Neonatal exposure to propofol affects BDNF but not CaMKII, GAP-43, synaptophysin and tau in the neonatal brain and causes an altered behavioural response to diazepam in the adult mouse brain

Behavioural Brain Research 223 (2011) 75–80

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Neonatal exposure to propofol affects BDNF but not CaMKII, GAP-43, synaptophysin and tau in the neonatal brain and causes an altered behavioural response to diazepam in the adult mouse brain Emma Pontén a,∗ , Anders Fredriksson b , Torsten Gordh a , Per Eriksson c , Henrik Viberg c a

Department of Surgical Sciences, Anaesthesiology and Intensive Care, Uppsala University, Sweden Department of Neuroscience, Psychiatry, Uppsala University, Sweden c Department of Environmental Toxicology, Uppsala University, Sweden b

a r t i c l e

i n f o

Article history: Received 4 February 2011 Received in revised form 7 April 2011 Accepted 11 April 2011 Keywords: Propofol BDNF Behaviour

a b s t r a c t Animal studies have shown that neonatal anaesthesia is associated with acute signs of neurodegeneration and later behavioural changes in adult animals. The anaesthetic effect of propofol is thought to be mediated by ␥-amino butyric acid (GABA)A receptors. The present study investigated the effects on proteins important for normal neonatal brain development (i.e. BDNF, CaMKII, GAP-43, synaptophysin and tau), and adult spontaneous motor and anxiety-like behaviours in response to diazepam, after neonatal exposure to propofol. Ten-day-old mice were exposed to 0, 10 or 60 mg/kg bodyweight propofol. Neonatal propofol exposure changed the levels of BDNF in the brain, 24 h after exposure, but did not alter any of the other proteins. Neonatal propofol exposure significantly changed the adult response to the GABA-mimetic drug diazepam, manifest as no change in spontaneous motor activity and/or reduced sedative effect and an extinguished effect on the reduction of anxiety-like behaviours in an elevated plus maze. Although no adult spontaneous behavioural changes were detected after neonatal propofol exposure, the exposure caused an adult dose-dependent decrease in the response to the GABA-mimetic drug diazepam. These changes may be due to neonatal alterations in BDNF levels. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Since the revelation that a combination of midazolam, isoflurane and nitrous oxide causes apoptosis in the developing rodent brain with behavioural consequences [1], it has been debated if and how neonatal exposure to anaesthesia alters the developing human brain. Furthermore, recent studies in humans indicate that anaesthesia before four years of age is associated with learning disabilities [2,3]. Propofol (2,6-diisopropylphenol) is a short-acting anaesthetic drug for intravenous use. Because of its lipophilic properties it is concentrated in lipid-rich tissues, such as the brain [4,5]. Propofol is commonly used in clinical practice and is classified as a non-barbiturate anaesthetic agent. Although propofol is not recommended for use in children under the age of three years, it is often used to treat this group of children, and in neonatology [6]. Propofol can cause neuroapoptosis in the infant developing mouse brain

∗ Corresponding author. Tel.: +46 706894842. E-mail addresses: [email protected], [email protected] (E. Pontén). 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.04.019

at a quarter of the dose required for anaesthesia in an infant mouse [7]. In vitro studies have shown that propofol induces growth cone collapse [8] and causes irreversible damage to ␥-amino butyric acid (GABA) neurons during brain cell maturation, but no damage has been observed in fully matured neurons [9]. The GABA-receptor system develops and modulates during the brain growth spurt (BGS) when rapid development of cells and synapses occurs. The BGS starts in the last trimester and lasts into the second year of life in humans. In rodents, BGS starts after birth and continues for about three weeks [10,11]. During the BGS there is a defined critical period when the developing brain is especially vulnerable to toxic agents [12,13], and this vulnerability has also been seen with anaesthetic agents [14]. Among the acute effects induced by different xenobiotics during this vulnerable period are neonatal apoptosis, altered cholinergic receptors and changes in proteins important for normal brain development. There are also later effects such as adult irreversible alterations in behaviour, habituation, learning and memory, and response to adult drug exposure. Many of these effects worsen with age [12,14–17]. In a previous study we demonstrated that the normal, anxiolytic effect of diazepam was eradicated in mice neonatally exposed to propofol [18]. The aim of this study was to further elucidate if expo-

