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The endogenous cannabinoid system controls extinction of aversive memories

GIOVANNI MARSICANO*, CARSTEN T. WOTJAK, SHAHNAZ C. AZAD, TIZIANA BISOGNO, GERHARD RAMMES, MARIA GRAZIA CASCIO, HEIKE HERMANN*, JIANRONG TANG, CLEMENTINE HOFMANN#, WALTER ZIEGLGNSBERGER, VINCENZO DI MARZO & BEAT LUTZ*

Nature 418, 530 - 534 (2002); doi:10.1038/nature00839

* Molecular Genetics of Behaviour, Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany
Neuronal Plasticity/Mouse Behaviour, Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany
Neuropharmacology Group, Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany
Clinic of Anaesthesiology, Klinikum Grosshadern, Ludwig-Maximilians-University, Marchioninistrasse 15, 81377 Munich, Germany
Endocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR, Via Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
Institute of Mammalian Genetics, GSF National Research Center for Environment and Health, Ingolstdter Landstrasse 1, 85764 Neuherberg-Munich, Germany
These authors contributed equally to this work
# Present address: Molecular Neurogenetics Group, Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany.

Correspondence and requests for materials should be addressed to B.L.
e-mail: lutz@mpipsykl.mpg.de

Acquisition and storage of aversive memories is one of the basic principles of central nervous systems throughout the animal kingdom1. In the absence of reinforcement, the resulting behavioural response will gradually diminish to be finally extinct. Despite the importance of extinction2, its cellular mechanisms are largely unknown. The cannabinoid receptor 1 (CB1)3 and endocannabinoids4 are present in memory-related brain areas5, 6 and modulate memory7, 8. Here we show that the endogenous cannabinoid system has a central function in extinction of aversive memories. CB1-deficient mice showed strongly impaired short-term and long-term extinction in auditory fear-conditioning tests, with unaffected memory acquisition and consolidation. Treatment of wild-type mice with the CB1 antagonist SR141716A mimicked the phenotype of CB1-deficient mice, revealing that CB1 is required at the moment of memory extinction. Consistently, tone presentation during extinction trials resulted in elevated levels of endocannabinoids in the basolateral amygdala complex, a region known to control extinction of aversive memories9. In the basolateral amygdala, endocannabinoids and CB1 were crucially involved in long-term depression of GABA (-aminobutyric acid)-mediated inhibitory currents. We propose that endocannabinoids facilitate extinction of aversive memories through their selective inhibitory effects on local inhibitory networks in the amygdala.

To study the involvement of the endogenous cannabinoid system in memory processing, we generated CB1-deficient mice (CB1-/-; see Supplementary Information). CB1-/- mice and CB1+/+ littermates were tested in auditory fear conditioning, which is highly dependent on the amygdala1 and enables the dissection of different phases of memory formation, including acquisition, consolidation and extinction. Mice were trained to associate a tone with a foot-shock (conditioning). After conditioning, animals froze when re-exposed to the tone. This response served as an indicator of aversive memory, and is gradually extinguished on repeated tone presentations. As the amygdala has a crucial role for extinction of aversive memories9, 10, we studied amygdala-dependent memory performance in the absence of possible confounding influences of the hippocampus by re-exposing the mice to the tone in an environment different from the conditioning context1. In this environment, neither CB1-/- nor CB1+/+ mice showed freezing without tone presentation 24 h after conditioning (data not shown). During the subsequent tone presentation, however, animals of both groups showed the same amount of freezing (Fig. 1a; d1, P > 0.05), pointing to an equally successful tonefoot-shock association. On repeated exposure to the tone, however, CB1+/+ and CB1-/- mice differed significantly in their freezing behaviour (genotype: F1,20 = 5.81, P < 0.05; genotype day interaction: F3,60 = 4.86, P < 0.005; Fig. 1a). In fact, CB1+/+ mice (F3,10 = 9.70, P < 0.0005), but not CB1-/- (F3,10 = 0.94, P = 0.433), showed extinction of freezing.

