1 % BSA in a 0.05 M phosphate buffer). Furthermore, prior to the RIA, the tracer peptides were iodated with the chloramine-T procedure, followed by HPLC purification (Hallberg et al., 2000). The 125I-MEAP and 125I-Dyn B were diluted in above-mentioned buffers to an activi-ty of 4000-5000 cpm/100 µL. The fraction and standards subjected to the MEAP assay were oxidized by incubating the samples in 100 µL, 1 M HAc and 10 µL 30 % H2O2 for 30 min in 37 °C and subsequently dried in the vac-uum centrifuge. All fractions were diluted with MeOH:HCl 0.1 M (1:1). The standards (25 µL) and samples (25 µL) were incubated with 100 µL antise-rum and 100 µL radiolabeled peptide for 24 h, 4 °C. The total and blank were conducted under the same conditions with 25 µL MeOH:HCl 0.1 M (1:1) and 100 µL gelatin buffer or 100 µL antiserum respectively.

The RIA technique used for MEAP is based on charcoal absorption, whilst the Dyn B RIA is based on double-antibody precipitation. Briefly, after incubation with 200 µL of charcoal suspension (2.5 % charcoal and 0.25 % dextran 70 in 0.05 M sodium phosphate buffer) for 10 min on ice, the MEAP samples were centrifuges 2 min at 12 000 × g. A volume of 300 µL of the supernatant was collected and analysed in a gamma-counter. For the Dyn B assay, samples were incubated with 50 µL Goat ant-rabbit IgG serum and 50 µL normal rabbit serum (Peninsula Laboratories LLC; San Carlos, CA) for 90 min in 4 °C, centrifuged 12 000 × g, 4 °C for 20 min. The super-natants were discarded and the radioactivity in the pellets analyzed.

Statistical analysis All the data sets obtained from the behavioral studies and the biochemical analysis were first tested for normality distribution using Shapiro Wilk’s test.

36

As the data sets did not follow a normal distribution, all the behavioral data were analyzed using non-parametric methods and are expressed as me-dian plus minimal and maximal values. Comparison between treatment groups (paper III and IV), regarding the behavioral data obtained from the WM and OF procedures was statistically analyzed using the Kruskal Wallis one-way ANOVA, followed by Dunn’s multiple comparison test. Perfor-mance with in each treatment group over time (paper III) was statistically analyzed using Friedman matched pair test.

Differences between treatment groups, regarding weight measurements, autoradiographic assays, ELISA and RIA studies (paper I-IV) followed a normal distribution and were therefore analyzed using the parametric one-way ANOVA, followed by Tukey’s post hoc test. The Student's t-test was used to compare the differences in basal levels and agonist induced [35S]GTPγS binding within each treatment group (paper I-III). Data, ana-lysed by parametric methods are presented as mean ± S.E.M. Furthermore, Grubb’s test was used to detect outliers. α-values less than 0.05 were consid-ered outliers and were removed. The correlation analysis performed between biochemical parameters were performed with the Pearsons parametric test (paper III and IV).

All statistical analyses were assessed using Graphpad Prism 5.0d and p-values less than 0.05 were considered as significant.

37

Results and discussion

GABAB receptors In the first study (paper I), male Sprague Dawley rats were subjected to rhGH (0.7 and 0.07 IU/kg) twice daily for 7 days. This treatment led to alter-ations in both density and functionality of GABAB receptors in several brain areas.

As seen in figure 10, the receptor density is increased in the cortical areas of primary motor corte and cingulate cortex area 2 in low dose treated ani-mals (0.07 IU/kg), but not in the high dose treated group (0.7 IU/kg). In con-gruence with our results, Walser and co-workers observed an increase of GABAB(1) gene transcript in the cerebral cortex after 6 days of GH treatment (Walser et al., 2011). Interestingly, in the same study, in hippocampus and dentate gyrus, two regions that are crucial for learning and memory no alter-ations in GABAB(1) gene transcript were observed. This is also in line with the observations found in our study.

Not only the motor cortex and cingulate cortex 2, but also the caudate pu-tamen and periaqueductal grey displayed an increased expression of GABAB

receptors only with the low dose treatment. Thus, all four regions seem to display a bell-shaped response (Cunningham et al., 1991, Gent et al., 2002). Bell-shaped curves have over the years been observed when studying GH. For example GH also induce bell-shape effects in cell proliferation and dif-ferentiation (Mustafa et al., 1997, Lyuh et al., 2007, Aberg et al., 2009).

Figure 10. The GABAB receptor density (niCi/mg) in Sprague Dawley male rats after 7 days of s.c. treatment with rhGH. Values are expressed as mean ± S.E.M. *p<0.05

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In the third study (paper III), male Sprague Dawley rats received GHB treatment (50 and 300 mg/kg) for 16 consecutive days. In addition these rats were submitted to behavioural tests in a WM.

These results demonstrate an over all down regulation of the GABAB re-ceptor system, in several brain regions after the treatment. For example as can be seen in figure 11 and 12, in the fields of hippocampus (CA1 and CA 3) and dentate gyrus there is a clear attenuation of both the GABAB receptor functionality and the density.

Figure 11. The effect on GABAB receptor density (nCi/mg) in brain regions associ-ated with learning and memory in rats pretreated with GHB for 16 days. CA1 = Fields of hippocampus 1, CA3 =Fields of hippocampus 3. Values are presented as mean ± S.E.M. *p<0.05, **p<0.01 and ***p<0.001.

Figure 12. Baclofen stimulated binding of [35S]GTPγS-binding autoradiography after 16 days of treatment with 50 mg/kg GHB and 300 mg/kg GHB. CA1 = Fields of hippocampus 1 and CA3 =Fields of hippocampus 3. Values are presented as mean ± S.E.M. *p<0.05 and **p<0.01.

The GABA receptor system has been proposed as a target for new treatments of memory impairments. Several research groups have reported that GABAB receptor antagonists can improve memory and learning in in several mam-mals by reducing the transcription factor CRE, which is associated in memory formation (Mondadori et al., 1993, Helm et al., 2005). Based on the reports using GABAB antagonists an effect of decreased GABAB density should theoretically lead to improved memory. We however observed a gen-eral down regulation of the GABAB receptor system is accompanied by a GHB induced cognitive impairment. Although more in line with our obser-vations, another study using intra-hippocampal injections of the GABAB

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receptor agonist baclofen demonstrated a reduced retention memory (Zarrindast et al., 2002).

The amygdala is a region predonimantly associated with processing of emotions, but also formulation of long-term memory (see review (LeDoux, 2000)). Both the rhGH (figure 13) and GHB treatment induced a clear down-regulation of GABAB receptor density and functionality in this area. These results may seem a bit inconsistent, since GHB is known to induce neurotox-ic effects, whilst GH exhibits neuroprotective capabilities (Svensson et al., 2008, Pedraza et al., 2009). On the other hand, the outcome may be ex-plained by the fact that that GHB have been reported to induce an increased GH release.

The fact that GHB is used for its relaxative and anxiolytic effects may partly be explained by these alterations seen in the amygdala (Freese et al., 2002, Cryan and Kaupmann, 2005)

The GABAB receptors have also attracted attention as targets for the de-velopment of new antidepressants (Nowak et al., 2006, Cryan and Slattery, 2010). GABAB(1) receptor knockout mice and GABAB receptor antagonist display antidepressant effects in a forced swim test, effects that are suggest-ed to involve the serotonergic system (Mombereau et al., 2004, Slattery et al., 2005, Cornelisse et al., 2007). Moreover, it has been reported that there is an over-representation of GHD patients suffering from depression (Deijen et al., 1998). An effect that may be counteracted with GH replace treatment since GH has an overall positive effect on the quality of life (Hull and Harvey, 2003).

Figure 13. The figure display GABAB receptor functionality (nCi/mg), after 7 days of rhGH treatment in male rats. Values are expressed as mean ± S.E.M. *p<0.05.

Although both receptor density and functionality in many regions were al-tered after treatment, there were no correlation between these effects i.e. density vs. functionality. A possible explanation is that the GABAB receptor consist of 2 receptor subunits, GABAB(1) and GABAB(2) (White et al., 1998). The GABAB(1) subunit is suggested to bind the specific ligand e.g. baclofen,

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whilst GABAB(2) is connected to the G-protein, inducing intracellular activa-tion. Therefore we believe in this study that the [3H]CGP54626 binding mainly indicates GABAB(1) levels, whilst the baclofen induced [35S]GTPγS binding measures both the subunits, hence receptor functionality.

For more detailed information regarding the results on GABAB receptor density and functionality in respectively study, please see paper I and III.

Mu and delta opioid receptors MOP and DOP receptors are widely distributed in the brain and in peripheral sensory neurons. The receptors are predominantly associated with analgesia and emotional responses, like depression and anxiety (Law and Loh, 1999, Filliol et al., 2000, Dietis et al., 2009). Several studies have also emphasized their importance in reward, drug addiction and cognition, including learning and memory (Burgess, 2008, Marinelli et al., 2009, Rudy, 2009, Le Merrer et al., 2011). In our study (paper II) we evaluated the MOP and DOP receptor functionali-ty following treatment with rhGH (0.07 IU/kg or 0.7IU/kg). The MOP recep-tor dose-dependently enhanced the receptor functionality in central amygda-la, laterodorsal hypothalamus, ventral posterolateral nucleus of thalamus as well as in the lateral globus pallidus (figure 14). In the latter region a signifi-cant difference was also observed between the two GH treatment groups. Regarding the mechanism of action, the MOP receptor may be directly acti-vated by the GH receptor or via indirect activation by its mediator IGF-1. Although the mechanisms behind the GH induced alterations are not fully clarified, it however seems clear that opioids and GH interact. For example, an acute dose of morphine down-regulates the transcript for the GH receptor and GHBP in hippocampus and the spinal cord (Thornwall-Le Greves et al., 2001). Furthermore, enhanced plasma levels of GH have been reported after acute opiate treatment in humans (Bartolome and Kuhn, 1983, Vuong et al., 2010). In the case of DOP receptor functionality, GH did not have an effect in our experimental model. Other studies performed on hypophysectomized rats have reported a significant down-regulation of the DOP receptor in cer-ebral cortex and cerebellum after GH treatment (Persson et al., 2003, Persson et al., 2005). The same research group also observed a decrease in the expression of large sized DOP receptors in the cerebral. The results also implicated that the large sized receptors consisted of a receptor dimer, whereas the smaller entities corresponded to monomeric forms of the recep-tors (Persson et al., 2005). The effects of GH on the DOP receptors needs to be further elucidated.

For more detailed information regarding the results on MOP and DOP re-ceptor density, please see paper II.

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Figure 14. Figures represent MOP receptor activity in specific brain areas after treatment with rhGH for 7 consecutive days. The method used is DAMGO induced [35S]GTPγS binding autoradiography on coronal brain sections. Values are ex-pressed as mean ± S.E.M. *p<0.05; **p<0.01 and ***p<0.001.

Insulin-like growth factor-1 receptor In paper (III) we demonstrate that long-term GHB treatment has a major impact on IGF-1 receptors in regions important for declarative and spatial learning and memory. In e.g. dentate gyrus and the fields of hippocampus CA1, the IGF-1 receptor density was significantly reduced (figure 15). At the end of the GHB treatment, behavioral test using MW were conducted. The results obtained indicated poorer spatial learning in GHB treated rats compared to controls.

