© Kalueff A.V., Minasyan A., Lou Y.-R., Lakssi I., Tuohimaa P., 2005

WHAT CAN WE LEARN FROM

the mutant mice about the possible link between neurosteroid Vitamin D and anxiety?

A.V. KALUEFF, A. MINASYAN, Y.-R. LOU, I. LAKSSI, P. TUOHIMAA

Medical School, University of Tampere, Tampere University Hospital, Tampere, Finland

Калуев А.В., Минасян А., Лоу Я-Р., Лаксси И., Туохимаа П.

Что можно узнать из поведения му-тантных мышей о возможной связи между нейростероидным гормоном витамином Д и тревожностью? // Психофармакол. и биол. наркол. 2005. Т. 5. № 2. С. 930-938. Медицинская школа и Университетский госпиталь, Тампере, Финляндия.

В статье рассматриваются собственные и литературные данные о поведенческих дисфункциях у мышей с нарушениями в системе витамина Д, вызванными мутацией ядерного рецептора (VDR) данного гормона. С использованием методов поведенческого фенотипирова-ния и этологического анализа показано, что мутация VDR приводит к целостным поведенческим

изменениям у мышеи, подтверждающим их тревожный поведенческий профиль. В работе анализируются поведенческие особенности мутантных мышей по гену УБИ и обсуждается возможная роль нейростероидного гормона витамина Д в патогенезе тревожности у человека и животных.

V

Ключевые слова: витамин Д, нейростероиды, тревожность, поведение.

Kalueff A.V., Minasyan A., Lou Y.-R., Lakssi I., Tuohimaa P. What can we learn from the mutant mice about the possible link between neurosteroid Vitamin D and anxiety? // Psychopharmacol. Biol. Narcol. 2005. Vol. 5. N 2. P. 930-938. Medical School, University of Tampere,

Tampere University Hospital, Tampere, Finland.

Here we analyse in detail our own and literature data showing behavioural anomalies in mice with genetically ablated VDR — nuclear receptor of a steroid hormone vitamin D (recently emerging as a potent neuro-active, or even neurosteroid, hormone). Using detailed behavioural phenotyping in a battery of tests and subsequent neuroethological analysis, we established that mice with ablated VDR generally display more anxiety, suggesting the potential role of the vitamin D/VDR system in anxiety disorder in both animals and humans.

Key words: vitamin D, neuro-steroids, anxiety, behaviour.

1. INTRODUCTION

Vitamin D hormone is essential for growth and dif ferentiation in a variety of organs, including the brain (Garcion et al., 2002; Carswell, 1997; Yoshizawa et al., 1997). Numerous data suggest that vitamin D plays an important role in the brain through induction of key CNS genes, modulation of neuroprotection, neuro-trophins release and activity of key neurotransmitter metabolism enzymes (Garcion et al., 2002; Carswell, 1997; Altemus et al., 1987). The functions of vitamin D are mediated through nuclear vitamin D receptors (VDR, a member of the nuclear receptors superfami-ly of ligand-activated transcription factors), which are widely distributed through the nervous system (Garcion et al., 2002; Yoshizawa et al., 1997). VDR is a 50-60 kDa protein, consisting of several functional domains responsible for ligand and DNA binding, heterodimerization, nuclear localization and transcriptional activation (Endo et al., 2003).

Evidence for the presence of vitamin D, the enzymes of its bioactivation and metabolism and functional VDR in the brain implies that this vitamin may have some function in the CNS as an autocrine or paracrine neurosteroid hormone (Garcion et al., 2002). VDR are found in key brain areas including the cortex, cerebellum and limbic system, all known to regulate behaviour (Prufer et al., 1999; Walbert et al., 2001; Langub et al., 2001). In humans, vitamin D deficiency has long been known to be accompanied by irritability, depression, psychoses and defects in mental development (Carswell, 1997). The psychotropic mood-elevating effects of vitamin D have also been well-documented in the literature (Gloth et al., 2001).