Figure 1 Impaired extinction of aversive memory in an auditory fear-conditioning task of CB1-/- mice (filled circles) as compared to their CB1+/+ littermates (open circles). Full legend

The identical behavioural performance of the two genotypes on day 1 indicates that acquisition and early consolidation processes do not involve CB1. However, it is possible that memory consolidation processes were not completed 24 h after conditioning, leaving open a potential involvement of CB1 in later phases of memory consolidation. To test this hypothesis, new groups of animals remained undisturbed after conditioning for 6 days, and mice from these groups were then exposed to the 60-s tones (Fig. 1b). Again, CB1-/- and CB1+/+ mice did not differ in their initial freezing response, but behaved in a significantly different way in the course of repeated tone presentations (genotype day interaction: F3,42 = 3.03, P < 0.05). Whereas CB1+/+ mice showed a decrease in freezing behaviour until day 11 (F3,27 = 3.73, P < 0.05), CB1-/- mice failed to extinguish the freezing response (F3,15 = 1.03, P = 0.404). A more detailed analysis of the freezing response in 20-s intervals confirmed the difference in extinction (genotype 20-s bin interaction: F11,154 = 2.60, P < 0.005; Supplementary Information). These differences were due to altered short-term and long-term extinction in CB1-/- mice but not to increased spontaneous recovery of the freezing response (genotype: F1,14 = 0.18, P = 0.675; genotype day interaction: F2,28 = 1.61, P = 0.217; Supplementary Information).

We next analysed whether the differences in memory extinction between the two genotypes could be attributed to alterations in sensory-motor abilities of CB1-/- mice, as cannabinoids are known to influence pain perception, emotionality and locomotion4, 11, 12. However, mice of either genotype showed the same pain sensitivity to a rising electric foot-shock defined as the shock intensity at which mice showed first signs of discomfort, that is, jumping and/or vocalization (Fig. 1c). Moreover, if the same animals were repeatedly exposed to the tone, there were no significant differences in freezing behaviour between the genotypes (genotype: F1,12 = 1.61, P = 0.228; genotype day interaction: F3,36 = 0.225, P = 0.878; Fig. 1d), indicating that CB1 deficiency does not affect foot-shock-induced behavioural sensitization or unconditioned freezing to the tone. Anxiety-related behaviour was analysed on an elevated plus maze. Animals of either genotype spent the same relative time on open arms of the maze (P > 0.05, t-test and U-test; Fig. 1e), and made the same relative number of entries into open arms (CB1+/+: 22.0 4.0%; CB1-/-: 21.1 7.6%, P > 0.05, t-test and U-test). In contrast, CB1-/- mice showed reduced exploratory activity (number of closed-arm entries: 11.6 1.1 in CB1+/+ mice compared with 6.5 1.2 in CB1-/- mice, P < 0.01, t-test). However, in an open-field locomotor activity test, no significant differences were found, including horizontal (Fig. 1f) and vertical locomotion, resting time, and time spent close to the walls of the box (data not shown).

The failure of CB1-/- mice to diminish their freezing response during a limited number of 60-s tone presentations (Fig. 1a, b) raises the question as to whether CB1-/- mice are able to extinguish aversive memories at all. Thus, conditioned CB1-/- and CB1+/+ mice were exposed to a stronger extinction protocol (3 min tone, six exposures; Fig. 1g). Both CB1+/+ (F17,119 = 15.01, P < 0.000001) and CB1-/- mice (F17,119 = 7.59, P < 0.000001) extinguished their freezing response over the course of repeated tone presentations. Nevertheless, extinction was still more pronounced in CB1+/+ as compared with CB1-/- mice (genotype: F1,14 = 5.30, P < 0.05). Notably, the most marked differences between CB1-/- and CB1+/+ mice were observed during acute tone presentation (short-term extinction). Therefore, CB1-/- mice might be primarily impaired in short-term extinction, with a resulting impairment in long-term extinction, assessed in the course of the subsequent extinction trials. Accordingly, spontaneous recovery was not different between the genotypes (genotype: F1,14 = 1.73, P = 0.208; genotype day interaction: F4,56 = 1.19, P = 0.323; Supplementary Information).

