Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin

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Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin Arafat Nasser a,b,∗ , Lisbeth B. Møller a , Jess H. Olesen c , Louise S. Konradsen b , Jesper T. Andreasen b a

Applied Human Molecular Genetics, Kennedy Center, Copenhagen University Hospital, Rigshospitalet, Glostrup, Denmark Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Copenhagen University, Copenhagen, Denmark c Department of Clinical Genetics, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark b

a r t i c l e

i n f o

Article history: Received 4 June 2014 Received in revised form 28 August 2014 Accepted 28 August 2014 Available online xxx Keywords: GCH1 Serotonin Dopamine Nitric oxide Anxiety Depression

a b s t r a c t Decreased tetrahydrobiopterin (BH4) biosynthesis has been implicated in the pathophysiology of anxiety and depression. The aim of this study was therefore to characterise the phenotype of homozygous hph-1 (hph) mice, a model of BH4 deficiency, in behavioural tests of anxiety and depression as well as determine hippocampal monoamine and plasma nitric oxide levels. In the elevated zero maze test, hph mice displayed increased anxiety-like responses compared to wild-type mice, while the marble burying test revealed decreased anxiety-like behaviour. This was particularly observed in male mice. In the tail suspension test, hph mice of both sexes displayed increased depression-like behaviours compared to wild-type counterparts, whereas the forced swim test showed a trend towards increased depressionlike behaviours in male hph mice, but significant decrease in depression-like behaviours in female mice. This study provides the first evidence that congenital BH4 deficiency regulates anxiety- and depressionlike behaviours. The altered responses observed possibly reflect decreased hippocampal serotonin and dopamine found in hph mice compared to wild-type mice, but also reduced nitric oxide formation. We propose that the hph-1 mouse may be a novel tool to investigate the role of BH4 deficiency in anxiety and depression. © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

1. Introduction Tetrahydrobiopterin (BH4) is a cofactor essential for the activity of tyrosine and tryptophan hydroxylases as well as for all isoforms of nitric oxide synthase (NOS), making BH4 a key player in the biosynthesis of NO and the monoamines dopamine, noradrenaline and serotonin (Werner et al., 2011). Given the pleiotropic effects

Abbreviations: BH4, tetrahydrobiopterin; NO, nitric oxide; NOS, nitric oxide synthase; GTP-CH1, guanosine triphosphate cyclohydrolase 1; DRD, DOPA-responsive dystonia; hph-1, hyperphenylalaninemia 1; WT, wild-type; hph, homozygous hph-1; DOPAC, deuterium-labelled 3,4-dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HTP, 5-hydroxytryptophan; HVA, homovanillic acid; VMA, Vanillylmandelic acid; DHBA, dihydroxybenzylamine; DA, dopamine; 5-HT, serotonin; NA, noradrenaline; MHPG, 3-methoxy-4-hydroxyphenylglycol; EZM, elevated zero maze; SAP, stretched-attend posture; MB, marble burying; TST, tailsuspension test; FST, forced swim test; ANOVA, analysis of variance; SEM, standard error of the mean; EPM, elevated plus maze. ∗ Corresponding author at: Applied Human Molecular Genetics, Kennedy Center, Copenhagen University Hospital, Rigshospitalet, Glostrup, Denmark. Tel.: +45 43260100; fax: +45 43431130; mobile: +45 43260108. E-mail address: [email protected] (A. Nasser).

of BH4 in various biological systems, BH4 has been implicated in several pathological conditions such as inherited neurological diseases, cardiovascular diseases and painful conditions (Tegeder et al., 2006; Werner et al., 2011; Nasser et al., 2013). Additionally, the BH4 pathway is thought to be involved in neuropsychiatric disorders such as depression and anxiety (Kealey et al., 2005; McHugh, 2011; Pan et al., 2011). Alterations in BH4 biosynthesis is usually assessed by quantification of total biopterin and neopterin, and clinical studies evaluating the association between these pterins and major depression have shown ambiguous results. Some report decreased plasma and urine BH4 in patients with depression compared to controls (Hashimoto et al., 1990; Abou-Saleh et al., 1995; Hoekstra et al., 2001), while others show no change in BH4 biosynthesis in plasma (Tiemeier et al., 2006), or even increased total biopterins in urine (Duch et al., 1984; Garbutt et al., 1985). Thus, the role of BH4 in depression appears equivocal. De novo synthesis of BH4 proceeds from GTP via three steps catalysed by guanosine triphosphate cyclohydrolase 1 (GTP-CH1), 6-pyruvoyltetrahydrobiopterin synthase and sepiapterin reductase, respectively. Mutations in the human GCH1 gene, coding for GTP-CH1, are associated with DOPA-responsive dystonia (DRD), a

http://dx.doi.org/10.1016/j.neures.2014.08.015 0168-0102/© 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Please cite this article in press as: Nasser, A., et al., Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin. Neurosci. Res. (2014), http://dx.doi.org/10.1016/j.neures.2014.08.015

