By priming brain circuits, associations between low-salience stimuli often guide future behavioral choices through a process known as mediated or inferred learning. However, the precise neurobiological mechanisms of these incidental associations are largely unknown. Using sensory preconditioning procedures, we show that type 1 cannabinoid receptors (CB 1 R) in hippocampal GABAergic neurons are necessary and sufficient for mediated but not direct learning. Deletion and re-expression of CB 1 R in hippocampal GABAergic neurons abolishes and rescues mediated learning, respectively. Interestingly, paired presentations of low-salience sensory cues induce a specific protein synthesis-dependent enhancement of hippocampal CB 1 R expression and facilitate long-term synaptic plasticity at inhibitory synapses. CB 1 R blockade or chemogenetic manipulations of hippocampal GABAergic neurons upon preconditioning affect incidental associations, as revealed by impaired mediated learning. Thus, CB 1 R-dependent control of inhibitory hippocampal neurotransmission mediates incidental associations, allowing future associative inference, a fundamental process for everyday life, which is altered in major neuropsychiatric diseases.

Thus, applying genetic, pharmacological, biochemical, imaging, electrophysiological, and chemogenetic approaches to sensory preconditioning procedures in mice, the present study shows how the physiological inhibition of specific hippocampal GABAergic neuronal populations by CB 1 R is crucial for incidental associations between low-salience stimuli, eventually leading to mediated learning.

Type 1 cannabinoid receptors (CBR) are key neuromodulatory elements of synapses, and they are the main targets of endogenous signaling molecules, the endocannabinoids, forming the so-called endocannabinoid system (ECS) (). Through CBR, the ECS has been involved in direct conditioning such as fear conditioning () or conditioned taste aversion (). Notably, the involvement of the ECS in direct conditioning appears to be more prominent in the modulation of behavioral expression of the acquired memory, rather than its formation (). Regarding hippocampus, CBR are mainly expressed in GABAergic neurons (), where they negatively control inhibitory neurotransmission (), thereby modulating synaptic plasticity () and cognitive processes (). Although hippocampal CBR have been implicated in the cognitive impairment produced by exogenous cannabinoids (), no study has addressed the physiological role of the ECS in higher-order learning such as sensory preconditioning.

Anatomical distribution of receptors, ligands and enzymes in the brain and the spinal cord: circuitries and neurochemistry.

Sensory preconditioning is a typical behavioral procedure to study mediated learning (). In this protocol, pairings of two low-salience stimuli (e.g., odors, tastes, lights, tones) are followed by classical conditioning of one of these stimuli with an aversive or appetitive unconditioned reinforcer (). As a result of these associations, subjects present aversion or preference to the stimulus never explicitly paired with the reinforcer, thereby allowing the evaluation of mediated learning (). Thus, three distinct and temporally successive processes occur in sensory preconditioning. First, an incidental association is formed between low-salience stimuli during the preconditioning phase. Second, direct association with a reinforcer increases the salience of one of the stimuli during the conditioning phase. Finally, exposing the subjects to either of the original stimuli (the one directly associated with the reinforcer and the one never associated) reveals the retrieval of direct and mediated memories, respectively. The neurobiological mechanisms of these connected but distinct processes are poorly understood. The hippocampus has been suggested to play an important role in the conditioning and retrieval phases of sensory preconditioning procedures (). However, its involvement in the formation of initial incidental associations leading to mediated learning is unknown.

Direct associative memories, in which a sensory stimulus is explicitly paired with a negative or rewarding outcome, can determine daily behavioral choices. Very often, however, human behavior is governed by mediated learning, based on previous events implying incidental associations between low-salience sensory cues (). In other words, we are often repulsed or attracted by stimuli never explicitly paired with negative or positive outcomes but previously associated with other stimuli paired with a specific aversive or rewarding meaning (). These processes are well conserved in all mammals, including rodents (). However, whereas the biological mechanisms underlying direct associative learning are under intense scrutiny (), much less is known about the neural substrates mediating sensory stimulus-stimulus associations leading to higher-order mediated learning ().

