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Behavioural Brain Research
j ourna l ho mepage: www.elsev ier .com/ locate /bbr
esearch report
B1 receptor activation in the nucleus accumbens core impairs contextual fearearning
odrigo Pedroza-Llinása, Mónica Méndez-Díaza, Alejandra E. Ruiz-Contrerasb, Óscar Prospéro-Garcíaa,∗
Grupo de Neurociencias, Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, México, D.F., MexicoLaboratorio de Neurogenómica Cognitiva, Departamento de Psicofisiología, Facultad de Psicología, Universidad Nacional Autónoma de México, México, D.F., Mexico
i g h l i g h t s
Pre-training anandamide infusion into the nucleus accumbens core (NAcC) impairs contextual fear conditioning.Pre-training anandamide infusion into the NAcC does not have an effect on cued fear conditioning.The behavioral effect of anandamide is reversed by co-infusion of AM251.Pre-training anandamide infusion into the nucleus accumbens shell does not have an effect on contextual fear conditioning.Post-training anandamide infusions into the NAcC does not impair contextual fear conditioning.
r t i c l e i n f o
rticle history:eceived 10 July 2012eceived in revised form4 September 2012ccepted 17 September 2012vailable online 24 September 2012
a b s t r a c t
Contextual fear conditioning is a behavioral model in which a subject learns that a specific context ispredictive of danger occurrence. There is evidence suggesting an important role for both the nucleusaccumbens (NAc) and the endocannabinoid system in contextual fear conditioning formation. Thepurpose of this study was to assess whether endocannabinoids within the NAc modulate fear memory for-mation. Pre-training anandamide (AEA) infusions into the NAc core (NAcC) of male Wistar rats decreased
eywords:ontextual fear conditioningucleus accumbensnandamidendocannabinoid systemB1
freezing behavior in the contextual fear-conditioning paradigm, as evaluated 24 h after training. How-ever, AEA did not induce any effect on the cued fear-conditioning paradigm. Likewise, AEA infusionsinto the NAc shell did not interfere with the contextual fear learning. AEA’s effect was blocked whenco-infused with AM251 (CB1R inverse agonist). Post-training AEA infusions failed to exert an effect oncontextual conditioning. These results suggest a cannabinergic regulation in the NAcC of the acquisitionof contextual fear conditioning.
. Introduction
Fear can be conceptualized as a set of behavioral and physiolog-cal responses generated to maximize survival in dangerous – andotentially lethal – situations [1–3]. This complex fear responsean be elicited by innately determined threatening stimuli (suchs the sight or the scent of a predator) or, through learning, toriginally neutral stimuli [1,4]. Thus, given certain circumstances,
subject learns to assign to an originally neutral stimulus – or con-ext – a danger-predicting status. A valuable model to study thisear learning process is the fear-conditioning paradigm.
∗ Corresponding author at: Departamento de Fisiología, Facultad de Medicina,niversidad Nacional Autónoma de México, Apdo. Postal 70-250, México, D. F.4510. Mexico. Tel.: +52 55 5623 2509; fax: +52 55 5623 2241.
Besides the well-known roles of the amygdala and the hip-pocampus in fear conditioning formation and extinction (reviewedin [5]), there is evidence supporting a role for the nucleus accum-bens (NAc) in contextual fear memory. The NAc is a structure ofthe basal forebrain, and is the major component of the ventralstriatum [6]. It comprises the limbic NAc shell (NAcS), and themotor NAc core (NAcC) [7]. The NAc has been associated to reward-ing behaviors, such as food ingestion [8,9] and addictive drugintake [8,10–12], as well as to locomotor activity [6,13]. Regardingcontextual fear learning, it has been found that pre-training, intra-NAcC administration of an anesthetic (bupivacaine) impairs theacquisition of contextual, but not cued, fear conditioning [14]. Sim-ilarly, excitotoxic lesions of the NAcC made before or after training
decrease conditioned freezing evoked by the training context, whenevaluated 48 h after training [15]. These studies suggest that theNAcC is part of a neurobiological circuit crucial for contextual fearmemory formation.
