Developmental exposure to nicotine atdifferent neonatal ages

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Developmental exposure to nicotine at different neonatal ages

Analysis of protein tau in the cerebral cortex

Min-Yu Wu

Degree project inapplied biotechnology, Master ofScience (2years), 2010 Examensarbete itillämpad bioteknik 30 hp tillmasterexamen, 2010

Biology Education Centre and Department ofEnvironmental Toxicology, Uppsala University Supervisor: Per Eriksson

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1 Preface

This master thesis is a part of the graduate study program in Applied Biotechnology, and was carried out at Department of Environmental Toxicology, University of Uppsala, Sweden.

The work has been financially supported by Swedish Research council for Environment, Agricultural Sciences and Spatial Planning.

To my supervisor, Professor Per Eriksson, for his approval, encouragement, and patience, to Associate Professor Henrik Viberg, for his support for protein analysis, I would like to express my heartfelt appreciation and gratitude.

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Abstract

Developmental exposure to nicotine at different neonatal ages Analysis of protein tau in the cerebral cortex

Min-Yu Wu

During brain growth spurt (BGS), spanning the first 3 -4 weeks after birth in rodents and reaching the peak around postnatal day 10, the brain of mouse develops and grows rapidly. It is known that neonatal exposure to nicotine during this period can lead to changes of

cholinergic receptors in both the neonatal and adult mice. Furthermore, it has been shown that mice neonatally exposed to nicotine and as adults to paraoxon develop cognitive defects and increased levels of tau protein. Tau protein, belonging to microtubule – associated proteins, is involved in the outgrowth and development of neuron in brain during BGS. Recent studies have shown that proteins important for normal brain development, such as GAP-43, CaMKII, synaptophysin and tau can be affected by environmental toxicants during neonatal life. The present study was undertaken to investigate whether tau protein can be affected in mice exposed to nicotine during different defined time periods of the neonatal brain development.

To achieve this the altered level of tau protein in mice exposed to nicotine neonatally and to paraoxon at adult age were compared to 10 days old mice were exposed to nicotine (66μg/

kg body weight.) or saline (10 ml/kg body weight) on 5 consecutive days twice daily when they were aged 3, 10, or 19 days. The animals were killed 12 hours after the last injection of nicotine and the cerebral cortex of the brain was analyzed for tau protein. The protein analysis showed that neonatal exposure to nicotine did not affect the levels of tau protein. There were no significant differences in the levels of tau in the cerebral cortex of the neonatal mouse at the three different developmental ages. However, the levels of tau protein increased in 4- month-old mice neonatally exposed to nicotine on postnatal days (PND) 10-14 and later exposed to paraoxon at 2 months of age. This suggests that nicotine has affected the

developing cholinergic system, but an additional exposure to a cholinergic agent is needed to affect the levels of tau protein.

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Introduction

Exposure to toxic agents present in our environment

Due to more hazardous contaminants in our environment, human beings have higher probabilities to be exposed to toxic agents starting at the fertilization throughout the whole life. During the gestational period, mammal’s embryo/foetus can be exposed through the maternal intake of toxic compounds. After birth, infants and newborns can be directly exposed to chemicals via inhalation, ingestion, mother’s milk, or through the skin.

In animal studies, it has been shown that both persistent and non-persistent xenobiotics can induce disruption of brain development when administrated during a period of rapid brain growth in the neonatal mouse. During this period it has been established that xenobiotics can cause persistent changes in behavior and brain receptors (Eriksson et al., 1992, 2002; Ahlbom et al., 1995; Eriksson, 1998; Viberg et al., 2003b). It is the presence of the chemicals during a defined period of neonatal brain development that induces persistent brain disorders (Eriksson, 1997).

