Cellular and behavioural effects of a new steroidal inhibitor of the N-methyl-d -aspartate receptor 3α5β-pregnanolone glutamate

Neuropharmacology 61 (2011) 61e68

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Cellular and behavioural effects of a new steroidal inhibitor of the N-methyl-D-aspartate receptor 3a5b-pregnanolone glutamate Lukas Rambousek a, c, e, Vera Bubenikova-Valesova a, c, *, Petr Kacer e, Kamila Syslova e, Jana Kenney a, Kristina Holubova a, Vera Najmanova e, Petr Zach d, Jan Svoboda a, Ales Stuchlik a, Hana Chodounska b, Vojtech Kapras b, Eva Adamusova a, Jirina Borovska a, Ladislav Vyklicky a, Karel Vales a, c a

Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4, Czech Republic Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, Prague 6, Czech Republic Prague Psychiatric Centre, Ustavni 91, Prague 8, Czech Republic d 3rd Medical Faculty, Charles University Prague, Ruska 87, Prague 10, Czech Republic e Institute of Chemical Technology, Technicka 5, Prague 6, Czech Republic b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 December 2010 Received in revised form 15 February 2011 Accepted 16 February 2011

Preclinical studies have demonstrated a considerable role for N-methyl-D-aspartate (NMDA) receptors in excitotoxicity and the concurrent neuroprotective effect of NMDA receptor antagonists. Because NMDA receptors are one of the most widespread receptors in the central nervous system, application of their antagonist often leads to serious side effects ranging from motor impairment to induction of schizophrenic-like psychosis. Therefore, we have initiated development and testing of a novel synthetic NMDA receptor antagonist derived from naturally occurring neurosteroids. 20-oxo-5b-pregnan-3a-yl-L-glutamyl-1-ester (3a5bP-Glu) is a novel synthetic steroidal inhibitor of the NMDA receptor. Our results show that 3a5bP-Glu preferentially inhibits tonically activated NMDA receptors, is able to cross the blood brain barrier, does not induce psychotomimetic symptoms (such as hyperlocomotion and sensorimotor gating deficit) and reduced an excitotoxic damage of brain tissue and subsequent behavioural impairment in rats. In particular, 3a5bP-Glu significantly ameliorated neuronal damage in the dentate gyrus and subiculum, and improved behavioural performance in active allothetic place avoidance tasks (AAPA, also known as the carousel maze) after bilateral NMDA-induced lesions to the hippocampi. These findings provide a possible new therapeutic approach for the treatment of diseases induced by NMDA receptor overactivation. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Neuroactive steroid 3a5b-Pregnanolone glutamate Glutamate excitotoxicity Neuroprotectivity Cognitive function

1. Introduction A number of preclinical studies have shown the remarkable ability of NMDA receptor antagonists to prevent excessive exocytosis of glutamate and neural damage (Choi et al., 1988; Choi, 1990; Rao et al., 2001; Schauwecker, 2010). However, from a clinical point of view, their therapeutic potential in stroke and traumatic brain injury treatment is rather limited (Lees et al., 2000). Since glutamate receptors are very abundant in the brain and provide important physiological functions, application of their antagonists can lead to wide variety of side effects, ranging from motor impairment to

* Corresponding author at: Prague Psychiatric Centre, Department of Biochemistry and Brain Pathophysiology, Ustavni 91, Prague 8-18103, Czech Republic. Tel.: þ420 266003173; fax: þ420 266003160. E-mail address: [email protected] (V. Bubenikova-Valesova). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.02.018

induction of psychotic symptoms (Shiigi and Casey, 1999; ManahanVaughan et al., 2008). In the past decade, biomedical research has focused on the role of neurosteroids in the pathogenesis of a number of neuropsychiatric diseases and the evaluation of their therapeutic potential (Gasior et al., 1999; Melcangi et al., 2008). A number of experimental studies with animal models have shown their potential in the therapeutic treatment of several disorders of the central nervous system, including ischemia (Li et al., 2001; Pringle et al., 2003), neurodegenerative disorders (Mellon et al., 2008), traumatic brain injury (Djebaili et al., 2005) and multiple sclerosis (Morrow, 2007). The naturally occurring compound 3a5b-pregnanolone sulfate (3a5bP-S) inhibits the activity of NMDA receptors in a use-dependent manner (Petrovic et al., 2005). Thus, sulfated neurosteroids and their analogues could be promising molecules in the therapy of central nervous system diseases. Nevertheless, enzymes constantly maintain the ratio between 3-hydroxy- neurosteroids and their sulfated esters in the brain. The enzyme steroid sulfohydrolase

