Tolerance and Antagonism to Allopregnanolone Effects in the Rat CNS

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Tolerance and Antagonism to Allopregnanolone Effects in the

Rat CNS

Şahruh Türkmen

Umeå University Medical Dissertations New Series No 1026 – ISSN 0346-6612 – ISBN 91-7264-073-1

From the Department of Clinical Sciences, Obstetrics and Gynecology Umeå University

Tolerance and antagonism to allopregnanolone effects in the rat CNS

Şahruh Türkmen

Umeå 2006

Department of Clinical science, Obstetrics & Gynecology. Umeå University SE-901 85 Umeå, Sweden

Cover illustration: Neuron

ISSN 0346-6612

ISBN 91-7264-073-1

Printed by Print & Media, Umeå University, 2006

To my family

……. Tolerance, a view as politically pernicious, philosophically false and scientifically ambiguous……..

Contents

Pages

ABSTRACT …………………………………………………………..6

LIST OF ORIGINAL PAPERS ……………………………………..7

ABBREVIATIONS …………………………………………………..8

1. INTRODUCTION …………………………………………………9 1.1. SEX HORMONES AND CNS ………………………………....9 1.1.1 Genomic and non-genomic mechanisms …………………..11 1.1.2 Neurosteroids and Neuroactive Steroid .…………………...11 1.1.3 Allopregnanolone ………………………………………….12 1.1.4 Allopregnanolone concentration …………………………..13 1.2. GABA-A RECEPTORS ………………………………………..15 1.2.1 Structure …………………………………………………....15 1.2.2 Distributions and functions ………………………………...17 1.2.3 GABA-A receptor agonist and antagonist steroids ...... ….18 1.2.4 Allopregnanolone and interaction with the GABA-A receptor ……………………………………………………20 1.3. CLINICAL CONDITIONS RELATED TO THE SEX SETROID HORMONES EFFECT IN THE BRAIN ………….21 1.4. TOLERANCE ………………………………………………….24 1.4.1 The GABA-A receptor and tolerance …………………….25 1.4.2 Changes in the GABA-A receptor during tolerance ……...26 1.4.3 Tolerance to allopregnanolone ……………………………27 1.5. LEARNING AND MEMORY .………………………………..28 1.5.1 Spatial learning and memory ……………………………..28 1.5.2 The GABA-A receptor modulators and spatial learning and memory ……………………………………………….29

2. AIMS ……………………………………………………………….31

3. METHODS…………………………………………………………32 3.1 In situ hybridization………………………………………….32 3.2 Immunohistochemistry……………………………………....32 3.3 Chloride ion uptake assay …………………………………...33 3.4 EEG threshold method ………………………………………33 3.5 Morris Water Maze test ……………………………………..35 3.6 Hormone assays ……………………………………………..37 3.7 Statistical analysis …………………………………………...37

4. RESULTS AND DISCUSSIONS …………………………………39 4.1 Antagonism of an allopregnanolone effect ………………….39 4.2 Chronic allopregnanolone tolerance …………………………42 4.3 Acute allopregnanolone tolerance and the GABA-A receptor subunits .……………………………………………46 4.4 Persistence of allopregnanolone tolerance .………………….50

5. GENERAL DISCUSSIONS .………………………………………55

6. CONCLUSIONS .…………………………………………………..57

7. ACKNOWLEDGMENTS .………………………………………...58

8. REFERENCES .…………………………………………………….60

APPENDIX - Papers I – IV

ABSTRACT Many studies have suggested a relationship between sex steroids and negative mental and mood changes in women. Allopregnanolone, a potent endogenous ligand of the GABA-A receptor and a metabolite of progesterone, is one of the most accused neuroactive steroids. Variations in the levels of neuroactive steroids that influence the activity of the GABA-A receptor cause a vulnerability to mental and emotional pathology. In women, there are physiological conditions in which allopregnanolone production increases acutely (e.g. stress) or chronically (e.g. menstrual cycle, pregnancy), thus exposing the GABA-A receptor to high allopregnanolone concentrations. In such conditions, tolerance to allopregnanolone probably develops. We have evaluated the 3β-hydroxy pregnane steroid UC1011 as a functional antagonist to allopregnanolone-induced negative effects in rats. In vivo, we used the Morris Water Maze (MWM) test of learning and, in vitro, we studied chloride ion uptake into cortical and hippocampal membrane preparations. The steroid UC1011 reduces the allopregnanolone-induced learning impairment in the MWM and the increase in chloride ion uptake induced by allopregnanolone. To detect whether chronic tolerance develops to an allopregnanolone-induced condition, male rats were pretreated with allopregnanolone injections for three or seven days. These rats were then tested in the Morris Water Maze for five days and compared with relevant controls. Rats with seven days’ allopregnanolone pretreatment experienced improved performance compared with the acutely allopregnanolone-exposed group, reflecting chronic tolerance development. To study the GABA-A receptor changes in acute allopregnanolone tolerance, we used the silent second (SS) anaesthesia threshold method. At acute tolerance, 90 minutes of anaesthesia, the abundance of the GABA-A receptor α4 subunit and the expression of the α4 subunit mRNA in the thalamus ventral-posteriomedial (VPM) nucleus were reduced. There was also a significant negative correlation between the increase in the allopregnanolone dose needed to maintain anaesthesia and the α4 mRNA in the VPM nucleus. We also investigated whether allopregnanolone tolerance was still present one or two days after the end of the anaesthesia-induced acute tolerance. Tolerance persisted to one day, but not two days, after the treatment and the α4 subunit mRNA expression in the VPM nucleus was negatively related to the allopregnanolone doses needed after one day. In conclusion, the current thesis shows that the substance UC1011 can reduce the allopregnanolone-induced negative effects in the water maze test. Chronic allopregnanolone tolerance can develop to the effects of allopregnanolone. Allopregnanolone tolerance persists one day after the induction of acute allopregnanolone tolerance. The GABA-A receptor α4 subunit in the thalamus might be involved in the development and persistence of acute tolerance to allopregnanolone.

Key words: Allopregnanolone, GABA-A receptor, UC1011, Spatial memory, Morris Water Maze, Tolerance, Silent second, mRNA

LIST OF ORIGINAL PAPERS:

I) 3beta-20beta-dihydroxy-5alpha-pregnane (UC1011) antagonism of the GABA potentiation and the learning impairment induced in rats by allopregnanolone. Turkmen S., Lundgren P., Birzniece V., Zingmark E., Backstrom T., and Johansson I.M. Eur J Neurosci. 2004: 20, 1604-1612.

II) Tolerance development to Morris water maze test impairments induced by acute allopregnanolone. Türkmen S., Löfgren M., Birzniece V., Bäckström T., and Johansson IM. Neuroscience. 2006: 139(2): 651-9

III) GABA-A receptor changes in acute allopregnanolone tolerance. Birzniece* V., Türkmen* S., Lindblad C., Zhu D., Johansson IM., Bäckström T., Wahlström G. Eur J Pharmacol. 2006: 535(1-3):125-34. * The first two authors have contributed equally in this work.

IV) Remaining anaesthetic allopregnanolone tolerance in male rat. Türkmen Ş., Wahlström G., Bäckström T., Johansson I.M. Manuscript.

These papers are reproduced with the permission of the copyright holders.

ABBREVIATIONS

ANOVA analysis of variance BZP benzodiazepine CC celite chromatography CNS central nervous system DG dentate gyrus DHEAS dehydroepiandrosterone sulphate DHP dihydroprogesterone DNA deoxyribonucleic acid EEG electroencephalography GABA γ-aminobutyric acid HPLC high performance liquid chromatography HRT hormone replacement therapy HSD hydroxysteroid dehydrogenase i.m. intramuscular i.p. intraperitoneal i.v. intravenous LSD least significant difference MDR maintenance dose rate MPA medroxyprogesterone acetate NMDA N-methyl-D-aspartate P450scc p450 side-chain cleavage enzyme PMDD premenstrual dysphoric disorder PMS premenstrual syndrome RIA radioimmunoassay SEM standard error of the mean SS silent second THP tetrahydroprogesterone UC1011 3β-20β-dihydroxy-5α-pregnane VPM ventral posteriomedial thalamic nucleus

1. INTRODUCTION

Many women report mental or emotional changes when the concentrations of ovarian hormones fluctuate during the menstrual cycle, pregnancy and the post-partum period. In addition, problems become apparent with exogenous steroid treatment such as contraceptive usage and hormone replacement therapy. Some women also point out that their cognitive performance varies to some degree with the hormonal changes in the menstrual cycle and at menopause. The sex steroid hormones, estrogen and progesterone, influence a variety of behaviours in vertebrates and have crucial effects on female reproductive functions such as the development and maturation of reproductive organs, carbohydrate metabolism and osmotic equilibrium (Whitfield et al., 1999). Animal studies have also shown that the steroid hormones of gonadal origin act on the foetal central nervous system (CNS) tissues and permanently alter the function and behaviours they will mediate in the future (Grady et al., 1965; Phoenix et al., 1959). After puberty, the female reproductive system undergoes cyclical changes, the menstrual cycle. The most obvious of these changes is the vaginal bleeding that occurs with the cyclic changes in hormonal production of the hypothalamus-pituitary-ovarian axis (Adashi, 1992). The mechanisms by which the ovarian hormones affect brain functions are not completely understood. In the present thesis, we investigated the mechanisms of steroid and receptor interactions and receptor changes at exposure to a neuroactive steroid.

1.1. SEX HORMONES AND CNS Glands secrete hormones, which trigger changes in gene expression in distant parts of the body. There are two main structural classes of hormones, 1) steroid hormones and 2) peptide hormones. Steroid hormones are synthesised in steroidogenic tissues such as the gonads, adrenal glands and placenta. Some steroid hormones are also synthesised in the nervous system (Baulieu and Robel, 1990; Speroff, 1999). The steroid hormones all contain the same structure on the A-ring with a keto group at the 3 position and a double bond between carbon 4 and 5; the only exception is the sex hormone estradiol. The precursor of steroid hormones (with the exception of retinoic

acid) is cholesterol. Moreover, with the exception of vitamin D, they all contain a basic skeletal structure of four fused rings, referred to as perhydrocyclopentanophenanthrene, and the same atomic numbering system as for cholesterol (Fig 1 B). Members of the cytochrome P450 superfamily and hydroxysteroid dehydrogenase (HSD) enzymes are involved in the biosynthesis of the steroid hormones (Mellon and Griffin, 2002; Serra, 1983; Speroff, 1999). The first step in steroidogenesis is the conversion of cholesterol to pregnenolone by the side-chain cleavage enzyme cytochrome P450scc located in the inner mitochondrial membrane (Speroff, 1999). Pregnenolone is then converted to progesterone by 3βHSD located in the endoplasmic reticulum (Speroff, 1999). From this point, progesterone can enter a range of biochemical pathways to synthesise a variety of steroid hormones, such as the main neuroactive steroid, allopregnanolone (Fig. 1).

A) Cholesterol

3αHSD-II/III 3α5α-THP(allopregnanolone) 5α- DHP P450scc 3α5β-THP 3βHSD 5α reductase-I/II (isoallopregnanolone) Pregnenolone

3αHSD 3βHSD-I/II 3α5β-THP (pregnanolone) 5β- DHP Progesterone 3β5β-THP 3βHSD 5β- reductase (isopregnanolone)

B) C)

Figure 1: A) One steroid synthesis pathway (progesterone derivates) (adapted from Compagnone and Mellon, 2000). B) The perhydrocyclopentanophenanthrene structure and numbering. C) The allopregnanolone (5α-pregnane-3α-ol-20-one) structure. DHP, dihydroprogesterone; THP, tetrahydroprogesterone; P450scc, P450 side-chain cleavage enzyme; HSD, hydroxysteroid dehydrogenase

1.1.1 Genomic and non-genomic mechanisms of action Steroid hormones are known to act through intracellular receptors, located in the nucleus and cytoplasm that acts as ligand-activated transcription factors in the regulation of gene expression (genomic mechanism). However, this classical mechanism of action is unable to account for a variety of rapid steroid effects that occur within milliseconds to seconds. Steroid hormones also have effects through membrane-bound receptors such as ion channels (non-genomic or non-nuclear mechanism), a mechanism in which receptor binding to DNA and changed RNA synthesis is not required (Baulieu and Robel, 1995; Frye et al., 1992; McEwen, 1994; Rupprecht, 2003). However, steroid actions on membrane-bound ion channels can secondarily activate the genome so that a change in gene expression occurs. This may be the case during tolerance development, for example, when the target receptor changes to be less sensitive to the drug of action (Biggio et al., 2003). A full understanding of these mechanisms has not yet been obtained. The estrogen receptor (ER) exists as an alpha (ERα) and a beta (ERβ) isoform; progesterone also has two intracellular receptors, PR-A and PR-B. It was long believed that steroid hormones acted exclusively through these classical receptors. However, both estrogen and progesterone can also affect the target cells by non-genomic action (Coiret et al., 2005; Duras et al., 2005). In most tissues co-expressing estrogen and progesterone receptors, estrogen controls the amount of progesterone receptors (up-regulation by estrogen) and thereby also the sensitivity to progestins (steroids with progesterone-like effects). The progesterone receptor is down-regulated by exposure to endogenous progesterone (Bouchard, 1999). The presence of these hormone receptors within different regions of the brain has been described in several studies (Genazzani et al., 2000; Jung-Testas et al., 1992; McEwen et al., 1982). The non-genomic actions of steroid hormones at membrane receptors are often associated with signalling pathways. Using such pathways, steroids rapidly regulate multiple cellular functions (Christ et al., 1995). The rapid time course and the insensitivity of the effect to transcription and protein synthesis inhibitors are essential conditions in the non-genomic action (for a review see Schmidt et al., 2000).