Extensive research has been done to describe not only the neuralircuitry, but also the neurotransmitters involved in fear mod-lation. In this context, the endocannabinoid system (eCBS) hasttracted special interest due to the report that mice lacking theannabinoid receptor 1 (CB1R) exhibit impaired extinction in theued fear-conditioning paradigm [16]. Regarding contextual fearonditioning, it is important to note that it has been reported thatystemic administration of WIN-55, 212-2 (a CB1R agonist) prior tohe acquisition session reduces freezing in the test memory session,erformed 24 h after training [17].
The eCBS in the brain is comprised by the pre-synapticeceptor (CB1R) the endogenous ligands (such as anandamide, 2-rachidonoylglycerol, and oleamide), the enzymes that synthesizeNAPE-PLD, DGL) and the enzymes that degrade (FAAH, MAGL)hese ligands [18,19]. The endocannabinergic system has beenmplicated in the regulation of food intake, in the sleep-wakingycle, and in learning and memory [17].
CB1R expression in the brain is abundant [20], including theAc [21], and particularly the NAcC [22]. CB1R is present in thelutamatergic terminals contacting GABAergic cell bodies [22,23].uch CB1R presence appears to be physiologically relevant for NAcxcitability. For example, in mouse NAc slices, the pharmacolog-cal activation of CB1R by WIN-55,212-2, significantly decreasesvoked glutamatergic transmission [22]. This effect has been con-rmed in in vivo preparations [24]. Therefore, given the presencef CB1R in the NAcC and the role of NAcC in contextual fearonditioning acquisition, the present study was aimed at inves-igating the function of the CB1R in the NAcC in contextual fearearning.
. Materials and methods
.1. Subjects
Male Wistar rats, weighing 250–300 g at the time of surgery, were used. Theyere kept in individual plastic cages, with free access to water and food, and main-
ained under a 12/12 regular light/dark cycle. All experiments were performeduring the light phase (between 12:00 and 16:00 h). Rats for this study werebtained from the animal facility of our institution. Animals were handled in accor-ance with current local animal care regulations (NOM-062-ZOO-1999). The ratsere housed in an animal facility different from the room where experiments tooklace.
.2. Surgery
Rats were subjected to surgery under anesthesia (pharmacological mixture:6 mg/kg ketamine + 0.26 mg/kg xylazine + 1.3 mg/kg acepromazine). They were
mplanted bilaterally with 23-gauge stainless steel cannulae, aimed at the NAcCA + 1.7; L ± 1.4; V − 6.8) or at the NAcS (A + 1.4; L ± 1.4; V − 7.8). Cannulae coor-inates were derived from [25]. Stylets were used to prevent the cannulae fromlogging. The tip of the cannulae was left 1 mm above the desired injection site. Ratsere allowed to recover for 4–6 days following surgery.
.3. Drugs and microinjections
Anandamide (N-arachidonoylethanolamine, AEA, Sigma–Aldrich) wassed as CB1R agonist. It was dissolved in 10% DMSO/PBS 0.1 M. AM2511-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole--carboxamide; Cayman Chemical Company) was used as a CB1R antagonist. Itas dissolved in 30% DMSO/0.2% Tween 80/PBS 0.1 M; this mixture has been used
efore as vehicle for cannabinoids with no significant effects by itself on behavior9,26].
Microinfusions were delivered to hand restrained, conscious animals. Styletsere withdrawn and 30-gauge injectors were inserted into cannulae. These injectorsere connected via polyethylene tubing to 10 �l Hamilton microsyringes, whichere driven by an automated microinfusion pump (KD Scientific). Total volume
nfused was 0.5 �l in 5 min, into either accumbal subregion. Injectors were left inlace for an additional minute, to allow diffusion of the solutions into the tissue.