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hydrolyses sulfate groups at the 3rd carbon position (Reed et al., 2005). Systemic administration of sulfated esters thus does not necessarily increase the ratio of sulfated/non-sulfated concentration in the brain (Wang et al., 1997). Furthermore 3a5bP-S do not sufficiently cross the blood-brain barrier because it is ionized molecule at physiological pH (Knapstein et al., 1968; Weaver et al., 1997; Bowen et al., 1999). Therefore, we have started the development and testing of a novel NMDA receptor antagonist derived from the 3a5bP-S (Stastna et al., 2009). The proposed synthetic molecule should analogue of the 3a5bP-S rasistant to sulfohydrolase activity. We selected a lead structure (Fig. 1), 20-oxo-5b-pregnan-3a-ylL-glutamyl-1-ester (3a5b-pregnanolone glutamate, 3a5bP-Glu). In the present study we show the distribution of 3a5bP-Glu and its penetration into the brain, its binding and action on native NMDA receptors in cultured hippocampal neurons, the effect of 3a5bP-Glu on spontaneous behaviours related to schizophrenia-like behaviour (prepulse inhibition of the acoustic startle response and locomotor activity in an open field test) and its effect against neuronal damage and behavioural impairment induced by intrahippocampal application of NMDA. 2. Materials and methods 2.1. Animals Adult male Long-Evans rats (weighting from 250 to 350 g) from a breeding colony of the Institute of Physiology AS CR, Prague were used in the study. Rats were kept in transparent Plexiglas cages measuring 25  25  50 cm located in an airconditioned animal room with a 12:12 hour light-dark cycle with the lights turned on beginning at 7:00 a.m. Animals had free access to water and food. All experiments were done in accordance with European Union regulations on animal care and protection, the Animal Protection Code of the Czech Republic and NIH guidelines. 2.2. 3a5bP-Glu synthesis 3a5bP-Glu was prepared by esterification of 3a-hydroxy-5b-pregnan-20-one (Steraloids INC, USA) with a protected glutamate (Boc-L-Glu(OBzl)-OH, Bachem AG, Germany) catalysed by DCC and DMAP. Esterification was followed by deprotection of the carboxy and amino groups. The structure was confirmed by IR, NMR, MS, and HR-MS spectra. Other reagents used were purchased from Sigma Aldrich (Germany). 2.3. 20-oxo-5b-pregnan-3a-yl-L-glutamyl-1-ester [9,12,12-3H] (3a5bP-Glu- d3) synthesis A deuterated analogue was prepared from 11a-hydroxy-progesterone (ACROS Organics, USA). This was converted by stereoselective hydrogenation to yield