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E. Pontén et al. / Behavioural Brain Research 223 (2011) 75–80

sure to propofol, during the BGS in mice, causes neonatal alterations in proteins (BDNF, CaMKII, GAP-43, synaptophysin and tau) active during brain development, and dose-related altered responses in adulthood to the GABAA agonist, diazepam. 2. Materials and methods

horseradish peroxidase-conjugated secondary antibody against mouse (074-1806, 1:20,000) or rabbit (KPL 074-1506, 1:20,000). Immunoreactive bands were detected using dura reagent (Pierce, Super SignalWestDura), an enhanced chemiluminescent substrate, with imaging on a LAS-1000 (Fuji Film, Tokyo, Japan). The intensity of bands was quantified using IR-LAS 1000 Pro (Fuji Film). In these experiments eight hippocampi, frontal and parietal cortices from each treatment group were used in the slot-blotting procedure.

2.1. Animals

2.5. ELISA (BDNF)

Forty-six pregnant NMRI (Naval Medical Research Institute) mice were purchased from B&K, Sollentuna, Sweden. Each group of offspring was adjusted within 48 h after birth to 8–10 mice and to contain pups of either sex in about equal number. The pups were kept together with their respective mother in a plastic cage in a room at temperature of 22 ± 1 ◦ C and a 12/12 h constant light/dark cycle (lights on within 06.00–18.00 h). At age 10 d, propofol 10 or 60 mg/kg bodyweight (bw) or control (0.9% NaCl) was administered subcutaneously to both sexes. Animals used for neonatal analysis of proteins in the brain were sacrificed 24 h after exposure. Only the male offspring were used for the neonatal protein studies and the adult behavioural recordings, since males might be more vulnerable [19], and to compare with previous studies. The animals for adult behavioural testing were weaned at the age of four weeks and only the males were raised in groups of 4–6 animals in a room for male mice only. Behavioural testing was conducted in young adults at age 60–70 d. Please see Fig. 1 for a graphic representation of the exposure and testing. Testing was carried out within 09:00–14:00 h. For the different adult behavioural tests, mice were randomly chosen and used in the spontaneous behaviour test or only once in the other tests. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) after approval from the local ethics committee (Uppsala University and Agricultural Research Council), and by the Swedish Committee for Ethical Experiments on Laboratory Animals.

Hippocampus, frontal cortex and parietal cortex were sonicated in 20 volumes (w/v) of ice-cold lysis buffer (137 mM NaCl, 20 mM Tris–HCl, pH 8.0, 1 mM phenylmethyl-sulfonyl fluoride, 0.5 mM sodium vanadate, 1% NP40, 10% glycerol, 10 ␮g/mL aprotinin and 1 ␮g/mL leupeptin). The homogenate was centrifuged for 20 min at 20,000 × g at 4 ◦ C, and the supernatant acidified (pH < 3) with HCl and neutralized back to pH 7.6 with NaOH. The Promega Emax TM ImmunoAssay System was used to determine the amount of BDNF in the samples according to the technical bulletin supplied by the distributor. In brief, BDNF from each sample was captured with a monoclonal antibody (mAb) against BDNF; and the captured BDNF then bound to a second, specific, polyclonal antibody (pAb) against BDNF. After washing, the amounts of specifically bound pAb were detected using a specific anti-IgY antibody conjugated to horseradish peroxidase as a tertiary reactant. Unbound conjugate was removed by washing and, following an incubation period with a chromogenic substrate, the colour change measured in a micro-plate reader at 450 nm. The amount of BDNF was proportional to the colour change generated and compared to a standard curve. The cross-reactivity to other neurotrophic factors was <3% and the purity of the anti-BDNF antibodies >95%. In these experiments, eight hippocampi, frontal and parietal cortices from each treatment group were used.

2.2. Treatment and drugs At 10 d postnatal, mouse pups were administered either propofol (Diprivan, 10 mg/mL; Astra, Södertälje, Sweden) 10 or 60 mg/kg bw or vehicle (0.9% NaCl) in a volume of 5 mL/kg bw, by subcutaneous injection in the neck. The doses of propofol were the same as used in a previous experiment showing neurodegeneration [18]. The doses of propofol were not anaesthetic but sedating [7], and normal skin colour and respiration were observed in the pups. The animals for neonatal protein studies were randomly chosen from 10 different litters (groups of offspring) and divided into two treatment groups (control and propofol 60 mg/kg bw). The part of the study concerning behaviour in adult animals consisted of three different groups (control and propofol 10 and 60 mg/kg bw), with each treatment group randomly derived from 12 different litters. Diazepam (Stesolid novum, 5 mg/mL; Alpharma AB, Stockholm, Sweden) 0.1, 0.5 or 1 mg/kg bw delivered subcutaneously was used in the behavioural testing of adult animals. 2.3. Neonatal protein measurements At 24 h after exposure to saline or 60 mg propofol/kg bw, the male mice were killed by decapitation and the brains dissected on an ice-cold glass plate. Hippocampus, frontal cortex and parietal cortex were collected and frozen in liquid nitrogen directly after the dissection. Thereafter, the brain regions were stored at −80 ◦ C until the analysis was carried out.