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disorder characterised by dystonia-related gait problems (Romstad et al., 2003). Besides this classical clinical manifestation, studies report increased prevalence of anxiety and major depression in DRD patients (Hahn et al., 2001; Van Hove et al., 2006; TrenderGerhard et al., 2009; Tadic et al., 2012). The hyperphenylalaninemia 1 (hph-1) mouse is a model of DRD generated by mutagenesis using N-ethyl-N -nitrosourea (Bode et al., 1988). Similar to humans, the hph-1 mice display substantial decrease in murine Gch1 mRNA expression accompanied by a great decrease in GTP-CH1 enzyme activity and BH4 biosynthesis in liver and plasma (Tatham et al., 2009; Nasser et al., 2013). Moreover, previous studies report that hph-1 mice exhibit lower concentrations of BH4 and monoamines in the brain (Hyland et al., 1996), as well as decreased NO formation in brain astrocytes and blood (Barker et al., 1998; Lam et al., 2007). The hph-1 mutation has been localised to an interval of 1.6–2.8 Mb on chromosome 14, containing the Gch1 gene (Khoo et al., 2004), although the exact location is still undefined. To elucidate if the reported affective disturbances in DRD patients translates back to the hph-1 mouse model of DRD, this study investigated if hph-1 mice display altered phenotype in behavioural tests related to anxiety and depression. The mouse elevated zero maze and marble burying test were used to assess anxiety-like behaviour, and forced swim and tail suspension tests to measure depression-like behaviour. Considering the role of monoamines in controlling affective states (Ressler and Nemeroff, 2000; Dell’Ossp et al., 2010), reduced levels of these neurotransmitters or their metabolites in DRD patients (Blau and Hoffmann, 1998; Hahn et al., 2001) may be implicated in the higher prevalence of affective disturbances in these patients. The hippocampus is a key element in the limbic brain circuits controlling affective states, and substantial evidence point to its role in both depression and anxiety disorders (Campbell and Macqueen, 2004; Adhikari, 2014). Therefore, to relate the behavioural responses to brain neurochemistry, hippocampal monoamine levels and plasma NO levels were measured in wild-type (WT) and hph-1 mice. To assess any sex-dependent effects of genotype, all tests were performed in male and female mice.

2. Materials and methods 2.1. Animals The breeding took place at Taconic Denmark. Initially, heterozygous hph-1 mice were backcrossed to C57BL/6JOlaHsd mice (Harlan Laboratories, UK) to produce a congenic strain. From this colony of mice, homozygous hph-1 (hph) and WT breeding pairs were generated by heterozygous breeding. For experiments, hph and WT mice were subsequently generated by homozygous breeding, no more than 6 generations (The Jackson Laboratory, 2007). Breeder genotypes were verified as previously described (Khoo et al., 2004). Experiments were performed on 7–10 weeks-old male and female mice, housed in colony cages with Tapvei 2HV bedding (L: 37 cm × W: 21 cm × H: 15 cm; at maximum 8 mice per cage) in temperature-controlled environments, with unrestricted access to standard diet (Altromin 1314 F, Brogaarden, Denmark) and tap water, and kept on a 12:12 h light-dark cycle (lights on at 06.00). Environmental enrichment included house, Tapvei s-bricks (Brogaarden), Ancare NES3600 nestlets (Brogaarden) and corn. The mice were delivered to the animal facility at an age of 4–5 weeks old. Behavioural testing was performed between 08.00 and 16.00. All animal experiments were done in compliance with international guidelines regarding care and use of laboratory animals (NIH publication No. 80-23, revised 1996) and with confirmed ethical approval by the Danish Animal Experiments Inspectorate, Ministry

of Food, Agriculture and Fisheries (autorisation no. 2011/561-121). Experimenters were blinded to the animal sex and genotype during all experimental procedures. All efforts were made to minimise animal suffering and to reduce the number of animals used. 2.2. General appearance, health and motor performance of hph mice The hph mice were observed for gross abnormalities by examining the animal’s physical appearance and presence of abnormal behaviours. The physical examination included weight, presence of whiskers, bald patches, piloerection, palpebral closure, exophthalmos, fur condition, footpad colour and respiratory infections (dark yellow crustiness around nostrils and eyes). Animals were then placed in clean cages and observed for 3 min and the following behaviours were studied: gait, rearing, immobility, wild running, sniffing, jumping, defecation and urination. Sensorimotor reflexes were examined by evaluating the postural, eye blink and ear twitch reflex. 2.3. Chemicals Deuterium-labelled 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5HIAA), 5-hydroxytryptophan (5HTP), homovanillic acid (HVA) and Vanillylmandelic acid (VMA) were all purchased from QMX Laboratories in UK. Dihydroxybenzylamine (DHBA), dopamine (DA), serotonin (5-HT), noradrenaline (NA), 3-methoxy-4-hydroxyphenylglycol (MHPG) as well as all other chemicals were purchased from Sigma–Aldrich in Denmark. 2.4. Determination of monoamines in hippocampus Mice (n = 6–9) were sacrified by cervical dislocation and the brains removed. The two hippocampi were immediately dissected out on ice, weighed and snap frozen in liquid nitrogen. Tissues were stored at −80 ◦ C until analysis. The hippocampal tissues were homogenised with 100 ␮L of ice-cold 0.1% (w/v) ascorbic acid and 0.01% (w/v) Na2 EDTA per 10 mg of wet tissue using a digital sonifier (Branson, United States). The amplitude was set at 10% and at least 3 cycles of 10 s was used. Monoamines were analysed as previously described (Song et al., 2012), with some modifications. Briefly, 160 ␮L hippocampal homogenates were mixed with 40 ␮L internal standard (400 nM d5 -DOPAC, d5 -5HIAA, d3 -5HTP, d3 -HVA, d3 -VMA, 400 nM DHBA and 50 ␮M ascorbic acid in 0.9% (w/v) NaCl) in amber-coloured glass tubes. Proteins were then precipitated with 600 ␮L ice-cold acetonitrile. Supernatants were transferred to new glass tubes containing 600 ␮L 150 mM Na2 CO3 and 5 mM H3 BO3 , pH 9.5. 50 ␮L 6% (v/v) benzoyl chloride in acetonitrile was added under vortexing and the samples were left to incubate in the dark for 1 h. Subsequently, samples were evaporated to dryness in a HETOVAC vacuum centrifuge. Salt pellets containing the analytes were then extracted with 400 ␮L 60% (v/v) ethanol and centrifuged for 10 min at 2600 × g. The clear supernatants were transferred to UPLC-vials and analysed on a Acquity UPLC system combined with a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA). 5 ␮L of samples and standards were injected on a BEH C18, 1.7 ␮, 2.1 mm × 100 mm column kept at 35 ◦ C with the following gradient at 500 ␮L/min: 0–0.5 min: 74A/26B; 0.5–8 min: 64A/36B; 8–18 min: 56A/44B; 18–20 min: 45A/55B; 20–22 min: 10A/90B; 22.01–25 min: 74A/26B. Buffer A: 0.1% HCOOH, 10 mM NH4 HCOO, Buffer B: 0.1% HCOOH in acetontrile. Analytes were detected with ESI+ in MRM mode with the MRM transitions and tune parameters similar to those listed previously (Song et al., 2012). The quantification was performed with an external 6-point standard curve of extracted saline standards (0.9% NaCl and 50 ␮M ascorbic acid)