Brain development

In mammals, the general maturation of central nervous system (CNS) can be roughly divided into two major parts. The first part is the early embryonic development of the brain. At this moment, the general shape of the brain takes form and the precursors to glia and neurons multiply. Malformation of the brain can be caused by exposure to xenobiotics during this period. In humans the embryonic period is constituted by 20% of the gestational period and the foetal period 80%. In contrast, in research animals such as the mouse and rat the

embryonic period constitutes about 80% and foetal period about 20% of the gestational. The second period of the brain development is the “brain growth spurt” (Davison and Dobbing, 1968). Series of rapid fundamental developmental changes occurs during this period

characterized by axonal and dendritic outgrowth, glia cells with accompanying myelinisation, together with cell, axonal and dendritic death (Kolb and Whishaw 1989). Besides that, during this period, the concentration of brain lipids and their biosynthesis increase. Also, mice and rats acquire motor and sensory faculties (Bolles and Woods 1964), and reach a peak in their spontaneous behavior (Campbell et al. 1969). In humans, this period begins during the third trimester of pregnancy and continues throughout the first two years of life. However, in mice

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and rats the BGS is neonatal, spanning the first 3-4 weeks of life, and the peak is around postnatal day (PND) 10.

Figure 1: Rate curves of brain growth in relation to birth in different species. Values are calculated at different time intervals for each species. (Data from Davidson and Dobbing, 1968, and Eriksson unpublished. Illustration by Ylva Stenlund)

It has been shown that exposure to both persistent and non-persistent environmental agents can induce irreversible disruption of adult brain function when given to neonatal mice (Eriksson, 1997). When toxicants are administrated to adult animals at similar doses, no apparent disturbances appear. There are also studies showing that the persistent effects can only be induced during a defied period of the neonatal development of the mouse brain, on postnatal day 10 (Eriksson et al., 1992; Ahlbom et al., 1995; Eriksson, 1998). Furthermore, this exposure to toxic agents can also increase susceptibility at adult age, which indicates that exposure to toxic agents during neonatal life can potentiate and/or modify reactions, to xenobiotics at adult age (Johansson et al., 1995; Johansson et al., 1996; Eriksson and Talts, 2002).

Cholinergic system

The cholinergic system, one of the major transmitter systems in brain, is associated with learning, memory, audition and vision (Karczmar 1975). According to studies, by using nicotinic or muscarinic antagonists to block the cholinergic transmission one can cause

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6 learning and memory impairments both in humans and animals (Fibiger et al., 1991;

Newhouse et al., 1992).

Catalysed by cholineacetyltransferase (ChAT), acetylcholine (ACh) is synthesized through a reaction between choline and acetyl coenzyme-A, and then ACh is stored in vesicles, which would be released into the synaptic cleft responding to the change of electrical potential. The enzyme acetylcholine esterase (AChE) regulates the amount of ACh in the synaptic cleft. Via hydrolysis, into choline and acetate, AChE splits the acetylcholine and choline is transported back into the presynaptic terminal for reuse in the synthesis of ACh.

In rodents, the ontogenesis of the cholinergic system takes place during the first 3 to 4 weeks after birth. During this period, enzymes such as ChAT and AChE, and the sodium-dependent choline uptake, and the cholinergic receptors increase in the various brain regions (Coyle and Yamamura, 1976; Kuhar et al., 1980; Falkeborn et al., 1983; Fiedler et al., 1987; Hohmann et al., 1995).

There are two classes of the cholinergic receptors, muscarinic and nicotinic (Dale 1914), which both are activated by acetylcholine but they belong to different gene families.

Muscarine and nicotine act as agonists for these two receptors.

Nicotine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs), belonging to gene superfamily of homologous receptors which includes γ-Aminobutyric acid (GABA), glycine and 5-hydroxy tryptamine, are believed to have a pentameric structure consisting of five membrane spanning regions around a central ion-channel (Karlin 2002). Nicotinic receptor binding sites are at least three different binding sites in human brain, including superhigh-, high-, and low- affinity sites (Nordberg et al. 1988).

In rodent brain, nine alpha (2-10) and three beta subunits (2-4) have been cloned and show regional distributions (reviewed by Lucas- Meunier et al., 2003). The functional receptor is a homomer unit formed by either alpha7, alpha8 or alpha9 subunit. Snake venom toxin alpha- bungarotoxin has a specific affinity to alpha7-10 subunit, and the subtype can correspond to nicotinic receptors low-affinity binding sites. Besides the homomeric receptor described,

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7 receptors formed from heteromeric combinations of α2-α5 subunits with β2-β4 subunits have been reported. The high affinity receptor in the brain is typicallyα4 and β2 subunits, and studies have demonstrated presynaptic localisation of this receptor in nigrostriatal dopaminergic neurons (Paterson and Nordberg 2000).