.4. Apparatus
Two experimental chambers were used. Chamber A, where training for bothontextual and cued fear conditioning was performed, consisted of a wooden box
in Research 237 (2013) 141– 147
(internal dimensions 50 cm high × 40.5 cm wide × 45 cm long). Two fans, embed-ded in the ceiling of this box, were operating during all experimental stages,providing ventilation and background noise to the animals. The illumination forChamber A was delivered by a 35-watt, dichroic halogen lamp, located at the cen-ter of the ceiling. The floor of the chamber was a grid, made of 6 mm-diameter,stainless-steel rods spaced 9 mm from each other (side to side), through whichfoot-shocks were delivered. The front door of the chamber had a one-way mir-ror (47 cm high × 39 cm wide) which permitted full view of the inside of thechamber.
Chamber B, where testing for cued fear conditioning was performed, consisted ofan aluminum box (internal dimensions 27.5 cm high × 36.5 cm wide × 35 cm long).Illumination in Chamber B was provided by a 4-watt fluorescent lamp. The floor ofthe chamber was also a grid made of 6 mm-diameter, stainless-steel rods spaced12 mm from each other (side to side). The front door of the chamber had a one-way mirror (25.5 cm high × 35.5 cm wide), allowing full view of the inside of thechamber. Both chambers were kept in the same room. Lights in this room wereturned off during experiments; hence, the animals could not see through the glassto the outside.
Electric shocks were delivered by a Grass S44 stimulator, through a GrassCCU1A constant current unit. Acoustic tones were delivered by a Grass S10CTCMclick-tone control module, through a 40-watt RadioShack amplifier, and a Real-istic stereo frequency equalizer. Both the stimulator and the tone modulewere controlled by an AMPI Master 8 pulse generator, driven in turn by aPC.
2.5. Behavioral procedures
2.5.1. Contextual fear conditioningAnimals were allowed to habituate to chamber A for 30 s. After this habituation
period, rats received a footshock 20 times through the grid floor of the chamber.Shocks were 1 mA in intensity, and 5 s in duration. The frequency of shock deliverywas at random intervals of 3–10 modules of 5 s each (i.e., minimum time betweenshocks was 15 s, maximum time was 50 s). Total time spent in the training chamberwas determined by the time needed for the PC software to deliver all the shocks,between 12 min and 15 min. After completion of the training session, the rats werereturned to their home cages. Twenty-four hours later, they were placed again intochamber A. They remained there for 10 min, without receiving footshocks. Rats werefilmed throughout this session. An experienced observer evaluated on-line the timerats spent in freezing behavior (defined as the total absence of movement exceptfor those necessary for breathing [4]). After each training or test session, the floor,grid, walls and front door of the experimental chamber were cleaned with a chlorinesolution. A plastic tray placed below the shock grid to collect excretions was emptiedand cleaned.
2.5.2. Cued fear conditioningAfter the 30 s of habituation in Chamber A, the rats were subjected to electric
footshocks for 20 times, being the intensity and duration of this stimuli and theduration of the session the same as in contextual conditioning. However, in thisparadigm the footshocks were accompanied by acoustic tones (parameters: fre-quency 2 kHz, volume 90 dB, and duration 10 s). The footshocks were delivered oncethe acoustic tone elapsed 5 s. After completion of the training session, the animalswere returned to their home cages. Twenty-four hours later, the rats were placed inChamber B, and given a minute to explore the new context. Freezing behavior wasassessed during this minute to detect any possible fear to the test context. Then,20 tones of the same characteristics as those used in training were delivered, withinter-tone intervals at random, as in the training session. During this test sessionthe footshocks were omitted. Freezing behavior was evaluated during each of thetones by an experienced observer. After each training or test session, the floor, grid,walls and front door of both experimental chambers were cleaned with a chlorinesolution.
2.6. Histology
After the experiments were completed, rats were given an i.p. overdose of pento-barbital, and perfused intracardially with PBS 0.1 M, followed by paraformaldehyde4%/PBS 0.1 M. Brains were removed and stored in the paraformaldehyde solu-tion at 4 ◦C. After tissue saturation, the brains were placed successively in sucrose18%, then 30% until tissue saturation. Forty �m-thick coronal slices were obtainedusing a cryostat. Mounted slices were stained with cresyl violet, and analyzedunder the light microscope. Cannulae placement was confirmed; all animalswith erroneous cannulae placement were discarded from statistical analysis (seeFig. 1).
