Chronic exposure to WIN55212-2 affects more potently spatial learning and memory in adolescents than in adult rats via a negative action on dorsal hippocampal neurogenesis

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Chronic exposure to WIN55,212-2 affects more potently spatial learning and memory in adolescents than in adult rats via a negative action on dorsal hippocampal neurogenesis

Oualid Abboussi^a,

⁎

, Abdelouahhab Tazi^b, Eleni Paizanis^c, Soumaya El Ganouni^a

aLaboratory of Biochemistry and Neurosciences, University Hassan 1^er. Settat, Morocco

bDepartment of Pharmacology, Faculty of Medicine and Pharmacy. Casablanca, Morocco

cMemory and Behavioural Plasticity Group, University Caen Basse-Normandie, Caen, France

a b s t r a c t a r t i c l e i n f o

Article history:

Received 8 October 2013

Received in revised form 20 February 2014 Accepted 20 February 2014

Available online 26 February 2014

Keywords:

Adolescence Cannabinoids Morris water maze Shuttle-box

Hippocampal neurogenesis

Several epidemiological studies show an increase in cannabis use among adolescents, especially in Morocco for being one of the major producers in the world. The neurobiological consequences of chronic cannabis use are still poorly understood. In addition, brain plasticity linked to ontogeny portrays adolescence as a period of vulnerability to the deleterious effects of drugs. The aim of this study was to investigate the behavioral neurogenic effects of chronic exposure to the cannabinoid agonist WIN55,212-2 during adolescence, by evaluating the emotional and cognitive performances, and the consequences on neurogenesis along the dorso-ventral axis of the hippocampus in adult rats. WIN55,212 was administered intraperitoneally (i.p.) once daily for 20 days to adolescent (27–30 PND) and adult Wistar rats (54–57 PND) at the dose of 1 mg/kg. Following a 20 day washout period, emotional and cognitive functions were assessed by the Morris water maze test and the two-way active avoidance test. Twelve hours after, brains were removed and hippocampal neurogenesis was assessed using the doublecortin (DCX) as a marker for cell proliferation. Our results showed that chronic WIN55,212-2 treatment significantly increased thigmotaxis early in the training process whatever the age of treatment, induced spatial learning and memory deficits in adolescent but not adult rats in the Morris water maze test, while it had no significant effect in the active avoidance test during multitrial training in the shuttle box. In addition, the cognitive deficits assessed in adolescent rats were positively correlated to a decrease in the number of newly generated neurons in dorsal hippocampus. These data suggest that long term exposure to cannabinoids may affect more potently spatial learning and memory in adolescent compared to adult rats via a negative action on hippocampal plasticity.

1. Introduction

Cannabis is the most commonly used illicit substances in the world, especially among adolescents and young adults[1,2]. In Morocco, being one of the major producers in the world, and despite all the repressive and prevention measures deployed, the social and health issues related to the consumption of these drugs took on a large scale since thirty years ago[3]. Several epidemiological studies report the increasing consumption of cannabis in all social strata, especially among adolescents due to its easy access, and the fact that users generally perceive these drugs as relatively harmless[3].

The psychoactive effects of cannabis are mediated primarily by the cannabinoid CB1 receptor which is the most abundant of the Gi/o protein coupled receptors[4]. The CB1 cannabinoid receptor is highly expressed in the limbic regions that are involved in other functions, in learning and memory processes [5]. Several studies have assessed the

neurobehavioral consequences of cannabis. These studies have demonstrated that chronic exposure to cannabinoids, both in humans and animal models, results in behavioral deﬁcits in several physiological do- mains including; locomotor activity, attention, working memory, visuo- spatial skills, and executive functioning[6–8]. While the majority of these behavioral studies have been conducted mainly in adults, very lit- tle is known about the long term effects of chronic cannabis use during adolescence[9].