These data are in agreement with the idea that hippocampal inhibitory neurotransmission plays a key role in the formation of incidental associations. However, generalized activation of GABAergic neurons by DREADD-Gq does not allow determination of whether there is a specific population of GABAergic neurons modulating mediated learning. In this context, it is important to note that CBR are expressed in specific interneuronal subpopulations in the hippocampus, CCK-positive but not in parvalbumin (PV)-positive basket cells (). CCK- and PV-positive interneurons are two functionally and anatomically distinct cell populations, which together encompass the large majority of inhibitory basket cells innervating somas and proximal dendrites of hippocampal pyramidal neurons (). Due to the additional expression of CCK in pyramidal hippocampal neurons, the specific targeting of CCK- and CBR-positive interneurons is technically very challenging and laborious (). Conversely, the use of PV-Cre mice () allows a reliable targeting of PV-positive interneurons, which represent approximately half of the hippocampal basket cells (). Therefore, we injected adeno-associated viruses expressing Cre-dependent DREADD-Gq into the hippocampi of PV-Cre mice, obtaining the localization of the receptor in PV-positive neurons (>85% colocalization, Figure 6 D), thereby generating PV-DREADD-Gq mice. First, we verified the functionality of the approach, by showing that CNO administration was able to increase the number of c-Fos-positive neurons in the hippocampus of these mice ( Figure S6 I). Strikingly, the specific activation of PV interneurons by CNO administration during preconditioning did not affect mediated learning ( Figures 6 D, S6 J, and S6K). These data suggest that specific PV-negative subpopulations of GABAergic hippocampal interneurons are involved in the development of incidental associations between low-salience stimuli, eventually enabling the successive formation of mediated learning and memory.

Anatomical distribution of receptors, ligands and enzymes in the brain and the spinal cord: circuitries and neurochemistry.

The data presented so far imply a key role of the inhibition of hippocampal GABAergic transmission during the processing of incidental associations between low-salience stimuli and related synaptic plasticity. Therefore, stimulation of GABAergic transmission in the hippocampus of mice expressing normal levels of CBR should impair these processes and, thereby, block mediated learning. To test this idea, an adeno-associated viral vector carrying Cre-dependent expression of an excitatory DREADD (DIO-hM3DGq, hereafter called DREADD-Gq) () was infused into the hippocampi of mice expressing the Cre recombinase in Dlx5/6-positive cells ( Figure 6 C), encompassing all GABAergic interneurons (GABA-Cre mice;) to obtain GABA-DREADD-Gq mice. We first verified that CNO administration was able to increase the number of cFos-positive neurons in the hippocampus of these mice ( Figure S6 E) and that CNO treatment did not alter mediated or direct aversion in GABA-Cre mice infused with a control Cre-dependent virus (GABA-mCherry; Figure S6 F). Notably, however, CNO administration during preconditioning fully blocked mediated, but not direct, aversion in GABA-DREADD-Gq mice ( Figures 6 C, S6 G, and S6H).

Considering the inhibitory role of CBR on hippocampal GABAergic neurotransmission () and the enhancement of I-LTD sensitivity induced by preconditioning training, lack of CBR-dependent inhibition of hippocampal GABAergic transmission during incidental associations might be responsible for the impairment of mediated learning found in CBR-KO, HC-CBR-KO, and GABA-CBR-KO mice. In other words, the lack of CBR-dependent control might induce excessive inhibitory transmission, impairing incidental learning. To test this hypothesis, we aimed at reducing hippocampal GABAergic transmission in GABA-CBR-KO mutant mice during preconditioning, i.e., during each odor-taste pairing, using a chemogenetic approach ( Figure 6 A). An adeno-associated viral vector carrying Cre-dependent expression of an inhibitory DREADD (DIO-hM4DGi, hereafter called DREADD-Gi) () was infused into the hippocampi of GABA-CBR-KO mice, leading to specific expression of DREADD-Gi in hippocampal GABAergic neurons to generate GABA-CBR-KO (DREADD-Gi) ( Figure 6 B). To control for the potential effects of CNO in virally infected mice lacking DREADD receptors (), GABA-CBR-WT mice were infused with a control AAV expressing the mCherry protein (GABA-CBR-WT [mCherry]). The DREADD ligand clozapine-N-oxide (CNO) enhanced hippocampal in vivo LTP in GABA-CBR-KO (DREADD-Gi) mice as compared to saline-treated mice ( Figure S6 A), thereby indicating the effectiveness of the chemogenetic approach. Importantly, however, CNO injections into GABA-CBR-WT (mCherry) mice did not affect the consumption during preconditioning ( Figure S6 B) and had no effect on mediated ( Figures 6 B and S6 C) or direct aversion ( Figures 6 B and S6 D), showing that the potential unspecific effects of the drug or its metabolites () were not present in our experimental conditions. Strikingly, however, the same treatment in GABA-CBR-KO (DREADD-Gi) mice fully rescued mediated learning ( Figures 6 B and S6 C), without altering preconditioning consumption or direct aversion ( Figures 6 B, S6 B, and S6D). These results suggest that an excess of inhibitory neurotransmission during the formation of incidental stimulus-stimulus associations might be the cause of the mediated learning impairment in GABA-CBR-KO mice.