Please cite this article in press as: Nasser, A., et al., Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin. Neurosci. Res. (2014), http://dx.doi.org/10.1016/j.neures.2014.08.015

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with the following standard levels 1, 1/4, 1/16, 1/64, 1/256, 0 made by sequential dilution of the standard at level 1 (1600 nM DOPAC, 5HIAA, HVA, VMA and 320 nM DA, 5-HT, NA, 5HTP, MHPG). DA and NA were related to DHBA as internal standard, while 5-HT was related to d3 -5HTP and MHPG to d3 -VMA. 2.5. Determination of nitrite/nitrate in plasma Mice (n = 9–11) were sacrified by decapitation and blood collected in tubes containing K2 -EDTA (Sigma–Aldrich). Blood was centrifuged at 1000 × g for 30 min and plasma stored at −20 ◦ C. On the day of analysis plasma was thawed at 4 ◦ C, ultrafiltered through Amicon Ultra Centrifugal filters (Ultracel-10k, Millipore, Denmark) pre-rinsed three times with MiliQ water and diluted ten times with MiliQ water. The total nitrite and nitrate concentrations were measured, as an index of NO production, following the instructions of the manufacturer (Cayman Chemical Company,United States). The samples were read on Mithras LB 940 Multilabel Reader (Berthold Technologies, Germany) using an excitation wavelenght of 360 nm and an emission wavelengt of 430 nm. 2.6. Elevated zero maze (EZM) test The EZM apparatus was a ring-shaped runway (inner diameter = 47 cm) consisting of 2 open and two closed quadrants elevated 50 cm above floor level. The runway had a black plexiglass floor (2.8 cm width). The closed quadrants were surrounded by an 11 cm high clear plexiglass wall, whereas the open quadrants were flanked by a 6 mm plexiglass lip. Testing took place under dim white light (5 lux at maze level). Mice (n = 14–16) were individually placed on the maze facing a closed quadrant and allowed 300 s exploration of the apparatus. The behaviour of the mouse was scored manually by an observer situated at least 1 m from the maze. Measures recorded were: latency to enter an open quadrant; number of entries into open quadrants; time spent in open quadrants; and number of stretched-attend postures (SAPs). A SAP is a type of risk assessment behaviour in the closed quadrant, where a mouse shows an elongated body posture with its hind legs being in the closed arms and its snout and forelegs stretched into the open area (Kaesermann, 1986). A subject was counted as moving into the open quadrant when all four legs fully crossed into the open quadrant. After each test, the maze was cleaned with water and dry tissue, and faecal boli removed. Mice were not placed back into their home cage after testing until all mice in each cage were tested. 2.7. Marble burying (MB) test The MB assay was performed with transparent cages (L: 37 cm × W: 21 cm × H: 15 cm) filled with 5 cm bedding and 20 glass marbles (1.5 cm diameter), equidistantly distributed throughout the cage. Marbles were placed in a 4 × 5 row manner with no marble more than 1 cm near the border of the cage. Testing was performed under normal ambient room lighting (>350 lux). Mice (n = 14–15) were individually placed into the cage for 30 min with a transparent cage placed on top of the test cage to prevent escape. A marble was considered buried when more than 2/3rds of it was covered by bedding. The number of buried marbles was counted manually by an observer. 2.8. Tail-suspension test (TST) Mice (n = 5–9) were suspended by the tail with adhesive tape placed ∼1 cm from the tip of the tail for 260 s. Immobility time during this period was recorded by a camera placed in front of the mouse and movement was automatically monitored (Biobserve, Germany). The behaviours rated were immobility: hanging by the