In congruence with our results several studies have demonstrated that GH- and IGF-1 impaired rats also display deteriorated spatial cognitive be-havior when tested in a WM (Charlton et al., 1988, Le Greves et al., 2006, Nieves-Martinez et al., 2010). Furthermore decreased levels of IGF-1 and IGF-1R in hippocampus following induced vascular dementia have been reported (Gong et al., 2012). Decreased levels of GH, IGF-1, IGFBP and IGF-1 mRNA have also been observed with increased age. The latter find-ings have been suggested to contribute to age-related decline in cognitive functions (Corpas et al., 1993, Sonntag et al., 1997, Lai et al., 2000, Strasburger et al., 2001). Interestingly, treatment with IGF-1 given to older rats has proven to restore hippocampus dependent memories assessed in a

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water maze (Markowska et al., 1998). In the neurodegenerative disease, Alzheimers’s disease (AD), stimulations of IGF-1 inhibits the formation of neurofibrillary tangles (Gasparini and Xu, 2003), oxidative stress (Garcia et al., 2006) and stimulates the clearance of Aβ (Carro and Torres-Aleman, 2004). Taken together, these findings strengthens the connection between GHB and IGF-1, since IGF-1 receptors have been down regulated in areas of hippocampus, and in addition these rats proved to have poorer spatial cogni-tive capabilities.

For more detailed information regarding the results on IGF-1 receptor density, please see paper III.

Figure 15. The effect on IGF-1 receptor density (niCi/mg) in brain regions associat-ed with learning and memory in rats pretreated with GHB for 16 days. CA1 = Fields of hippocampus 1, CA3 =Fields of hippocampus 3. Values are presented as mean ± S.E.M. *p<0.05, **p<0.01 and ***p<0.001.

Insulin-like growth factor-1 in plasma After 7 days of GHB (50 mg/kg and 300 mg/kg) the concentration of IGF-1 in plasma was determined (paper IV). All treatment groups displayed ap-proximately 2 µg IGF-1/ml plasma (figure 16), a concentration correspond-ing well to levels in another study using male rats (Gronbladh et al., 2012). In our study, no differences between treatment groups were encountered. Other groups have reported, enhanced levels of IGF-1 in serum following GHB treatment (Murphy et al., 2007) as well as increased GH secretion fol-lowing GHB administration in both humans and rats (Takahara et al., 1977, Takahara et al., 1980, Van Cauter et al., 1997, Volpi et al., 2000),. Thus it seems that several studies report increased IGF-1 levels in connection to GHB use, the lacking effect in our study may be explained by the impact of the treatment length and the doses given.

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Figure 16. IGF-1 plasma levels in male Sprague Dawley rats treated with saline, 50 mg/kg GHB or 300 mg/kg GHB for 7 days. Values are presented in as mean ± S.E.M.

Opioid peptides The endogenous opioid peptides Dyn B and MEAP, were analyzed in dis-sected brain regions as biomarkers for prodynorphin and proenkephlin after GHB treatment (50 mg/kg or 300 mg/kg) for 7 days (paper IV).

The results demonstrate that treatment with the higher dose of GHB (300 mg/kg) resulted in a 38 % increase of ir MEAP levels in the frontal cortex compared to controls (figure 17). In congruence with our findings, GHB have in a previous study demonstrated to enhance mRNA expression of the precursor proenkephalin in that specific discrete region (Schmidt-Mutter et al., 1999a). Moreover the observed alteration of cortical MEAP, corroborate well with other drugs of abuse. For example, an acute high dose of metham-phetamine increases the levels of Met-enkephalin in the frontal cortex (Alburges et al., 2001). Moreover, repeated treatment with supra therapeutic doses of anabolic androgenic steroids (AAS) increases the levels of MEAP in the regions of hypothalamus, striatum and PAG (Johansson et al., 2000). The fact that GHB induces alterations in MEAP levels in the frontal cortex may implicate that this peptide may be involved in GHB related reward and addiction.

Figure 17. The MEAP concentration (fmol/mg) in male Sprague Dawley rats after 7 days of GHB treatment. Values are expressed as mean ± S.E.M. *p<0.05.

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Although, no significant alterations were observed regarding ir Dyn B levels between treatment groups, an imbalance between the two peptides (Dyn B and MEAP) was registered in amygdala, hippocampus and hypothalamus in the GHB receiving groups. In controls on the other hand, the levels of the two peptides correlated well in the above written areas (figure 18). We have reported similar patterns in a previous study on AAS, where the levels of Dyn B and MEAP were positively correlated in control, but the AAS treat-ment seemed to create an imbalance between the two peptides (Johansson et al., 2000).

Figure 18. Correlation between ir MEAP and ir Dyn B levels in a) amygdala, b) hippocampus and c) hypothalamus in rats pretreated with GHB for 7 days. P-values less than 0.05 are considered significant.

The water maze The spatial cognitive capability in rats treated with GHB was evaluated us-ing the WM regime. Rats treated with the higher dose of GHB performed poorer in both the acquisition trial (figure 19) and probe trial (figure 20). Animals submitted to the high-dose GHB treatment demonstrated signifi-cantly longer escape latency i.e. time to reach the platform on the last day of

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acquisition (day 4) compared to controls. On the probe-day (day 5), when the platform was removed, the same group made significantly fewer visits to the target quadrant and also spent less time there. There were no differences regarding swim speed, swim distance nor thigmotaxis between any treatment groups, at any time, which indicates that GHB did not affect the motoric system. Our findings are in line with studies, where rats have received GHB intraperitoneally for approximately 5 days (Sircar et al., 2008, Sircar et al., 2010). The GHB induced cognitive impairments, however seem to be age- and gender related, since in adult female rats GHB treatment did not cause any significant impairment measured in the WM. However adolescent fe-male rats treated performed significantly poorer than controls in the WM paradigm (Sircar and Basak, 2004, Sircar et al., 2010).

Figure 19. Escape latency in the WM, i.e. time to find the hidden platform in se-conds (s), after long-term treatment with GHB. Values are presented in mean as mean ± S.E.M. *p<0.05 control vs. GHB300 mg/kg, ap<0,05, aap<0,01, aaap<0,001 (control group), bp<0,05, bbp<0,01 (GHB 50 mg/kg), p<0,05 and ccp<0,001 (GHB 300 mg/kg) compared to the first learning trial, on day 1.

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Figure 20. The figures display observed behavior in male rats following treatment with GHB (50 mg/kg and 300 mg/kg) and saline during probe trial in the WM. Pa-rameters presented are the number of visits to the target quadrant in %, and the total time spent in that same area in seconds (s). Values are presented as median plus minimal and maximal values. *p<0.05 and ***p<0.001 in controls vs. GHB 300 mg/kg.

The Open field The open field test was used to evaluate whether GHB had an impact on spontaneous explorative behavior and locomotor activity. In the last study (paper IV), the OF test was conducted 60 min after the last administration of GHB (50 mg/kg, GHB 300 mg/kg or saline) and lasted for 10 min. The re-sults obtained regarding general parameters i.e. velocity, track length or ac-tivity did not demonstrate any significant differences between treatment groups (Table 1). Furthermore, no significant differences between treatment groups were detected regarding duration, number of visits and latency in respectively zone. Thus, the results demonstrate that GHB, did not induce any motoric effects that could be detected in the OF, results that are in con-gruence with our results generated form the WM (paper III), where neither swim speed, swim length or thigmotaxis differed between treatment groups.

Table 1. General parameters observed in the OF arena following 6 days of GHB treatment.

Saline GHB 50 mg/kg GHB 300 mg/kg Significance

Velocity (cm/s) 7.3 (5.5 - 8.8) 7.2 (5.1 – 8.9) 7.3 (4.9 – 9.8) ns

Track length (cm) 4387 (3279 – 5275) 4274 (3069 – 5351) 4348 (2919 – 5855) ns

Activity (%) 31.5 (26.1 – 36.3) 31.8 (24.3 – 38.3) 32.4 (23.5 – 38.2) ns

Other groups using mice have however displayed significant hypolocomo-tion 10 min after GHB treatment. The reduced locomotion was explained by GHBs sedative onset 10 min after administration. Moreover, the reported motoric effects were abolished after 60-120 min, results that are more in line

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with ours (Itzhak and Ali, 2002). Nevertheless, the differences in GHB re-sponse might also reflect spices differences in the mechanism of action.

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Summarizing conclusion

This thesis reports that treatment with GH and GHB affect transmittor sys-tems in the Sprague Dawley male rat brain that are associated with cognitive processes and the development of drug dependence. Furthermore, evidence for a GHB induced decline in learning and memory are presented. The main outcomes from the studies included in this thesis are:

• Treatment with rhGH for one week induces significant alterations in

density and functionality of GABAB receptor in brain regions that are involved in cognitive function.

• The functionality of MOP receptors, but not DOP receptors func-

tionality is altered in male rat brain following one week of rhGH treatment. The observed alterations were detected in brain regions that are essential for learning and memory.

• GHB impairs spatial learning and memory in rats. The deficient

cognitive parameters were verified in the well-established WM par-adigm, by prolonged escape latency during acquisition trials and fewer visits to the target quadrant during probe test. In addition, GHB significantly down regulated GABAB receptor density and functionality, in several regions that are crucial for e.g. learning and memory. In these areas, alterations of IGF-1 receptor density were also observed.

• GHB treatment increases the level of MEAP in the frontal cortex,

and also creates an imbalance between MEAP and Dyn B in the amygdala, hippocampus and hypothalamus, brain structures that are important for the development of addiction and cognitive functions.

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Populärvetenskaplig sammanfattning

Missbruket av beroendeframkallande substanser, såsom alkohol och anabola androgena steroider har ökat under de senaste decennierna. En direkt följd av missbruket är fysiska och psykiska skador hos den enskilda individen, men det leder även ökade påfrestningar på samhället i stort, fram för allt på grund av ökad kriminalitet, ökat våld och förstörelse. Missbruk av droger ger stör-ningar på kroppens så kallade belöningssystem, det nätverk i hjärnan som förmedlar vällust och njutning. Vidare verkar vissa droger ge skador på nervvävnaden i hjärnan och därmed ge upphov till bl.a. problem med inlär-ning och minne. Detta gäller bland annat för alkohol, opioider och gamma-hydroxybutyrat (GHB). På senare tid pekar dock ny forskning på att man ska kunna reversera dessa drog-relaterade skador med hjälp av tillväxthormon (GH) men även dess främsta mediator insulinliknande tillväxtfaktor-1 (IGF-1). Dessa hormon har en positiv inverkan på inlärning och minne, detta ge-nom egenskaper som förmedlar celltillväxt och verkar skyddande för celler.

Det övergripande syftet med dessa studier är att inbringa en fördjupad kunskap kring GHs positiva och GHBs negativa inverkan på inlärning, minne och belöningssystemet. Studierna inkluderar beteende där den spatiala förmågan samt motorik har studerats i råtta som har behandlats med GHB. Beteendestudier har vidare kompletterats med biokemiska analyser.

Resultaten visar att GH har en mätbar påverkan på vissa hjärnregioner, såsom främre hjärnbarken. I denna region och även andra områden som är relevanta för inlärning och minne observeras även tydliga GHB-inducerade förändringar. Vidare visar beteendestudierna att GHB ger en tydlig försäm-ring av den spatiala inlärnings- och minnesförmågan.

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Acknowledgements

The research presented in this thesis was carried out at Uppsala University, Department of Pharmaceutical Bioscience, the Division of Biological Re-search on Drug Dependence.

The Swedish Research Council, Swedish Council for Working Life and Social Research (FAS), Swartz Foundation and Apotekarsocieteten, support-ed this work financially.