Nicotinic receptors are present particularly in the thalamus, cortex, hippocampus, striatum, and cerebellum (Court et al., 2000, Paterson and Nordberg, 2000).

Muscarinic receptors

The muscarinic receptors, a heterogeneous group of receptors, coupled to G-protein and exhibit a slow response time, belong to a superfamily of structurally related proteins

possessing seven transmembrane spanning regions. Generally, two classes of the muscarinic receptors subtypes can be characterized. According to Hammer et al., (1980), the M1-like receptors which stimulate the phophoinositol pathway are classified as M1, M3 and M5. The M2-like receptors including M2 to M4, act by inhibiting adenylate cyclase (reviewed by Lucas-Meunier et al., 2003). The classic muscarinic antagonists, QNB and atropine for

instances, do not distinguish between the subtypes, but bind to all equally well (Cooper, 1996).

The tau protein

During development there are proteins important for neonatal development of the brain.

Recently studies have shown some proteins such as brain derived neurotrophic factor (BDNF), calcium/calmodulin-dependent protein kinase II (CaMKII), and growth associated protein – 43 (GAP-43) and synaptophysin, are involved with neuronal survival, growth and

synaptogenesis during brain development period. Changes in the levels of those proteins may affect the development of the brain, and also clarify possible mechanisms of brain damage that xenobiotics might cause (Viberg et al., 2008). Among those proteins, tau protein has shown to have multiple bio-functions and playing a crucial role during brain development (Viberg H. 2009).

Tau protein, first discovered in Marc Kirschner's laboratory at Princeton University

(Weingarten et al., 1975) and belonging to the family of microtubule associated proteins, are plentiful in neurons in the brain, but are scarce in other parts. Stabilization of microtubules and promotion of tubulin assembly into microtubules by interacting with tubules are the main

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8 duties of tau proteins, and by using either isoforms or phosphorylation is the way that how tau proteins control microtubule stability. In brain tissue six tau isoforms exists, the number of binding domains can be used to distinguish between the different tau isoforms. The

phosphorylation of tau proteins can be regulated by a host of kinases such as protein kinase N (PKN). Tau proteins structurally have two domains; one is projection domain covering the amino-terminal, and the other is microtubule-binding domain covering the carboxyl-terminal.

The biological function of tau proteins is the involvement in the outgrowth of neural processes and development of neuronal polarity, and in maintenance of the normal morphology of neurons. Besides that, tau proteins play multiple roles not only in normal architecture but also in signal transduction by interacting with other functional and structural proteins (Wang and Liu, 2008). Tau proteins also regulate the formation of microtubule in cells, cellular processes, cytokines, mitosis, and vesicular transport, cellular motion, and maintain and determine the cell shape as well (Weingarten et al., 1975; Vila- Ortiz et al., 2001). Recent studies also show that tau proteins participate in the regulation of cell viability by phosphorylation (Li et al. 2007). Furthermore, some studies indicate that tau proteins are involved in the pathogenesis of Alzheimermer’s disease and other diseases because of hyperphosphorylation leading to straight filaments and the self-assembly of tangles of paired helical filaments (Alonso et al., 2001).

Nicotine

Nicotine is one of the most frequently used substances (Henningfield and Woodson 1989), and has an influence on human health as a component in tobacco, causing vasoconstriction, increasing heart rate and many other physiological effects. Another application of nicotine is its use as an insecticide which causes unorthodox up-regulation of nicotinic receptors leading to over-stimulation and blockage of cholinergic synapses associated with motor nerves. In people smoking cigarettes it is presumed that nicotine can activate reward mechanisms in the CNS (James and Nordberg 1995). It is also known that nicotine stimulates the nicotinic receptors directly, and then acetylcholine, dopamine and other neurotransmitters can be released (Wonnacott et al. 1989). An earlier study by Nordberg et al., (1991) have shown that neonatal administration of nicotine (66 µg(-)nicotine base/kg body weight s.c., twice daily between days 10-16, altered the susceptibility of adult mice where the mice displayed a hypoactive condition in response to an acute nicotine injection, whereas neonatal mice

administrated saline displayed a hyperactive condition. Furthermore, it has also been seen that