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tail without engaging in any active behaviour and climbing: active upward movements of the entire body. 2.9. Forced swim test Mice (n = 15–23) were individually placed in a beaker (16 cm in diameter) filled with 20 cm water maintained at 23.5–24.5 ◦ C. Total swim distance and time immobile during a 360 s test period was automatically recorded by a camera mounted above the cylinders and stored on a computer equipped with Ethovision XT (Noldus, Holland). 2.10. Locomotor activity test Mice (n = 8–18) were individually placed for 30 min in transparent cages (L: 37 cm × W: 21 cm × H: 15 cm) with a thin layer of bedding. Total locomotor distance and velocity were automatically recorded by a camera mounted above the arenas and stored on a computer equipped with Ethovision XT (Noldus). 2.11. Experimental setup Each mouse was exposed to more than one behavioural test. The order of behavioural testing was always; (1) locomotor activity test, (2) MB, (3) EZM, (4) FST and (5) TST. The animals were allowed at least 2 days to rest between each test to minimise carryover effects between assays. 2.12. Statistical analysis Data were analysed using two-tailed unpaired t-test or two-way analysis of variance (ANOVA) with genotype and sex as independent variables, followed by pair-wise comparisons on the predicted means using the Fisher’s LSD test. Log or square root transformations were done where appropriate to obtain normality and variance homogeneity, a requirement of the ANOVA approach. Data are presented as mean + SEM. p values less than 0.05 were considered statistically significant. Statistical analysis were performed with SigmaPlot 11.0 (Systat Software Inc., United States). 3. Results 3.1. General appearance, health and motor performace of hph mice Table 1 shows the general physical appearance and behavioural responses of hph mice compared with WT mice. The physical examination revealed normal physcial appearance of hph mice (i.e. weight, whiskers and fur), normal behaviours (i.e. gait in straight line and rearing) and intact sensory reflexes (i.e. eye blink reflexes). We have previously shown that hph mice exhibit normal motor performance and absence of dystonia-like symptoms (Nasser et al., 2013). Here, spontaneous locomotor activity was evaluated in a novel open field. Fig. 1 shows the spontaneous locomotor activity of hph and WT mice. The two-way ANOVA showed no significant overall effect of genotype on both distance moved and velocity (F1,52 = 1.239, p = 0.271 and F1,52 = 2.15, p = 0.149, respectively), no significant overall difference of sex (F1,52 = 0.920, p = 0.342 and F1,52 = 0.39, p = 0.533, respectively) and no significant genotype × sex interaction (F1,52 = 0.627, p = 0.432 and F1,52 = 0.560, p = 0.458, respectively). Pair-wise comparisons revealed no difference between any of the groups.

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Table 1 General physical appearance, motor and sensory responses of wild-type and hph mice. Wild-type

hph

Physical appearance Weight (g) Whiskers (% with) Bald patches (% with) Piloerection (% with) Palpebral closure (% with) Exophthalmos (% with) Normal fur (% with) Pink footpads (% with) Respiratory infections (% with)

22.8 (0.5) 100 0 0 0 0 100 100 0

22.5 (0.3) 100 0 0 0 0 100 100 0

General behavioural observations Gait in straight line (%) Constant circling (%) Rearing (%) Frozen immobility (%) Wild running (%) Sniffing (%) Jumping (%) Defecation (%) Urination (%)

100 0 100 0 0 100 10 50 0

100 0 100 0 0 100 0 50 40

Sensorimotor reflexes % with normal response postural reflex Eye blink reflex Ear twitch reflex

100 100 100

100 100 100

Motor responses Hanging wire test Hind-paw clasping Rotarod test Locomotor activity test

na na na n

na na na n

overall effect of sex (F1,27 = 0.132, p = 0.720) and no significant genotype × sex interaction (F1,27 = 0.132, p = 0.720). Pairwise comparisons revealed a significant decrease in hippocampal DA in both male and female hph mice compared to their WT counterparts (p = 0.014 and p = 0.003, respectively). Moreover, the results revealed decreased concentrations of the DA metabolites HVA and DOPAC in hph mice compared to WT mice (data not shown). Fig. 2c shows the NA levels in hippocampus from hph and WT mice. The two-way ANOVA showed no significant overall effect of genotype on NA (F1,27 = 0.223, p = 0.640), no significant overall effect of sex (F1,27 = 2.43, p = 0.146) and no significant genotype × sex interaction (F1,27 = 2.43, p = 0.146). Pair-wise comparisons revealed no statistically significant differences in NA levels between any of the groups (p > 0.05). Additionally, the no sex or genotype differences were observed in the concentrations of the NA metabolite MHPG (data not shown). 3.3. Plasma NO levels in hph mice Fig. 3 shows the NO levels in plasma from hph and WT mice. No difference between sex was observed (data not shown), hence data obtained from male and female mice were pooled. Two-tailed unpaired t-test revealed significantly lower NO plasma levels in hph mice compared to WT mice (t18 = 2.81, p = 0.016). 3.4. Behavioural phenotype of hph mice in EZM

Animals were 8 weeks old. n = 10 in each group. Data for body weight present mean (SEM). n, normal response. a For results refer to Nasser et al. (2013).