5b-steroid, followed by protection of the 3- and 20- oxo groups and mild oxidation of the 11a-OH group. Deuterium exchange at enolisable positions was then conducted under basic conditions using MeOD as an economical and convenient source of deuterium. Deuterated 11-ketone was reduced with LiAlH4 to fix deuterium atoms. Oxygen functionality was removed using the Barton-McCombie reaction. The sequence was then finished using acid catalysed deprotection followed by stereoand regioselective reduction of the 3-keto group into a tri-deuterated analogue of the parent pregnanolone. Coupling with protected glutamic acid followed by deprotection gave the desired [9,12,12-3H] pregnanolone glutamate at >89% isotopical purity. The overall yield of this sequence was 13%. Ratios and positions of deuterium labels were determined by MS and NMR analyses. All other reagents used were purchased from Sigma Aldrich (Germany). 2.4. Hippocampal cultures Primary dissociated hippocampal cultures were prepared from 1- to 2-day-old postnatal rats. Animals were decapitated, and hippocampi were dissected. Trypsin digestion followed by mechanical dissociation was used to prepare cell suspensions. Neuronal cultures were maintained in NeurobasalÔ-A (Invitrogen, USA) medium supplemented with glutamine (0.5 mM) and B-27 Serum-Free Supplement (Invitrogen, USA). 2.5. Electrophysiological recording from cultured cells Experiments were performed on cultured hippocampal neurons prepared as described above. Whole-cell voltage-clamp recordings were made with a patchclamp amplifier (Axopatch 1D; Axon Instruments, Inc., Foster City, CA, USA) after a capacitance and series resistance (<10 MU) compensation of 80e90%. Agonistinduced responses were low-pass filtered at 1 kHz with an 8-pole Bessel filter (Frequency Devices, Haverhill, MA, USA), digitally sampled at 5 kHz and analysed using pClamp software version 9 (Axon Instruments, USA). Patch pipettes (3e4 MU) pulled from borosilicate glass were filled with a Csþ-based intracellular solution containing 125 mM gluconic acid, 15 mM CsCl, 5 mM EGTA, 10 mM HEPES, 3 mM MgCl2, 0.5 mM CaCl2, and 2 mM ATP-Mg salt (pH-adjusted to 7.2 with CsOH). The extracellular solution (ECS) contained 160 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 10 mM glucose, 0.2 mM EDTA and 0.7 mM CaCl2 (pH-adjusted to 7.3 with NaOH). Glycine (10 mM), an NMDA receptor co-agonist, was present in the control and test solutions. The drugs were purchased from Sigma Aldrich (Germany) or Tocris Cookson Ltd. (Avonmouth, UK). 3a5bP-Glu solutions were made from a freshly prepared 20 mM stock in DMSO. Control experiments were performed in extracellular solution containing DMSO at the same concentration present in steroid containing solutions. A microprocessor-controlled fast-perfusion system, with a time constant of solution exchange around cells of 10 ms, was used to apply test and control solutions (Vyklicky et al., 1990). Results are presented as mean  S.E.M. with n equal to the number of cells studied. 2.6. Determination of 3a5bP-Glu plasma and tissue levels 3a5bP-Glu levels were determined in plasma and tissue samples. On the day of the experiment, rats were divided into groups based on their scheduled sacrification time (15, 30, 45, 60, 90, 120, 180 min and 24 or 48 h post-injection) and administered with a single dose 1 mg/kg, i.p. of 3a5bP-Glu dissolved in hydroxypropyl-b-cyclodextrine (b-CD, 72 mM saline solution, SigmaeAldrich, Germany) adjusted pH7.4 by 1 M NaOH. Each group consisted of four rats. Plasma and tissue (brain, lung, liver, heart and kidney) were isolated. 2.6.1. Plasma We added 10 ng of the internal standard (3a5bP-Glu-d3), 0.5 mL of acetonitrile and 0.25 mL of methanol to a 0.5 mL aliquot of rat plasma, followed by ultrasonication for 20 min and centrifugation at 6500 g for 10 min. The supernatant was filtered using a 0.2 mm PTFE filter. 2.6.2. Tissue The tissue was weighted and the 3a5bP-Glu- d3 internal standard (10 ng/1 g tissue) was added to the whole tissue and the sample was homogenised. The tissue was then extracted with methanol:acetonitrile (1:1), sonicated for 20 min and centrifuged at 6500 g for 10 min. The supernatant was filtered using a 0.2 mm PTFE filter, evaporated to dryness and reconstituted in 1 mL of methanol and 9 mL of acetonitrile. The sample was then centrifuged at 6500 g for 5 min and the supernatant was filtered. The final solution was evaporated and reconstituted in 1 mL of methanol.

Fig. 1. Chemical structure of 3a5bP-Glu (An L-glutamic acid residue replaces the sulfate group at position 3 on the steroidal ring A), 3a5bP-HS and 3a5bP-S.

2.6.3. Analysis An HPLC-MS system equipped with a Luna C18 column (5 mm  20 mm  2 mm; Phenomenex, USA), using a methanol/water mixture (70:30) as the eluent at a flow rate of 150 mL/min was used. The column was heated to 25  C. The injection volume was 20 mL. The HPLC system was directly coupled to a mass spectrometer Agilent 6320 Ion Trap (Agilent, USA) equipped with an electrospray ion source operated in ionization mode ESIþ for 3a5bP-Glu and 3a5bP-Glu-d3. Selective reaction

