Behavioural disturbances and altered Fos protein expression in adult rats after chronic pubertal cannabinoid treatment

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Behavioural disturbances and altered Fos protein expression in adult rats after chronic pubertal cannabinoid treatment Nico Wegener⁎, Michael Koch Brain Research Institute, Department of Neuropharmacology, University of Bremen, PO Box 330440, 28334 Bremen, Germany

A R T I C LE I N FO

AB S T R A C T

Article history:

Cannabis is one of the world's most popular recreational drugs. However, little is known

Accepted 21 November 2008

about long-lasting cellular and neurobehavioural effects of chronic cannabinoid intake,

Available online 3 December 2008

especially during puberty where cannabis use among humans is commonly initiated. This study in rats investigates the long-term effect of pubertal cannabinoid treatment on

Keywords:

prepulse inhibition (PPI), locomotor activity and on anxiety in the elevated-plus maze during

Cannabis

adulthood. Furthermore, changes in adult basic neuronal activity, assessed by c-Fos

WIN 55,212-2

immunoreactivity (Fos IR), and a potentially altered Fos expression after acute treatment

Development

with dopaminergic drugs was evaluated. Chronic treatment with the synthetic cannabinoid

Prepulse inhibition

full agonist WIN 55,212-2 (WIN; 1.2 mg/kg) was carried out over 25 days of the rats' puberty

Elevated-plus maze

and subsequent behavioural testing was conducted in adult animals. Finally, Fos IR was

c-Fos

evaluated in several brain regions under basal conditions and after acute administration of haloperidol (0.1 mg/kg) and apomorphine (2 mg/kg). Chronic WIN treated animals exhibited a lasting disruption of PPI. These rats were also more active in the open field and less anxious in the elevated-plus maze than their vehicle treated controls. Additionally, when comparing Fos IR in selected brain regions, these animals displayed altered basal neuronal activity and responded differently to acute application of haloperidol or apomorphine. Taken together, these results indicate that chronic stimulation of the cannabinoid receptor CB1 during the rats' puberty not only leads to persistent behavioural changes but also to cellular long-term adaptations within brain regions critical for drug of abuse or neuropsychiatric diseases. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Products of the hemp plant Cannabis sativa, marijuana and hashish, belong to the most widely used illicit substances worldwide and cannabis potency as well as the rate of abuse are increasing since the early 1990s (UNODC, 2007). Despite its long history as a drug for both recreational and medicinal purposes, very little is known about potentially persistent effects of chronic cannabis use, such as long-term neurobe-

havioural disturbances and/or neuronal adaptations. Nowadays, most cannabis users start in their mid to late teens which coincides with major neuronal changes in the central nervous system (Monshouwer et al., 2005; Hall, 2006; EMCDDA, 2007). Hence concern has been growing about the possible adverse effects of cannabis on physical and/or mental health. It has been shown in humans that cannabinoid exposure especially during pubertal development induces lasting behavioural and morphological alterations (Pope et al., 2003;

⁎ Corresponding author. E-mail address: [email protected] (N. Wegener). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.11.081

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Arseneault et al., 2004; Caspi et al., 2005). For example, Ehrenreich et al. (1999) found that early-onset cannabis users (before the age of 17 years) displayed a disruption of attentional functions. Additionally, another study revealed that young consumers had a higher percentage of white matter relatively to whole-brain volume (Wilson et al., 2000). In both studies no such persistent effects were found in control subjects, i.e. long-term cannabis users who had initiated use after the age of 17. These findings are supported by animal studies, which offer better possibilities for a controlled investigation of the neuroactive components of cannabis' impact on brain maturation. Treatment of rats with the synthetic cannabinoid receptor agonist WIN 55,212-2 (WIN) or CP 55,940 throughout pubertal development from postnatal day (pd) 40 to pd65 (Schneider, 2008) led to lasting behavioural alterations in adulthood such as attentional deficits, impaired memory and altered social behaviour. Prepubertal (pd 15–40) or adult (Npd70) cannabinoid treatment had no such effects or affected the rats′ behaviour only marginally (Schneider and Koch, 2003, 2005; O'Shea et al., 2004, 2006). Interestingly, in a CB1 receptor radioligand binding study Rodriguez de Fonseca et al. (1993) found that the endocannabinoid system, which consists of endogenous cannabinoids (e.g. anandamide) and at least two cannabinoid receptors, the neuronal CB1 receptor and the CB2 receptor (which is mainly expressed in the immune system), displays its developmental peak at the onset of puberty. Additionally, during this period numerous neurodevelopmental alteration take place, e.g. myelination, synaptic pruning and the maturation of neurotransmitter systems such as the dopaminergic and glutamatergic system (Andersen et al., 2000; Spear, 2000; Schneider, 2008). It has been a matter of debate whether especially adolescent cannabis consumption increases the risk for certain neuropsychiatric diseases such as schizophrenia (Arseneault et al., 2004; Caspi et al., 2005). Increased levels of endocannabinoids were found in the cerebrospinal fluid of schizophrenic patients (Leweke et al., 1999, 2007) as well as alterations in prefrontal CB1 receptor density (Dean et al., 2001), indicating a connection between early cannabis use and schizophrenia. Additionally, there seems to be an association between time and frequency of cannabis use and subsequent use of other illicit drugs. This cannabis “gateway hypothesis” is currently subject to controversial discussions, but human studies indicate that young cannabis users are more likely to use psychostimulants, hallucinogens or opioids than those who have never used cannabis (Ferrguson et al., 2006). Further evidence comes from experimental animal models which showed that cannabinoids might induce lasting neuronal alterations that could affect the stimulant and/or reinforcing values of other drugs of abuse (Pistis et al., 2004; Ellgren et al., 2007). Therefore, both human and animal studies indicate puberty as a developmental period during which the immature organism is vulnerable to the long-term adverse effects of exogenous cannabinoids. The aim of the present study in rats was to identify longterm effects of pubertal WIN treatment on sensorimotor gating, motor activity, and anxiety during adulthood. Additionally, altered adult neuronal activity in response to chronic