2.7. Statistics
For data analysis, a one-way ANOVA test was used in all contextual fear con-ditioning experiments. The Tukey post hoc test was employed to further assessdifferences between groups. For the cued fear conditioning experiment, an ANOVAtest for repeated measures was used, followed by a Tukey post hoc test for unequal
R. Pedroza-Llinás et al. / Behavioural Brain Research 237 (2013) 141– 147 143
Fig. 1. Localization of drugs injection sites in the brain. Top panel depicts a coronalsection of the rat brain (A + 1.7 relative to bregma), showing the location of NAcC.Bottom panel depicts a more caudal, coronal section (A + 1.4), showing the location ofNAcS. For both panels, the left hemisphere is a scheme of the rat brain; the black dotsrepresent drug injection sites (as observed in actual brain samples). These schemeshave been adapted from [25]. For both panels, the right hemisphere is a micrographof a representative brain slice, stained with cresyl violet, showing the lesion madebp
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Fig. 2. Effect of AEA administration in the NAcC before contextual fear conditioningtraining. Freezing behavior is plotted as percentage of total test session duration(mean + SEM). Every dose of AEA (56 �M, 5.6, 11.4 and 56 mM) infused into theNAcC significantly reduced freezing behavior compared to both intact and vehicle
y injection needles. See Section 2.2 for full description of cannulae and injectorslacement coordinates.
’s. For all experiments, significance was accepted if p < 0.05. Data were analyzedsing the SPSS PC software
. Results
For both contextual and cued fear conditioning, an intact groupno surgical intervention, one for each paradigm) was includedn order to assess the behavior of the rats in the most possiblehysiological conditions. For contextual fear conditioning, 18 intactnimals were included in all subsequent statistical analyses wherereezing behavior to the training context was evaluated. The freez-ng behavior observed in these rats was 50.08% ± 6.27 (percentagef total duration of the test session, mean ± SEM). For the cuedear conditioning experiment, 10 intact rats were included in thetatistical analysis. These rats showed 78.47% ± 7.79 of freezing tooth tone and context tests (percentage of total duration of the testession, mean ± SEM).
One-way ANOVA and Tukey tests did not reveal differencesetween intact and vehicle groups from all contextual fear con-itioning experiments (F(4, 47) = 1.61). Also, when comparing the
otal time spent in freezing behavior during the entire durationf the respective test session, from all the control groups (intactnd vehicle) used in all the experiments (cued and contextual
groups (* p < 0.05). Inset schematizes the behavioral procedure followed. The blackarrow represents AEA or vehicle administration.
conditioning), there were no significant differences found betweengroups (F(6, 64) = 2.73).
3.1. Pre-training infusion of AEA in the NAcC impairs formation ofcontextual fear conditioning
As seen in Fig. 2, every dose of AEA infused into the NAcC hasbeen successful in significantly reducing freezing, compared toboth vehicle and intact groups. One-way ANOVA (F5,53 = 9.74) andTukey post hoc test revealed that the AEA 56 �M (n = 10), 5.6 mM(n = 11), 11.4 mM (n = 8), and 56 mM (n = 4) groups were differentfrom both vehicle (n = 9) and intact groups. No differences werefound between the intact and vehicle groups. Thus, the infusion ofanandamide into the NAcC impairs the acquisition of contextualfear conditioning.