I would like to express my sincere gratitude to all of you who has contribute to this thesis:

Associate professor Mathias Hallberg, my supervisor, for given me the opportunity to be a PhD student, for guiding me in the field of neuroscience and for given me the chance to teach. Also, I would like to thank you for all the interesting discussions, from research to cool aps.

Professor Fred Nyberg, my co-supervisor, for your support, for given me

the opportunity to go to conferences and fruitful scientific discussions. My “three musketeers” and present co-workers Alfhild Grönbladh, Anna

Carlsson and Erika Brolin for all your valuable comments on this thesis, for your every-day-support and for being great friends! I don’t know what I would have done without you.

Britt-Marie Johansson for being a wonderful and fun roommate, for your

excellent skills in the lab and for your warm personality. Agneta Bergström, for always helping me with LOA or PhD related problems and questions.

All past and present members at the Division of Biological Research on

Drug Dependence, the Division of Neuropharmacology, Addiction and Be-haviors, the Division of Biochemical Pharmacology and the Division of Drug Safety and Toxicology for providing a good working and social envi-ronment. A special thank to Sadia Oreland for a great friendship and all the laughter, Loudin Daura for your good friendship and lovely cakes, Helén Andersson, my good and strong training partner for your “peps”, Inga Hoff-mann and Anneli Wennman for Monday yoga, Sara Palm for helping me

51

with RIA and fika-talks, Shima Momeni and Linnéa Granholm for your “af-ter-noon-laughter”, Richard Henriksson for yo’ girl and Rebecca Fransson for helping me getting started with my thesis.

Magnus Jansson for helping me with the layouts of the manuscripts, and

over all computer-related problems. Kjell Åkerlund, Agneta Hortlund, Marianne Danersund, Elisabeth Jons-

son, Annika Bokström and Marina Rönngren for practical assistance and administrational matters.

Raili Engdahl, Lena Norgren, Ingrid Nylander, Anne-Lie Svensson and

Lena Bergström for your valuable scientific knowledge and help in the lab. Familjen Eklund-Axelsson för att jag alltid känner mig välkommen hos

er! Madde för vår djupa vänskap, för alla långa telefonsamtal och dina mäs-terliga bullar och kakor. Jag hoppas att du tycker att jag nu har skött mig;)

De bästa ”norbaggarna”, Mari, Gustavo, Ada och Adrian Zaera Holo!

Tack Mari för att du tog mig an när jag var en ung vilsen svenska som var-ken förstod hotten-tott språk eller kunde laga kaffe. Inte trodde jag då att vi fortfarande skulle stå varandra så nära. Gustavo som introducerade mig för spansk tapas, pata negra och goda viner – jag hoppas på flera gris-ben.

Apotekartjejerna för alla skratt, hetsiga diskussioner och tjej-helger. Utan

er hade jag aldrig fått prova på lerduve-skytte, lekt Fångarna på fortet eller lagat mat med proffs-kock.

Gymnasieligan, ingen nämnd, ingen glömd. Är otroligt lottad som får till-

höra ett så stort och härligt gäng. Hoppas på många fler äventyr med er! Övriga vänner och bekanta som livar upp min tillvaro! Jag vill tacka min underbara familj för all kärlek och värme, allt roligt vi

gjort tillsammans, resan till Sydamerika, otaliga slalomsvängar och mys i Källviken. Mamma och pappa, för att ni alltid tror på mig, stöttar och pushar, för servicen med god mat och lån av lille-KA när tiden tryter. Världens bästa lillebror Martin och Anna för att ni är så omtänksamma, roliga och förstå-ende. Sist och minst, lille Knut, för att du verkligen förgyller fasters tillvaro och för dina ”spontana applåder”. Johansson-Urby, Ni betyder så otroligt mycket för mig och upptar en stor del av mitt hjärta!

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References

(1985) Degenerative neurologic disease in patients formerly treated with

human growth hormone. Report of the Committee on Growth Hormone Use of the Lawson Wilkins Pediatric Endocrine Society, May 1985. J Pediatr 107:10-12.

Abanades S, Farre M, Segura M, Pichini S, Barral D, Pacifici R, Pellegrini M, Fonseca F, Langohr K, De La Torre R (2006) Gamma-hydroxybutyrate (GHB) in humans: pharmacodynamics and pharmacokinetics. Ann N Y Acad Sci 1074:559-576.

Aberg ND, Johansson I, Aberg MA, Lind J, Johansson UE, Cooper-Kuhn CM, Kuhn HG, Isgaard J (2009) Peripheral administration of GH induces cell proliferation in the brain of adult hypophysectomized rats. J Endocrinol 201:141-150.

Absalom N, Eghorn LF, Villumsen IS, Karim N, Bay T, Olsen JV, Knudsen GM, Brauner-Osborne H, Frolund B, Clausen RP, Chebib M, Wellendorph P (2012) alpha4betadelta GABA(A) receptors are high-affinity targets for gamma-hydroxybutyric acid (GHB). Proc Natl Acad Sci U S A 109:13404-13409.

Addolorato G, Caputo F, Capristo E, Bernardi M, Stefanini GF, Gasbarrini G (1999) A case of gamma-hydroxybutyric acid withdrawal syndrome during alcohol addiction treatment: utility of diazepam administration. Clin Neuropharmacol 22:60-62.

Akil H, Mayer DJ, Liebeskind JC (1976) Antagonism of stimulation-produced analgesia by naloxone, a narcotic antagonist. Science 191:961-962.

Akil H, Owens C, Gutstein H, Taylor L, Curran E, Watson S (1998) Endogenous opioids: overview and current issues. Drug Alcohol Depend 51:127-140.

Alburges ME, Keefe KA, Hanson GR (2001) Unique responses of limbic met-enkephalin systems to low and high doses of methamphetamine. Brain Res 905:120-126.

Almodovar-Fabregas LJ, Segarra O, Colon N, Dones JG, Mercado M, Mejias-Aponte CA, Vazquez R, Abreu R, Vazquez E, Williams JT, Jimenez-Rivera CA (2002) Effects of cocaine administration on VTA cell activity in response to prefrontal cortex stimulation. Ann N Y Acad Sci 965:157-171.

53

Amit T, Hochberg Z, Waters MJ, Barkey RJ (1992) Growth hormone- and prolactin-binding proteins in mammalian serum. Endocrinology 131:1793-1803.

Anderson IB, Kim SY, Dyer JE, Burkhardt CB, Iknoian JC, Walsh MJ, Blanc PD (2006) Trends in gamma-hydroxybutyrate (GHB) and related drug intoxication: 1999 to 2003. Ann Emerg Med 47:177-183.

Andriamampandry C, Taleb O, Kemmel V, Humbert JP, Aunis D, Maitre M (2007) Cloning and functional characterization of a gamma-hydroxybutyrate receptor identified in the human brain. FASEB J 21:885-895.

Andriamampandry C, Taleb O, Viry S, Muller C, Humbert JP, Gobaille S, Aunis D, Maitre M (2003) Cloning and characterization of a rat brain receptor that binds the endogenous neuromodulator gamma-hydroxybutyrate (GHB). FASEB J 17:1691-1693.

Asa SL, Coschigano KT, Bellush L, Kopchick JJ, Ezzat S (2000) Evidence for growth hormone (GH) autoregulation in pituitary somatotrophs in GH antagonist-transgenic mice and GH receptor-deficient mice. Am J Pathol 156:1009-1015.

Avena NM, Rada P, Hoebel BG (2008) Underweight rats have enhanced dopamine release and blunted acetylcholine response in the nucleus accumbens while bingeing on sucrose. Neuroscience 156:865-871.

Ayyar VS (2011) History of growth hormone therapy. Indian journal of endocrinology and metabolism 15 Suppl 3:S162-165.

Azcoitia I, Perez-Martin M, Salazar V, Castillo C, Ariznavarreta C, Garcia-Segura LM, Tresguerres JA (2005) Growth hormone prevents neuronal loss in the aged rat hippocampus. Neurobiol Aging 26:697-703.

Barbaccia ML, Colombo G, Affricano D, Carai MA, Vacca G, Melis S, Purdy RH, Gessa GL (2002) GABA(B) receptor-mediated increase of neurosteroids by gamma-hydroxybutyric acid. Neuropharmacology 42:782-791.

Bartolome MB, Kuhn CM (1983) Endocrine effects of methadone in rats; acute effects in adults. Eur J Pharmacol 95:231-238.

Beleen C, Martinez-Fuentes AJ, Gracia-Navarro F (2012) Role of SST, CORT and ghrelin and its receptors at the endocrine pancreas. Frontiers in endocrinology 3:114.

Belujon P, Grace AA (2011) Hippocampus, amygdala, and stress: interacting systems that affect susceptibility to addiction. Ann N Y Acad Sci 1216:114-121.

Benavides J, Rumigny JF, Bourguignon JJ, Cash C, Wermuth CG, Mandel P, Vincendon G, Maitre M (1982a) High affinity binding sites for gamma-hydroxybutyric acid in rat brain. Life Sci 30:953-961.

Benavides J, Rumigny JF, Bourguignon JJ, Wermuth CG, Mandel P, Maitre M (1982b) A high-affinity, Na+-dependent uptake system for gamma-hydroxybutyrate in membrane vesicles prepared from rat brain. J Neurochem 38:1570-1575.

54

Bengtsson BA, Eden S, Lonn L, Kvist H, Stokland A, Lindstedt G, Bosaeus I, Tolli J, Sjostrom L, Isaksson OG (1993) Treatment of adults with growth hormone (GH) deficiency with recombinant human GH. J Clin Endocrinol Metab 76:309-317.

Berridge KC, Robinson TE (1998) What is the role of dopamine in reward: hedonic impact, reward learning, or incentive salience? Brain Res Brain Res Rev 28:309-369.

Bhattacharya I, Boje KM (2006) Potential gamma-hydroxybutyric acid (GHB) drug interactions through blood-brain barrier transport inhibition: a pharmacokinetic simulation-based evaluation. Journal of pharmacokinetics and pharmacodynamics 33:657-681.

Black J, Houghton WC (2006) Sodium oxybate improves excessive daytime sleepiness in narcolepsy. Sleep 29:939-946.

Bliss TV, Cooke SF (2011) Long-term potentiation and long-term depression: a clinical perspective. Clinics (Sao Paulo) 66 Suppl 1:3-17.

Bliss TV, Gardner-Medwin AR (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the unanaestetized rabbit following stimulation of the perforant path. J Physiol 232:357-374.

Bliss TV, Lomo T (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 232:331-356.

Blumenfeld M, Suntay GR, Harmel M (1962) Sodium gamma-hydroxybutyric acid: a new anesthetic adjuvant.

Bodnar RJ (2012) Endogenous opiates and behavior: 2011. Peptides. Bohannon NJ, Corp ES, Wilcox BJ, Figlewicz DP, Dorsa DM, Baskin DG

(1988) Localization of binding sites for insulin-like growth factor-I (IGF-I) in the rat brain by quantitative autoradiography. Brain Res 444:205-213.

Bowery NG, Bettler B, Froestl W, Gallagher JP, Marshall F, Raiteri M, Bonner TI, Enna SJ (2002) International Union of Pharmacology. XXXIII. Mammalian gamma-aminobutyric acid(B) receptors: structure and function. Pharmacol Rev 54:247-264.

Bozarth MA, Wise RA (1984) Anatomically distinct opiate receptor fields mediate reward and physical dependence. Science 224:516-517.