WIN as well as dopaminergic drugs was assessed via c-Fos immunoreactivity (Fos IR). The acoustic startle response (ASR) can be elicited in both humans and rodents by a sudden, intense noise pulse and is characterised by a fast contraction of facial and skeletal muscles as well as by an arrest of ongoing behaviours (Koch, 1999). If the startling stimulus is shortly preceded by a weaker, non-startling stimulus (prepulse), the magnitude of the ASR is reduced. This phenomenon is called prepulse inhibition (PPI) and is thought to be due to a pre-attentive filter or sensorimotor gating mechanism regulated by a modulatory circuitry involving cortico-limbic brain structures (Swerdlow et al., 2001; Koch and Fendt, 2003). Interestingly, a reduced PPI can be observed in certain neuropsychiatric disorders, e.g. schizophrenia (Swerdlow et al., 2006). In humans and rodents PPI deficits can be induced by various drugs (reviewed in Geyer et al., 2001) as well as CB1 receptor agonists (Schneider and Koch, 2002; Wegener et al., 2008). However, studies investigating the long-term effects of pubertal cannabinoid treatment on sensorimotor gating ability are rare and provide inconsistent results. One previous study from our laboratory has shown that chronic pubertal but not adult WIN treatment led to persistent PPI deficits in Wistar rats (Schneider and Koch, 2003). In contrast, one other study found no such differences when using Sprague–Dawley rats and a slightly different experimental setup (Bortolato et al., 2005). Therefore, we strived to replicate our previous data as well as to add more information about the behavioural consequences of chronic cannabinoid exposure during a critical developmental stage. Besides PPI regulating circuits, the CB1 receptor is densely expressed in regions which initiate and coordinate movement, e.g. the cerebellum and the basal ganglia, and can also be found in limbic structures, e.g. amygdala, which is known to mediate anxiety-like behaviour (Herkenham et al., 1991; Tsou et al., 1998). To assess the impact of pubertal WIN on adult locomotor activity and anxiety we also tested the rats' behaviour in the open field and in the elevated-plus maze. Finally, altered neuronal activity as consequence of chronic pubertal cannabinoid treatment was evaluated in adult rats. It was suggested that expression of the proto-oncogene c-fos may serve as a marker for neuronal activity (Dragunow and Faull, 1989; Sagar et al., 1988). The Fos protein is a well characterised member of a family of immediate early genes (IEG) which can bind to DNA, thereby influencing the transcription of specific target genes (Sheng and Greenberg, 1990). Altered expression of such IEGs would lead to altered neuronal activity and ultimately to changes in the neural circuits in which those neurons operate. Psychostimulant drugs such as cocaine or amphetamine, but also antipsychotic drugs are known to induce a specific pattern of Fos activation in the striatum and nucleus accumbens (NAc) (reviewed in Harlan and Garcia, 1998; Sumner et al., 2004). Previous studies examining the effects of acute Δ9-THC or its synthetic analogues on Fos IR have also shown a consistent distribution of Fos expression, e.g. in the caudate–putamen, NAc, ventral tegmental area (VTA), and amygdala (McGregor et al., 1998; Miyamoto et al., 1997; Arnold et al., 2001; Patel and Hillard, 2003; Rubino et al., 2007). However, little is known about possible persistent changes in neuronal activity after chronic pubertal CB1 receptor

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stimulation. A matter of particular interest is the question of whether WIN treatment during a critical phase of brain development also leads to an altered neuronal response to certain drugs of abuse in adulthood. Such drugs commonly target the mesolimbic and mesoaccumbal dopamine (DA) system. Therefore, in this study the neuronal effects of the DA receptor agonist apomorphine and the DA receptor antagonist haloperidol were assessed via Fos quantification.

2.

Results

2.1.

PPI of the ASR

sity: F2,88 = 28.9; P N 0.001] even though less distinctive (Tukey's t-tests: 68 dB: P = 0.015; 72 dB: P = 0.014) (Fig. 1D). No significant differences between the ASR amplitudes of controls and WIN treated rats were found on pd80 or pd105 (data not shown).

2.2.

Locomotor activity

The t-tests for open field behaviours showed a significant increase in general activity on pd85 (Fig. 1B). Chronic WIN treated animals travelled a longer distance (P = 0.006), were more active (P = 0.001), spent more time in the center of the arena (P = 0.035), and exhibited increased rearing behaviour (P = 0.035).