Adolescence is a critical phase for brain development characterized by a plethora of plastic neuronal changes which include; a sprouting and pruning of synapses, myelination of nerveﬁbers, changes in neuro- transmitter concentrations and their receptor levels in brain areas essential for the homeostasis of brain physiology[10,11]. The impor- tance of the endocannabinoidsignalling in brain development is well established. Endocannabinoids are fundamental cues controlling cell proliferation, migration and differentiation[12]. Interestingly, the role of endocannabinoids in shaping brain organization is operational during both pre and post-natal life[12–14]. Suchﬁndings suggest that long-term cannabinoid exposure during adolescence may produce

⁎ Corresponding author.

E-mail address:oualid.ab@gmail.com(O. Abboussi).

Contents lists available atScienceDirect

Pharmacology, Biochemistry and Behavior

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p h a r m b i o c h e m b e h

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maladaptive plasticity in brain structures involved in learning and memory, such as the hippocampus.

The role of adult neurogenesis in hippocampal plasticity has received considerable attention recently because of its important role in improv- ing and maintaining cognitive functions[15–18]. This developmental process which was described in multiple species including; rodents, non-human primates and, most recently, in humans[19–22]continue throughout adult life, in both the dorsal and ventral parts of the hippocampus[23]. The hippocampal neurogenesis starts when the proliferat- ing neuroprogenitors of the dentate gyrus exit cell cycle and begin expressing neuronal markers, such as the doublecortin (DCX)[24].

Within approximately one month, the neural hippocampal progenitor cells located in the subgranular zone of the dentate gyrus give rise to neuroblasts that migrate into the granule cell layer, where they differ- entiate into functional hippocampal granule cells and some glial cells [25–27]. This form of adult neurogenesis is modulated by several intrin- sic and/or extrinsic factors including genetic background, neurotrans- mitters (such as dopamine and serotonin), behavior, stress and drugs [25,28–31]. However, the neural mechanisms underlying the cognitive impairments and brain structure's aberrations induced by chronic activation of CB1 receptors remain less clear[9,32,33]. Despite the high frequency of cannabis use, and the great pharmaceutical interest in developing new therapies from CB1 agonists[34], the effect of chronic activation of cannabinoid receptors (CB1) on neural cell proliferation and functioning in adolescent and adult rats, and their long-term developmental and neurobehavioral consequences have not received their due interest. Therefore, the aim of the present study is to investigate the effects of a potent synthetic cannabinoid receptor agonist (WIN55,212-2) on learning and memory performances and hippocampal neurogenesis by quantifying the expression of a reliable molecular marker of neurogenesis, the DCX, in the dorsal and the ventral parts of the hippocampus[35,36]. Our hypothesis is that the deleterious effects of cannabinoids on cognitive functions may result from serious impairments in the hippocampal plasticity[37].

2. Materials and methods

2.1. Animals

A total of 42 adolescent (27–30 PND) and adult (54–57 PND) male Wistar rats were used in all the experiments (Fig. 1). They were housed in groups of four in each cage, with free access to food and water, and kept at a constant temperature (22 ± 2 °C) under a 12 h/12 h light– dark cycle (light beginning at 7:00 a.m.). All experiments were carried out during the light phase between 9 a.m. and 5 p.m.

This study was conducted in conformity with approved institutional protocols, and carried out in accordance with the guidelines for the treatment of animals in behavioral research and teaching[38].

2.2. Drug treatments

WIN55,212-2 (Tocris Bioscience, France), a potent aminoalkylindole CB1 receptor agonist (Ki = 62.3 and 3.3 nM for human cloned CB1 and CB2 receptors respectively), was dissolved in saline solution (0.9% NaCl solution) containing 5% Tween 80 and 5% DMSO.

Animals were assigned to 2 groups: adolescent and adult groups.

They were treated for 20 days with WIN55,212-2 (1 mg/kg, according to a preliminary study from our laboratory and others[39–41]) or vehicle. The last drug treatment was followed by a 20 day drug washout period,ﬁrst to allow the adolescent groups to reach adulthood, and then to exclude the confounding effects of drug withdrawal (Fig. 1).