Our behavioural data clearly indicate an involvement of the endogenous cannabinoid system in extinction of aversive memories. However, the life-long absence of CB1 could result in developmental defects leading to the phenotype observed. It, furthermore, precludes any temporal dissection of the involvement of the endogenous cannabinoid system in different stages of memory formation. Thus, we treated wild-type C57BL/6J mice with the CB1 antagonist SR141716A (ref. 13), either before conditioning, or before the first extinction trial. Systemic application of SR141716A 20 min before the first extinction trial impaired both short-term and long-term extinction of the freezing response as compared with both vehicle-treated controls and animals treated with SR141716A before conditioning (treatment time interaction: F10,160 = 2.72, P < 0.005), with no difference between the two latter treatments and with a similar performance of all three groups in the beginning of the first extinction trial (Fig. 2a). These data largely confirm the phenotype of CB1-/- mice (Fig. 1a, b, g), indicating that endocannabinoids have only a negligible function in memory acquisition, consolidation and recall (indicated by the similar performance at the beginning of the first extinction trial), but selectively interfere with extinction of the freezing response to the tone. Mice treated with SR141716A before the first extinction trial showed an attenuated extinction of freezing not only during the first tone presentation (short-term extinction) but also in the absence of pharmacological treatment during the first 60 s of tone presentation at day 6 (long-term extinction). Spontaneous recovery of the behavioural performance from the end of the first (day 1) to the beginning of the second tone presentation session (day 6) was not different among the three groups (F2,34 = 0.29, P = 0.744; Supplementary Information). Together, these findings support the idea that CB1 might be particularly important for the extinction of acute responses to the tone (short-term extinction), which, in turn, relates to behavioural extinction over repeated tone presentations (long-term extinction), without affecting spontaneous recovery of the behavioural performance. Accordingly, the CB1 antagonist had to be present at the time of tone presentation (that is, during aversive memory recall) in order to interfere with memory extinction, as SR141716A failed to affect extinction if administered immediately at the end of the extinction trial (data not shown) or 10 min later (Fig. 2b).

Figure 2 CB1 antagonist SR141716A impairs short-term and long-term extinction, but not acquisition and consolidation of aversive memories. Full legend

These observations, together with the pharmacokinetics of SR141716A (ref. 14), led us to assume that presentation of the tone during the extinction trial causes an instantaneous rise in endocannabinoid levels. To confirm this assumption, we measured in C57BL/6J mice levels of the two major endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), in brain punches of the medial prefrontal cortex (mPFC) and the basolateral amygdala complex (BLAC), both of which are thought to have central roles in extinction of aversive memories9, 15. In those animals forming an association between tone and foot-shock, levels of AEA and 2-AG were significantly higher in the BLAC at the end of tone presentation of the extinction trial on day 1, as compared with animals with unpaired tone and foot-shock presentation on the previous day and with animals with paired tone and foot-shock presentation but no re-exposure to the tone (Fig. 3). There were no significant differences in levels of AEA and 2-AG in the mPFC, suggesting a specific involvement of endocannabinoids in extinction processes within the BLAC. Data of the two control groups indicate that both a successful tonefoot-shock association and re-exposure to the tone are required to trigger the acute increase of endocannabinoid levels.

