Accepted Manuscript Intra-accumbal blockade of endocannabinoid CB1 receptors impairs learning but not retention of conditioned relief Jorge R. Bergado Acosta, Miriam Schneider, Markus Fendt PII: DOI: Reference:
S1074-7427(17)30101-6 http://dx.doi.org/10.1016/j.nlm.2017.06.001 YNLME 6695
To appear in:
Neurobiology of Learning and Memory
Received Date: Revised Date: Accepted Date:
3 February 2017 2 June 2017 12 June 2017
Please cite this article as: Bergado Acosta, J.R., Schneider, M., Fendt, M., Intra-accumbal blockade of endocannabinoid CB1 receptors impairs learning but not retention of conditioned relief, Neurobiology of Learning and Memory (2017), doi: http://dx.doi.org/10.1016/j.nlm.2017.06.001
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Neurobiology of Learning & Memory Short Communication
Intra-accumbal blockade of endocannabinoid CB1 receptors impairs learning but not retention of conditioned relief Jorge R. Bergado Acostaa, Miriam Schneiderb, Markus Fendta,c,*
Institute for Pharmacology and Toxicology, Otto-von-Guericke University Magdeburg, Germany
Department of Psychology, Heidelberg University, Germany
Center of Behavioral Brain Sciences, Otto-von-Guericke University Magdeburg, Germany
* Corresponding author at: Institute for Pharmacology and Toxicology, Otto-von-Guericke University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany. Fax: +49 391 67 15869. E-mail address: [email protected]
2 ABSTRACT Humans and animals are able to associate an environmental cue with the feeling of relief from an aversive event, a phenomenon called relief learning. Relief from an aversive event is rewarding and a relief-associated cue later induces an attenuation of the startle magnitude or approach behavior. Previous studies demonstrated that the nucleus accumbens is essential for relief learning. Here, we asked whether accumbal cannabinoid type 1 (CB1) receptors are involved in relief learning. In rats, we injected the CB1 receptor antagonist/inverse agonist SR141716A (rimonabant) directly into the nucleus accumbens at different time points during a relief learning experiment. SR141716A injections immediately before the conditioning inhibited relief learning. However, SR141716A injected immediately before the retention test was not effective when conditioning was without treatment. These findings indicate that accumbal CB1 receptors play an important role in the plasticity processes underlying relief learning.
Keywords: Backward conditioning; nucleus accumbens; rats; rimonabant; SR141716A; startle response.
A basic motivation of animals and humans is to avoid potentially threatening events since this can have critical impact on the well-being or survival of the organism (Lang, Davis, & Öhman, 2000; LeDoux, 2012). While experiencing a threatening event is highly aversive, the relief from such an incident is rewarding (Leknes, Lee, Berna, Andersson, & Tracey, 2011; Seymour et al., 2005). Notably, animals and humans can associate this rewarding feeling with environmental cues, a phenomenon entitled ‘Relief Learning’ (Denny, 1971; Gerber et al., 2014). In experimental paradigms of relief learning, a conditioned relief stimulus induces behavioral changes such as approach behavior or attenuation of the startle response (Andreatta et al., 2012; Navratilova et al., 2012; Yarali et al., 2008). These behavioral changes are usually observed in the presence of appetitive stimuli (e.g., Conzelmann et al., 2009; Friederich et al., 2006; Lang, Bradley, & Cuthbert, 1990; Schmid, Koch, & Schnitzler, 1995; Schneider & Spanagel, 2008). A series of studies in humans and rodents demonstrated that the nucleus accumbens (NAC), a central part of the brain reward system (e.g., Ikemoto, 2007), is crucial for relief learning in mammals (Andreatta et al., 2012; Bergado Acosta, Kahl, Kogias, Uzuneser, & Fendt, 2017; Bruning, Breitfeld, Kahl, BergadoAcosta, & Fendt, 2016; Kahl & Fendt, 2016; Leknes, Lee, Berna, Andersson, & Tracey, 2011; Mohammadi, Bergado Acosta, & Fendt, 2014; Mohammadi & Fendt, 2015; Navratilova et al., 2012). At the level of the NAC, the endocannabinoid (eCB) system, and in particular cannabinoid type 1 (CB1) receptors, has been reported to act as a fundamental mediator of the encoding of reward and incentive cues by allowing neural synchrony and rhythmicity patterns to emerge during reinforcement processes (Hernandez & Cheer, 2012). The eCB system is an evolutionarily ancient and widely distributed neuromodulatory system (Elphick, 2012) which is critically involved in the regulation and modulation of a plethora of neurophysiological processes, such as motor control, emotional homeostasis, memory storage, or reward processing (Kano, Ohno-Shosaku, Hashimotodani, Uchigashima, & Watanabe, 2009; Moreira & Lutz, 2008). The present study was performed to address the hypothesis whether accumbal CB1 receptors are involved in relief learning. In experiment 1, we submitted animals to different conditioning procedures to demonstrate that the startle attenuation that is observed in relief conditioning experiments is due to the associative status of the conditioned relief stimulus. In two further experiments, we injected the CB1 receptor antagonist/inverse agonist SR141716A (rimonabant) directly into the NAC of rats. In experiment 2, we evaluated the role of accumbal CB1 receptors on the acquisition of conditioned relief, i.e. injections were performed immediately before relief learning and the retention test on learned relief was performed without injections. In experiment 3, the role of accumbal CB1 receptors on the
4 expression of conditioned relief was evaluated, i.e. rats were submitted to relief learning without any treatment and SR141716A injections were performed immediately before the retention test on learned relief.