(D) Top, representative micrograph image showing the mCHERRY viral expression, the PV interneurons and the merged image in the CA1 region of the hippocampus of PV-CRE mice (see Figure S6 for further details; scale bar, 100 μm). Middle bottom, effect of chemogenetic activation of hippocampal PV neurons during preconditioning on mediated (middle) and direct (bottom) aversion in PV-DREADD-Gq mice. Note the lack of impairment after CNO treatment.p < 0.05;p < 0.01;

(C) Top, representative micrograph image showing the viral expression of Cre-dependent DREADD-Gq in the hippocampus of GABA-CRE mice. Middle bottom, effect of chemogenetic activation of hippocampal GABAergic neurons during preconditioning on mediated (middle) and direct (bottom) aversion in GABA-DREADD-Gq mice (see Figure S6 for further details; scale bar, 300 μm). Note mediated learning impairment after CNO treatment.

(B) Top, representative micrograph image showing the viral expression of Cre-dependent DREADD-Gi in the hippocampus of GABA-CBR-KO mice. Middle bottom, effect of chemogenetic inhibition of hippocampal GABAergic neurons during preconditioning on mediated (middle) and direct (bottom) aversion in GABA-CBR-KO mice (see Figure S6 for further details; scale bar, 300 μm). Note the reversal of the mediated learning impairment in the mutants after CNO treatment.

Altogether, these results show that exposure to three odor-taste pairings increases protein synthesis-dependent expression of CB 1 R, CREB phosphorylation, in vivo LTP, and cellular sensitivity to endocannabinoid-dependent I-LTD in the hippocampus. However, whereas the effects on CREB phosphorylation and LTP are also triggered by the mere exposure to low-salience stimuli such as odors or tastes alone, the enhancements of CB 1 R expression and I-LTD amplitude and sensitivity are specifically related to preconditioning odor-taste pairings, suggesting their involvement in the formation of incidental associations and the expression of mediated learning.

Hippocampal CBR in GABAergic neurons control inhibitory transmission and plasticity, such as short-term depolarization-induced suppression (DSI) or long-term depression (I-LTD) of hippocampal evoked inhibitory post-synaptic currents (eIPSCs;). Hippocampal slices were prepared from mice exposed to three odor-taste pairings or control conditions, and inhibitory synaptic plasticity was analyzed. Preconditioning altered neither basal inhibitory neurotransmission as assessed by measuring miniature IPSCs ( Figure S5 F) nor short-term plasticity as determined by DSI measurements ( Figure S5 G). As previously described (), we observed that the application of two HFS trains induced a reliable I-LTD in 71% of pyramidal neurons from naive control mice ( Figures 5 D and 5E). Odor-taste preconditioning procedures involve limited access to water (1 hr per day;; see Star Methods ). Surprisingly, only 26% of pyramidal neurons in hippocampal slices from water-restricted control mice only exposed to plain water underwent I-LTD after HFS ( Figure 5 E), indicating an impact of limited access to water on this form of synaptic plasticity. However, paired odor-taste exposures in equally water-restricted mice rescued I-LTD expression to levels undistinguishable from naive animals in both amplitude ( Figure 5 D) and percentage of responsive cells ( Figure 5 E). Importantly, these effects were not present in mice exposed either to taste or odor alone ( Figures 5 D and 5E).

CBR activation participates in the regulation of synaptic transmission and plasticity (). Therefore, we asked whether odor-taste pairings impacted in vivo hippocampal long-term potentiation (LTP) induced by high-frequency stimulation (HFS) of the Schaffer collateral–CA1 pathway (). Exposure to three odor-taste pairings enhanced in vivo hippocampal LTP in wild-type mice, but not in GABA-CBR-KO ( Figures 5 B, 5C, and S5 E). However, control experiments indicated that exposure to the taste alone also increased in vivo hippocampal LTP in WT, but not in GABA-CBR-KO mice ( Figures 5 B and 5C).

Intracellular CBR signaling involves many different pathways, including extracellular-regulated kinases (ERKs), mechanistic target of rapamycin (mTOR), and the cAMP response element-binding protein (CREB) (). Neither the phosphorylation of ERKs nor the activation of the mTOR pathway was affected by exposure to three odor-taste pairings (data not shown). Conversely, CREB phosphorylation was enhanced after three pairings, but not after extended training ( Figure S5 D). However, whereas exposure to taste alone did not affect CREB phosphorylation, odor alone promoted it ( Figure S5 D).