Bradbury AF, Smyth DG, Snell CR (1976) Lipotropin: precursor to two biologically active peptides. Biochem Biophys Res Commun 69:950-956.

Bramness JG, Haugland S (2011) [Abuse of gamma-hydroxybutyrate]. Tidsskrift for den Norske laegeforening : tidsskrift for praktisk medicin, ny raekke 131:2122-2125.

Brancucci A, Berretta N, Mercuri NB, Francesconi W (2004) Presynaptic modulation of spontaneous inhibitory postsynaptic currents by gamma-hydroxybutyrate in the substantia nigra pars compacta. Neuropsychopharmacology 29:537-543.

55

Brantl V, Gramsch C, Lottspeich F, Mertz R, Jaeger KH, Herz A (1986) Novel opioid peptides derived from hemoglobin: hemorphins. Eur J Pharmacol 125:309-310.

Burgess N (2008) Spatial cognition and the brain. Ann N Y Acad Sci 1124:77-97.

Burman P, Broman JE, Hetta J, Wiklund I, Erfurth EM, Hagg E, Karlsson FA (1995) Quality of life in adults with growth hormone (GH) deficiency: response to treatment with recombinant human GH in a placebo-controlled 21-month trial. J Clin Endocrinol Metab 80:3585-3590.

Burman P, Deijen JB (1998) Quality of life and cognitive function in patients with pituitary insufficiency. Psychother Psychosom 67:154-167.

Burman P, Hetta J, Wide L, Mansson JE, Ekman R, Karlsson FA (1996) Growth hormone treatment affects brain neurotransmitters and thyroxine [see comment]. Clin Endocrinol (Oxf) 44:319-324.

Carro E, Torres-Aleman I (2004) The role of insulin and insulin-like growth factor I in the molecular and cellular mechanisms underlying the pathology of Alzheimer's disease. Eur J Pharmacol 490:127-133.

Carter LP, Koek W, France CP (2009) Behavioral analyses of GHB: receptor mechanisms. Pharmacology & therapeutics 121:100-114.

Carter LP, Richards BD, Mintzer MZ, Griffiths RR (2006) Relative abuse liability of GHB in humans: A comparison of psychomotor, subjective, and cognitive effects of supratherapeutic doses of triazolam, pentobarbital, and GHB. Neuropsychopharmacology 31:2537-2551.

Castelli MP, Ferraro L, Mocci I, Carta F, Carai MA, Antonelli T, Tanganelli S, Cignarella G, Gessa GL (2003) Selective gamma-hydroxybutyric acid receptor ligands increase extracellular glutamate in the hippocampus, but fail to activate G protein and to produce the sedative/hypnotic effect of gamma-hydroxybutyric acid. J Neurochem 87:722-732.

Caton R (1893) NOTES of a CASE of ACROMEGALY TREATED by OPERATION. British medical journal 2:1421-1423.

Chambliss KL, Gibson KM (1992) Succinic semialdehyde dehydrogenase from mammalian brain: subunit analysis using polyclonal antiserum. The International journal of biochemistry 24:1493-1499.

Chang KJ, Cuatrecasas P (1979) Multiple opiate receptors. Enkephalins and morphine bind to receptors of different specificity. J Biol Chem 254:2610-2618.

Chang KJ, Hazum E, Cuatrecasas P (1980) Multiple opiate receptors. Trends in neurosciences 3.

Charlton HM, Clark RG, Robinson IC, Goff AE, Cox BS, Bugnon C, Bloch BA (1988) Growth hormone-deficient dwarfism in the rat: a new mutation. J Endocrinol 119:51-58.

56

Cheramy A, Nieoullon A, Glowinski J (1977) Stimulating effects of gamma-hydroxybutyrate on dopamine release from the caudate nucleus and the substantia nigra of the cat. J Pharmacol Exp Ther 203:283-293.

Cipolli C, Galliani I (1987) Addiction time and intellectual impairment in heroin users. Psychol Rep 60:1099-1105.

Clark RE, Broadbent NJ, Squire LR (2005) Hippocampus and remote spatial memory in rats. Hippocampus 15:260-272.

Coculescu M (1999) Blood-brain barrier for human growth hormone and insulin-like growth factor-I. J Pediatr Endocrinol Metab 12:113-124.

Corkin S, Amaral DG, Gonzalez RG, Johnson KA, Hyman BT (1997) H. M.'s medial temporal lobe lesion: findings from magnetic resonance imaging. J Neurosci 17:3964-3979.

Cornelisse LN, Van der Harst JE, Lodder JC, Baarendse PJ, Timmerman AJ, Mansvelder HD, Spruijt BM, Brussaard AB (2007) Reduced 5-HT1A- and GABAB receptor function in dorsal raphe neurons upon chronic fluoxetine treatment of socially stressed rats. J Neurophysiol 98:196-204.

Corpas E, Harman SM, Blackman MR (1993) Human growth hormone and human aging. Endocrine reviews 14:20-39.

Cosman D, Lyman SD, Idzerda RL, Beckmann MP, Park LS, Goodwin RG, March CJ (1990) A new cytokine receptor superfamily. Trends in biochemical sciences 15:265-270.

Cremer CM, Palomero-Gallagher N, Bidmon HJ, Schleicher A, Speckmann EJ, Zilles K (2009) Pentylenetetrazole-induced seizures affect binding site densities for GABA, glutamate and adenosine receptors in the rat brain. Neuroscience 163:490-499.

Cruz HG, Ivanova T, Lunn ML, Stoffel M, Slesinger PA, Luscher C (2004) Bi-directional effects of GABA(B) receptor agonists on the mesolimbic dopamine system. Nat Neurosci 7:153-159.

Cryan JF, Kaupmann K (2005) Don't worry 'B' happy!: a role for GABA(B) receptors in anxiety and depression. Trends Pharmacol Sci 26:36-43.

Cryan JF, Slattery DA (2010) GABAB receptors and depression. Current status. Adv Pharmacol 58:427-451.

Cunningham BC, Ultsch M, De Vos AM, Mulkerrin MG, Clauser KR, Wells JA (1991) Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science 254:821-825.

Cushing H (1909) III. Partial Hypophysectomy for Acromegaly: With Remarks on the Function of the Hypophysis. Annals of surgery 50:1002-1017.

Darnell JE, Jr., Kerr IM, Stark GR (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415-1421.

Daughaday WH (2000) Growth hormone axis overview--somatomedin hypothesis. Pediatr Nephrol 14:537-540.

Daughaday WH, Trivedi B (1991) Clinical aspects of GH binding proteins. Acta endocrinologica 124 Suppl 2:27-32.

57

de Vos AM, Ultsch M, Kossiakoff AA (1992) Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255:306-312.

Degenhardt L, Darke S, Dillon P (2002) GHB use among Australians: characteristics, use patterns and associated harm. Drug Alcohol Depend 67:89-94.

Deijen JB, de Boer H, Blok GJ, van der Veen EA (1996) Cognitive impairments and mood disturbances in growth hormone deficient men. Psychoneuroendocrinology 21:313-322.

Deijen JB, de Boer H, van der Veen EA (1998) Cognitive changes during growth hormone replacement in adult men. Psychoneuroendocrinology 23:45-55.

Derkach VA, Oh MC, Guire ES, Soderling TR (2007) Regulatory mechanisms of AMPA receptors in synaptic plasticity. Nature reviews Neuroscience 8:101-113.

Diana M, Mereu G, Mura A, Fadda F, Passino N, Gessa G (1991) Low doses of gamma-hydroxybutyric acid stimulate the firing rate of dopaminergic neurons in unanesthetized rats. Brain Res 566:208-211.

Dietis N, Guerrini R, Calo G, Salvadori S, Rowbotham DJ, Lambert DG (2009) Simultaneous targeting of multiple opioid receptors: a strategy to improve side-effect profile. Br J Anaesth 103:38-49.

Doherty JD, Hattox SE, Snead OC, Roth RH (1978) Identification of endogenous gamma-hydroxybutyrate in human and bovine brain and its regional distribution in human, guinea pig and rhesus monkey brain. J Pharmacol Exp Ther 207:130-139.

Dore S, Kar S, Rowe W, Quirion R (1997) Distribution and levels of [125I]IGF-I, [125I]IGF-II and [125I]insulin receptor binding sites in the hippocampus of aged memory-unimpaired and -impaired rats. Neuroscience 80:1033-1040.

Elliott S, Lowe P, Symonds A (2004) The possible influence of micro-organisms and putrefaction in the production of GHB in post-mortem biological fluid. Forensic science international 139:183-190.

ElSohly MA, Salamone SJ (1999) Prevalence of drugs used in cases of alleged sexual assault. J Anal Toxicol 23:141-146.

Enhamre E, Carlsson A, Gronbladh A, Watanabe H, Hallberg M, Nyberg F (2012) The expression of growth hormone receptor gene transcript in the prefrontal cortex is affected in male mice with diabetes-induced learning impairments. Neurosci Lett 523:82-86.

Erhardt S, Andersson B, Nissbrandt H, Engberg G (1998) Inhibition of firing rate and changes in the firing pattern of nigral dopamine neurons by gamma-hydroxybutyric acid (GHBA) are specifically induced by activation of GABA(B) receptors. Naunyn Schmiedebergs Arch Pharmacol 357:611-619.

Evans HM, Long JA (1922) Characteristic Effects upon Growth, Oestrus and Ovulation Induced by the Intraperitoneal Administration of Fresh

58

Anterior Hypophyseal Substance. Proc Natl Acad Sci U S A 8:38-39.

Falleti MG, Maruff P, Burman P, Harris A (2006) The effects of growth hormone (GH) deficiency and GH replacement on cognitive performance in adults: a meta-analysis of the current literature. Psychoneuroendocrinology 31:681-691.

Ferrara SD, Zotti S, Tedeschi L, Frison G, Castagna F, Gallimberti L, Gessa GL, Palatini P (1992) Pharmacokinetics of gamma-hydroxybutyric acid in alcohol dependent patients after single and repeated oral doses. Br J Clin Pharmacol 34:231-235.

Filliol D, Ghozland S, Chluba J, Martin M, Matthes HW, Simonin F, Befort K, Gaveriaux-Ruff C, Dierich A, LeMeur M, Valverde O, Maldonado R, Kieffer BL (2000) Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses. Nat Genet 25:195-200.

Frank SJ (2002) Receptor dimerization in GH and erythropoietin action--it takes two to tango, but how? Endocrinology 143:2-10.

Fraser RA, Harvey S (1992) Ubiquitous distribution of growth hormone receptors and/or binding proteins in adenohypophyseal tissue. Endocrinology 130:3593-3600.

Freese TE, Miotto K, Reback CJ (2002) The effects and consequences of selected club drugs. Journal of substance abuse treatment 23:151-156.

Fryklund L, Sievertsson H (1978) Primary structure of somatomedin B: a growth hormone-dependent serum factor with protease inhibiting activity. FEBS Lett 87:55-60.

Fryklund LM, Bierich JR, Ranke MB (1986) Recombinant human growth hormone. Clinics in endocrinology and metabolism 15:511-535.

Fuh G, Cunningham BC, Fukunaga R, Nagata S, Goeddel DV, Wells JA (1992) Rational design of potent antagonists to the human growth hormone receptor. Science 256:1677-1680.

Fuller DE, Hornfeldt CS (2003) From club drug to orphan drug: sodium oxybate (Xyrem) for the treatment of cataplexy. Pharmacotherapy 23:1205-1209.