3.2. Hippocampal monoamine levels in hph mice Fig. 2a shows the 5-HT levels in hippocampus from hph and WT mice. The two-way ANOVA showed a significant overall effect of genotype on 5-HT (F1,27 = 10.483, p = 0.003), no significant overall effect of sex (F1,27 = 0.038, p = 0.848) and no significant genotype × sex interaction (F1,27 = 0.038, p = 0.848). Pairwise comparisons revealed a significant decrease in hippocampal 5-HT in both male and female mice compared to their WT counterparts (p = 0.025 and p = 0.036, respectively). Furthermore, the concentrations of the 5-HT metabolites 5-HIAA and 5-HTP were also found to be decreased in hph mice compared to WT mice (data not shown). Fig. 2b shows the DA levels in hippocampus from hph and WT mice. The two-way ANOVA showed a significant overall effect of genotype on DA (F1,27 = 17.495, p < 0.001), no significant

12000

Total distance (cm)

(b)

14000

WT

8

hph

WT

hph

6

10000

Velocity cm/s

(a)

Fig. 4a shows the latency to enter the open quadrants in the EZM test. The two-way ANOVA showed no significant overall effect of genotype (F1,53 = 0.92, p = 0.343), no significant overall effect of sex (F1,53 = 0.32, p = 0.573) and a near-significant genotype × sex interaction (F1,53 = 3.11, p = 0.084). Pair-wise comparisons revealed no significant differences between groups, although there was a trend towards longer latency in male hph mice compared to male WT mice (p = 0.057). Fig. 4b shows the number of entries into the open quadrants. The two-way ANOVA showed no significant overall effect of genotype (F1,55 = 1.12, p = 0.294), no significant overall effect of sex (F1,55 = 0.238, p = 0.628) and no significant genotype × sex interaction (F1,55 = 0.364, p = 0.549). Pair-wise comparisons revealed no difference between any of the groups. Fig. 4c shows the percentage time spent in the open quadrants. The two-way ANOVA showed no significant overall effect of genotype (F1,55 = 0.482, p = 0.491), a significant overall effect of sex (F1,55 = 16.003, p < 0.001) and a significant genotype × sex interaction (F1,55 = 8.469, p = 0.005). Pair-wise comparisons revealed that female WT mice spent significantly less time in the open quadrants than male WT mice (p < 0.001), whereas no sex difference was seen in hph mice (p = 0.439). Moreover, male hph mice spent

8000 6000 4000

4

2

2000 0

0 Male

Female

Male

Female

Fig. 1. Spontaneous locomotor activity of hph mice. (a) Total distance (cm) moved and (b) movement velocity (cm/s). The hph mice showed no difference in locomotor activity in a novel open field compared to WT mice (p = 0.271). Moreover, no difference between sex was found (p = 0.342). n = 8–18. Two-way ANOVA with pair-wise comparisons using the Fisher’s LSD test. Data are presented as mean + SEM.

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(a)

Plasma NOx

Hippocampal 5-HT

125

% of WT

100

*

*

WT hph

60 40

(b)

Male

Female

120

% of WT

*

*

80

hph

3.5. Behavioural phenotype of hph mice in MB Fig. 5 shows the MB behaviour of female and male hph mice and WT mice. The two-way ANOVA showed a significant overall effect of genotype (F1,54 = 9.120, p = 0.004), no significant overall effect of sex (F1,54 = 1.525, p = 0.222), and no significant genotype × sex interaction (F1,54 = 0.887, p = 0.351). Pair-wise comparisons revealed that male hph mice buried significantly lower number of marbles compared to male WT mice (p = 0.009), whereas the corresponding difference in female mice did not reach statistical significance (p = 0.134).

**

60 40 20 Male

WT

Fig. 3. NO levels in plasma of WT and hph mice. NO was measured as the total concentration of nitrite and nitrate. No difference in sex was observed, hence data from male and female mice was pooled. A significant decrease in NO production was found in hph mice compared to WT mice (* p = 0.016). n = 9–11. Two-tailed unpaired t-test. Data are presented as mean + SEM.

WT hph

100

Female

3.6. Behavioural phenotype of hph mice in TST

(c)

Hippocampal NA 120

WT hph

100

% of WT

50

0

Hippocampal DA

0

75

25

20 0

100

% of WT

120

80

5

80 60 40 20 0

Male

Female

Fig. 2. Monoamine levels in hippocampus of WT and hph mice. (a) Serotonin (5-HT), (b) dopamine (DA) and (c) noradrenaline (NA). The hph mice exhibited significantly lower 5-HT (p = 0.003) and DA (p < 0.001) levels, but intact NA levels (p = 0.640), when compared to WT mice. Moreover, no difference between sex was found (p > 0.05). *p < 0.05 and **p < 0.01 versus WT mice. n = 6–9. Two-way ANOVA with pair-wise comparisons using the Fisher’s LSD test. Data are presented as the percent of WT levels and the error bar represents + SEM.

significantly less time in the open quadrants compared to male WT mice (p = 0.018), while an opposite tendency was observed in females, although this did not reach statistical significance (p = 0.107). Fig. 4d shows the number of SAPs. The two-way ANOVA showed a significant overall effect of genotype (F1,55 = 10.30, p = 0.002), no significant overall effect of sex (F1,55 = 0.205, p = 0.653) and no significant genotype × sex interaction (F1,55 = 0.058, p = 0.811). Pair-wise comparisons revealed that both male and female hph mice displayed more SAPs compared to WT controls (p = 0.021 and p = 0.036, respectively).