First, we investigated whether incidental learning specifically modulates CBR expression in the hippocampus. Immunoblotting experiments revealed that 3 odor-taste pairings, but not the same number of presentations of odor alone, taste alone, or unpaired odor-taste, specifically resulted in enhanced hippocampal expression of CBR protein ( Figures 5 A, S5 A, and S5B). Extended preconditioning training is known to suppress the expression of mediated learning (), suggesting that increasing the number of stimuli associations could trigger cellular and molecular changes. Intriguingly, we observed that hippocampal CBR expression was normalized by extended preconditioning training (six odor-taste pairings) ( Figures 5 A and S5 B), further suggesting that increased levels of hippocampal CBR protein are reliably associated to the expression of mediated learning. To investigate the molecular mechanisms of this effect, we asked whether the enhancement of CBR expression was due to new protein synthesis during the preconditioning phase. The administration of the protein synthesis inhibitor Anisomycin (18 mg/kg, i.p.;) before each odor-taste pairing blunted the increase of CBR expression ( Figure S5 C).

(E) Top, traces of eIPSCs before and after HFS in representative recordings resulting in I-LTD (left) or no I-LTD (right). I-LTD was defined as a ≥15% reduction of eIPSCs as compared to baseline. Bottom, black bar histograms representing the proportion of cells displaying I-LTD in the same groups as described in (D).p < 0.05;p < 0.01 andp < 0.001 as compared to Water or KO conditions.p < 0.05 andp < 0.01 as compared to baseline. NS, not significant. For statistical details and n, see Tables S1 and S2

(D) Left, time course plots showing eIPSCs amplitude before and after HFS in hippocampal slices obtained from non-water-restricted mice (Naive), or mice that received water, three odor-taste pairings (3 OT pairings), odor alone (Odor), or taste alone (Taste). Right, averaged eIPSCs recorded 15–20 min after HFS in the same groups.

(C) Bar histogram representing LTP amplitude during the last 3 min of recording in the same groups as described in (B).

(B) Summary traces (top) and time course plots of normalized fEPSPs recorded in vivo before (1) and after (2) high-frequency stimulation (HFS) in the CA1 hippocampal region of GABA-CB 1 R-WT mice (left) and GABA-CB 1 R-KO littermates (right). Animals were anesthetized for recording immediately after the last exposure to water, odor-taste (3 OT pairings), taste alone, or odor alone, respectively.

(A) Optical densitometric quantification of CBR levels in mice that received water, three odor-taste pairings (three OT pairings), odor alone (Odor), taste alone (Taste), unpaired odor-taste exposures (Unp. OT), or six odor-taste pairings (6 OT) (see Figure S5 for the representative gels and controls).

The results showed above suggest a key role of the hippocampus and of GABAergic neurons in the modulation of odor-taste mediated learning. Thus, we next aimed at determining whether preconditioning recruits classical hippocampal mechanisms associated with memory formation, such as protein synthesis and synaptic plasticity, and the involvement of inhibitory transmission in these processes.

Altogether, these data indicate that, within distinct cell type-specific populations of CBR (), those present in GABAergic neurons are specifically involved in odor-taste mediated learning.

Anatomical distribution of receptors, ligands and enzymes in the brain and the spinal cord: circuitries and neurochemistry.

The large majority of hippocampal CBR are present in a specific subpopulation of GABAergic interneurons, namely the cholecystokinin (CCK)-expressing perisomatic basket cells (). To study the role of this specific population of CBR, we first used conditional mutant mice lacking the receptor from forebrain GABAergic neurons (Dlx5/6-CBR-KO mice, generally and hereafter called GABA-CBR-KO;). Notably, these animals displayed a specific impairment of mediated aversion independently of the sensory modality ( Figures 4 A, 4B, S4 A, and S4B), accompanied by normal direct aversion ( Figures 4 C, 4D, S4 C, and S4D). This impairment might be due to a delayed formation of mediated learning. However, when exposed to an extended preconditioning training (six odor-taste pairings;), GABA-CBR-KO mice were still unable to display mediated aversion ( Figures S4 E and S4F), strongly suggesting that “GABAergic” CBR are necessary for the formation of incidental learning independently of the training intensity. In addition, “rescue” mice carrying expression of the CBR protein exclusively in forebrain GABAergic neurons (GABA-CBR-RS) () displayed normal mediated aversion, both in the taste or odor modalities, in contrast to littermates globally lacking CBR expression (STOP-CBR mice; Figures 4 E, 4F, S4 G, and S4H;), with no effect on direct aversion ( Figures 4 G, 4H, S4 I, and S4J).