1.1.2 Neurosteroids and Neuroactive Steroids The term “neurosteroid” refers to steroids formed in the nervous system. The name was created by Dr. E.E. Baulieu and colleagues, when they showed

that the brain has the capacity to synthesise its own steroids in situ (Corpechot et al., 1981). Neurosteroids can be synthesised de novo in the brain from cholesterol or from steroid precursors that cross the blood-brain barrier. The closely related term “neuroactive steroids” includes all the steroids that are neuronal modulators (Gasior et al., 1999; Paul and Purdy, 1992). The terms “neurosteroid” and “neuroactive steroid” are often used interchangeably. The term “GABAergic steroid” is used for the group of steroids that act on the γ-amino butyric acid type-A (GABA-A) receptor (Majewska, 1990). Neurosteroids can have non-genomic actions (Paul and Purdy, 1992), thereby rapidly altering neuronal excitability by binding and modulating membrane receptors for inhibitory or excitatory neurotransmitters (Rupprecht and Holsboer, 1999). A non-genomic neurosteroid effect is the interaction between the GABA-A receptor and progesterone metabolites, such as the neurosteroid allopregnanolone (Gee, 1988; Majewska et al., 1986).

1.1.3 Allopregnanolone The neurosteroid allopregnanolone (3α-hydroxy-5α-pregnanone-20-one or tetrahydroprogesterone) is an A-ring reduced pregnane steroid and one of the main circulating metabolites of progesterone (Fig. 1). Allopregnanolone is synthesised in the corpus luteum of the ovary, in the adrenal cortex and in the central nervous system (Holzbauer et al., 1985; Ottander et al., 2005; Paul and Purdy, 1992). Allopregnanolone is an allosteroic modulator, which, at nanomolar concentrations potentiates the GABA effect at the GABA-A receptor. It is assumed that the modulatory action of allopregnanolone is mediated by binding to the GABA-A receptor (Majewska et al., 1986; Rupprecht, 2003). Recently, one preliminary study reported the presence of two distinct allopregnanolone binding sites on the GABA-A receptor (Hosie, 2005). Allopregnanolone is synthesised from progesterone by a two-step reaction (Fig. 1A). The first step is irreversible and is mediated by 5α- reductase that converts progesterone to 5α-hydroxyprogesterone (5α- pregnanedione). The second step is reversible and is mediated by 3αHSD that converts 5α-hydroxyprogesterone to allopregnanolone (Karavolas and Hodges, 1990; Krieger and Scott, 1984; Mellon and Griffin, 2002). The positive allosteric modulation of the GABA-A receptors by allopregnanolone exerts sedative, hypnotic, anaesthetic, anxiolytic and anticonvulsant effects, at least in animals (Arafat et al., 1988; Backstrom et al., 2003a; Bitran et al., 1991; Carl et al., 1990; Frye and Duncan, 1994; Lancel et al., 1997; Reddy, 2004). In humans, allopregnanolone may play a critical role in neuropsychiatric disorders, in particular the disruption of

memory processes and sleep (Reddy, 2002; Schumacher et al., 2003). Moreover, the possibility has to be considered that the neurosteroid allopregnanolone might not only selectively modulate the GABA-A receptor but might also affect the NMDA and oxytocin receptors, which could induce clinical effects other than those induced by the potentiation of GABA-A receptors (Johansson and Le Greves, 2005; Landgren et al., 1998; Widmer et al., 2003).

1.1.4 Allopregnanolone concentrations In both humans and animals, the allopregnanolone levels in brain and plasma change during various physiological and pathological conditions, i.e. stress, pregnancy and the menstrual cycle (in animals, the estrus cycle) (Bixo et al., 1997; Bixo et al., 1986; Paul and Purdy, 1992). Allopregnanolone is rapidly redistributed from the brain and the first compartment half-life is very short (Purdy et al., 1990b). However, the long-term clearance is slow, probably due to a large accumulation in fat tissue (Timby et al., 2005; Zhu et al., 2004). In ovariectomised female rats, the progesterone half-life and clearance are around 75 and 165 min (respectively) (Gangrade et al., 1992). The pharmacokinetics of medroxyprogesterone acetate, a progestin, are affected by ageing, i.e. increased substance bioavailability and, as a result, lower medroxyprogesterone acetate doses are suggested for women of more advanced age (<60) (Jarvinen et al., 2004). In post-menopausal women, plasma concentrations of allopregnanolone increased in a dose-dependent manner and their time course closely followed the time course of the progesterone concentrations reached after treatment. The peak plasma concentrations of allopregnanolone after injecting (i.m.) the 25, 50 and 100 mg doses of progesterone was 5 ng/ml, 8.07 ng/ml, and 10.3 ng/ml and these peaks occurred seven, four and six hours after injections (respectively) (de Wit et al., 2001). In this female category, a low oral dose (20 mg) of progesterone 12 hours after treatment produces an allopregnanolone concentration comparable to that achieved physiologically in premenopausal women (Andreen et al., 2006), while the allopregnanolone plasma concentrations were increased to 15.9 and 19.5 ng/ml after two hours when 400 or 800 mg (respectively) progesterone were administered vaginally (Andreen et al., 2005). In women with regular menstruations a single dose of progesterone (100 mg; i.m.), increased the peak plasma concentration of allopregnanolone to 12.83 ng/ml after three hours which then decreases to 10.2 ng/ml by 13 hours (de Wit et al., 2001). The brain and plasma concentrations of allopregnanolone are partly regulated by independent mechanisms (Paul and Purdy, 1992). The brain,

like the gonads and adrenals, is a steroidogenic organ which is able to produce allopregnanolone, since the enzymes 5α-reductase and 3αHSD that are needed for the production of allopregnanolone from progesterone are present in many brain areas, notably in the cortex and the hippocampus (Cheney et al., 1995; Compagnone and Mellon, 2000). The brain concentration of allopregnanolone is significantly higher than the plasma level in both cycling and pregnant female rats and both brain and plasma levels of allopregnanolone temporally follow those of progesterone (Corpechot et al., 1993). In humans, allopregnanolone accumulates in the brain and increases in plasma during the luteal phase of the menstrual cycle (Bixo et al., 1997). The serum allopregnanolone level does not differ significantly between young fertile women in the follicular phase and age- matched men (0.25 vs. 0.23 ng/ml), whereas in women during the luteal phase the allopregnanolone level is significantly higher (approximately 1.2- 3.8 ng/ml). In non-pregnant women, plasma pregnanolone and allopregnanolone concentrations of between 25 to 50 ng/ml cause sedation and 168-223 ng/ml cause anaesthesia (Carl et al., 1990; Sundstrom et al., 1999; Timby et al., 2005). In postmenopausal women, the serum allopregnanolone level does not change with age, while a progressive decrease is observed in men, reaching the lowest level after 60 years of age (Bicikova et al., 1998; Genazzani et al., 1998). The serum allopregnanolone level increases during pregnancy, with the highest levels (> 50 ng/ml) at term (Luisi et al., 2000). The allopregnanolone concentrations in maternal and foetal sera are similar in 20-week pregnant women (3.2 ng/ml), while levels about three times lower are present in the amniotic fluid (Bicikova et al., 2002). In the plasma of intact adult male rats, allopregnanolone is barely detectable, whereas the plasma concentrations in intact cycling female rats on random days of the estrus cycle are much higher (approximately 2 ng/ml) (Corpechot et al., 1993). The allopregnanolone concentration in the brain of male rats is 0.6-1.2 ng/g protein, while the concentration in the female rat brain (whole) on random days of the estrus cycle is approximately 11 ng/g protein (Corpechot et al., 1993). The allopregnanolone concentration in the rat brain cortex can reach 3-10 ng/g protein after swimming stress (Paul and Purdy, 1992; Purdy et al., 1991) and 30 ng/g protein 30 minutes after acute electroshock (Barbaccia et al., 1997). The allopregnanolone concentration in the brain differs between regions and depends on the phase of estrus cycle, i.e., in the cortex, 4 ng/g protein in pro-estrus and 6 ng/g protein in the estrus phase of the cycle has been measured (Paul and Purdy, 1992; Purdy et al., 1991). In pregnant rats, the allopregnanolone concentration in plasma shows

a maximum increase on day 19 (approximately 11 ng/ml) and returns to control values on day 21, while, in the brain cortex, the allopregnanolone value is greatly increased (> 30 ng/g protein) (Corpechot et al., 1993). It has also been indicated that there is a significant regional and interspecies diversity in the progesterone metabolic pathways and allopregnanolone accumulation in the brain, in which the incubation of rat brain slices with progesterone leads to the accumulation of 5α-DHP and allopregnanolone, whereas the incubation of mouse brain slices leads to the accumulation of 5α-DHP and 20α-DHP and that of monkey brain slices leads to the accumulation of only 20α-DHP (Korneyev et al., 1993). In the MWM test, the allopregnanolone concentration 511 ng/g in the rat frontal cortex impaired learning (eight minutes after an i.v. injection), the brain levels then decreased by 30-60% during the next 12 min (Johansson et al., 2002). Rats remain anaesthetised as long as the allopregnanolone level in the brain frontal cortex is above 955 ng/g (Korneyev and Costa, 1996; Wang et al., 1996). The anti-anxiety effect of allopregnanolone in the cortex has been determined to be in the range of 9.5-10.5 ng/g (Bitran et al., 1993).

1.2 GABA-A RECEPTORS 1.2.1 Structure GABA is the major inhibitory neurotransmitter in the CNS. GABA is released from GABAergic neurons after synthesis from glutamate by the enzyme glutamic acid decarboxylase (GAD). GABA activates three classes of receptors, the GABA-A, GABA-B and GABA-C receptors. In this paper, only the GABA-A receptor will be discussed, as allopregnanolone modulates this receptor. The transmembrane heteropentameric GABA-A receptor is composed of five subunits that belong to different subunit classes. So far, six α, four β, four γ, one δ, one ε, one π and one θ subunit have been identified. Some of the subunits also exist as splice variants, such as the γ2 subunit that is expressed as γ2S (short form) and γ2L (long form). There is 30-40% sequence identity between the GABA-A receptor subunit families and the other ligand gated ion channels (i.e. glycine receptor) and all the sequences within each subunit family are homologous, with about 70- 80% amino acid sequence identity. The subunits form a symmetrical structure around the ion channel, each subunit contributing to the wall of the channel (Fig. 2) (DeLorey and Olsen, 1992; Olsen and Tobin, 1990; Schofield et al., 1987). The pentameric combination of the GABA-A receptors most often includes two α, two β and one γ subunit. The α1, β2/3, and γ2 subunits co-exist in many GABA-A receptors and the composition of

the receptor varies between different brain regions (Backus et al., 1993; Benke et al., 1994; Chang et al., 1996). It is assumed that GABA-A receptor expression on the cell surface is controlled by a dynamic mechanism. The GABA-A receptor subunits are translated on the endoplasmic reticulum and then assemble into receptor complexes. Unassembled receptor subunits are degraded, whereas assembled receptors are targeted via the Golgi apparatus to the plasma membrane. Surface receptors cluster at synapses. Receptors can also dissociate from the synaptic clusters and re-enter the mobile pool by endocytosis (Barnes, 2001). After the translation in the endoplasmic reticulum, GABA-A receptors slowly appear on the cell surface (Gorrie et al., 1997). It has been suggested that GABA-A receptor endocytosis is a mechanism that underlies tolerance development in physical dependence on benzodiazepines, a GABA-A receptor positive modulator substance (Poisbeau et al., 1997; Tehrani et al., 1997).

Figure 2: GABA-A receptor structure and chloride ion channel.

The GABA-A receptor contains specific binding sites for at least GABA, picrotoxin, barbiturates, benzodiazepines and anaesthetic steroids (Fig. 2). These substances bind to the GABA-A receptor and regulate the gating (opening and closing) of the chloride ion channel. The GABA-A receptor exhibits distinct pharmacological and electrophysiological properties depending on the subunit composition (Mehta and Ticku, 1999; Olsen and Tobin, 1990; Whiting et al., 1999). The localisation of the various binding sites has been investigated. The muscimol binding site (a GABA analogue) is located at the interface of the α and β subunits of the receptor (Pregenzer et al., 1993). The β subunit has also been suggested as a requirement for the GABA-A receptor chloride channel blockers TBPS (t-

butylbicyclophosphorothionate) and picrotoxin (Slany et al., 1995). The benzodiazepine binding site is located at the interface of the α and γ subunits of GABA-A receptors (Wong et al., 1992).

1.2.2 Distributions and functions The individual subunits exhibit distinct distributions throughout the nervous system and the expression of multiple subunits in the same neurons shows the existence of a large variety of GABA-A receptor subunits in the brain neurons. This suggests that different receptor subunits might have specific functions (Fritschy et al., 1998; Wisden et al., 1992). Studies to determine the function of the GABA-A receptor subunits indicate that the sedative, amnesic and probably ataxic and partly anticonvulsive actions of the benzodiazepines are mediated by the receptors containing the α1 subunit, whereas the benzodiazepine anxiolytic action takes place via receptors that contain the α2 subunit (McKernan et al., 2000). The specific localisation of the α5 subunit in the hippocampus indicates that the memory-impairing effects of benzodiazepines are possibly mediated by the α5 subunit. This conclusion is supported by the recent finding that α5 subunit-deficient animals display increased learning and memory abilities (Collinson et al., 2002; Fritschy et al., 1998; McKernan et al., 2000). An increase in α4 subunit level is associated with conditions characterised by increased neuronal excitability, increased seizure susceptibility and higher levels of anxiety (Frye and Bayon, 1999; Gallo and Smith, 1993; Gulinello et al., 2001). On the other hand, the receptors in the brain containing the α6 subunit are exclusively located in cerebellar granule cells and, like the α4 subunit, make the GABA-A receptors insensitive to benzodiazepines (Khan et al., 1996; Mehta and Shank, 1995). It is suggested that the GABA-A receptors containing the α6 subunit are responsible for the motor control of the body (Korpi et al., 1993). The γ2 subunit is involved in synaptic targeting and clustering and allopregnanolone sensitivity. The loss of the GABA-A receptor clusters results in the loss of the synaptic clustering molecule gephyrin and synaptic GABAergic function (Essrich et al., 1998). In the GABA-A receptor assembly, the γ2 subunit also enhances inhibitory synaptic transmission by reducing the desensitisation of the GABA-A receptor ion channel (Tia et al., 1996). In contrast to synaptic receptors that contain the γ2 subunits, receptors containing the δ subunit are located extrasynaptically and mediate tonic inhibition. The γ2 and δ subunits compete for assembly with some of the α and β subunits in the GABA-A receptor complex, to regulate the balance of synaptic and extrasynaptic receptors (Luscher and Keller, 2004; Peng et al.,

2002). Functionally, the δ subunit affects the sensitivity of the GABA-A receptors to neurosteroids (Mihalek et al., 1999). It has been suggested that the β2 subunit is present in about 50% of the GABA-A receptors in the CNS (McKernan and Whiting, 1996) and the β2 subunit enhances the GABA-A receptor-evoked response to some anaesthetic drugs (e.g. etomidate) but not to the neurosteroid allopregnanolone (Belelli et al., 2002; Reynolds et al., 2003).