Fig. 6a shows the immobility time in the TST. The twoway ANOVA showed a significant overall effect of genotype (F1,24 = 16.89, p < 0.001), no significant overall difference of sex (F1,24 = 0.26, p = 0.615) and no significant genotype × sex interaction (F1,24 = 1.29, p = 0.268). Pair-wise comparisons revealed that male and female hph mice spent significantly more time immobile than WT counterparts (p = 0.033 and p = 0.002, respectively). Fig. 6b shows the climbing behaviour in the TST. The twoway ANOVA revealed a significant overall effect of genotype (F1,24 = 13.38, p = 0.001), no significant overall difference of sex (F1,24 = 0.792, p = 0.382) and no significant genotype × sex interaction (F1,24 = 0.10, p = 0.754). Pair-wise comparisons revealed that male and female hph mice spent significantly less time climbing compared to WT controls (p = 0.018 and p = 0.014, respectively). 3.7. Behavioural phenotype of hph mice in FST Fig. 7a shows the swimming behaviour of hph and WT mice. The two-way ANOVA showed no significant overall effect of genotype on both distance (F1,69 = 0.78, p = 0.382) and velocity (F1,69 = 0.417, p = 0.521), a significant overall effect of sex on distance (F1,69 = 11.72, p = 0.001) and velocity (F1,69 = 12.1, p < 0.001), and a significant genotype × sex interaction on distance (F1,69 = 11.13, p = 0.001) and velocity (F1,69 = 9.27, p = 0.003). Pair-wise comparisons revealed that female hph mice exhibited significantly higher swim distance (p < 0.001) and velocity (p < 0.001) than male hph mice, whereas no sex difference was seen in WT mice (p = 0.952 and p = 0.763, respectively). The significant genotype × sex interaction is borne out by subsequent pair-wise comparisons showing that male hph mice displayed a trend towards lower swim distance (p = 0.064) and velocity (p = 0.071) compared to male WT mice, whereas female hph mice

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Fig. 4. Behavioural phenotype of hph mice in the elevated zero maze test. (a) Latency (s); male hph mice showed a trend towards longer latency to enter the open arena than male WT mice (p = 0.057), a difference that was not significant in hph female mice. (b) Number of entries; male and female mutant mice displayed no significant difference in the number of entries into the open arena. (c) Time in open arena (%); male hph mice spend significantly less time in the open arena than male WT mice (*p = 0.018). In addition, a statistical significant sex difference in time in open arena was found in WT animals (### p < 0.001). (d) Number of SAPs: male and female hph mice exhibited lower numbers of SAPs compared to WT counterparts (*p = 0.021 and *p = 0.036, respectively). n = 14–16. Two-way ANOVA with pair-wise comparisons using the Fisher’s LSD test. Data are presented as mean + SEM. NS, non-significant.

showed significantly higher swim distance (p = 0.007) and velocity (p = 0.017) compared to female WT mice. Fig. 7b shows the immobility time in the FST. The twoway ANOVA showed no significant overall effect of genotype (F1,69 = 0.47, p = 0.495), a significant overall difference of sex (F1,69 = 9.89, p = 0.002) and a significant genotype × sex interaction (F1,69 = 9.20, p = 0.003). Pair-wise comparisons revealed that female hph mice displayed significantly lower immobility time compared to male hph mice (p < 0.001), while no sex difference was found between WT animals (p = 0.937). Furthermore, the significant genotype × sex interaction is borne out by the pair-wise comparisons revealing that female hph mice were significantly less immobile than female WT mice (p = 0.017), while there was a near-significant

Fig. 5. Marble burying behaviour of hph mice. The number of marbles buried was recorded during 30 min. The data show that hph-1 mice buried significantly lower numbers of marbles than WT mice (p = 0.004). Pairwise comparisons revealed significant differences between male hph-1 mice and male WT mice (**p = 0.009), and a non-significant difference between female mice (p = 0.134). No difference in marble burying behaviour was found between sex (p = 0.222). n = 14–15. Two-way ANOVA with pair-wise comparisons using the Fisher’s LSD test. Data are presented as mean + SEM. NS, non-significant.

trend towards higher immobility time in male hph mice compared to male WT mice (p = 0.077). 4. Discussion In this study, we used genetically modified male and female hph mice exhibiting a mutation in the Gch1 locus to study the consequence of congenital BH4 deficiency on anxiety- and depression-like behaviours. We found that the behavioural phenotype of mutant mice was distinct from that of WT counterparts, although the differences appeared both test- and sex-dependent. 4.1. Hippocampal monoamine levels in hph mice Male and female hph mice displayed lower levels of hippocampal 5-HT (25% decrease) and DA (44% decrease) as well as their metabolites, when compared to WT mice. Differences in hippocampal NA level were not statistically significant, showing only 17% NA decrease in male hph mice and 8% NA increase in female hph mice. These findings indicate that the greatest influence of BH4 deficiency in hph-1 mice is on 5-HT and DA biosynthesis, consistent with previous work showing reduced brain 5-HT, DA and NA levels in hph-1 mice, with the order of decrement being 5-HT > DA > NA (Hyland et al., 1996). Considering the role of hippocampus in both anxiety and depression, and that deficiencies in monoamine signalling are associated with affective disorders, the present findings suggest a role for reduced 5-HT and DA in the anxiety- and depression-like phenotypes of hph mice. In order to verify that the behavioural alterations are indeed owing to monoamine and BH4 defiency in the brain, further studies should examine the effects of supplementation of monoamine precursors and BH4. It is known that hph-1 mutants exhibit a transient neonatal hyperphenylalaninemia peaking at day 6 and remaining elevated