(E–H) Liquid consumption under conditions of mediated taste (E) or odor (F) aversion and direct odor (G) or taste aversion (H) in mice carrying exclusive expression of CBR in forebrain GABAergic neurons (GABA-CBR-RS) and in littermates lacking CBR expression (STOP-CBR).p < 0.05;p < 0.01;p < 0.001 (mCS+ versus mCS− or CS+ versus CS). For statistical details, see Tables S1 and S2

(A–D) Liquid consumption under conditions of mediated taste (A) and odor (B) aversion and direct odor (C) and taste (D) aversion in mice lacking CB 1 R in forebrain GABAergic neurons (GABA-CB 1 R-KO) and wild-type littermates (GABA-CB 1 R-WT).

Anatomical distribution of receptors, ligands and enzymes in the brain and the spinal cord: circuitries and neurochemistry.

Inactivation and functional imaging experiments have suggested the involvement of the hippocampus in mediated learning (), but the role of hippocampal CBR in these processes have never been investigated. To address this issue, we injected adeno-associated viruses (AAVs) expressing Cre recombinase or control green fluorescent protein (GFP) into the hippocampus of CBR-flox mice () to generate mice lacking CBR in the hippocampus and control littermates with normal expression of the receptor (called HC-CBR-KO and HC-CBR-WT, respectively; Figures 3 A and S3 A). HC-CBR-KO mice displayed an impairment of mediated, but not direct, aversion in the odor-taste protocol, regardless of the sensory modality ( Figures 3 B, 3C, and S3 B–S3E). The lack of mediated learning after three odor-taste pairings was not due to slower incidental learning as mice undergoing extended preconditioning using six odor-taste pairings () did not show mediated aversion ( Figures S3 F and S3G). We next investigated whether hippocampal CBR are sufficient to allow mediated learning. Notably, the viral re-expression of CBR in the hippocampus of CBR-KO mice (HC-CBR-RS mice; Figures 3 D and S3 A;) fully rescued mediated learning ( Figures 3 E, S3 H, and S3I). Altogether, these data show that hippocampal CBR are necessary and sufficient for mediated learning.

(E) Effects of exclusive hippocampal re-expression of CBR on mediated taste (left) and direct odor (right) learning.p < 0.05;p < 0.01 (mCS+ versus mCS− or CS+ versus CS−). For statistical details and n, see Tables S1 and S2

(B and C) (B) Effects of specific hippocampal deletion of CB 1 R on mediated taste (left) and direct odor (right) learning and (C) on mediated odor (left) and direct taste (right) learning.

Next, we evaluated whether CBR activation was required also in another sensory preconditioning protocol using different sensory stimuli (visual and auditory cues) and unconditioned stimulus (food reward). To this aim, we adapted an existing paradigm originally developed in rats using operant chambers and visual/auditory cues ( Figure 2 H). Mice treated with vehicle or Rimonabant (1 mg/kg, i.p.) were exposed to two preconditioning sessions consisting in pairings between a light and a tone (preconditioned cues), and to presentations of a click alone (unpaired cue). Next, the light was paired with food delivery and the visits of the food magazine in response to tone and click presentations were evaluated in the absence of the food reward (test session). Vehicle-treated mice displayed mediated conditioning as shown by a discrimination index indicating the increased visits of the food magazine during tone presentations as compared to the click ( Figure 2 I). Interestingly, mediated learning was specifically prevented by the Rimonabant treatment during the preconditioning phase ( Figure 2 I), whereas both vehicle- and Rimonabant-treated mice exhibited comparable levels of direct conditioning to the light ( Figures 2 J and S2 K). Altogether, these results indicate that mediated learning relies on CBR activation specifically during the formation of incidental associations, regardless of the sensory modalities used and of the nature (aversive or appetitive) of the reinforcers.

Long-lasting absence of CBR in CBR-KO mice might induce chronic alterations, eventually causing the observed phenotype in mediated learning. To test whether CBR are acutely required during incidental associations, we administered the CBR antagonist Rimonabant (1 mg/kg, i.p.) before each session of preconditioning ( Figures 2 A, S1 A, and S1B). This treatment impaired mediated learning, both in the taste and odor modalities, without affecting either direct aversions ( Figures 2 B–2E and S2 A–S2D). Importantly, Rimonabant treatment did not alter the total liquid consumption during preconditioning ( Figure S2 E), nor did it affect the lack of taste-induced odor conditioning during odor-taste pairings ( Figures S2 F–S2H). Importantly, Rimonabant administration immediately before the test did not affect mediated odor aversion ( Figures 2 F, 2G, S2 I, and S2J) or mediated taste aversion (), suggesting that CBR activation is specifically required for the formation of incidental associations, but not for the expression of mediated aversion.