L. Rambousek et al. / Neuropharmacology 61 (2011) 61e68 monitoring mode was used (3a5bP-Glu 448/283; CID energy ¼ 17 eV); ([9,12,123H] 3a5bP-Glu-d3 451/286; CID energy ¼ 17 eV) because of its extremely high degree of selectivity and the stable-isotope-dilution assay was used because of its high precision in quantification. The method was characterised with a low imprecision (9.7%); an acceptable inaccuracy (9.6%), a low limit of detection (LOD  2 pg/mL and a low limit of quantification (LOQ ¼ 10 pg/mL). Results are presented as the mean  S.E.M. with n equal to the number of samples. 2.7. Prepulse inhibition of acoustic startle response (PPI) 3a5bP-Glu dissolved in b-CD was acutely applied to rats at doses of 1 and 10 mg/ kg i.p., 60 min befor test session. Day before test session rats were pre-trained by five minuts of acclimatization period and five single startel pulses (125 dB, 40 ms). The test session was composed of a mixture of the following types of trials that were presented against a constant 75 dB background noise: a) trials in which a prepulse stimulus has intensities 83 or 91 dB preceded a startle-eliciting pulse stimulus with 30, 60 or 120 milliseconds with duration 20 ms, b) trials in which no discrete stimulus other than the constant background noise was presented (ns [no-stimulus] trials), and c) trials in which startle pulse were presented alone 125 dB (40 ms) (startle pulse). All stimuli and background noise employed in the experiment consisted of broadband white noise. After a 5-minute period of acclimatization to the background noise, 72 discrete trials were presented according to a variable intertrial interval with a mean of 14 s (ranged from 4e20 s). The first (block 1) and last block (block 3) consisted of 6 consecutive startle pulse trials. The middle block (block 2) consisted of 60 trials, i.e., 12 trials of each of the 5 conditions, presented in a pseudorandomized order. The test session lasted approximately 20 min. PPI was calculated as the difference between the average values of the single pulse and prepulse-pulse trials and was expressed as a percentage of PPI [100  (mean response for prepulse-pulse trials/startle response for single pulse trials)  100]. Data from the four single pulse trials at the beginning of the test session were not included in the calculation of PPI and acoustic startle response values. Animals showing average startle amplitudes lower than 10 mV were removed from the calculation of PPI and were marked as non-responders (about 3% of the total number). The number of removed animals did not differ between treatment groups. Data were tested for significant differences using the one-way ANOVA, followed by the Tukey post hoc test. Results are presented as the mean  S.E.M. 2.8. Locomotor activity in a novel environment Animals received 3a5bP-Glu dissolved in b-CD at doses of 1 or 10 mg/kg i.p. 60 min prior to the test. Locomotor activity, expressed as the total distance travelled during 30 min in a box (68 cm  68 cm  30 cm) located in a soundproof room, was measured using a video tracking system for automation of the behavioural experiments (Noldus, Netherlands, EthoVision Colour Pro-Version 3.1), as previously described (Bubenikova-Valesova et al., 2007). Data were tested for significant differences using the one-way ANOVA, followed by the Tukey post hoc test. Results are presented as the mean  S.E.M.

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NMDA into the dorsal hippocampus. At each time point, 3a5bP-Glu was injected at 0.1 mg/kg, 1 mg/kg or 10 mg/kg i.p. of body weight dissolved in b-CD. Controls received buffered saline solution infusions into the hippocampus and a b-CD solution i.p. The apparatus for the AAPA task was developed in our laboratory and is described in detail elsewhere (Bures et al., 1998; Cimadevilla et al., 2001; Stuchlik et al., 2008). Rats were trained seven days after surgery in four 20 min sessions. The sessions took place over a period of two consecutive days with two sessions per day. In healthy rats this training procedure was found to be long enough to induce optimal spatial avoidance of the punished sector and the data from the fourth session was close to asymptotic levels of performance (Stuchlik et al., 2008). Thus, only data from session four were analysed. The following parameters were used: 1) Number of entrances into the punished region, a measure of the total effective sector avoidance 2) Maximum time between two entrances (Max avoidance time), a measure of the ability of an animal to avoid the sector, i.e. a measure of spatial memory and attention; 3) Total path, a non-cognitive parameter measuring the total path length travelled during a session. Data were analysed using the One-way Between Subjects ANOVA test followed by the Tukey post-hoc test. All p-values were accepted at <0.05. Results are presented as the mean  S.E.M.

3. Results 3.1. 3a5bP-Glu is a use-dependent inhibitor of NMDA receptors We used the patch clamp technique to study the effect of 3a5bPGlu on NMDA receptors expressed in hippocampal neurons. Fig. 2A shows whole-cell currents induced by 100 mM NMDA applied in the presence of 10 mM glycine (no added Mg2þ) and reduced Ca2þ in cultured rat hippocampal neurons and the response to the presence of 100 mM 3a5bP-Glu. At this concentration of 3a5bP-Glu responses induced by NMDA application were inhibited by 54  9% (n ¼ 7). This effect of 3a5bP-Glu was dose dependent (3e300 mM) with a mean IC50 value of 69.1  9.9 mM (n ¼ 7) (Fig. 2B). Application of 3a5bP-Glu in the absence of NMDA receptor agonist, even at a concentration of 300 mM, had no effect on whole-cell recorded currents (not shown). We have shown previously that 3a5bP-S is a use-dependent and voltage-independent NMDA receptor inhibitor (Petrovic et al.,