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Fig. 6. Behavioural phenotype of hph mice in the tail suspension test. (a) immobility time (s); the data show that male and female hph mice spent significantly more time immobile than WT controls (*p = 0.033 and **p = 0.002, respectively). (b) time spent climbing (s); male and female hph mice spent significantly lower time climbing as compared to WT controls (*p = 0.018 and *p = 0.014, respectively). No sex difference was found in any of the behaviours (p > 0.05). n = 5–9. Two-way ANOVA with pair-wise comparisons using the Fisher’s LSD test. Data are presented as mean + SEM.

until the age of 3 weeks (McDonald and Bode, 1988). Since hyperphenylalaninemia affects both behaviour and brain monoamine levels (Pascucci et al., 2013), the possibility that hyperphenylalaninemia contributes to the reduced brain monoamine levels and the changes in anxiety- and depression-like behaviours in hph-1 mice should be considered. Notably, mice used in this study were >7 weeks old, and human studies show no effect of hyperphenylalaninemia on monoamine levels after normalisation of plasma phenylalanine (Guttler and Lou, 1986). While this would suggest that monoamine deficiency is a direct result of decreased cofactor availability, it cannot be ruled out that hyperphenylalaninemia early in life could have long-term effects on monoamine levels and behaviour. 4.2. Phenotype of hph mice in the EZM and MB The increased anxiety-like response of hph mice, particularly male mice, observed in the EZM possibly reflects the decreased 5-HT and DA levels observed. However, since BH4 is also a cofactor in the formation of NO, reduced NO levels in hph mice may also contribute to the altered emotional response. Indeed, hph mice in this study displayed ∼75% lower plasma nitrite and nitrate concentrations, an index of NO production, compared to WT mice, which is in line with other studies showing markedly reduced NO levels in hph-1 mice (Barker et al., 1998; Lam et al., 2007). In the elevated plus maze test (EPM, the precurser to the EZM), neuronal NO synthase knock-out mice displayed increased anxiety-like behaviours (Weitzdoerfer et al., 2004), and studies in both rats and mice have reported anxiogenic-like effects of NOS inhibitors in the EPM (De Oliveira et al., 1997; Vale et al., 1998; Zarrindast et al., 2013). While this could indicate that the increased anxiety-like behaviour observed in hph mice is linked to low NO levels, other studies have shown anxiolytic-like properties of NOS inhibitors (Guimarães et al., 1994; Volke et al., 1995; Faria et al., 1997; Spiacci et al., 2008). In some studies, the anxiolytic-like effect of NOS inhibitors was U-shaped, with low doses reducing and high doses promoting anxiety-like responses. It should be mentioned that most studies have tested the acute effects of NOS inhibition. The consequence of long-lasting NO reduction exhibited by hph mice is perhaps more comparable to neuronal NOS knock-out mice where increased (Weitzdoerfer et al., 2004) and decreased (Zhang et al., 2010) anxiety-like responses have been reported. Further studies are needed to ascertain if persistent and marked reduction of brain production of NO in hph mice contributes to the anxiogenic-like responses observed in the EZM test. In the MB test, hph mice showed reduced digging compared to WT mice, in particular males, indicating a reduced anxiety-like response in this paradigm. Since NOS inhibitors exert anxiolyticlike effects in the MB test (Umathe et al., 2009; Krass et al., 2010), decreased brain NO production in hph mice could explain the

anxiolytic-like effects observed in the MB test. It is notable that the response of hph mice, as well as the reported effects of NOS inhibitors, in the MB test appears to contradict the evidence from the EPM and EZM. The reasons for the opposite direction in these behavioural paradigms is not immediately clear. Rodent tests of anxiety show qualitative differences, in which each test reflect overlapping, but partially distinct anxiety-like traits (Cryan and Holmes, 2005; Thomas et al., 2009), and the generation of distinct anxiety-like behaviours might involve different neuroanatomical and neurochemical pathways (Cheeta et al., 2000). Specifically, behaviours in the EZM are contingent on rodent’s inherent motivation to explore novel environments (Sousa et al., 2006). The MB test may have a closer resemblance with compulsive behaviour rather than reflecting anxiety per se, which was recently re-classified to no longer be defined as an anxiety disorder (DSM-5). Irrespective of the construct that the MB test is purported to measure, it is highly sensitive to drugs that enhance 5-HT neurotransmission. For instance, selective serotonin re-uptake inhibitors, which are used as first-line treatment in anxiety disorders and obsessive–compulsive disorder (Koen and Stein, 2011), consistently decrease digging behaviour in the MB test (Hirano et al., 2005; Egashira et al., 2007; Albelda and Joel, 2012). Considering that hph mice showed lower 5-HT levels than WT mice, the reduced digging behaviour in the MB test is unlikely to be explained by changes in 5-HT levels, and more likely to result from the decreased NO production, given the evidence that NOS inhibition reduces digging behaviour (Umathe et al., 2009; Krass et al., 2010). 4.3. Phenotype of hph mice in the TST and FST In the TST, both male and female hph mice exhibited increased depression-like phenotypes compared to WT mice. Additionally, the trend towards decreased swimming behaviour in male hph mice suggest a heigthened depression-like phenotype of hph mice. These findings may reflect decreased biosynthesis of 5HT and DA, since both genetic and pharmacological depletion of these monoamines produces depression-like responses in the TST ´ (OLeary et al., 2007; Beaulieu et al., 2008; Savelieva et al., 2008) and FST (Nagakura et al., 2009; Mosienko et al., 2012). Several studies have reported antidepressant-like effects of both pharmacological (Harkin et al., 1999; da Silva et al., 2000; Heiberg et al., 2002; Volke et al., 2003) and genetic (Salchner et al., 2004; Zhou et al., 2007) inhibition of NOS in TST and FST. However, similar to the EZM test, NO seems to have a dual role in these tests (da Silva et al., 2000; Inan et al., 2004). For instance administration of the NO precursor L-arginine evoked increased and decreased immobility time at low and high doses in FST, respectively, and inhibition of NOS decreased and increased immobility time following injection of low and high doses, respectively (Inan et al., 2004). NO is a crucial signalling molecule in many physiological systems and its dual effects is also