Gahete MD, Duran-Prado M, Luque RM, Martinez-Fuentes AJ, Quintero A, Gutierrez-Pascual E, Cordoba-Chacon J, Malagon MM, Gracia-Navarro F, Castano JP (2009) Understanding the multifactorial control of growth hormone release by somatotropes: lessons from comparative endocrinology. Ann N Y Acad Sci 1163:137-153.

Galicia M, Nogue S, Miro O (2011) Liquid ecstasy intoxication: clinical features of 505 consecutive emergency department patients. Emerg Med J 28:462-466.

Gallimberti L, Ferri M, Ferrara SD, Fadda F, Gessa GL (1992) gamma-Hydroxybutyric acid in the treatment of alcohol dependence: a double-blind study. Alcohol Clin Exp Res 16:673-676.

59

Gallimberti L, Spella MR, Soncini CA, Gessa GL (2000) Gamma-hydroxybutyric acid in the treatment of alcohol and heroin dependence. Alcohol 20:257-262.

Galloway GP, Frederick SL, Staggers FE, Jr., Gonzales M, Stalcup SA, Smith DE (1997) Gamma-hydroxybutyrate: an emerging drug of abuse that causes physical dependence. Addiction 92:89-96.

Garcia J, Ahmadi A, Wonnacott A, Sutcliffe W, Nagga K, Soderkvist P, Marcusson J (2006) Association of insulin-like growth factor-1 receptor polymorphism in dementia. Dementia and geriatric cognitive disorders 22:439-444.

Gardner EL (2005) Endocannabinoid signaling system and brain reward: emphasis on dopamine. Pharmacol Biochem Behav 81:263-284.

Gasparini L, Xu H (2003) Potential roles of insulin and IGF-1 in Alzheimer's disease. Trends in neurosciences 26:404-406.

Gent J, van Kerkhof P, Roza M, Bu G, Strous GJ (2002) Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis. Proc Natl Acad Sci U S A 99:9858-9863.

Gibney J, Wallace JD, Spinks T, Schnorr L, Ranicar A, Cuneo RC, Lockhart S, Burnand KG, Salomon F, Sonksen PH, Russell-Jones D (1999) The effects of 10 years of recombinant human growth hormone (GH) in adult GH-deficient patients. J Clin Endocrinol Metab 84:2596-2602.

Gilchrist FJ, Murray RD, Shalet SM (2002) The effect of long-term untreated growth hormone deficiency (GHD) and 9 years of GH replacement on the quality of life (QoL) of GH-deficient adults. Clin Endocrinol (Oxf) 57:363-370.

Glamsta EL, Morkrid L, Lantz I, Nyberg F (1993) Concomitant increase in blood plasma levels of immunoreactive hemorphin-7 and beta-endorphin following long distance running. Regul Pept 49:9-18.

Gobaille S, Schmidt C, Cupo A, Herbrecht F, Maitre M (1994) Characterization of methionine-enkephalin release in the rat striatum by in vivo dialysis: effects of gamma-hydroxybutyrate on cellular and extracellular methionine-enkephalin levels. Neuroscience 60:637-648.

Gold BI, Roth RH (1977) Kinetics of in vivo conversion of gamma-[3H]aminobutyric acid to gamma-[3H]hydroxybutyric acid by rat brain. J Neurochem 28:1069-1073.

Goldenberg N, Barkan A (2007) Factors regulating growth hormone secretion in humans. Endocrinology and metabolism clinics of North America 36:37-55.

Goldstein A, Fischli W, Lowney LI, Hunkapiller M, Hood L (1981) Porcine pituitary dynorphin: complete amino acid sequence of the biologically active heptadecapeptide. Proc Natl Acad Sci U S A 78:7219-7223.

60

Goldstein A, Tachibana S, Lowney LI, Hunkapiller M, Hood L (1979) Dynorphin-(1-13), an extraordinarily potent opioid peptide. Proc Natl Acad Sci U S A 76:6666-6670.

Gong X, Ma M, Fan X, Li M, Liu Q, Liu X, Xu G (2012) Down-regulation of IGF-1/IGF-1R in hippocampus of rats with vascular dementia. Neurosci Lett 513:20-24.

Gouzoulis-Mayfrank E, Thimm B, Rezk M, Hensen G, Daumann J (2003) Memory impairment suggests hippocampal dysfunction in abstinent ecstasy users. Progress in neuro-psychopharmacology & biological psychiatry 27:819-827.

Griffiths S, Scott H, Glover C, Bienemann A, Ghorbel MT, Uney J, Brown MW, Warburton EC, Bashir ZI (2008) Expression of long-term depression underlies visual recognition memory. Neuron 58:186-194.

Gronbladh A, Johansson J, Nostl A, Nyberg FJ, Hallberg M (2012) Growth hormone improves spatial memory and reverses certain anabolic androgenic steroid-induced effects in intact rats. J Endocrinol.

Gustafson K, Hagberg H, Bengtsson BA, Brantsing C, Isgaard J (1999) Possible protective role of growth hormone in hypoxia-ischemia in neonatal rats. Pediatr Res 45:318-323.

Hallberg M, Johansson P, Kindlundh AM, Nyberg F (2000) Anabolic-androgenic steroids affect the content of substance P and substance P(1-7) in the rat brain. Peptides 21:845-852.

Hanci M, Kuday C, Oguzoglu SA (1994) The effects of synthetic growth hormone on spinal cord injury. Journal of neurosurgical sciences 38:43-49.

Harper C, Matsumoto I (2005) Ethanol and brain damage. Current opinion in pharmacology 5:73-78.

Harris GC, Aston-Jones G (2003) Critical role for ventral tegmental glutamate in preference for a cocaine-conditioned environment. Neuropsychopharmacology 28:73-76.

Harris GC, Wimmer M, Byrne R, Aston-Jones G (2004) Glutamate-associated plasticity in the ventral tegmental area is necessary for conditioning environmental stimuli with morphine. Neuroscience 129:841-847.

Harvey S, Hull KL (1997) Growth hormone. A paracrine growth factor? Endocrine 7:267-279.

Hauser KF, Houdi AA, Turbek CS, Elde RP, Maxson W, 3rd (2000) Opioids intrinsically inhibit the genesis of mouse cerebellar granule neuron precursors in vitro: differential impact of mu and delta receptor activation on proliferation and neurite elongation. Eur J Neurosci 12:1281-1293.

Hechler V, Bourguignon JJ, Wermuth CG, Mandel P, Maitre M (1985) gamma-Hydroxybutyrate uptake by rat brain striatal slices. Neurochem Res 10:387-396.

61

Hechler V, Gobaille S, Bourguignon JJ, Maitre M (1991) Extracellular events induced by gamma-hydroxybutyrate in striatum: a microdialysis study. J Neurochem 56:938-944.

Hechler V, Gobaille S, Maitre M (1989) Localization studies of gamma-hydroxybutyrate receptors in rat striatum and hippocampus. Brain Res Bull 23:129-135.

Hechler V, Weissmann D, Mach E, Pujol JF, Maitre M (1987) Regional distribution of high-affinity gamma-[3H]hydroxybutyrate binding sites as determined by quantitative autoradiography. J Neurochem 49:1025-1032.

Helm KA, Haberman RP, Dean SL, Hoyt EC, Melcher T, Lund PK, Gallagher M (2005) GABAB receptor antagonist SGS742 improves spatial memory and reduces protein binding to the cAMP response element (CRE) in the hippocampus. Neuropharmacology 48:956-964.

Henschen A, Lottspeich F, Brantl V, Teschemacher H (1979) Novel opioid peptides derived from casein (beta-casomorphins). II. Structure of active components from bovine casein peptone. Hoppe-Seyler's Zeitschrift fur physiologische Chemie 360:1217-1224.

Herington AC (1994) New frontiers in the molecular mechanisms of growth hormone action. Mol Cell Endocrinol 100:39-44.

Herkenham M, Pert CB (1982) Light microscopic localization of brain opiate receptors: a general autoradiographic method which preserves tissue quality. J Neurosci 2:1129-1149.

Herrington J, Carter-Su C (2001) Signaling pathways activated by the growth hormone receptor. Trends in endocrinology and metabolism: TEM 12:252-257.

Herz A (1997) Endogenous opioid systems and alcohol addiction. Psychopharmacology (Berl) 129:99-111.

Hibell B, Guttormsson U, Ahlström S, Balakireva O, Bjarnason T, Kokkevi A, Kraus L (2011)

Substance Use Among Students in 36 European Countries. In: The 2007 ESPAD Report.

Horseman ND, Yu-Lee LY (1994) Transcriptional regulation by the helix bundle peptide hormones: growth hormone, prolactin, and hematopoietic cytokines. Endocrine reviews 15:627-649.

Hu S, Sheng WS, Lokensgard JR, Peterson PK (2002) Morphine induces apoptosis of human microglia and neurons. Neuropharmacology 42:829-836.

Hughes J, Smith TW, Kosterlitz HW, Fothergill LA, Morgan BA, Morris HR (1975) Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258:577-580.

Hull KL, Harvey S (2003) Growth hormone therapy and Quality of Life: possibilities, pitfalls and mechanisms. J Endocrinol 179:311-333.

Itzhak Y, Ali SF (2002) Repeated administration of gamma-hydroxybutyric acid (GHB) to mice: assessment of the sedative and rewarding effects of GHB. Ann N Y Acad Sci 965:451-460.

62

Johansson JO, Larson G, Andersson M, Elmgren A, Hynsjo L, Lindahl A, Lundberg PA, Isaksson OG, Lindstedt S, Bengtsson BA (1995) Treatment of growth hormone-deficient adults with recombinant human growth hormone increases the concentration of growth hormone in the cerebrospinal fluid and affects neurotransmitters. Neuroendocrinology 61:57-66.

Johansson P, Hallberg M, Kindlundh A, Nyberg F (2000) The effect on opioid peptides in the rat brain, after chronic treatment with the anabolic androgenic steroid, nandrolone decanoate. Brain Res Bull 51:413-418.

Jones KA, Borowsky B, Tamm JA, Craig DA, Durkin MM, Dai M, Yao WJ, Johnson M, Gunwaldsen C, Huang LY, Tang C, Shen Q, Salon JA, Morse K, Laz T, Smith KE, Nagarathnam D, Noble SA, Branchek TA, Gerald C (1998) GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2. Nature 396:674-679.

Jones S, Bonci A (2005) Synaptic plasticity and drug addiction. Current opinion in pharmacology 5:20-25.

Kalivas PW (2004) Glutamate systems in cocaine addiction. Current opinion in pharmacology 4:23-29.

Kalivas PW (2009) The glutamate homeostasis hypothesis of addiction. Nature reviews Neuroscience 10:561-572.

Kameda SR, Frussa-Filho R, Carvalho RC, Takatsu-Coleman AL, Ricardo VP, Patti CL, Calzavara MB, Lopez GB, Araujo NP, Abilio VC, Ribeiro Rde A, D'Almeida V, Silva RH (2007) Dissociation of the effects of ethanol on memory, anxiety, and motor behavior in mice tested in the plus-maze discriminative avoidance task. Psychopharmacology (Berl) 192:39-48.

Kaminski T, Smolinska N (2012) Expression of orexin receptors in the pituitary. Vitamins and hormones 89:61-73.

Kar S, Seto D, Dore S, Chabot JG, Quirion R (1997) Systemic administration of kainic acid induces selective time dependent decrease in [125I]insulin-like growth factor I, [125I]insulin-like growth factor II and [125I]insulin receptor binding sites in adult rat hippocampal formation. Neuroscience 80:1041-1055.