2.3. Behavioral assays

2.3.1. Spatial learning and memory task

Spatial learning and memory were tested using the Morris water maze paradigm (MWM)[42]. Animals were trained in a circular swimming pool (170 cm in diameter and 50 cm in deep). The maze was placed in a room surrounded by constant visual cues (posters, doors, win- dows…) andﬁlled with opaque water (22 ± 2 °C) to a depth of 35 cm.

The water maze was divided virtually into four equal quadrants (north, south, east, and west). In the center of the north quadrant a platform (12 cm in diameter) was placed at 1–2 cm beneath the surface of the water.

Animals were given four trials per day forﬁve consecutive days. Each trial began from one of the four starting positions which are changed across days. During each trial, the rat was placed in the water facing the wall of the maze and given 90 s to locate the platform. If the animal fails to locate the platform, it was gently guided and allowed 20 s on top of the platform. After each trial, the animal is placed in a holding cage containing dry towels for 30 s before the next trial.

On the 6th day following the last day of training, a probe trial was conducted to assess retention of the platform location. During this trial, the platform is removed from the maze. Rats were given 90 s of free swimming. The animals which remember the position of the platform are expected to spend more time in the goal quadrant (north), and to cross it several times.

A webcam connected to a tracking system software Any-Maze (Stoelting) was used to analyze the swim path of each animal, the thigmotaxis (time of swimming in the outer 10% close to the pool walls), the time spent in goal quadrant, and the latency toﬁnd the hidden platform.

2.3.2. Two-way active avoidance learning

Emotional memory was assessed in a Shuttle Box LE916 (58 × 36 × 30.5 cm), which consists of two similar compartments equipped with in- dependent electriﬁable gridﬂoors, separated by a plank with a square hole (8 × 10 cm) in the center. Rats were allowed an easy access from one compartment to the other to avoid an electric shock (0.5 mA and

Rats Treated during adolescence: Win (n=10),

control (n=10)

Rats Treated during adulthood: Win (n=11),

control (n=11)

Washout

MWM test Win55

1mg/kg or Vehicle

IHC analysis

24h 12h

54-57 PND 74-77 94-97 >97

Adult (271.2±7.6 g)

27-30 PND 47-50 67-70 >70

Adolescent

(82±3.3 g) Adult (261.2±9.1 g)

Shuttle box test

Fig. 1.Schematic representation of the experimental design used to assess the behavioral effects of chronic administration of WIN55,212-2 (1 mg/kg) during adolescence and adulthood in Morris water maze (MWM) and shuttle box tests, followed by immunohistochemical (IHC) analysis. (PND: post natal days).

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5 s of duration)delivered by the gridﬂoor (unconditioned stimulus) directly after a visual and vocal stimulus (conditioned stimulus) generated in the compartment in which the animal is detected by a weight transducer located above the grid.

A conditioned stimulus followed by an unconditioned stimulus is considered as a trial. The trials were given with a 25 s intertrial interval [43], and grouped into sessions consisting of 20 trials each.

Before testing, rats were placed in the apparatus for 10 min to explore the apparatus and to be familiar with the learning environment [44]. The behavioral assay lastedﬁve days, with one session per day. The number of active avoidance that reﬂects the learning performance of the animals was recorded.

2.3.3. Immunohistochemical staining

Twelve hours after the last session of each group, the rats were deeply anesthetized with pentobarbital sodium (60 mg/kg, i.p.) transcardially perfused with sodium nitrite solution (NaCl 0.9%, NaNO20.1%), followed by 4% paraformaldehyde (PFA) freshly prepared in phosphate sodium buffer (PBS 1×, pH 7.4). Brains were removed from the skulls, postﬁxed overnight in PFA 4%. Sets of serial coronal sections along the axis of the rostrocaudal hippocampus were cut into 40-μm-thickness on a vibrat- ing blade microtome (Leica VT 1000S; Leica Microsystems, Nussloch, Germany), collected in PBS 1× and stored at−20 °C in cryoprotectant solution (glycerol 30% vol/vol, ethylene glycol 30% vol/vol, 40% vol/vol PBS 1×).