Methods and materials Subjects
Male Sprague-Dawley rats (250-350 g) were used for the present experiment. They were kept in groups of 4 to 6 animals per cage under a light:dark cycle of 12h:12h (lights on 6:00 am) and had free access to water and food. All experiments and surgeries were done during the light phase. The experiments were performed in accordance with international guidelines for the use of animals in experiments (2010/63/EU) and were approved by the local ethical committee (Landesverwaltungsamt Sachsen-Anhalt, Az. 42502-2-1309 UniMD).
A startle system with eight chambers (SR-LAB, San Diego Instruments, USA) was used. Each chamber was equipped with a loudspeaker (50 dB SPL background noise), a light cue (5 s, ca. 1000 lux) and a transparent animal enclosure (9 cm x 16 cm). As startle stimuli noise burst with a duration of 40 ms and an intensity of 96 dB SPL were used. As aversive stimuli, scrambled electric stimuli (0.5 s, 0.4 mA) were administered via a floor grid. The delivery of the startle, light and electric stimuli was controlled by the SR-LAB software. The responses to the startle stimuli and to the electric stimuli were measured by piezoelectric motion sensors underneath of the animal enclosure and further analyzed by the SR-LAB software. The mean sensor output in the time window 10-30 ms after startle stimulus onset was used as the startle magnitude, whereas the mean output during the whole stimulus period was used to quantify the response to the electric stimulus.
For experiment 2 and 3, guide cannulas were implanted for intracranial injections. The animals were anesthetized with an isoflurane/oxygen mixture (2.0-2.5%) and fixed into a stereotaxic apparatus. The skull was exposed and stainless steel guide cannulas (custommade; diameter: 0.7 mm, length: 8.0 mm) were bilaterally implanted aiming at NAC: 1.2 mm rostral, ± 1.5 mm lateral, and 7.4 mm ventral to the bregma (Paxinos & Watson, 2014). Cannulas were fixed with dental cement and anchoring screws.
5 Experiment 1: To evaluate potential unconditioned effects of the light CS, forty rats were placed into the startle boxes. Following 5 minutes of acclimation time, 10 startle stimuli were presented with an inter-trial interval of 30 s to habituate the animals. Subsequently, 20 further startle stimuli were presented, 10 of them without the light CS (startle alone trials) and 10 of them upon presentation of the light CS (CS-startle trials). The order of the trials with and without light CS was pseudo-randomized. The mean startle magnitudes on startle alone trials and on CS-startle trials, as well as the difference, were calculated. The following day, rats were assigned to four groups with varying conditioning protocols: The group “CS only” was exposed to 15 presentations of the 5s-light CS, i.e. no electric stimuli were delivered. The group “ISI 0” received 15 electric stimuli, directly followed by a 5s-light CS (without any inter-stimulus interval). The group “ISI 3” was submitted to our established relief conditioning protocol, i.e. 15 presentations of an electric stimulus followed by the 5slight that was presented 3 s after the onset of the US. The last group “random” received randomized US and CS presentation, i.e. US and CS could also coincide. On the third day, the test of the first day was repeated.