2.6. Spontaneous motor activity Male mice were randomly chosen from the three treatment groups designated for spontaneous behaviour testing (n = 8) and observed for spontaneous behaviour at an age of 60 d, measured in a specialized test cage (Rat-O-Matic; ADEA Electronic, Uppsala, Sweden) as previously described [20]. The following three variables were measured. Locomotion was registered with a low grid of infrared beams, i.e. horizontal movement. Rearing was registered throughout the time when at least one high-level infrared beam was interrupted, i.e. the number of counts registered was proportional to the amount of time spent rearing (vertical movement). Total activity was measured by a sensor mounted on a lever with a counterweight with which the cage was constantly in contact. The sensor registered all types of vibration of the test cage, such as those produced by locomotion and rearing as well as shaking, tremors, scratching and grooming. All three behavioural parameters were measured on one occasion over three consecutive 20-min periods. 2.7. Diazepam-induced motor activity Randomly chosen male mice were observed for diazepam-induced behaviour in the above described test cage at age 65 d. Half-an-hour before testing, the mice were subcutaneously injected with either saline or diazepam 1 mg/kg bw and put back to the home cages. The six groups saline–saline, saline–diazepam, propofol (10 mg)–saline, propofol (10 mg)–diazepam, propofol (60 mg)–saline, and propofol (60 mg)–diazepam; for neonatal and adult, respectively (n = 8) were observed for three consecutive 20-min periods in the test cages with the same three variables as for the spontaneous motor activity test. 2.8. Elevated plus-maze test

2.4. Slot blot (CaMKII, GAP-43, synaptophysin and tau) Hippocampus, frontal cortex and parietal 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 and 0.1% SDS) with the addition of 5 ␮L protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Calbiochem) per mL of RIPA cell lysis buffer. The homogenate was then centrifuged for 15 min at 14 000 × g and 4 ◦ C. The supernatant was collected and stored at −80 ◦ C until use. The protein content of the supernatant was measured using the bicinchoninic acid assay method (Pierce). The supernatant was diluted in sample buffer (120 mM KCl, 20 mM NaCl, 2 mM NaHCO3 , 2 mM MgCl2 , 5 mM HEPES, pH 7.4, 0.05% Tween 20 and 0.2% NaN3 ). Of the total protein content, 4 ␮g of CaMKII and GAP-43, 3 ␮g of synaptophysin and 3.5 ␮g of tau were diluted in sample buffer to a final volume of 200 ␮L. The diluted supernatant was then applied in duplicates to a nitrocellulose membrane (0.45 mm, Bio-Rad) soaked in 1× TBS [NaCl (0.9%), Tris–HCl (42.1 mM) and Tris–Base (7.5 mM)], using a Bio-Dot SF microfiltration apparatus (Bio-Rad). The membranes were fixed in a 25% isopropanol and 10% acetic acid solution, washed in 1× TBS, and blocked for 1 h at room temperature in 5% non-fat dry milk containing 0.03% Tween 20. The membranes were then incubated overnight at 4 ◦ C with either a mouse monoclonal synaptophysin antibody (VWR, Stockholm; 1:10,000), a mouse monoclonal CaMKII antibody (Upstate Millipore, Stockholm; 1:5000), a rabbit monoclonal GAP-43 antibody (Chemicon AB5220, Millipore, 1:5000) or a mouse monoclonal tau antibody (SDS, Stockholm; 1:1000). Immunoreactivity was detected using a