2.3. As expected from our previous studies, the ANOVA revealed a significant main effect of chronic pubertal WIN pretreatment [F 1,88 = 11.3; P = 0.002] and prepulse intensity [F 2,88 = 8.4; P < 0.001] on PPI when measured in adult rats on pd80. Post hoc Tukey's t-tests showed that PPI was disrupted at all three prepulse intensities of 68 (P = 0.003), 72 (P < 0.001), and 76 dB (P = 0.028) (Fig. 1A). This effect was persistent throughout adulthood, since the PPI deficit was still evident on pd105 [factor pretreatment: F1,88 = 5.7; P = 0.021/factor prepulse inten-

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Elevated-plus maze

Pubertal WIN treatment significantly reduced the percent time spent in closed arm exploration on pd90 (P = 0.009) while at the same time increasing the residence on the open arm in these animals when compared to vehicle treatment (P = 0.001) (Fig. 1C). Additionally, when comparing absolute number of arm entries WIN pretreated rats visited the open arm more often (P = 0.024) than controls while at the same time no difference in the total number of arm entries was found (data not shown).

Fig. 1 – Effects of chronic pubertal vehicle (n = 22) or cannabinoid treatment (n = 24) on prepulse inhibition (%PPI) (A, D), motor activity in the open field arena (B), and anxiety in the elevated-plus maze (C). Tests were performed on postnatal day (pd) 80, 85, 90, and 105. Data are means ± SEM. Pubertal WIN induced a persistent PPI deficit which was still evident 40 days after the last WIN injection. Furthermore, chronic WIN treated rats exhibited increased general motor activity and spent more time on the open, unprotected arm of the elevated-plus maze (asterisks, P < 0.05).

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Table 1 – Absolute counts of Fos labeled cells (±SEM) in various brain regions (distance from Bregma in mm) of chronic pubertal vehicle (Control) or WIN treated (WIN) rats after acute administration of either vehicle (VEH; n = 5) apomorphine (APO; 2 mg/kg; n = 4) or haloperidol (HAL; 0.1 mg/kg; n = 5) on postnatal day (pd) 108 Region

Bregma

Acute treatment VEH

APO

HAL

Control

WIN

Control

WIN

Control

WIN

Cortical regions 1. vmPFC 2. dmPFC

+ 2.20 + 1.70

22 ± 5 161 ± 17

53 ± 17 94 ± 16

59 ± 9 138 ± 16

9±2 78 ± 13

73 ± 13 128 ± 27

184 ± 37⁎△ 200 ± 25△

Ventral striatum 3. NAcC 4. NAcSh

+ 1.20 + 1.20

59 ± 14 122 ± 31

113 ± 12⁎ 125 ± 24

37 ± 7 150 ± 10

47 ± 8△ 97 ± 25

100 ± 11△ 141 ± 21

112 ± 17 139 ± 12

Dorsal striatum 5. mCPU 6. lCPU

+ 0.48 + 0.48

109 ± 14 85 ± 22

53 ± 9⁎ 39 ± 13

40 ± 9△ 20 ± 3

16 ± 3 35 ± 7

61 ± 15 46 ± 16

76 ± 12 44 ± 13

Amygdala 7. CeA 8. BLA

− 2.56 − 2.56

45 ± 12 22 ± 5

45 ± 10 28 ± 7

63 ± 12 60 ± 10

27 ± 8 15 ± 5⁎

115 ± 19△ 99 ± 17△

12 ± 5⁎ 12 ± 3⁎

Hippocampal area 9. dHIP 10. DG 11. vHIP

− 3.60 − 3.60 − 4.80

41 ± 6 22 ± 4 55 ± 11

42 ± 9 55 ± 11⁎ 9 ± 4⁎

62 ± 4 51 ± 4△ 21 ± 4△

19 ± 5⁎ 28 ± 8 28 ± 5

43 ± 6 17 ± 3 8±3

60 ± 6 28 ± 3 46 ± 6⁎△

Significant effects of peripubertal pretreatment are indicated by asterisk and significant effects of acute treatment by triangles (P < 0.05). Ventral/ dorsal medial prefrontal cortex (vmPFC, dmPFC), medial/lateral caudate/putamen (mCPu, lCPu), nucleus accumbens core/shell (NAcC, NAcSh), central nucleus of amygdala (CeA), basolateral amygdala (BLA), dorsal/ventral hippocampus (dHIP, vHIP), dentate gyrus (DG).

2.4.

Regional Fos IR

Fos counts for the 11 brain regions are shown in Table 1. Representative photomicrographs of Fos IR in selected brain

regions of pubertal WIN or vehicle treated rats after acute administration of dopaminergic drugs are presented in Fig. 2. Two-way ANOVAs revealed a significant interaction of the factors pretreatment and acute treatment in seven of the

Fig. 2 – Representative photomicrographs showing Fos labelled neurons of rats chronically treated with either vehicle (VEH) or WIN (WIN) during puberty after acute adult vehicle (VEH), apomorphine (APO), or haloperidol (HAL) administration. (A) nucleus accumbens core (NAcC), (B) dorsal hippocampus (dHIP), (C) ventral medial prefrontal cortex (vmPFC). Scale bar = 250 μm.