Figure 3 Re-exposure to the tone 24 h after conditioning causes increased endocannabinoid levels in the basolateral amygdala complex (BLAC) but not the medial prefrontal cortex (mPFC) of C57BL/6J mice. Full legend

If the endogenous cannabinoid system is activated during tone presentation, how exactly does it facilitate memory extinction? To answer this question, we performed a series of electrophysiological experiments in the BLAC of brain slices from CB1-/- and CB1+/+ mice. Basic electrical properties were similar in CB1-/- and CB1+/+ littermates, including input resistance and resting membrane potential (data not shown). High-frequency stimulation (HFS) in the lateral amygdala close to the external capsule induced long-term potentiation (LTP) in the basolateral amygdala of both genotypes (Fig. 4a). This effect was significantly more pronounced in CB1-/- than in CB1+/+ mice (potentiation of population spike amplitude to 147 11% in CB1-/- compared with 117 8% in CB1+/+ mice, n = 9, P < 0.05). However, we failed to affect basal synaptic transmission and LTP induction in wild-type slices superfused with SR141716A (5 M; data not shown). This indicates that the enhanced LTP in CB1-/- mice might reflect long-term developmental adaptations to life-long absence of CB1, and cannot be easily attributed to the lack of CB1 during LTP induction. Low-frequency stimulation with 900 pulses at 1 Hz (LFS 1) of the same pathway induced a persistent decrease in excitatory synaptic transmission (long-term depression, LTD) in both CB1-/- and CB1+/+ mice with no difference between genotypes (depression of population spike amplitude to 75 7% in CB1-/- compared with 80 7% in CB1+/+ mice, n = 9, P > 0.05; Fig. 4a).

Figure 4 Endogenous cannabinoid system and synaptic plasticity in the basolateral amygdala. Full legend

As several recent studies indicate an involvement of CB1 in GABA-mediated synaptic transmission in hippocampus16, 17 and amygdala6, we next looked for possible differences in this process within the basolateral amygdala of CB1-/- and CB1+/+ mice. Low-frequency stimulation with 100 pulses at 1 Hz (LFS 2) of the lateral amygdala close to the external capsule induced a significant suppression of isolated GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) in principal neurons of the basolateral amygdala of CB1+/+ mice. This suppression lasted for more than 20 min (hereafter called long-term depression of IPSCs, LTDi, to 66.7 5.4%, n = 8, P < 0.05; Fig. 4b). Importantly, LTDi was blocked in CB1+/+ mice by SR141716A (5 M; Fig. 4b), showing an acute involvement of the endocannabinoid system in the development of LTDi. The involvement of CB1 in LTDi was confirmed in CB1-/- mice in which LTDi was completely abolished (to 110.1 13.8%, n = 8, P < 0.01 compared with CB1+/+; Fig. 4b). Consistent with previous reports16, 17, suppression of GABA-mediated synaptic transmission also increased paired-pulse facilitation (PPF) in CB1+/+ (P < 0.05) but not in CB1-/- mice (Fig. 4c), indicating a local CB1-dependent decrease in GABA release from axon terminals in CB1+/+ slices.

Extinction of aversive memories is thought to be an active mnemonic process2. As a new memory, it shares several attributes with other steps of memory formation9, 10, 18; however, there is increasing evidence that some cellular pathways are specifically involved in extinction, but not in acquisition or consolidation of fear memories15, 19, 20. We demonstrated a specific involvement of CB1-mediated neurotransmission in extinction of aversive memories. In principle, the enhanced excitatory synaptic plasticity in CB1-/- mice (LTP; Fig. 4a) might explain the prolonged maintenance of aversive memories observed in these animals (Fig. 1a, b, g). However, an enhanced LTP is expected to coincide with an increased initial freezing response in the first extinction trial21, which was not observed in CB1-/- mice. Accordingly, acute blockade of CB1 by a selective antagonist failed to affect LTP induction as well as acquisition and consolidation of the aversive memory. In contrast, the same approach revealed a significant involvement of CB1 in extinction (Fig. 2a). Tone-induced recall of the aversive memory was accompanied by an activation of the endocannabinoid system within the BLAC (Fig. 3), which possibly leads to a decrease of GABA-mediated transmission in a CB1-dependent manner (LTDi; Fig. 4b, c).