1.2.3 GABA-A receptor agonist and antagonist steroids Certain steroids interact directly with the GABA-A receptor (GABA- steroids), as either allosteroic agonists or antagonists. Allopregnanolone and tetrahydrodeoxycorticosterone (5α-pregnan-3α,21-diol-20-one, or THDOC) display GABA agonistic features (Harrison et al., 1987; Majewska et al., 1986). Plasma and brain concentrations of the neurosteroids allopregnanolone and THDOC increase during stress, but this increase can be prevented by adrenalectomy. Allopregnanolone and THDOC potentiate the binding of the GABA-A receptor agonist muscimol and the benzodiazepine flunitrazepam and they allosterically inhibit the binding of the convulsant TBPS. Both neurosteroids also stimulate chloride uptake and currents in synaptoneurosomes and neurons (Majewska, 1990; Reddy, 2002; Schwartz et al., 1987). Positive GABA modulator steroids display different potencies in different regions of the brain, since the regionally variable subunit composition of the GABA-A receptor is reflected in the neurosteroid response (Jussofie, 1993b; Schwabe et al., 2005). The reduction of testosterone by 5α-reductase generates 5α-dihydrotestosterone, which is then converted to 3α-androstanediol (3α-Diol) by 3αHSD. 3α-androstanediol is also a powerful GABA-A receptor-modulating neurosteroid, with allopregnanolone-like properties (Frye et al., 1996; Martini et al., 1993; Reddy and Kulkarni, 2000). Pregnenolone sulphate (PS) and dehydroepiandrosterone sulphate (DHEAS) are also naturally occurring GABA-A active steroids (Paul and Purdy, 1992). PS exhibits mixed agonistic features, increasing muscimol binding at nanomolar concentrations and reducing it at micromolar concentrations, but it behaves as an allosteroic GABA-A receptor antagonist (Majewska et al., 1988). In pre-optic cells, PS has no effect by itself on GABA-A receptor activation by spontaneously released GABA but inhibits the allopregnanolone-induced chloride flux enhancement (Haage et al., 2005). PS can also activate the glutamate receptor and is therefore excitatory in two ways. The neurosteroid DHEAS reversibly blocks GABA-induced currents and behaves as an allosteroic GABA antagonist at the GABA-A receptor

(Majewska et al., 1990). The mechanism of the antagonistic neurosteroids is not completely clear. Neuroactive steroids, which are antagonists against GABA-A receptor steroid agonists, also exist. Studies show that 3β-pregnane steroids are non- competitive GABA antagonists (Maitra and Reynolds, 1998; Wang et al., 2002). The 3β-hydroxy isomer of allopregnanolone, isoallopregnanolone (3β-hydroxy-5α-pregnane-20-one, Fig. 1), however, is not active by itself at the GABA-A receptor with GABA concentrations of less than 15 μM (Wang et al., 2002). The action of isoallopregnanolone is dependent on GABA-A receptor activation (Lundgren et al., 2003; Wang et al., 2000). Isoallopregnanolone functions as an allopregnanolone antagonist at the GABA-A receptor in the brain (Lundgren et al., 2003; Wang et al., 2000) and with receptors expressed in the Xenopus Lavis oocytes (Wang et al., 2002). Isoallopregnanolone is produced in the brain in a similar way to allopregnanolone, with the exception that the 3βHSD enzyme is needed instead of the 3αHSD enzyme (Weir et al., 2004). In vitro, isoallopregnanolone selectively inhibits the allopregnanolone-induced chloride ion uptake into rat cortical tissue (Lundgren et al., 2003). Isoallopregnanolone is also able to block the allopregnanolone-induced inhibition of the population spike in hippocampus slices (Wang et al., 2000). The fact that a 3β-pregnane steroid also functions as an allopregnanolone antagonist in vivo was demonstrated for the first time in Paper I of this thesis. Recently, the in vivo inhibitory action of isoallopregnanolone was shown on the anaesthetic action of allopregnanolone (Backstrom et al., 2005). The neuroactive steroid epipregnanolone also has an in vivo inhibitory impact on the neuroactive steroid effects on operant ethanol self- administration in rats (O'Dell et al., 2005). Different results relating to the glucocorticoid effects on GABAergic neurons in the CNS have been reported (Morrow et al., 1990b; Woodbury, 1952). One study showed that corticosterone, cortisol, cortisone and their reduced metabolites at low nanomolar concentrations potentiate TBPS binding at the GABA-A receptor and slightly reduce it at high nanomolar and micromolar concentrations (Majewska, 1987; Majewska et al., 1985). Contrary to these results, studies in our laboratory showed that 3α-hydroxy 5α-reduced metabolites of both cortisol and cortisone reduce GABA- mediated chloride ion uptake by themselves. However, when interactions between allopregnanolone, THDOC and the glucocorticoid metabolites were studied, an enhancement of the allopregnanolone effect was noted (Stromberg et al., 2005).

1.2.4 Allopregnanolone interaction with the GABA-A receptor The neurosteroid allopregnanolone enhances the GABA effect on the GABA-A receptor. This can be observed at nanomolar concentrations of the neurosteroid. The threshold concentrations for enhancement is 10-30 nM, but at higher concentrations (μM) allopregnanolone may in neurons also directly open the GABA-A receptor chloride channel (Belelli and Lambert, 2005). However, neurosteroid interactions with the GABA-A receptor are not completely understood. It is suggested that the allopregnanolone enhancement of GABA at the GABA-A receptor results from an increase in opening frequency and burst duration (Macdonald and Olsen, 1994; Majewska, 1992). In clinical and other in vivo situations, the effects of allopregnanolone on the CNS via the GABA-A receptor can be divided into three categories: 1) direct effects on the receptor function; 2) induction of tolerance; and 3) abstinence (withdrawal) (Backstrom et al., 2003a). The direct effect of allopregnanolone on mood and behaviour displays a biphasic pattern. At high concentrations, allopregnanolone induces a general enhancement of the GABA-A receptor and thereby induces sedative, hypnotic and even anaesthetic effects in both human and animals (Carl et al., 1990; Norberg et al., 1987; Sundstrom and Backstrom, 1998). In certain individual, however, a low concentration of allopregnanolone induces the loss of impulse control, negative mood and aggression/irritability (Backstrom et al., 2003a; Miczek et al., 2003). The modulatory effect of allopregnanolone depends on hormonal variations (Reddy and Kulkarni, 1999). In addition, studies of recombinant GABA-A receptors provide evidence of a subunit selective heterogeneity of steroid sensitivity, in which the γ3 subunit increases the maximum efficacy of allopregnanolone compared with other γ subunit subtypes (Maitra and Reynolds, 1999). Another study also indicates that both the α and γ subunits are important determinants of allopregnanolone sensitivity (Maitra and Reynolds, 1998). The δ subunit predominantly co-assembles with the α4 and α6 subunits, the second of which is limited to cerebellar granule cells (Huh et al., 1996; Quirk et al., 1995). Disruption of the δ subunit produces a considerable attenuation of behavioural responses to neuroactive steroids but not to other neuromodulatory drugs (Mihalek et al., 1999). It has been shown that the expression of GABA-A receptor subunits changes during the estrus cycle. In late diestrus, the number of α4, β1 and δ subunit immunopositive cells increases in the midbrain of rats. Quick changes in the expression level of subunits at the beginning of the estrogen peak (late diestrus) also indicate that the expression of the GABA-A receptor can

change within a few hours (Landgren et al., 1998; Lovick et al., 2005; Reddy and Kulkarni, 1999). In the hippocampal CA1 region, for example, but not in the dentate gyrus region the effect of allopregnanolone changes with the estrus cycle (Landgren et al., 1998). Phosphorylation is suggested as an additional mechanism involved in the dynamic regulation of allopregnanolone interaction with the GABA-A receptors (Brussaard et al., 2000; Fancsik et al., 2000; Harney et al., 2003; Lambert et al., 2003). The outcome of GABA-A receptor phosphorylation depends on the nature of the kinase, the subunit composition of the receptor and the residues that are phosphorylated. In magnocellular oxytocin neurons of the hypothalamus, the promotion of phosphorylation suppresses the inhibitory allopregnanolone response, whereas, in the hippocampus, phosphorylation increases the interaction between allopregnanolone and the GABA-A receptor (Harney et al., 2003; Koksma et al., 2003).

1.3 CLINICAL CONDITIONS RELATED TO THE EFFECT OF SEX HORMONES IN THE FEMALE BRAIN The sex steroid hormones act on neuronal and glial cells, modulating neurotransmitter synthesis, neurotransmitter receptor expression and synaptic transmission (Compagnone and Mellon, 2000). The sex steroids may therefore exert clinically significant effects on the central nervous system. For example, the menstrual cycle influences the occurrence of seizures in some female epileptics and the performance of certain cognitive tests (Backstrom, 1976; Postma et al., 1999; Reddy, 2004). However, the appearance of mood and behavioural symptoms during the menstrual cycle, oral contraceptive usage, hormone replacement therapy, pregnancy and postpartum has been extensively reported. The mechanisms underlying these symptoms are not clear.

Pre-Menstrual Dysphoric Disorder (PMDD): one of the most obvious interactions between sex steroid hormones and the CNS is found in the premenstrual syndrome (PMS). The term “premenstrual dysphoric disorder” is used for a more severe condition, when the psychological symptoms are so disruptive that they impair the function of the woman and there is a need for treatment (AmericanPsychiatricAssociation., 1994). PMDD is a psychoneuroendocrine disorder characterised by cyclical changes in physical, psychological and behavioural symptoms that are related to the formation of the corpus luteum and fluctuations in gonadal hormones (Backstrom et al., 2003b; Sundstrom Poromaa et al., 2003). The etiology of

PMS is not yet clearly known, but the ovarian steroids, as well as changes in GABAergic and serotoninergic functions, are involved (AC et al., 2005; Backstrom et al., 1983; Halbreich, 1990; Hammarback et al., 1985; Hammarback et al., 1991; Sundstrom Poromaa et al., 2003). In one study, different estrogen and progesterone levels and a lower luteal phase allopregnanolone concentration have been shown and the possibility of the defective synthesis of GABA agonist steroids in PMDD was put forward (Monteleone et al., 2000). The majority of studies, including those from our laboratory, show that pregnanolone, PS, 5α-DHP, allopregnanolone and estradiol plasma levels, in the luteal phase of PMS patients and control subjects, are similar. However, within individuals, there is a relationship between symptom severity and the serum concentrations of estradiol, allopregnanolone and PS (Wang et al., 1996). PMDD patients during the luteal phase less sensitive to the effects of neurosteroids, benzodiazepines and alcohol, and show reduced GABA-A receptor sensitivity to these substances in the CNS (Nyberg et al., 2004; Sundstrom et al., 1997; Sundstrom and Backstrom, 1998). In rats, it has been shown that short-term exposure to allopregnanolone reduces the sensitivity to benzodiazepines, with changes in the composition of the GABA-A receptor and in association with increased anxiety (Gulinello et al., 2001). Similar changes are suggested as the basic mechanism behind PMDD.

Pregnancy and postpartum mood disorders: pregnancy results in marked alterations in both the psychology and the physiology of women. Memory impairment, cognitive changes and mood changes during pregnancy and in the postpartum period have been documented (Davey, 1995). Estrogen increases 1,000 times during pregnancy and progesterone levels also rise compared with the level before conception (Brett and Baxendale, 2001; Casey, 1993). Allopregnanolone is the progesterone metabolite that mainly increases during pregnancy (Morrow et al., 1987; Paul and Purdy, 1992). Chronic allopregnanolone treatment results in reduced receptor responses to this neurosteroid (Yu et al., 1996b). If this down-regulation occurs during pregnancy, the rapid postpartum fall in progesterone metabolites will increase the excitability (hormone withdrawal effect), due to the down- regulated receptor. This could be an explanation for postpartum mood disorders and increased seizure attacks during labour and delivery among women with epilepsy (Tomson, 1997; Yu et al., 1996a; Yu et al., 1996b). The memory deficit might be related to allopregnanolone rather than to progesterone itself (Brett and Baxendale, 2001; Johansson et al., 2002). It has also been suggested that hormone-related phenomena such as PMDD are

related to the occurrence of postpartum mood disorders (Bloch et al., 2005). The etiology of postpartum mood disorders might then be related to differential neurosteroid sensitivity.

Oral contraceptive usage: negative mood symptoms are well-known side- effects of oral contraceptives (OC) in women (Rosenberg and Waugh, 1998). Women who experience adverse mood symptoms during OC usage appear to be the same women as those with PMDD (Cullberg, 1972). It appears that women with mild PMDD might benefit from OC, whereas women with severe PMDD develop more negative mood symptoms as a result of the treatment (Backstrom, 1996). This might be caused by a different brain sensitivity to ovarian hormones, in which the GABA-A receptor is less sensitive in women with PMDD compared with controls (Sundstrom Poromaa et al., 2003). It has also been shown that long-term daily administration of OC induces a persistent and marked decrease in the synthesis and accumulation of progesterone and the metabolite allopregnanolone, and at least in the rats changes the GABA-A receptor composition in the brain (Follesa et al., 2001).