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investigated brain regions reflecting that acute treatment with dopaminergic drugs affected Fos IR in dependence of pubertal pretreatment (vmPFC: F(2,22) = 4.47, P = 0.023; dmPFC: F(2,22) = 4.29, P = 0.027; dHIP: F(2,22) = 6.54, P = 0.006; vHIP: F(2,22) = 18.62, P < 0.001; DG: F(2,22) = 6.40, P = 0.006; BLA: F(2,22) = 7.23, P = 0.004; CeA: F(2,22) = 6.20, P=0.007). Albeit no such effect was seen in the NAcC ANOVA showed a statistical effect of the main factors pretreatment (F(1,22) =9.22, P=0.006) and acute treatment (F(2,22) =20.08, P<0.001). Subsequent t-tests revealed altered baseline Fos IR in several brain regions of animals which had received an acute vehicle injection on pd108 and were chronically treated with WIN during puberty. Increased baseline Fos IR was evident in NAcC and DG (P = 0.003; P = 0.005), while in mCPu and vHIP (P = 0.015; P < 0.001) baseline Fos expression was reduced when compared to chronic vehicle treated rats. Throughout the brain, significant differences in Fos IR were observed in both, acute haloperidol and apomorphine treatment when compared to the corresponding acute vehicle treated controls. Within the chronic vehicle treated group haloperidol increased Fos IR in NAcC (P = 0.021), BLA (P < 0.001), and CeA (P = 0.008). Apomorphine increased Fos IR in DG (P = 0.048) while in mCPu (P = 0.017) and vHIP (P = 0.013) less Fos IR was seen. Following pubertal WIN treatment haloperidol altered Fos IR in other brain areas than in control animals. Increased Fos expression was evident in dmPFC and vHIP (P = 0.028; P = 0.003), whereas vmPFC showed reduced Fos IR (P = 0.004). Apomorphine altered Fos IR in NAcC, where a significant reduction (P = 0.003) was observed. When comparing the neuronal consequences of pubertal WIN pretreatment on acute drug administration, haloperidol led to an increased Fos IR in vmPFC (P = 0.005) and vHIP (P < 0.001) and decreased Fos IR in the amygdala (BLA: P < 0.001; CeA: P < 0.001) compared to vehicle pretreated controls. WIN pretreatment also affected the response to acute apomorphine so that dHIP and BLA (P = 0.002; P = 0.028) showed less Fos expression.

3.

Discussion

This study was designed to test whether chronic pubertal cannabinoid treatment persistently affects behaviour and neuronal activity of adult rats. Therefore, we used the PPI paradigm to test the effect of chronic CB1 receptor stimulation on sensorimotor gating, the elevated-plus maze to measure anxiety-like behaviour, and the open field arena to evaluate locomotor activity. Additionally, immunoreactivity of the cFos protein, a marker of neuronal activity was analysed in several brain regions to test if the animals' drug history affected their basal Fos IR or their neuronal response to acute administration of dopaminergic drugs, respectively. The present data show that chronic pubertal treatment (pd40–65) with the synthetic cannabinoid CB1 receptor agonist WIN led to several behavioural alterations in adult animals, including long-lasting disruptions of sensorimotor gating, increased motor activity, and reduced anxiety-like behaviour. Additionally, when comparing Fos IR in selected brain regions, these animals displayed altered basal neuronal activity and responded differently to acute application of haloperidol or apomorphine.

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Our results of the behavioural assessment of pubertal cannabinoid treatment in adult rats is consistent with several other studies in humans (Ehrenreich et al., 1999; Pope et al., 2003; Arseneault et al., 2004) and animals (Schneider and Koch, 2003, O'Shea et al., 2004, 2006; Schneider et al., 2008) identifying puberty as a vulnerable period for the adverse effects of chronic cannabinoids. Puberty and adolescence are overlapping time periods, with puberty being a part of adolescence, and represent important developmental periods during which an adolescent matures into an adult. In the rat, the exact period of puberty can be determined by specific external physical signs. In male rats balano preputial separation (BPS), defined as complete separation of prepuce from gland penis (balano), normally occurs around pd40 (Korenbrot et al., 1977; Fernandez-Fernandez et al., 2005) and is therefore considered as a physiological sign for the onset of puberty. For the male rat a juvenile period was suggested for pd22–36, followed by a peripubertal period from pd36 until, after pubertal development from pd40, fertility is reached around pd60, indicated by the presence of mature spermatozoa in the vas deferens (Ojeda and Urbanski, 1994). However, there is an ongoing discussion on this matter and opinions slightly differ about the precise time windows of certain developmental periods (for a thorough review see Schneider, 2008). The different components of the endocannabinoid system are present from the early stages of gestation and it was shown that the endocannabinoid system, including receptors and endogenous ligands, plays a vital role during neurodevelopment (Rodriguez de Fonseca et al., 1993; Fernandez-Ruiz et al., 1999, 2000, reviewed in Schneider, 2008). Interestingly, a thorough radioligand binding study by Rodriguez de Fonseca et al. (1993) found a progressive increase of CB1 receptor density in several brain regions gradually starting from pd10 and reaching a maximum around the onset of the rats' puberty (pd40). During the pubertal period CB1 receptor density seems to decrease until finally reaching adult values on pd70. Considering that the activity of the endocannabinoid system seems to be highest around puberty it is conceivable that this specific developmental period is vulnerable to the consequences of chronic exogenous cannabinoid exposure. PPI is an operational measure for sensorimotor gating mechanisms and is regulated by a cortico-limbic-striatopallidal circuit (Koch, 1999; Swerdlow et al., 2001). Dopamine (DA) as well as glutamate (Glu) seem to play a major role in regulating PPI in humans and other animals as could be demonstrated in several studies (for review see Geyer et al., 2001). For example, systemic administration of DA agonists such as apomorphine or amphetamine disrupts PPI in rats, which is probably due to an overactivation of the mesoaccumbal DA system. It is well known that acute cannabinoid treatment affects PPI in rats (Schneider and Koch, 2002; Martin et al., 2003; Wegener et al., 2008). A recent microinjection study from our laboratory identified the mPFC and the vHIP as important sites of acute cannabinoid action in inducing deficits of sensorimotor gating. CB1 receptor stimulation probably modulates GABA and/or Glu release within these areas, thereby directly or indirectly affecting neuronal PPI-regulating key structures, such as NAc and VTA (Wegener et al., 2008). Since the endocannabinoid system plays a major role in the maturation of other neurotransmitter systems,