The role of GABA-mediated transmission for extinction is, however, controversial22, 23. Within the amygdala, CB1 immunoreactivity was detected in a distinct subset of GABA-containing interneurons of the BLAC6 (one of the sites where aversive memories might be formed and stored24), but not in the central nucleus of the amygdala6 (the principal output site of the amygdala1). Taking into consideration that principal neurons of the BLAC and neurons of the central nucleus of the amygdala might be inversely correlated in their activities25, 26, we propose that the CB1-mediated decrease of activity of local inhibitory networks within the BLAC leads to a disinhibition of principal neurons and finally to extinction of the freezing response. The selective and locally restricted inhibition of GABA-mediated transmission might not be easily reproduced by systemic administration of GABA-interfering drugs22, 23. Thus, future studies will have to confine such treatments to the BLAC to validate that CB1-mediated inhibition of GABA-mediated transmission is indeed crucially involved in the extinction of aversive memories mediated by CB1. It remains to be shown whether CB1 is not only involved in extinction of aversive memories but also in adaptation to aversive situations in general and/or in extinction of memories, independently from their emotional value.

Overall, our findings suggest that the endogenous cannabinoid system could represent a therapeutic target for the treatment of diseases associated with inappropriate retention of aversive memories or inadequate responses to aversive situations, such as post-traumatic stress disorders2, phobias, and certain forms of chronic pain11.

Methods

Animals Adult male C57BL/6JOlaHsd mice (68 weeks; HarlanWinkelmann) and male CB1-/- and CB1+/+ littermates (1016 weeks; see Supplementary Information) were housed individually with an inverse 12/12 h light/dark cycle (lights off at 8:00) for at least 2 weeks before starting the experiments.

Behavioural studies Experimental procedures were approved by the Committee on Animal Health and Care of local Government. Experiments were performed between 9:00 and 14:00. Animal's behaviour was analysed in a blind fashion with regards to genotype and drug treatment. Data were analysed by analysis of variance (ANOVA) followed by Fisher's least significant difference test for planned comparisons, Mann-Whitney U-test or unpaired Student's t-test. A P-value of <0.05 was considered statistically significant. Experimental procedures for pain threshold and unconditioned freezing, elevated plus maze and open field are described in Supplementary Information.

Fear conditioning For conditioning, animals were placed into conditioning chambers (MED Associates). After 3 min, a 20-s tone (9 kHz, 80 dB) was presented that co-terminated with a 2-s electric foot-shock (0.7 mA). In pharmacological experiments animals received a 1-s shock to avoid ceiling effects in the freezing response due to the combination of foot-shock and injection stress. Animals were returned to their home cages 60 s after shock application. At the given time points after conditioning, animals were placed into transparent plexiglas cylinders that differed from the conditioning context, and a 60-s or 180-s tone was presented 3 min later (extinction trials). Animals were returned to their home cages after another 60 s. Mice were experimentally naive except for the stronger extinction protocol, where they had been tested on the elevated plus maze 5 days before. Freezing behaviour (defined as the absence of all movements except for respiration) was quantified from videotapes by trained observers that were blind to genotype and drug treatment, and data were normalized to the respective observation periods.

Pharmacological treatment SR141716A (NIMH Chemical Synthesis and Drug Supply Program) was dissolved in vehicle solution (1 drop of Tween-80 in 3 ml 2.5% dimethylsulphoxide in saline). SR141716A (3 mg per kg body weight) and vehicle were injected subcutaneously at 20 ml per kg body weight under light isofluran anaesthesia.