2.9. NMDA-induced lesion All animals were anaesthetised in a specialised initial chamber using isofluran. During surgery animals were fixed using a stereotactic apparatus and anaesthetised with isofluran (3.5%) propelled by atmospheric air. Small openings in the skull were made using a micro-drill and subsequently 2 mL of NMDA (90 mM NMDA in phosphate buffer, SigmaeAldrich, Germany) was bilaterally applied (1 mL each) at a flow rate of 0.2 mL/min into the dorsal hippocampi and 2 min after the end of the infusion the needle was retracted. The application coordinates (4 mm AP from bregma, 2.5 mm ML and 4 mm below the skull surface) were measured in relation to a stereotaxic atlas (Paxinos and Watson, 2004). 2.10. Fluoro-Jade B staining after NMDA-induced hippocampal lesion Animals were divided into three groups: NMDA-lesioned animals, NMDAlesioned animals treated with 3a5bP-Glu dissolved in b-CD (1 mg/kg i.p.) 30 min prior and 30 min after lesion, and sham-operated animals. Three animals from each group were sacrificed and perfused transcardially using Ringer’s solution (5 min) followed by a 4% solution of paraformaldehyde (10 min) in Ringer’s solution (pH 7.2) at intervals of 1day following NMDA-lesion induction. After removal from the cranial cavity, brains were kept in the same fixative solution. For Fluoro-Jade B (Millipore, USA) DAPI staining (SigmaeAldrich, Germany), brains were rinsed five times in 0.1 M phosphate-buffered saline (pH 7.4) and left overnight in 30% sucrose. Brains were then frozen and embedded in medium for frozen tissue and cryo-sectioned into 40 mm slices. Staining protocols were as described in Bubenikova-Valesova et al. (2006). 2.11. Active allothetic place avoidance (AAPA, Carousel Maze) Rats were randomly assigned into experimental groups according to the 3a5bPGlu application protocol used: 30 min, 3 hours and 24 hours after application of

Fig. 2. The effect of 3a5bP-Glu on native NMDA receptors. A. Whole-cells responses evoked in cultured hippocampal neurons by application of 100 mM NMDA (indicated by an open bar) and simultaneous application of 100 mM 3a5bP-Glu (indicated by a filled bar). Note the slow kinetics of the onset and offset of inhibition. B. Doseeresponse relationship of the effect of 3a5bP-Glu on NMDA induced responses. The relative inhibition induced by 3a5bP-Glu (3, 30, 100 and 300 mM) was fit using nonlinear regression (I ¼ 1/(1 þ [3a5bP-Glu]/IC50; where IC50 is the concentration of 3a5bP-Glu that produces 50% inhibition of the NMDA-evoked current and [3a5bP-Glu] is the neurosteroid concentration. Results from each cell were independently fit to the nonlinear regression and IC50 values were averaged (IC50 ¼ 69.1  9.9; n ¼ 7).

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2005). Next experiments were aimed to test whether the inhibitory action of 3a5bP-Glu depends or is independent upon receptor activation. Two experimental protocols for 3a5bP-Glu and NMDA applications were used: (1) co-application of 3a5bP-Glu and NMDA after the onset of a response to NMDA and (2) sequential application of NMDA after pre-application of 3a5bP-Glu for 30 s. Fig. 3 shows responses induced by co-application of 100 mM NMDA and 50 mM 3a5bP-Glu on cultured hippocampal neurons. At concentrations close to the steroid IC50 value inhibition was 54%. After cessation of 3a5bP-Glu application the response to NMDA recovered on a slow timescale (within seconds). Subsequent experiments were designed to test the effect of 3a5bP-Glu on the response to NMDA when 3a5bP-Glu is preapplied in the absence of NMDA receptor agonist. As seen in Fig. 3 (right trace), the response to 100 mM NMDA observed after preapplication of 50 mM 3a5bP-Glu was significantly faster than that seen after co-application of 3a5bP-Glu and NMDA while onset kinetics were similar to that induced by control applications of 100 mM NMDA (without neurosteroid pre-application) (see overlaid responses in Fig. 3). Similar differences in NMDA receptor response onset kinetics were observed in five additional cells after either co- or pre-application of 3a5bP-Glu. These results show that the inhibitory action of both 3a5bP-Glu and 3a5bP-S depends upon receptor activation, and that both neurosteroids are use-dependent inhibitors of NMDA receptors. 3.2. 3a5bP-Glu plasma and tissues levels Time course experiments show the highest 3a5bP-Glu plasma concentration at the 15-min time point after i.p. injection (cmax ¼ 675  20.02 ng/ml). The plasma t1/2 of 3a5bP-Glu was 267 min.