(I and J) (I) Effect of daily preconditioning administrations of Rimonabant (1 mg/kg, i.p.) and its vehicle on mediated tone learning, expressed as discrimination ratio, and (J) on the conditioning to light in C57BL/6-J mice (see Star Methods for further details).p < 0.05;p < 0.01; ***p<0.001 (mCS+ versus mCS- or CS+ versus CS-).p < 0.001 (vehicle- versus Rimonabant-treated mice). For statistical details and n, see Tables S1 and S2

(F and G) Effect of acute administration of Rimonabant (1 mg/kg, i.p.) and its vehicle before testing on mediated odor (F) and direct taste aversion (G) in C57BL/6-N mice.

(D and E) Effect of daily preconditioning administrations of Rimonabant (1 mg/kg, i.p.) and its vehicle on mediated odor (D) and direct taste aversion (E) in C57BL/6-N mice.

(B and C) Effect of daily preconditioning administrations of the CB 1 R antagonist Rimonabant (1 mg/kg, i.p.) and its vehicle on mediated taste (B) and direct odor aversion (C) in C57BL/6-N mice.

Mice were exposed to a preconditioning phase with three odor-taste pairings (sucrose and maltodextrin as tastes; banana and almond as odors), followed by a conditioning phase consisting in the devaluation of one of the two stimuli ( Figures 1 A, S1 A, and S1B). First, we controlled that the potential salience of taste stimuli () was not sufficient to induce any type of observable direct conditioning in our experimental protocol ( Figures S1 C and S1D). Then we observed that wild-type mice consumed lower amounts of tastes or odors that were indirectly (preconditioned) or directly devaluated, indicating the formation of reliable mediated and direct aversion learning, respectively ( Figures 1 B–1E and S1 E–S1H). Interestingly, global CBR knockout mice (CBR-KO) displayed impaired mediated aversion independently of the sensory modality ( Figures 1 B, 1C, S1 E, and S1F), still maintaining normal expression of direct learning ( Figures 1 D, 1E, S1 G, and S1H), thereby demonstrating that CBR are specifically required for mediated learning.

(D and E) Liquid consumption under conditions of direct odor (D) or taste (E) aversion in CBR-KO mice and CBR-WT.p < 0.05;p < 0.001 (mCS+ versus mCS− or CS+ versus CS−). For statistical details and n, see Tables S1 and S2

(B and C) Liquid consumption under conditions of mediated taste (B) or odor (C) aversion in CB 1 R-KO mice and wild-type littermates (CB 1 R-WT).

Discussion

Bornstein et al., 2017 Bornstein A.M.

Khaw M.W.

Shohamy D.

Daw N.D. Reminders of past choices bias decisions for reward in humans. Shohamy and Wagner, 2008 Shohamy D.

Wagner A.D. Integrating memories in the human brain: hippocampal-midbrain encoding of overlapping events. Wimmer and Shohamy, 2012 Wimmer G.E.

Shohamy D. Preference by association: how memory mechanisms in the hippocampus bias decisions. Bornstein et al., 2017 Bornstein A.M.

Khaw M.W.

Shohamy D.

Daw N.D. Reminders of past choices bias decisions for reward in humans. Shohamy and Wagner, 2008 Shohamy D.

Wagner A.D. Integrating memories in the human brain: hippocampal-midbrain encoding of overlapping events. Wimmer and Shohamy, 2012 Wimmer G.E.

Shohamy D. Preference by association: how memory mechanisms in the hippocampus bias decisions. Gewirtz and Davis, 2000 Gewirtz J.C.

Davis M. Using pavlovian higher-order conditioning paradigms to investigate the neural substrates of emotional learning and memory. Parkes and Westbrook, 2011 Parkes S.L.