3.2. Pre-training infusion of AEA in the NAcC does not interferewith cued fear conditioning
As seen in Fig. 3, none of the doses infused into the NAcC signif-icantly reduced freezing behavior during the presentation of tonesin a novel context. The number of subjects per group was as follows:56 �M: n = 10; 5.6 mM: n = 9; 56 mM: n = 8; vehicle: n = 9. Panel A ofFig. 3 shows freezing to the test context during the first minute oftraining session. One-way ANOVA and Tukey post hoc test failed tofind significant differences between groups during this first minute(F(4, 41) = 1.81). Panel B of Fig. 3 depicts freezing to the tone whenmade present in the test context. For clarity purposes, data fromeach two successive tones were combined into one; statistical anal-ysis was performed on the data clustered in this fashion. ANOVA forrepeated measures failed to find a significant interaction betweenfactors group and tone (F(36, 369) = 0.90). Likewise, no differenceswere found between groups (F(4, 41) = 0.63). Nevertheless, differ-ences were found between tones (F(9, 369) = 1.19). Accordingly, theTukey post hoc test revealed that tone [3,4] was different fromtones [13,14] to [19,20], tone [5,6] was different from tones [11,12]to [19,20], tone [7,8] was different from tones [15,16] to [19,20],and tone [19,20] was different from tones [9,10] to [13,14]. Panel C
of Fig. 3 shows the total time of freezing behavior displayed bythe animals to all twenty tones. No significant differences werefound between groups (F(4,41) = 2.12). These results suggest a lack
144 R. Pedroza-Llinás et al. / Behavioural Brain Research 237 (2013) 141– 147
Fig. 3. Effect of AEA administration in the NAcC before the cued fear conditioningtraining. Data is presented in all three panels as seconds of freezing behavior. PanelA shows the freezing evoked in the context test during the first minute of exposureto such context. Panel B shows the freezing evoked by tones; data from each twosuccessive tones is combined into one. Panel C shows the total freezing time duringafg
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Fig. 4. Effect of AM251 alone or combined with an effective dose of AEA adminis-tered in the NAcC before contextual fear conditioning training. The CB1R antagonistAM251 was found to be ineffective on its own to affect behavior at 2.8 mM. How-
ll the 20 tones delivered. The dotted line marks the maximum time the rats couldreeze to the presence of all tones. No significant differences were found betweenroups in A and C, or between groups per time point in B.
f effect of anandamide on CB1 receptor in the NAcC for the cuedear conditioning acquisition.
.3. Disruptive effect of AEA on contextual fear learning is blockedy coadministration of antagonist AM251 in the NAcC
In order to confirm that the effect of AEA in the NAcC onontextual fear learning is due to interaction of endocannabinoidsith the CB1R in the NAcC, a pre-training co-administration of
EA and the selective CB1 inverse agonist AM251 was performed.s seen in Fig. 4, an ineffective dose of AM251 (2.8 mM; n = 9) onehavior was able to block freezing reduction when co-infusedith an effective dose of AEA (56 �M, n = 9), as revealed by the lack
ever, when infused together with AEA (AM + AEA), the disruptive effect of theendocannabinoid on behavior was blocked. No significant differences were foundbetween groups. The black arrow represents drug administration.
of significant differences between groups found by the one-wayANOVA test (F(3, 40) = 1.11). Thus, the infusion of AM251 preventsthe anandamide-induced conditioning impairment.
3.4. Pre-training infusion of AEA does not block contextual fearlearning when infused into the NAcS
Given the anatomical and functional division of the NAc, itwas decided to investigate whether the disruptive effect of AEAon contextual fear was restricted to the NAcC. The lowest, mosteffective (regarding contextual fear learning disruption) dose usedin the NAcC was administered in the NAcS, immediately beforetraining. As depicted in Fig. 5, 56 �M delivered into the NAcS ofthe experimental animals (n = 13) did not cause an effect differentfrom vehicle administered to control animals (n = 11). The one-wayANOVA and Tukey tests did not reveal a significant effect of pharma-cological treatments on freezing behavior (F(2,39) = 3.20). Therefore,the anandamide-induced behavioral effect seen in the NAcC wasnot found in the NAcS.