Karin M, Theill L, Castrillo JL, McCormick A, Brady H (1990) Tissue-specific expression of the growth hormone gene and its control by growth hormone factor-1. Recent progress in hormone research 46:43-57; discussion 57-48.

Kato H, Faria TN, Stannard B, Roberts CT, Jr., LeRoith D (1993) Role of tyrosine kinase activity in signal transduction by the insulin-like growth factor-I (IGF-I) receptor. Characterization of kinase-deficient IGF-I receptors and the action of an IGF-I-mimetic antibody (alpha IR-3). J Biol Chem 268:2655-2661.

Kauer JA, Malenka RC (2007) Synaptic plasticity and addiction. Nature reviews Neuroscience 8:844-858.

63

Kaupmann K, Malitschek B, Schuler V, Heid J, Froestl W, Beck P, Mosbacher J, Bischoff S, Kulik A, Shigemoto R, Karschin A, Bettler B (1998) GABA(B)-receptor subtypes assemble into functional heteromeric complexes. Nature 396:683-687.

Kelley AE (2004) Memory and addiction: shared neural circuitry and molecular mechanisms. Neuron 44:161-179.

Kelly PA, Ali S, Rozakis M, Goujon L, Nagano M, Pellegrini I, Gould D, Djiane J, Edery M, Finidori J, et al. (1993) The growth hormone/prolactin receptor family. Recent progress in hormone research 48:123-164.

Kieffer BL, Gaveriaux-Ruff C (2002) Exploring the opioid system by gene knockout. Prog Neurobiol 66:285-306.

Knudsen K, Greter J, Verdicchio M (2008) High mortality rates among GHB abusers in Western Sweden. Clin Toxicol (Phila) 46:187-192.

Knudsen K, Jonsson U, Abrahamsson J (2010) Twenty-three deaths with gamma-hydroxybutyrate overdose in western Sweden between 2000 and 2007. Acta anaesthesiologica Scandinavica 54:987-992.

Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656-660.

Koob GF (2006) The neurobiology of addiction: a neuroadaptational view relevant for diagnosis. Addiction 101 Suppl 1:23-30.

Koob GF, Sanna PP, Bloom FE (1998) Neuroscience of addiction. Neuron 21:467-476.

Lai M, Hibberd CJ, Gluckman PD, Seckl JR (2000) Reduced expression of insulin-like growth factor 1 messenger RNA in the hippocampus of aged rats. Neurosci Lett 288:66-70.

Lai Z, Roos P, Zhai O, Olsson Y, Fholenhag K, Larsson C, Nyberg F (1993) Age-related reduction of human growth hormone-binding sites in the human brain. Brain Res 621:260-266.

Lai ZN, Emtner M, Roos P, Nyberg F (1991) Characterization of putative growth hormone receptors in human choroid plexus. Brain Res 546:222-226.

Lasarge CL, Banuelos C, Mayse JD, Bizon JL (2009) Blockade of GABA(B) receptors completely reverses age-related learning impairment. Neuroscience 164:941-947.

Laursen T, Jorgensen JO, Christiansen JS (2008) The management of adult growth hormone deficiency syndrome. Expert Opin Pharmacother 9:2435-2450.

Laviola L, Natalicchio A, Giorgino F (2007) The IGF-I signaling pathway. Current pharmaceutical design 13:663-669.

Law PY, Loh HH (1999) Regulation of opioid receptor activities. J Pharmacol Exp Ther 289:607-624.

Le Greves M, Zhou Q, Berg M, Le Greves P, Fholenhag K, Meyerson B, Nyberg F (2006) Growth hormone replacement in hypophysectomized rats affects spatial performance and

64

hippocampal levels of NMDA receptor subunit and PSD-95 gene transcript levels. Exp Brain Res 173:267-273.

Le Merrer J, Plaza-Zabala A, Del Boca C, Matifas A, Maldonado R, Kieffer BL (2011) Deletion of the delta opioid receptor gene impairs place conditioning but preserves morphine reinforcement. Biol Psychiatry 69:700-703.

Le Roith D, Bondy C, Yakar S, Liu JL, Butler A (2001a) The somatomedin hypothesis: 2001. Endocrine reviews 22:53-74.

Le Roith D, Scavo L, Butler A (2001b) What is the role of circulating IGF-I? Trends in endocrinology and metabolism: TEM 12:48-52.

LeDoux JE (2000) Emotion circuits in the brain. Annual review of neuroscience 23:155-184.

Lee HK, Kirkwood A (2011) AMPA receptor regulation during synaptic plasticity in hippocampus and neocortex. Semin Cell Dev Biol 22:514-520.

LeRoith D, Roberts CT, Jr. (1991) Insulin-like growth factor I (IGF-I): a molecular basis for endocrine versus local action? Mol Cell Endocrinol 77:C57-61.

LeRoith D, Werner H, Beitner-Johnson D, Roberts CT, Jr. (1995) Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocrine reviews 16:143-163.

Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI (1987) Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537-543.

Li CH, Dixon JS, Liu WK (1969) Human pituitary growth hormone. XIX. The primary structure of the hormone. Arch Biochem Biophys 133:70-91.

Li F, Tsien JZ (2009) Memory and the NMDA receptors. N Engl J Med 361:302-303.

Li Q, Kuhn CM, Wilson WA, Lewis DV (2007) Effects of gamma hydroxybutyric acid on inhibition and excitation in rat neocortex. Neuroscience 150:82-92.

Liechti ME, Kunz I, Greminger P, Speich R, Kupferschmidt H (2006) Clinical features of gamma-hydroxybutyrate and gamma-butyrolactone toxicity and concomitant drug and alcohol use. Drug Alcohol Depend 81:323-326.

Lingenhoehl K, Brom R, Heid J, Beck P, Froestl W, Kaupmann K, Bettler B, Mosbacher J (1999) Gamma-hydroxybutyrate is a weak agonist at recombinant GABA(B) receptors. Neuropharmacology 38:1667-1673.

Lord JA, Waterfield AA, Hughes J, Kosterlitz HW (1977) Endogenous opioid peptides: multiple agonists and receptors. Nature 267:495-499.

Louagie HK, Verstraete AG, De Soete CJ, Baetens DG, Calle PA (1997) A sudden awakening from a near coma after combined intake of

65

gamma-hydroxybutyric acid (GHB) and ethanol. J Toxicol Clin Toxicol 35:591-594.

Luscher C, Huber KM (2010) Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease. Neuron 65:445-459.

Lyuh E, Kim HJ, Kim M, Lee JK, Park KS, Yoo KY, Lee KW, Ahn YO (2007) Dose-specific or dose-dependent effect of growth hormone treatment on the proliferation and differentiation of cultured neuronal cells. Growth Horm IGF Res 17:315-322.

Maitre M, Cash C, Weissmann-Nanopoulos D, Mandel P (1983) Depolarization-evoked release of gamma-hydroxybutyrate from rat brain slices. J Neurochem 41:287-290.

Maitre M, Hechler V, Vayer P, Gobaille S, Cash CD, Schmitt M, Bourguignon JJ (1990) A specific gamma-hydroxybutyrate receptor ligand possesses both antagonistic and anticonvulsant properties. J Pharmacol Exp Ther 255:657-663.

Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1988) Anatomy of CNS opioid receptors. Trends in neurosciences 11:308-314.

Mao J, Sung B, Ji RR, Lim G (2002) Neuronal apoptosis associated with morphine tolerance: evidence for an opioid-induced neurotoxic mechanism. J Neurosci 22:7650-7661.

Maren S (2005) Synaptic mechanisms of associative memory in the amygdala. Neuron 47:783-786.

Margolis EB, Toy B, Himmels P, Morales M, Fields HL (2012) Identification of rat ventral tegmental area GABAergic neurons. PLoS One 7:e42365.

Marinelli PW, Funk D, Harding S, Li Z, Juzytsch W, Le AD (2009) Roles of opioid receptor subtypes in mediating alcohol-seeking induced by discrete cues and context. Eur J Neurosci 30:671-678.

Markowska AL, Mooney M, Sonntag WE (1998) Insulin-like growth factor-1 ameliorates age-related behavioral deficits. Neuroscience 87:559-569.

Martial JA, Hallewell RA, Baxter JD, Goodman HM (1979) Human growth hormone: complementary DNA cloning and expression in bacteria. Science 205:602-607.

Martin WR, Eades CG, Thompson JA, Huppler RE, Gilbert PE (1976) The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog. J Pharmacol Exp Ther 197:517-532.

Maruff P, Falleti M (2005) Cognitive function in growth hormone deficiency and growth hormone replacement. Horm Res 64 Suppl 3:100-108.

Maxwell R, Roth RH (1972) Conversion of 1,4-butanediol to -hydroxybutyric acid in rat brain and in peripheral tissue. Biochem Pharmacol 21:1521-1533.

Mazarr-Proo S, Kerrigan S (2005) Distribution of GHB in tissues and fluids following a fatal overdose. J Anal Toxicol 29:398-400.

66

McGauley G, Cuneo R, Salomon F, Sonksen PH (1996) Growth hormone deficiency and quality of life. Horm Res 45:34-37.

McGinty JF (2007) Co-localization of GABA with other neuroactive substances in the basal ganglia. Progress in brain research 160:273-284.

McMahon CD, Radcliff RP, Lookingland KJ, Tucker HA (2001) Neuroregulation of growth hormone secretion in domestic animals. Domestic animal endocrinology 20:65-87.

Miotto K, Darakjian J, Basch J, Murray S, Zogg J, Rawson R (2001) Gamma-hydroxybutyric acid: patterns of use, effects and withdrawal. Am J Addict 10:232-241.

Mitchell JB, Stewart J (1990) Facilitation of sexual behaviors in the male rat associated with intra-VTA injections of opiates. Pharmacol Biochem Behav 35:643-650.

Moller N, Jorgensen JO (2009) Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects. Endocrine reviews 30:152-177.

Molnar T, Fekete EK, Kardos J, Simon-Trompler E, Palkovits M, Emri Z (2006) Metabolic GHB precursor succinate binds to gamma-hydroxybutyrate receptors: characterization of human basal ganglia areas nucleus accumbens and globus pallidus. J Neurosci Res 84:27-36.

Mombereau C, Kaupmann K, Froestl W, Sansig G, van der Putten H, Cryan JF (2004) Genetic and pharmacological evidence of a role for GABA(B) receptors in the modulation of anxiety- and antidepressant-like behavior. Neuropsychopharmacology 29:1050-1062.

Mondadori C, Jaekel J, Preiswerk G (1993) CGP 36742: the first orally active GABAB blocker improves the cognitive performance of mice, rats, and rhesus monkeys. Behav Neural Biol 60:62-68.

Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11:47-60.

Morris RG, Anderson E, Lynch GS, Baudry M (1986) Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 319:774-776.

Morris RG, Garrud P, Rawlins JN, O'Keefe J (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297:681-683.

Mott DD, Lewis DV, Ferrari CM, Wilson WA, Swartzwelder HS (1990) Baclofen facilitates the development of long-term potentiation in the rat dentate gyrus. Neurosci Lett 113:222-226.

Muller EE, Locatelli V, Cocchi D (1999) Neuroendocrine control of growth hormone secretion. Physiological reviews 79:511-607.

Munir VL, Hutton JE, Harney JP, Buykx P, Weiland TJ, Dent AW (2008) Gamma-hydroxybutyrate: a 30 month emergency department review. Emerg Med Australas 20:521-530.

Murphy KD, Rose MW, Chinkes DL, Meyer WJ, 3rd, Herndon DN, Hawkins HK, Sanford AP (2007) The effects of

67

gammahydroxybutyrate on hypermetabolism and wound healing in a rat model of large thermal injury. The Journal of trauma 63:1099-1107.