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alterations within the endocannabinoid system induced by chronic pubertal cannabinoid exposure are paralleled by changes in other transmitter systems, e.g. the dopaminergic system. Previous studies demonstrated that DA D1/D2 receptor density in rat striatum and prefrontal cortex peaked at the onset of puberty and declined by 58–75% upon reaching adulthood (Teicher et al., 1995; Andersen et al., 2000). In the present study a long-lasting PPI deficit in drug-free adult rats was evident after chronic pubertal WIN treatment. Even though there are only few studies on the effects of chronic cannabinoid treatment on sensorimotor gating, their results suggest persistent changes within the dopaminergic system for such behavioural alterations. First, Wu and French (2000) showed that VTA DA neurons from animals that received twice-daily injections of Δ9-THC for 14 days were characterised by an increased firing rate in response to acute Δ9THC, thus enhancing mesoaccumbal DA release. Second, a comparable chronic cannabinoid treatment also modified the structure of dendrites (increased length and branching) in the shell of the NAc and the mPFC, regions implicated in PPI circuitry and DA-mediated mechanisms of reward (Kolb et al., 2006). These neuronal changes seem to reflect a persistent reorganisation of synaptic organisation in the affected brain regions and were similar to that produced by chronic exposure to certain drugs of abuse such as amphetamine or cocaine (Robinson and Kolb, 1999). Third, a previous behavioural study from our laboratory adds more evidence for an involvement of long-term alterations within the dopaminergic system in adult sensorimotor gating deficits. Schneider and Koch (2003) showed that chronic pubertal but not adult WIN treatment led to persistent PPI deficits and also observed that this deficit could be reversed by the DA D2 receptor antagonist haloperidol 85 days after chronic treatment was ceased. Since in our study no effects of pubertal WIN treatment on ASR magnitude were found, we assume that the observed PPI deficit reflects a genuine sensorimotor gating disturbance and is not due to altered processing of the startle stimulus. Another behavioural finding of this study was an increased motor activity in the open field in adult rats after pubertal chronic WIN treatment. The rats travelled a longer distance, were more active, spent more time in the center of the arena, and exhibited increased rearing behaviour when compared to controls. Cannabinoids are classically known to produce potent effects on movement (Chaperon and Thiebot, 1999). The neuronal localisation of the CB1 receptor is consistent with these effects since a prominent expression pattern of the receptor was shown in areas controlling motor behaviour, such as the basal ganglia and the cerebellum (Herkenham et al., 1991; Tsou et al., 1998). Several recent reports suggest that acute cannabinoid administration affects locomotion in a dose-related bi-phasic manner via CB1 dependent regulation of glutamatergic and GABAergic systems within the basal ganglia (Rodriguez de Fonseca et al., 1998). Cannabinoids major acute effect in movement is hypoactivity and catalepsy (Chaperon and Thiebot, 1999) while an increase in motor activity has been associated with low doses of cannabinoid receptor agonists (Sanudo-Pena et al., 2000; Drews et al., 2005). In contrast to our result, previous studies, where rats were chronically treated with cannabinoid receptor agonists

through either perinatal, adolescent, or young adult development, found no such effect on locomotor activity in the open field (Biscaia et al., 2003; O'Shea et al., 2004, 2006; Schneider et al., 2005). Even in a previous study from our laboratory, where chronic pubertal WIN treatment and subsequent behavioural testing procedures were comparable, no effect of cannabinoid pretreatment could be observed in adult rats (Schneider and Koch, 2003). The only explanation we can offer for this discrepancy is that slight procedural differences may account for our present result. For example, in the studies of O'Shea et al. (2004, 2006) increasing doses of CP 55,940 were administered for 21 consecutive days from pd35, thus beginning treatment 5 days earlier than in this study, whereas also in one of these experiments female rats were used. Additionally, with respect to the study of Schneider and Koch (2003), where in contrast to this study testing was started on pd75 with an operant behaviour task, variations in the sequence of the applied behavioural tests might be responsible for the different effects on locomotor activity. However, lasting alterations of endocannabinoid functioning within regions relevant for movement might also account for the observed enhanced activity of the rats in this study. It was shown by in situ hybridisation that chronic treatment with CP 55,940 reduced cannabinoid receptor mRNA by 33% in the caudate–putamen (Rubino et al., 1994). Additionally, Castelli et al. (2007) observed the persistence of long-term modifications within the endocannabinoid system (reduced FAAH and increased AEA levels) e.g. in the striatum of male adult rats exposed to cannabinoid agonists in the prenatal period. Since rodents have a natural aversion against open spaces and an increased time spent in the center of the open field arena could be observed in our study, it is conjectured that pubertal WIN pretreatment leads to anxiolytic-like responses in adulthood. Similar to locomotion and exploratory behaviour, bi-directional effects of acute cannabinoids on anxiety have been reported, with low doses having anxiolytic and high doses exerting anxiogenic effects (reviewed in Chaperon and Thiebot, 1999; Viveros et al., 2005; Patel and Hillard, 2006). The elevated-plus maze, a test validated for anxiolytic and anxiogenic compounds, exploits the aversion of rodents for novel, high and open spaces (File, 1987). Here, we report decreased anxiety after pubertal chronic WIN treatment in the elevated-plus maze, as evidenced by increased residence on the open/unprotected arm when compared to controls while leaving total number of arm entries unaffected. Few animal studies have been devoted to possible alterations of anxiety-like behaviour after chronic cannabinoid administration and the possible mechanisms involved in the anxiolytic-like response induced by cannabimimetics have not been clearly elucidated. O'Shea et al. (2004) showed an adult decrease in social interaction with a novel conspecific, i.e. increased social anxiety, after chronic adolescent cannabinoid exposure for 21 days (see also Schneider et al., 2008). A subsequent study by the same group revealed a modest reduction of anxiety in the emergence test only in adolescent CP 55,940 treated rats but not in perinatal or adult treatment groups (O'Shea et al., 2006). However, one study by Biscaia et al. (2003) in Wistar rats found a sex-dependent decrease in the level of anxiety in the open field and in the