Hormone Replacement Therapy (HRT): negative mood changes are clinically well-known side effects of combined HRT (estrogen + progesterone) and constitute a major problem in relation to compliance. Women on estrogen-only HRT do not develop negative mood changes (Hammarback et al., 1985). All progestagens (hormones with a similar effect to that of progesterone) appear to be able to induce negative mood changes (Backstrom, 1996). In fact, various studies of the effect of estrogen hormone therapy have shown significant improvements in mood in postmenopausal women (Montgomery et al., 1987; Rasgon et al., 2002). In contrast, negative mood symptoms begin shortly after adding progesterone to the treatment and continue to rise in severity until the progesterone treatment has been ended (Bjorn et al., 2000; Hammarback et al., 1985; Panay and Studd, 1997). In postmenopausal women with a previous PMDD history, the lower dose of progestagen increased negative mood symptoms (Andreen et al., 2003; Bjorn et al., 2002). Menopausal women with higher pre-treatment serum estradiol levels reported more negative effects on mood than women with low estrogen levels when given a combination of estrogen-progesterone (Klaiber et al., 1997). On the other hand, another study proposed that, together with the progesterone, a higher dose of estrogen caused significantly more negative mood symptoms than a lower dose (Bjorn et al., 2003). Allopregnanolone is the main metabolite of progesterone and the

chronic administration of allopregnanolone results in the down-regulation of the GABA-A receptor. In addition, these receptors become less sensitive to neurosteroids (Yu et al., 1996a). As a result, in HRT, reduced sensitivity to neurosteroids caused by repeated exposure to progestagen is suggested as a possible mechanism underlying negative mood changes (Andreen et al., 2005; Bjorn et al., 2002). However, the previously held belief that estrogen has a positive effect on memory function and performance has not been confirmed in recent controlled studies. The results of the Women’s Health Initiative Memory Study (WHIMS) revealed that estrogen therapy alone did not reduce dementia or the incidence of mild cognitive impairment; on the contrary, there was an increased risk from estrogen plus progestin therapies (Shumaker et al., 2004; Shumaker et al., 2003).

1.4 TOLERANCE It is known that regular drug users and alcohol drinkers generally become able to tolerate larger amounts of drug or ethanol after repeated exposure. Pharmacologically, tolerance is defined as a decrease over time in the ability of the drug to produce the same degree of pharmacological effect. Tolerance is classified with respect to: 1) possible mechanisms (pharmacodynamic, or pharmacokinetic) and 2) rate of development (acute, chronic, or rapid) (Kalant et al., 1971). Pharmacodynamic tolerance (functional tolerance, or receptor site tolerance) includes reductions in the sensitivity of the target tissue to the same degree of drug exposure. This type of tolerance usually tends to reduce the clinical drug effects, as the duration of drug exposure proceeds and behavioural tolerance is generally of this type. On the other hand, pharmacokinetic tolerance (dispositional or metabolic tolerance) comprises changes in drug absorption, distribution, excretion and metabolism, which lead to reductions in the concentration and duration of drugs in the target tissues. The term usually describes the phenomenon of increased drug clearance. In acute tolerance, there is a reduction in sensitivity to a drug within the duration of a single exposure to the drug (intra-sessional), while, in chronic tolerance, a decrease in sensitivity to a drug occurs after multiple exposures to the drug (inter-sessional). “Rapid tolerance” is a term suggested by Kalant and colleagues and is described as a decrease in sensitivity to a drug after the duration of a single exposure to that drug (Kalant, 1993). A distinction is also made between tolerance to a specific drug, to related drugs (selective or homologous cross-tolerance), or to drugs from unrelated drug classes

(heterologous cross-tolerance) (Greenblatt and Shader, 1978; Kalant et al., 1971).

1.4.1 The GABA-A receptor and tolerance The role of the GABA-A receptors in the development of tolerance to positive modulator substances is mostly investigated using benzodiazepines (BZPs) and ethanol. Both substances produce effects by interacting with the GABA-A receptors. BZPs are widely prescribed for the treatment of disorders, such as anxiety and insomnia, but tolerance to their effects develops rapidly. After a long period of BZP usage, withdrawal symptoms can also appear following the cessation of the intake of the drug (Harris et al., 1992; Roy-Byrne, 2005). Withdrawal symptoms take place when there is a decline in the blood and tissue concentration of BZP or another dependence-forming substance to which an individual has been continuously exposed. Symptoms of BZP withdrawal are time limited (1-2 weeks), but the duration varies according to the drug and the individual (Roy-Byrne, 2005). The first report of acute tolerance was in relation to ethanol (Mellanby, 1919). Acute tolerance development has also been reported for many other GABA modulatory substances, such as barbiturates and BZPs in both humans and animals, and for the neurosteroid allopregnanolone in rats (Bolander and Wahlstrom, 1988; Dripps et al., 1956; Ihmsen et al., 2004; Kroboth et al., 1993; Zhu et al., 2004). The time needed for tolerance to develop varies. One day of continuous intracerebroventricular pentobarbital exposure reduces the duration of the loss of righting reflex, but tolerance to the hypothermic effects of thiopental and barbital progressed after seven days (Saunders et al., 1990). Increased sensitivity to pentylenetetrazol-induced seizures was first observed after three days of pentobarbital exposure. The same study proposed that the acute effects of barbiturates on the GABA-A receptor are reversible and the pharmacokinetic and pharmacodynamic tolerances are separable (Saunders et al., 1990). It is well established that, following chronic exposure to benzodiazepines and alcohol, there are changes in GABAergic neurotransmission. These changes contribute to the symptoms of tolerance, dependence and withdrawal. However, the nature and mechanism of these changes are not clear (Allison and Pratt, 2003; Kan et al., 2004; O'Brien C, 2005). The treatment of cortical neurons in cultures with benzodiazepines results in GABA-A receptor down-regulation. Although the exact molecular mechanisms responsible for the changes in GABA-A receptor function induced by the positive modulator exposure remain unclear, post-

translational modifications, receptor trafficking, effects on receptor density and changes in subunit expression have been shown (Devaud et al., 1997; Grobin et al., 1998; Kumar et al., 2002). GABA-A receptor down-regulation is supposed to take place in the following order; desensitisation (tachyphylaxis) that is characterised by decreased receptor currents during continuous GABA exposure, the sequestration (endocytosis) of subunit polypeptides and the uncoupling of allosteric interactions between the GABA and benzodiazepine binding sites, subunit polypeptide degradation and the repression of subunit gene expression (Barnes, 1996). Consequently, the end point of these changes can be observed as changes in GABA-A receptor subunit mRNA expression and finally BZP tolerant condition.

1.4.2 Changes in the GABA-A receptor during tolerance Chronic treatment with positive allosteroic modulators that act at different sites of the GABA-A receptor results in changes in the sensitivity and expression of the receptor subunit mRNA (Holt et al., 1996; Mhatre and Ticku, 1992; Morrow et al., 1990a). In rats, seven days of flunitrazepam treatment induces tolerance and quantitative in situ hybridisation showed that the α1 and β3 subunit mRNAs are decreased, while the β2 subunit mRNA increased in the hippocampus of the tolerant animals (Tietz et al., 1999). In another study, seven days of diazepam treatment increased the α4, β1 and γ3 subunit mRNA levels and these changes are sustained at 14 days’ treatment, along with increases in α3 and α5 and a decrease in γ2 subunit mRNA (Holt et al., 1996). The treatment of rats with diazepam continuously (s.c.) for three weeks reduced the α1 subunit mRNA level in the hippocampus but not in the cortex or cerebellum. The α5 subunit mRNA level was reduced in the cerebral cortex and hippocampus, while the γ2 subunit mRNA level only decreased in the cortex (Wu et al., 1994). The acute sedative effect of diazepam is mediated via the GABA-A receptor α1 subunit, while it is suggested that tolerance to the sedative action of diazepam is associated with the decreased α5 subunit level in the hippocampal dentate gyrus (van Rijnsoever et al., 2004). These studies revealed that, for the induction of tolerance, the chronic activation of the GABA-A receptors by diazepam is sufficient and changes in subunit mRNAs could possibly account for the mechanism of tolerance development. However, tolerance develops to some, but not all, behavioural actions of BZPs. As mentioned above, tolerance to the anxiolytic action of BZP can develop, but little or no tolerance develops to the anterograde amnesia after chronic treatment in humans or animals (Ghoneim et al., 1981; Hughes et al., 1984). In rats, it has been shown that tolerance can develop to

the amnesic effect of diazepam on spatial learning using the Morris Water Maze (MWM) test. The authors revealed that the amnesic effect of diazepam is not secondary to sedation and that the amnesic and sedative effects are independent factors (McNamara and Skelton, 1997). The development of ethanol tolerance and dependence is associated with alterations in many properties of the GABA-A receptors throughout the brain and changes the expression of distinct GABA-A receptor subunit mRNAs and peptides in various brain regions. The levels of mRNA for the GABA-A receptor α1, α2, and α3 subunits are reduced in the cerebral cortex, while the α4, β1, β2, β3, and γ1 subunit peptide and mRNA levels are increased in the cerebral cortex following chronic ethanol exposure (Devaud et al., 1997; Mahmoudi et al., 1997; Matthews et al., 1998; Mhatre and Ticku, 1992). Phosphorylation is also proposed as a mechanism that underlies tolerance development. Although the molecular mechanism is not understood, it is clear that the interaction of positive modulator substances with the GABA-A receptor can be regulated by the relative activity of kinases and phosphatases (Brussaard and Koksma, 2002; Koksma et al., 2003; Song and Messing, 2005).

1.4.3 Tolerance to allopregnanolone Repeated exposure to allopregnanolone results in the development of tolerance to the anticonvulsant effect but not to the somnogenic effect (Czlonkowska et al., 2001; Damianisch et al., 2001). Another study proposed genotype-dependent differences in the mechanism of allopregnanolone tolerance (Palmer et al., 2002). Long-term exposure of cultured cortical neurons to allopregnanolone produces a down-regulation of the GABA and TBPS binding sites and the uncoupling of the GABA-A receptor complex (Yu and Ticku, 1995b). This is associated with a reduction in the efficacy of the GABA-induced chloride ion influx and reduced efficacy of other positive modulators. In these studies, chronic allopregnanolone treatment reduced the α2, and α3, β2 and β3 subunit mRNA levels but did not alter the γ2 subunit mRNA level in cortical neurons (Yu et al., 1996a; Yu et al., 1996b; Yu and Ticku, 1995a; 1995b). Short-term exposure to allopregnanolone and the discontinuation of neurosteroid after long-term exposure (sudden decrease in the neurosteroid level) results in the increased expression of the GABA-A receptor α4 subunit mRNA (Follesa et al., 2000; Gulinello et al., 2001). The increased expression of the α4 subunit correlates with increased anxiety and the reduced sensitivity of the GABA-A receptors to classical benzodiazepine

agonists and to zolpidem, as well as a distinct effect by flumazenil and other positive and negative modulators (Barnard et al., 1998; Gulinello et al., 2001). Our laboratory has used electroencephalography (EEG) to show that acute allopregnanolone tolerance can develop after just 60 minutes of i.v infusion. Using this threshold method, tolerance was accompanied by increased allopregnanolone concentrations in the hippocampus and the brain stem (Zhu et al., 2004). To the best of our knowledge, there are no earlier studies of GABA-A receptor subunit mRNA expression in acute tolerance to allopregnanolone or any other GABA-A receptor active substance.

1.5 LEARNING AND MEMORY Learning is defined as the acquisition of information or skills, while memory refers to the process of recalling what has been learnt (Gazzaniga et al., 2002). Learning is subdivided into several types, such as simple learning (perceptual learning), associative learning (conditioned learning or stimulus- response learning) and more complex learning such as spatial learning (Gazzaniga et al., 2002). Different brain systems are involved in different types of learning and memory. In rodents, lesions of the hippocampus impair the learning of spatial tasks, in addition to many other abilities, e.g. a subset of configural tasks and working memory tasks (O'Keefe and Nadal, 1978; Olton and Papas, 1979; Scoville and Milner, 1957). In women, cognitive disturbances are reported when a high progesterone level is present in the brain (Brett and Baxendale, 2001). It is also known that progesterone has potent anaesthetic properties and a dampening effect on brain excitability (Backstrom et al., 1984). These rapid negative effects of progesterone are mediated by the neuroactive metabolite allopregnanolone (Paul and Purdy, 1992). As a result, the primary action of progesterone on memory and learning may be due to its metabolite allopregnanolone, which has a highly specific action and increases neuronal inhibition via the GABA-A receptor.

1.5.1 Spatial learning and memory Spatial learning is a behavioural task which requires the animal or person to use multiple pieces of information simultaneously. The hippocampus, striatum, basal forebrain, cerebellum and neocortex are brain regions that are known to be involved in this learning (Cain and Saucier, 1996). The Morris Water Maze (MWM) is one of the most frequently used tests for studies of spatial learning; it takes advantage of the fact that rats are good swimmers

(Cain and Saucier, 1996; Morris, 1984). The technique of this test is described in detail in the method part of the current thesis. Several neurotransmitters and receptor systems are proposed in the learning and memory mechanisms, with the percentage of significance calculated as follows: glutamatergic (93%), GABA (81%), dopamine (81%), acetylcholine (81%), serotonin (55%) and nor-epinephrine (48%) (Myhrer, 2003). GABA is suggested as a critical neurotransmitter with an impact on performance in the water maze task (Myhrer, 2003). GABA, opioids and biogenic amines are thought to be either harmful or not to have any effect on this kind of learning (McNamara and Skelton, 1993; Myhrer, 2003).