Experiment 2: Five days after implantation of the guide cannulas, a startle baseline test was performed. Rats were placed into the animal enclosure and after 5 minutes of acclimation, 10 startle stimuli were presented with an inter-trial interval of 30 s. One day later, twenty-three rats received injections of either 0.3 µl vehicle (0.9% saline, 8.3% Tween 80, 1.6% ethanol) or 0.9 µg/0.3 µl SR141716A (dissolved in vehicle) solution. The dose and the vehicle for SR141716A were chosen based on previous publications (Malinen & Hyytia, 2008; Manzanares, Corchero, & Fuentes, 1999). Immediately after the injections, relief conditioning was performed as described above (experiment 1, group “ISI 3”). During this phase, locomotor response to foot shocks was also measured. The following day, a retention test on conditioned relief was performed without any injections. The retention test was identical to the test used in experiment 1.
Experiment 3: As in experiment 2, first a baseline test was performed for twelve rats. The next day, rats were relief conditioned without treatment. Immediately before the retention test one day later, either 0.3 µl vehicle or 0.9 µg/0.3 µl SR141716A solution was injected into the NAC. A further day later, the animals were re-conditioned. For the second retention test, a cross-over design was used, i.e. animals that received vehicle before the first retention test now received SR141716A and vice versa.
The rats of experiments 2 and 3 were sacrificed after the behavioral experiments. The brain was removed, sectioned and Nissl-stained to verify injection sites into the NAC. Only animals with bilateral injections into the NAC shell and core regions were included into final analyses. The injection sites of these animals are shown in Figure 1.
Statistical analyses were performed with Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA). Data were normally distributed (D’Agostino & Pearson omnibus normality test) and two-way ANOVAs were performed using trial type (startle alone, CS-startle), conditioning protocol (experiment 1) and treatment (experiment 2+3) as factors. The statistical threshold was set to P < 0.05.
+ 1.2 NAC CPu
NAC 1 mm
Fig. 1. Reconstruction of the injection sites into the NAC on frontal brain sections (Paxinos & Watson, 2014). Filled circles correspond to vehicle injections before relief conditioning, filled diamonds to SR141716A injections before relief conditioning, and filled triangles to vehicle and SR141716A before the retention test. Abbreviations: aca, anterior part of the anterior commissure; CPu, caudate putamen; Ctx, cortex; ec, external capsule; LSI, intermediate part of the lateral septum; NAC, nucleus accumbens. Values represent the anterior distance to bregma (mm).
Experiment 1: Associative character of the relief CS
Figure 2A depicts the startle magnitudes in the two different startle trial types, i.e. in the absence (startle alone) or presence (CS-startle) of the light stimulus, before the animals were submitted to different conditioning protocols. Light stimulus did not affect the startle magnitude (t-test: t = 0.42, P = 0.67). This was different in the startle tests which were performed after the rats have been submitted to different conditioning protocols. An ANOVA revealed a significant interaction of conditioning protocol and trial type (F(3,44) = 2.86, P = 0.048; factor protocol: F(3,44) = 0.59, P = 0.63; factor trial type: F(1,44) = 10.04, P = 0.0005). Post-hoc comparison of trial types within the different groups demonstrated significant startle attenuation after the relief conditioning protocol (ISI 3: t = 4.04, P = 0.001) but not after “CS only” (t = 0.64, P = 0.95), “ISI 0” (t = 1.65, P = 0.37) and “random” (t = 0.79, P = 0.90). For the following two experiments, only the relief conditioning protocol (“ISI 3”) was used.
B e fo r e
s ta rtle m a g n itu d e [± S E M ]
A f t e r d if f e r e n t c o n d it i o n i n g p r o t o c o l s s t a r t le a lo n e C S - s ta r t le
d if fe r e n c e
-1 0 0 p re
C S o n ly
IS I 0
IS I 3
ra n d o m
Fig. 2. Effects of the light CS on startle magnitude before (A) and after (B) submitting rats to different conditioning protocols. Shown are startle magnitudes in the absence (black bars) and presence (open bars) of the light stimulus, as well as the difference (hatched bars). The light stimulus did neither affect the startle magnitude before conditioning nor after conditioning with the “CS only”, “ISI 0” and “random” protocols. However, after relief conditioning (“ISI 3”), a significant attenuation of the startle magnitude was found. ** P < 0.01 comparison startle alone vs. CS-startle (Sidak’s multiple comparison tests after significant effects in ANOVA).