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9 neonatal exposure to the nicotine can increase adult susceptibility to other cholinergic agents, such as the AChE inhibitor paraoxon, where non-toxic doses of paraoxon can induce

persistent cognitive deficit in the adult animals neonatally exposure to nicotine, and that this effect might worsen with age as well (Ankarberg et al. 2004, Feiya Luo, 2009). This neonatal exposure to nicotine and later adult exposure to paraoxon have also been shown to increase tau protein in adult mice showing cognitive deficit.

Aims

The aim of this thesis was to investigate possible changes of tau protein in mice exposed to nicotine during different defined time periods of the neonatal brain development. An

additional objective was to compare these observations with the altered level of tau proteins in mice neonatally exposed to nicotine and later as adults exposed to paraoxon in adult.

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Materials and Method

Nicotine was obtained from Sigma, U.S.A. Pregnant NMR1 mice were obtained from B&K, Sollentuna, Sweden. The animals were kept in plastic cages in a room with an ambient room temperature of 22 and a 12/12 hours light dark cycle and supplied with standardized pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. Each liter was kept with its respective dam, and was adjusted to 8-12 animals within 48 hours by killing excess pups.

Treatment

Neonatal mice were given (-)nicotine, 66 μg nicotine-base (nicotine-bi(+)- tartrate, Sigma, USA) per kg body weight s.c twice daily on 5 consecutive days, when they were aged 3, 10 , or 19 days. The control mice received in the same manner, 10 ml/kg b. wt. of saline vehicle.

Each treatment group consisted of mice from 3-4 different litters.

After treatment, the female mice were sacrificed about 12 hours after the last nicotine injection by decapitation, and the brains were dissected on an ice-cold glass plate. The cerebral cortex was collected, and was frozen at -80℃ until protein assays were performed.

Protein analysis Slot-blot analysis for tau

Cerebral cortex were homogenized in a RIPA cell lysis buffer(50 mM Tris-HCL, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1％ Triton X - 100, 20 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1％ sodium deoxycholate) with the addition of 0.5% protease inhibitor cocktail (Proteases Inhibitor Cocktail Set III, Calbiochem ). The homogenate was then centrifuged at 14,000× g for 15 min at 4℃. The protein content of the supernatant was measured using the BCA method (bucinchoninic acid assay) (Pierce), and then the

supernatant was stored at -80℃ until use.

In the slot-blotting procedure 3.5µg of protein was diluted to a final volume of 200 µl with sample buffer (120mM KCL, 20 mM NaCl, 2 mM NaHCO3 , 2 mM MgCl2, 5 mM HEPES, pH 7.4, 0.05％ Tween20, 0.2％ NaN3). With the use of Bio- Dot SF microfiltration apparatus (Bio- Rad), the protein was transferred onto nitrocellulose membrane and subsequently fixed in 25% isoporpanol, 10% acetic acid. The membrane was washed and blocked with 5% non-

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11 fat dry milk containing 0.03% Tween-20 for 1h room temperature. The membranes were then incubated overnight at 4℃ with a mouse monoclonal tau antibody (Santa Cruz sc-32274, 1:1000). Immunoreactivity was detected using a horseradish peroxidase conjugated secondary antibody against mouse (074-1806, 1:20,000). Immunoreactive bands were detected using an enhanced chemiluminescent substrate (Pierce, Super Signal West Dura) with imaging on a LAS-1000 (Fuji film, Tokyo, Japan). The intensity of bands was quantified using IR-LAS 1000 Pro (Fuji film).

Statistical analysis

Tau proteins levels in control and nicotine-treated animals were determined using a two-tailed Student’s t-test.

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Results and Discussion

Neonatal exposure to nicotine (66 µg/ kg body weight) on PND 3 – 7, PND 10 – 14 or on PND 19 -23 did neither cause any clinical signs of toxic symptoms in the animals nor were there any significant differences in body weight gain between the saline and nicotine treated mice, regardless age-category (Fig.2).