1.5.2 The GABA-A receptor modulators and spatial learning and memory GABA-A antagonistic steroids have been found to enhance learning and memory processes, while GABA-A agonistic steroids are most often found to impair these processes (Darnaudery et al., 2000; Johansson et al., 2002; Mayo et al., 1993; Reddy and Kulkarni, 1998) GABA-A receptor active substances, like benzodiazepines (BZP), are widely used as sedative, anxiolytic and anticonvulsant drugs, but unwanted memory deficits frequently occur during treatment. These effects are mediated by the activation of the GABA-A receptor, e.g. in the hippocampus, and the enhancement of inhibitory GABAergic transmission (Barker et al., 2004; Macdonald and Barker, 1978; McNamara et al., 1993). The stimulation of GABA-A receptors with BZPs reduces the neuronal firing of glutamate neurons, which correlates with the spatial memory impairment induced by BZPs (Riedel, 1996). BZP induces GABA-A receptor-mediated synaptic inhibition and affects the NMDA receptors and Ca+ influx. In other words, an interactive mechanism may conduct and regulate the effect of BZPs on spatial learning, by the concurrent affection of the NMDA and glutamate receptors (McNamara et al., 1993). It is known that the excitability of the hippocampal neurons is influenced by powerful cholinergic inputs that in turn have an effect on cognition. Allopregnanolone participates in the GABAergic modulation of the central cholinergic function, (Landgren et al., 1998) and inhibits basal acetylcholine release from the prefrontal cortex and hippocampus, but not from the striatum, in a dose-dependent manner (Dazzi et al., 1996). Gonadal hormones influence the brain organisation and a variety of reproductive or non-reproductive behaviours, including some cognitive abilities (Williams and Meck, 1991). Dendritic spines are believed to be important sites for the enhancement of synaptic efficacy and networks involved in learning and neural plasticity (Hering and Sheng, 2001). The

dendritic spine density in the hippocampus declines slowly as estrogen levels fall, a decline that is much more rapid if progesterone is administered (Woolley and McEwen, 1993). This rapid down-regulation of dendritic spines is probably due to the action of progesterone at the progesterone receptor (PR), as this effect can be inhibited by the progesterone antagonist RU 38486 that exerts its effect on PR (Woolley and McEwen, 1993). There is controversy about the effect of allopregnanolone on learning and memory, but in the majority of studies allopregnanolone impairs spatial learning and memory in animals. It is even possible to observe a progressive memory impairment, a few minutes after an injection of allopregnanolone, due to a robust increase in allopregnanolone levels in both the brain and plasma of rats (Freeman et al., 1993; Johansson et al., 2002; Ladurelle et al., 2000; Paul and Purdy, 1992). The mechanism of the effect of allopregnanolone on memory and learning is not yet clear. Apparently, however, this neuroactive steroid act as a positive allosteric modulator of the GABA-A receptor and, like BZPs, increases the frequency and duration of the GABA-gated chloride channel opening. The dose-dependent potentiation of the GABA-evoked Cl¯ currents is expressed as an inhibitory effect on neurons (Paul and Purdy, 1992), especially in the hippocampus, one of the major brain areas involved in learning (Landgren et al., 1998). In rodents, the activity of individual hippocampal pyramidal cells is correlated with the animals’ location in the environment (called place cells) (Muller et al., 1987; O'Keefe and Dostrovsky, 1971). The application of allopregnanolone to the hippocampus prolongs the GABA-mediated inhibitory postsynaptic currents from hippocampal neurons, altering the hippocampal neurophysiology with the inhibition of hippocampal pyramidal neurons (Harrison et al., 1987; Landgren et al., 1998; Tokunaga et al., 2003). Acute allopregnanolone administration to rats, and consequent changes in hippocampal neurobiology, selectively impairs the use of spatial reference memory, while saving the non-spatial memory (Matthews et al., 2002). The selectivity of allopregnanolone in relation to spatial memory tasks is similar to the deficits found following acute ethanol administration (Matthews and Morrow, 2000).

2. AIMS

1. To test whether a 3β-hydroxy pregnane steroid can block the negative effects of allopregnanolone on learning in the Morris Water Maze test (Paper I).

2. To study the mechanism of the 3β-hydroxy pregnane steroid antagonism on the negative effects of allopregnanolone on learning (Paper I).

3. To evaluate whether chronic tolerance develops to the negative effects of allopregnanolone on learning (Paper II).

4. To study the mechanism behind acute allopregnanolone tolerance, by quantifying GABA-A receptor subunit mRNA expression in different brain areas (Paper III).

5. To evaluate whether acute tolerance induced by allopregnanolone anaesthesia persists after the end of treatment and whether persistence is related to changes in GABA-A subunit mRNA expression (Paper IV).

3. METHODS

3.1 In situ hybridisation This technique can be used to determine the location and for quantification of the expression level of specific RNA or DNA in tissues, cells or chromosomes using specific labelled nucleic acid probes. The nucleic acid hybridisation techniques are based on the fact that, in appropriate conditions, complementary sequences of single-stranded nucleic acid species spontaneously anneal to form a double-stranded hybrid (duplex). To study the distribution or level of expression of specific genes or gene transcripts, isotopic or non-isotopic markers (enzymatic or photo-chemical) can be incorporated into the probe (Wilkinson, 1995). The sensitivity of in situ hybridisation exceeds that of Southern and Northern blotting techniques, as the target mRNA is then diluted by the relatively large-scale homogenisation of tissue. With in situ hybridisation, mRNA species present at low levels and expressed in few cells can be analysed. In our studies, oligonucleotide probes labelled with the 35S-isotope were used for the determination of the GABA-A receptor α1, α2, α4, α5, α6, β2, γ2(L+S) and δ subunit mRNA expression (Wisden et al., 1992). Denatured oligonucleotide probes for the subunit in 100 μl of hybridisation mixture were used on each slide containing three consecutive fixated sections of half the brain from the same animal. Slides were hybridised over night, followed by washes, and they were then exposed to Hyperfilm Biomax MR-1 film for two to 12 days. The slides were then dipped in Kodak NTB2 photoemulsion, exposed at +4ºC for 0.5 to six months, after which sections were counterstained with Pyronin-Y. Using a special image analysing system (MCID M4 analysing system- Imaging Research Inc. Ontario, Canada), we counted the silver grains in fixed microscopic areas. Counting was performed in blind conditions.

3.2 Immunohistochemistry This technique is a combination of microscopy and immunology, in which an antibody with very specific binding for its antigen is used and the reaction site can be detected by attaching a specific probe to the antigen-antibody complex. Using this technique, antigens in any position within the cell can be identified. There are numerous immunohistochemical techniques that can be used to localise antigens. The selection of a suitable technique is based on parameters such as the type of specimen under investigation and the degree of sensitivity required (Beesley, 1993).

We used a Streptoavidin-Biotin immunohistochemistry method for the GABA-A receptor α4 subunit protein (Paper III). Sections were fixated and blocked with normal swine serum. This was followed by incubation with the primary antibody (1:20 goat anti rat α4) for two hours. Subsequently, slides were incubated with secondary antibody (swine anti-goat-biotinylated substance), followed by avidin/HRP. Sections were then incubated with diaminobenzidine/H2O2 and counterstained with Meyer’s hematoxylin. Usual problems, such as unspecific labelling (background), too weak signals (method problem) and wrong results (specificity) (Beesley, 1993), were overcome by changing the type of blocking substance and incubation time with the primary antibody. Using the x40 magnification lens of the light microscope (BX51M- Olympus, Japan), positively stained cells containing the α4 subunit were counted in three distinct random fields of the ventral posteriomedial nucleus region of the thalamus in each of the three sections. The average number of positive cells per slide (animal) was calculated by dividing the sum of cells by the number of areas that were analysed.

3.3 Chloride ion uptake assay Allopregnanolone potentiates the GABA-induced chloride ion influx through the GABA-A receptor (Majewska et al., 1986). The chloride ion uptake assay can be used to determine the GABA-A receptor function and modulation by drugs. Briefly, in this study (Paper 1), microsacs were prepared by the homogenisation of cortical or hippocampal tissue + buffer in a glass-Teflon homogeniser, filtration through a nylon mesh and purification by centrifugation (as described by Lundgren et al., 2003). A Skatron (Lier, Norway) twelve-channel cell harvester and 96-well microplates were used to perform the chloride ion uptake assays. Wells were filled by adding steroids, GABA, buffer and [36Cl-]. By adding homogenate to the wells, the up-take reaction was started and, after five seconds, the reaction was terminated by washes with buffer containing picrotoxin. After rapid filtration of the well content through a fibreglass filter, captured [36Cl-] was measured with a liquid scintillation spectroscope. Each chloride influx test was performed in quadruplicate determinations. The data are expressed as net chloride ion uptake (i.e. minus the basal uptake in the homogenate).

3.4 EEG threshold method In Papers III and IV, acute allopregnanolone tolerance and changes in CNS sensitivity to allopregnanolone were evaluated with the electroencephalographic (EEG) burst suppression threshold test. This

method is constituted on the induction of a burst suppression of one second or more in the EEG (silent second, SS) by the i.v. infusion of an anaesthetic drug. The dose needed to induce the criterion of a “silent second” (SS) is defined as the threshold dose of the anaesthetic (Fig. 3).

After 1-2 min infusion

Gradually decreased frequency

First Silent Second after 3-5 min

Figure 3: The EEG of a rat before and during continuous infusion of an anaesthetic drug (adapted from (Korkmaz and Wahlstrom, 1997).

The period of burst suppressions (including SS) is a reversible CNS depression that is induced by the test drug. Factors like the chemical properties of the drug and vehicle (e.g. water solubility) and age of the animals affect the results of the test. To minimise the infusion volume and to follow the time, an infusion volume rate is calculated. The optimal infusion rate must be used, i.e. the rate at which the lowest dose is needed to obtain an SS; for allopregnanolone, the dose rate is 4 mg/kg/min (Zhu et al., 2001). In addition, some CNS depressant drugs (e.g. ethanol) are not able to induce SS and some drugs (e.g. barbiturates) may induce convulsive-like episodes during the excitatory phase (inhibition of the CNS inhibition) of the induction (Wahlstrom and Norberg, 1984). The EEG was recorded from subcutaneous stainless steel sutures placed in a bifrontal configuration (Korkmaz and Wahlstrom, 1997).

In this thesis (Papers III and IV), allopregnanolone was infused i.v. into one of the tail veins with a syringe pump, until the first SS was recorded in the EEG. When an SS was recorded, the infusion was immediately stopped, while the EEG recording continued. Immediately after the first SS detection, the control animals were decapitated. In the other rats, the next infusion of allopregnanolone started when no SS had been recorded for a period of one minute. The method of stopping and re-starting the infusion continued for a fixed time (30 and 90 min) and was then ended at an SS and the rats were immediately decapitated. The allopregnanolone doses needed to maintain anaesthesia over time (maintenance dose rate, MDR, mg/kg/min) were calculated from the accumulated doses during three different intervals of 20 min. The three intervals were 10-30 minutes (MDR1), 40-60 minutes (MDR2) and 70-90 minutes (MDR3). The calculated individual difference between MDR3 and MDR1 (ΔMDR) was used as an in vivo measure of acute tolerance. In Paper IV, rats in two groups were allowed to recover after the 90 minutes of anaesthesia and, one or two days later, the rats were decapitated immediately after a new SS induction with allopregnanolone.

3.5 Morris Water Maze test The Morris Water Maze test was developed by Richard Morris (Morris, 1984) and is one of the most frequently used tests for evaluating the capacity of rodents to learn and retrieve spatially encoded information. In humans, a computerised (virtual) version of the Morris Water Maze (VMWM) test is used for the laboratory testing of spatial ability (Astur et al., 1998; Hamilton et al., 2002). In animals, the Morris Water Maze test can be used in two different spatial tasks; a place-learning task that requires reference memory (remembering the location of the platform from the previous day) and a matching-to-place task that requires both reference memory and working memory (learning and remembering the new location) (Whishaw, 1985). In our studies, we used a place-learning version of the test. Technically, water provides the in-maze environment during the process and escape from water is used for the motivation of learning. Two advantages of the MWM test over other mazes are 1) the rat wants to escape so it searches and 2) in water, there are no local cues (e.g. scent trails). The task requires that the rat swims in a pool until it finds and climbs onto a small platform which is hidden, slightly submerged in the water. In the place-learning version, the platform place is fixed, whereas the platform place in the matching-to-place task is moved to a new location each day (Whishaw, 1985). The animal is placed in the pool at various points near the wall and,

to successfully complete the performance the animal must learn the exact location of the hidden platform in relation to the visual extra-maze cues in the room. The spatial memory is measured on a probe trial, in which the platform is removed. Animals that have learnt the position of the platform will spend more time in the area of the pool where the platform was previously placed. The MWM test with a visible platform can also be used for simple (stimulus-response) learning and the examination of visual sharpness (Gerlai, 2001). In Papers I and II, the water maze was a round black pool that was filled with clear tap water. The circular escape platform made of transparent Plexiglas was placed in the middle of the north-east quadrant. To reduce the stress effect of the test, we daily handled and habituated the rats for five days, until two days before the experiment. This included the same procedures as in the experiment, except for injections. Studies of place navigation with a constant location of the hidden platform were performed. Animals received allopregnanolone or a vehicle injection eight minutes before the test. They were then given four trials every day from four different starting positions, which were varied randomly from day to day but were the same for all the rats every day. The training took place for five or six days. The trial numbers per day (four) was optimal, as the effect of allopregnanolone in the brain is short lasting, because of a short half-life, and more trials in one day would have affected the results as the allopregnanolone level would have diminished. We already knew that the allopregnanolone concentrations in the brain tissue at eight minutes were 1.5 to 2.5 times higher than at 20 minutes after the allopregnanolone injections in the MWM test (Johansson et al., 2002). In each trial, the rats had a maximum of 120 s to search for the platform. Rats that did not find the platform were guided there by hand. All the rats were allowed to sit on the platform for 30 s and were then picked up and dried with a towel for 30 s, until the start of the next trial. The performance of rats was evaluated with an overhead camera connected to an image analyser (HVS imaging, Hampton, UK) with the water maze software program HVS Water 2020. The parameters latency, path length to reach the platform, swimming speed and thigmotaxis are prominent factors for evaluating the learning condition and the motor ability in the MWM test. Latency and path length were used as representatives of learning, while swimming speed was used as an index of changes in spontaneous locomotor activity and sedation (Gallagher et al., 1993; Lindner, 1997; Morris, 1984). It has been suggested that high thigmotaxis shows a low ability to engage in the procedural part of the

navigational strategies in the MWM, but it can also be seen as a sign of fear and anxiety (Devan et al., 1999; Simon et al., 1994). In Paper II, to test spatial memory in rats, a probe test was carried out one day after the last training session (day 6), without any injections before the test. The rats swam for 60 s with no platform present and the time spent in the different quadrants was analysed.