The immunohistochemical staining was performed on free-ﬂoating sections rinsed in PBS 1 × three times for 5 min. After incubation in PBS 1 ×/3% H2O2/methanol 10%, followed by three washes for 5 min by PBS 1 ×, they were immersed for 1 h in a mixture of BSA (bovine serum albumin) 3%, 0.3% Triton X100 in 1× PBS (blocking buffer) before incubation overnight at 4 °C with the primary anti-DCX antibody (poly- clonal-goat 1:100 Santa Cruz Biotechnology, USA) diluted in blocking buffer. The day after, the sections were thoroughly rinsed in 1 × PBS (4 washes of 10 min) before being placed for 2 h at room temperature in blocking buffer, supplemented with the biotinylated anti-goat antibody 1:200 (Vector Laboratories, Burlingame, CA). They were then rinsed 3 times (10 min for each) in 1 × PBS before incubation for 2 h at room temperature with the“avidin–biotin complex” (A and B 1:200, Vectastain Elite kit, Vector Laboratories, Burlingame, CA) in 0.1% Triton X-100/PBS 1× mixture. Afterwards sections were rinsed 3 times for 10 min in 1× PBS, andﬁnally revealed by incubation in 3,3- diaminobenzidine 0.05%/H₂O₂0.01% in PBS 1× for 5 min followed by 3 washes of 10 min in 1 × PBS. Sections were then mounted on

“super frost”slides, air dried, rinsed in double distilled water, and dehydrated after a cresyl violet counterstaining, by incubation in increasing concentrations of alcohol (70%, 90% and 100%) solutions before being cover slipped using a polymerizing resin (Eukitt, Kindler, Freiburg, Germany).

2.3.4. Cell counting

Morphological quantiﬁcations were performed blindly using a high resolution slide scanner Olympus VS120-SL (20 × objective), DCX- labeled cells were counted separately in the dorsal (interaural 3.70 to 6.20 mm); and ventral (interaural 2.28 to 3.70 mm) hippocampus[45].

In every 10th section at 320μm intervals in a total of 10 sections per animal (n = 20) as described by Banasr et al.[46]. Granule cell layer (GCL) volume was calculated according to the Cavalieri principle[47].

The area of the GCL in each section was computed using a package ImageJ software 1.42q (Wayne Rasband National Institutes of Health, USA) and the total number of DCX-positive cells per granule cell layer (GCL) volume was determined.

2.3.5. Data analysis

Repeated-measures analysis of variance (two-way ANOVA) with treatment (vehicle or WIN55,212-2) and days of testing as dependent variables was used to analyze latency, thigmotaxis, and active avoidances.

When significant, a post hoc LSD (least significant difference) test was performed to compare treatment groups. While one-way ANOVA was used for the effects of WIN55,212-2 treated animals during adolescence and adulthood on Probe Trial Performance and DCX expression. Rela- tionships between behavioral performances and immunohistochemical data were evaluated by simple linear regression analysis. The level of significance was set atpb0.05. Statistical analyses were carried out using StatView software (SAS Institute Inc., Cary, NC, USA). All analyses were conducted separately in adolescent and adult rats.

3. Results

3.1. Morris water maze

Theﬁrst objective of this study was to establish whether chronic exposure to WIN55,212-2 (1 mg/kg, for 20 days followed by 20 days washout period) would affect the anxiety-like behavior and acquisition of spatial memory in adolescent and adult rats tested in MWM task.

The results of WIN55,212-2 effect on spatial memory acquisition and thigmotaxis in adolescent and adult rats are summarized inFigs. 2 and 3 respectively. The time spent to locate the escape platform was grouped according to days (1 session per day, 4 trials per session).