0 2 4 6 8 10 12 14

3-7days 10-14 days 19-23 days

body weight (g) Control Group

treatment group 3-7days 10-14days 19-23days

Figure 2. Body weight gain in neonatal female mice exposed to nicotine (66 µg/kg body weight) or saline on PND 3-7, 10-14, or 19-23.

The effects of neonatal exposure to nicotine (66 µg nicotine/kg body weight) on tau protein levels in the cerebral cortex were analyzed in neonatal mice at different developmental stages.

The levels of tau protein 24 hrs after the last injection of nicotine on PND 3 -7, 10 – 14 or 19 – 23 indicated no significant difference in the levels of tau between control animals and nicotine exposed animals, regardless age-category (Fig. 3).

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(a) (b) (c)

Fig.3. Protein levels of tau in cerebral cortex of neonatal female mice exposed to nicotine (66 µg/kg body weight) on PND 3 – 7, 10 – 14 or 19 – 23. The protein level of tau was analyzed 24 hrs after last injection. (a) Levels of tau after neonatal exposure to nicotine on PND 3 – 7. (b) Levels of tau after neonatal exposure to nicotine on PND 10 – 14. (c) Levels of tau after neonatal exposure to nicotine on PND 19 -23. The data were subjected to Student’s t-test and the significant difference is α= 0.05. The height of the bars represents the mean value ± S.D.

Many studies have shown that environmental agents can affect the cholinergic receptors, including muscarinic and nicotinic receptors, in adult animals neonatally exposed to those agents during a defined critical period of neonatal development, namely around PND10 (Eriksson et al., 2008 ). Some of these studies have also shown that tau can be altered during the neonatal period (Viberg et al., 2009). Therefore, both tau and nicotinic receptors could be crucial during the brain developmental period.

Tau proteins, microtubule-associated proteins, are critical to the axon of the neurons and are essential for the maintenance of the stability of microtubules formed previously and for the promotion of microtubule assembly (Wand and Liu, 2008). The ontogeny of tau has been studied in the neonatal mouse (Viberg H. 2009).

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Fig.4. Postnatal ontogeny of tau shown as the amount of tau in brain tissues in untreated mice on PND 1, 3, 7, 10, 14, and 28, expressed as percent of the amount of tau on PND 1 (Viberg H. 2009)

The amount of tau in brain tissue of mice was studied from PND 1 to PND 28 and tau increased in the early neonatal period whereas during the last part of the period decreased gradually. The peak of the amount of tau was between PND 7 and 10 in the cerebral cortex and whole brain, and the level decreased after the peak. On PND 28, the level of tau was detected even below the level on PND 1 (see fig.4, from Viberg 2009). Another study have also indicated that from PND 9 and 12 the level of tau protein maintained constant, and then decreased by PND 20 (Vila-Ortiz et al., 2001). This timing seems to have some connection with its function that promotes microtubule assembly, neuritis stabilization, and facilitation of axon outgrowth during this time (Drubin et al., 1985), and therefore tau may be less needed after synaptic stabilization and the protein concentration would decrease in the brain of mice (Vila-Ortiz et al., 2001). In other words, high levels of tau protein might be only required during the period connected with the brain growth spurt at which axon grow and synapses are formed (Jacobson wt al., 1986; Vila-Ortiz et al., 2001). The ontogeny of nicotine receptors also shows an increase during the neonatal period. During embryonic and postnatal

development the level of mRNA of α7 nAChR is higher than in the adult rodent cortex and other brain regions indicating that nicotinic receptors may play an important role during the development period (Adams et al., 2002). However, studies have shown that around birth there is a significant decrease in ³H – nicotine binding sites, but from PND 1 there is a continual increase and the adult levels are reached on PND 28. It is known that various nicotinic subtypes have different developmental patterns. The peak of cortical α4 mRNA is observed on PND 14, while the peak of α7 mRNA is on PND 7 (Shacka et al., 1998), and decreased by the end of the second postnatal week in the cortex (Bina et al., 1995) suggesting that between PND 7-14 is a significant increase of expression for nicotinic receptors. From

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15 these data it is noticed that both tau and nicotinic receptors share similar ontogeny in some extent on PND 7-14. During this time, ACh plays as an important role in the developmental processes, and so do the nicotinic receptors that can act as guiders during development.