3.6 Hormone assays Allopregnanolone in serum (Paper III) or plasma (Paper IV) was extracted with diethyl ether and the extracts then evaporated to dryness under nitrogen gas. Neurosteroids in tissues were extracted by ethanol (99.5%) for seven days at +4ºC. The recovery of steroids from the tissue using this procedure has previously been shown to be 100% (Bixo et al., 1984). Plasma and serum samples were analysed in triplicate and brain samples were analysed in duplicate. Allopregnanolone in extracts was purified by either preparative high performance liquid chromatography (HPLC) (Papers I and IV) or celite chromatography (CC) (Paper III) and measured by radioimmunoassay (RIA). The allopregnanolone antiserum used for RIA analyses was raised against 3α-hydroxy-20-oxo-5α-pregnane-11-yl-carboxymethyl ether coupled to bovine serum albumin. The sensitivity of the assay was 25 pg, the intra- assay coefficient of variation 7% and the inter-assay coefficient of variation 8% (Purdy et al., 1990a). Evaluations of the repeatability, reproducibility and sensitivity of the HPLC purification were tested using aliquots spikes with a known amount of steroid, 100, 200 and 300 pg (0.03, 0.06 and 0.12 pmol). The recovery of steroid after the HPLC separation was 79% for brain tissue and 72% for plasma, while the recovery was 85% for brain tissue and 78% for plasma with CC. The test samples were therefore corrected for this loss. Our comparison of the two methods revealed that the allopregnanolone concentration that was obtained was the same with both chromatographic methods (unpublished data).

3.7 Statistical analysis Repeated measures analysis of variance (ANOVA) was used when the data from individual rats were obtained several times. With significant results (p≤0.05), the Least Significant Differences (LSD) post-hoc test was used. In the MWM test, the factors were days by treatment (groups). Group parameters found to be significantly different (ANOVA+LSD, p≤0.05) were further analysed two by two with the Mann-Whitney U-test to identify the days on which the groups were significantly different. The dose-response

data were fitted to the sigmoidal dose-response equation: Y = Minimal response + (Maximum response – Minimal response)/(1+10^((LogEC50- X)*HillSlope). The independent factors were the concentration of enhancing substance (GABA, allopregnanolone) and the concentration of antagonising substance (0–30 µM UC1011) when the chloride ion uptake parameters were evaluated. In the EEG-threshold studies, the between-subject factors were groups and the time of onset of anaesthesia (i.e. am, pm and mid-time in Paper IV). To compare the mean value of a variable with independent observations (between groups), we used one-way ANOVA (number of groups >2) or Student’s t-test (number of groups =2), e.g. MDRs, ΔMDR, SSs and ΔSS. However, to determine how responses were affected by the two independent factors treatment and time of onset of anaesthesia, we used two-way ANOVA. Linear regression coefficients (b) and parametric correlation coefficients (r) were used to evaluate simple statistical relationships in the data set. For Paper III, the chi-square test was used to test significance in frequency tables. The significance level used was p ≤ 0.05. All the values in the text and tables are displayed as means ± SEM. The SPSS statistical package versions 10.0 and 11.0 were used.

4. RESULTS AND DISCUSSIONS

The current thesis investigated changes in the sensitivity (tolerance) and plasticity of the GABA-A receptors and the opportunity to inhibit negative behavioural effects induced by allopregnanolone.

4.1 Antagonism of an allopregnanolone effect, Paper I To evaluate the 3β-hydroxypregnane steroid UC1011 (3β-20β-dihydroxy- 5α-pregnane) as a blocker of allopregnanolone-induced effects, both the in vivo MWM test of learning and the in vitro chloride ion uptake into cortical and hippocampal membrane preparations (microsacs) were used. The rats were injected every day either with UC1011 20 mg/kg, allopregnanolone 2 mg/kg, allopregnanolone: UC1011 2:6, 2:8 and 2:20 mg/kg, or with vehicle (10% 2-hydroxypropyl-β-cyclodextrin). As the MWM test is hippocampus dependent, we analysed the effect of UC1011 with both cortex- and hippocampus-derived membranes. The increase in chloride ion uptake by the cortical and hippocampal microsacs induced by allopregnanolone was antagonised by the UC1011 dosages of 10 and 30 µM. UC1011 in a concentration of 3-30 µM did not display any significant inhibitory effect on the chloride ion uptake induced by 10 µM GABA alone.

A B 220 200 1000 nM Allo 180 190 160 160 1000 nM Allo uptake uptake -

- 140 Cl

Cl 150 nM Allo (% of control) 130 (% of control) 120

100 100 0 1 3 10 30 Max 0 1 3 10 30 Max [UC1011] (µM) [UC1011] (µM)

Figure 4: The antagonistic effect of UC1011 on two concentrations of allopregnanolone in cortical microsacs (A) and of one concentration of allopregnanolone in hippocampal microsacs (B). The inhibition of allopregnanolone-stimulated chloride ion uptake was performed in the presence of 10 µM of GABA and different concentrations of UC1011. The chloride ion uptake in the presence of 10 μM of GABA was taken as 100%.

In Figure 4, the antagonistic effect of UC1011 against two concentrations of allopregnanolone with cortical microsacs and of one concentration of allopregnanolone with hippocampal microsacs is shown. These studies showed that UC1011 significantly reduces the allopregnanolone-induced increase in chloride ion uptake seen in the presence of GABA in both the brain regions that were analysed.

Benzodiazepines are also positive modulators of the GABA-A receptor that, like allopregnanolone, have negative effects on memory and learning and these effects are mediated by the activation of BZP-specific sites within the GABA-A receptor complex (Lister, 1985). The enhancement of spatial learning by the benzodiazepine receptor antagonists and inverse agonists helped to provide an understanding of this mechanism (Raffalli-Sebille and Chapouthier, 1991). Allopregnanolone reduces spatial learning in the MWM test (Johansson et al., 2002). Most of the animals injected with allopregnanolone eight minutes before the MWM test preferred to swim close to the pool wall (thigmotaxis); this effect is not caused by the impairment of the motor function, as the swimming speed is unaffected (Johansson et al., 2002). Using the parameters escape latency (i.e. time to reach the platform, in seconds), path length (cm), swimming speed (cm/s) and thigmotaxis, the performance of rats in the MWM test was evaluated for all the training days. 6 or 8 mg UC1011/kg injected, together with allopregnanolone (2 mg/kg) eight minutes before the swimming test, did not improve learning compared with allopregnanolone alone (data not shown). However, with an increased dosage of UC1011 (20 mg/kg), it was possible to reduce the negative effects on learning that were induced by allopregnanolone in the MWM test. In rats injected with the mixture of allopregnanolone and UC1011 (2:20 mg/kg, respectively), the parameters latency and thigmotaxis were significantly reduced (both p<0.05) on days 3-6, compared with the allopregnanolone- injected group (Fig. 5). The improved swimming performance was not due to improved motor performance, as there was no significant difference in swimming speed between the groups. In the MWM test, learning that there is an opportunity to escape from the water is a strong incentive factor that motivates the animals to leave the pool wall. This condition is achieved by searching the pool and acquiring the information about the maze. Only after leaving the wall can a true spatial learning of the platform position take place (Morris, 1984). As a result, the allopregnanolone-treated rats are stuck in an early phase of the task.

A B 120 100

100 Allopregnanolone 80 80 Allo:UC1011 Vehicle 60 (s) 60 UC1011 (%)

latency 40 40 thigmotaxis 20 20 0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Day Day

Figure 5: MWM test. Allopregnanolone (2 mg/kg), allopregnanolone and UC1011 (2:20 mg/kg respectively), UC1011 (20 mg/kg), or vehicle was injected (i.v) eight minutes before the start of each swimming session. The data shown are mean ± SEM for all four swimming trials each day A) Latency – time to find the hidden platform. B) Thigmotaxis – the fraction of the total path during which the rats swam close to the pool wall.

In the group injected with allopregnanolone and UC1011 (2:20 mg/kg) the concentration of allopregnanolone was lower in the investigated brain regions than the levels in the group only injected with allopregnanolone. The differences were statistically significant in the striatum and cortex. We know that high concentrations of allopregnanolone within the brain are needed to produce severe negative effects on learning. In an earlier study, we found no learning deficit 20 min after the injection, vs. vehicle-injected rats, and the allopregnanolone concentration in different brain regions was then decreased by 30-60% compared with the levels eight minutes after the injection (Johansson et al., 2002). In the present study, we found decreased levels of allopregnanolone within the cortex and striatum, but not in the hippocampus, after injections of allopregnanolone together with UC1011 (20 mg/kg). Changes in the brain concentration of allopregnanolone might be related to the inhibitory effects of UC1011 on allopregnanolone and GABA-A receptor binding and/or changes in the allopregnanolone metabolism that resulting in changes in allopregnanolone concentration in the brain tissue. In spite of this, we know that 3β-hydroxysteroids are not direct antagonists of 3α- hydroxypregnane steroids, such as allopregnanolone, but are instead non- competitive, probably state-dependent functional inhibitors of GABAA receptor potentiation (Wang et al., 2002). The present study shows that UC1011 can reduce the harmful effect of allopregnanolone on learning. However, to produce a positive effect byUC1011, we needed a ten times higher concentration of the drug than the concentration of allopregnanolone that was infused. It is probable that the

allopregnanolone-induced decrease in learning is caused by increased GABA-A activation and that the reduction of the allopregnanolone effect by UC1011depends on reduced GABA-A activation. The effects of allopregnanolone on cognitive functions may be the cause of cognitive disturbances noted by women with premenstrual dysphoric disorder, as their problems occur during the luteal phase of the menstrual cycle when the allopregnanolone concentration is high (Man et al., 1999). It is therefore essential to find a substance that antagonises the adverse effects of allopregnanolone. We also need more studies to determine whether UC1011 can meet the criteria required for a drug that can inhibit the negative effects produced by allopregnanolone.

4.2 Chronic allopregnanolone tolerance, Paper II To study the cognitive effects of repeated administration of allopregnanolone, two experiments with differences in pre-treatment time and injection method were performed. In Experiment 1, the three-days pre- treatment period consisted of i.v. injections (in the tail vein) with allopregnanolone (2 mg/kg) dissolved in 10% 2-hydroxypropyl-β- cyclodextrin (vehicle) two times a day (the 3xAllo+Allo group), with allopregnanolone (2 mg/kg) in the morning and vehicle (1 ml/kg) in the evening (the 3xAllo once+Allo group), or with vehicle (1 ml/kg) twice a day (the 3xVeh+Allo and 3xVeh+Veh groups). In Experiment 2, the rats were injected twice a day during the seven-day pre-treatment period (i.p.) with allopregnanolone (20 mg/kg) dissolved in sesame oil (the 7xAllo+Allo group) or with sesame oil (1.33 ml/kg, the 7xVeh+Allo and 7xVeh+Veh groups). After pre-treatment, the animals were subjected to the MWM test for five consecutive days. In the first experiment, the group pre-treated with allopregnanolone twice a day (3xAllo+Allo) experienced a less negative effect from the allopregnanolone that was injected before swimming. The rats in this allopregnanolone-pretreated group had significantly lower latency and thigmotaxis and shorter path length in comparison with the acute allopregnanolone group (Fig. 6). However, the 3xAllo+Allo group also had significantly higher values for the parameters latency, path length and thigmotaxis than the control group (3xVeh+Veh). The group pre-treated with allopregnanolone once a day (3xAllo once+Allo) did not differ from the acute allopregnanolone-treated group (3xVeh+Allo) and only differed significantly from the control rats.

A) B)

Latency Thigmotaxis

120 100 3 x Veh.+Allo 100 3 x Allo once+Allo 75 80 3 x Allo+Allo s

3 x Veh.+Veh. % 60 50

40 25 20

0 0 0 1 2 3 4 5 0 1 2 3 4 5 Day Day

Figure 6: Data from the MWM test after three days of i.v. pretreatment. (A) Latency to escape; (B) Thigmotaxis (path swum close to wall), during the five days of training with a hidden platform. Allopregnanolone 2 mg/kg or vehicle was injected before the four trials each day. The maximum time allowed during one trial was 120 seconds.

Chronic tolerance is defined as a change in the dose-response relationship produced by multiple exposure to a drug (Kalant et al., 1971) and a given dose therefore has less effect as a result of the previous administrations. These findings suggest that pre-treatment once a day for three days was not able to induce tolerance of the acute allopregnanolone effect during the MWM testing. Rats pre-treated with allopregnanolone twice daily (3xAllo+Allo) showed incomplete tolerance and had a swimming performance between that of the 3xVeh+Allo and 3xVeh+Veh groups. The differences in performance might depend on the short treatment duration and the fact that an insufficient steroid concentration was reached in rats injected just once a day. The development of tolerance towards a given drug is dependent on factors like the length of exposure, the dose and the route of delivery of the drug (Kalant et al., 1971). In the next experiment, with longer pre-treatment, the route of administration was changed to i.p., since the tail veins are unable to tolerate excessive i.v injections during pre-treatment. However, as in Experiment 1, i.v. injections were given during the MWM test. Sesame oil was used as a vehicle for intra-peritoneal injections. This produces a slow and continuous absorption of the steroid, creating optimal conditions for tolerance development, as the duration of drug exposure is prolonged (Chien, 1981). When allopregnanolone is injected intra-peritoneally, the neurosteroid is taken up into the portal circulation and the metabolism of the drug in the liver begins before the drug reaches the brain. A larger amount of steroid is therefore required to induce an effect (Holzbauer, 1976). For this reason, the

dosage of allopregnanolone was changed accordingly. In Experiment 1, we used a fixed dose of allopregnanolone (2 mg/kg) during both pre-treatment and MWM testing. Previous studies revealed that allopregnanolone at this dosage produces an acute negative effect in the MWM (Johansson et al., 2002). Spatial memory in the water maze has been studied after i.p injections and the dosage of 17 mg of allopregnanolone/kg significantly impaired memory when given i.p. However, memory was significantly poorer when 20 mg of allopregnanolone/kg was used (Matthews et al., 2002). So, in experiment 2, we used the dosage of 20 mg of allopregnanolone /kg i.p. given during pre-treatment. The group that was pre-treated with two i.p. injections of allopregnanolone a day for seven days (the 7xAllo+Allo group) had a significantly better performance than the acute allopregnanolone group (7xVeh+Allo) that was pre-treated with the vehicle. The rats in the 7xAllo+Allo group improved their performance, while the 7xVeh+Allo group did not learn to locate the platform. The 7xAllo+Allo group learnt the task almost as well as the controls (7xVeh+Veh), as they were significantly different only on the first day (p<0.01 for all parameters, Fig. 7). In the MWM test, learning is expected after fewer than 20 trials (in this setting, five days) and the first two days of the test are accepted as acquisition of the escape behaviour (Morris, 1984).