In this test the animal’s anxiety-like behaviour was observed, based on the assumption that normal mice prefer a closed environment to an open space. The elevated plus maze test was based on the method described by Lister [21]. The plus maze apparatus was made of plywood and had two opposite open arms (white floor with no wall, 30 cm × 6 cm) and two opposite closed arms (black floor with walls, 30 cm × 6 cm × 30 cm) mounted 50 cm above the smooth floor. The male animals were transferred to the testing laboratory in their home cages at least 60 min before the elevated plus maze testing. The mice, age 70 d, were randomly chosen from the three neonatal treatment groups and subcutaneously administered diazepam (0.1, 0.5 or 1 mg/kg bw) or vehicle (0.9% NaCl) 30 min before testing. The 12 groups of mice (n = 7) were placed on the central platform of the apparatus facing either of the closed arms. A video motility system (TSE, Bad-Hamburg, Germany) was used to monitor the behaviour of the animals. The number of entries into the open and closed arms and the time spent in each category of arms were measured for 5 min. Arm entry was defined as all four paws present in the arm. The maze apparatus was cleaned after each trial. 2.9. Statistical analysis 2.9.1. Analysis of neuroproteins The relative levels of BDNF, CaMKII, GAP-43, synaptophysin and tau in eight animals treated with propofol was compared with levels in eight control animals using Student’s t-test.

E. Pontén et al. / Behavioural Brain Research 223 (2011) 75–80

Exposure1

Protein analysis2

10

11

Spontaneous behaviour3

Behaviour after diazepam4

60

65

77

Elevated plus maze5

70

Post natal day

Fig. 1. Graphic representation of study design. (1 ) Ten days after birth the pups were injected subcutaneously with either 60 or 10 mg/kg bw propofol or 0.9% NaCl. (2 ) Twentyfour hours later pups from the 60 mg/kg propofol and control groups were sacrificed for protein analysis. BDNF, CaMKII, Gap-43 synaptophysin and tau was measured in frontal and parietal cortex and hippocampus (n = 8). (3 ) At 60 d after birth spontaneous behaviour was tested on eight animals from each exposure group (all three groups tested). (4 ) Five days later, at 65 d, behaviour was tested with the same method as at 60 d, but with diazepam 1 mg/kg bw or saline (control) administered 30 min before testing. This is done for all three exposure groups – a total of six groups tested (n = 8). (5 ) At 70 d after birth elevated plus maze was tested 30 min after 0.1, 0.5 or 1.0 mg/kg bw diazepam or control with saline. This is tested for all three exposure groups – a total of 12 groups tested (n = 7). All animals were tested only once.

2.9.3. Elevated plus maze The percentage time spent in the open arms and the percentage entries into the open arms in the elevated plus maze were analysed using a one-way ANOVA. Pair-wise testing between groups was performed using a Tukey HSD test [23].

800

Locomotion mean

2.9.2. Spontaneous and diazepam-induced behaviour The data were subjected to a split-plot analysis of variance (ANOVA), and pairwise testing between treated groups and the control group was performed using a Tukey HSD (honestly significant difference) test [22].

600 400

A

200 0 0 - 20

20 - 40

3. Results

3.1. Protein levels of BDNF, CaMKII, GAP-43, synaptophysin and tau in frontal cortex, parietal cortex and hippocampus after neonatal propofol exposure

1800

Rearing mean

There were no clinical signs of dysfunction in the propofoltreated mice throughout the experimental period, nor were there any significant deviations in body weight in propofol-treated compared with vehicle-treated mice.

1350 900 A

450 0

3.2. Spontaneous motor activity after neonatal propofol exposure After exposure to 10 or 60 mg propofol/kg bw at 10 d postnatal, the spontaneous behavioural variables of locomotion, rearing and total activity in 60-d-old male mice showed no significant treatment × time-period interactions: F(4,42) = 0.81, F(4,42) = 0.59 and F(4,42) = 0.60, respectively (Fig. 2). There was a distinctive decrease in activity in the control group and the two treatment groups during the 60-min test period, for all three spontaneous behavioural variables. Pair-wise testing between propofol-exposed groups and control animals showed no significant differences.