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plus maze after chronic administration of CP 55,940 for 11 days from pd35–45. The anxiolytic-like effects of chronic cannabimimetics might depend on long-term alterations within the opioid system (König et al., 1996; Biscaia et al., 2003). Perinatal exposure to cannabinoids is able to alter the development of opioidergic neurons (Fernandez-Ruiz et al., 1999) and chronic cannabinoid treatment for 18 days increased proenkephalin mRNA in several brain regions of adult rats, e.g. in hypothalamic and limbic areas implicated in the regulation of anxiety (Manzanares et al., 1998). Therefore, it seems likely that there is a critical time-window during which the effects of chronic CB1 receptor stimulation produces the long-term anxiolytic effects observed in this study, possibly mediated via lasting alterations within the opioid system. Besides the behavioural alterations induced by pubertal WIN treatment, our study also revealed persistent changes of neuronal activity assessed by c-Fos protein quantification in several brain regions under basic conditions and in response to dopaminergic drugs. The expression of the Fos protein, a product of the IEG family which acts as a transcription factor, is increased as a result of somatodendritic depolaristation and can therefore be considered as a marker of increased neuronal excitation (Morgan and Curran, 1986, 1988; Harlan and Garcia, 1998). Acute administration of drugs of abuse produces a rapid but transient induction of several Fos family members e.g. in the NAc (Nestler, 2001). Patel and Hillard (2003) showed that acute CP 55,940 increased Fos expression in VTA neurons, an effect which was inhibited by pretreatment with the CB1 receptor antagonist SR141716, providing strong support for a CB1 receptor mediated effect. Additionally, in another study a Δ9-THC induced Fos IR in the NAc was blocked by the DA D1 receptor antagonist SCH 23390 possibly reflecting a cannabinoid mediated action on DA release in the NAc with increased DA levels caused by Δ9-THC leading to increased Fos IR (Miyamoto et al., 1996). In addition to our behavioural results which showed lasting alterations of sensorimotor gating, locomotor activity and anxiety, in rats which had received acute vehicle on pd108, baseline Fos IR was altered in the NAcC, mCPu, DG, and vHIP depending on the rats' drug history. It is well known that the NAc, the striatum and the hippocampus play roles in regulating PPI and locomotion. The repeated exposure to a drug of abuse would eventually lead to altered gene expression and neuronal activity and ultimately to changes within neural circuits in which those neurons operate, which might be demonstrated by stable changes of behaviour as seen in this study (Nestler, 2001). We therefore conclude that CB1 receptor stimulation throughout the rats' puberty leads to lasting alterations of neuronal excitability within neuronal circuits regulating behaviour. There are at least three other studies reporting long-term effects of prolonged cannabinoid treatment on neuronal Fos expression. For example, Singh et al. (2006) treated rats with Δ9-THC during perinatal development. They found a widespread stimulation of basal Fos IR in adult rats but also reported an increase in the addictive properties of heroin. An earlier study of the same group, where rats received 21 injections of Δ9-THC throughout juvenile development, revealed that preexposure to a cannabinoid either inhibited

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or stimulated heroin induced Fos IR in several brain regions (Singh et al., 2005). Furthermore, Miyamoto et al. (1997) found a desensitisation of the cannabinoid dependent induction of Fos protein expression in the rat NAc and striatum after repeated Δ9-THC administration for 3 days. In our study, the rats' drug history affected the neuronal response to acute apomorphine or haloperidol challenge. When comparing the effects of acute apomorphine or haloperidol treatment on Fos IR in pubertal vehicle or WIN treated rats, a generally altered neuronal Fos expression pattern for each drug was evident depending on pubertal pretreatment. For example, acute apomorphine altered Fos IR in the striatum and hippocampus of pubertal vehicle treated rats, while in WIN treated animals only Fos expression in the NAcC was affected. Haloperidol, however, increased Fos IR in the NAcC and amygdala in control animals but affected prefrontal and hippocampal Fos IR in the WIN group. Interestingly, chronic WIN treated rats not only showed a higher baseline Fos IR in the NAcC, a key structure of the mesolimbic reward system, but within this region also responded differently to dopaminergic drugs. This result implicates an altered response to drugs of abuse, as e.g. Singh et al. (2005, 2006) reported for heroin. Hence, together with these studies, our study shows that brain function seems to be altered weeks after cannabinoid exposure has ceased and raises the possibility that an increased vulnerability of neuronal integrity to prolonged CB1 receptor stimulation exists during certain phases of brain development. In conclusion, our results indicate that chronic stimulation of the cannabinoid CB1 receptor with the synthetic cannabinoid receptor agonist WIN during puberty not only leads to lasting changes in sensorimotor gating, motor activity and anxiety, but also to long-term changes on a cellular level, possibly affecting the adult organism's response to certain drugs of abuse. These persistent alterations might be due to an imbalance in various neurotransmitter systems, e.g. the dopaminergic, opioid, and/or endocannabinoid system, induced by chronic WIN during a critical phase of brain development. Furthermore, the change in Fos IR may represent a neuronal correlate for the effects of pubertal WIN exposure on behavioural alterations observed during adulthood. Consistent with previous results we therefore conclude puberty in rats as a vulnerable period for the adverse effects of cannabinoids.