3a5bP-Glu rapidly entered the brain (Tmax ¼ 60 min, cmax ¼ 246  6.80 ng/g brain, t1/2 ¼ 371 min) after i.p injection (1 mg/kg) and exponentially decreased to a mean of 113 ng/g after 2 h, 60 ng/ g after 3 h, 6 ng/g after 24 h and 0.3 ng/g after 48 h. Fig. 4 shows a time course (0e180 min) of 3a5bP-Glu levels in plasma and the brain. Data shown represent the mean  SD of four animals. The peak level and t1/2 for other tissues was as follows: heart (Tmax ¼ 120 min, cmax ¼ 35  2.25 ng/g, t1/2 ¼ 631 min), liver (Tmax ¼ 30 min, cmax ¼ 42  2.77 ng/g, t1/2 ¼ 645 min) and kidney (Tmax ¼ 180 min, cmax ¼ 57  7.49 ng/g, t1/2 ¼ 1680 min). Data represent the mean  SD of three animals. There were no detectable levels (
Fig. 3. 3a5bP-Glu is a use-dependent inhibitor of NMDA receptor channels. Examples of recordings obtained from cultured hippocampal neurons. On the left) response to co-application of 50 mM 3a5bP-Glu and 100 mM NMDA after the onset of the response to the agonist was inhibited by 54% and recovered from the inhibition on a slow timescale. On the right) the onset of the response to the application of 100 mM NMDA made immediately after pre-application of 50 mM 3a5bP-Glu for 30 s was rapid, similar to the control NMDA response on the left. To illustrate the difference in the time course of responses to NMDA made after co-application of 3a5bP-Glu and NMDA (b; boxed area) and that made after pre-application of 3a5bP-Glu (indicated in red), both responses are shown superimposed on an expanded timescale. The rise time of responses to NMDA made after pre-application of 3a5bP-Glu (indicated in red) was similar to that of the control response to 100 mM NMDA (a; boxed area) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 4. Levels of 3a5bP-Glu in plasma and the brain after 1 mg/kg i.p. The highest levels of 3a5bP-Glu were found at 15 min in plasma and 60 min in the brain after administration of 3a5bP-Glu. The maximum concentration in the brain was 246 ng/g of tissue.

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Fig. 5. Effect of acute application of 3a5bP-Glu at dose 1 and 10 mg/kg i.p. on prepulse inhibition of the acoustic startle response. There was no significant effect of 3a5bP-Glu on PPI.

and small interneurons in layer IV. Minor damage was also observed in the caudatoputamen and reticular nucleus in the thalamus, while the dorsal hippocampus was less stained than the ventral and supracomissural hippocampus. Application of 3a5bP-Glu resulted in reduction of morphological signs of tissue damage in the pyramidal cells of the dentate gyrus, CA1-CA3 subfields and the subiculum (Fig. 7). Intermediate and medial portions of the entorhinal cortex and the piriform cortex were found to contain barely quantifiable lesions in layers II and III ranging as far as the olfactory tubercle. The most prominent absence of tissue damage was observed in the whole neocortex (the whole frontal, prefrontal, parietal and occipital fields, including the functional cortical areas-the visual, acoustic and sensorimotor cortex). A decrease in staining was also observed (although staining was not completely absent) in the caudatoputamen and thalamus. In all cases, co-localization of Fluoro-Jade B and DAPI staining was imaged using a Zeiss AxioVision Imaginer Z1.

Cognitive deficit was also assessed using the ‘Maximum Time Avoided’ parameter (Fig. 8B). A One-way ANOVA test revealed a significant group effect F(10,53) ¼ 5.42, P < 0.0001). As a subsequent post-hoc test specified, NMDA lesion induced deficit (P < 0.05, compared to the sham group) was blocked by 3a5bP-Glu only at a dose of 10 mg/kg administered 3 h post-surgery. To some extent, 3a5bP-Glu administration affected locomotory behaviour. After calculating the significance of any group effect (F(10,53) ¼ 6.47, P < 0.0001), a Tukey post-hoc test found that untreated excitotoxic hippocampal lesions resulted in a mild, but insignificant hypolocomotion compared to sham operated rats. In addition, nearly all doses of 3a5bP-Glu treatment 24 h after surgery (with the exception of 0.1 mg/kg) induced sedative effect. To a lesser extent, this was also apparent when administrating 1 mg/ kg 3 h after surgery (Fig. 8C).