Total activity mean

0 - 20

Neonatal exposure to 60 mg propofol/kg bw, at 10 d postnatal, changed the levels of BDNF in frontal cortex, parietal cortex and hippocampus, measured 24 h after exposure, compared to control animals (Table 1). In frontal cortex, the levels of BDNF increased 242.8% (P < 0.001) compared to the controls (from 1.17 ± 0.34 to 4.00 ± 1.57 ng/g). In parietal cortex, exposure to 60 mg propofol/kg bw at 10 d postnatal, decreased the level of BDNF by 34.7%, (P < 0.001) compared to controls (from 10.18 ± 1.49 to 6.64 ± 0.57 ng/g). In hippocampus, exposure to 60 mg propofol/kg bw at 10 d postnatal, significantly increased the level of BDNF by 52.2% (P < 0.01) compared to control (from 2.77 ± 0.61 to 4.21 ± 1.23 ng/g). Neonatal exposure to 60 mg propofol/kg bw, at 10 d postnatal, did not alter the protein levels of CaMKII, GAP-43, synaptophysin or tau in frontal cortex, parietal cortex or hippocampus, measured 24 h after exposure, compared to control animals (Table 1). In parietal cortex there was a trend towards increased in levels of GAP-43, since two separate slot-blot analyses of the samples showed Pvalues between 0.05 and 0.10.

40 - 60 Vehicle Propofol low Propofol high

20 - 40

40 - 60

20 - 40

40 - 60

6000

4000 A

2000

0 0 - 20

Time periods (min) Fig. 2. Spontaneous motor activity of adult mice treated with 0, 10 or 60 mg propofol/kg bw or vehicle at 10 d postnatal and tested in the activity test chambers at age 60 d. Values represent means ± SD (n = 8) of locomotor, rearing and total activity counts over three consecutive 20-min periods.

3.3. Diazepam-induced motor activity after neonatal propofol exposure Mice neonatally exposed to saline or 10 or 60 mg propofol/kg bw, were injected with one subcutaneous shot of saline or 1.0 mg diazepam/kg bw at age 65 d and tested for motor activity 30 min later in the same way as for the spontaneous behaviour testing. Split-plot ANOVA indicated significant effects of treatment × time-period interactions for diazepam-induced locomotion, rearing and total activity (Fig. 3): F(10, 84) = 18.05, F(10, 84) = 18.34 and F(10, 84) = 27.06, respectively. In mice exposed neonatally to saline or 10 or 60 mg propofol/kg bw and injected with saline as adults there were no differences in locomotion, rearing and total activity.

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Table 1 Relative levels of BDNF, CaMKII, GAP-43, synaptophysin and tau, expressed as percentage of control, in frontal cortex, parietal cortex and hippocampus at 24 h after exposure to 60 mg propofol/kg bw at 10 d postnatala . Protein

Frontal cortex Control

BDNF CaMKII GAP-43 Synaptophysin Tau

100 100 100 100 100

± ± ± ± ±

Parietal cortex Propofol

29.1 20.5 14.8 14.4 20.6

342.8 104.5 103.8 98.7 101.9

± ± ± ± ±

Hippocampus

Control 134.4*** 23.1 20.3 19.9 17.2

100 100 100 100 100

± ± ± ± ±

Propofol

14.1 22.4 9.7 13.9 18.1

65.3 100.9 112.1 108.5 101.3

± ± ± ± ±

Control 5.6*** 27.6 13.7b 22 1 11.6

100 100 100 100 100

± ± ± ± ±

Propofol

22.1 22.3 12.3 19.71 19.3

152.2 94.3 95.1 111.3 106.2

± ± ± ± ±

44.4** 38.8 9.1 21.7 20.8

Mean ± SD is presented. The statistical evaluation was made with Student’s t-test: *** P < 0.001 and ** P < 0.01. The statistical evaluation with Student’s t-test generated a P-value = 0.068; and in a subsequent slot-blot of the same samples the control animals showed 100 ± 9.9 and the propofol-treated animals showed 114.4 ± 18.8, giving a P-value = 0.081. a

b

75

300

A

150

A

0 0 - 20

20 - 40

Vehicle + Saline Vehicle + Diazepam Propofol low + Saline Propofol low + Diazepam Propofol high + Saline Propofol high + Diazepam

Rearing mean

1800

1350

900

40 - 60

60

A Vehicle Propofol low Propofol high

B A

A

D

B C

45

C D

30

15

0

A

50

450

A

0 0 - 20

20 - 40

40 - 60

6000

Total activity mean

% of entries into the open arms

450

4000 A A

2000

% of time spent on the open arms

Locomotion mean

600

40

A

A

30

D D

D

20

10

0

0 0 - 20

20 - 40

40 - 60

Time periods (min) Fig. 3. Diazepam-induced behaviour, locomotion, rearing and total activity, in adult mice neonatally exposed to 0, 10 or 60 mg propofol/kg bw. Half-an-hour prior to testing the mice were administered either saline or diazepam 1 mg/kg bw. The mice were tested for three consecutive 20-min periods in the test cages. Values represent means ± SD (n = 8). Statistical significance is indicated by ‘A’ (P < 0.01) versus all other treatment groups.