Westbrook R.F. Role of the basolateral amygdala and NMDA receptors in higher-order conditioned fear. 1 R-dependent control of discrete subpopulations of hippocampal GABAergic interneurons is necessary and sufficient for the processing of incidental stimulus-stimulus associations. Accordingly, incidental associations induce a specific protein synthesis-dependent increase of hippocampal CB 1 R expression, which is accompanied by an enhancement of hippocampal sensitivity to CB 1 R-dependent synaptic plasticity. Thus, our data are compatible with a scenario in which incidental associations of different stimuli would trigger CB 1 R activity and induce CB 1 R-dependent synaptic plasticity, which would “prime” hippocampal circuits allowing mediated learning. In this context, our data reveal that the CB 1 R-dependent control of hippocampal inhibitory synaptic neurotransmission is a key element of high-order cognitive processes, which allow the flexible use of the complex and changing patterns of sensory information that characterize daily individual experiences. Thus, the hippocampal ECS appears to enhance the repertoire of possible behavioral choices dictated by previous experiences, thereby increasing the survival potential of individuals. Our daily behavioral choices are mainly based on past experiences. These behaviors can depend on direct associative memories, where sensory stimuli are directly associated with specific aversive or rewarding situations. However, they often originate from previous incidental stimulus-stimulus associations between low-salience sensory cues, which are able to assign new value to stimuli that were never directly reinforced (). Stimulus-stimulus associations can be evaluated through the sensory preconditioning paradigm in humans () and animals (). Using this task, our data strongly suggest that the CBR-dependent control of discrete subpopulations of hippocampal GABAergic interneurons is necessary and sufficient for the processing of incidental stimulus-stimulus associations. Accordingly, incidental associations induce a specific protein synthesis-dependent increase of hippocampal CBR expression, which is accompanied by an enhancement of hippocampal sensitivity to CBR-dependent synaptic plasticity. Thus, our data are compatible with a scenario in which incidental associations of different stimuli would trigger CBR activity and induce CBR-dependent synaptic plasticity, which would “prime” hippocampal circuits allowing mediated learning. In this context, our data reveal that the CBR-dependent control of hippocampal inhibitory synaptic neurotransmission is a key element of high-order cognitive processes, which allow the flexible use of the complex and changing patterns of sensory information that characterize daily individual experiences. Thus, the hippocampal ECS appears to enhance the repertoire of possible behavioral choices dictated by previous experiences, thereby increasing the survival potential of individuals.

Yiannakas and Rosenblum, 2017 Yiannakas A.

Rosenblum K. The Insula and Taste Learning. Wheeler et al., 2013 Wheeler D.S.

Chang S.E.

Holland P.C. Odor-mediated taste learning requires dorsal hippocampus, but not basolateral amygdala activity. 1 R signaling impairs mediated learning when either tastes or odors are used as cues to elicit aversion. Thus, we can conclude that our odor-taste preconditioning paradigm is suitable to identify the formation of incidental associations between stimuli that are of low salience for the mice. In line with this idea, the general implication of the ECS in incidental learning extends also to other sensory preconditioning paradigms, in which low-salience auditory and visual stimuli are used and appetitive behavior is evaluated. Thus, the common involvement of the ECS in different experimental conditions suggests that similar mechanisms might underline higher-order cognitive processes independently of the sensory modalities used and of the nature (aversive or appetitive) of the reinforcer. Future studies will address this intriguing hypothesis that would lead to a unified vision of complex higher-order learning processes in the brain. Importantly, these mechanisms do not appear to be limited to specific sensory modalities of the stimuli. Despite the fact that tastes can also act as primary reinforcers (), gustatory cues have been widely used in sensory preconditioning protocols as low-salience stimuli (). Important control experiments showed that these cues are unable to elicit direct odor conditioning per se in our experimental conditions. Moreover, inhibition of CBR signaling impairs mediated learning when either tastes or odors are used as cues to elicit aversion. Thus, we can conclude that our odor-taste preconditioning paradigm is suitable to identify the formation of incidental associations between stimuli that are of low salience for the mice. In line with this idea, the general implication of the ECS in incidental learning extends also to other sensory preconditioning paradigms, in which low-salience auditory and visual stimuli are used and appetitive behavior is evaluated. Thus, the common involvement of the ECS in different experimental conditions suggests that similar mechanisms might underline higher-order cognitive processes independently of the sensory modalities used and of the nature (aversive or appetitive) of the reinforcer. Future studies will address this intriguing hypothesis that would lead to a unified vision of complex higher-order learning processes in the brain.

Voss et al., 2017 Voss J.L.

Bridge D.J.

Cohen N.J.

Walker J.A. A closer look at the hippocampus and memory. Iordanova et al., 2011 Iordanova M.D.

Good M.

Honey R.C. Retrieval-mediated learning involving episodes requires synaptic plasticity in the hippocampus. Talk et al., 2002 Talk A.C.

Gandhi C.C.