3.5. The effect of 3a5bP-Glu on AAPA performance after intrahippocampal lesion induction by NMDA

We hypothesised that if 3a5bP-Glu specifically inhibits the NMDA receptor, the drug could also be neuroprotective in in vivo studies. We have observed that 3a5bP-Glu is a use- dependent NMDA receptor inhibitor similar to the endogenous steroid 3a5bpregnanolone sulfate (Petrovic et al., 2005), and that both inhibitors decrease NMDA-induced currents on NMDA receptors expressed in hippocampal neurons. We confirmed the use-dependent action of 3a5bP-Glu on this subtype of glutamate receptors following methods used in the study of 3a5b-pregnanolone sulfate (Petrovic et al., 2005; Kussius et al., 2009). Our neurosteroid inhibits only NMDA receptors which are active and therefore could protect neurons against excitotoxic damage induced by overactivation of the NMDA receptor. Pharmacokinetic data indicate that 3a5bP-Glu is rapidly absorbed after i.p. administration and penetrates through the bloode brain barrier with a Tmax of 60 min. Moreover the drug is eliminated by a first-order process and it is not accumulated in the brain or other tissues. Pharmacokinetic properties of pregnanolone hemisuccinate (3a-ol-5b-pregnan-20-one hemisuccinate; 3a5bP-HS, Fig. 1) show similar good penetration through the blood brain barrier with a Tmax of 10 min. However there is no evidence about the distribution to the other tissues. The higher 3a5bP-Glu Tmax may be related to the different pKa value and manner of dissolution. We used b-CD (FDA approved excipient) for the dissolution of the steroid instead of DMSO which was used in the other study. The bCD/3a5bP-Glu complex may interact with lipoproteins in the plasma and blood brain barrier as well and thus influence the final pharmacokinetic properties. Inhibition of NMDA receptors by non-competitive antagonists has been linked to schizophrenia-like behaviour (BubenikovaValesova et al., 2008). Therefore, we measured two types of

3a5bP-Glu ameliorated hippocampal lesion induced cognitive spatial deficit as assessed by the number of entrances into the punished region (Fig. 8A). A Post-hoc test, measuring the significance of group effect (F(10,53) ¼ 5.19, P < 0.0001), revealed a considerable increase in ‘Number of Entrances’ after an NMDA induced lesion (P < 0.05). However, 3a5bP-Glu ameliorated this deficit when given 30 min after surgery (at a dose of 0.1 or 1 mg/kg, P < 0.05 for both) or at a dose of 10 mg/kg given 3 h post-surgically (P < 0.05). When applied 24 h after surgery, no dose tested was found to be effective.

Fig. 6. Effect of 3a5bP-Glu at doses 1 and 10 mg/kg i.p. administration on locomotion generated during 30 min in an open-field apparatus. There is no significant effect of 3a5bP-Glu on locomotor activity in a novel environment.

4. Discussion

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Fig. 7. A representative picture of the CA3 region of the dorsal hippocampus 1 day after NMDA lesion induction (Part A) and the effect of 3a5bP-Glu (1 mg/kg i.p. 30 min prior and 30 min post lesion administration) (Part B) Administration of 3a5bP-Glu decreased NMDA-induced damage of neurons. DAPI blue colour, Fluoro-Jade B green colour or matched staining. Arrows indicate the Fluro-Jade B positive neurons. Images obtained using a Zeiss Axioplan2 microscope imaging system (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

behaviours as models of psychotic symptoms: locomotor activity in a novel environment and information processing deficiency (prepulse inhibition of the acoustic startle response) (Geyer and Swerdlow, 2001; van den Buuse, 2010). We have found that neither behaviourally active dose used in the AAPA task nor a high dose of 3a5bP-Glu (10 mg/kg) induced schizophrenia-like behaviour. Similarly, 3a5bP-HS did not affect locomotion up to a dose of 20 mg/kg in rats (Sadri-Vakili et al., 2003). The reasons for this convenient safety profile compared with other NMDA antagonists e for instance MK-801, phencyclidine, is unclear, but a distinct mode of action of neurosteroids such as 3a5bP-HS on NMDA receptors may be possible. These neurosteroids have a usedependent but voltage- independent effect on NMDA receptors, possibly mediated by binding to the extracellular side of the NMDA receptor (Park-Chung et al., 1997; Sedlacek et al., 2008). The overall goal of this study was to measure the possible neuroprotective effect of 3a5bP-Glu in rats after NMDA-induced lesions in the hippocampi. First, we detected dead neurons using Fluoro-Jade B and DAPI staining in NMDA-lesioned rats (Schmued and Hopkins, 2000). We found that administration of 3a5bP-Glu pre- and post-lesion reduced the size of neurodegeneration induced by NMDA in several areas of the brain. It is well known that