Repeated measure ANOVA analyses showed a signiﬁcant effect of days on all groups. All animals learned to locate the hidden platform after 5 days of testing in less than 20 s (Fadolescents(1,86) = 100.002, pb0.0001, F_adults(1,86) = 139.214,pb0.0001,Fig. 2). A main effect of drug treatment (Fadolescents (1,86) = 34.42, p b0.0001) and

0 10 20 30 40 50 60 70 80

1 2 3 4 5

Adults Vehicle Adults Win55 212-2

Escape latency (sec)

Days

B

0 10 20 30 40 50 60 70 80

1 2 3 4 5

Adolescents Vehicle Adolescents Win55 212-2

Days

***

A

***

Fig. 2.The effect of chronic administration (i.p.) of WIN55,212-2 (1 mg/kg) during adolescence (A) and adulthood (B) on spatial memory acquisition in Morris water maze expressed by escape latency. Results are presented as means ± SEM. ***pb0.001 (post hoc Fisher's LSD).

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treatment–day interaction (Fadolescents(4,86) = 7.853,pb0.0001) on spatial acquisition were observed in WIN55,212-2-treated rats during adolescence, as they spent longer time to reach the platform compared to their respective control group, while there was no signiﬁcant treatment effect among rats treated during adulthood (Fadults(1,86) = 1.960,pb0.1649).

One way ANOVA analysis of probe trial (Fig. 4) revealed that retention of spatial memory was impaired in WIN55,212-2-treated rats during adolescence compared to the vehicle. Furthermore, Fisher's LSD post hoc comparison showed a signiﬁcant increase in the latency to swim over the previous location of the escape platform (p= 0.0002,Fig. 4A), no clear preference to swim in the goal quadrant containing the platform during learning sessions (p= 0.0004,Fig. 4B), and a decrease in time spent in the goal quadrant (p= 0.0005,Fig. 4C). On the contrary, no signiﬁcant effects on WIN55,212-2-treated rats during adulthood compared to their corresponding control group (p= 0.4032, p= 0.3186,p= 0.1197 respectively) were observed.

Thigmotaxis data analysis (Fig. 3) revealed a marked decline in time spent hugging the wall of the water maze in WIN55,212-2-treated rats during adolescence and adulthood, presenting a main effect of drug treatment (Fadolescents(1,86) = 13.627, p= 0.0004, Fadults(1,86) = 26.451, pb0.0001), days (Fadolescents(1,86) = 76.66,p = 0.0004, Fadults(1,86) = 212.051,pb0.0001), and treatment–day interaction (Fadolescents(4,86) = 16.465,p= 0.0001, Fadults(4,86) = 5.5442, pb0.0002). Post hoc LSD analysis comparing WIN55,212-2-treated rats to their respective control groups signiﬁcantly conﬁrmed the

main effect of WIN55,212-2treatment that occurred particularly during the 1st and the 2nd day of testing (p≤0.0013,Fig. 3).

3.2. Active avoidance task

Active avoidance performance was assessed using the same groups of animals. All rats successfully learned to avoid the electric shock (Fig. 5); there was no signiﬁcant Win55 212-2-treatment effect, (Fadolescents(1,20) = 0.072,p= 0.7911) (Fadults(1,20) = 0.353, p=0.5588), nor treatment–day interaction (Fadolescents(4,20) = 0.513, p= 0.7261) (Fadults(4,20) = 1.750,p= 0.8854). Only a main effect of days (Fadolescents(1,20) = 104.789,pb0.0001) (Fadults(1,20) = 94.795,pb0.0001) among rats treated during adolescence and adulthood was observed.

0 10 20 30 40 50 60 70 80

Day1 Day2 Day3 Day4 Day5

Thigmotaxis (sec)

***

B

0 10 20 30 40 50 60 70 80

Day1 Day2 Day3 Day4 Day5

***

A

Thigmotaxis (sec)

Fig. 3.The effect of chronic administration (i.p.) of WIN55,212-2 (1 mg/kg) during adolescence (A) and adulthood (B) on thigmotaxis expressed by the time of swimming in the outer 10% close to walls of the Morris water maze. Results presented by means ± SEM.