4.

Experimental procedures

4.1.

Subjects

A total of 46 naïve first-offspring male Wistar rats (Hannover strain) from our own breeding colony were used for pubertal cannabinoid treatment. The litters were culled to eight pups directly after birth. In case of less than eight male pups, females were used to fill up the litter. Main treatment groups were recruited from nine litters. To avoid litter effects, equal proportions of rats of each litter were assigned to the different treatment groups. After weaning on postnatal day (pd) 21, male pups were removed from the breeding cages and housed in a different room in groups of 4–6 in Macrolon cages (type IV)

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under standard conditions on a 12 h light–dark schedule (lights on 0700–1900). They received free access to tap water and were fed ad libitum until pd40 after reaching a body weight of 180 g. Then they were maintained on their experimental body weight of 220–270 g by controlled feeding of 12 g rodent chow/rat/day. The experiments were performed in accordance with the NIH ethical guidelines for the care and use of laboratory animals for experiments, and were approved by the local animal care committee.

4.2.

Drugs

WIN 55,212-2 (Sigma-Aldrich, Steinheim, Germany) was dissolved in Tween80 and diluted in 0.9% saline (1% Tween80/ saline) and administered intraperitoneally (i.p.) at a dose of 1.2 mg/kg. Apomorphine (Sigma, Steinheim, Germany) was dissolved in water with 0.1% ascorbic acid and injected subcutaneously (s.c.) at a dose of 2 mg/kg. Haloperidol (0.1 mg/kg; Haldol Janssen, Neuss, Germany) was diluted in saline and administered i.p. All drugs were freshly prepared before being used in an injection volume of 1 ml/kg.

4.2.1.

Experimental procedure

Pubertal chronic treatment with either WIN (n = 24) or its vehicle (n = 22) lasted from pd40 to pd65 throughout the rats puberty (Schneider, 2008). During this period, rats received 20 injections in an irregular injection schedule (10 times one injection, 5 times two injections, and 10 times no injection per day). In order to exclude withdrawal effects after chronic WIN treatment a rest period of 15 days was allowed before adult behavioural testing began. Testing included analysis of sensorimotor gating (PPI of the ASR) on pd80 and pd105, locomotor activity (pd85), and anxiety on the elevated-plus maze (pd90). Finally, after behavioural experiments were finished Fos IR was examined on pd108.

4.3.

Behavioural testing

PPI of the ASR was measured using an eight-unit automated SRLab startle system (San Diego Instruments, San Diego, USA). Startle-boxes consisted of non-restrictive plexiglass cylinders (9 cm in diameter) resting on a platform, under which a piezoelectric sensor was mounted, inside of a sound-attenuated and ventilated chamber. Vibrations of the cylinder caused by the whole-body ASR were transduced into analogue signals and then digitised and stored by a computer using the SRLab software (San Diego Instruments). At the beginning of each session animals were placed into the startle chambers and during a 5 min acclimatisation period exposed to a 60 dB white background noise, delivered through speakers above the animal, which continued for the remainder of the session. A total of 70 trials were delivered with an average intertrialinterval of 25 s. The first and last five trials consisted of single 20 ms pulse-alone white noise stimuli with an intensity of 105 dB sound pressure level (SPL). The middle 60 trials consisted of ten 105 dB pulse-alone trials, 30 prepulse–pulse trials, ten prepulse trials (76 dB), and ten nostim trials, during which no stimuli were presented. The different trial-types

were delivered in a pseudorandom order. Prepulse–pulse trials consisted of a single 105 dB pulse preceded by 100 ms by a white noise prepulse of either 68, 72, or 76 dB (duration 20 ms, 0 ms rise/fall time). The percentage of PPI (%PPI) induced by each of the three prepulse intensities was calculated as: [100(startle amplitude on prepulse–pulse trial)/(startle amplitude on pulse-alone trial)]. Locomotor activity was measured in an infrared-beam operated open field arena (44.7 × 44.7 × 44 cm, ActiMot-System, TSE, Bad Homburg, Germany) for 35 min. The open field boxes were evenly illuminated by room light and through the plexiglass walls of the arena extra maze cues were visible. At the beginning of the test session, the rat was placed in the center of the test chamber, and distance travelled (m), time spent in center (sec), activity (%), and number of rearings were recorded. Between subjects the arena was cleaned with a 70% ethanol solution. The elevated-plus maze used to assess the rats' anxiety consisted of four arms elevated 80 cm above the floor. Each arm (12 cm wide, 76 cm long) was joined to the others by a central square (12 cm × 12 cm) in a cross-like disposition. A wall (30 cm in height) enclosed two opposite arms, while the other two arms were open. Placing the rat on the platform facing an open arm started the test. During the next 10 min the number of entries in open or closed-arms and the accordant time spent was recorded. The rats were considered to have entered an arm when all four limbs were located in an arm of the maze. The experimenter monitored the movements of the rats via a video camera mounted above the maze and a TV-screen outside the experimental room. In between animals the alleys of the maze were cleaned with a 70% ethanol solution.