In animals, vitamin D deficiency produces behavioural alterations including decreased exploration and maze performance (Altemus et al., 1987; also see recent interesting behavioural data in: Burne et al. 2004). Collectively, this suggests that vitamin D could be an important factor controlling behaviour in animals and

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humans. As such, various vitamin D and VDR-related disorders may therefore be a risk factor for abnormal emotional behaviour in both animals and humans. In the present paper, we will discuss data suggesting that genetic ablation of VDR in mice may strongly affect their behaviours, especially anxiety.

2. INCREASED ANXIETY IN VDR NULL MUTANT MICE: PILOT STUDY IN A BATTERY OF TESTS

In our pioneering studies (Kalueff et al., 2004a,b) we assessed the behavioural performance of VDR null mutant mice tested in several tests of anxiety and activity. Subjects were adult mice (aged 24—52 weeks) bred in the University of Tampere (Finland). VDR gene mutant mice were bred in Tampere from the line initially generated in the University of Tokyo (Japan). 129S1 mouse substrain was used as a genetic background for these mutant mice.

Since genetically modified animals often demonstrate motor and sensory abnormalities which may non-specifically affect behaviour (Crawley, 1999), we first tested the general locomotion and sensory abilities of VDR null mutant mice (Kalueff et al., 2004a). Assessment of locomotor activity in the actometer test revealed that VDR null mutant, heterozygous and the wild type mice had similar baseline levels of horizontal and vertical activity. All three groups had unaltered major sensory (visual/olfactory) abilities as assessed on the food- and novel object-finding tests, and motor coordination (Kalueff et al., 2004a-c). In addition, all three groups seemed to have unaltered vestibular system as assessed on the horizontal bar test (although this does not preclude some other forms of vestibular anomalies).

However, marked behavioural alterations in VDR null mutants were found when the mice were subjected to a battery of anxiety tests. In the holeboard test, VDR null mutants demonstrated decreased horizontal and vertical activity, compared to the wild type and heterozygous mice. In the open field, compared to the wild type and heterozygous control groups, VDR null mutants demonstrated decreased horizontal and vertical exploration, as well as increased duration of grooming. In the elevated plus maze, compared to the wild type and heterozygous groups, VDR null mutant mice demonstrated a dramatic decrease in vertical activity as well as longer latency to leave the central area. Finally, in the light-dark transition test, VDR null mutant mice showed less exploration of the light compartment than did their littermates from both control groups (Kalu-eff et al., 2004a).

Calcium levels

Since vitamin D plays an important role in the regulation of Ca++ homeostasis, a key factor likely to affect motor performance in mutant mice can be dysregulation

of Ca++ homeostasis, which is usually associated with vitamin D-related disorders (Carswell, 1997; Langub et al., 2001). To test this hypothesis, in a separate experiment we measured plasma Ca++ level in these mutant mice. Indeed, despite a special rich Ca++ diet, they mice had generally lower plasma Ca2+ (2.04 ± 0.08 mmol/l, P < 0.05; n = 6), compared to 2.28 ± 0.10 mmol/l in the wild-type (n = 3) and 2.27 ± 0.48 mmol/l in heterozygous (n = 7) mice (Kalueff et al., 2004b).

3. ANXIOGENIC PROFILE IN THE PROTECTED ELEVATED PLUS MAZE

In a separate study, to further assess anxiety in VDR mutants, we subjected these mice to the protected elevated plus maze test. This modification of the regular elevated plus maze was made from plywood and consisted of two open arms with small side-walls (20 x 10 x 0.5 cm) and two enclosed arms (20 х 10 x 30 cm) extending from a common central region (10 x 10 cm) elevated to a height of 50 cm. The opposite walls of both enclosed arms were interconnected in the central region, forming two «doors» (7 x 7 cm each) leading to the open arms. Since this modification of the maze used smaller arms, lower height, opaque walls, interconnected walls of the closed arms, and special protective 0.5-cm walls in the open arms, this model generally utilizes less anxiogen-ic stimuli (compared to the transparent apparatus used in the previous experiments), and therefore was considered to be less stressful.