***pb0.001 (post hoc Fisher's LSD).

0 2 4 6 8 10 12 14

Adolescents Adults

Vehicle

Win55 212-2

Latency (sec)

A

***

0 1 2 3 4 5 6 7 8 9 10

Adolescents Adults

Vehicle

Win55212-2

Number of crossing

B

***

0 10 20 30 40 50 60 70

Adolescents Adults

Vehicle Win55 212-2

% Time spent in goal quadrant

***

C

Fig. 4.The effect of chronic administration (i.p.) of WIN55,212-2 (1 mg/kg) during adolescence and adulthood on spatial memory retrieval expressed by latency (A), number crossing (B) and time spent in the goal quadrant (C) in Morris water maze. Results are presented as means ± SEM. ***pb0.001.

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3.3. Immunohistochemical results

Quantification of DCX-positive cells revealed a significant effect of chronic administration of Win55 212–2 on hippocampal neurogenesis in rats treated at adolescence (p= 0.0252) with a main effect on the dorsal part (pb0.0003) and non on the ventral part (p= 0.6981) as was confirmed by ANOVA post hoc analysis (Fig. 6A). In WIN55,212- 2-treated rats during adulthood, no significant effects were found, as they exhibited similar rate of DCX-positive cell along the rostrocaudal axis in comparison to their respective control group(p= 0.3803, Fig. 6B).

3.4. Correlation between spatial learning and hippocampal neurogenesis The above results indicated that chronic treatment of adolescent rats by Win55 212-2 treatment impairs spatial learning and memory in Morris water maze test, and reduces the number of DCX-positive cell expressed in the dorsal part of the hippocampus, which can suggest a relationship between task performance and neurogenesis. Therefore, we analyzed the correlation between behavioral performance tested in the Morris water maze and cell proliferation using simple regression analysis. A positive correlation was found between the exhibited performances during acquisition and the rate of DCX-positive cell in the dorsal

hippocampus. Analysis of variance indicated that the linear regression lineﬁt to the data was statistically signiﬁcant (r(10) = 0.66,p= 0.0003), showing that the effect of chronic treatment on spatial learning and memory in adolescents is linked to a decrease in the neurogenesis in the dorsal hippocampus (Fig. 7).

4. Discussion

The purpose of the current study was to examine whether chronic exposure to cannabinoids may persistently and differentially affect emotional and cognitive behaviors and neuronal activity in adolescent and adult rats. In line with our hypothesis, chronic exposure to the cannabinoid receptor agonist WIN55,212-2 at the dose of 1 mg/kg during adolescence but not during adulthood, leads to long-term deleterious effects on spatial learning and memory in Morris water maze paradigm.

This effect is likely to be underlined by an attenuation of dorsal hippocampal neurogenesis.

4.1. Effects on anxiety-like behavior

We found that long-term exposure to WIN55,212-2during adolescence (28–48 PND) and adulthood (75–95 PND) significantly increased time of thigmotaxis during thefirst and second days of Morris water maze testing carried out 20 days after the last treatment. This was demonstrated by a longer time spent by treated animals hanging on the wall of the MWM in comparison to their respective control groups. After the 2nd day of the training session, all animals demonstrated a significant reduction in thigmotaxis across days.

Thigmotaxis is a reliable indicator of anxiety-like behavior during the Morris water maze testing in rodents. Anxiolytic agents are known to reduce the total duration of thigmotaxis[48,49]while anxiogenic stimuli such as systemic corticosteroids administration conversely increase thigmotaxis[50–52]. Additionally, a correlation between the performances in the elevated plus maze and thigmotaxis was reported[51].