Measurement of endocannabinoids C57BL/6JOlaHsd mice were randomly assigned to three groups (n = 20 each). On the conditioning day, two groups were conditioned as described before (paired). The remaining group received the foot-shock first and a 20 s tone 3 min later (unpaired). On the next day, all animals were placed into the cylinders, but only one of the paired groups and the unpaired group were exposed to a 3-min tone. Immediately after the end of the tone (or equivalent time in cylinder), animals were killed, brains were quickly removed and snap-frozen in isopentane/dry ice. mPFC and BLAC were punched from the frozen brain using a cryocut and cylindric brain punchers (Fine Science Tools, internal diameter 2.0 mm and 0.8 mm, respectively). Length of punches was approximately 1.6 mm for mPFC (start: bregma +2.8 mm27) and 1.2 mm for BLAC (start: bregma -1.0 mm27). Brain tissue of mPFC and bilateral BLAC, respectively, of 5 mice was pooled to obtain a single data point. Tissues (1015 mg per data point) were dounce-homogenized with chloroform/methanol/Tris-HCl 50 mM, pH 7.4 (1/1/1 by volume) containing 5 pmol of octa-deuterated (d8)-anandamide and 50 pmol of d8-2-arachidonoylglycerol (Cayman Chemicals) as internal standards. Lipid-containing organic phase was dried down, weighed and pre-purified by open-bed chromatography on silica gel, and analysed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) using a Shimadzu high-performance liquid chromatography (HPLC) apparatus (LC-10ADVP) coupled to a Shimadzu quadrupole mass spectrometer (LCMS-2010) via a Shimadzu APCI interface. Mass spectrometry analyses were carried out in the selected ion-monitoring (SIM) mode as described previously28. Temperature of the APCI source was 400 C; HPLC column was a Phenomenex (5 m, 150 4.5 mm) reverse phase column, eluted as described28. Anandamide (retention time of 14.5 min) and 2-AG (retention time of 17.0 min) quasi-molecular ions were quantified by isotope dilution with the above-mentioned deuterated standards28 and their amounts in pmols normalized per mg of lipid extract. Data were statistically evaluated by ANOVA.

Electrophysiology Brain slices were prepared essentially as described29. IPSCs and population spikes were evoked by square pulse stimuli (0.066 Hz, 512 mA, 200 s) delivered by means of bipolar tungsten electrodes positioned within the lateral amygdala close to the external capsule. Population spikes were recorded in the basolateral amygdala close to lateral amygdala using glass microelectrodes (23 M) filled with artificial cerebrospinal fluid (ACSF)29. HFS (five trains at 100 Hz for 1 s, 10-s interstimulus interval) was applied to induce LTP, and LFS1 (900 pulses at 1 Hz) was applied to induce LTD. Whole-cell GABA-mediated currents were isolated by adding NBQX (0.005 mM) and D-(-)-2-amino-5-phosphopentanoic acid (AP5; 0.05 mM) to ACSF (bubbled with 95% O2/5% CO2; pH 7.3), and were recorded from visually identified somata of principal neurons of the basolateral amygdala30 by glass electrodes (4.55 M)16 containing (in mM): Mg-ATP 2, CsCH3SO3 100, CsCl 60, EGTA 0.2, HEPES 10, MgCl2 1, QX314 5 and Na3GTP 0.3 (pH 7.3). Patch clamp experiments were performed at 24 1 C at a holding potential of -70 mV. LTDi was induced by 100 stimuli at 1 Hz (LFS 2). PPF was induced as described30. Data are expressed as means s.e.m. We tested significance using the Student's t-test.

Supplementary information accompanies this paper.

Received 24 December 2001; accepted 16 April 2002

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Acknowledgements. S.C.A., T.B. and G.R. contributed equally to this work. We thank S. Bourier, B. Brachvogel and W. Wurst for feeder cells and technical support; K. Pfeffer for E14 embryonic stem cells; K. Rajewsky for Cre deleter mouse line; B. Lscher for FRT-flanked PGK-neo cassette; M. Wiedemann, H. Dietrich, B. Wlfel, A. Daschner, F. Fezza and A. Rippl for technical assistance, mouse breeding and genotyping; A. Mederer for help with behavioural experiments; F. Holsboer for continuous support; E. Gll for secretarial work; and C. Behl for critically reading the manuscript.

Competing interests statement. The authors declare that they have no competing financial interests.

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