Since our previous experiments have shown 1) similar behavioural profiles of heterozygous and wild type animals in the behavioural tests including elevated plus maze, 2) robust behavioural differences of both these groups from the VDR mutant mice, and 3) no sex differences between VDR mutant male and females (own unpublished observation), in this experiment we compared 10 VDR null mutant mice (6 males, 4 females) to 10 conrol mice (4 heterozygous and 1 wild type male, 3 heterozygous and 2 wild type female mice) tested in the protected elevated plus maze. The following behaviours were recorded for 5 min: head dipping from the open arms; the number of open, closed and total arm entries, vertical rears, vegetative behaviours (urination episodes + defecation boli), the number of grooming bouts and time spent grooming (s).

Overall, in the protected elevated plus maze, the VDR null mutant mice demonstrated significantly lower exploration as assessed by head dipping behaviour, open entries and vertical activity (Fig. 1) compared to their controls. These behavioural responses are generally consistent with higher anxiety profile in the VDR null mutant animals. In addition, as can be seen in Fig. 1, these mutant mice showed fewer closed entries and total entries, unaltered defecation/urination scores and grooming bouts, and a tendency to more grooming duration (P > 0.05, U-test). Importantly, the ratio

in the central part of the test, and spend more time in the center. In addition, 129 mice are known to show unusual center > open > closed arm preference in the plus maze. In line with this, Burne et al. (2005) showed that wild type and mutant mice spent 53 and 59% test time in the center, respectively (also see similar phenomenon in the Y-maze in the same study). This observation clearly indicates robust «background» effect of their findings which, in our opinion, may «mask» all potential mutation-evoked behavioural alterations in these mice, and therefore may not be interpreted as the lack of anxiety. The use of the protected elevated plus maze (where the visible cental area is lacking due to the presence of walls interconnecting the opposite closed arms, thus representing a part of the closed arms area) allowed us to minimize the impact of the cental area on mouse behaviours (Fig. 1) and obtain less confounded plus maze data. Indeed, as can be seen in Fig. 1, the robust reduction of arm exploration and head dips clearly supports the notion that VDR null mutant mice display high anxiety. Furthermore, while the control mice showed almost equal number of open and closed entries (most likely reflecting strong «background» effect), the VDR null mutants displayed a tendency to fewer open entries, lower open/closed and open/total entries, also consistent with their high anxiety phenotype reported previously (Kalueff et al., 2004a).

Another behavioural feature of all 129 mice is the robust tendency to avoid enclosed arms of the maze — the phenomenon which can be mistakenly interpreted as «anxiolysis». Taken together, this again indicates that the elevated plus maze may not be a valid model to study behaviours of VDR mutant mice, especially possessing portions of the 129 strain genome.

Similar conclusion may apply to the holeboard test — another highly sensitive behavioural paradigm widely used in behavioural neurogenetics (Crawley, 1999; Kalueff and Tuohimaa, 2004a). Both groups studying VDR mutant mice (Kalueff et al., 2004a, Burne et al., 2005) failed to detect any alteration in the specific anxiety-sensitive exploratory activity — nose poking. Inconsistent with traditional anxiety phenotype, this observation allowed Burne et al. (2005) to question the idea of high anxiety phenotype of these mice reported in our experiments. However, this notion does not consider the important role of whiskers in nose-poking behaviours in rodents, and the fact that VDR mutant mice (but not their wild type counterparts) have no whiskers. Clearly, animals without whiskers will differ in the holeboard test, demonstrating altered nose-poking activity. In addition, specific behavioural features of «background» 129 mice (e. g., high initial anxiety, frequent freezing and general hypoactivity of this strain; see Kalu-eff and Tuohimaa, 2004b for details) may also dramatically affect their hole-poking activity, thus, making this well-validated behavioural paradigm inappropriate for the study of VDR mutant mice. Taken together, this suggests that the «lack» of anxiety response of VDR mutant mice tested in the holeboard test may

be due to poor ability of this model to detect anxiety in these animals.