A) Latency B) Thigmotaxis 7 x Veh.+Allo 120 100 7 x Allo+Allo 100 7 x Veh.+Veh. 80 80 ↓ * ↓ ↓ 60 s 60 % ↓ ↓ 40 ↓ 40 * 20 20

0 0 0 1 2 3 4 5 0 1 2 3 4 5 Day Day

Figure 7: Data from the MWM test after seven days of pre-treatment (i.p.). A) Latency to escape and B) Thigmotaxis during the five days of training with a hidden platform. Allopregnanolone 2 mg/kg or vehicle was injected and four trials per rat were performed each day. The maximum time allowed during one trial was 120 seconds.

In this experiment, a probe test was carried out on day 6. The test results confirmed that pre-treatment with allopregnanolone improves the acute negative effects of the neurosteroid in the MWM test. The 7xAllo+Allo group spent an equal amount of time in the quadrant with the former platform position as did the control rats (group 7xVeh+Veh), while the

7xVeh+Allo group spent significantly less time in the right quadrant (p<0.05, Fig. 8). Probe test

50 Quad NE (former platform position) 40 Quad SE

30 Quad SW (opposite to former platform) Quad NW 20 % of total time total of % 10

0 7 x Allo+Allo 7 x Veh+Allo 7 x Veh+Veh Groups

Figure 8: Data from the memory test performed in Experiment 2. The rats swam for 60 seconds without any platform being present in the pool.

However, the 7xAllo+Allo group did not recall the platform position as well as the 7xVeh+Veh group of rats. These findings suggest that seven days of pre-treatment induced a clear-cut but no total chronic tolerance to the negative effect of the allopregnanolone given before the MWM test (Fig. 8). In animals, it has previously been shown that tolerance to the anticonvulsant activity of allopregnanolone develops after five days of intracerebroventricular repeated administration (Czlonkowska et al., 2001), whereas the effects of allopregnanolone on sleep were not changed after the same treatment time with daily i.p. injections (Damianisch et al., 2001). In fact, it is known that tolerance does not always develop at an identical speed to all the effects of a given drug and the drug exposure length needed for tolerance development can vary depending on the method and dosage (Damianisch et al., 2001; Kokate et al., 1998; LeBlanc et al., 1969; Mendelson, 1964; Mendelson et al., 1964; Wikler, 1956; Wikler et al., 1956). Experience from studies with other substances acting on the GABA- A receptor shows similar time frames for tolerance development as allopregnanolone in the MWM test. For instance, cats and rabbits develop tolerance to the hypnotic effects of some barbiturates after two to seven days’ treatment (Gluckman and Gruber, 1952; Jaffe and Sharpless, 1965), while rats and mice show tolerance after one to five days (Aston, 1965, 1966; Rumke, 1963). In humans, tolerance of the hypnotic action of barbiturates has been reported to occur after three to 8.5 days (Belleville and Fraser, 1957).

In this study, we demonstrated for the first time that repeated allopregnanolone treatment, given over several days, can generate tolerance towards the spatial learning impairment produced by acute allopregnanolone administration.

4.3 Acute allopregnanolone tolerance and the GABA-A receptor subunits, Paper III Possible changes in GABA-A receptor subunit mRNA expression levels during the development of acute allopregnanolone tolerance were studied. Allopregnanolone (3 mg/ml dissolved in 10% hydroxypropyl-β- cyclodextrin) was infused (i.v.) during EEG recording. After different time intervals (first SS, 30 min or 90 min of anaesthesia), the last infusion period to SS was followed by decapitation. Samples from blood, different brain regions, muscle and fat tissue were analysed. Half the brain was used for the determination of GABA-A receptor subunit protein and mRNA expression levels.

100 90 30 min 80 90 min 70 60 50

(mg/kg) 40 30

Cumulative dose 20 10 MDR1 MDR2 MDR3 0 0 10 20 30 40 50 60 70 80 90 100 Duration of anaesthesia (min)

Figure 9: Cumulative allopregnanolone doses given to maintain anaesthesia at the silent second level in the 30-min and 90-min groups. A theoretical straight line is shown for the cumulative doses without tolerance development. The time periods used for calculating maintenance dose rates (MDR) are indicated.

The development of acute tolerance to allopregnanolone was detected using the EEG-threshold anaesthesia method (Korkmaz and Wahlstrom, 1997). The initial sensitivity to allopregnanolone (threshold dose) for the first silent second induction did not differ between the three experimental groups (control, 30-min and 90-min groups). In the 90-minute group, the dose rate of allopregnanolone needed to maintain anaesthesia (MDR) was significantly (p<0.001) increased during the time period 65-85 min (MDR3) compared with the period 10-30 min (MDR1) and the period 35-55 min

(MDR2, Fig. 9), indicating that acute tolerance had developed in the MDR3 period. The difference between MDR3 and MDR1 (ΔMDR) is a direct in vivo measurement of the magnitude of acute tolerance, since this shows the increase in allopregnanolone dose that is needed to maintain SS anaesthesia. The allopregnanolone concentrations in certain regions of the brain in the 90-min group were significantly higher than in the SS group (Fig. 10). These regions were the hippocampus, MTH (midbrain, thalamus and hypothalamus) and MOP (medulla oblongata and pons). Allopregnanolone concentrations in serum and fat in the 90-min group and in fat in the 30-min group were higher when compared with the SS group (Fig. 10).

160 * 120 SS (control) mol/l)

μ 30 min 80 * 90 min * * * (nmol/g or Allopregnanolone 40 *

0 . m H ex u ala . o fat d b llum scle hipp MT MOP g e serumcort mu striat my a ereb c Figure 10: Allopregnanolone concentrations in serum, different brain areas, muscle and fat tissue in the different groups. * – significant difference versus the controls in group; SS ⁪ – significant difference versus 30-min group. hipp = Hippocampus; MTH = Midbrain, thalamus and hypothalamus; MOP = Medulla oblongata and Pons; b.o. = Olfactory bulb.

In a previous study, acute tolerance to hexobarbital produced increased drug concentrations in the cortex and brainstem as early as at 30 min of anaesthesia vs. controls (Bolander and Wahlstrom, 1988). In old rats but not in young ones, propofol brain concentrations increased compared with controls after 60 min of anaesthesia in the hippocampus, striatum, brainstem and cerebellum, but not in the cortex (Larsson and Wahlstrom, 1996). Substance accumulation in fat tissue has been observed in all the studies in which acute tolerance has been detected using the EEG-threshold method. From the allopregnanolone concentration data, the hippocampus, thalamus and hypothalamus were found to be the most important brain regions for the development of acute allopregnanolone tolerance. We therefore performed

in situ hybridisation in order to analyse mRNA expression in the areas that are most probably important for tolerance development. GABA-A receptor subunit expression patterns were similar to those that have been found before, with only slight differences from Wisden (Wisden et al., 1992).

A) B)

α4 protein * * 40 50 38 ) 36

4 mRNA4 VPM 34

α 25 / X40 area grains/cell ( 32

GABA-A 30 number of positive cells 28 0 0.0 0.1 0.2 0.3 0.4 0.5 Control 30 min 90 min Allopregnanolone dose difference (ΔMDR)

Figure 11: A) Linear regression plot for the GABA-A receptor α4 mRNA expression in the ventral posteriomedial nucleus of the thalamus and the ΔMDR (increase in allopregnanolone dose per minute needed to maintain the anaesthesia; mg/kg/min) in the rats in the 90-min group. B) The number of GABA-A receptor α4 protein positive cells (mean ± S.E.M in the ventral posteriomedial nucleus of the thalamus, in X40 magnification areas from the light microscope. * p < 0.05 vs. the groups’ SS.

A statistically significant decrease in the GABA-A receptor α4 subunit mRNA and protein level was detected in the thalamic VPM nucleus in the tolerant 90-min group when compared with the SS group (post-hoc, p<0.05) and the 30-min group (post-hoc, p<0.05; Fig. 11). There was also a negative correlation between the ΔMDR and the expression of the GABA-A receptor α4 subunit in the VPM of the thalamus (r = -0.74, b = -20.76, d.f. = 7, p<0.05). This means that greater tolerance developed in the animals with the smallest amount of the GABA-A receptor α4 subunit in the VPM of the thalamus. It has been shown that the α4 subunit containing GABA-A receptors is highly plastic and is rapidly altered in response to changes in neuronal activity (Sur et al., 1999). The change in the α4 subunit mRNA and protein levels and the increase in allopregnanolone levels in the thalamus region indicates that this part of the brain is involved in the development of acute allopregnanolone anaesthesia tolerance. The thalamus has often been called “the switchboard of the brain”. This part of the brain plays an

important role in modulating and synchronising the signal traffic between regions and is probably also involved in anaesthesia and tolerance of GABA-A receptor active anaesthetic compounds (Reynolds et al., 2003). In line with our results, there are data which indicate that a four-day application of allopregnanolone to developing neuronal cells reduces the GABA-A receptor α4 subunit in a concentration-dependent manner (Grobin and Morrow, 2000). However, in pregnant rats, when there are high allopregnanolone levels for a long period, no change in α4 subunit expression can be found in the cortex or hippocampus. On the seventh day postpartum (withdrawal from allopregnanolone), an increase in this subunit was detected in the hippocampus, while the thalamus was not investigated (Concas et al., 1999). With cerebellar granule cell cultures, progesterone application for five days does not change the amount of the α4 subunit, but, six hours after withdrawal, an increase was found, followed by a decrease at 12 h to 24 h after withdrawal (Follesa et al., 2000). On the other hand, an increase in GABA-A receptor α4 subunit mRNA expression has been shown in the hippocampus in relation to increased anxiety after three days of progesterone or allopregnanolone treatment and 24 h after progesterone withdrawal (Gulinello et al., 2001). With benzodiazepines, an increase in the GABA-A receptor α4 subunit in the anteroventral thalamus was obtained by triazolam treatment for four weeks (Ramsey-Williams and Carter, 1996). Interestingly, GABA-A receptor α4 subunit expression in the cortex varies depending on intermittent (increase) or continuous (decrease) supplementation of diazepam for a two-week period (Arnot et al., 2001).The α4 subunit expression therefore varies depending on the drug used, or the time of application. The GABA-A receptor β2 subunit mRNA expression in the hippocampus of the tolerant rats (group 90 min) did not differ from that in the control group. However, during the course of the development of acute tolerance, the amount of β2 subunit mRNA decreased in the 30-minute group (V-shaped changes), at a time point when the acute tolerance has not yet developed. A study with ethomidate, a selective positive GABA-A receptor modulator, demonstrated that the sedative and a component of the hypnotic action of this anaesthetic is mediated by β2 subunit-containing receptors (Reynolds et al., 2003). A change in β2 mRNA expression is therefore probably involved as part of the anaesthesia but not necessarily in the tolerance mechanism. For the GABA-A receptor α1, α2, α5, α6 and δ subunits, no difference in the amount of mRNA was found between groups in the brain regions that were studied.

In the 90-minute group, the increase in allopregnanolone doses needed to maintain anaesthesia for 65-85 min was significantly positively correlated with the α2 mRNA in different hippocampal subregions. The in vivo measurement of acute tolerance (ΔMDR) also correlated with α2 mRNA in the CA1 region (r = 0.71, b = 14.14, d.f. = 7, p<0.05). In vitro GABA-A receptors containing the α2 subunit have been shown to react less to allopregnanolone, compared with receptors including the α1, α3, or α6 subunits (Belelli et al., 2002). The higher amount of the α2 mRNA in the hippocampus of tolerant rats might therefore help to explain the need for a higher allopregnanolone dose to induce anaesthesia for 65-85 min. However, it is also possible that other mechanisms might be involved in the development of tolerance, e.g. phosphorylation which changes the steroid sensitivity of the receptor, independently of any change in subunit composition (Harney et al., 2003; Koksma et al., 2003). Our finding of the involvement of GABA-A receptor subunits in the development of acute allopregnanolone tolerance is of great importance for identifying the mechanism of steroid tolerance.

4.4 Persistence of allopregnanolone tolerance, Paper IV In this study, we investigated whether tolerance persisted after the interruption of the exposure following the induction of acute allopregnanolone tolerance by 90 minutes of allopregnanolone anaesthesia at the EEG-threshold level. All the groups were anaesthetised with allopregnanolone (3 mg/ml dissolved in 10% hydroxypropyl-β - cyclodextrin) to the anaesthesia level of the silent second (SS), but the exposure patterns were different: 1) Control group with anaesthesia to the first silent second (SS1 group), 2) 90 minutes of anaesthesia (LAn group), 3) 90 minutes of anaesthesia + anaesthesia to the silent second level (SS2) one day later (SS2;D1 group), 4) 90 minutes of anaesthesia + SS2 two days later (SS2;D2 group). In the SS2; D1 group and the SS2; D2 group, the 90-minute anaesthesia period was induced in either the morning (am subgroup) or the afternoon (pm subgroup), whereas in the LAn group the onset of anaesthesia took place between the am and pm onset (mid-time). In the SS1 group, anaesthesia was always induced around 12 pm. The data are therefore analysed for anaesthesia onset in the three subgroups, am, pm and mid-time. All the rats were decapitated between 11 am and 12.30 pm. Samples from blood, different brain regions, muscle and fat tissue were analysed for

allopregnanolone content. Half the brain was used for the determination of GABA-A receptor subunit mRNA expression levels. Acute tolerance to allopregnanolone developed in all groups with 90 minutes of anaesthesia (LAn, SS2;D1 and SS2;D2 groups). Acute tolerance to allopregnanolone was also established in all subgroups (am, pm and mid-time) and the subgroups individually showed a significant increase in MDR (p<0.01 - 0.001, Fig. 13A). Unexpectedly, we found a significant interaction between the time of onset of anaesthesia and MDR (p<0.05), in which ΔMDR (MDR3-MDR1) was significantly lower in the am subgroup compared with the pm subgroup (p<0.01). However, the MDRs showed no significant difference between the subgroups (Fig. 13B).