From earlier studies it has been seen that mice neonatally exposed to nicotine between PND 3-7, 10-14, or 19-23 only high affinity binding site could be observed in cerebral cortex, while in mice given just saline both high affinity and low affinity binding site was observed

(Eriksson et al., 2000). Furthermore, the data of adult mice strikingly showed that the low affinity binding site of adult mice neonatally exposed to nicotine between PND 10-14 still could not be observed; however, the low affinity binding site was observed in adult mice neonatally exposed to nicotine between PND 3-7 or 19-23, and also in saline- treated mice (see table 1). This previous study indicated that nicotinic low affinity binding sites in the cerebral cortex would be affected by neonatal exposure to nicotine and that the persistence would last to adult age. This study also indicated that the change of nicotinic receptors in mice exposed to nicotine on PND 10-14 was irreversible while in the other age categories these changes were reversible (See table 1).

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16 Treatment

(66 μg/kg bw)

High affinity %;

k (nM)

Low affinity %;

k (μM)

High affinity %;

k (nM)

Low affinity %;

k (μM)

Age Neonatal Adult

Day 3-7 NaCl Nicotine

78± 7; 5.2 95± 1; 20

22±7; 18 - -

76±4; 3.4 87±1; 6.6

24±4; 1.7 13±1; 2.0 Day 10-14

NaCl Nicotine

71±15; 5.0 97±4; 17

29±15; 5.0 - -

83±7; 3.4 100±3; 8.0

17±7; 1.6 - - Day 19-23

NaCl Nicotine

87±13; 4.0 96±1; 8.2

13±13; 7.3 - -

90±3; 7.7 86±4; 7.0

10±3; 5.2 14±4; 5.4

Table 1 Developmental neurotoxicity of nicotine –Effects on nicotinic receptors (high and low affinity binding sites) in the cerebral cortex of neonatal and adult mice

Adapted from Eriksson et al. (2000)

Although changes can be observed in nicotinic receptors in neonatal mice exposed to nicotine at dose of 66 µg/ kg body weight (see table 1), the present study showed that levels of tau protein was not affected in the nicotine treated mice. Studies in adult animals have indicated that exposure to nicotine can cause phosphorylation of tau in transgenic mice that have synaptic dysfunction and showing long term deficits in synaptic plasticity (Oddo et al., 2004).

Treating these one-month-old 3xTg – AD mice with nicotine chronically in the drinking water resulted in an increase of phosphorylated tau (Oddo wt al., 2005). From an in vitro study, where a human neuroblastoma cell line SH-SY5Y were chronically exposed with AChE- inhibitors, tacrine, donepezil, and galantamine, an increased amount of phosphorylated tau was observed (Hellstrom-Lindahl et al., 2000); however, the mechanism of the increased phosphorylation of tau is still unclear. From another in vitro study in PC12 M1 cells (Sadot et al., 1996) it has been seen that phosphorylation of tau decreased when the activation of mAChR increased. This indicates that changes in cholinergic activity can lead to both an increase and a decrease in phosphorylation of tau. There are also reports that indicate that

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17 highly phosphorylated tau would be more resistant to degradation by protease than less

phosphorylated tau, and both a reduced sensitivity of tau to proteolysis and an increase of tau expression can increase the total levels of tau protein. In the presents study the total amount of tau was measured and whether there could be changes in the amount of phosphorylated tau remains to be investigated.