The animals showed the same motor activity behaviour profile observed for animals at age 60 d. In mice neonatally treated with saline and injected with 1.0 mg diazepam/kg bw as adults all three variables, locomotion, rearing and total activity were altered compared to the controls receiving saline both neonatally and as adults. During the first 20min period they showed significantly lower activity (P < 0.01), and during the last 20-min period significantly higher activity (P < 0.01), compared to controls (saline–saline). Animals treated neonatally with propofol (10 or 60 mg/kg bw) did not show the same altered activity in locomotion, rearing or total activity after the adult diazepam injection as for animals treated neonatally with control (saline).

SAL

DIA 0.1

DIA 0.5

DIA 1.0

Pretreatment 30 min before test Fig. 4. Diazepam-induced behaviour in the elevated plus maze in adult mice neonatally exposed to 0, 10 (low) or 60 (high) mg propofol/kg bw. Half-an-hour prior to testing the mice were administered either saline or diazepam (0.1, 0.5 or 1 mg/kg bw). Percentage of entries into open arms and percentage of time spent in open arms are displayed. Values represent means ± SD (n = 7). Letters in upper indicate P < 0.01. A – diazepam versus saline pre-treated mice neonatally exposed for the same treatment; B – diazepam versus saline and other diazepam pre-treated group neonatally exposed for the same treatment; C – propofol versus vehicle neonatal treatment pretreated with the same treatment; and D – propofol versus vehicle and the other propofol neonatal treatment pretreated with the same treatment.

3.4. Diazepam-induced behaviour in elevated plus maze after neonatal propofol exposure Mice neonatally exposed to saline or 10 or 60 mg propofol/kg bw were subcutaneously injected with saline or 0.1, 0.5 or 1.0 mg diazepam/kg bw at age 70 d and then tested in the elevated plus maze 30 min later (Fig. 4). There were significant effects in mice exposed neonatally to propofol in response to adult diazepam treatment regarding enter-

E. Pontén et al. / Behavioural Brain Research 223 (2011) 75–80

ing [F(11, 72) = 22.32], and time spent in [F(11, 72) = 27.54], the open arms. In animals treated neonatally with saline and as adults with the two higher doses of diazepam (0.50 or 1.0 mg/kg bw) the number of times of entering, and time spent in, the open arms increased significantly (Fig. 4) – interpreted as a pharmacological inhibition of the reluctance/anxiety towards entering open spaces. In contrast, animals neonatally exposed to propofol showed a significantly lower number of entries and time spent in the open arms after adult diazepam treatment, compared to animals exposed to neonatal saline and adult diazepam, effects that were dose–response related. This effect of propofol was more pronounced with the higher neonatal dose (60 mg/kg bw), and there were no significant increase in entries and time spent in open arms, compared to animals exposed to saline both as neonates and adults. 4. Discussion Propofol is used in neonates and toddlers, even though it is not recommended for use in children under the age of three years. Animal studies have shown that propofol causes neuroapoptosis in the infant developing mouse brain [7], growth cone collapse [8] and damage to GABA neurons during brain cell maturation. The present study showed that neonatal propofol exposure altered the neonatal levels of BDNF in different areas of the brain, altered the adult response to diazepam (e.g. by reducing its effect) and caused an up-regulation in GABAA expression throughout the adult brain. The study supports earlier findings on propofol as a possible neurotoxicant in the developing brain. In the neonatal brain, the propofol treatment did not alter the levels of CaMKII, GAP-43, synaptophysin and tau in frontal cortex, parietal cortex or hippocampus. In previous studies, we found that exposure to certain chemicals, including the anaesthetic drug ketamine [14], during the BGS, can cause alterations in one or more of these four proteins, which undergo marked ontogeny during the neonatal period. These proteins are involved in several important developmental processes and we speculate that alterations in their levels could have consequences later in life. Several recent studies have shown that neonatal exposure to different xenobiotics, including environmental contaminants (e.g. brominated flame retardants and fluorinated compounds) and pharmaceutical agents (e.g. ketamine) can cause adult behavioural disturbances [14,24–26] that are preceded by neonatal alterations in the levels of one or more of these proteins, and always by a hippocampal elevation of CaMKII [14,27–30]. In the present study, there were no alterations in the levels of these proteins, suggesting that no adult behavioural disturbances should be seen. This was also the case, since propofol-treated animals showed the same spontaneous behaviour as the control animals. This further supports our previous study of neonatal neurodegeneration and adult behaviour after neonatal propofol exposure in which there were no detectable changes in adult spontaneous behaviour after neonatal propofol exposure to the same doses (10 or 60 mg propofol/kg bw) as in the present study [18]. In the present study, diazepam was sedating and thus decreased activity in the control animals as expected. In mice exposed neonatally to propofol (10 or 60 mg/kg bw) and treated with diazepam as adults, the spontaneous motor activity was not changed following diazepam treatment, indicating that neither the spontaneous motor activity nor the sedating effect of diazepam was affected. Interestingly, the result from the elevated plus maze clearly showed that animals treated neonatally with saline and with diazepam as adults lost some anxiety-like behaviour. However, animals exposed neonatally to propofol and treated with diazepam as adults showed that anxiety-like behaviour was significantly affected as they entered the open arms less frequently and spent less time there. Furthermore, the highest neonatal propofol dose (60 mg/kg bw, for