A) B)

100 am Subgroup * am Subgroup pm Subgroup * * 1.00 pm Subgroup mid-time Subgroup 75 mid-time 0.75 50

(mg/kg) 0.50

25 Cumulative dose Cumulative

rate (mg/kg/min) rate 0.25 MDR1 MDR2 MDR3 Allopregnanolone dose 0 0 10 20 30 40 50 60 70 80 90 0.00 123 Duration of anaesthesia (min) MDR10-30 MDR40-60 MDR70-90

Figure 13: Allopregnanolone cumulative dose and maintenance dose rate (MDR) during the 90-min threshold testing: A) cumulative dose in the am, pm and mid-time subgroups and B) the maintenance dose rate in the am, pm and mid-time subgroups. The maintenance dose rate in the 70- to 90-minute interval (MDR3) was different compared with the maintenance dose rate in the 40- to 60-minute interval (MDR2) and the maintenance dose rate in the 10- to 30-minute interval (MDR2). * MDR3 differences from MDR1 and MDR2 are significant (p<0.05).

The initial CNS sensitivity to allopregnanolone was determined by the amount of allopregnanolone needed to induce anaesthesia to reach the first SS (SS1). The allopregnanolone SS1 dose did not differ between groups (p>0.05; Fig. 14). To investigate whether tolerance remained one day and two days after the long anaesthesia, a new anaesthesia was induced in the SS2;D1 and SS2;D2 groups of rats. The amount of allopregnanolone needed to induce the first SS during the long anaesthesia (SS1) and the dose needed to induce SS one or two days after the long anaesthesia (SS2) showed a significant difference in the SS2;D1 group (ΔSS, p<0.05, Fig. 14)., Between the SS2;D1 and SS2;D2 groups, the ΔSS value did not differ (p<0.05). These findings suggest that tolerance only persisted for one day. The time of

day of the onset of anaesthesia (am, pm and mid-time) had no effect on the persistence of tolerance.

* SS1 LAn 10 SS2;D1 SS2;D2

Dose of Dose (mg/kg) 5 Allopregnanolone

0 SS1 SS2 ΔSS (SS2-SS1)

Figure 14: Persistence of acute tolerance. The dose of allopregnanolone needed for the induction of the first SS (SS1) during the 90-min anaesthesia and for the induction of SS2 in the same individual rats in the SS2;D1 and SS2;D2 groups. The SS2;D1 dose was significantly higher than the SS1 dose in the same animals. The ΔSS (SS2-SS1) value in the SS2;D1 group did not differ significantly from the ΔSS in the SS2;D2 group. * The difference is significant (p<0.05).

The allopregnanolone concentrations in the LAn group were significantly higher in the MTH, brain stem and fat tissue compared with the other groups (p<0.01, 0.01 and 0.001 respectively; Fig. 15). The time of day of the onset of anaesthesia had no influence on the allopregnanolone concentrations in the brain regions. * 80 SS1 LAn * SS2;D1 60 SS2;D2 * 40 nmol/g -nmol/L

[Allopregnanolone] 20

0 x H at T um F M Corte in stem Muscle Serum Striatum a Br Hippocamp. Cerebell Figure 15: Allopregnanolone concentrations in tissues and serum. The LAn group was decapitated after a 90-minute long anaesthesia, while the SS1, SS2;D1 and SS2;D2 groups were decapitated after the induction of anaesthesia up to the SS criterion. The values are presented as the mean ± SEM. * Between groups, there is a significant overall difference, p<0.05.

It is suggested that allopregnanolone produces a regional difference in responsiveness, in agreement with the variability in the subunit composition of GABA-A receptors in different brain regions (Jussofie, 1993a; 1993b). The normal level of allopregnanolone in the rat brain cortex is 1.3- 8.6 nmol/kg (Purdy et al., 1991) (Johansson et al., 2002) and the amount of drug producing the loss of the righting reflex is with some drugs sufficient for tolerance development (Dingemanse et al., 1990): With allopregnanolone, this is achieved with an concentration of 3 μmol/kg (Korneyev and Costa, 1996). The SS occurs at a deeper anaesthesia than the loss of righting reflex (Korkmaz and Wahlstrom, 1997) and, in the present study, the allopregnanolone concentration in the cortex of all the groups was at least nine times higher than the concentration required to induce the loss of the righting reflex (> 27 µmol/kg). The allopregnanolone concentration in the SS2;D1 group was not higher than the concentration in the control group (SS1), except for in fat. It is possible that the methods that were used were not sensitive enough to detect localised changes in allopregnanolone concentrations in the grossly dissected brain regions. A detailed study of the rat brain uptake of progesterone and its metabolites pregnanolone and pregnanedione revealed that the higher steroid concentration in the brain was not associated with a stronger anaesthesia effect (Raisinghani et al., 1968). Taken together with our results, it can be concluded that the allopregnanolone concentration in tissue may change after acute tolerance development. There was no significant difference between groups in the expression of different GABA-A receptor subunits in the hippocampus (α2) and VPM (α2, α4, δ, γ2). However, the α4 subunit mRNA in the VPM of the individual animals correlated negatively both with the dose of allopregnanolone needed to induce SS2 in the SS2;D1 group (r=-0.88, b=-0.33, DF=8, p<0.01) and with ΔSS (SS2-SS1, r=-0.70, b=-0.27, DF=8, p<0.05). There were no similar correlations in the non-tolerant SS2;D2 group. These recorded negative correlations are very similar to the correlation already reported in Paper III. In the present study, the change in α4 subunit mRNA expression in the thalamic VPM regions (6% changed) was not significant between groups. The α4 subunit containing receptors in normal rats account for 20% of the total thalamic GABA-A receptors (Sur et al., 1999) and it therefore appears that a small variation in α4 level could have a significant functional role. In Paper III, a 10% change in the amount of the α4 subunit (both mRNA and protein) was, however, found to be significant. Animals anaesthetised in the afternoon (pm subgroup) required a higher amount of allopregnanolone for the maintenance of anaesthesia than the

animals anaesthetised in the morning. Moreover, in the pm subgroup, the allopregnanolone penetration to the brain (Q-values) was lower than in the am and mid-time subgroups. One possible explanation is based on the diurnal changes in the glucocorticoid level that may influence tolerance development. It has been shown that high levels of glucocorticoid in the brain change the GABA level (Sherman and Gebhart, 1974; Singh et al., 1979) and can influence the allopregnanolone effect and finally augment the function of the brain GABA system (Schwartz et al., 1987; Stromberg et al., 2005). The circadian rhythm of the glucocorticoids in rats shows the highest plasma level at the beginning of the dark period, followed by a decrease to the lowest level in the middle of this period (Atkinson and Waddell, 1997). In the current study, the am rats were anaesthetised at the time of the highest glucocorticoid level, as we used a reversed light-dark period, and the pm rats were anaesthetised at the time of the lowest glucocorticoid secretion. To our knowledge, no detailed study of the time course of the acquisition and subsequent loss of allopregnanolone tolerance has been conducted. The present work shows for the first time that acute allopregnanolone tolerance develops progressively over a period of minutes (< 90 min) and reverts to normal several hours (< 48 h) after the end of the neurosteroid treatment.

5 GENERAL DISCUSSION

The present thesis shows that the negative effects induced by allopregnanolone on spatial learning can be reduced by using a 3β- hydroxypregnane steroid (UC1011). Numerous studies have evidenced the existence of alterations in learning and memory processes during the course of female life. Cognitive disturbances are reported in pregnancy and during the luteal phase of the menstrual cycle, when high circulating levels of progesterone and allopregnanolone are found (Brett and Baxendale, 2001; Man et al., 1999). In animals, treatment with the neurosteroid allopregnanolone impairs learning and memory (Frye and Sturgis, 1995; Johansson et al., 2002). It would therefore be of great importance to discover or synthesise a drug that is effective against allopregnanolone-induced cognitive disturbances. UC1011 appears to be a promising substance in this field, as it can reduce the allopregnanolone-induced learning impairment and GABA potentiation.

The sensitivity to a substance can decrease, i.e. tolerance can be induced during a single administration of the substance (acute tolerance) or after multiple exposures to the substance (chronic tolerance) (Kalant et al., 1971). Many of the behavioural and physiological effects of allopregnanolone are similar to those of ethanol and other positive modulators of the GABA-A receptor, such as benzodiazepines and barbiturates (Majewska et al., 1986; Paul and Purdy, 1992; Vanover et al., 1999). In contrast to benzodiazepines and ethanol, less is known about the tolerance that might result from exposure to allopregnanolone. In women, the levels of allopregnanolone or allopregnanolone-like substances are high during the luteal phase of the menstrual cycle, pregnancy, use of oral contraceptives and hormone replacement therapy. Tolerance may therefore develop and may cause the appearance of mood and behavioural symptoms during these periods (see Section 1.3). Our results show that one prolonged exposure to allopregnanolone can cause acute tolerance development in rats following less than 90 minutes’ exposure. Moreover, tolerance persists to one day after the induction of acute allopregnanolone tolerance. The time of day of the onset of anaesthesia may also play an important role in the development of allopregnanolone tolerance by influencing the neurosteroid impact in the

CNS. We also succeeded in developing chronic tolerance to allopregnanolone after seven days of repeated exposure. The activity of steroids has been suggested to depend on the subunit composition of the GABA-A receptor. Lambert and co-workers have clearly demonstrated a role for the subunit composition of the receptor in neuroactive steroid/GABA-A receptor interactions (Lambert et al., 2003). However, it is not clear which GABA-A receptor subunits are involved in the development of allopregnanolone tolerance. Accordingly, we analysed the expression of GABA-A receptor subunits in the brain and its correlation with tolerance development after 90 minutes of allopregnanolone anaesthesia. In the tolerant group, both the mRNA expression and protein level of the GABA-A receptor α4 subunit in the ventral posteriomedial nucleus of the thalamus was lower than in non-tolerant rats and the in vivo measurements of tolerance (ΔMDR and ΔSS) showed negative correlations with the actual amount of α4 mRNA in the ventral posteriomedial nucleus of the thalamus. As receptors containing the α4 subunit display higher GABA sensitivity than most other receptors containing α subunit (Knoflach et al., 1996), a decrease in α4 subunit expression level can then be suggested as one of the pathways changing the effect of allopregnanolone during tolerance development and the maintenance of tolerance. Finally, the strength of the present thesis is that it addresses the questions of allopregnanolone tolerance and changes in GABA-A receptor function, as this may be related to the cognitive impairment and emotional changes in certain periods of female life.

6. CONCLUSIONS

1. The increase in chloride ion uptake induced by allopregnanolone can be inhibited by UC1011.

2. UC1011 significantly improves the allopregnanolone-induced impairment of learning in the water maze test, probably via an effect on the GABA-A receptor.

3. The negative effects of allopregnanolone in the water maze can be improved after pre-treatment with allopregnanolone for several days.

4. Repeated exposure to allopregnanolone induces the development of tolerance to the effects of allopregnanolone.

5. Acute allopregnanolone tolerance can be induced and the GABA-A receptor α4 subunit in the thalamus might be involved in the development of tolerance.

6. Tolerance persists to one day but not to two days after the end of a long allopregnanolone anaesthesia inducing acute tolerance.

7. The time of day of the onset of anaesthesia with allopregnanolone is of importance for the development of tolerance.

7. ACKNOWLEDGEMENTS

I wish to express my deepest gratitude to all the people who have supported me throughout this work and especially to:

Associate Professor Inga-Maj Johansson, my main supervisor, for her excellent academic supervision and for introducing me to the field of molecular biology, tack för din eminenta guidning genom vetenskapens gåta

Professor Torbjörn Bäckström, my co-supervisor, for accepting me as a student and introducing and inspiring me to work in the exciting world of research. The opportunity to work with you was something that really helped me to acquire a view of the way science works and opened my mind and encouraged me to work with enthusiasm

Professor Göran Wahlström, for fruitful collaboration and sharing great research experiences and ideas

Professor Ann Lalos, head of the Department of Obstetrics and Gynaecology, for friendly support

Dr. Per Lundgren, for collaboration and friendly guidance in all areas Monica Isaksson, for exceptional and excellent technical laboratory assistance Elizabeth Zingmark and Agneta Andersson, for all the help in the laboratory with the steroid analysis Tobias Näslund, Birgit Ericsson and Carina Jonsson, for all the administrative help during my research studies at the Department of Obstetrics and Gynaecology My friends and co-workers, Charlotte Lindblad, Magnus Löfgren, Vita Birzniece, Mozibur Rahman, Jessica Strömberg, Magdalena Taube, Gianna Ragagnin, Di Zhu, Ming-de Wang, and all wonderful people at the Department of Obstetrics and Gynaecology To all the people at UKBF 3B, especially Jamshid Pourarzi, for always helping out and sharing their experience

To my sister and brother in low, Shahla and Hooshmand, for unfailing help and for trying to make my life easy in Umeå

Finally, to my family, Melek, Atlan and Aral, this thesis would have not been possible to finish without their love and understanding, and to my parents, Kerim and Arazbakht, for their unreserved and endless support throughout the whole of my life.

This study was carried out at the Department of Clinical Science, Obstetrics and Gynaecology, Umeå University, and was supported by the EU Structural Programme, objective 1, the Swedish Research Council, Medicine (project No. 72X-11198), a “Spjutspets” grant from Norrland University Hospital, and by Umeå University Foundations.

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APPENDIX -Papers I-IV

Umeå University Medical Dissertations New Series No 1026 – ISSN 0346-6612 – ISBN 91-7264-073-1

Edited by the Dean of the Faculty of Medicine

Umeå University WWW.UMU.SE 2006