administration of non-competitive antagonists such are phencyclidine and MK-801 damage neurons in the cerebral cortex of rats (Olney et al., 1991). In contrast, administration of 3a5bP-Glu does not induce neurodegeneration visualised by Fluoro-Jade B staining. Next we evaluated the pro-cognitive effects of 3a5bP-Glu on AAPA performance in rats with NMDA-induced lesions of the hippocampi. Intrahippocampal microinjection of NMDA significantly worsened performance of rats in the AAPA task as measured by the ‘Number of Entrances’ and ‘Maximum Time Avoided’ parameters. Simultaneously, NMDA-lesioned rats had decreased locomotor activity during performance of the AAPA task. The AAPA test is dependent on an intact hippocampus (Cimadevilla et al., 2001) and can be used to test cognitive coordination in certain configurations (Wesierska et al., 2005; Kubik et al., 2006). Administration of 3a5bP-Glu at specific doses administered at particular time intervals after NMDA injection ameliorated such behavioural deficits in rats assayed in the AAPA task compared with ‘lesionedonly’ animals. Such protective action on the behavioural consequences of excitatory damage is often observed after application of drugs inhibiting the NMDA receptor such as MK-801 (McDonald et al., 1990), however, many such drugs are excluded from the search for potential human neuroprotectants due to their

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significant side-effects ranging from sensory and motor disturbances to induction of schizophrenia-like symptoms; in fact, some of these drugs are used to induce animal models of this disorder (Bubenikova-Valesova et al., 2008). The neuroprotective and pro-cognitive effect of 3a5bP-Glu is in accordance with several reports aimed at testing potential neuroprotective properties of other neuroactive steroid derivatives such as 3a5bP-HS. For example, systemic administration of 3a5bP-HS after focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduced infarct size in mice (Weaver et al., 1997). Even when 3a5bP-HS was applied 30 min after the onset of ischemia the volume of cortical infarct was reduced by 39 %. This study also reported evidence supporting 3a5bHS neuroprotectivity in vitro. NMDA-induced cell damage was dose-dependently attenuated by co-application of 3a5bP-HS (Weaver et al., 1997). In another study, 3a5bP-HS significantly improved ischemia induced behavioural deficit in rabbits if administered up to 30 min following ischemia using an irreversible spinal cord ischemia model (Lapchak, 2004). The same authors evaluated the neuroprotective properties of 3a5bHS following embolic stroke if given 5 min after embolization (Lapchak, 2006). An additional synthetic analogue (3a-ol-5b-pregnan-20-one, L-Valine ester hydrochloride) showed good solubility in water (0.33 mg/mL) and was capable of reducing edema in an animal model of traumatic brain injury (MacNevin et al., 2009). These results together with our present study suggest that neurosteroid substances and their derivatives may act protectively against excitotoxic damage to the brain and subsequent behavioural disturbances, via a mechanism related to the action of these substances on NMDA receptors. Similarly to 3a5bP-HS, our 3a5bP-Glu derivate shows good penetration into the brain with no adverse effects such as schizophrenia. Moreover we demonstrated the selective action of 3a5bPGlu on the NMDA receptor in vitro as well as in vivo. Moreover, our derivate was shown to be more potent in comparison with 3a5bPHS, with the lowest effective dose only 0.1 mg/kg. Our efforts have centred on understanding steroid action in animal models of human diseases and disorders. We show that a synthetic analogue of the endogenous steroid 3a5b-pregnanolone sulfate has a protective effect both at the cellular and behavioural level on NMDA-induced memory impairment and hypothesise that this could open new directions in the search for steroid-based substances which could be used to treat severe neurological and psychiatric disorders with NMDA receptor pathology such as ischemia, neurodegenerative disorders and schizophrenia.

Acknowledgements This work was supported by the IGA Ministry of Health NS10365, NR 9180-3 and GACR (309/07/0271; 203/08/1498; 309/ 09/0286), Research Project of the AS CR AV0Z 50110509, EC FP6 PHOTOLYSIS (LSHM-CT-2007-037765) and The Ministry of Education, Youth and Sports of the Czech Republic (1M0157 and LC554). Hana Chodonska was supported by Research Project Z4 055 0506.

References Fig. 8. Effect of 3a5bP-Glu administration 30 min, 3 h, or 24 h after surgery on NMDAinduced lesions of the dorsal hippocampi in a behavioural spatial paradigm, the Active Allothetic Place Avoidance (AAPA) task. Cognitive performance was assessed using ‘Number of Entrances’ (A), and ‘Maximum Time Avoided’ (B) parameters, while locomotor activity was assessed by measuring the total path length elapsed during a session (C). Data are presented from the last day of training. Number of animals in a group is given at the column base. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group; #P < 0.05, ##P < 0.01 compared to the NMDA group.

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