Numerous studies have shown that during the neonatal brain development in mouse the cholinergic system is vulnerable to nicotine and other cholinergic agents such as

organophosphorus compounds leading to behavior disturbances and changes in nicotinic and muscarinic receptors (Ankarberg et al., 2001; Eriksson, 1997; Eriksson et al., 2000; Liu et al., 1999; Moser and Padilla, 1998; Zheng et al., 2000). Besides that, neonatal exposure to low doses of toxicants can increase the susceptibility to xenobiotics at adult age. This increase in susceptibility was seen at doses that had no marked effects when given to adult control animals (Eriksson and Talts, 2000; Ankarberg et al., 2004). Particularly, mice exposed to nicotine on PND 10-14 showed behavior disturbance when adult mice were challenge to nicotine, in a nicotine-induced behavior test. However, this reaction was not observed in mice exposed on PND 3-7 or 19-23 (Eriksson, 2000). A recent study has shown that this early exposure to nicotine and later adult exposure to the oranophosphorous compound, paraoxon, can alter the levels of tau in cerebral cortex in conjunction with cognitive defects.

Protein analysis of tau in the cerebral cortex of adult brain in mice neonatally exposed to nicotine and at adult age exposed to paraoxon revealed a significant change in this exposure group. The levels of tau protein was remarkable increased in mice neonatally exposed to nicotine and exposed to paraoxon at adult age, whereas mice neonatally exposed to nicotine and saline as adults or neonatally to saline and to paraoxon as adults no significant change in adult tau levels was seen (Fig. 5).

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Tau

Saline-Saline Nicotine-Saline

Nicotine-OP 0

200 400 600 800

1000 A

B

Cerebral cortex

Protein level (% of control)

Tau

Saline -Saline

Saline-OP Nicotine-OP 0

100 200 300 400

A B

Cerebral cortex

Protein level (% of control)

Fig.5. Levels of tau protein in the adult brain of 4-month-old mice neonatally exposed to nicotine (66 µg/kg body weight) and receiving to paraoxon (0.3 mg/kg body weight) at an adult age of 2 months. (Unpublished data from Eriksson et al.2009).

The change in tau in this exposure group may be due to that exposure to nicotine during the neonatal period the nicotinic receptors of mice might be changed and thereby also the potentiality of phosphorylation of tau might be increased. At adult age, when the mice neonatally exposed to nicotine and adults exposed to OP, the amount of tau was increased.

However, for other groups exposed to OP at adult age not showing the increase of tau a possible explanation might be the dose of OP given to the adult mouse. Studies, both in vivo and in vitro experiments, have shown that the amount of phosphorylated tau can be increased after treating with nicotine agonists and cholinergic inhibitors (Chalmers et al., 2009.

Hellstrom-Lindahl et al., 2000). The dose of OP used in the studies by Ankarberg (2004) and Feiya (2009) was low and caused about 30% inhibition of AChE and did not cause any behavioral disturbances in the adult mice neonatally receiving saline, but it affected adult mice neonatally exposed to nicotine leading to the behavior disturbance (Ankarberg et al, 2004, Feiya Luo 2009) suggesting exposure to OP at doses not causing direct effects can have negative influence on animals neonatally exposed to nicotine with an already altered function of the cholinergic system. Furthermore, the reaction to cholinergic agents can also differ depending on developmental period. It can be seen in Oddo’s study, the age-related and regional-dependent manner affected the alternation of conformation and phosphorylation of tau proteins (Oddo et al., 2003). The present studies do not distinguish between

phosphorylated tau and tau protein and whether there can be a change in either form of tau remains to investigate.

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Concluding remarks

The present study shows that neonatal exposure to nicotine does not affect the levels of tau protein in the cerebral cortex of the neonatal mouse. However, it is known that during brain growth spurt, the nicotinic receptors are vulnerable to affects by cholinergic agents during a defined critical period of neonatal development leading to irreversible changes in cholinergic function and behavioral deficits in adult animals. It is also noticed that the nicotinic receptors can be affected, especially the low affinity binding site, and that an increase in tau can be seen in mice neonatally exposed to nicotine and later as adult exposed to paraoxon. Regarding the relationship between phosphorylated tau protein and Alzheimer’s disease (AD) (Thathiah and Strooper, 2009), and that the cholinergic system always is affected in AD suggest that further studies are needed to see whether early changes in phosphorylated tau can be seen and

whether this can have consequences for adult exposure to toxic agents affecting the cholinergic system.

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Developmental exposure to nicotine atdifferent neonatal ages