Taken together, the increase in thigmotaxis seen in our treated animals early in the training process is unlikely to reflect a change in behavioral strategy to locate the platform. Rather, it would most likely reflect an anxiogenic-like effect of WIN55,212-2. This conclusion is corroborated by other studies employing the openfield and social interaction tests to measure anxiety-like outputs, which report that chronic exposure to cannabinoids such as THC or synthetic cannabinoids (such as WIN55,212; CP55,940 or HU210) leads to increased anxiety- like behavior[53–57]. Our study shows that in line with this, both adolescents and adults are equally sensitive to the deleterious effects of chronic exposure to cannabinoids.

4.2. Effects on cognition

The obtained results concerning the effects of chronic exposure to cannabinoids on cognitive behavior suggest that chronic exposure to WIN55,212-2 impairs more potently spatial learning and memory performance in adolescent than in adult rats. Animals treated during adolescence consistently performed poorly during the learning process and spatial memory retention in comparison to their respective control group. On the contrary, no signiﬁcant differences were observed in rats treated during adulthood.

However, no signiﬁcant effect of chronic exposure to cannabinoids during adolescence and adulthood on the acquisition of contextual information in a fear conditioning paradigm in the shuttle box was observed. This test is an operational measure for implicit memory in which the performances depend on the integrity of the hippocampus and the amygdala[58–60]. Therefore, the lack of effect of WIN55,212-2 in the shuttle-box test may be explained by a differential effect of chronic exposure to cannabinoids during adolescence on the development of hippocampal neuronal circuitries and/or on the fact that the pro- cessing of distinct stimuli (such as a vocal signal) requires the 0

2 4 6 8 10 12 14 16

1 2 3 4 5

Number of Active Avoidances

Days

B

0 2 4 6 8 10 12 14 16

1 2 3 4 5

Days

Number of Active Avoidances

A

Fig. 5.The effect of chronic administration (i.p.) of Win55,212-2 (1 mg/kg) during adolescence (A) and adulthood (B) on two-way active avoidance acquisition in shuttle-box.

Results presented by means ± SEM. *pb0.05.

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amygdala, but not the hippocampus[61]. Nevertheless, our results suggest that chronic exposure to cannabinoids during adolescence may have differential effects on learning and memory depending on the aversivity of the context.

In agreement with our results, Quinn and O'shea suggest that chronic exposure to cannabinoids during adolescence (28–50 PND) but not during adulthood (70–90 PND) causes greater lasting memory deﬁcits in hippocampal-dependent tests[53,56]. However, May et al. reported no signiﬁcant lasting effects of chronic exposure to THC during

adolescence on either spatial or non-spatial learning in Sprague– Dawley rats[62]. Likewise Higuera-Matas et al. found no signiﬁcant changes in anxiety and working memory tested using MWM and novel object recognition tests in adult Wistar rats treated at adolescence (28–38 PND) with the cannabinoid agonist CP 55,940 (0.4 mg/kg)[63].

The discrepancies in these reports could be related to the different agonists used (WIN55,212-2 a full agonist while THC acts as a partial agonist), the duration of the treatment, or the duration of abstinence between the end of the chronic treatment and the behavioral testing and the speciﬁc test employed (see for review[64]). Generally, ourﬁnd- ings are in agreement with the residual deleterious effects on adult attention and working memory assessed in early human cannabis users (b16 years)[65–67].

4.3. Effects on hippocampal neurogenesis

Interestingly, the deleterious effect of chronic exposure to WIN55,212- 2 during adolescence on spatial learning and memory resembles the effects of hippocampal lesions on this form of memory[68–70]suggest- ing that cannabinoid exposure during adolescence may alter memory formation via an interference with the normal maturation and/or function of the hippocampus. It has long been known that the hippocampus is one of the brain areas with the highest density of CB1 receptors and a large amount of endocannabinoids (anandamide and 2-arachidonoylglycerol) which are responsible for the establishment of long-term potentiation through modulation of cAMP/PKA activity, a puta- tive mechanism for synaptic plasticity (for review, see[71]). Moreover, the endocannabinoid system plays a major role in axonal sprouting, hippocampal neural plasticity and adult neurogenesis[72–74]. Thus, Interaural