Matzel L.D. Hippocampal function during behaviorally silent associative learning: dissociation of memory storage and expression. Wheeler et al., 2013 Wheeler D.S.

Chang S.E.

Holland P.C. Odor-mediated taste learning requires dorsal hippocampus, but not basolateral amygdala activity. Wimmer and Shohamy, 2012 Wimmer G.E.

Shohamy D. Preference by association: how memory mechanisms in the hippocampus bias decisions. The hippocampus has been suggested to be necessary for associating and temporarily maintaining an internal record of different stimuli simultaneously presented (), making it a key brain region for the integration of different sensory information. Accordingly, previous studies supported the importance of the hippocampus in sensory preconditioning both in humans and in animals (). However, these studies focused on the role of the hippocampus during the second phase of the procedure (conditioning) or during the retrieval test, when incidental associations have already formed. Thus, these studies underlined the role of the hippocampus in enabling the value of the reinforcer to spread across the associated and not explicitly reactivated item, rather than in the processing and recording of incidental associations per se. Conversely, the present results reveal that the hippocampus is also involved in the preconditioning phase, when the coincidence of low-salience stimuli is recorded and stored for future potential use. Specifically, our data provide an unforeseen physiological link between hippocampal GABAergic signaling and associative memory between low-salience events.

The cellular and molecular mechanisms underlying ECS-dependent control of incidental learning in the hippocampus appear to be quite complex. Exposure to low-salience stimuli enhances in vivo hippocampal LTP and CREB phosphorylation. However, these effects are not specific to incidental associations, because they are also induced by the mere exposure to gustatory or olfactory stimuli alone, respectively. On the other hand, the simultaneous presentation of stimuli induces specific molecular and cellular effects. Incidental learning causes a protein synthesis-dependent increase of CB 1 R expression and higher hippocampal sensitivity to ECS-dependent plasticity of inhibitory neurotransmission. The increased sensitivity of hippocampal pyramidal neurons to undergo I-LTD might be due to the increase of CB 1 R expression. However, the fact that preconditioning does not alter other forms of ECS-dependent plasticity, such as DSI, suggests that the enhanced I-LTD sensitivity might result from other mechanisms, such as, for instance, increased endocannabinoid mobilization following HFS. Whereas enhancement of hippocampal CB 1 R expression and I-LTD sensitivity are specific to odor-taste preconditioning (i.e., they do not occur under exposure to odors or tastes alone), the increased in vivo LTP induced by both odor-taste and taste alone is abolished in GABA-CB 1 R-KO mice. Differently from I-LTD, this ECS-dependent phenomenon and the enhanced phosphorylation of CREB cannot be linked to increased CB 1 R expression levels, because they occur in conditions where these levels are not changed (i.e., under exposure to single stimulus). Future experiments will address these complex molecular and cellular interactions. However, the present data allow speculating that incidental learning might occur via a “two-step” hippocampal CB 1 R-dependent process: (1) First, animals might record the presence of stimuli independently of their possible association. CB 1 R-dependent increase of LTP might underline this process, at least for taste-related information. (2) The simultaneous presence of odors and tastes then triggers additional processes, again involving the ECS (increased CB 1 R expression and I-LTD sensitivity). In this frame, step (1) might represent a preliminary event (stimulus detection) that is necessary to further develop step (2), which would underline the “actual” incidental learning. Future studies will address these intriguing possibilities.

Busquets-Garcia et al., 2017b Busquets-Garcia A.

Soria-Gómez E.

Redon B.

Mackenbach Y.

Vallée M.

Chaouloff F.

Varilh M.

Ferreira G.

Piazza P.V.

Marsicano G. Pregnenolone blocks cannabinoid-induced acute psychotic-like states in mice. McLaren and Mackintosh, 2002 McLaren I.P.

Mackintosh N.J. Associative learning and elemental representation: II. Generalization and discrimination. 1 R. Thus, it will be extremely interesting to investigate the causes and the consequences of these molecular adaptations in the training-dependent behavioral switch from mediated learning to “reality testing” responses ( McDannald and Schoenbaum, 2009 McDannald M.

Schoenbaum G. Toward a model of impaired reality testing in rats. Our previous data revealed that extended preconditioning training (six odor-taste pairings instead of three) suppresses mediated learning, installing the so-called “reality testing,” through which the animals are able to successfully discriminate between stimuli that they initially reacted to in the same way (). Notably, extended training also normalizes the hippocampal expression of CBR. Thus, it will be extremely interesting to investigate the causes and the consequences of these molecular adaptations in the training-dependent behavioral switch from mediated learning to “reality testing” responses ().