Finally, the importance of assessing general physiological status, locomotor, vestibular and sensory systems is widely recognized as a key part of behavioural analysis of mutant animals (Crawley et al., 1999). VDR deficiency in knockout mice has recently been reported to yield aberrant musculoskeletal development as well as rickets-like bone malformations (Yoshizawa et al., 1997; Endo et al., 2003). Using different behavioural tests we show that, despite these abnormalities, VDR null mutant mice exhibited unaltered baseline motor activity, movements coordination and sensory abilities. This observation is in line with previously published works showing no obvious motor and neurological abnormalities in VDR-deficient mice (Li et al., 1997; Yoshizawa et al., 1997). In addition, special Ca++-rich diet used in our study (known to normalize all major physiological functions and reduce or eliminate muscular abnormalities in VDR mutants) also led to sufficient normalization of Ca++ plasma levels in mice tested in our behavioural experiments (Kalueff et al., 2004b).

In contrast, Burne et al. (2005) found aberrant motor functions in VDR null mutant mice, including poor rotaroid performance and a shorter stride length, suggesting that these mice may have severely impaired muscular and motor finctions rather than anxiety phenotype. We shall note, however, one principally important methodological difference between the two studies. While we used special Ca++/Ph-rich diet with 20% lactose (Kalueff et al., 2004b), Burne et al. (2005) used water supplemented with 2mM Ca++. Since lactose markedly increases Ca++ absorbtion from food, we may suggest that our diet was more effective to normalize mineral homeostasis in VDR mutant mice. Indeed, while our null mutant mice showed only moderate 11% hypocalcemia (2.04 vs 2.28 mmol/l in controls), the mice used by Burne et al. (2005) displayed markedly lower plasma Ca++ (1.7 vs. 2.5 mmol/ l in controls). In our opinion, this 0.8-mmol/l difference in Ca++ levels between both genotypes may be considered as severe hypocalcemia (32%), representing a serious factor non-specifically impeding all behaviours in VDR null mutant mice. Therefore, motor impairments seen by Burne et al. (2005) may indeed be due to Ca++-dependent imbalance in these mice, possibly due to insufficient Ca++ supplementation. However, these data to not contradict our findings. Indeed, the difference in plasma Ca++ between genotypes observed in our experiments (0.24 mmol/l) was almost 3-fold lower than in (Burne et al., 2005). We may suggest that this mild hypocalcemia may not lead to dramatical behavioural impairment in the VDR mutant group (Kalueff et al., 2004a), therefore enabling more specific assessment of behavioural phenotypes of these mutant mice. However, both cited studes support the notion that mineral homeostasis affected by VDR genetic ablation may strongly affect animal behavioural performance.

Leaving the central zone

Movements during the novelty exploration

Different spatial strategies used by the VDR mutant (left) and control mice (right) in the unfamiliar novel arena

these behavioural observations are consistent with high anxiety phenotype reported for mice lacking VDR.

5. HABITUATION IN VDR NULL MUTANT MICE

In their recent comprehensive study, Burne et al. (2005) described the phenomenon of markedly impaired within-session habituation in VDR null mutant mice subjected to the open field novelty test. In contrast, the wild type controls demonstrated gradual reduction of their horizontal activity measures (distance traveled) over a 5-min test interval. Interestingly, these mutant mice also showed reduced habituation in the accoustic startle chambers, suggesting that VDR may be involved in the regulation of habituation (Burne et al., 2005).

Analysing these interesting behavioural observations, we speculated that impaired open field haitua-tion in VDR mutant mice are related to their altered exploratory strategies, and may be explained by underlying anxiety phenotype. Given our recent data showing altered spatial strategies of these mice (Fig. 2), this possibility seems indeed likely. For example, while unimpaired within-trial habituation implies relatively normal exploration strategies (commonly seen in various rodent species in the open field tests), poor habituation in this test (especially accompanied by lover exploratory activity, e.g., Burne et al., 2005), in our opinion, may reflect abnormal «anxious» novelty-coping strategy. Can this be the case for our VDR mutant mice? Indeed, it is possible to assume that VDR mutants do not reduce their exploration during the test because they are already highly anxious and do not want to fully explore the potentially dangerous

environment. In contrast, they may prefer to use a highly conservative «cautious» strategy maintaining their exploration at a «safe» constantly low levels during the test.