3.5. Post-training infusion of AEA into the NAcC does not impaircontextual fear memory
To further elucidate the role of the NAcC in contextual fear mem-ory, AEA was infused immediately after training, searching for apossible role of the NAcC on short-term memory or memory con-solidation. As shown in Fig. 6, the infusion of AEA did not have aneffect on freezing behavior, as assessed by the one-way ANOVA test(F(2,29) = 3.18) and Tukey post hoc test (AEA group: n = 7; vehicle:n = 7). This result indicates that the impairment of fear condition-ing provoked by the anandamide infusion into the NAcC is not dueto disruption of later stages of memory formation.
4. Discussion
There is an extensive literature implicating the NAc in rewardand addiction. However, mounting evidence also suggests animportant role for this structure in aversive behavior [27], suchas fear conditioning. In this regard, it is known that the NAc,
R. Pedroza-Llinás et al. / Behavioural Bra
Fig. 5. Effect of AEA administered in the NAcS before the contextual fear condi-tioning training. The effective dose of AEA in the NAcC did not significantly reducefva
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reezing behavior when infused into the NAcS (AEA), compared to both intact andehicle groups. Inset schematizes the behavioral procedure followed. The blackrrow represents drug or vehicle administration.
articularly the core subregion, is necessary for the contextualear conditioning acquisition, as revealed by transitory pharmaco-ogical inactivation [14] and by excitotoxic lesions [15]. Of specialnterest is the study by Levita and co-workers, since they assessed
hether pre- or post-training lesions of the NAcC could impairontextual fear conditioning. They found that both pre- and post-raining lesions impaired contextual conditioning [15]. Similarly,e have found in our experiments that pre-training infusions of
nandamide into the NAcC impair memory formation (Fig. 2);
owever, post-training infusions failed to exert a significantffect on contextual fear memory (Fig. 6). The pharmacologicalffect of AEA on memory acquisition could not be explained by aotential interference with pain perception, or any other condition
ig. 6. Effect of AEA administered in the NAcC after the contextual fear condition-ng training. Administration of AEA after contextual fear conditioning training intohe NAcC did not reduce freezing behavior in the test session. Inset schematizes theehavioral procedure followed. The black arrow represents drug or vehicle admin-
stration.
in Research 237 (2013) 141– 147 145
that could induce a possible change of motivational value ofthe aversive procedure (i.e., any part of the conditioning beingperceived as reinforcing, or even lacking a motivational value, dueto CB1R activation) given that the cued fear conditioning (Fig. 3)was unaffected by AEA infusions. This is true for the endocannabi-noid modulation into the NAcC; however, regarding the NAcS, itcould be modulating the cued fear memory through cannabinoidneurotransmission. In summary, our experiments support thenotion that the eCBS in the NAcC may play an important role incontextual fear memory acquisition, but not in later phases ofmemory formation (i.e., short-term memory or consolidation).
There were no behavioral differences in the cued fear condition-ing between groups. However, when analyzing the behavior of allgroups across time, it was found that the middle tones are differentto the last tones (i.e., (3,4) to (7,8) tones being different from (15,16)to (19,20) tones). This could be explained as an effect of memoryextinction. In other words, the CS (tone) diminished its associativestrength to the US (shock) due to the repetitive presentation of theCS alone. Thus, this CS fails to predict the onset of the US, resultingin a decrease in the production of conditioned responses (freezing).
It is important to notice that the freezing total time shown byall control groups from all experiments are not significantly dif-ferent. According to the Rescorla-Wagner theory of learning, theassociative strength of a given CS will depend on the accuracy withwhich that CS predicts a US. Given that a phasic tone predicts moreaccurately the occurrence of shock than a context that is constantlypresent, the association formed to the US would be stronger to thetone than to the context. Hence, the tones would elicit more freez-ing than the context [28]. However, the aforementioned analysisshows that, in this study, the fear acquired as a response to thetones can be assumed to be equivalent to the fear acquired as aresponse to the context. Therefore, the differential effects obtainedwith AEA administration on the contextual versus the cued condi-tioning could not be due to an effect on the level of conditioning; i.e.a differential effect of the endocannabinoid in ‘weak’ versus ‘strong’conditioning.