6.20

Interaural 3.70

Interaural 2.28

Dorsal Ventral

C

Ventral sections Dorsal sections

Control WIN55,212-2

D

A B

Fig. 6.Box plots showing individual jitter values of the expression of DCX-positive cells in dorsal (DH) and ventral (VH) hippocampal subregions of animals (n = 20) undergoing chronic WIN55,212-2 (1 mg/kg) treatment (i.p.) during adolescence (A) and adulthood (B). Results are presented as means ± SEM, *pb0.05, ***pb0.001.. (C) Illustration of dorsal and ventral hippocampus sections used for DCX cell quantiﬁcation[40], and (D) representative photomicrographs of positive DCX-labeled cells in dorsal and ventral hippocampal sections from treated animals during adolescence.

DCX labelled cells/1mm³ 4

5 6 7 8 9 10 11 12 13 14

20000 22000 24000 26000 28000 30000 32000 34000 r= 0.66, p=0.0003

Fig. 7.Correlation between escape latency during spatial training and number of DCX- labeled cells in dorsal hippocampus for rats treated during adolescence.

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the long-term deleterious effects induced by adolescent exposure to WIN55,212-2 on spatial learning and memory could result from a perturbation in the normal development and/or maturation of the neural hippocampal circuitries. In light of these facts we investigated the effects of chronic exposure to cannabinoids during adolescence on hippocampal neurogenesis. Our results showed that chronic exposure to WIN55,212-2 during adolescence but not during adulthood reduced cell proliferation in the hippocampus as demonstrated by a speciﬁc decrease in the number of positive-DCX cells expressed in the dorsal part of the hippocampus in comparison to the control group. No significant changes were observed in the ventral part of the hippocampus.

This decrease in cell proliferation in the dorsal part of the hippocampus was positively correlated with the behavioral deficits of the animals in spatial learning in the Morris water maze. These results suggest that chronic exposure to WIN55,212-2 may differentially alter hippocampal- dependent learning during spatial training in the Morris water maze via a negative action on dorsal hippocampus neurogenesis. Interestingly, functional segregation between the dorsal and the ventral parts of the hippocampus is well established with the dorsal hippocampus mediating primarily cognitive functions while the ventral hippocampus is more involved in emotional and neuroendocrine functioning[75,76]. This sub-regional functional segregation along the dorsal–ventral axis of the hippocampus could account as well for the lack of effects on the acquisition of contextual information in a fear conditioning paradigm in the shuttle box. Few studies have investigated the effect of chronic exposure to WIN55,212-2 during adolescence on hippocampal neurogenesis. In line with ourfindings Rueda et al. have shown that in vivo and in vitro chronic administration of an endogenous cannabinoid (the anandamide) significantly inhibits the new born neurons in the rat dentate gyrus through attenuation of the Rap1/B-Raf/ERK pathway[77]. In contrast, other reports suggest that chronic exposure to cannabinoids actually promote hippocampal neurogenesis (for review[14,78]). Wen Jiang et al. have shown that repeated administration of a potent synthetic cannabinoid HU210 (100μg/kg) for 10 days increased the hippocampal neurogenesis[79]. Similarly, pharmacological and genetic disruption of the endogenous cannabinoid signaling enhances adult hippocampal neurogenesis[80–82]. The inconsistencies between all these studies might be related principally to the methodological differences, the opposing effects induced by high and low doses of cannabinoids used [83,84], and the duration between treatment and the assessment of adult hippocampal neurogenesis[9].

5. Conclusion

In conclusion, our study adds to the increasing awareness of the vulnerability of the adolescent brain to the deleterious effects of cannabinoids that affect potently spatial learning and memory in a hippocampal- dependent task[85,86]. This effect could be mediated through a negative action on cell proliferation in the dorsal part of the hippocampus. Further investigations with careful consideration to the phylogenetic differences in hippocampus organization between rats and human[87]are however, required for a better understanding of the neuronal mechanisms underlying the adverse effects of cannabis on brain development.

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