In order to investigate this problem in detail, in a separate experiment we replicated the original Burne et al. (2005) habituation experiments, ethologically dissecting a wide spectrum of exploratory and non-exploratory open field behaviours in 10 VDR null mutant mice. We also chose to use a longer, 10-min observation time, allowing us to more fully assess the mouse habituation responses. The following behavioural measures (per min) were recorded for 10 min:

1) horizontal exploration (squares visited with 4 paws);

2) total vertical exploration (including «protected» and «unprotected» vertical rears);

3) «protected» vertical rears (wall-leanings with forepaws touching the walls);

4) «unprotected» vertical rears (with forepaws in the air);

5) stopping activity (the number of exploratory stops > 2 s);

6) grooming activity frequency (the number of grooming episodes); and

7) grooming duration (s).

Overall, our experiments using a 10-min open field test confirm impaired habituation in the VDR null mutant mice previously described for horizontal activity in a shorter 5-min version of the test (Burne et al., 2005). As can be seen in Fig. 3, during the first 5 min and during the whole 10-min test, these mice maintained constant exploratory activity (13-15 squares/ min, «9-12% of total activity scored). In addition, a strikingly constant (7-8 stops/min) level of stopping activity was observed in this group during the test (data not shown). Since stops represent an important exploratory behaviour in rodents, including mice (Golani et al., 1993; Drai et al., 2001) subjected to novel open arenas, the lack of their habituation in the VDR mutant mice may again support the idea of their abnormal exploratory strategy in the open field and other similar anxiety tests.

In contrast, total vertical activity of these mutant animals was very low at the beginning of the test (2-4 squares/min, «3-5%) but gradually increased over the time reaching 9-14% (min 3-10). Similar effects were observed for other vertical activity measures (wall leanings, «unprotected» rears, Fig. 3). This suggests that the initial anxiety immediately after novelty exposure was very high (dramatically reducing vertical exploration of the VDR mutant group) but gradually decreased as the animals become more familiar with the environment.

On one hand, this observation further confirms our hypothesis that robust drop of vertical activity is the most specific pattern of emotional reactivity in the VDR mutant mice (Kalueff et al., 2004a). On the other hand, this clearly demonstrates that VDR mutant mice become more motivated to explore (and therefore less anxious) as they become more familiar with the environment. Interestingly, this phenomenon was

most robust for «unprotected» vertical rears reflecting a typical exploratory activity particularly sensitive to anxiety. Indeed, this behaviour reached maximal levels (»15-17%) by the end of the test (min 910), Fig. 3. Finally, grooming activity measures (frequency and duration) varied widely during the test, failing to reveal any behavioural tendency (data not shown). This observation is in line with our hypothesis (see further chapter in this book) that grooming in the VDR null mutant mice may represent a separate behavioural domain unlikely to reflect their anxiety (Kalueff et al., 2004c).

Taken together, these observations suggest that that within-trial habituation is impaired in the VDR null mutant mice, as assessed by their horizontal and stopping activity in the open field test. In contrast, vertical activity consistently increased with time, consistent with a gradual reduction of the initially high anxiety baseline. Collectively, these findings further support our earlier notion that VDR null mutant mice generally display high anxiety in various novelty-based anxiety tests (Kalueff et al., 2004a).

6. GENERAL CONCLUSION

In general, our study revealed that VDR mutant mice displayed consistent anxiety-like decreased exploration when subjected to anxiety tests (Fig. 1, 2). Notably, VDR null mutants react to stress by a robust decrease of vertical activity. Overall, these results indicate that VDR null mutant mice display a higher anxiety level compared to the wild type and heterozygous groups. Importantly, these findings are consistent with data from the literature showing that in rodents stress and anxiety are associated with decreased exploratory behaviours (Crawley, 1999; Prut and Belzung, 2003). Furthermore, since alterations in vertical activity reflect emotional components of rodent behaviour (Prut and Belzung, 2003; Thiel et al., 1999), our results are in line with earlier studies (Al-themus et al., 1987) linking vitamin D deficiency to inhibition of open field vertical activity in rats.

Fig. 3.