Mustafa IA, Cole B, Wanebo HJ, Bland KI, Chang HR (1997) The impact of histopathology on nodal metastases in minimal breast cancer. Arch Surg 132:384-390; discussion 390-381.

Nelson T, Kaufman E, Kline J, Sokoloff L (1981) The extraneural distribution of gamma-hydroxybutyrate. J Neurochem 37:1345-1348.

Nestler EJ (2001) Neurobiology. Total recall-the memory of addiction. Science 292:2266-2267.

Niall HD, Hogan ML, Tregear GW, Segre GV, Hwang P, Friesen H (1973) The chemistry of growth hormone and the lactogenic hormones. Recent progress in hormone research 29:387-416.

Nicholson KL, Balster RL (2001) GHB: a new and novel drug of abuse. Drug Alcohol Depend 63:1-22.

Nieves-Martinez E, Sonntag WE, Wilson A, Donahue A, Molina DP, Brunso-Bechtold J, Nicolle MM (2010) Early-onset GH deficiency results in spatial memory impairment in mid-life and is prevented by GH supplementation. J Endocrinol 204:31-36.

Nilsson A, Isgaard J, Lindahl A, Dahlstrom A, Skottner A, Isaksson OG (1986) Regulation by growth hormone of number of chondrocytes containing IGF-I in rat growth plate. Science 233:571-574.

Nilsson AG, Svensson J, Johannsson G (2007) Management of growth hormone deficiency in adults. Growth Horm IGF Res 17:441-462.

Nissbrandt H, Engberg G (1996) The GABAB-receptor antagonist, CGP 35348, antagonises gamma-hydroxybutyrate- and baclofen-induced alterations in locomotor activity and forebrain dopamine levels in mice. J Neural Transm 103:1255-1263.

Nowak G, Partyka A, Palucha A, Szewczyk B, Wieronska JM, Dybala M, Metz M, Librowski T, Froestl W, Papp M, Pilc A (2006) Antidepressant-like activity of CGP 36742 and CGP 51176, selective GABAB receptor antagonists, in rodents. Br J Pharmacol 149:581-590.

Nyberg F (2000) Growth hormone in the brain: characteristics of specific brain targets for the hormone and their functional significance. Front Neuroendocrinol 21:330-348.

Nyberg F (2006) Growth hormone and insulin-like growth factor-1 in human cerebrospinal fluid. In "The Somatotrophic Axis in Brain Function". San Diego, CA Elsevier Academic Press.

Nyberg F (2007) Growth Hormone and Brain Function. Growth Hormone Therapy in Pediatrics - 20 Years of KIGS 450-450.

Nyberg F (2009) The role of the somatotrophic axis in neuroprotection and neuroregeneration of the addictive brain. Int Rev Neurobiol 88:399-427.

Nyberg F, Hallberg M (2011) Localization of neuropeptides by radioimmunoassay. Methods Mol Biol 789:191-201.

68

Nyberg F, Sanderson K, Glamsta EL (1997) The hemorphins: a new class of opioid peptides derived from the blood protein hemoglobin. Biopolymers 43:147-156.

Palatini P, Tedeschi L, Frison G, Padrini R, Zordan R, Orlando R, Gallimberti L, Gessa GL, Ferrara SD (1993) Dose-dependent absorption and elimination of gamma-hydroxybutyric acid in healthy volunteers. Eur J Clin Pharmacol 45:353-356.

Pan W, Yu Y, Cain CM, Nyberg F, Couraud PO, Kastin AJ (2005) Permeation of growth hormone across the blood-brain barrier. Endocrinology 146:4898-4904.

Pasternak GW, Goodman R, Snyder SH (1975) An endogenous morphine-like factor in mammalian brain. Life Sci 16:1765-1769.

Paxinos G, Watson C (1997) The rat brain in stereotaxic coordinates San Diego, CA, USA: Academic Press.

Pedraza C, Garcia FB, Navarro JF (2009) Neurotoxic effects induced by gammahydroxybutyric acid (GHB) in male rats. Int J Neuropsychopharmacol 12:1165-1177.

Perez E, Chu J, Bania T (2006) Seven days of gamma-hydroxybutyrate (GHB) use produces severe withdrawal. Ann Emerg Med 48:219-220.

Persson AI, Aberg ND, Oscarsson J, Isaksson OG, Ronnback L, Frick F, Sonesson C, Eriksson PS (2003) Expression of delta opioid receptor mRNA and protein in the rat cerebral cortex and cerebellum is decreased by growth hormone. J Neurosci Res 71:496-503.

Persson AI, Thorlin T, Eriksson PS (2005) Comparison of immunoblotted delta opioid receptor proteins expressed in the adult rat brain and their regulation by growth hormone. Neurosci Res 52:1-9.

Pert CB, Snyder SH (1973) Opiate receptor: demonstration in nervous tissue. Science 179:1011-1014.

Postel-Vinay MC (1996) Growth hormone- and prolactin-binding proteins: soluble forms of receptors. Horm Res 45:178-181.

Postel-Vinay MC, Finidori J, Sotiropoulos A, Dinerstein H, Martini JF, Kelly PA (1995) [Growth hormone receptor. Structure and signal transduction]. Annales d'endocrinologie 56:209-212.

Raben MS (1957) Preparation of growth hormone from pituitaries of man and monkey. Science 125:883-884.

Raben MS (1958) Treatment of a pituitary dwarf with human growth hormone. J Clin Endocrinol Metab 18:901-903.

Ratomponirina C, Gobaille S, Hode Y, Kemmel V, Maitre M (1998) Sulpiride, but not haloperidol, up-regulates gamma-hydroxybutyrate receptors in vivo and in cultured cells. Eur J Pharmacol 346:331-337.

Reimunde P, Quintana A, Castanon B, Casteleiro N, Vilarnovo Z, Otero A, Devesa A, Otero-Cepeda XL, Devesa J (2011) Effects of growth hormone (GH) replacement and cognitive rehabilitation in patients with cognitive disorders after traumatic brain injury. Brain injury : [BI] 25:65-73.

69

Rodgers J, Ashton CH, Gilvarry E, Young AH (2004) Liquid ecstasy: a new kid on the dance floor. Br J Psychiatry 184:104-106.

Rollero A, Murialdo G, Fonzi S, Garrone S, Gianelli MV, Gazzerro E, Barreca A, Polleri A (1998) Relationship between cognitive function, growth hormone and insulin-like growth factor I plasma levels in aged subjects. Neuropsychobiology 38:73-79.

Roos P, Fevold HR, Gemzell CA (1963) Preparation of Human Growth Hormone by Gel Filtration. Biochim Biophys Acta 74:525-531.

Rosen MI, Pearsall HR, Woods SW, Kosten TR (1997) Effects of gamma-hydroxybutyric acid (GHB) in opioid-dependent patients. Journal of substance abuse treatment 14:149-154.

Roskam WG, Rougeon F (1979) Molecular cloning and nucleotide sequence of the human growth hormone structural gene. Nucleic Acids Res 7:305-320.

Roth RH, Delgado JM, Giarman NJ (1966) Gamma-butyrolactone and gamma-hydroxybutyric acid. II. The pharmacologically active form. International journal of neuropharmacology 5:421-428.

Roth RH, Giarman NJ (1969) Conversion in vivo of gamma-aminobutyric to gamma-hydroxybutyric acid in the rat. Biochem Pharmacol 18:247-250.

Rudy JW (2009) Context representations, context functions, and the parahippocampal-hippocampal system. Learn Mem 16:573-585.

Rumigny JF, Cash C, Mandel P, Vincendon G, Maitre M (1981) Evidence that a specific succinic semialdehyde reductase is responsible for gamma-hydroxybutyrate synthesis in brain tissue slices. FEBS Lett 134:96-98.

Saal D, Dong Y, Bonci A, Malenka RC (2003) Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons. Neuron 37:577-582.

Salmon WD, Jr., Daughaday WH (1957) A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. The Journal of laboratory and clinical medicine 49:825-836.

Sartorio A, Molinari E, Riva G, Conti A, Morabito F, Faglia G (1995) Growth hormone treatment in adults with childhood onset growth hormone deficiency: effects on psychological capabilities. Horm Res 44:6-11.

Scharf MB, Hauck M, Stover R, McDannold M, Berkowitz D (1998a) Effect of gamma-hydroxybutyrate on pain, fatigue, and the alpha sleep anomaly in patients with fibromyalgia. Preliminary report. J Rheumatol 25:1986-1990.

Scharf MB, Lai AA, Branigan B, Stover R, Berkowitz DB (1998b) Pharmacokinetics of gammahydroxybutyrate (GHB) in narcoleptic patients. Sleep 21:507-514.

Schenk S, Valadez A, Worley CM, McNamara C (1993) Blockade of the acquisition of cocaine self-administration by the NMDA antagonist MK-801 (dizocilpine). Behav Pharmacol 4:652-659.

70

Schep LJ, Knudsen K, Slaughter RJ, Vale JA, Megarbane B (2012) The clinical toxicology of gamma-hydroxybutyrate, gamma-butyrolactone and 1,4-butanediol. Clin Toxicol (Phila) 50:458-470.

Schmahl HJ, Dencker L, Plum C, Chahoud I, Nau H (1996) Stereoselective distribution of the teratogenic thalidomide analogue EM12 in the early embryo of marmoset monkey, Wistar rat and NMRI mouse. Archives of toxicology 70:749-756.

Schmidt-Mutter C, Gobaille S, Muller C, Maitre M (1999a) Prodynorphin and proenkephalin mRNAs are increased in rat brain after acute and chronic administration of gamma-hydroxybutyrate. Neurosci Lett 262:65-68.

Schmidt-Mutter C, Muller C, Zwiller J, Gobaille S, Maitre M (1999b) Gamma-hydroxybutyrate and cocaine administration increases mRNA expression of dopamine D1 and D2 receptors in rat brain. Neuropsychopharmacology 21:662-669.

Schuler V, Luscher C, Blanchet C, Klix N, Sansig G, Klebs K, Schmutz M, Heid J, Gentry C, Urban L, Fox A, Spooren W, Jaton AL, Vigouret J, Pozza M, Kelly PH, Mosbacher J, Froestl W, Kaslin E, Korn R, Bischoff S, Kaupmann K, van der Putten H, Bettler B (2001) Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1)). Neuron 31:47-58.

Schwartz RH, Milteer R, LeBeau MA (2000) Drug-facilitated sexual assault ('date rape'). South Med J 93:558-561.

Scoville WB, Milner B (1957) Loss of recent memory after bilateral hippocampal lesions. J Neurol Neurosurg Psychiatry 20:11-21.

Scoville WB, Milner B (2000) Loss of recent memory after bilateral hippocampal lesions. 1957. The Journal of neuropsychiatry and clinical neurosciences 12:103-113.

Sharma HS, Nyberg F, Gordh T, Alm P, Westman J (1997) Topical application of insulin like growth factor-1 reduces edema and upregulation of neuronal nitric oxide synthase following trauma to the rat spinal cord. Acta Neurochir Suppl 70:130-133.

Shippenberg TS, Chefer VI, Thompson AC (2009) Delta-opioid receptor antagonists prevent sensitization to the conditioned rewarding effects of morphine. Biol Psychiatry 65:169-174.

Sim LJ, Selley DE, Childers SR (1995) In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding. Proc Natl Acad Sci U S A 92:7242-7246.