Please cite this article in press as: Nasser, A., et al., Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin. Neurosci. Res. (2014), http://dx.doi.org/10.1016/j.neures.2014.08.015

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such changes are both sex- and test-dependent. Despite their face value similarity, the FST and TST measure activity involving partially different neuronal mechanisms (Bai et al., 2001; Renard et al., 2003). These tests have shown differential sensitivities to drugs exploiting both monoamine (Bai et al., 2001; Renard et al., 2003) and non-monoamine mechanisms (Andreasen and Redrobe, 2009; Andreasen et al., 2013). Moreover, baseline and drug responses in these two tests depend on both genotype and sex (Andreasen and Redrobe, 2009). Studies employing more translational models of depression are needed to further charachterize the phenotype and to clarify the translational value of hph-1 mice in relation to depression. 4.4. Motor performance of hph mice The hph-1 mouse model is proposed to be a model of DRD. This is based on comparable biochemical features such as decreased GCH1 expression and GTP-CH1 activity, as well as reduced biosynthesis of BH4 and monoamines (Hyland et al., 2003). The prominent phenotype of DRD patients is gait problems due to dystonia. Therefore, reduced BH4 in mice may also yield effects on motor behaviour, which can confound the anxiety- and depression-like behaviours in these animals. In the current study, locomotor activity of hph mice was evaluated in a novel open field, and here both distance travelled and velocity was not different between genotypes, indicating intact spontaneous motor function in mutant mice. We have previously reported that hph showed no difference in the rotarod test and the hanging wire test as well as lack of dytonia-like symptoms compared to WT mice (Nasser et al., 2013). Also, hph mice exhibited normal physical features, general behavioural observations and sensory reflexes. Based on these observations, the differences in behavioural responses observed are not confounded by effects on motor performance or general condition of the animals. 4.5. Summary and conclusion

Fig. 7. Behavioural phenotype of hph mice in the forced swim test. (a) Total distance (m) swimmed and swimming velocity (cm/s); male hph mice showed lower swimming behaviour compared to male WT mice that was almost statistical significant (p = 0.064), whereas female hph mice displayed significantly higher swimming behaviour than female WT mice (**p = 0.007). Female hph mice also manifested with significantly higher swimming behaviour than male hph mice (### p < 0.001). (b) Immobility time (s); female hph mice were significantly less immobile than female WT mice (*p = 0.017), whereas a trend for higher immobility time was seen in male hph mice compared to male WT mice (p = 0.077). Female hph mice also displayed significantly lower immobility time compared to male hph mice (### p < 0.001). n = 15–23. Two-way ANOVA with pair-wise comparisons using the Fisher’s LSD test. Data are presented as mean + SEM. NS, non-significant.

well-documented in other conditions such as pain (Miclescu and Gordh, 2009). Further studies are needed to deliniate the relative roles of NO and monoamines in mediating the genotype differences in FST and TST phenotype observed here. Whereas the TST response suggested a depression-like phenotype of both male and female hph mice, the FST response was sex-dependent: male hph mice showed a depression-like trend, corroborating the TST results, but female hph mice exhibited significantly less depression-like responses compared to female WT mice in the FST. We did not find sex differences in hippocampal biosynthesis of monoamines or NO production in plasma. Given that behavioural changes in hph-1 mice are attributable to reductions in monoamines and/or NO, the current data suggest that

Several studies have reported increased prevalence of anxiety and major depression in DRD patients (Hahn et al., 2001; Van Hove et al., 2006; Trender-Gerhard et al., 2009; Tadic et al., 2012). The current study demonstrated that the affective distubances seen in DRD patients partly translates back to the hph-1 mice. Though, the direction of the responses was both sex- and test-dependent and less uniform than that observed in DRD patients. Therefore further studies are needed to evaluate if this mouse model is applicable as a tool to reveal the role of BH4 in affective disturbances associated with DRD. In conclusion, the present study provides, to our knowledge, the first evidence that inherited BH4 deficiency is associated with altered anxiety- and depression-like behaviours in mice. We suggest that this is probably related to changed availability of brain 5-HT, DA and NO. Finally, we propose that the hph-1 mouse model may be used to provide insights into the role of congenital BH4 deficiency in affective disorders. Conflict of interest The authors declare that they have no financial or non-financial conflict of interests. Acknowledgements This work was supported by a research grant from Augustinus Foundation to Arafat Nasser and by a research grant from Lundbeck Foundation (Vort J.nr. R83-A7690) to Lisbeth B. Møller and Arafat Nasser. The foundations had no involvement in the study design or

Please cite this article in press as: Nasser, A., et al., Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin. Neurosci. Res. (2014), http://dx.doi.org/10.1016/j.neures.2014.08.015

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in the collection, analysis and interpretation of the data. Moreover, they had no impact on the preparation of this paper.

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Please cite this article in press as: Nasser, A., et al., Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin. Neurosci. Res. (2014), http://dx.doi.org/10.1016/j.neures.2014.08.015