Experiment 2: SR141716A injections before relief conditioning
8 Injections of the CB1 receptor antagonist/inverse agonist SR141716A into the NAC before relief conditioning impaired the acquisition of conditioned relief (Fig. 3B) without affecting locomotor response to the foot shock (Fig. 3A; see also below). An ANOVA showed a significant effect of trial type (F(1,21) = 21.51; P < 0.0001) but no main effects of SR141716A injections (F(1,21) = 1.69; P = 0.21). Importantly, there was a significant interaction between treatment and trial type (F(1,21) = 5.08; P = 0.035) indicating that the pretraining SR141716A injections blocked the effect of the relief CS. This is confirmed by post-hoc Sidak’s multiple comparisons tests demonstrating a significant startle magnitude attenuation by the relief CS after vehicle injections (t = 4.27; P = 0.0007) but not after SR141716A injections (t = 2.02; P = 0.11). Notably, SR141716A injections did not affect the locomotor response to the foot shock US (Fig. 3A; t test: t = 0.14, P = 0.89), suggesting that CB1 receptors are not involved in US perception.
Experiment 3: SR141716A injections before retention test on conditioned relief
In the third experiment, animals were conditioned without treatment and SR141716A were injected before the retention test on conditioned relief. Clearly, these injections had no effects (Fig. 3C). An ANOVA revealed a significant main effect of trial type (F (1,11) = 8.73; P = 0.01) but no main effects of SR141716A treatment (F(1,11) = 1.17; P = 0.30) and no interaction between treatment and trial type (F (1,11) = 0.07; P = 0.80). This was confirmed by post-hoc Sidak’s multiple comparisons test demonstrating significant startle magnitude attenuation by the relief CS after both vehicle (t = 3.42; P = 0.01) and SR141716A injections (t = 3.06; P = 0.02).
F o o t s h o c k r e a c t i v it y
P r e t e s t in g in je c t io n s
v e h ic le 2000
P r e t r a i n in g in j e c t i o n s
B s ta rtle m a g n itu d e [± S E M ]
lo c o m o to r re s p o n s e [+ S E M ]
s t a r t le a lo n e C S - s ta r t le d if fe r e n c e
-1 0 0 -1 0 0
-2 0 0 v e h ic le
v e h ic le
Fig. 3. Effects of SR141716A injections into the NAC on relief learning. (A) Injections before the relief conditioning procedure had no effect on locomotor response to foot shocks but (B) blocked startle attenuation by the relief CS, indicating SR141716A affects relief memory acquisition. (C) Injections before the retention test did not affect startle attenuation by the relief CS. ** p < 0.01 comparison startle alone vs. CS-startle, # p < 0.05 comparison with vehicle (Sidak’s multiple comparison tests after significant effects in ANOVA).
Taken together, the present study shows that accumbal CB1 receptors are involved in the acquisition but not expression of conditioned relief. The latter finding, together with the observation that locomotor response to foot shocks was not affected by intra-accumbal SR141716A injections, implies that blockade of relief learning is not caused by general deficits in sensory processing of the US or CS after blockade of accumbal CB1 receptors. In the current study we used the acoustic startle response, a bivalent and translational paradigm (Fendt & Koch, 2013) to measure the behavioral effects of the CS. After relief learning, the relief CS robustly attenuates the startle magnitude. A startle attenuation is typically seen when animals or humans are exposed to appetitive stimuli (reviewed in Koch, 1999; Lang, Bradley, & Cuthbert, 1990). In our first experiment, we demonstrated that this startle attenuation is based on an associative status of the CS since startle attenuation was only observed in animals which were submitted to a relief conditioning protocol (Fig. 2A, group “ISI 3”) and not in unconditioned animals (Fig. 2A) or after sham conditioning (Fig. 2B). Sham conditioning refers to protocols in which the CS was not associated with the US during the conditioning phase, i.e. either the CS was presented without the US (Fig. 2B, group “only CS”) or the CS and the US were randomly presented during the conditioning phase (Fig. 2B, group “random”). Similar findings were previously published (Andreatta et al., 2012; Davis & Astrachan, 1978), however, our findings add that presenting the light CS immediately after the US offset, i.e. without any delay (Fig. 2B, group “ISI 0”), does lead to robust startleattenuating effects. Most probably, the still scaring effects of the foot shock compete with the relieving effects of the foot shock offset at such early time points after the foot shock which then leads to ambiguous emotional associations. Taken together, our first experiment supports that the idea that the startle-attenuating effects of a relief CS are due to its association with the relief from the foot shock (cf. Andreatta et al., 2012; Gerber et al., 2014). Of note is that startle attenuation can also be observed after safety learning, i.e. the