Although AEA is a well-proven CB1 agonist, it may interact withother kind of receptors, among them TRPV-1 receptor [29–31].This ionotropic receptor is present in the NAc [32]. Moreover, itis known that mice lacking TRPV-1 show impaired acoustic tone-and contextual-conditioned freezing behavior under highly aver-sive experimental conditions [33]. To determine if the AEA-inducedcontextual fear impairment was due to a specific interaction withCB1R, the high affinity [34] inverse agonist AM251 was used toblock AEA effects on contextual fear conditioning. Which wasindeed the case, since AM251 appears to compete for CB1R andblock AEA interaction with CB1R (Fig. 4). The successful block-ade of AEA-induced fear impairment by AM251 confirms that thecannabinergic effect on behavior is due to CB1R facilitation, and notto the activation of any other kind of receptor.
An important caveat to be considered is that the anandamide-mediated memory deficit shown here is, primarily, a pharmacolog-ical effect. This means that the natural-occurring, endocannabinoidneuromodulation in the NAc during contextual fear learningremains to be assessed: none of the experiments reported inthis work directly measured such endogenous biological response.However, our results suggest that a cannabinergic regulation in theNAc core for the acquisition of contextual fear memory may exist.Therefore, the NAc-hippocampus relation is of special interest. Ithas been reported that the hippocampus sends excitatory projec-tions, via the ventral subiculum (vSub), to both NAc subregions,although the most densely innervated is the NAcS [35–38]. Addi-
tionally, projections from the basolateral amygdala (BLA) and thevSub converge on the distal dendrites and spines of NAc neurons[39]. Moreover, it has been shown in vivo that high frequency stim-ulation of the BLA induces either a short-duration decrease or a
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46 R. Pedroza-Llinás et al. / Behaviour
ong-lasting increase in NAc neuron response; these BLA-evokedesponses are attenuated when the vSub is pharmacologicallynactivated, suggesting that both terminals interact synergistically40]. Accordingly, if the projections arriving at the NAc from theSub are functionally relevant for behavior, it may be hypothesizedhat the NAc-hippocampus connection plays an important role inontextual fear memory formation, hence, the disruption of thisnteraction results in contextual fear memory impairment. Inter-stingly, it has been found that excitotoxic or electrolytic lesions ofhe vSub reduces freezing behavior evoked by a context [41]. Addi-ionally, it has been found that systemic, chronic administration ofhe CB1-R agonist WIN 55,212-2 in adolescent rats (post-natal days5–60) impaired short-term memory in the Morris water mazeask and in the object recognition memory task, and the LTP in theSub-NAc pathway; this effect was observed up to 10 days afterast drug infusion [42]. Furthermore, it has been previously demon-trated that the NAc may play an important role in spatial memory:xcitotoxic lesions made in the NAc of rats impaired acquisitionf spatial tasks, such as the T-maze and the Morris water maze43]. These aforementioned studies support the results obtainedn the present work, and contribute to the notion that there coulde a cannabinergic regulation of the inputs arriving from the hip-ocampus and from the amygdala into the NAc, and particularly
nto the NAcC, that mediates contextual fear memory formation.f the CB1R is activated, the influence of these excitatory inputs
ould be attenuated, and the contextual conditioning would beherefore reduced. This idea is reinforced by the fact that, as haseen mentioned previously, CB1-R activation reduces stimulation-voked responses within the NAc [22]. However, this proposedode of action of CB1 modulation of contextual fear conditioning
cquisition remains speculative, since there is no direct evidenceupporting that, in fact, such cannabinergic modulation exists dur-ng contextual fear learning. Further work is needed to shed lightn this issue.
In conclusion, this study indicates that endocannabinoids in theAcC may play a crucial role in modulating the formation of con-
extual aversive experiences, complementing their widely shownole as promoters of reward.
cknowledgements
This research was supported by Grant IN208010 from DGAPA-NAM to OPG, IN217311 to AERC and Grants 80148 from CONACyTnd PICSA10-115 to MMD. Authors acknowledge Edith Monroy foreviewing the English version of this manuscript.
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