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which there was no significant increase in entries and time spent in open arms), compared to animals exposed to saline both as neonates and adults, indicated that the diazepam treatment does not erase or close down the anxiety-like behaviour as effectively in animals treated neonatally with high doses of propofol. The adult susceptibility/reaction to diazepam was changed in propofol-treated animals. These results might indicate an altered GABAA receptor function since explorative behaviour and possibly sedation after benzodiazepines were affected. We previously reported that neonatal exposure to a chemical can alter the adult response to exposure to the same chemical or other chemicals. For example, neonatal exposure to nicotine altered the response to adult nicotine exposure, manifest as hypoactivity after an adult nicotine injection, instead of the normal response of hyperactivity [12], and these changes were also related to neonatal and adult changes in nicotinic receptors. Furthermore, we also reported increased susceptibility or altered response in adults after neonatal exposure to alcohol and certain environmental pollutants [24,31,32]. The levels of BDNF can influence the composition of GABAA subunits. In status epilepticus models in rodents, BDNF levels are increased, leading to an up-regulation of alpha 1 subunits in adult rodents [33]. However in neonates, the alpha subunits decreased in another study making interpretation difficult [34]. After anaesthesia, during the BGS, the levels of BDNF decrease, possibly by suppressing tissue plasminogen activator that in several steps activate BDNF [35], and it seems likely that this also changes the GABAA subunit composition, thus explaining the altered response to substances directed to the GABAA receptor. Studies examining the subunit expression of the GABAA receptor after anaesthesia may clarify this interesting topic. Compared to human requirements for anaesthesia the doses of propofol were high in the present study. However, translating doses between species is difficult, making the results difficult to interpret. Adequate depth of anaesthesia requires 200 mg/kg in infant mice [7]; therefore, 10 and 60 mg/kg bw are sub-anaesthetic doses. These doses are so low that respiration and circulation should not be affected. Hypoxia and hypovolemia are theoretical confounders of all studies of adverse effects of neonatal anaesthesia, but no one has yet detected hypoxia and ischemia at these doses [36–38]. Our experiments showed that neonatal propofol exposure significantly changed the adult response to the GABA-mimetic drug diazepam, manifest as no change in spontaneous motor activity and/or reduced sedative effect and an extinguishing effect on the reduction of anxiety-like behaviours. All these effects were observed in animals showing normal spontaneous behaviour. One possible mechanism behind these findings could be the neonatal alterations in the levels of BDNF, which is known to affect both the GABA receptors and the process of neurodegeneration through the induction of apoptosis. Further studies of the mechanisms of propofol treatment and/or other anaesthetic drugs during brain development are needed to gather new information for the safe use of these drugs. References [1] Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, et al. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003;23:876–82. [2] Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, et al. Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology 2009;110:796–804. [3] DiMaggio C, Sun LS, Kakavouli A, Byrne MW, Li G. A retrospective cohort study of the association of anesthesia and hernia repair surgery with behavioral and developmental disorders in young children. J Neurosurg Anesthesiol 2009;21:286–91. [4] Kahraman S, Zup SL, McCarthy MM, Fiskum G. GABAergic mechanism of propofol toxicity in immature neurons. J Neurosurg Anesthesiol 2008;20:233–40.

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