4.4.

Fos IR

To quantify adult neuronal activity via Fos protein expression, chronically WIN or vehicle treated rats were assigned to three different testing groups receiving either one injection of apomorphine (chronic vehicle, n = 4; chronic WIN, n = 4), haloperidol (chronic vehicle, n = 5; chronic WIN, n = 5), or vehicle (chronic vehicle, n = 5; chronic WIN, n = 5). Two habituation days were given immediately prior to the test day in order to minimise any Fos IR due to novelty of testing, handling or injection procedures. On habituation days the animals were subjected to exactly the same procedure to those used on the test day except that they were given only saline injections. On testing day individual rats were removed from their homecage, kept in a small Macrolon cage (type III) and injected with either apomorphine, haloperidol, or vehicle. For the next 2 h the rat was individually kept in a dark and quiet room.

4.5.

Immunohistochemistry

Immediately afterwards, animals were deeply anaesthetised with chloral hydrate and transcardially perfused with 250 ml phosphate buffered saline (PBS; pH 7.4) followed by 500 ml 4% paraformaldehyde in 0.1 M PB (pH 7.4). After perfusion the brains were removed and cryoprotected in 30% sucrose solution for 72 h at 4 °C. Three series of 40 µm coronal brain sections were cut on a cryostat and collected in PBS. One of

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these series was mounted on gelatine-coated slides and Nisslstained with thionin. The second series was processed for Fos IR and first underwent four steps of washing (5 min each) in PBS. To block non-specific antibody binding and inhibit endogenous peroxidase activity, free-floating sections were preincubated for 60 min in blocking buffer (10% normal goat serum, 0.1% Triton X-100 in PBS and 0.15% H2O2). Sections were then incubated for 48 h at 4 °C in blocking buffer containing rabbit α-c-Fos serum (Calbiochem, Darmstadt, Germany) diluted 1:2000. After washing, sections were placed in PBS containing 1% bovine serum albumine (PBSA, pH 7.4) for 60 min. Thereafter, they were directly placed in biotinylated secondary antibody solution (1:2000 in PBSA; Goat α-rabbit; DAKO, Glostrup, Denmark) for 48 h at 4 °C. Next, after washing the sections, they were preincubated in PBSA for 60 min and then left for 24 h in a solution of PBSA and a Avidin/Biotinylated enzyme complex (1:1000; ABC-Kit, Vector Laboratories Inc., Burlingame, USA) at room temperature. On the last day, sections were again washed and transferred to Tris-buffered saline (TBS, pH 7.6). To visualise peroxidase activity, they were then preincubated in a TBS solution containing 0.05% 3,3diaminobenzidine tetrahydrochloride and 0.07% Imidazol for 10 min. Thereafter, ammonium nickel sulfate (0.3%) and 0.01% H2O2 was added for 3 min and the reaction terminated by washing in PBS. Finally, the immunostained sections were mounted onto gelatine-coated slides, air-dried, dehydrated in alcohols, cleared in xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany). Controls for labelling specificity included omitting the primary antibody in preliminary tests. The third series of sections was kept frozen for reserve.

4.6.

89

Counting of labelled cells

The nuclei of Fos IR cells were quantified using a Zeiss Axiophot microscope (Göttingen, Germany) equipped with the digital camera RT Slider Spot and the image analysis software Metamorph 4.6 (both from Visitron Systems GmbH, Puchheim, Germany) in 11 different brain regions or subregions with reference to the rat brain atlas of Paxinos and Watson (1998). Counts were bilaterally made within frames of 0.15 mm2 when viewed under the 100 times magnification by an observer who was blind to group assignment. Placement of the frames in each region is presented in Fig. 3. Fos IR was counted when the nucleus appeared round or oval, was completely filled, and dark in color. No stereological counting techniques were used. Double counting errors were excluded since the Fos labelled sections were 120 μm apart.

4.7.

Statistical analysis

The descriptive statistics is based on means and variance and is indicated by the standard error of the mean (±SEM). All analyses were performed with the statistical software SigmaStat (version 2.03 for Windows). A value of P < 0.05 was considered to represent a significant effect. Two-way repeated measure analysis of variance (ANOVA) followed by post-hoc Tukey t-tests for pairwise comparison were performed to analyse the effect chronic pubertal WIN on PPI (factors: pretreatment and prepulse intensity). Effects on ASR magnitude, locomotor activity in the open field and anxiety behaviour in the elevated-plus maze were evaluated by t-tests. Fos IR in different brain regions after acute injection

Fig. 3 – Schematic diagrams of coronal sections of the rat brain (Paxinos and Watson, 1998). Fos positive neurons were counted within frames (shaded in grey) and numbered areas correspond to the brain regions listed in Table 1. Numbers represent caudal and rostral distance (mm) from Bregma, respectively.

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of dopaminergic drugs was evaluated via two-way ANOVA (factors: pretreatment and acute treatment) followed by posthoc Bonferroni t-tests.

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