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

Sircar R, Basak A (2004) Adolescent gamma-hydroxybutyric acid exposure decreases cortical N-methyl-D-aspartate receptor and impairs spatial learning. Pharmacol Biochem Behav 79:701-708.

71

Sircar R, Basak A, Sircar D (2008) Gamma-hydroxybutyric acid-induced cognitive deficits in the female adolescent rat. Ann N Y Acad Sci 1139:386-389.

Sircar R, Basak A, Sircar D, Wu LC (2010) Effects of gamma-hydroxybutyric acid on spatial learning and memory in adolescent and adult female rats. Pharmacol Biochem Behav 96:187-193.

Sjogren P, Thomsen AB, Olsen AK (2000) Impaired neuropsychological performance in chronic nonmalignant pain patients receiving long-term oral opioid therapy. J Pain Symptom Manage 19:100-108.

Skottner A, Clark RG, Robinson IC, Fryklund L (1987) Recombinant human insulin-like growth factor: testing the somatomedin hypothesis in hypophysectomized rats. J Endocrinol 112:123-132.

Slattery DA, Desrayaud S, Cryan JF (2005) GABAB receptor antagonist-mediated antidepressant-like behavior is serotonin-dependent. J Pharmacol Exp Ther 312:290-296.

Smith MT (2000) Neuroexcitatory effects of morphine and hydromorphone: evidence implicating the 3-glucuronide metabolites. Clin Exp Pharmacol Physiol 27:524-528.

Smith PF (2003) Neuroprotection against hypoxia-ischemia by insulin-like growth factor-I (IGF-I). IDrugs : the investigational drugs journal 6:1173-1177.

Snead OC, 3rd (1987) gamma-Hydroxybutyric acid in subcellular fractions of rat brain. J Neurochem 48:196-201.

Snead OC, 3rd (1996) Presynaptic GABAB-and gamma-hydroxybutyric acid-mediated mechanisms in generalized absence seizures. Neuropharmacology 35:359-367.

Snead OC, 3rd, Gibson KM (2005) Gamma-hydroxybutyric acid. N Engl J Med 352:2721-2732.

Snead OC, 3rd, Liu CC, Bearden LJ (1982) Studies on the relation of gamma-hydroxybutyric acid (GHB) to gamma-aminobutyric acid (GABA). Evidence that GABA is not the sole source for GHB in rat brain. Biochem Pharmacol 31:3917-3923.

Solway J, Sadove MS (1965) 4-Hydroxybutyrate: a clinical study. Anesth Analg 44:532-539.

Sonntag WE, Lynch CD, Cooney PT, Hutchins PM (1997) Decreases in cerebral microvasculature with age are associated with the decline in growth hormone and insulin-like growth factor 1. Endocrinology 138:3515-3520.

Sovago J, Dupuis DS, Gulyas B, Hall H (2001) An overview on functional receptor autoradiography using [35S]GTPgammaS. Brain Res Brain Res Rev 38:149-164.

Spain JW, Newsom GC (1991) Chronic opioids impair acquisition of both radial maze and Y-maze choice escape. Psychopharmacology (Berl) 105:101-106.

Spanagel R, Herz A, Shippenberg TS (1992) Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. Proc Natl Acad Sci U S A 89:2046-2050.

72

Squire LR, Clark RE, Knowlton BJ (2001) Retrograde amnesia. Hippocampus 11:50-55.

Staud R (2011) Sodium oxybate for the treatment of fibromyalgia. Expert Opin Pharmacother 12:1789-1798.

Strasburger CJ, Bidlingmaier M, Wu Z, Morrison KM (2001) Normal values of insulin-like growth factor I and their clinical utility in adults. Horm Res 55 Suppl 2:100-105.

Sumnall HR, Woolfall K, Edwards S, Cole JC, Beynon CM (2008) Use, function, and subjective experiences of gamma-hydroxybutyrate (GHB). Drug Alcohol Depend 92:286-290.

Svensson AL, Bucht N, Hallberg M, Nyberg F (2008) Reversal of opiate-induced apoptosis by human recombinant growth hormone in murine foetus primary hippocampal neuronal cell cultures. Proc Natl Acad Sci U S A 105:7304-7308.

Takahara J, Yunoki S, Hosogi H, Yakushiji W, Kageyama J, Ofuji T (1980) Concomitant increases in serum growth hormone and hypothalamic somatostatin in rats after injection of gamma-aminobutyric acid, aminooxyacetic acid, or gamma-hydroxybutyric acid. Endocrinology 106:343-347.

Takahara J, Yunoki S, Yakushiji W, Yamauchi J, Yamane Y (1977) Stimulatory effects of gamma-hydroxybutyric acid on growth hormone and prolactin release in humans. J Clin Endocrinol Metab 44:1014-1017.

Talamantes F, Ortiz R (2002) Structure and regulation of expression of the mouse GH receptor. J Endocrinol 175:55-59.

Terenius L (1973) Stereospecific interaction between narcotic analgesics and a synaptic plasm a membrane fraction of rat cerebral cortex. Acta pharmacologica et toxicologica 32:317-320.

Terenius L, Wahlstrom A (1975) Search for an endogenous ligand for the opiate receptor. Acta physiologica Scandinavica 94:74-81.

Thai D, Dyer JE, Benowitz NL, Haller CA (2006) Gamma-hydroxybutyrate and ethanol effects and interactions in humans. Journal of clinical psychopharmacology 26:524-529.

Thomas MJ, Kalivas PW, Shaham Y (2008) Neuroplasticity in the mesolimbic dopamine system and cocaine addiction. Br J Pharmacol 154:327-342.

Thornwall-Le Greves M, Zhou Q, Lagerholm S, Huang W, Le Greves P, Nyberg F (2001) Morphine decreases the levels of the gene transcripts of growth hormone receptor and growth hormone binding protein in the male rat hippocampus and spinal cord. Neurosci Lett 304:69-72.

Tzanela M, Wagner C, Tannenbaum GS (1997) Recombinant human growth hormone-binding protein fails to enhance the in vivo bioactivity of human growth hormone in normal rats. Endocrinology 138:5316-5324.

73

Tzschentke TM, Schmidt WJ (1995) N-methyl-D-aspartic acid-receptor antagonists block morphine-induced conditioned place preference in rats. Neurosci Lett 193:37-40.

Ungless MA, Whistler JL, Malenka RC, Bonci A (2001) Single cocaine exposure in vivo induces long-term potentiation in dopamine neurons. Nature 411:583-587.

van Amsterdam JG, Brunt TM, McMaster MT, Niesink RJ (2012) Possible long-term effects of gamma-hydroxybutyric acid (GHB) due to neurotoxicity and overdose. Neuroscience and biobehavioral reviews 36:1217-1227.

Van Cauter E, Plat L, Scharf MB, Leproult R, Cespedes S, L'Hermite-Baleriaux M, Copinschi G (1997) Simultaneous stimulation of slow-wave sleep and growth hormone secretion by gamma-hydroxybutyrate in normal young Men. J Clin Invest 100:745-753.

van Nieuwpoort IC, Drent ML (2008) Cognition in the adult with childhood-onset GH deficiency. Eur J Endocrinol 159 Suppl 1:S53-57.

Van Wyk JJ, Underwood LE, Hintz RL, Clemmons DR, Voina SJ, Weaver RP (1974) The somatomedins: a family of insulinlike hormones under growth hormone control. Recent progress in hormone research 30:259-318.

Varela M, Nogue S, Oros M, Miro O (2004) Gamma hydroxybutirate use for sexual assault. Emerg Med J 21:255-256.

Vayer P, Mandel P, Maitre M (1985) Conversion of gamma-hydroxybutyrate to gamma-aminobutyrate in vitro. J Neurochem 45:810-814.

Verstraete AG (2004) Detection times of drugs of abuse in blood, urine, and oral fluid. Therapeutic drug monitoring 26:200-205.

Vicini JL, Buonomo FC, Veenhuizen JJ, Miller MA, Clemmons DR, Collier RJ (1991) Nutrient balance and stage of lactation affect responses of insulin, insulin-like growth factors I and II, and insulin-like growth factor-binding protein 2 to somatotropin administration in dairy cows. J Nutr 121:1656-1664.

Vickers MD (1968) Gamma hydroxybutyric acid. Clinical pharmacology and current status. Proceedings of the Royal Society of Medicine 61:821-824.

Volpi R, Chiodera P, Caffarra P, Scaglioni A, Malvezzi L, Saginario A, Coiro V (2000) Muscarinic cholinergic mediation of the GH response to gamma-hydroxybutyric acid: neuroendocrine evidence in normal and parkinsonian subjects. Psychoneuroendocrinology 25:179-185.

Vuong C, Van Uum SH, O'Dell LE, Lutfy K, Friedman TC (2010) The effects of opioids and opioid analogs on animal and human endocrine systems. Endocrine reviews 31:98-132.

Walser M, Hansen A, Svensson PA, Jernas M, Oscarsson J, Isgaard J, Aberg ND (2011) Peripheral administration of bovine GH regulates the expression of cerebrocortical beta-globin, GABAB receptor 1, and the Lissencephaly-1 protein (LIS-1) in adult hypophysectomized rats. Growth Horm IGF Res 21:16-24.

74

White JH, Wise A, Main MJ, Green A, Fraser NJ, Disney GH, Barnes AA, Emson P, Foord SM, Marshall FH (1998) Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature 396:679-682.

Whitlock JR, Heynen AJ, Shuler MG, Bear MF (2006) Learning induces long-term potentiation in the hippocampus. Science 313:1093-1097.

Wine RN, McPherson CA, Harry GJ (2009) IGF-1 and pAKT signaling promote hippocampal CA1 neuronal survival following injury to dentate granule cells. Neurotox Res 16:280-292.

Wise RA (2008) Dopamine and reward: the anhedonia hypothesis 30 years on. Neurotox Res 14:169-183.

Wong JH, Dukes J, Levy RE, Sos B, Mason SE, Fong TS, Weiss EJ (2008) Sex differences in thrombosis in mice are mediated by sex-specific growth hormone secretion patterns. J Clin Invest 118:2969-2978.

Wood DM, Nicolaou M, Dargan PI (2009) Epidemiology of recreational drug toxicity in a nightclub environment. Substance use & misuse 44:1495-1502.

Zadina JE, Hackler L, Ge LJ, Kastin AJ (1997) A potent and selective endogenous agonist for the mu-opiate receptor. Nature 386:499-502.

Zarrindast MR, Bakhsha A, Rostami P, Shafaghi B (2002) Effects of intrahippocampal injection of GABAergic drugs on memory retention of passive avoidance learning in rats. J Psychopharmacol 16:313-319.

Zhai Q, Lai Z, Roos P, Nyberg F (1994) Characterization of growth hormone binding sites in rat brain. Acta Paediatr Suppl 406:92-95.

Zhang Y, Landthaler M, Schlussman SD, Yuferov V, Ho A, Tuschl T, Kreek MJ (2009) Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin. Neuroscience 158:474-483.

Zhou Q, Liu Z, Ray A, Huang W, Karlsson K, Nyberg F (1998) Alteration in the brain content of substance P (1-7) during withdrawal in morphine-dependent rats. Neuropharmacology 37:1545-1552.

Zhou YC, Waxman DJ (1999) Cross-talk between janus kinase-signal transducer and activator of transcription (JAK-STAT) and peroxisome proliferator-activated receptor-alpha (PPARalpha) signaling pathways. Growth hormone inhibition of pparalpha transcriptional activity mediated by stat5b. J Biol Chem 274:2672-2681.

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