Habituation in VDR null mutant

mice tested in the open field test for 10 min

Interestingly, the behavioural responses of heterozygous (+/-) mice were very similar to the wild type mice (Kalueff et al., 2004a,b). These behavioural observations are consistent with earlier data showing that heterozygous mice, possessing 50% of mutated VDR genes, show similar VDR gene mRNA expression, have no overt abnormalities and phenotypically are indistinguishable from the wild type animals (Yoshizawa et al., 1997; Li et al., 1997). This is also in line with our recent findings showing no behavioural differences between these two genotypes in self-grooming activity and some other behaviours (Kalueff et al., 2005). Together, our findings demonstrate that genetic ablation of VDR in mice is associated with increased anxiety-like behaviours, and that this effect can only be seen in VDR null mutant animals.

How can loss of VDR lead to increased anxiety? Vitamin D has been implicated in a number of physiological processes in the brain, including the modulation of brain neurotransmitters such as acetylcholine and catheholamines (review, Garcion et al., 2002; Carswell, 1997), mediators that have long been known to be involved in the regulation of emotional behaviours. The widespread VDR distribution in the brain suggests its functional properties in the CNS (Prufer et al., 1999; Walbert et al., 2001; Langub et al., 2001). Importantly, the highest brain VDR concentration has been found in the limbic system, the key emotiogenic brain structure, and its extensions in the brain (Walbert et al., 2001; Langub et al., 2001).

Given our findings in mutant mice, we can suggest that genetic ablation of VDR in the brain, especially in the emotiogenic limbic structures, may disrupt vitamin D-VDR signalling pathways which, associated with disturbed modulation of neurotransmitters in these regions, may cause the increased anxiety level seen in our experiments.

Our results directly link VDR deficiency to increased anxiety symptoms and indicate that the vitamin D-VDR system significantly affects emotional behaviour. Defects of this system may directly lead to emotional disorders, as has already been speculated (Garcion et al., 2002; Carswell, 1997; Gloth et al.,

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Table 1

Comparison of the available published studies characterizing VDR mutant mice

Behaviours and tests Studies

Kalueff et al., 2004a,b,c Burne et al., 2005

Vertical exploration

• Open field (") (-)

• Elevated plus maze (") (-)

• Light-dark box (") N/a

• Holeboard (") (-)

Horizontal activity

• Open field Trend: (-) (-)

• Elevated plus maze 0 (-)

• Light-dark box 0

• Holeboard (-) (-)

• Y-maze N/a (-)

Additional exploratory behaviours

• Hole-poking (holeboard) 0 Trend: (-)

• Head-dips (plus maze) N/a 0

• Open time (plus maze) N/a 0

• Y-maze arm alternation N/a (-)

Other anxiety-sensitive behaviours

• Urination 0* (_)**

• Defecation 0 *** 0**

• Accoustic startle response N/a (-)

• Marble burrying N/a (-)

Other behaviours

• Memory in Y-maze N/a 0

• Digging (bedding test) N/a (-)

• Open field habituation **** N/a (-)

• Accoustic response habituation N/a (-)

• Self-grooming *** (+) 0 *****

• Pre-pulse inhibition ****** N/a (-)

N/a — not assessed. (+) — activation, (-) — reduction, 0 — no effects. * Unpublished data from (Kalueff et al., 2004a) study. ** Open field data. *** Open field, holeboard, plus maze, light-dark box data. **** Horizontal activity (distance traveled). ***** Holeboard test data. ****** To acoustic startle response (at long 256 ms, but not short 8-64 ms intervals).

2001). Our studies allow us to introduce the vitamin D-related hypothesis of anxiety-related disorders, suggesting that anxiety may be a key disorder provoked by abnormal neuroendocrine vitamin D/VDR system (Kalueff et al., 2004a). For the development of effective treatments for such disorders it is therefore necessary to increase our knowledge on the central function of this system. We may also suggest that new psychotrop-ic drugs can be created based on targeting the vitamin D/DVR system.

Acknowledgements

This research was supported by research grants from CIMO, EVO, Tampere University and the Academy of Finland.

The authors are greatly indebted to Prof. S.Kato (University of Tokyo, Japan) for providing the knockout mice for our research. Authors also thank Dr. A. Galeeva (University of Tampere, Finland) for providing the protected elevated plus maze for our studies.

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