learning that a CS predicts the absence of the US (Mohammadi, Bergado Acosta, & Fendt, 2014). Safety learning occurs when the CS is presented explicitly unpaired to the US during the conditioning phase (Ostroff, Cain, Bedont, Monfils, & LeDoux, 2010; Pollak et al., 2008; Rogan, Leon, Perez, & Kandel, 2005; Schiller, Levy, Niv, LeDoux, & Phelps, 2008) or when the CS signals that an already fear conditioned stimulus is not followed by an aversive event ("conditioned inhibition"; e.g., Falls, 1993; Fendt, 1998; Josselyn, Falls, Gewirtz, Pistell, & Davis, 2005; Myers & Davis, 2004). Notably, there is a neural dissociation between safety and relief learning. A number of studies showed that the NAC is crucial for relief learning (Andreatta et al., 2012; Bruning, Breitfeld, Kahl, Bergado-Acosta, & Fendt, 2016; Kahl &
10 Fendt, 2016; Mohammadi, Bergado Acosta, & Fendt, 2014; Mohammadi & Fendt, 2015; Navratilova et al., 2012) but seems to play no or only a minor role for safety learning (Josselyn, Falls, Gewirtz, Pistell, & Davis, 2005; Mohammadi, Bergado Acosta, & Fendt, 2014). The working hypothesis of the present study was based on the findings that the NAC is crucially involved in relief learning (see citations above) and that the eCB system modulates reward-related learning processes (e.g., Cohen, Perrault, Voltz, Steinberg, & Soubrie, 2002; Moreira & Lutz, 2008). In the present study, we observed only an effect of accumbal SR141716A injections when it was injected before the conditioning phase, indicating that CB1 receptors only play a role in the acquisition of conditioned relief but not during the expression of conditioned relief. The NAC is made up of two regions, the core and the shell region (Zahm & Brog, 1992). The NAC shell receives direct input from neurons in the ventral tegmental area which are activated by the offset of electric stimuli (Brischoux, Chakraborty, Brierley, & Ungless, 2009). Since this response timing coincides with the time period of relief from the aversive stimulus, we hypothesized that the shell region is critical for relief learning (Bergado Acosta, Kahl, Kogias, Uzuneser, & Fendt, 2017) and tried to perform the injections of the present study into the shell region. Most of our injection sites were indeed nicely located in the shell region (see Fig. 1) but our dataset was too small to allow a meaningful comparison of the efficiency of injection into the shell and core region, respectively. Since we injected a volume of 0.3 µl that may diffuse approximately 0.5 mm (Martin, 1991), our injections most probably affect both the shell and the core regions of the NAC. How do eCBs and CB1 receptors modulate neural activity within the NAC? It is well established that eCBs are retrograde messengers that are able to suppress both excitatory and inhibitory transmission (Kano, Ohno-Shosaku, Hashimotodani, Uchigashima, & Watanabe, 2009). Therefore, the eCB system is considered a ubiquitous regulator of synaptic transmission in the brain which mediates numerous forms of short- and long-term plasticity (Castillo, Younts, Chàvez, & Hashimotodani, 2012). In the NAC, CB1 receptors are exclusively expressed on fast-spiking interneurons which form GABA-ergic synapses to medium spiny neurons (Winters et al., 2012). The medium spiny neurons are described to be a site for synaptic and structural plasticity within the NAC (e.g., Lee et al., 2006), particularly for plasticity based on the interaction of dopamine D1 and NMDA receptors (Renteria, Maier, Buske, & Morrisett, 2017; Surmeier, Ding, Day, Wang, & Shen, 2007). Indeed, a recent study of our group indicates that the acquisition of relief learning is dependent on an interaction of accumbal dopamine D1 and NMDA receptors (Bergado Acosta, Kahl, Kogias, Uzuneser, & Fendt, 2017). Together, our findings suggest that the SR141716A injections in the present
11 study interfere with dopamine D1/NMDA receptor-dependent plasticity in medium spiny neurons of the NAC. Taken together, the present study shows that temporary blockade of CB1 receptors within the NAC blocks the acquisition of relief learning. This suggests an important regulatory role of accumbal CB1 receptors in plasticity processes underlying relief learning (e.g., dopamine D1 and NMDA receptor interaction).
Acknowledgements This study was funded by the Deutsche Forschungsgemeinschaft (SFB779/B13). The authors are grateful to Evelyn Kahl, Dana Meyer and Kathrin Freke for excellent technical assistance and Judith Kreutzmann for language editing and helpful comment to the manuscript.
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Relief learning is associating a stimulus with the offset of an aversive event
We tested whether accumbal CB1 receptors are involved in relief learning
Injections of a CB1 receptor antagonist blocked relief learning in rats
Already established relief memory was not affected by these injections
antagonist of CB1 receptors
blockade of relief learning